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Tillyer, C. R., S. Rakhorst e C. M. Colley. "Multicomponent analysis for alkaline phosphatase isoenzyme determination by multiple linear regression". Clinical Chemistry 40, n.º 5 (1 de maio de 1994): 803–10. http://dx.doi.org/10.1093/clinchem/40.5.803.
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Abstract Alkaline phosphatase (EC 3.1.3.1) isoenzymes in serum may be determined by multicomponent analysis of the enzyme activities in the presence of multiple inhibitors. To determine inhibition coefficients of the isoenzymes, we used multiple linear regression analysis to compare alkaline phosphatase activities in the presence of known inhibitors with electrophoretically determined isoenzyme activities in plasma and serum samples. All possible combinations of exactly determined and overdetermined linear systems of inhibitors were ranked according to their prediction error to select an optimum set. The best multicomponent system for prediction included the use of levamisole, phenylalanine, and heat inhibition at 56 degrees C and 65 degrees C to determine bone, hepatic, intestinal, and placental isoenzymes. Consideration of the hepatic isoenzyme as liver and macromolecular fractions resulted in significantly worse predictions. Error analysis involving repeat determinations and a simplex optimization of the inhibition coefficients indicated that the inaccuracy of the comparison electrophoretic method may have been a major factor affecting poor isoenzyme prediction in some samples.
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Dunaway, G. A., T. P. Kasten, T. Sebo e R. Trapp. "Analysis of the phosphofructokinase subunits and isoenzymes in human tissues". Biochemical Journal 251, n.º 3 (1 de maio de 1988): 677–83. http://dx.doi.org/10.1042/bj2510677.
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The 6-phosphofructo-1-kinase (PFK) subunits and isoenzymes were studied in human muscle, heart, brain, liver, platelets, fibroblasts, erythrocytes, placenta and umbilical cord. In each tissue, the subunit types in the native isoenzymes were characterized by immunological titration with subunit-specific antibodies and by column chromatography on QAE (quaternary aminoethyl)-Sephadex. Further, the subunits of the partially purified native isoenzymes were resolved by SDS/polyacrylamide-gel electrophoresis, identified by immunoblotting, and quantified by scanning gel densitometry of silver-stained gels and immunoblots. Depending on the type of tissue, one to three subunits were detected. The Mr values of the L, M and C subunits regardless of tissue were 76,700 +/- 1400, 82,500 +/- 1640 and 86,500 +/- 1620. Of the tissues studied, only the muscle PFK isoenzymes exhibited one subunit, which was the M-type subunit. Of the other tissues studied, the PFK isoenzymes contained various amounts of all three subunits. Considering the properties of the native PFK isoenzymes, it is clear that, in human tissues, they are not simply various combinations of two or three homotetrameric isoenzymes, but complex mixtures of homotetramers and heterotetramers. The kinetic/regulatory properties of the various isoenzyme pools were found to be dependent on subunit composition.
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Yoshikuni, Keiko, Takashi Matsuda, Janka Poracova, Akemi Sakai, Keiko Shimada, Noriko Tabuchi e Kiyoh Tanishima. "Phylogenic Study of Denaturation of Lactate Dehydrogenase Isoenzymes from Different Species by High and Low Temperature". Annals of Clinical Biochemistry: International Journal of Laboratory Medicine 38, n.º 5 (setembro de 2001): 548–53. http://dx.doi.org/10.1177/000456320103800513.
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We studied the influence of storage at different temperatures on lactate dehydrogenase (EC 1.1.1.27; LD) isoenzymes from different tissues and different species, and analysed biochemical and biophysical mechanisms of denaturation during storage. Isoenzymes obtained from tissue extracts of mammals, poultry, reptiles, amphibians and fish were shown to have their own denaturation ranges at low temperatures by post-treatment assays and transition temperature analysis. These ranges were between − 10 and − 20°C for most vertebrate LD isoenzymes. Circular dichroism analysis indicated that the denaturation of LD isoenzymes was probably caused by a change in the hydrophobic interactions in the molecule. At higher temperatures, LD-1 isoenzyme was more thermostable than LD-5 from the same animal species, except for rats, the LD-5 activity of which was more thermostable than the LD-1 activity. These findings indicate that variable effects of storage of samples and reference materials at low temperatures should be considered, and that it is necessary to establish LD isoenzyme standards for animal clinical laboratory investigations.
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Giannoulaki, E. E., D. L. Kalpaxis, C. Tentas e P. Fessas. "Lactate dehydrogenase isoenzyme pattern in sera of patients with malignant diseases." Clinical Chemistry 35, n.º 3 (1 de março de 1989): 396–99. http://dx.doi.org/10.1093/clinchem/35.3.396.
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Abstract Total lactate dehydrogenase (LDH; EC 1.1.1.27) activity and the percentage distribution of LDH isoenzymes were determined in 127 patients with malignant diseases. A shift in the isoenzyme patterns was observed toward the M-type, with an increase in the percentage of LDH-4 and LDH-5 isoenzymes and a slight increase in total LDH activity of all patients. Serum samples from 68 of the patients contained an abnormal isoenzyme of LDH, "LDH-1 ex," that, on agarose gel electrophoresis at pH 8.6, migrated between albumin and LDH-1 isoenzyme. Chemotherapy, radiotherapy, or surgical removal of the tumor was accompanied by disappearance of this abnormal isoenzyme. The heat stability of LDH-1 ex isoenzyme appears to be similar to that of LDH-1 but greater than that of the other LDH isoenzymes. Statistical analysis of these data demonstrated a significant correlation between malignancy and the appearance of LDH-1 ex isoenzyme (P less than 0.001). In contrast, the relationship between LDH-1 ex isoenzyme and metastasis or anatomical location of the malignancy is not statistically important (P less than 0.1).
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Bar, Jair, Stuart Spencer, Shethah Morgan, Laura Pike, David Cunningham, Jane D. Robertson, Juliane M. Jürgensmeier e Glenwood D. Goss. "Correlation of lactate dehydrogenase (LDH) isoenzyme profile with outcome in advanced colorectal cancer (CRC) patients (pts) treated with chemotherapy and bevacizumab (BEV) or cediranib (CED)." Journal of Clinical Oncology 30, n.º 15_suppl (20 de maio de 2012): e13541-e13541. http://dx.doi.org/10.1200/jco.2012.30.15_suppl.e13541.
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e13541 Background: There is a clinical need for predictive biomarkers of efficacy of VEGF signaling inhibitors (VSIs). LDH is a tetramer that can include any combination of the M and H subunits. LDH4 and 5 isoenzymes are composed mostly of the M subunit and are more abundant in hypoxic conditions. We speculated that the levels of LDH isoenzymes in pts serum may correlate with outcome of pts treated with VSIs. HORIZON I trial enrolled pts that were randomized to mFOLFOX6 with BEV or CED. A retrospective exploratory analysis was performed on baseline serum samples of these pts. Methods: Total serum LDH was tested on fresh samples during the conduct of the trial. About 2 years after the trial, frozen samples stored at -70 degrees were tested for total serum LDH and LDH isoenzymes, measured by agarose electrophoresis and a colorimetric enzymatic assay. Relative levels of M and H subunits were derived based on the known structure of each isoenzyme. Progression free survival (PFS) and overall survival (OS) were compared by subgroups of total LDH and LDH isoenzyme levels. P values were not calculated for these exploratory analyses. Results: Baseline total LDH levels from fresh serum were available for 207 pts. Total and isoenzyme LDH levels were available for frozen serum samples of 189 pts. Total LDH in the frozen and fresh samples correlated (R=0.9). Distant isoenzymes (e.g. LDH1 and 5) were negatively correlated. High M/H subunits ratio correlated with poor OS (HR=1.804, 95%CI 1.24-2.620). A non-significant trend for better OS to CED-treated vs. BEV-treated pts was seen in pts with high M/H ratio (e.g., CED 30mg vs BEV HR=0.685, 95%CI 0.382-1.23). Conclusions: Evaluation of LDH isoenzymes is feasible using serum samples kept frozen for 2 years. A negative correlation is seen between hypoxic-related and oxic-related isoenzymes. In CRC pts treated with a VSI, LDH isoenzyme hypoxia-associated profile correlates with poorer outcome. LDH isoenzyme profile as a possible predictive biomarker for benefit from CED vs. BEV requires further investigation.
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Pompeu, Georgia Bertoni, Priscila Lupino Gratão, Victor Alexandre Vitorello e Ricardo Antunes Azevedo. "Antioxidant isoenzyme responses to nickel-induced stress in tobacco cell suspension culture". Scientia Agricola 65, n.º 5 (2008): 548–52. http://dx.doi.org/10.1590/s0103-90162008000500015.
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Exposure to nickel (Ni) at high concentrations can lead to production of reactive oxygen species (ROS) resulting in oxidative damage at the cellular level. We investigated the antioxidative responses of Nicotiana tabacum cv BY-2 cell suspension to Ni stress (0.075 and 0.75 mM NiCl2) over a 72 h period with special attention to potential alterations in isoenzymes of superoxide dismutase (SOD), catalase (CAT) and glutathione reductase (GR). Two main SOD isoenzymes were observed, a Mn-SOD (band I) and a Fe-SOD (band II), as well as one CAT isoenzyme and four GR isoenzymes. Activity staining analysis revealed that CAT activity plays a major role in the early response to Ni-induced oxidative stress, particularly when the Ni concentration used was low, whilst a specific GR isoenzyme appears to respond to the Ni-induced oxidative stress when a much higher Ni concentration was used to induce the stress for the same period of treatment. These results illustrate the importance and advantages of determining individual isoenzyme activities.
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Kochhar, Sunita, Christopher B. Watkins, Patricia L. Conklin e Susan K. Brown. "A Quantitative and Qualitative Analysis of Antioxidant Enzymes in Relation to Susceptibility of Apples to Superficial Scald". Journal of the American Society for Horticultural Science 128, n.º 6 (novembro de 2003): 910–16. http://dx.doi.org/10.21273/jashs.128.6.0910.
