Journal articles: 'Sheep liver enzyme purification'

1

Çiftci, M., V. Turkoglu, and S. Aldemir. "Effects of some antibiotics on glucose 6-phosphate dehydrogenase in sheep liver." Veterinární Medicína 47, No. 10 - 11 (March 30, 2012): 283–88. http://dx.doi.org/10.17221/5836-vetmed.

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In vitro effects of penicillin, sulbactam, cefazolin, and amikacine on the activity of the enzyme glucose-6-phosphate dehydrogenase in sheep liver were investigated. Glucose 6-phosphate dehydrogenase was purified from sheep liver, using a simple and rapid method. The purification consisted of two steps, preparation of homogenate and 2’, 5’-ADP Sepharose 4B affinity chromatography. As a result of the two consecutive procedures, the enzyme, having the specific activity of 11.76 EU/mg proteins, was purified with a yield of 35.72% and 1.913 fold. In order to control the enzyme purification SDS polyacrylamide gel electrophoresis (SDS-PAGE) was done. SDS-PAGE showed a single band for the enzyme. In addition, I50 values of the antibiotics were determined by plotting activity % vs. antibiotic concentrations. I50 values were 17.71 mM for penicillin, 27.38 mM for sulbactam, 28.88 mM for cefazolin, and 30.59 mM for amikacine.

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Zaahkouk, Samir A. M., Doaa A. Darwish, Hassan M. M. Masoud, Mohamed M. Abdel-Monsef, Mohamed S. Helmy, Sayed S. Esa, Abdel-Hady M. Ghazy, and Mahmoud A. Ibrahim. "Purification and Characterization of Xanthine Oxidase from Liver of the Sheep (Ovis Aries)." Journal of Antioxidant Activity 1, no. 4 (March 15, 2019): 8–18. http://dx.doi.org/10.14302/issn.2471-2140.jaa-19-2699.

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Xanthine oxidase is a commercially important enzyme with wide area of medical applications to develop diagnostic kits. Xanthine oxidase was extracted, purified and characterized from sheep liver (SLXO). The purification procedure involved acetone precipitation and chromatography on DEAE-cellulose and Sephacryl S-300 columns. The sheep liver xanthine oxidase was homogeneously purified 31.8 folds with 3.5 U/mg specific activity and 24.1% recovery. SLXO native molecular weight was 150 kDa and on SDS-PAGE appeared as single major band of 75 kDa representing a homodimer protein. Isoelectric focusing of the purified SLXO resolved into two closely related isoforms with pI values of 5.6 and 5.8. The apparent Km for xanthine oxidase at optimum pH 7.6 was found to be 0.9 mM xanthine. FeCl2 and NiCl2 increased the activity of SLXO, while CuCl2 and ZnCl2 were found to be potent inhibitors of the purified enzyme. Allopurinol inhibits SLXO competitively with one binding site on the purified molecule and Ki value of 0.06 mM.

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KRISHNA RAO, J. V., Junutula R. JAGATH, Balasubramanya SHARMA, N. APPAJI RAO, and H. S. SAVITHRI. "Asp-89: a critical residue in maintaining the oligomeric structure of sheep liver cytosolic serine hydroxymethyltransferase." Biochemical Journal 343, no. 1 (September 24, 1999): 257–63. http://dx.doi.org/10.1042/bj3430257.

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Aspartate residues function as proton acceptors in catalysis and are involved in ionic interactions stabilizing subunit assembly. In an attempt to unravel the role of a conserved aspartate (D89) in sheep-liver tetrameric serine hydroxymethyltransferase (SHMT), it was converted into aspargine by site-directed mutagenesis. The purified D89N mutant enzyme had a lower specific activity compared with the wild-type enzyme. It was a mixture of dimers and tetramers with the proportion of tetramers increasing with an increase in the pyridoxal-5′-phosphate (PLP) concentration used during purification. The D89N mutant tetramer was as active as the wild-type enzyme and had similar kinetic and spectral properties in the presence of 500 μM PLP. The quinonoid spectral intermediate commonly seen in the case of SHMT was also seen in the case of D89N mutant tetramer, although the amount of intermediate formed was lower. Although the purified dimer exhibited visible absorbance at 425 nm, it had a negligible visible CD spectrum at 425 nm and was only 5% active. The apo-D89N mutant tetramer was a dimer unlike the apo-form of the wild-type enzyme which was present predominantly as a tetramer. Furthermore the apo mutant dimer could not be reconstituted to the holo-form by the addition of excess PLP, suggesting that dimer-dimer interactions are weak in this mutant. The recently published crystal structure of human liver cytosolic recombinant SHMT indicates that this residue (D90 in the human enzyme) is located at the N-terminal end of the fourth helix of one subunit and packs against K39 from the second N-terminal helix of the other symmetry related subunit forming the tight dimer. D89 is at the interface of tight dimers where the PLP 5′-phosphate is also bound. Mutation of D89 could lead to weakened ionic interactions in the tight dimer interface, resulting in decreased affinity of the enzyme for the cofactor.

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Demirdag, Ramazan, Emrah Yerlikaya, and Omer Irfan Kufrevioglu. "Purification of carbonic anhydrase-II from sheep liver and inhibitory effects of some heavy metals on enzyme activity." Journal of Enzyme Inhibition and Medicinal Chemistry 27, no. 6 (October 10, 2011): 795–99. http://dx.doi.org/10.3109/14756366.2011.615744.

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Camadan, Yasemin, Hasan Özdemir, and İlhami Gulcin. "Purification and characterization of dihydropyrimidine dehydrogenase enzyme from sheep liver and determination of the effects of some anaesthetic and antidepressant drugs on the enzyme activity." Journal of Enzyme Inhibition and Medicinal Chemistry 31, no. 6 (January 13, 2016): 1335–41. http://dx.doi.org/10.3109/14756366.2015.1132710.

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6

Rip, J. W., M. B. Coulter-Mackie, C. A. Rupar, and B. A. Gordon. "Purification and structure of human liver aspartylglucosaminidase." Biochemical Journal 288, no. 3 (December 15, 1992): 1005–10. http://dx.doi.org/10.1042/bj2881005.

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We have recently diagnosed aspartylglucosaminuria (AGU) in four members of a Canadian family. AGU is a lysosomal storage disease in which asparagine-linked glycopeptides accumulate to particularly high concentrations in liver, spleen and thyroid of affected individuals. A lesser accumulation of these glycopeptides is seen in the kidney and brain, and they are also excreted in the urine. The altered metabolism in AGU results from a deficiency of the enzyme aspartylglucosaminidase (1-aspartamido-beta-N-acetylglucosamine amidohydrolase), which hydrolyses the asparagine to N-acetylglucosamine linkages of glycoproteins and glycopeptides. We have used human liver as a source of material for the purification of aspartylglucosaminidase. The enzyme has been purified to homogeneity by using heat treatment, (NH4)2SO4 fractionation, and chromatography on concanavalin A-Sepharose, DEAE-Sepharose, sulphopropyl-Sephadex, hydroxyapatite, DEAE-cellulose and Sephadex G-100. Enzyme activity was followed by measuring colorimetrically the N-acetylglucosamine released from aspartylglucosamine at 56 degrees C. The purified enzyme protein ran at a ‘native’ molecular mass of 56 kDa in SDS/12.5%-PAGE gels, and the enzyme activity could be quantitatively recovered at this molecular mass by using gel slices as enzyme source in the assay. After denaturation by boiling in SDS the 56 kDa protein was lost with the corresponding appearance of polypeptides alpha,beta and beta 1, lacking enzyme activity, at 24.6, 18.4 and 17.4 kDa respectively. Treatment of heat-denatured enzyme with N-glycosidase F resulted in the following decreases in molecular mass; 24.6 to 23 kDa and 18.4 and 17.4 to 15.8 kDa. These studies indicate that human liver aspartylglucosaminidase is composed of two non-identical polypeptides, each of which is glycosylated. The N-termini of alpha,beta and beta 1 were directly accessible for sequencing, and the first 21, 26 and 22 amino acids respectively were identified.

