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1
Oppert, B., K. Hartzer, and M. Zuercher. "Digestive proteinases in Lasioderma serricorne (Coleoptera: Anobiidae)." Bulletin of Entomological Research 92, no. 4 (August 2002): 331–36. http://dx.doi.org/10.1079/ber2002179.
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AbstractThe cigarette beetle, Lasioderma serricorne (Fabricius), is a common pest of stored foods. A study of digestive proteinases in L. serricorne was performed to identify potential targets for proteinaceous biopesticides, such as proteinase inhibitors. Optimal casein hydrolysis by luminal proteinases of L. serricorne was in pH 8.5–9.0 buffers, although the pH of luminal contents was slightly acidic. Results from substrate and inhibitor analyses indicated that the primary digestive proteinases were serine proteinases. The most effective inhibitors of caseinolytic hydrolysis were from soybean (both Bowman Birk and Kunitz), with some inhibition by chymostatin, N-TOSYL-L-phenylalanine chloromethyl ketone, and leupeptin. Casein zymogram analysis identified at least eight proteolytic activities. Activity blot analyses indicated one major proteinase activity that hydrolysed the trypsin substrate N-α-benzoyl-L-arginine ρ-nitroanilide, and three major proteinase activities that hydrolysed the chymotrypsin substrate N-succinyl ala-ala-pro-phe ρ-nitroanilide. The absence of cysteine, aspartic, and metallo proteinases in L. serricorne digestion was evidenced by the lack of activation by thiol reagents, alkaline pH optima, and the results from class-specific proteinase inhibitors. The data suggest that protein digestion in L. serricorne is primarily dependent on trypsin- and chymotrypsin-like proteinases.
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Jankangram, Wichuda, and Sunthorn Chooluck. "In silico Analysis and Characterization of a Putative Aspartic Proteinase Inhibitor, IA3 from Lachancea kluyveri." Trends in Sciences 20, no. 3 (January 19, 2023): 6377. http://dx.doi.org/10.48048/tis.2023.6377.
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Aspartic proteinases play important role in various physiological and biological processes. Understanding catalytic mechanisms of these enzymes could lead to crucial medical and biological applications. Two types of aspartic proteinase inhibitors have been identified i.e. small molecule inhibitor and naturally occurring peptides. Most of aspartic proteinases are highly susceptible to inhibition by a series of small, non-proteinaceous natural products; pepstatin. However, only limited number of naturally-occurring polypeptide inhibitors of aspartic proteinases has so far been discovered. One among these inhibitors is Saccharomyces cerevisiae IA3. The S. cerevisiae Proteinase A (ScPrA) is solely and potently inhibited by this small polypeptide at sub-nanomolar level. It was proven that, not only IA3 has no detectable effect against a wide range of aspartic proteinases, but it was also shown to be cleaved as a substrate of these non-target enzymes. Bioinformatics analysis of the IA3 structure has been undertaken in this study. Database searching for sequence homology from available fungal genome data bank using the Basic Local Alignment Search Tool (BLAST) revealed that 4 structurally related, IA3-like proteins have successfully been identified. The amino acid sequence of IA3-like proteins from Lachancea kluyveri share highest degree of similarity toward wild-type IA3. The Lk-IA3-like gene was synthesized using a PCR-based gene synthesis method. Protein expression in E. coli, protein purification and characterization of Lk-IA3-like by enzyme kinetic assays were performed. The results indicated that Lk-IA3-like protein inhibits ScPrA at the Ki value of 190 ± 0.11 nM and possesses no inhibitory effect toward AfPrA (Aspergillus fumigatus proteinase A) or HuCatD (Human cathepsin D). HIGHLIGHTS Aspartic proteinases enzyme family is crucially involved in various physiological and biological processes including in the pathogenesis of numerous diseases which make them a possible target for drug design Inhibitors of aspartic proteinases are not only applied for human disease treatment but also extended to plant and other economically important animals Aspartic proteinase from cerevisiae (Proteinase A) is solely and potently inhibited by a small peptideinhibitor, IA3 Bioinformatics analysis of the naturally occurring aspartic proteinases inhibitor, IA3 structure has been undertaken in this research and putative IA3-like proteins have successfully been identified IA3-like proteins from Lachancea kluyveri were produced in coli and enzyme inhibition assays revealed that Lk-IA3-like protein inhibits ScPrA at the Ki value of 190 ± 0.11 nM GRAPHICAL ABSTRACT
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Rosenthal, P. J., K. Kim, J. H. McKerrow, and J. H. Leech. "Identification of three stage-specific proteinases of Plasmodium falciparum." Journal of Experimental Medicine 166, no. 3 (September 1, 1987): 816–21. http://dx.doi.org/10.1084/jem.166.3.816.