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The activities and isoenzyme patterns of guaiacol-dependent peroxidase (POX), ascorbate peroxidase (APX), superoxide dismutase (SOD) and catalase (CAT) were studied in yellow- and red-fruited crab apple [Malus (L.) Mill.] selections from a `White Angel' × `Rome Beauty' cross that show differential susceptibility to the physiological storage disorder, superficial scald. There were no consistent relationships between total enzyme activities and scald incidence, high activities of the enzymes being detected in selections with both high and low susceptibilities to scald. However, additional individual isoforms of some antioxidant enzymes were detected in the scald-resistant selections when compared with scald-susceptible selections. In a native gel system, four guaiacol-dependent POX isoenzymes were detected in both yellow and red scald-resistant selections compared with only two in scald-susceptible selections. Similarly, for anodic acidic POX assayed using benzidine, six isoenzymes were detected in both yellow and red scald-resistant selections compared with five in yellow and four in red susceptible selections. Ten SOD isozymes were detected in scald-resistant yellow-fruited selections compared with only five faint bands in scald-susceptible selections, but similar patterns were not detectable for red-fruited selections. Differences in the presence of various isoenzymes for CAT and APX were also detected among the selections, but associations with scald susceptibility were also affected by fruit color or were inconsistent. The presence or absence of individual isoenzymes may be a better indication of scald resistance or susceptibility than the total enzyme activities. Isoenzyme analyses, especially of POX, could be useful to breeders for the early detection of scald resistance/susceptibility in apples.
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Singh, S. V., A. Kurosky e Y. C. Awasthi. "Human liver glutathione S-transferase ψ. Chemical characterization and secondary-structure comparison with other mammalian glutathione S-transferases". Biochemical Journal 243, n.º 1 (1 de abril de 1987): 61–67. http://dx.doi.org/10.1042/bj2430061.
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The isolation and chemical characterization of the anionic human liver glutathione S-transferase (GST) psi (pI 5.5) are described and compared with other GST isoenzymes reported for rat and human. Amino acid compositional analysis, substrate specificity and isoelectric focusing indicated that GST psi is a unique isoenzyme form of GST. Strikingly, however, amino acid sequence analysis of the N-terminal region indicated that GST psi was identical with GST mu in the first 23 amino acid residues reported. It is likely that these two enzyme forms are at least partially structurally related. In order to investigate further the genetic relationship of GST psi to other reported GST isoenzymes, secondary-structure analysis was performed. Despite substantial differences in the N-terminal-region amino acid sequences of some of the GST isoenzymes, the secondary structure of all the isoenzymes is highly conserved at their N-termini. The general uniformity of the secondary structure of this enzyme class at their N-termini strongly indicated that the observed diversity of these isoenzymes probably occurred as a result of a mechanism of gene duplication followed by divergence rather than a mechanism of convergent evolution.
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Maekawa, M., K. Sudo, K. Iwahara e T. Kanno. "Lactate dehydrogenase inhibition by immunoglobulin G in human serum." Clinical Chemistry 32, n.º 7 (1 de julho de 1986): 1347–49. http://dx.doi.org/10.1093/clinchem/32.7.1347.
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Abstract Low lactate dehydrogenase (LD; EC 1.1.1.27) activity and an abnormal LD pattern in electrophoretograms of LD isoenzymes in the sera of two patients were caused by inhibition of LD by immunoglobulin G. One of these showed inhibitor activity in the serum upon direct analysis, while the other showed activity only after the immunoglobulin was stripped from the LD. As judged from the LD isoenzyme patterns in serum, the LD inhibitor appeared to act against M subunits. However, quantification of binding affinities to each isolated isoenzyme showed that the LD inhibitor had a stronger effect on LD isoenzymes 2 and 3 (H3M1 and H2M2, respectively).
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Segil, N., A. Shrutkowski, M. B. Dworkin e E. Dworkin-Rastl. "Enolase isoenzymes in adult and developing Xenopus laevis and characterization of a cloned enolase sequence". Biochemical Journal 251, n.º 1 (1 de abril de 1988): 31–39. http://dx.doi.org/10.1042/bj2510031.
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As part of a study of glycolysis during early development we have examined the pattern of expression of enolase isoenzymes in Xenopus laevis. In addition, the nucleotide sequence of a cDNA clone coding for the complete amino acid sequence of one enolase gene (ENO1) in X. laevis was determined. X. laevis ENO1 shows highest homology to mammalian non-neuronal enolase. Analysis of enolase isoenzymes in X. laevis by non-denaturing electrophoresis on cellulose acetate strips revealed five isoenzymes. One form was present in all tissues tested, two additional forms were expressed in oocytes, embryos, adult liver and adult brain, and two further forms were restricted to larval and adult muscle. Since enolase is a dimer, three different monomers (gene products) could account for the observed number of isoenzymes. This pattern of enolase isoenzyme expression in X. laevis differs from that of birds and mammals. In birds and mammals the most acidic form is neuron-specific and there is only one major isoenzyme expressed in the liver. RNAase protection experiments showed the presence of ENO1 mRNA in oocytes, liver and muscle, suggesting that it codes for a non-tissue-restricted isoenzyme. ENO1 mRNA concentrations are high in early oocytes, decrease during oogenesis and decrease further after fertilization. Enolase protein, however, is maintained at high concentrations throughout this period.
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DUJARDIN, J. C., A. L. BAÑULS, J. P. DUJARDIN, J. AREVALO, M. TIBAYRENC e D. LE RAY. "Comparison of chromosome and isoenzyme polymorphism in geographical populations of Leishmania (Viannia) peruviana". Parasitology 117, n.º 6 (dezembro de 1998): 547–54. http://dx.doi.org/10.1017/s0031182098003357.
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Five chromosomes and 17 isoenzyme loci were analysed in 4 allopatric populations of Leishmania (Viannia) peruviana, and molecular distances calculated with 2 estimators, Chromosomal Size Difference Index and Jaccard Distance. Chromosome and isoenzyme data were in overall concordance: 13/30 isolates clustered similarly on the dendrograms constructed from the different estimators, and a significant correlation (P<0·001) was observed between the molecular distances calculated from the two sets of characters. This indicates an evolutionary association between chromosomal size polymorphism and isoenzymes. Chromosomes have a faster molecular clock than isoenzymes; twice as many genotypes were identified by chromosome analysis and significant size differences (for a total of up to 500 kb for 5 chromosomes together) were observed within a given zymodeme. Chromosomes most likely represent better indicators of genetic drift than isoenzymes, as suggested by the higher correlation between both estimators of chromosomal size-polymorphism and eco-geography. Some chromosomes might present an adaptive response to environmental variation.
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Palmieri, Gianna, Paola Giardina, Carmen Bianco, Bianca Fontanella e Giovanni Sannia. "Copper Induction of Laccase Isoenzymes in the Ligninolytic Fungus Pleurotus ostreatus". Applied and Environmental Microbiology 66, n.º 3 (1 de março de 2000): 920–24. http://dx.doi.org/10.1128/aem.66.3.920-924.2000.
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ABSTRACT Pleurotus ostreatus is a white rot basidiomycete that produces several extracellular laccase isoenzymes, including phenol oxidase A1b (POXA1b), POXA2, and POXC. POXC was the most abundant isoenzyme produced under all of the growth conditions examined in this study. Copper was the most efficient inducer of laccase activity among the putative inducers tested. The amounts of all of the previously described laccase isoenzymes increased substantially in copper-supplemented cultures. Under these conditions expression of POX isoenzymes was regulated at the level of gene transcription. It is worth noting that poxa1b mRNA was the most abundant induced transcript at all of the growth times analyzed, and the amount of this mRNA increased until day 7. The discrepancy between thepoxa1b transcript and protein amounts can be explained by the presence of a high level of the protein in P. ostreatuscellular extract, which indicated that the POXA1b isoenzyme could be inefficiently secreted and/or that its physiological activity could occur inside the cell or on the cell wall. Moreover, the POXA1b isoenzyme behaved uniquely, as its activity was maximal on the second day of growth and then decreased. An analysis performed with protease inhibitors revealed that the loss of extracellular POXA1b activity could have been due to the presence of specific proteases secreted into the copper-containing culture medium that affected the extracellular POXA1b isoenzyme.
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Cousineau, J. C., A. K. Anderson, H. A. Daubeny e D. J. Donnelly. "Characterization of Red Raspberry Cultivars and Selections Using Isoenzyme Analysis". HortScience 28, n.º 12 (dezembro de 1993): 1185–86. http://dx.doi.org/10.21273/hortsci.28.12.1185.
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Isoenzyme staining of horizontal starch gels was used to characterize 23 cultivars and three advanced selections of red raspberry (Rubus idaeus L.). The genotypes were separable using the enzymes malate dehydrogenase, phosphoglucoisomerase, phosphoglucomutase, and triose phosphate isomerase. In addition, staining for isocitrate dehydrogenase and shikimate dehydrogenase revealed polymorphisms in some cultivars. By combining these results with those obtained for 78 previously tested cultivars, 75 of the 104 (72%) genotypes tested were uniquely characterized using the six isoenzymes.
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Pereira-Lorenzo, Santiago, Ana M. Ramos-Cabrer, Javier Ascasíbar-Errasti e Juan Piñeiro-Andión. "Analysis of Apple Germplasm in Northwestern Spain". Journal of the American Society for Horticultural Science 128, n.º 1 (janeiro de 2003): 67–84. http://dx.doi.org/10.21273/jashs.128.1.0067.