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Leoncini, R., R. Pagani, A. Casella, and E. Marinello. "Rat liver L-threonine deaminase: Properties and purification." Bioscience Reports 5, no. 6 (June 1, 1985): 499–508. http://dx.doi.org/10.1007/bf01116949.

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A new method of purification of rat liver L-threonine deaminase has been developed, and the results obtained are compared with values obtained by other authors. Some properties of this enzyme (pH optimum, temperature optimum, thermal stability, specificity, etc.) have been examined and we found that the enzyme is inhibited by carbonate ions, that L-cysteine (a competitive inhibitor) is also an inactivator of the enzyme and that it is bound to the enzyme in a ratio of 0.25 mole of cysteine per mole of enzyme, supporting the hypothesis that the enzyme consists of 4 subunits.

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8

Jeffery, D., D. M. Rutherford, P. D. J. Weitzman, and G. G. Lunt. "Purification and partial characterization of 4-aminobutyrate:2-oxoglutarate aminotransferase from sheep brain and locust ganglia." Biochemical Journal 249, no. 3 (February 1, 1988): 795–99. http://dx.doi.org/10.1042/bj2490795.

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We report here the first purification to homogeneity of 4-aminobutyrate: 2-oxoglutarate aminotransferase (EC 2.6.1.19) (GABA-T) from an invertebrate source (locust) and its initial comparison with that of GABA-T from mammalian brain (sheep). The enzyme from both organisms was found to be a dimer of similar-sized subunits, with a native Mr of approx. 97,000. The pI of GABA-T from the locust was 6.7 and that of the sheep enzyme was 5.5. Michaelis constants for 4-aminobutyric acid (GABA) and 2-oxoglutarate were respectively 0.79 +/- 0.16 mM and 0.27 +/- 0.08 mM for the locust enzyme and 2.2 +/- 0.24 mM and 0.22 +/- 0.11 mM for the sheep enzyme. 5-(Aminomethyl)-3-isoxazolol (muscimol) was a competitive inhibitor of both enzymes, whereas 5-amino-1,3-cyclohexadienylcarboxylic acid (gabaculine) acted as a potent suicide substrate. However, 3-aminopropane-1-sulphonic acid, diaminobutyric acid, 1,2,3,4-tetrahydro-1-methyl-3-pyridinecarboxylic acid (isoguvacine), beta-(aminomethyl)-4-chlorobenzenepropanoic acid (baclofen), bicuculline and picrotoxin did not inhibit either enzyme at concentrations below 100 mM. Polyclonal antisera raised against GABA-T from the sheep failed to cross-react with the enzyme from locust in either an Ouchterlony immunodiffusion plate or a competitive enzyme-linked immunosorbent assay. The purification procedures differed considerably. Ion-exchange chromatography, which was found suitable for the purification of GABA-T from the sheep, was ineffective with locust enzyme, which was finally purified by hydrophobic-interaction chromatography and chromatofocusing.

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9

De Jongh, K. S., P. J. Schofield, and M. R. Edwards. "Kinetic mechanism of sheep liver NADPH-dependent aldehyde reductase." Biochemical Journal 242, no. 1 (February 15, 1987): 143–50. http://dx.doi.org/10.1042/bj2420143.

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The kinetic mechanism of the major sheep liver aldehyde reductase (ALR1) was studied with three aldehyde substrates: p-nitrobenzaldehyde, pyridine-3-aldehyde and D-glucuronate. In each case the enzyme mechanism was sequential and product-inhibition studies were consistent with an ordered Bi Bi mechanism, with the coenzymes binding to the free enzyme. Binding studies were used to investigate the interactions of substrates, products and inhibitors with the free enzyme. These provided evidence for the binding of D-glucuronate, L-gulonate and valproate, as well as NADP+ and NADPH. The enzyme was inhibited by high concentrations of D-glucuronate in a non-competitive manner, indicating that this substrate was able to bind to the free enzyme and to the E X NADP+ complex at elevated concentrations. Although the enzyme was inhibited by high pyridine-3-aldehyde concentrations, there was no evidence for the binding of this substrate to the free enzyme. Sheep liver ALR1 was inhibited by the ionized forms of alrestatin, sorbinil, valproate, 2-ethylhexanoate and phenobarbitone, indicating the presence of an anion-binding site similar to that described for the pig liver enzyme, which interacts with inhibitors and substrates containing a carboxy group. Sorbinil, valproate and 2-ethylhexanoate inhibited the enzyme uncompetitively at low concentrations and non-competitively at high concentrations, whereas phenobarbitone and alrestatin were non-competitive and uncompetitive inhibitors respectively. The significance of these results with respect to inhibitor and substrate binding is discussed.

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10

Macias, Pedro, and M. Carmen Pinto. "Purification and Partial Characterization of Rat Liver Lipoxygenase." Zeitschrift für Naturforschung B 42, no. 10 (October 1, 1987): 1343–48. http://dx.doi.org/10.1515/znb-1987-1020.

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Abstract Lipoxygenase was purified from rat liver cytosolic fraction by a method involving two successive chromatographic steps on Sephacryl S-200 and Phenyl Sepharose CL-4B. The enzyme has a molecular weight of 96 Kdal and it seems to be composed of two identical subunits. Chromatofocusing of the enzyme revealed a single band of activity at pi 6.3. The enzyme activity of the purified fraction showed maximum activity at pH 7.0 with a Km for linoleic acid of 1.4 μM and is competitively inhibited by the specific lipoxygenase inhibitor nordihydroguaiaretic acid. The purified enzyme shows absorption and fluorescence spectra similar to those of lipoxygenase from other sources. However, the molecular weight of lipoxygenase purified from liver is found to be different from that of the enzyme from polymorphonuclear leukocytes. It is suggested that there are different isoenzymes of lipoxygenases in mammals.

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11

Falany, C. N., M. E. Vazquez, and J. M. Kalb. "Purification and characterization of human liver dehydroepiandrosterone sulphotransferase." Biochemical Journal 260, no. 3 (June 15, 1989): 641–46. http://dx.doi.org/10.1042/bj2600641.

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A form of sulphotransferase capable of sulphating dehydroepiandrosterone and other steroids was purified from cytosol prepared from human liver. Dehydroepiandrosterone sulphotransferase was purified 621-fold when compared with the activity in cytosol using DEAE-Sepharose CL-6B and adenosine 3′,5′-bisphosphate-agarose affinity chromatography. During affinity chromatography, dehydroepiandrosterone sulphation activity could be resolved from p-nitrophenol sulphation activity catalysed by phenol sulphotransferase by using a gradient of adenosine 3′-phosphate 5′-phosphosulphate. The purified enzyme was most active towards dehydroepiandrosterone but was capable of conjugating a number of other steroids, including pregnenolone, androsterone and beta-oestradiol. No activity towards p-nitrophenol or dopamine, substrates for the phenol sulphotransferase, was observed with the pure enzyme. A single band with a subunit molecular mass of 35 kDa was observed by Coomassie Blue staining following SDS/polyacrylamide-gel electrophoresis of the purified enzyme. A molecular mass of 68-70 kDa was calculated for the active form of the enzyme by chromatography on Sephacryl S-200, suggesting that the active form of the enzyme is a dimer.