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We have identified and characterized three stage-specific proteinases of Plasmodium falciparum that are active at neutral pH. We analyzed ring-, trophozoite-, schizont-, and merozoite-stage parasites by gelatin substrate PAGE and characterized the identified proteinases with class-specific proteinase inhibitors. No proteinase activity was detected with rings. Trophozoites had a 28 kD proteinase that was inhibited by inhibitors of cysteine proteinases. Mature schizonts had a 35-40 kD proteinase that also was inhibited by cysteine proteinase inhibitors. Merozoite fractions had a 75 kD proteinase that was inhibited by serine proteinase inhibitors. The stage-specific activity of these proteinases and the correlation between the effects of proteinase inhibitors on the isolated enzymes with the effects of the inhibitors on whole parasites suggest potential critical functions for these proteinases in the life cycle of malaria parasites.
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Sever, Natasa, Metka Filipic, Joze Brzin, and Tamara T. Lah. "Effect of Cysteine Proteinase Inhibitors on Murine B16 Melanoma Cell Invasion in vitro." Biological Chemistry 383, no. 5 (May 15, 2002): 839–42. http://dx.doi.org/10.1515/bc.2002.088.
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Abstract Various types of proteinases are implicated in the malignant progression of human and animal tumors. Proteinase inhibitors may therefore be useful as therapeutic agents in antiinvasive and antimetastatic treatment. The aims of this study were (1) to estimate the relative importance of proteinases in B16 cell invasion in vitro using synthetic, classspecific proteinase inhibitors and (2) to assess the inhibitory effect of some naturally occurring cysteine proteinase inhibitors. Serine proteinase inhibitor reduced invasiveness by up to 24%, whereas inhibition of aspartic proteinases reduced invasion by 11%. Synthetic inhibitors of cysteine proteinases markedly impaired invasion: cathepsin B inhibitors, particularly Ca 074Me, inhibited invasion from 20 40%, whereas cathepsin L inhibitor Clik 148 reduced invasion by 11%. The potato cysteine proteinase inhibitor PCPI 8.7 inhibited invasion by 21%, whereas another potato inhibitor, PCPI 6.6, and the mushroom cysteine proteinase inhibitor clitocypin had no effects. As the inhibitors that inhibited cathepsin B were in general more efficient at impairing the invasiveness, we conclude that of the two cysteine proteinases, cathepsin B plays a more important role than cathepsin L in murine melanoma cell invasion.
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Garciacarreno, F. L., L. E. Dimes, and N. F. Haard. "Substrate-Gel Electrophoresis for Composition and Molecular Weight of Proteinases or Proteinaceous Proteinase Inhibitors." Analytical Biochemistry 214, no. 1 (October 1993): 65–69. http://dx.doi.org/10.1006/abio.1993.1457.
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Hiemstra, P. S. "Novel roles of protease inhibitors in infection and inflammation." Biochemical Society Transactions 30, no. 2 (April 1, 2002): 116–20. http://dx.doi.org/10.1042/bst0300116.