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Spain is the 15th largest apple (Malus ×domestica Borkh.) producer in the world with production depending mainly on foreign cultivars. During the 1970s, a germplasm bank of local cultivars was established in Galicia with the aim of preserving the local resources of northwestern Spain. A total of 408 accessions were studied using 89 morphological characters, with 15 corresponding to phenology, 46 to fruit, 7 to flowers, 11 to leaves, 6 to pests and 4 to diseases. Three variable isoenzymes, PGM E.C.5.3.1.9, PGI E.C.2.7.5.1 and EST E.C.2.7.5.1, were analyzed for 405 accessions and 27 commercial cultivars. The main objectives of this work were 1) to evaluate the inter- and intracultivar variability using morphological characters and isoenzymes, 2) to classify the accessions according to the main sources of variability, and 3) to identify repetitions in the germplasm bank. Principal component analysis (PCA) revealed six main sources of variability in the following order: size of fruit, color of skin, acidity, sweetness, harvest time, and attractiveness. The PCA analysis across 350 accessions produced 42 morphological groups. The 3 isoenzymes produced 190 genotype clusters. Combining morphological classification with the isoenzyme genotypes, we found 31 groups of synonyms involving 82 accessions and 8 more possible groups involving 17 accessions. This result allows the elimination of 53 repetitive accessions from the germplasm bank. Six commercial cultivars were identified as the progenitors of eighteen accessions: `Reineta Blanca' of seven, `Reina de Reinetas' of two, `Reineta de Caux' of eight and `Golden Delicious', `Golden 4187' or `Ozark Gold' of one each. Because inter- and intracultivar variability was high and names given by the growers were not reliable, the suggested selection strategy is to select individual clones among and within cultivars to exploit both the inter- and intracultivar genetic variability.
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YOSHIMURA, Kazuya, Yukinori YABUTA, Masahiro TAMOI, Takahiro ISHIKAWA e Shigeru SHIGEOKA. "Alternatively spliced mRNA variants of chloroplast ascorbate peroxidase isoenzymes in spinach leaves". Biochemical Journal 338, n.º 1 (8 de fevereiro de 1999): 41–48. http://dx.doi.org/10.1042/bj3380041.
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We have previously shown that stromal and thylakoid-bound ascorbate peroxidase (APX) isoenzymes of spinach chloroplasts arise from a common pre-mRNA by alternative splicing in the C-terminus of the isoenzymes [Ishikawa, Yoshimura, Tamoi, Takeda and Shigeoka (1997) Biochem. J. 328, 795–800]. To explore the production of mature, functional mRNA encoding chloroplast APX isoenzymes, reverse transcriptase-mediated PCR and S1 nuclease protection analysis were performed with poly(A)+ RNA or polysomal RNA from spinach leaves. As a result, four mRNA variants, one form of thylakoid-bound APX (tAPX-I) and three forms of stromal APX (sAPX-I, sAPX-II and sAPX-III), were identified. The sAPX-I and sAPX-III mRNA species were generated through the excision of intron 11; they encoded the previously identified sAPX protein. Interestingly, the sAPX-II mRNA was generated by the insertion of intron 11 between exons 11 and 12. The use of this insertional sequence was in frame with the coding sequence and would lead to the production of a novel isoenzyme containing a C-terminus in which a seven-residue sequence replaced the last residue of the previously identified sAPX. The recombinant novel enzyme expressed in Escherichia coli showed the same enzymic properties (except for molecular mass) as the recombinant sAPX from the previously identified sAPX-I mRNA, suggesting that the protein translated from the sAPX-II mRNA is functional as a soluble APX in vivo. The S1 nuclease protection analysis showed that the expression levels of mRNA variants for sAPX and tAPX isoenzymes are in nearly equal quantities throughout the spinach leaves grown under normal conditions. The present results demonstrate that the expression of chloroplast APX isoenzymes is regulated by a differential splicing efficiency that is dependent on the 3´-terminal processing of ApxII, the gene encoding the chloroplast APX isoenzymes.
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Iverson, A. J., A. Bianchi, A. C. Nordlund e L. A. Witters. "Immunological analysis of acetyl-CoA carboxylase mass, tissue distribution and subunit composition". Biochemical Journal 269, n.º 2 (15 de julho de 1990): 365–71. http://dx.doi.org/10.1042/bj2690365.
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Changes in the mass and subunit structure of liver acetyl-CoA carboxylase (ACC) accompany altered nutrition in vivo. Enzyme activity in different tissues and cell lines is also, in part, determined by variations in both total mass and ACC isoenzyme composition. ACC isoenzyme mass and hetero/homo-isoenzyme association were quantified by three sandwich e.l.i.s.a. assays, i.e. an avidin-based assay that measured total isoenzyme mass and two antibody-sandwich assays which measure polypeptide association. Results from the avidin-based assay reveal that the two major isoenzymes, of molecular mass 265 kDa (ACC 265) and 280 kDa (ACC 280), are present in markedly variable concentration in several rat and mouse tissues and in cell lines of rat and mouse origin. Hepatic ACC mass has been reported to be distributed between mitochondrial and cytosolic fractions and to undergo only a change in subcellular distribution without alteration in total mass on induction/repression of activity in vivo [Roman-Lopez, Shriver, Joseph & Alfred (1989) Biochem. J. 260, 927-930]. However, in the present study, immunoblotting and e.l.i.s.a. analysis reveals that, in rat liver, the mass of both isoenzymes is predominantly cytosolic in distribution, is markedly diminished on fasting and rises 6-8-fold on refeeding of a high-carbohydrate diet. These data support the results of several other investigations of hepatic ACC mass, and are consistent with known nutritionally altered changes in ACC mRNA content. By the two antibody-sandwich e.l.i.s.a. assays, isoenzyme complexes either composed of both ACC 280 and 265 or with multiple copies of ACC 265 are detectable in rat liver enzyme; their concentration varies independently of total ACC mass with the nutritional state of the rat, being lowest in fasting and highest on fasting/refeeding. E.l.i.s.a. analysis, applicable to crude tissue/cell extracts, provides a simple, sensitive and quantitative measurement of ACC mass and subunit composition. Its use may permit needed quantitative insight into the role of variable total ACC and isoenzyme mass and of alterations in ACC subunit composition that occur in vivo or in isolated cells in response to a variety of hormonal and nutritional influences.
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Cousineau, J. C., e D. J. Donnelly. "Identification of Raspberry Cultivars in Vivo and in Vitro Using Isoenzyme Analysis". HortScience 24, n.º 3 (junho de 1989): 490–92. http://dx.doi.org/10.21273/hortsci.24.3.490.
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Abstract Isoenzyme analysis of leaf tissue was used to characterize and separate 18 greenhouse-grown red raspberry (Rubus idaeus L.) cultivars and two purple raspberry (R. ×neglectus Peck) cultivars. Five enzymes were found to be useful: Phosphoglu-coisomerase (PGI), phosphoglucomutase (PGM), malate dehydrogenase (MDH), triose phosphate isomerase (TPI), and isocitrate dehydrogenase (IDH). Fourteen of the 20 cultivars were uniquely separated using these enzymes. The remaining six were assigned to three different groups. Staining for additional isoenzymes may lead to the separation of cultivars within these groups. The effect of tissue type on isoenzyme banding patterns was determined by comparing different tissues from three cultivars. No differences were noted, except in the case of the enzyme PGM, for which differences in the relative densities of the bands were seen. For PGM, the best patterns were obtained from mature tissues (leaves, stems, or petioles). Three cultivars were micro-propagated from shoot tips in vitro and were subsequently analyzed by isoenzyme staining. The culture environment was not found to have any effect on isoenzyme phenotype. This result suggests that isoenzyme analysis can be used to identify raspberry cultivars grown in vitro.
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Viallard, J. L., M. R. Ven Murthy e B. Dastugue. "Rapid electrophoretic determination of neuron-specific enolase isoenzymes in serum." Clinical Chemistry 32, n.º 4 (1 de abril de 1986): 593–97. http://dx.doi.org/10.1093/clinchem/32.4.593.
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Abstract This assay procedure for each of the two neuron-specific enolases (alpha gamma and gamma gamma) and the non-neuronal enolase (alpha alpha) in serum involves two steps: electrophoretic separation of the three isoenzymes--alpha alpha, alpha gamma, and gamma gamma--on cellulose acetate, and bioluminescence measurement of total enolase activity. From these data, the activity concentrations (U/L) of the three isoenzymes in serum are calculated. Both measurement steps are based on the enzymatic activity of enolase and thus differ from the immunological methods currently in use, which require the availability of specific antibodies. The method is rapid (approximately 30 min for both steps) and requires only 10 microL of serum for the complete analysis. Studies of normal children and adults, and of patients suffering from neuroblastoma and small-cell lung cancer, show that it is suitable for clinical use. Furthermore, the fact that both neuron-specific isoenzymes of enolase can be systematically separated is an advantage over immunological techniques in determining isoenzyme patterns for pathological samples.
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Cousineau, Johanne C., e Danielle J. Donnelly. "Use of Isoenzyme Analysis to Characterize Raspberry Cultivars and Detect Cultivar Mislabeling". HortScience 27, n.º 9 (setembro de 1992): 1023–25. http://dx.doi.org/10.21273/hortsci.27.9.1023.
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Isoenzyme staining was used to characterize 55 of 78 raspberry cultivars (Rubus idaeus L., R. × neglectus Peck, and R. occidentalis L.). Six enzymes were needed to achieve this characterization: isocitrate dehydrogenase, malate dehydrogenase, phosphoglucoisomerase, phosphoglucomutase, shikimic acid dehydrogenase, arid triose phosphate isomerase. The 23 cultivars that were not uniquely characterized were grouped into eight groups of two and two groups of three and four. Two of these groups comprised black raspberry cultivars, all of which were similar isozymically. Isoenzymes could not distinguish between the cultivar Willamette and a spine-free mutant of the cultivar. Analysis of cultivars obtained from several sources revealed that raspberry cultivar mislabeling exists but is not very prevalent. Regular isoenzyme analysis of raspberry cultivars held by germplasm repositories, certified and other propagators, and breeders is both feasible and advisable for early detection of cultivar mislabeling.