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Aydin, Fatih, Vedat Turkoglu, and Zehra Bas. "Purification and characterization of angiotensin-converting enzyme (ACE) from sheep lung." Molecular Biology Reports 48, no. 5 (May 2021): 4191–99. http://dx.doi.org/10.1007/s11033-021-06432-8.

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13

de Groen, P. C., G. D. LeSage, P. S. Tietz, and N. F. LaRusso. "Purification and immunological quantification of rat liver lysosomal glycosidases." Biochemical Journal 264, no. 1 (November 15, 1989): 115–23. http://dx.doi.org/10.1042/bj2640115.

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Although lysosomal enzyme activities are known to vary in response to numerous physiological and pharmacological stimuli, the relationship between lysosomal enzyme activity and enzyme concentration has not been systematically studied. Therefore we developed radioimmunoassays for two lysosomal glycosidases in order to determine lysosomal enzyme concentration. beta-Galactosidase and beta-glucuronidase were purified from rat liver 2780-fold and 1280-fold respectively, by using differential centrifugation, affinity chromatography, ion-exchange chromatography and molecular-sieve chromatography. Polyclonal antibodies to these enzymes were raised in rabbits, and two radioimmunoassays were established. Antibody specificity was shown by: (i) selective immunoprecipitation of enzyme activity; (ii) identical bands of purified enzyme on SDS/polyacrylamide-gel electrophoresis and immunoelectrophoresis; (iii) single immunoreactive peaks in molecular-sieve chromatography experiments. Sensitivities of the assays were such that 15 ng of beta-galactosidase and 45 ng of beta-glucuronidase decreased the ratio of bound to free radiolabel by 50%; minimal detectable amounts of immunoreactive enzymes were 2 ng and 10 ng respectively. The assays were initially used to assess the effects of physiological perturbations (i.e. fasting and age) on enzyme concentrations in rat liver; these experiments showed that changes in enzyme concentrations do not always correlate with changes in enzyme activities. This represents the first report of radioimmunoassays for lysosomal glycosidases. The results suggest that these radioimmunoassays provide useful technology for the study of regulatory control mechanisms of the concentrations of lysosomal glycosidases in mammalian tissues.

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GOODALL, GREGORY J., WALTHER JOHANNSSEN, JOHN C. WALLACE, and D. BRUCE KEECH. "Sheep Liver Propionyl-CoA Carboxylase: Purification and Some Molecular Properties." Annals of the New York Academy of Sciences 447, no. 1 Biotin (June 1985): 396–97. http://dx.doi.org/10.1111/j.1749-6632.1985.tb18456.x.

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Askar, Kasim Abass, A. Caleb Kudi, and A. John Moody. "Purification of Soluble Acetylcholinesterase from Sheep Liver by Affinity Chromatography." Applied Biochemistry and Biotechnology 165, no. 1 (April 16, 2011): 336–46. http://dx.doi.org/10.1007/s12010-011-9254-7.

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Tollersrud, O. K., and N. N. Aronson. "Purification and characterization of rat liver glycosylasparaginase." Biochemical Journal 260, no. 1 (May 15, 1989): 101–8. http://dx.doi.org/10.1042/bj2600101.

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1. Rat liver glycosylasparaginase [N4-(beta-N-acetylglucosaminyl)-L-asparaginase, EC 3.5.1.26] was purified to homogeneity by using salt fractionation, CM-cellulose and DEAE-cellulose chromatography, gel filtration on Ultrogel AcA-54, concanavalin A-Sepharose affinity chromatography, heat treatment at 70 degrees C and preparative SDS/polyacrylamide-gel electrophoresis. The purified enzyme had a specific activity of 3.8 mumol of N-acetylglucosamine/min per mg with N4-(beta-N-acetylglucosaminyl)-L-asparagine as substrate. 2. The native enzyme had a molecular mass of 49 kDa and was composed of two non-identical subunits joined by strong non-covalent forces and having molecular masses of 24 and 20 kDa as determined by SDS/polyacrylamide-gel electrophoresis. 3. The 20 kDa subunit contained one high-mannose-type oligosaccharide chain, and the 24 kDa subunit had one high-mannose-type and one complex-type oligosaccharide chain. 4. N-Terminal sequence analysis of each subunit revealed a frayed N-terminus of the 24 kDa subunit and an apparent N-glycosylation of Asn-15 in the same subunit. 5. The enzyme exhibited a broad pH maximum above 7. Two major isoelectric forms were found at pH 6.4 and 6.6. 6. Glycosylasparaginase was stable at 75 degrees C and in 5% (w/v) SDS at pH 7.0.

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LINDSTAD, I. Rune, Peter KÖLL, and John S. McKINLEY-McKEE. "Substrate specificity of sheep liver sorbitol dehydrogenase." Biochemical Journal 330, no. 1 (February 15, 1998): 479–87. http://dx.doi.org/10.1042/bj3300479.

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The substrate specificity of sheep liver sorbitol dehydrogenase has been studied by steady-state kinetics over the range pH 7-10. Sorbitol dehydrogenase stereo-selectively catalyses the reversible NAD-linked oxidation of various polyols and other secondary alcohols into their corresponding ketones. The kinetic constants are given for various novel polyol substrates, including L-glucitol, L-mannitol, L-altritol, D-altritol, D-iditol and eight heptitols, as well as for many aliphatic and aromatic alcohols. The maximum velocities (kcat) and the substrate specificity-constants (kcat/Km) are positively correlated with increasing pH. The enzyme-catalysed reactions occur by a compulsory ordered kinetic mechanism with the coenzyme as the first, or leading, substrate. With many substrates, the rate-limiting step for the overall reaction is the enzyme-NADH product dissociation. However, with several substrates there is a transition to a mechanism with partial rate-limitation at the ternary complex level, especially at low pH. The kinetic data enable the elucidation of new empirical rules for the substrate specificity of sorbitol dehydrogenase. The specificity-constants for polyol oxidation vary as a function of substrate configuration with D-xylo > d-ribo > L-xylo > d-lyxo ≈ l-arabino > D-arabino > l-lyxo. Catalytic activity with a polyol or an aromatic substrate and various 1-deoxy derivatives thereof varies with -CH2OH >-CH2NH2 >-CH2OCH3 ≈-CH3. The presence of a hydroxyl group at each of the remaining chiral centres of a polyol, apart from the reactive C2, is also nonessential for productive ternary complex formation and catalysis. A predominantly nonpolar enzymic epitope appears to constitute an important structural determinant for the substrate specificity of sorbitol dehydrogenase. The existence of two distinct substrate binding regions in the enzyme active site, along with that of the catalytic zinc, is suggested to account for the lack of stereospecificity at C2 in some polyols.

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Fernley, R. T., J. P. Coghlan, and R. D. Wright. "Purification and characterization of a high-Mr carbonic anhydrase from sheep parotid gland." Biochemical Journal 249, no. 1 (January 1, 1988): 201–7. http://dx.doi.org/10.1042/bj2490201.