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Ahmad, Riaz, Nazia Nazam, Arshad Khan e Mumtaz Alam. "Significance of Serum Lactate Dehydrogenase and its Isoenzymes During Post-Burn Follow-Up". Journal of Medical Biochemistry 28, n.º 3 (1 de julho de 2009): 176–83. http://dx.doi.org/10.2478/v10011-009-0018-7.
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Significance of Serum Lactate Dehydrogenase and its Isoenzymes During Post-Burn Follow-Up The present study aims to evaluate the role of lactate dehydrogenase (LDH) isoenzymes in thermal burns. A total of 18 patients of both genders with 20 to 50% total burn surface area (TBSA), admitted to the Burn Ward of JN Medical College and Hospital was assessed. These patients were subjected to general and systemic examinations. The sera collected at day 1, 2, 5 and 10 during follow-up of burn patients were used for LDH quantitation. PAGE profiles showed significant differences in the levels of LDH isoenzymes in all the burn subjects (P=0.05). Software analysis of gel-scans showed the presence of five isoenzyme bands of which LDH-1 and -2 are the least contributors. During follow-up, it was observed that the ranking of LDH isoenzymes approaches control values at day 2 in 20% TBSA patients, while in the remaining cases it occurs at day 5. 3D-densitograms indicated high activity of LDH in 50% of TBSA patients even at day 10; however, the relative ranking of these isoenzymes was similar to control values (LDH-4>- 5>-3>-1>-2). We were of the opinion that the high activity of LDH enzyme is due to the enzyme-immunoglobulin-G (LDH-IgG) complex, but surprisingly we did not observe this complex in 50% of burn patients at any of the durations. Therefore, it is suggested that LDH isoenzymes play a role in the pathophysiology of the disease and can be an asset to ascertain the invisible tissue damage. Moreover, the high activity of LDH in 50% of burns is due to some unknown mechanism and not due to the binding of LDH with IgG.
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Cousineau, Johanne C., e Danielle J. Donnelly. "Genetic Analysis of Isoenzymes in Raspberry". Journal of the American Society for Horticultural Science 117, n.º 6 (novembro de 1992): 996–99. http://dx.doi.org/10.21273/jashs.117.6.996.
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The inheritance of five isoenzymes was studied in red and purple raspberry F1 progenies (Rubus idaeus L. and Rubus × neglectus Peck). Isocitrate dehydrogenase (IDH; EC 1.1.1.42) was a dimeric enzyme present in the cytosol and coded for by one locus (Idh-1). Three of the four crosses analyzed at this locus had deviations from expected ratios, possibly caused by its linkage to a recessive lethal gene. Malate dehydrogenase (MDH; EC 1.1.1.37), phosphoglucoisomerase (PGI; EC 5.3.1.9), and triose phosphate isomerase (TPI; EC 5.3.1.1) were dimeric enzymes with two loci. Each of these three enzymes was present in an organelle and in the cytosol for locus 1 and 2, respectively. Phosphoglucomutase (PGM; EC 2.7.5.1) was monomeric with two loci, Pgm-1 and Pgm-2, located in an organelle and the cytosol, respectively. Each allele at Pgm-1 resulted in the formation of two bands. Although most progenies analyzed supported Mendelian inheritance of polymorphic loci (except for Idh-1), there was a higher than expected number of aberrant segregation ratios observed (18.4%). Analysis of 85 pairs of jointly segregating loci revealed a possible linkage group consisting of Mdh-2, Tpi-2, and Pgm-1.
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NOVELLI, G., F. CATIZONE e B. DALLAPICCOLA. "Analysis of isoenzymes in trophoblast cells". Cell Biology International Reports 10, n.º 3 (março de 1986): 149. http://dx.doi.org/10.1016/s0309-1651(86)80003-0.
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Scott, Joseph W., Farris L. Poole e Michael W. W. Adams. "Characterization of Ten Heterotetrameric NDP-Dependent Acyl-CoA Synthetases of the Hyperthermophilic ArchaeonPyrococcus furiosus". Archaea 2014 (2014): 1–9. http://dx.doi.org/10.1155/2014/176863.
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The hyperthermophilic archaeonPyrococcus furiosusgrows by fermenting peptides and carbohydrates to organic acids. In the terminal step, acyl-CoA synthetase (ACS) isoenzymes convert acyl-CoA derivatives to the corresponding acid and conserve energy in the form of ATP. ACS1 and ACS2 were previously purified fromP. furiosusand haveα2β2structures but the genome contains genes encoding three additionalα-subunits. The ten possible combinations ofαandβgenes were expressed inE. coliand each resulted in stable and activeα2β2isoenzymes. Theα-subunit of each isoenzyme determined CoA-based substrate specificity and between them they accounted for the CoA derivatives of fourteen amino acids. Theβ-subunit determined preference for adenine or guanine nucleotides. The GTP-generating isoenzymes are proposed to play a role in gluconeogenesis by producing GTP for GTP-dependent phosphoenolpyruvate carboxykinase and for other GTP-dependent processes. Transcriptional and proteomic data showed that all ten isoenzymes are constitutively expressed indicating that both ATP and GTP are generated from the metabolism of most of the amino acids. A phylogenetic analysis showed that the ACSs ofP. furiosusand other members of the Thermococcales are evolutionarily distinct from those found throughout the rest of biology, including those of other hyperthermophilic archaea.
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Redling, Stephanie, Imke L. Pfaff, Michael Leitges e Volker Vallon. "Immunolocalization of protein kinase C isoenzymes α, βI, βII, δ, and ε in mouse kidney". American Journal of Physiology-Renal Physiology 287, n.º 2 (agosto de 2004): F289—F298. http://dx.doi.org/10.1152/ajprenal.00273.2003.
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Localization of protein kinase C (PKC) isoenzymes α, βI, βII, δ, and ε was studied employing Western blot analysis and immunohistochemical methods including confocal laser-scanning microscopy in the kidney of two mice strains, namely, C57BL/6 and 129/Sv, which have recently been used as genetic backgrounds for respective knockout mice. Immunoblot analysis identified immunoreactive bands for each isoenzyme in total kidney cell extracts. Isoenzyme expression sites were identical for both strains. Glomeruli expressed PKC-α, -βI, and -ε. The latter isoenzme was also detected in apical aspects of proximal convoluted but not in proximal straight tubules. In contrast to rats, neither PKC-α nor PKC-βI was detectable in the proximal tubule. Immunofluorescence was observed in luminal membranes of medullary (MTAL) and cortical thick ascending limbs for PKC-βI and in MTAL for PKC-ε. The cortical collecting duct expressed PKC-α, -βI, and -δ in intercalated cells only. In the outer medullary collecting duct, PKC-α and -βI were detectable in principal cells, whereas PKC-δ was found in intercalated cells. In the inner medullary collecting duct, PKC-α, -βI, and -βII were detected. As described for the rat, the expression of PKC-βII was otherwise restricted to cortical and medullary interstitial cells. The specificity of all labeling was confirmed in respective PKC isoenzyme knockout mice. In summary, distinct expression patterns were shown for PKC isoenzymes α, βI, βII, δ, and ε in the mouse kidney.
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Lessard, F., e R. Dion. "Analysis for alpha-amylase isoenzymes by automated isoelectric focusing." Clinical Chemistry 35, n.º 10 (1 de outubro de 1989): 2116–18. http://dx.doi.org/10.1093/clinchem/35.10.2116.
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Abstract We describe the fractionation of alpha-amylase (EC 3.2.1.1) isoenzymes by use of the Pharmacia "Phastsystem" electrophoresis apparatus. The separation is done by isoelectric focusing on "Phastgel IEF 5-8." A complete run, including staining, takes 4 h and can easily resolve eight isoenzymes. Samples can be analyzed in a routine laboratory with use of conventional reagents.
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Ivashchenko, D. V., A. S. Osipov, E. V. Nazarova, M. A. Ovchinnikova, N. I. Buromskaya, V. V. Smirnov, P. V. Shimanov et al. "Efficacy and safety of antipsychotics in adolescents with an acute psychotic episode in relation to the activity of CYP3A and CYP2D6 isoenzymes". Neurology, Neuropsychiatry, Psychosomatics 12, n.º 6 (12 de dezembro de 2020): 11–18. http://dx.doi.org/10.14412/2074-2711-2020-6-11-18.
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Antipsychotics are a first-line treatment for psychotic disorders. The cytochrome P450 isoenzymes CYP3A4/5 and CYP2D6 metabolize most antipsychotics. The activity of these isoenzymes in children changes with maturation, so it is different from that in adults. Objective: to study the associations of CYP3A and CYP2D6 isoenzyme activity parameters with the efficacy and safety of antipsychotics in adolescents with an acute psychotic episode. Patients and methods. The investigation enrolled 53 adolescents with an acute psychotic episode who took antipsychotics. The observation period lasted 14 days. The CGAS, PANSS, UKU SERS, SAS, and BARS scales were used to evaluate the efficiency and safety of the therapy on day 14. The activity of CYP3A and CYP2D6 isoenzymes was measured by determining the metabolic ratios of the concentrations of endogenous substrates of the isoenzymes and their metabolites in a morning urine sample on days 1 and 14 of the study. The activity of CYP3A was assessed by the 6-beta hydroxycortisol/cortisol ratio; that of CYP2D6 was measured by the 6-hydroxy-1,2,3,4-tetrahydro-beta-carboline/pinoline ratio. The influence of carriage of polymorphic variants CYP2D6*4,*9,*10, CYP3A4*22, CYP3A5*3 on the activity of isoenzymes was excluded by removing their carriers from the analysis. Results and discussion. The investigators revealed an association of certain antipsychotic-induced undesirable symptoms with CYP3A and CYP2D6 activity parameters. On day 1, a lower CYP2D6 activity was initially observed in patients with tremor (0.54 [0.34; 1.34] vs 1.25 [0.91; 1.75]; p=0.023). Also, patients with any documented adverse reaction (ARs) to therapy had initially a decreased CYP3A activity (1.2 [0.85; 2.29] vs 2.55 [1.44; 4.83]; p=0.047) and an enhanced CYP3A activity during therapy (the activity difference between day 14 and day 1 was 0.28 [-0.28; 2.32] vs -1.3 [-3.47; 0.66]; p=0.042). Conclusion. The initially reduced activity of CYP2D6 and CYP3A isoenzymes is a significant predictor of antipsychotic-induced ARs in adolescents with an acute psychotic episode. The predictive role of CYP2D6 and CYP3A activity levels in the efficacy of antipsychotics has not been confirmed.