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Approximately half the carbonic anhydrase activity of sheep parotid-gland homogenate is derived from a high-Mr protein [Fernley, Wright & Coghlan (1979) FEBS Lett. 105, 299-302]. This enzyme has now been purified to homogeneity, and its properties were compared with those of the well-characterized sheep carbonic anhydrase II. The protein has an apparent Mr of 540,000 as measured by gel filtration under non-denaturing conditions and an apparent subunit Mr of 45,000 as measured by SDS/polyacrylamide-gel electrophoresis. After deglycosylation with the enzyme N-glycanase the protein migrates with an apparent Mr of 36,000 on SDS/polyacrylamide-gel electrophoresis. The CO2-hydrating activity was 340 units/mg compared with 488 units/mg for sheep carbonic anhydrase II measured under identical conditions. This enzyme does not, however, hydrolyse p-nitrophenyl acetate. The enzyme contains 0.8 g-atom of zinc/mol of protein subunit. The peptide maps of the two carbonic anhydrases differ significantly from one another, indicating they are not related closely structurally. Unlike the carbonic anhydrase II isoenzyme, which has a blocked N-terminus, the high-Mr enzyme has a free glycine residue at its N-terminus.

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19

Dickinson, F. M. "Studies on the mechanism of sheep liver cytosolic aldehyde dehydrogenase." Biochemical Journal 225, no. 1 (January 1, 1985): 159–65. http://dx.doi.org/10.1042/bj2250159.

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The dissociation of the aldehyde dehydrogenase X NADH complex was studied by displacement with NAD+. The association reaction of enzyme and NADH was also studied. These processes are biphasic, as shown by McGibbon, Buckley & Blackwell [(1977) Biochem. J. 165, 455-462], but the details of the dissociation reaction are significantly different from those given by those authors. Spectral and kinetic experiments provide evidence for the formation of abortive complexes of the type enzyme X NADH X aldehyde. Kinetic studies at different wavelengths with transcinnamaldehyde as substrate provide evidence for the formation of an enzyme X NADH X cinnamoyl complex. Hydrolysis of the thioester relieves a severe quenching effect on the fluorescence of enzyme-bound NADH.

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RODRIGO, Lourdes, Fernando GIL, Antonio F. HERNANDEZ, Anabel MARINA, Jesus VAZQUEZ, and Antonio PLA. "Purification and characterization of paraoxon hydrolase from rat liver." Biochemical Journal 321, no. 3 (February 1, 1997): 595–601. http://dx.doi.org/10.1042/bj3210595.

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Paraoxonase (paraoxon hydrolase), an enzyme that hydrolyses paraoxon (O,O-diethyl O-p-nitrophenyl phosphate), is located in mammals primarily in the serum and liver. Although considerable information is available regarding serum paraoxonase, little is known about the hepatic form of this enzyme. The present work represents the first study on the purification of rat liver paraoxonase. This enzyme has been purified 415-fold to apparent homogeneity with a final specific activity of 1370 units/mg using a protocol consisting of five steps: solubilization of the microsomal fraction, hydroxyapatite adsorption, chromatography on DEAE-Sepharose CL-6B, non-specific affinity chromatography on Cibacron Blue 3GA and anion exchange on Mono Q HR 5/5. The presence of Ca2+ and Triton X-100 in the buffers throughout the purification procedure was essential for maintaining enzyme activity. SDS/PAGE of the final preparation indicated a single protein-staining band with an apparent Mr of 45000. N-terminal and internal amino acid sequences were determined and compared with those of paraoxonases from human and rabbit serum and mouse liver, showing a high similarity. The pH profile showed optimum activity at pH 8.5. The pH stability and heat inactivation of the enzyme were also studied. The Km for liver paraoxonase was 1.69 mM.

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Brindle, N. P., V. A. Zammit, and C. I. Pogson. "Regulation of carnitine palmitoyltransferase activity by malonyl-CoA in mitochondria from sheep liver, a tissue with a low capacity for fatty acid synthesis." Biochemical Journal 232, no. 1 (November 15, 1985): 177–82. http://dx.doi.org/10.1042/bj2320177.

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The characteristics of inhibition of carnitine palmitoyltransferase (CPT) I by malonyl-CoA were studied for the enzyme in mitochondria isolated from sheep liver, a tissue with a very low rate of fatty acid synthesis. Malonyl-CoA was as potent in inhibiting the sheep liver enzyme as in inhibiting the enzyme in rat liver mitochondria. CPT I in guinea-pig liver mitochondria was also similarly inhibited. The inhibition showed the same time-dependent characteristics previously established for the rat liver enzyme. Methylmalonyl-CoA was as effective an inhibitor of CPT I as malonyl-CoA in sheep liver mitochondria, but did not affect CPT I activity in mitochondria from rat or guinea-pig liver. The concentrations of malonyl-CoA required to inhibit CPT I in sheep liver mitochondria in vitro were similar to those found in freeze-clamped sheep liver samples (about 7 nmol of malonyl-CoA/g wet wt.). In sheep liver cells the content of malonyl-CoA was only one-tenth of that observed in vivo when glucose only was added to the incubation medium. Inclusion of acetate and/or insulin increased the malonyl-CoA content about 10-fold, to values similar to those observed in vivo. The rate of fatty acid synthesis in sheep liver cells was about 1% of that observed in rat liver, but was correlated with the concentrations of malonyl-CoA in the cells under various incubation conditions. These observations are discussed in relation to (i) the regulatory role of malonyl-CoA in tissues that have a low capacity for fatty acid synthesis, and (ii) the utilization by sheep liver of propionate as a gluconeogenic precursor.

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Bielicki, J., and J. J. Hopwood. "Human liver N-acetylgalactosamine 6-sulphatase. Purification and characterization." Biochemical Journal 279, no. 2 (October 15, 1991): 515–20. http://dx.doi.org/10.1042/bj2790515.

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Human N-acetylgalactosamine 6-sulphatase (EC 3.1.6.14), which is involved in the lysosomal degradation of the glycosaminoglycans keratan sulphate and chondroitin 6-sulphate, was purified more than 130,000-fold in 2.8% yield from liver by an eight-step column procedure. One major form was identified with a pI of 5.7 and a native molecular mass of 62 kDa by gel filtration. When analysed by SDS/PAGE, dithioerythritol-reduced enzyme contained polypeptides of molecular masses 57 kDa, 39 kDa and 19 kDa, whereas non-reduced enzyme contained a major polypeptide of molecular mass 70 kDa. It is proposed that active enzyme contains either the 57 kDa polypeptide or disulphide-linked 39 kDa and 19 kDa polypeptides. Minor amounts of other enzyme forms separated during the chromatofocusing step and the Blue A-agarose step were not further characterized. Purified N-acetylgalactosamine 6-sulphatase was inactive towards 4-methylumbelliferyl sulphate, but was active, with pH optima of 3.5-4.0, towards 6-sulphated oligosaccharide substrates. Km values of 12.5 and 50 microM and Vmax. values of 1.5 and 0.09 mumol/min per mg were determined with oligosaccharide substrates derived from chondroitin 6-sulphate and keratan sulphate respectively. Sulphate, phosphate and chloride ions were inhibitors of enzyme activity towards both substrates, with 50 microM-Na2SO4 giving 50% inhibition towards the chondroitin 6-sulphate trisaccharide substrate.

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Pristupa, Z. B., C. M. McAuley, and K. G. Scrimgeour. "Folylpolyglutamate synthetase from beef liver: purification and some properties." Biochemistry and Cell Biology 69, no. 8 (August 1, 1991): 556–60. http://dx.doi.org/10.1139/o91-082.

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The polypeptide chain of folylpolyglutamate synthetase from beef liver has been isolated and partially characterized. This polypeptide has an apparent molecular weight of 73 000. Its amino-terminal residue is blocked. Amino acid analysis agrees with the hydrophobic properties and the pi (6.0) of this cytosolic enzyme. Polyclonal antibodies to the denatured enzyme have been prepared.Key words: folylpolyglutamate synthetase, folates, polyglutamates, electrophoresis, antisera.

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Furukawa, K., and S. Roth. "Co-purification of galactosyltransferases from chick-embryo liver." Biochemical Journal 227, no. 2 (April 15, 1985): 573–82. http://dx.doi.org/10.1042/bj2270573.

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Two galactosyltransferases with nearly identical Mr values were purified 5000-7000-fold from microsomal membranes of chick-embryo livers by using several affinity columns. One enzyme transfers galactose from UDP-galactose to form a β-(1→4)-linkage to GlcNAc (N-acetylglucosamine) or AsAgAGP [asialo-agalacto-(alpha 1-acid glycoprotein)]. The other enzyme forms a β-(1→3)-linkage to AsOSM [asialo-(ovine submaxillary mucin)]. Both enzymes were solubilized (85%) from a microsomal pellet by using 1% Triton X-100 in 0.1 M-NaCl. The supernatant activities were subjected to DEAE-Sepharose chromatography and four affinity columns: UDP-hexanolamine-Sepharose, alpha-lactalbumin-Sepharose, GlcNAc-Sepharose and either AsAgAGP-Sepharose or AsOSM-Sepharose. The AsAgAGP enzyme [(1→4)-transferase] and the AsOSM enzyme [(1→3)-transferase] behave identically on the DEAE-Sepharose and UDP-hexanolamine-Sepharose columns, and similarly on the alpha-lactalbumin-Sepharose column. Final separation of the two enzymes, however, could only be achieved on affinity columns of their immobilized respective acceptors. Both purified enzymes migrate as a single band on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis after silver staining, and both have an apparent Mr of 68 000. The enzymes were radioiodinated and again subjected to sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Radioautographic analyses showed only one, intensely radioactive, band. Activity stains performed for both transferases after cellulose acetate electrophoresis indicate that, with this system too, both activities have identical mobilities, and co-migrate, as well, with the major, silver-stained, protein band. Kinetic studies with the purified enzymes show that the Km value for GlcNAc, for the (1→4)-transferase, is 4mM; for the (1→3)-transferase the Km value for AsOSM is 5mM, in terms of GalNAc (N-acetylgalactosamine) equivalents. Both enzymes have a Km value of 25 microM for UDP-galactose.

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25

Somers, Donald O'N, Janos Hajdu, and Margaret J. Adams. "A two-step purification procedure for sheep liver 6-phosphogluconate dehydrogenase." Protein Expression and Purification 2, no. 5-6 (October 1991): 385–89. http://dx.doi.org/10.1016/1046-5928(91)90098-4.

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Wang, Qinding, Manji Sun, Han Zhang, and Cuifen Huang 1. "Purification and properties of soman-hydrolyzing enzyme from human liver." Journal of Biochemical and Molecular Toxicology 12, no. 4 (1998): 213–17. http://dx.doi.org/10.1002/(sici)1099-0461(1998)12:4<213::aid-jbt3>3.0.co;2-o.

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27

Stark, Annika, and Johan Meijer. "Purification and characterization of multifunctional enzyme from mouse liver peroxisomes." Comparative Biochemistry and Physiology Part B: Comparative Biochemistry 108, no. 4 (August 1994): 471–80. http://dx.doi.org/10.1016/0305-0491(94)90100-7.

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28

Hirshey, S. J., and C. N. Falany. "Purification and characterization of rat liver minoxidil sulphotransferase." Biochemical Journal 270, no. 3 (September 15, 1990): 721–28. http://dx.doi.org/10.1042/bj2700721.

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Minoxidil (Mx), a pyrimidine N-oxide, is used therapeutically as an antihypertensive agent and to induce hair growth in patients with male pattern baldness. Mx NO-sulphate has been implicated as the agent active in producing these effects. This paper describes the purification of a unique sulphotransferase (ST) from rat liver cytosol that is capable of catalysing the sulphation of Mx. By using DEAE-Sepharose CL-6B chromatography, hydroxyapatite chromatography and ATP-agarose affinity chromatography, Mx-ST activity was purified 240-fold compared with the activity in cytosol. The purified enzyme was also capable of sulphating p-nitrophenol (PNP) at low concentrations (less than 10 microM). Mx-ST was purified to homogeneity, as evaluated by SDS/PAGE and reverse-phase h.p.l.c. The active form of the enzyme had a molecular mass of 66,000-68,000 Da as estimated by gel exclusion chromatography and a subunit molecular mass of 35,000 Da. The apparent Km values for Mx, 3′-phosphoadenosine 5′-phosphosulphate and PNP were 625 microM, 5.0 microM and 0.5 microM respectively. However, PNP displayed potent substrate inhibition at concentrations above 1.2 microM. Antibodies raised in rabbits to the pure enzyme detected a single band in rat liver cytosol with a subunit molecular mass of 35,000 Da, as determined by immunoblotting. The anti-(rat Mx-ST) antibodies also reacted with the phenol-sulphating form of human liver phenol sulphotransferase, suggesting some structural similarity between these proteins.

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Pajares, M. A., M. Villalba, and J. M. Mato. "Purification of phospholipid methyltransferase from rat liver microsomal fraction." Biochemical Journal 237, no. 3 (August 1, 1986): 699–705. http://dx.doi.org/10.1042/bj2370699.

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Phospholipid methyltransferase, the enzyme that converts phosphatidylethanolamine into phosphatidylcholine with S-adenosyl-L-methionine as the methyl donor, was purified to apparent homogeneity from rat liver microsomal fraction. When analysed by SDS/polyacrylamide-gel electrophoresis only one protein, with molecular mass about 50 kDa, is detected. This protein could be phosphorylated at a single site by incubation with [alpha-32P]ATP and the catalytic subunit of cyclic AMP-dependent protein kinase. A less-purified preparation of the enzyme is mainly composed of two proteins, with molecular masses about 50 kDa and 25 kDa, the 50 kDa form being phosphorylated at the same site as the homogeneous enzyme. After purification of both proteins by electro-elution, the 25 kDa protein forms a dimer and migrates on SDS/polyacrylamide-gel electrophoresis with molecular mass about 50 kDa. Peptide maps of purified 25 kDa and 50 kDa proteins are identical, indicating that both proteins are formed by the same polypeptide chain(s). It is concluded that rat liver phospholipid methyltransferase can exist in two forms, as a monomer of 25 kDa and as a dimer of 50 kDa. The dimer can be phosphorylated by cyclic AMP-dependent protein kinase.

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30

Lund, K., D. K. Merrill, and R. W. Guynn. "Purification and subunit structure of phosphoglycerate dehydrogenase from rabbit liver." Biochemical Journal 238, no. 3 (September 15, 1986): 919–22. http://dx.doi.org/10.1042/bj2380919.