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Wyss, M., D. Maughan e T. Wallimann. "Re-evaluation of the structure and physiological function of guanidino kinases in fruitfly (Drosophila), sea urchin (Psammechinus miliaris) and man". Biochemical Journal 309, n.º 1 (1 de julho de 1995): 255–61. http://dx.doi.org/10.1042/bj3090255.
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Purification and biophysical characterization of mitochondrial creatine kinase (Mi-CK) from sperm of the sea urchin Psammechinus miliaris, as well as gel-permeation chromatography of human heart Mi-CK demonstrate that these two Mi-CK isoenzymes form highly symmetrical octameric molecules with an M(r) of approx. 350,000, a value similar to that found for all other Mi-CK isoenzymes investigated so far. The absolute evolutionary conservation of this oligomeric form from sea urchins to mammals points both to its essentiality for Mi-CK function and to an important role of octameric Mi-CK in the energy metabolism of tissues and cells with high and fluctuating energy demands. To investigate whether a similar physiological principle also operates in an even more distantly related animal phylum, the arginine kinase (ArgK) isoenzyme system of Drosophila flight muscle was investigated with two independent subcellular fractionation procedures and subsequent analysis of the fractions by SDS/PAGE, immunoblotting and native isoenzyme electrophoresis. In contrast with a previous report [Munneke and Collier (1988) Biochem. Genet. 26, 131-141], strong evidence against the occurrence of a Mi-ArgK isoenzyme in Drosophila was obtained. The findings of the present study are discussed in the context of CK and ArgK function in general and of structural and bioenergetic differences between vertebrate striated muscles and arthropod flight muscles.
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Hoesch, R. M., e T. D. Boyer. "Purification and characterization of hepatic glutathione S-transferases of rhesus monkeys. A family of enzymes similar to the human hepatic glutathione S-transferases". Biochemical Journal 251, n.º 1 (1 de abril de 1988): 81–88. http://dx.doi.org/10.1042/bj2510081.
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Thirteen forms of glutathione S-transferase were purified from the livers of female rhesus monkeys (Macaque mulatta). Most (74.7%) of the activity in the hepatic cytosol adhered well to the GSH affinity column and could be eluted only with the addition of GSH to the eluting buffer. The predominant isoenzymes (n = 5) in this ‘high-affinity’ fraction had alkaline pI values (greater than 9.0) and contained a subunit with an Mr value of 24,000. All of these isoenzymes had high organic peroxidase activity and, on the basis of amino acid analysis, substrate specificities and affinity for non-substrate ligands, appear to belong to the family of glutathione S-transferases that have been termed alpha [Mannervik, Alin, Guthenberg, Jensson, Tahir, Warholm & Jörnvall (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 7202-7206]. Also within the high-affinity fraction was an isoenzyme with an acidic (5.8) pI value. This acidic isoenzyme was composed of a unique subunit (Mr 23,000). The N-terminal sequence (ten residues) of this acidic enzyme was identical with that of a human form that is referred to as pi. The predominant form of enzyme in the ‘low-affinity’ (eluted from the GSH affinity column with an increase in buffer pH) fraction was a homodimer of a 26,000-Mr subunit. It had an alkaline pI (greater than 9.0) but it lacked organic peroxidase activity. The N-terminal sequence (ten residues) of this enzyme was identical with that of a human enzyme referred to as mu. The substrate specificities and affinity for non-substrate ligands of this monkey enzyme also were similar to those of the human enzyme. In conclusion, the liver cytosol of rhesus monkeys contains a number of glutathione S-transferase isoenzymes that are very similar to the human hepatic enzymes.
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Medh, R. D., M. Saxena, S. S. Singhal, H. Ahmad e Y. C. Awasthi. "Characterization of a novel glutathione S-transferase isoenzyme from mouse lung and liver having structural similarity to rat glutathione S-transferase 8-8". Biochemical Journal 278, n.º 3 (15 de setembro de 1991): 793–99. http://dx.doi.org/10.1042/bj2780793.
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In mouse lung, glutathione S-transferase (GST, EC 2.5.1.18) isoenzymes belonging to the three major known classes, Alpha, Mu and Pi, have been previously characterized, along with an isoenzyme (pI 5.7) that could not be identified with the Alpha, Mu or Pi classes of GSTs. In the present studies we have demonstrated that this isoenzyme is also expressed in liver. Its structural, kinetic, and immunological properties have been determined and compared with those of the three classes of GSTs. GST 5.7 has a subunit molecular mass of 23 kDa, which is intermediate between that of the previously characterized Alpha (25 kDa) and Pi (22.5 kDa) class GST subunits of mouse lung. Comparison of peptide maps of GST 5.7 with those representative of Alpha, Mu and Pi class GST isoenzymes of mouse lung showed that it had a distinct peptide fragmentation pattern. Kinetic and immunological properties of GST 5.7 were also distinct from other mouse GST isoenzymes belonging to the Alpha, Mu or Pi classes. N-Terminal amino-acid-sequence analysis of a 6 kDa fragment generated by CNBr digestion of mouse lung GST 5.7 revealed a 15-residue sequence that was distinct from sequences of known Alpha, Mu and Pi class mouse GSTs. The sequence, however, matched with the sequence of rat GST 8-8 between amino acid residues 106 and 120 with a 73% identity. The 6 kDa and 12 kDa fragments generated by CNBr digestion of mouse liver GST 5.7 also gave sequences which matched with those of rat GST 8-8 between positions 106 and 120 and 167 and 186, with a high degree of identity. These studies suggest that mouse GST 5.7 structurally corresponds to rat GST 8-8 and belongs to the Alpha class.
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Schultz, Carolyn J., Meier Hsu, Barbara Miesak e Gloria M. Coruzzi. "Arabidopsis Mutants Define an in Vivo Role for Isoenzymes of Aspartate Aminotransferase in Plant Nitrogen Assimilation". Genetics 149, n.º 2 (1 de junho de 1998): 491–99. http://dx.doi.org/10.1093/genetics/149.2.491.
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Abstract Arabidopsis contains five isoenzymes of aspartate aminotransferase (AspAT) localized to the cytosol, chloroplast, mitochondria, or peroxisomes. To define the in vivo function of individual isoenzymes, we screened for Arabidopsis mutants deficient in either of the two major isoenzymes, cytosolic AAT2 or chloroplastic AAT3, using a native gel activity assay. In a screen of 8,000 M2 seedlings, three independent mutants deficient in cytosolic AAT2 (aat2) and two independent mutants deficient in chloroplastic AAT3 (aat3) were isolated. Mapping of aat2 and aat3 mutations and the five AspAT genes (ASP1–ASP5) established associations as follows: the mutation affecting aat2 maps with and cosegregates with ASP2, one of two expressed genes for cytosolic AspAT; the mutation affecting aat3 maps to the same location as the ASP5 gene encoding chloroplastic AspAT. Phenotypic analysis of the aat2 and aat3 mutants revealed a dramatic aspartate-related phenotype in one of the mutants deficient in cytosolic AAT2. The aat2-2 mutant displays an 80% reduction in levels of aspartate transported in the phloem of light-grown plants, and a 50% reduction in levels of asparagine transported in dark-adapted plants. These results indicate that cytosolic AAT2 is the major isoenzyme controlling aspartate synthesized for nitrogen transport in the light, and that this aspartate pool is converted to asparagine when plants are dark adapted.
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Lebherz, H. G., T. Burke, J. E. Shackelford, J. E. Strickler e K. J. Wilson. "Specific proteolytic modification of creatine kinase isoenzymes. Implication of C-terminal involvement in enzymic activity but not in subunit-subunit recognition". Biochemical Journal 233, n.º 1 (1 de janeiro de 1986): 51–56. http://dx.doi.org/10.1042/bj2330051.
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We are using the isoenzymes of creatine kinase (CK) to investigate the effect of specific proteolytic modification on the abilities of enzyme subunits to establish precise subunit-subunit recognition in vitro. Previous work by others has shown that treatment of the MM isoenzyme of rabbit CK with Proteinase K results in a specific proteolytic modification and inactivation of the enzyme. In the present work, we show that both the MM and BB isoenzymes of chicken CK are also specifically modified by Proteinase K, resulting in over 98% loss of catalytic activity and approx. 10% decreases in subunit molecular masses of the enzymes. Similar reactions appear to occur when the isoenzymes are treated with Pronase E. Limited amino acid sequence analysis of intact and Proteinase K-modified MM-CK suggests that the proteolytic modification results from a single peptide-bond cleavage occurring between alanine residues 328 and 329, about 50 amino acid residues from the C-terminal end; the active-site cysteine residue was recovered in the large protein fragment of modified M-CK subunits. Proteolytically modified M-CK and B-CK subunits were able to refold and reassociate into dimeric structures after treatment with high concentrations of LiCl and at low pH. Thus the proteolytically modified CK subunits retain their ability to refold and to establish precise subunit-subunit recognition in vitro.
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Arenas, J., V. Diaz, G. Liras, E. Gutierrez, I. Santos, A. Martinez e J. M. Culebras. "Activities of creatine kinase and its isoenzymes in serum in various skeletal muscle disorders." Clinical Chemistry 34, n.º 12 (1 de dezembro de 1988): 2460–62. http://dx.doi.org/10.1093/clinchem/34.12.2460.
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Abstract We studied possible correlations between anatomopathological and clinical features and the values for total creatine kinase (CK; EC 2.7.3.2) and its isoenzymes, including the proportion of CK-MB, in a population displaying several neuromuscular pathologies. Although we observed no specific isoenzyme pattern associated with the different myopathies, we found isoenzyme analysis useful in studying the histopathological evolution of illness. We also considered whether the pathology was regenerative or nonregenerative, and what type of fiber (I or II) was involved. High CK-MB percentages (greater than 6%) were associated with regenerative and type I fiber myopathies, with regenerative type tissues being the principal factor associated with an increasing proportion of CK-MB. Studying the changes in CK-MB percentage in serum appears to be useful in discriminating neuromuscular from myocardial pathologies.
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Maekawa, Masato, Motoko Inomata, Masao S. Sasaki, Akihiro Kaneko, Mineko Ushiama, Kokichi Sugano, Jun Takayama e Takashi Kanno. "Electrophoretic Variant of a Lactate Dehydrogenase Isoenzyme and Selective Promoter Methylation of the LDHA Gene in a Human Retinoblastoma Cell Line". Clinical Chemistry 48, n.º 11 (1 de novembro de 2002): 1938–45. http://dx.doi.org/10.1093/clinchem/48.11.1938.
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Abstract Background: Lactate dehydrogenase (LD), a tetrameric product of the genes LDHA and LDHB, may be increased in sera of cancer patients. A variant isoenzyme with electrophoretic mobility between LD2 and LD3 (LD2ex) has been described in patients, but its molecular nature is largely unknown. Methods: A newly established retinoblastoma cell line, NCC-RbC-51 (R51), showed an isoenzyme pattern with only two bands, LD1 and LD2ex. We investigated the isoenzymes by Northern blot, Western blot, and methylation analysis and PCR. Results: Northern blot analysis revealed that R51 cells expressed no wild-type/somatic LDHA mRNA, but did express a small amount of LDHA-related mRNA with a slightly higher molecular mass. Western blot analysis confirmed the anti-LDHA-reactive protein with a 3-kDa higher molecular mass. Treatment of R51 cells with the demethylating agent 5-aza-2′-deoxycytidine restored the expression of the LD2, -3, -4, and -5 isoenzymes. PCR analysis of sodium bisulfite-treated genomic DNA revealed that the CpG island in the promoter region around exon a of the LDHA gene was completely methylated. Reverse transcription-PCR analysis and direct sequencing revealed that R51 cells expressed a RNA with the sequence of the human homolog of a murine testis-specific variant that has exon 0 as the 5′ noncoding sequence. LDHB was expressed normally in R51 cells. Conclusions: The somatic LDHA in R51 cells is transcriptionally silenced by promoter hypermethylation around exon a, leaving only LDHB to be expressed normally and a testis-specific variant transcript of LDHA containing exon 0. LD2ex possibly results from tetramerization of three wild-type LDHB molecules and one variant LDHA product.
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ICHIMURA, Michio, Rika EIKI, Keiko OSAWA, Satoshi NAKANISHI e Hiroshi KASE. "KS-505a, an isoform-selective inhibitor of calmodulin-dependent cyclic nucleotide phosphodiesterase". Biochemical Journal 316, n.º 1 (15 de maio de 1996): 311–16. http://dx.doi.org/10.1042/bj3160311.
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The effects of KS-505a, a novel microbial metabolite, on the activity of calmodulin-dependent cyclic nucleotide phosphodiesterase (CaM-PDE) were investigated. (1) KS-505a potently inhibited the purified 61 kDa isoenzyme of CaM-PDE from bovine brain and required much higher doses to inhibit the purified 59 kDa isoenzyme of CaM-PDE from bovine heart. The inhibition of both isoenzymes was observed only in the presence of calcium-activated calmodulin (Ca2+/CaM). The IC50 values for the 61 and 59 kDa isoenzymes were 0.17 and 13 μM respectively with 20 μM cAMP as a substrate. (2) Kinetic analysis indicated that the inhibitory mode of KS-505a for the 61 kDa isoenzyme was competitive with respect to Ca2+/CaM; the Ki for KS-505a was 0.089 μM. The inhibition was not competitive with respect to the substrates cAMP or cGMP. (3) KS-505a did not interfere with the interaction between Ca2+/CaM and n-phenyl-1-naphthylamine, a hydrophobic fluorescent probe, nor was it adsorbed to CaM-conjugated gels in the presence of Ca2+, thereby indicating that KS-505a does not bind to Ca2+/CaM. (4) Trypsin-activated 61 kDa isoenzyme, which lacked the Ca2+/CaM-binding domain, was not inhibited by KS-505a at less than micromolar concentrations. Taken together, these results suggest that KS-505a apparently bound to a site in the Ca2+/CaM-binding domain of the 61 kDa isoenzyme and selectively inhibited Ca2+/CaM-activated 61 kDa isoenzyme activity. (5) In rat hippocampal slices, KS-505a at 10 μM increased the intracellular cAMP concentration to approximately three times the basal level, whereas in rat striatal slices it had no effect on the cAMP concentration at concentrations of 1.0–10 μM, suggesting that each CaM-PDE isoenzyme functions differentially in these regions. These results demonstrate that KS-505a is a highly potent selective inhibitor both in vitro and in vivo and distinguishes between subfamily members within the CaM-PDE family.
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Bogaards, J. J. P., B. van Ommen e P. J. V. van Bladeren. "Purification and characterization of eight glutathione S-transferase isoenzymes of hamster. Comparison of subunit composition of enzymes from liver, kidney, testis, pancreas and trachea". Biochemical Journal 286, n.º 2 (1 de setembro de 1992): 383–88. http://dx.doi.org/10.1042/bj2860383.
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Eight dimeric isoenzymes of glutathione S-transferase (GST) were purified from liver, kidney and testis of the Syrian golden hamster, using S-hexylglutathione affinity chromatography and chromatofocusing. The isoenzymes were characterized according to their substrate selectivity, physical properties and amino acid sequence analysis. Thus a classification into Alpha, Mu and Pi classes was made in analogy with GSTs of other species. Two Alpha-class GSTs were purified, termed A1A1 (pI 8.9) and A1A2 (pI 8.6). Four Mu-class subunits were detected (M1-M4), all forming homodimers, with M2 and M3 also forming a heterodimer. The isoelectric points ranged from 5.9 to 8.6. One Pi-class isoenzyme was purified and termed P1P1 (pI 6.8). Using h.p.l.c. analysis, the subunit composition was determined in a number of organs. The major subunits in liver were A1 and M1. Subunit A1 was also the major subunit in the kidney. Subunit M1 was not detected in kidney, while subunit P1 was not found in the liver. Pancreas and trachea contained predominantly the Pi-class subunit, P1. GST in the testis was mostly of the Mu class. The major subunit was M4, and subunits M2 and M3 were exclusively detected in the testis.
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DEWASTE, Valérie, Valérie POUILLON, Colette MOREAU, Stephen SHEARS, Kazunaga TAKAZAWA e Christophe ERNEUX. "Cloning and expression of a cDNA encoding human inositol 1,4,5-trisphosphate 3-kinase C". Biochemical Journal 352, n.º 2 (24 de novembro de 2000): 343–51. http://dx.doi.org/10.1042/bj3520343.
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Inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] 3-kinase catalyses the phosphorylation of Ins(1,4,5)P3 to Ins(1,3,4,5)P4. cDNAs encoding two isoenzymes of Ins(1,4,5)P3 3-kinase (3-kinases A and B) have been described previously. In the present study, we report the cloning of a full-length 2052bp cDNA encoding a third human isoenzyme of the Ins(1,4,5)P3 3-kinase family, referred to as isoform C. This novel enzyme has a calculated molecular mass of 75.207kDa and a Km for Ins(1,4,5)P3 of 6µM. Northern-blot analysis showed the presence of a transcript of approx. 3.9kb in various human tissues. Inositol trisphosphate 3-kinase C demonstrates enzymic activity when expressed in DH5αF′ bacteria or COS-7 cells. Calcium alone decreases the Ins(1,4,5)P3 3-kinase activity of the 3-kinase C isoenzyme in transfected COS-7 cells. This inhibitory effect is reversed in the presence of calmodulin. The recombinant bacterial 3-kinase C can be adsorbed on calmodulin–Sepharose in the presence of calcium. The present data show that Ins(1,4,5)P3 3-kinase C: (i) shares a conserved catalytic domain of about 275 amino acids with the two other mammalian isoforms, (ii) could be purified on a calmodulin–Sepharose column and (iii) could be distinguished from the A and B isoenzymes by the effects of calcium and of calmodulin.
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Rahayu, Sri, Sutiman Bambang Sumitro, T. Susilawati e Soemarno Soemarno. "ANALISIS ISOENZIM UNTUK MEMPELAJARI VARIASI GENETIK SAPI BALI DI PROVINSI BALI". Berkala Penelitian Hayati 12, n.º 1 (31 de dezembro de 2006): 1–5. http://dx.doi.org/10.23869/bphjbr.12.1.20061.
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The aim of this research was to know Bali cattle genetic variation according to the band pattern of isoenzyme. Esteraseand Malate dehydrogenase Isoenzymes of Bali cattle were studied. Using the native-PAGE method, the analysis has been made of the genetic structure and variation of the bali cattle population. Isoenzyme isolated from leucocytes cell using homogenation method by adding Phosphat Buffer Saline (PBS). A hundred sample of Bali cattles were taken in Mambang, Slemadeg and Kuwumkeladi. The result of this research indicate that from the 2 different enzyme, 3 loci were detected, and 1 of them was polymorphic (MDH-2). There was null allele phenomenom in MDH-2 locus. The loci polymorphic proportion of three population were 0,333. Chi-Square analysis of three population were 1.251–1.560. The heterozygosity value of three population (Mambang, Slemadeg and Kuwumkeladi) were 0.098, 0.111 and 0.118, respectively.