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D-3-Phosphoglycerate dehydrogenase (EC 1.1.1.95) was purified from rabbit liver by (NH4)2SO4 fractionation, DEAE-Sephacel chromatography, affinity chromatography on AMP-agarose and molecular-sieve h.p.l.c. The purified enzyme was homogeneous as judged by SDS/polyacrylamide-slab-gel electrophoresis. On the basis of molecular-sieve h.p.l.c. and SDS/polyacrylamide-gel electrophoresis, the enzyme is a tetramer composed of subunits of Mr 60,000.

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31

Sunaga, H., H. Sugimoto, Y. Nagamachi, and S. Yamashita. "Purification and properties of lysophospholipase isoenzymes from pig gastric mucosa." Biochemical Journal 308, no. 2 (June 1, 1995): 551–57. http://dx.doi.org/10.1042/bj3080551.

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Two lysophospholipases, named gastric lysophospholipases I and II (enzymes I and II), were purified 3730- and 2680-fold from pig gastric mucosa. The preparations showed 22 and 23 kDa single protein bands on SDS/PAGE respectively. Both enzymes lacked transacylase activity and appeared to exist as monomers. Their activities were not affected by Ca2+, Mg2+ or EDTA. Enzyme I was most active at pH 8.5 and hydrolysed a variety of lysophospholipids including acidic lysophospholipids and the acyl analogue of platelet-activating factor, whereas enzyme II was most active at pH 8 and its activity was confined to lysophosphatidylcholine and lysophosphatidylethanolamine. When 1-palmitoylglycerophosphocholine was used as substrate, enzymes I and II showed half-maximal activities at 11 and 12 microM respectively. The enzymes exhibited no phospholipase B, lipase or general esterase activity. Enzyme II was significantly inhibited by lysophosphatidic acid whereas enzyme I was only moderately inhibited. Peptide mapping with V8 protease and papain revealed structural dissimilarity between the two enzymes. Antiserum raised against enzyme I did not recognize enzyme II, but did recognize the small-sized lysophospholipase purified from rat liver. Anti-(enzyme II) consistently did not cross-react with enzyme I or the liver enzyme. These antisera specifically recognized neither the 60 kDa lysophospholipase transacylase purified from liver nor any peritoneal macrophage protein. Thus gastric mucosa contains two different small-sized lysophospholipases: one is closely related to the small-sized lysophospholipase of liver, but the other appears to be a novel isoform.

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DEMİR, Yeliz, Bülent ŞENGÜL, Bülent ERGUN, and Şükrü BEYDEMİR. "Alcohol Dehydrogenase from Sheep Liver: Purification, Characterization and Impacts of Some Antibiotics." Journal of the Institute of Science and Technology 7, no. 3 (September 12, 2017): 151–59. http://dx.doi.org/10.21597/jist.2017.173.

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33

Chinchetru, Miguel A., Jose A. Cabezas, and Pedro Calvo. "Purification and characterization of a broad specificity β-glucosidase from sheep liver." International Journal of Biochemistry 21, no. 5 (January 1989): 469–76. http://dx.doi.org/10.1016/0020-711x(89)90126-2.

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34

Tayefi-Nasrabadi, Hossein, and Reza Rahmani. "Partial Purification and Characterization of Rhodanese from Rainbow Trout (Oncorhynchus mykiss) Liver." Scientific World Journal 2012 (2012): 1–5. http://dx.doi.org/10.1100/2012/648085.

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Cyanide is one of the most toxic substances present in a wide variety of food materials that are consumed by animals. Rhodanese, a ubiquitous enzyme, can catalyse the detoxification of cyanide by sulphuration reaction. In this study, rhodanese was partially purified and characterized from the liver tissue homogenate of the rainbow trout. The enzyme was active in a broad range of pH, from 5 to 12. The optimal activity was found at a high pH (pH 10.5), and the temperature optimum was25∘C. The enzyme was heat labile, losing > 50% of relative activity after only 5 min of incubation at40∘C. TheKmvalues for KCN and Na2S2O3as substrates were 36.81 mM and 19.84 mM, respectively. Studies on the enzyme with a number of cations showed that the activity of the enzyme was not affected by Sn2+, but Hg2+, Ba2+, Pb2+, and Ca2+inhibited and Cu2+activated the enzyme with a concentration-dependent manner.

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Cvetanović, M., M. Moreno de la Garza, V. Dommes, and W. H. Kunau. "Purification and characterization of 2-enoyl-CoA reductase from bovine liver." Biochemical Journal 227, no. 1 (April 1, 1985): 49–56. http://dx.doi.org/10.1042/bj2270049.

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Mitochondrial 2-enoyl-CoA reductase from bovine liver was purified and characterized. A simple three-step purification was developed, involving ion-exchange chromatography to separate the bulk of the NADPH-dependent 2,4-dienoyl-CoA reductase, followed by chromatography on Blue Sepharose and adenosine 2',5'-bisphosphate-Sepharose. Homogeneous enzyme with a subunit Mr of 35 500 is obtained in 35% yield. The Mr of the native enzyme, determined by three different methods, yielded values that suggest that the enzyme is dimeric. NADPH is required as cofactor, and cannot be replaced by NADH. The activity of the purified enzyme towards 2-trans-double bonds in 2-monoene and 2,4-diene structures was investigated. 2-Enoyl-CoA reductase reduced the double bonds in a series of 2-trans-monoenoyl-CoA esters with different chain lengths, but did not exhibit significant activity towards 2-trans-double bonds of 2,4-dienoyl-CoA esters. This result is discussed in the light of analogous observations with enoyl-CoA hydratase.

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Fujiwara, K., S. Takeuchi, K. Okamura-Ikeda, and Y. Motokawa. "Purification and cDNA cloning of lipoate-activating enzyme from bovine liver." Biochemical Society Transactions 28, no. 5 (October 1, 2000): A326. http://dx.doi.org/10.1042/bst028a326c.

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37

Kumagai, Hiroshi, Ryo Kon, Toshiya Hoshino, Tomoko Aramaki, Masuhiro Nishikawa, Seiyu Hirose, and Kazuei Igarashi. "Purification and properties of a decapping enzyme from rat liver cytosol." Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology 1119, no. 1 (February 1992): 45–51. http://dx.doi.org/10.1016/0167-4838(92)90232-3.

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38

Maellaro, E., B. Del Bello, L. Sugherini, A. Santucci, M. Comporti, and A. F. Casini. "Purification and characterization of glutathione-dependent dehydroascorbate reductase from rat liver." Biochemical Journal 301, no. 2 (July 15, 1994): 471–76. http://dx.doi.org/10.1042/bj3010471.

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GSH-dependent enzymic reduction of dehydroascorbic acid to ascorbic acid has been studied in rat liver cytosol. After gel filtration of cytosol on Sephadex G-100 SF, dehydroascorbate reductase activity was recovered in two distinct peaks, one corresponding to glutaredoxin (an enzyme already known for its dehydroascorbate reductase activity) and another, much larger one, corresponding to a novel enzyme different from glutaredoxin. The latter was purified to apparent homogeneity. The purification process involved (NH4)2SO4 fractionation, followed by DEAE-Sepharose, Sephadex G-100 SF and Reactive Red chromatography. SDS/PAGE of the purified enzyme in either the presence or absence of 2-mercaptoethanol demonstrated a single protein band of M(r) 31,000. The M(r) determined by both Sephadex G-100 SF chromatography and h.p.l.c. was found to be approx. 48,000. H.p.l.c. of the denatured enzyme gave an M(r) value identical with that obtained by SDS/PAGE (31,000). The apparent Km for dehydroascorbate was 245 microM and the Vmax. was 1.9 mumol/min per mg of protein; for GSH they were 2.8 mM and 4.5 mumol/min per mg of protein respectively. The optimal pH range was 7.5-8.0. Microsequence analysis of the electro-transferred enzyme band showed that the N-terminus is blocked. Data on internal primary structure were obtained from CNBr-and N-chlorosuccinimide-derived fragments. No significative sequence similarity was found to any of the protein sequences contained in the Protein Identification Resource database.