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Tillyer, C. R. "Error estimation in the quantification of alkaline phosphatase isoenzymes by selective inhibition methods." Clinical Chemistry 34, n.º 12 (1 de dezembro de 1988): 2490–93. http://dx.doi.org/10.1093/clinchem/34.12.2490.
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Abstract A method for calculating, by selective inhibition, the activities in serum of isoenzymes of alkaline phosphatase (EC 3.1.3.1) originating from bone, liver, intestine, and placenta produced results for a sample of patients for which the imprecision was five- to 25-fold higher than that of the alkaline phosphatase method used--too imprecise for routine clinical use. Error analysis by direct calculation and by Monte Carlo estimation revealed that the algorithm used in the method completely accounted for the increase in imprecision of the isoenzyme estimations. I recommend that all methods involving such algorithms or the principle of multicomponent analysis should undergo a thorough error analysis by use of the techniques described here, to obtain an estimate of the increase in imprecision that is ascribable to the particular numerical technique used.
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Morikawa, Toshinobu. "Monosomic analysis of leaf peroxidase Px0 and Px9 loci in hexaploid oats". Genome 30, n.º 2 (1 de abril de 1988): 99–102. http://dx.doi.org/10.1139/g88-017.
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Inheritance of the peroxidase isoenzymes of the flag leaf blade was examined by isoelectrofocusing in the hexaploid oats Avena byzantina cv. Kanota, Avena fatua ssp. compacta, and Avena sativa cv. Cherokee. Two independent peroxidase loci (Px0 and Px9) were detected in the F2 from the 'Kanota' × compacta cross. The Px0a derived from compacta expressed the highest peroxidase activity and was accompanied by a post-transcriptionally modified form or mozyme. A monosomic analysis of the Px0 and Px9 loci revealed that they were located on chromosomes 18 and 6, respectively. Phenotypic expression of the peroxidases varied in each genotye at the Px0 and Px9 loci. Phenotypes of the homozygote (Px0aPx0a) and the hemizygote (Px0a—) were similar to each other. The heterozygote (Px0aPx0b) had half the enzymatic activity of the others. Px9b of compacta was functional as a suppressor but that of 'Cherokee' was nonfunctional.Key words: monosomic analysis, peroxidase loci, isoenzyme, hexaploid oats.
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Naito, Eiji, Roka Shimada e Masashi Yuki. "Diagnostic utility of measuring lactate dehydrogenase levels and its isoenzyme activities for the evaluation of malignancy in feline pleural effusion and ascitic fluid". Open Veterinary Journal 12, n.º 5 (2022): 735. http://dx.doi.org/10.5455/ovj.2022.v12.i5.19.
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Background: Lactate dehydrogenase (LDH) isoenzymes may be useful in the differential diagnosis of pleural effusion (PE) and ascitic fluid (AF) aetiologies in cats since tissue damage induces their release, changing the pattern of their activity. Aim: The present study aimed to determine the diagnostic utility of measuring lactate dehydrogenase levels and isoenzyme activities in PE or AF in cats with malignancy. Materials and Methods: The results of LDH levels and isoenzyme activities in the serum, PE and AF of 29 cats were compared among malignant group, infectious group, and non-malignant, non-infectious group. A receiver operating characteristic (ROC) analysis was performed to assess the accuracy in diagnosing feline malignancy. Results: In PE or AF, there were significant differences in LDH level and LDH isoenzyme activities among the three groups. The combination of LDH level and LDH-1 activity in PE or AF had the highest area under the ROC (AUC) values for discriminating malignant effusion from non-malignant effusion. The AUC of the combination of LDH level and LDH-1 activity in PE or AF was 0.874. The sensitivity and specificity of using the combination of LDH level (cut-off: <2,269 U/L) and LDH-1 activity (cut-off: <4.8%) in PE or AF for predicting malignancy with the highest AUC value were 94.4% and 72.7%, respectively. Conclusion: Our results suggest that the combination of LDH level and LDH-1 activity in PE or AF is a potential factor for diagnosing malignancy. Considering that LDH isoenzymes can be measured inexpensively and easily, LDH tests can be readily accommodated in veterinary clinical practice.
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Di Ilio, C., A. Aceto, T. Bucciarelli, S. Angelucci, M. Felaco, A. Grilli e G. Federici. "Glutathione transferase isoenzymes from human prostate". Biochemical Journal 271, n.º 2 (15 de outubro de 1990): 481–85. http://dx.doi.org/10.1042/bj2710481.
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By using affinity-chromatography and isoelectric-focusing techniques, several forms of glutathione transferase (GSTs) were resolved from human prostate cytosol. All the three major classes of GST, i.e. Alpha, Mu and Pi, are present in human prostate. However, large inter-individual variation in the qualitative and quantitative expression of different isoenzymes resulted in the samples investigated. The most abundant group of prostate isoenzymes showed acid (pI 4.3-4.7) behaviour and were classified as Pi class GSTs on the basis of their immunological and structural properties. Immunohistochemical staining of Pi class GSTs was prevalently distributed in the epithelial cells surrounding the alveolar lumen. Class Mu GSTs are also expressed, although in small amounts and in a limited number of samples, by human prostate. The major cationic isoenzyme purified from prostate, GST-9.6; (pI 9.6; apparent subunit molecular mass of 28 kDa), appears to be different from the cationic GST alpha-epsilon forms isolated from human liver and kidney as evidenced by its structural, kinetical and immunological properties. This enzyme, which accounts for about 20-30% (on protein basis) of total amount of GSTs, is expressed by only 40% of samples. GST-9.6 has the ability to cross-react in immunoblotting analysis with antisera raised against rat liver GST 2-2, rather than with antisera raised against members of human Alpha, Mu and Pi class GSTs. Although prostate GST-9.6 shows close relationship with the human skin GST pI 9.9, it does not correspond to any other known human GST.
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Higashiyama, M., S. Doi, N. Tomita, T. Monden, M. Murotani, Y. Kawasaki, T. Kobayashi, T. Shimano, M. Ogawa e S. Takai. "Immunohistochemical analysis of amylase isoenzymes in thyroid cancer." Journal of Clinical Pathology 44, n.º 2 (1 de fevereiro de 1991): 144–46. http://dx.doi.org/10.1136/jcp.44.2.144.
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Marretto, Jesila Pinto M., e Sonia G. Andrade. "Biochemical behavior of Trypanosoma cruzi strains isolated from mice submitted to specific chemotherapy". Revista da Sociedade Brasileira de Medicina Tropical 27, n.º 4 (dezembro de 1994): 209–15. http://dx.doi.org/10.1590/s0037-86821994000400002.
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To investigate the influence of chemotherapy on the biochemical beha vior of Trypanosoma cruzi strains, three groups of mice were infected with one of three strains of T. cruzi of different biological and isoenzymic patterns (Peruvian, 21 SF and Colombian strains). Each group was subdivided into subgroups: 1 - treated with nifurtimox; 2 - treated with benznidazole and 3 - untreated infected controls. At the end of treatment, that lasted for 90 days, xenodiagnosis, sub inoculation of blood into new born mice and haemoculture were performed as tests of cure. From the positive tests, 22 samples of T. cruzi were isolated from all subgroups. Electrophoretic analysis of the isoenzymes PGM, GP1, ALAT and AS AT failed to show any difference between parasite strains isolated from treated and untreated mice, which indicates that no detectable clonal selection or parasite genetic markers alterations concerning the isoenzymes analysed have been determined by treatment with drugs of recognized antiparasitic effect, suggesting stability of the phenotypic characteristics of the three biological types of T. cruzi strains.
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Kazmierczak, S. C., W. J. Castellani, F. Van Lente, E. D. Hodges e B. Udis. "Effect of reticulocytosis on lactate dehydrogenase isoenzyme distribution in serum: in vivo and in vitro studies". Clinical Chemistry 36, n.º 9 (1 de setembro de 1990): 1638–41. http://dx.doi.org/10.1093/clinchem/36.9.1638.
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Abstract We investigated the effect of reticulocytosis on the lactate dehydrogenase (LD; EC 1.1.1.27) isoenzyme LD1/LD2 ratio in patients with and without evidence of hemolytic disease. Analysis of sera from patients with reticulocytosis and in vivo hemolysis showed a mean LD1/LD2 ratio of 0.92 compared with a ratio of 0.69 in patients with in vivo hemolysis and normal reticulocyte counts. Determination of LD isoenzymes in erythrocyte lysate revealed significantly increased LD1/LD2 ratios for patients with marked reticulocytosis compared with those for patients with normal-to-minimal increases in reticulocytes. Finally, separation of mature erythrocytes and reticulocytes by flow cytometry revealed marked differences in the LD1/LD2 isoenzyme distribution between these two cell types. The ability of hemolysis to cause a "flipped" LD1/LD2 ratio is dependent on the proportion of the hemolyzed cells that are reticulocytes.
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Reddy, M. K., G. D. Heda e J. K. Reddy. "Purification and characterization of α-amylase from rat pancreatic acinar carcinoma. Comparison with pancreatic α-amylase". Biochemical Journal 242, n.º 3 (15 de março de 1987): 681–87. http://dx.doi.org/10.1042/bj2420681.
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alpha-Amylase was purified to apparent homogeneity from normal pancreas and a transplantable pancreatic acinar carcinoma of the rat by affinity chromatography on alpha-glucohydrolase inhibitor (alpha-GHI) bound to aminohexyl-Sepharose 4B. Recovery was 95-100% for both pancreas and tumour alpha-amylases. They were monomeric proteins, with Mr approx. 54000 on SDS/polyacrylamide-gel electrophoresis. Isoelectric focusing of both normal and tumour alpha-amylases resolved each into two major isoenzymes, with pI 8.3 and 8.7. Tumour-derived alpha-amylase contained two additional minor isoenzymes, with pI 7.6 and 6.95 respectively. All four tumour isoenzymes demonstrated amylolytic activity when isoelectric-focused gels were treated with starch and stained with iodine. Two-dimensional electrophoresis, on SDS/10-20%-polyacrylamide-gradient gels after isoelectric focusing, separated each major isoenzyme into doublets of similar Mr values. Pancreatic and tumour-derived alpha-amylases had similar Km and Ki (alpha-GHI) values, but the specific activity of the tumour alpha-amylase was approximately two-thirds that of the normal alpha-amylase. Although amino acid analysis and peptide mapping with the use of CNBr, N-chlorosuccinimide or Staphylococcus aureus V8 proteinase gave comparable profiles for the two alpha-amylases, tryptic-digest fingerprint patterns were different. Antibodies raised against the purified pancreatic alpha-amylase and tumour alpha-amylase respectively showed only one positive band on immunoblotting after gel electrophoresis of crude extracts of rat pancreas and carcinoma, at the same position as that of the purified enzyme. More than 95% of the alpha-amylase activity in the pancreas and in the tumour was absorbed by an excess amount of either antibody, indicating that normal and tumour alpha-amylases are immunologically identical. The presence of additional isoenzymes in the carcinoma, and dissimilarity of tryptic-digest patterns, may reflect an alteration in gene expression or in the post-translational modification of this protein in this heterogeneously differentiated transplantable pancreatic acinar carcinoma.
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Hultberg, B., A. Isaksson, A. Lindgren, B. Israelsson e L. Brattström. "β-Hexosaminidase Isoenzymes A and B in Middle-Aged and Elderly Subjects: Determinants of Plasma Levels and Relation to Vascular Disease". Annals of Clinical Biochemistry: International Journal of Laboratory Medicine 33, n.º 5 (setembro de 1996): 432–37. http://dx.doi.org/10.1177/000456329603300506.
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Plasma/serum β-hexosaminidase (Hex) activity is known to be increased in chronic alcoholism, liver disorders, pregnancy and diabetes mellitus. Hex activity also shows an association with risk factors for vascular disease and heredity for arteriosclerosis. There are several isoenzymes of Hex. Using an enzyme immunoassay for Hex isoenzymes (Hex A and Hex B) we studied possible determinants of Hex isoenzymes and their relation to vascular disease in randomly invited ( n = 244) 35–95-year-old men and women. In both sexes there were significant age-related increases in Hex activities and men exhibited higher activity of both isoenzymes. Both Hex isoenzymes correlated with age, systolic blood pressure, serum triglycerides and liver enzymes, whereas Hex A was distinguished from Hex B by its stronger correlation with blood glucose. In multiple linear regression analysis Hex A was explained to 20·7% by blood glucose, age, serum aspartate aminotransferase and glutamyl transpeptidase. Hex B was explained to 14% by age, serum glutamyl transpeptidase and serum triglycerides. There was no significant increase in Hex isoenzymes in subjects with hypertension, diabetes mellitus or myocardial disease, nor did current smokers exhibit any increase of these enzymes compared to non-smokers. The main conclusion is that liver function, as reflected by the level of liver enzymes and glucose metabolism, is the major determinant for Hex isoenzymes in plasma.
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MITCHELL, Alyson E., Dexter MORIN, Jeffrey LAKRITZ e A. Daniel JONES. "Quantitative profiling of tissue- and gender-related expression of glutathione S-transferase isoenzymes in the mouse". Biochemical Journal 325, n.º 1 (1 de julho de 1997): 207–16. http://dx.doi.org/10.1042/bj3250207.
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Cytosolic glutathione S-transferase (GST) isoenzymes from brain, heart, lung, liver, kidney and gonads of male and female CD-1 mice were identified and quantified with a combination of affinity purification, electrospray ionization MS, Edman microsequencing, Western blot analysis and reverse-phase HPLC. The three principal hepatic GST subunits, mGSTA3 (25271 Da), mGSTP1 (23478 Da), and mGSTM1 (25839 Da), were isolated from liver, lung, kidney, testes and female heart, whereas brain, ovaries and male heart contained mGSTM1 and mGSTP1. Additional isoenzymes were detected in tissues, including mu class subunits mGSTM2 (25580 Da) and mGSTM3 (25570 Da), an N-terminally blocked Alpha subunit (25480 Da) assigned as mGSTA4, and proteins of molecular masses 25490, 22540, 24493, 24378 and 25383 Da. Distinct gender differences in expression of GST subunits were observed for liver, heart, kidney and gonads, whereas GST expression was similar in brain and lung for both genders. In contrast with patterns of expression in liver (high ratio of mGSTA3 to mGSTP1 in females relative to males), mGSTP1 was the most abundant subunit in female gonads, whereas mGSTA3 was not present in detectable quantities. The profile of GST expression in kidney was similar to that in liver; however, male kidneys expressed 30% more soluble GST than female kidneys. A striking gender-related difference in GST expression was found in cardiac tissue, where female animals expressed 50% more soluble GST than male tissues, and the GST isoenzyme with the greatest documented activity towards lipid hydroperoxides, mGSTA3, was present in female tissue yet absent from male tissue. These results point to complex gender- and tissue-dependent expression of individual mouse GST isoenzymes.
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Zoellner, H. F., e N. Hunter. "Histochemical identification of the vascular endothelial isoenzyme of alkaline phosphatase." Journal of Histochemistry & Cytochemistry 37, n.º 12 (dezembro de 1989): 1893–98. http://dx.doi.org/10.1177/37.12.2584695.
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Alkaline phosphatase (AP) is a widely studied membrane bound ecto-enzyme with an extensive distribution in nature. Three major human isoenzymes have been defined and can be distinguished on the basis of their differential sensitivity to specific inhibitors. Despite the voluminous literature describing AP, the physiological role of this enzyme is unclear. Microvascular endothelium is strongly AP positive and may provide a convenient model for study of the role of AP in vitro. This report describes the use of freeze-substitution and high-resolution plastic embedding techniques to identify the isoenzyme of endothelial AP by quantitative analysis of the relative inhibition by specific inhibitors of AP, using human gingival tissues and a number of rat tissues. Endothelial AP is found to be the liver/bone/kidney isoenzyme, indicating kidney as a credible source of enzyme for further experimental work investigating the role of AP.
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Zhou, Yixing, Michele R. Wing, John Sondek e T. Kendall Harden. "Molecular cloning and characterization of PLC-η2". Biochemical Journal 391, n.º 3 (25 de outubro de 2005): 667–76. http://dx.doi.org/10.1042/bj20050839.
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PLC (phospholipase C) isoenzymes catalyse the conversion of PtdIns(4,5)P2 into the Ca2+-mobilizing second messenger, Ins(1,4,5)P3, and the protein kinase C-activating second messenger, diacylglycerol. With the goal of identifying additional mammalian PLC isoenzymes, we screened the NCBI non-redundant database using a BLAST algorithm for novel sequences with homology with the conserved PLC catalytic core. Two unique sequences corresponding to two unknown PLC isoenzymes were identified, and one of these, designated PLC-η2, was cloned and characterized. Most of the coding sequence of PLC-η2 was constructed from two ESTs (expressed sequence tags), which included an overlapping sequence that was confirmed by multiple ESTs and mRNAs. 5′-RACE (rapid amplification of cDNA ends) also identified an upstream exon not deduced from available EST or mRNA sequences. Sequence analysis of PLC-η2 revealed the canonical domains of a PLC isoenzyme with an additional long C-terminus that contains a class II PDZ-binding motif. Genomic analyses indicated that PLC-η2 is encoded by 23 exons. RT-PCR (reverse transcriptase-PCR) analyses illustrated expression of PLC-η2 in human retina and kidney, as well as in mouse brain, eye and lung. RT-PCR with exon-specific primers also revealed tissue-specific expression of four splice variants in mouse that represent alternative use of sequences in exons 21, 22 and 23. PLC-η2-specific antisera recognized one of these splice variants as an approx. 155 kDa species when expressed in COS-7 cells; PLC-η2 natively expressed in 1321N1 human astrocytoma cells also migrated as an approx. 155 kDa species. PLC activity was observed in vitro and in vivo for three different constructs of PLC-η2, each containing possible alternatively spliced first exons. Co-expression of PLC-η2 with Gβ1γ2 dimers of heterotrimeric G-proteins resulted in marked stimulation of inositol lipid hydrolysis. Thus PLC-η2 may in part function downstream of G-protein-coupled receptors.
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Bialer, M. G., D. E. Bruns e T. E. Kelly. "Muscle enzymes and isoenzymes in Emery-Dreifuss muscular dystrophy". Clinical Chemistry 36, n.º 3 (1 de março de 1990): 427–30. http://dx.doi.org/10.1093/clinchem/36.3.427.
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Abstract Emery-Dreifuss muscular dystrophy (EDMD) is a rare X-linked muscular dystrophy. Creatine kinase (CK) activity usually is increased in serum of affected males, but results for aldolase and lactate dehydrogenase (LD) in serum have been inconsistent, as have those for CK in carrier females. There have been few studies of CK-MB or LD isoenzyme-1 (LD-1) in EDMD. We measured CK, CK-MB, LD, LD-1, and aldolase activity in sera of 84 members of two large families with EDMD. DNA analysis had been carried out on all subjects. Although CK, LD, and aldolase activities were significantly increased in affected males, CK activity was the most consistently increased and was the least subject to artifactual increases. Mean CK-MB in serum was mildly increased, but LD-1 was within the normal reference interval, suggesting that CK-MB is increased in skeletal muscle in EDMD, as has been found in other forms of dystrophy. CK decreased with age in affected males. We saw no significant increases of muscle enzymes or isoenzymes in 33 EDMD carriers studied, of whom 19 were obligate carriers and 14 had been identified by DNA analysis.