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Boopathy, R., and A. S. Balasubramanian. "Purification and characterization of sheep platelet cyclo-oxygenase Acetylation by aspirin prevents haemin binding to the enzyme." Biochemical Journal 239, no. 2 (October 15, 1986): 371–77. http://dx.doi.org/10.1042/bj2390371.

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Arachidonate cyclo-oxygenase (prostaglandin synthetase; prostaglandin endoperoxide synthetase; EC 1.14.99.1) was purified from sheep platelets. The purification procedure involved hydrophobic column chromatography using either Ibuprofen-Sepharose, phenyl-Sepharose or arachidic acid-Sepharose as the first step followed by metal-chelate Sepharose and haemin-Sepharose affinity chromatography. The purified enzyme (Mr approximately 65,000) was homogeneous as observed by SDS/polyacrylamide-gel electrophoresis and silver staining. The enzyme was a glycoprotein with mannose as the neutral sugar. Haemin or haemoglobin was essential for activity. The purified enzyme could bind haemin exhibiting a characteristic absorption maximum at 410 nm. The enzyme after metal-chelate column chromatography could undergo acetylation by [acetyl-3H]aspirin. The labelled acetylated enzyme could not bind to haemin-Sepharose, presumably due to acetylation of a serine residue involved in the binding to haemin. The acetylated enzyme also failed to show its characteristic absorption maximum at 410 nm when allowed to bind haemin.

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40

Williams, D. E., M. Dutchuk, and M. Y. Lee. "Purification and characterization of a microsomal cytochrome P-450 IIB enzyme from sheep lung." Xenobiotica 21, no. 7 (January 1991): 979–89. http://dx.doi.org/10.3109/00498259109039537.

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41

Freeman, C., P. R. Clements, and J. J. Hopwood. "Human liver N-acetylglucosamine-6-sulphate sulphatase. Purification and characterization." Biochemical Journal 246, no. 2 (September 1, 1987): 347–54. http://dx.doi.org/10.1042/bj2460347.

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Human N-acetylglucosamine-6-sulphate sulphatase was purified at least 50,000-fold to homogeneity in 78% yield from liver with a simple three-step four-column procedure, which consists of a concanavalin A-Sepharose/Blue A-agarose coupled step, chromatofocusing and Cu2+-chelating Sepharose chromatography. In all, four forms were isolated and partially characterized. Forms A and B, both with a pI greater than 9.5 and representing 30% and 60% respectively of the recovered enzyme activity, were separated by hydroxyapatite chromatography of the enzyme preparation obtained from the Cu2+-chelating Sepharose step. Both forms A and B had native molecular masses of 75 kDa. When analysed by SDS/polyacrylamide-gel electrophoresis, form A consists of a single polypeptide of molecular mass 78 kDa, whereas form B contained 48 kDa and 32 kDa polypeptide subunits. Neither form A nor form B was taken up from the culture medium into cultured human skin fibroblasts. The two other forms (C and D), with pI values of 5.8 and 5.4 respectively, represented approx. 7% and 3% of the total recovered enzyme activity. The native molecular masses of forms C and D were 94 kDa and approx. 75 kDa respectively. Form C contained three polypeptides with molecular masses of 48, 45 and 32 kDa. N-Acetylglucosamine-6-sulphate sulphatase activity was measured with a radiolabelled disaccharide substrate derived from heparin. The development of this substrate enabled the isolation and characterization of N-acetylglucosamine-6-sulphate sulphatase to proceed efficiently. Forms A, B and C had pH optima of 5.0, Km values of 11.7, 14.2 and 11.1 microM respectively and Vmax. values of 105, 60 and 53 nmol/min per mg of protein respectively. The molecular basis of the multiple forms of this sulphatase is not known. It is postulated that the differences in structure and properties of the four enzyme forms are due to differences in the state of processing of a large subunit.

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42

Bielicki, J., C. Freeman, P. R. Clements, and J. J. Hopwood. "Human liver iduronate-2-sulphatase. Purification, characterization and catalytic properties." Biochemical Journal 271, no. 1 (October 1, 1990): 75–86. http://dx.doi.org/10.1042/bj2710075.

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Human iduronate-2-sulphatase (EC 3.1.6.13), which is involved in the lysosomal degradation of the glycosaminoglycans heparan sulphate and dermatan sulphate, was purified more than 500,000-fold in 5% yield from liver with a six-step column procedure, which consisted of a concanavalin A-Sepharose-Blue A-agarose coupled step, chromatofocusing, gel filtration on TSK HW 50S-Fractogel, hydrophobic separation on phenyl-Sepharose CL-4B and size separation on TSK G3000SW Ultrapac. Two major forms were identified. Form A and form B, with pI values of 4.5 and less than 4.0 respectively, separated at the chromatofocusing step in approximately equal amounts of recovered enzyme activity. By gel-filtration methods form A had a native molecular mass in the range 42-65 kDa. When analysed by SDS/PAGE, dithioerythritol-reduced and non-reduced form A and form B consistently contained polypeptides of molecular masses 42 kDa and 14 kDa. Iduronate-2-sulphatase was purified from human kidney, placenta and lung, and form A was shown to have similar native molecular mass and subunit components to those observed for liver enzyme. Both forms of liver iduronate-2-sulphatase were active towards a variety of substrates derived from heparin and dermatan sulphate. Kinetic parameters (Km and Kcat) of form A were determined with a variety of substrates matching structural aspects of the physiological substrates in vivo, namely heparan sulphate, heparin and dermatan sulphate. Substrate with 6-sulphate esters on the aglycone residue adjacent to the iduronic acid 2-sulphate residue being attack were hydrolysed with catalytic efficiencies up to 200 times above that observed for the simplest disaccharide substrate without a 6-sulphated aglycone residue. The effect of incubation pH on enzyme activity towards the variety of substrates evaluated was complex and dependent on substrate aglycone structure, substrate concentration, buffer type and the presence of other proteins. Sulphate and phosphate ions and a number of substrate and product analogues were potent inhibitor of form A and form B enzyme activities.

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43

Rawal, N., R. Rajpurohit, W. K. Paik, and S. Kim. "Purification and characterization of S-adenosylmethionine-protein-arginine N-methyltransferase from rat liver." Biochemical Journal 300, no. 2 (June 1, 1994): 483–89. http://dx.doi.org/10.1042/bj3000483.

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A protein methylase I (S-adenosylmethionine-protein-arginine N-methyltransferase; EC 2.1.1.23), with a high specificity for recombinant heterogeneous nuclear ribonucleoprotein particle (hnRNP) protein A1, was purified from rat liver. The purification method is simple and rapid; a single initial step of DEAE-cellulose DE-52 chromatography resulted in a 114-fold enrichment from the cytosol, and subsequent Sephadex G-200 chromatography and f.p.l.c. yielded a homogeneous preparation. Ouchterlony double-immunodiffusion analysis indicated that the rat liver enzyme is immunologically different from an analogous enzyme from the calf brain, nuclear protein/histone-specific protein methylase I [Ghosh, Paik and Kim (1988) J. Biol. Chem. 263, 19024-19033; Rajpurohit, Lee, Park, Paik and Kim (1994) J. Biol. Chem. 269, 1075-1082]. The purified enzyme has a molecular mass of 450 kDa on Superose chromatography and 110 kDa on SDS/PAGE, indicating that it is composed of four identical-size subunits. The Km values for protein A1 and S-adenosyl-L-methionine were 0.54 x 10(-6) and 6.3 x 10(-6) M respectively. S-Adenosyl-L-homocysteine and sinefungin were effective inhibitors of the enzyme with Ki values of 8.4 x 10(-6) M and 0.65 x 10(-6) M respectively. Bivalent metal ions such as Zn2+, Mn2+ and Ni2+ were particularly toxic to the enzyme; at 1 mM Zn2+, 99% of the activity was inhibited. In addition, 50% of the enzyme activity was lost by treatment with 0.12 mM p-chloromercuribenzoate, indicating a requirement for a thiol group for enzyme activity. Glycerol, a compound often used to prevent enzyme inactivation, inhibited over 80% of the activity when present in the reaction mixture at a concentration of 20%.

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Loomes, K. M., and T. M. Kitson. "Reaction between sheep liver mitochondrial aldehyde dehydrogenase and various thiol-modifying reagents." Biochemical Journal 261, no. 1 (July 1, 1989): 281–84. http://dx.doi.org/10.1042/bj2610281.

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Sheep liver mitochondrial aldehyde dehydrogenase reacts with 2,2′-dithiodipyridine and 4,4′-dithiodipyridine in a two-step process: an initial rapid labelling reaction is followed by slow displacement of the thiopyridone moiety. With the 4,4′-isomer the first step results in an activated form of the enzyme, which then loses activity simultaneously with loss of the label (as has been shown to occur with the cytoplasmic enzyme). With 2,2′-dithiodipyridine, however, neither of the two steps of the reaction has any effect on the enzymic activity, showing that the mitochondrial enzyme possesses two cysteine residues that must be more accessible or reactive (to this reagent at least) than the postulated catalytically essential residue. The symmetrical reagent 5,5′-dithiobis-(1-methyltetrazole) activates mitochondrial aldehyde dehydrogenase approximately 4-fold, whereas the smaller related compound methyl l-methyltetrazol-5-yl disulphide is a potent inactivator. These results support the involvement of mixed methyl disulphides in causing unpleasant physiological responses to ethanol after the ingestion of certain antibiotics.

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45

Van Veldhoven, P. P., P. Van Rompuy, J. C. T. Vanhooren, and G. P. Mannaerts. "Purification and further characterization of peroxisomal trihydroxycoprostanoyl-CoA oxidase from rat liver." Biochemical Journal 304, no. 1 (November 15, 1994): 195–200. http://dx.doi.org/10.1042/bj3040195.

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The acyl-CoA oxidase, catalysing the peroxisomal desaturation of the CoA-ester of trihydroxycoprostanic acid, a bile acid intermediate, has been purified to homogeneity from rat liver. Its native molecular mass, as determined by gel filtration and native gel electrophoresis, was 120 and 175 kDa respectively, suggesting a homodimeric protein consisting of 68.6 kDa subunits. If isolated in the presence of FAD, the enzyme showed a typical flavoprotein spectrum and contained most likely 2 mol of FAD per mol of enzyme. The cofactor, however, was loosely bound. The enzyme acted exclusively on 2-methyl-branched compounds, including pristanoyl-CoA and 2-methylhexanoyl-CoA if albumin was present. Important parameters to obtain a pure and active enzyme were the following: (1) using chromatographic separations like hydrophobic interaction and metal affinity, which allow the presence of high salt concentrations, conditions which stabilize the oxidase; (2) avoiding dialysis and (NH4)2SO4 precipitation; (3) including, when appropriate, FAD, dithiothreitol and a diol-compound in the solvents; and (4) carefully monitoring the removal of other acyl-CoA oxidases which possess the same native molecular mass and subunit size.

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46

Pineau, T., P. Galtier, C. Bonfils, J. Derancourt, and P. Maurel. "Purification of a sheep liver cytochrome P-450 from the P450IIIA gene subfamily." Biochemical Pharmacology 39, no. 5 (March 1990): 901–9. http://dx.doi.org/10.1016/0006-2952(90)90206-z.

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47

Chooback, Lilian, Nancy E. Price, William E. Karsten, John Nelson, Paula Sundstrom, and Paul F. Cook. "Cloning, Expression, Purification, and Characterization of the 6-Phosphogluconate Dehydrogenase from Sheep Liver." Protein Expression and Purification 13, no. 2 (July 1998): 251–58. http://dx.doi.org/10.1006/prep.1998.0896.

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48

Ramsay, R. R., J. P. Derrick, A. S. Friend, and P. K. Tubbs. "Purification and properties of the soluble carnitine palmitoyltransferase from bovine liver mitochondria." Biochemical Journal 244, no. 2 (June 1, 1987): 271–78. http://dx.doi.org/10.1042/bj2440271.

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A new carnitine palmitoyltransferase (CPT) was purified to homogeneity from bovine liver mitochondria which were 96% free of peroxisomal contamination, as judged by catalase and glutamate dehydrogenase activities. The enzyme is easily removed from mitochondria, without the use of detergent. It is monomeric (Mr 63,500), unlike other preparations of CPT from mitochondria, and is most active with myristoyl-CoA and palmitoyl-CoA. The Km values are between 0.8 and 4 microM for a range of substrates from hexanoyl-CoA to stearoyl-CoA; these are much lower than values reported for other purified CPT preparations. The Km for L-carnitine is 185 microM measured with palmitoyl-CoA, and does not vary greatly with the chain length. This is also lower than the values reported for other CPT preparations, but higher than those cited for the medium-chain transferases. Kinetic and inhibitor studies were consistent with a rapid-equilibrium random-order mechanism. 2-Bromopalmitoyl-CoA, which is an inhibitor of the outer CPT, inhibited the enzyme competitively with palmitoyl-CoA as the variable substrate, when added without preincubation. If the enzyme was preincubated with 2-bromopalmitoyl-CoA and carnitine, the activity did not reappear after gel filtration of the protein. The inhibitor was bound in a 1:1 stoichiometry per subunit of enzyme.

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Sindelar, P., T. Chojnacki, and C. Valtersson. "Phosphatidylethanolamine:dolichol acyltransferase. Characterization and partial purification of a novel rat liver enzyme." Journal of Biological Chemistry 267, no. 29 (October 1992): 20594–99. http://dx.doi.org/10.1016/s0021-9258(19)36728-6.

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50

Fujiwara, Kazuko, Shinji Takeuchi, Kazuko Okamura-Ikeda, and Yutaro Motokawa. "Purification, Characterization, and cDNA Cloning of Lipoate-activating Enzyme from Bovine Liver." Journal of Biological Chemistry 276, no. 31 (May 29, 2001): 28819–23. http://dx.doi.org/10.1074/jbc.m101748200.

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Journal articles on the topic 'Sheep liver enzyme purification'

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