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The local balance between proteinase inhibitors and proteinases determines local proteolytic activity. Various studies have demonstrated the importance of serine proteinase inhibitors in regulating the activity of serine proteinases that are released by leucocytes during inflammation. Recently it has been shown that these inhibitors may also display functions that are distinct from those associated with the inhibition of leucocyte-derived proteinases. In this review the results of selected studies focusing on three inhibitors of neutrophil elastase, i.e. α1-proteinase inhibitor, secretory leucocyte proteinase inhibitor and elafin, are presented, with the aim of illustrating their possible involvement in the regulation of inflammation, host defence against infection, tissue repair and extracellular matrix synthesis.
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Ikeda, T. "Involvement of cysteine proteinases in excystment of Paragonimus ohirai metacercariae induced by sodium cholate and A23187." Journal of Helminthology 77, no. 1 (March 2003): 21–26. http://dx.doi.org/10.1079/joh2002144.
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AbstractThe involvement of intrinsic proteinases in the excystment of Paragonimus ohirai metacercariae was studied in in vitro excystment induced by sodium (Na) cholate, a bile salt and A23187, a Ca2+ ionophore. The effects of various proteinase inhibitors on the in vitro excystment were examined and similar inhibitory profiles were obtained. Benzyloxycarbonyl-L-leucyl-L-leucinal (Z-Leu-Leu-H), a cysteine proteinase inhibitor and 4-(2-aminoethyl)-benzenesulfonyl fluoride (Pefabloc SC), a serine proteinase inhibitor completely inhibited excystment, while L-3-carboxy-2,3-trans-epoxypropionyl-leucylamido (4-guanidino)-butane (E-64), a cysteine proteinase inhibitor and leupeptin, a cysteine/serine proteinase inhibitor permitted partial excystment at a lower rate, but inhibited it from proceeding from the partial excystment stage. In secretions released from metacercariae during excystment, proteinase activities detected towards various fluorogenic peptidyl substrates were almost completely inhibited by Z-Leu-Leu-H and E-64, but not by Pefabloc SC. Sodium cholate induced a higher secretion of cysteine proteinases and a higher rate of excystment than A23187. Profiles of cysteine proteinase activities towards five peptidyl substrates detected were markedly different among the two secretions and the lysate of newly excysted juveniles. Newly excysted juveniles released cysteine proteinases with similar activity profiles and levels to metacercariae induced by Na cholate-incubation, whereas the release of cysteine proteinases was reduced compared with metacercariae induced by A23187-incubation. These results provide valuable information about the involvement of intrinsic proteinases in metacercarial excystment.
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Bózner, P., and P. Demeš. "Proteinases inTrichomonas vaginalisandTritrichomonas mobilensisare not exclusively of cysteine type." Parasitology 102, no. 1 (February 1991): 113–15. http://dx.doi.org/10.1017/s0031182000060418.
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SUMMARYHigh molecular weight proteinases ofTrichomonas vaginalis(with apparentMrvalues 142 and > 220 kDa) andTritrichomonas mobilensis(Mr67, 86, 104 and 120 kDa), optimally active at pH 8, were analysed in gelatin-containing polyacrylamide gels. All of these proteinases were resistant to serine-, aspartic- as well as cysteine proteinase inhibitors. Both proteolytic bands inT. vaginalisand two proteinases inT. mobilensis(67 and 104 kDa) were inhibited by EDTA and EGTA suggesting that they belong to the metallo-proteinase class. The 67 kDa proteinase ofT. mobilensiswas inhibited also byo-phenanthroline. The other two bands ofT. mobilensis(86, 120 kDa) were not classified to any proteinase group since they appeared to be resistant to the chelating agents tested in this study.
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Jakobs, K. H., and K. Aktories. "Stimulation of high-affinity GTPase by trypsin and trypsin-like proteinases in membranes of human platelets." Biochemical Journal 249, no. 3 (February 1, 1988): 639–43. http://dx.doi.org/10.1042/bj2490639.
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The influence of various proteinases on GTP hydrolysis was studied in membranes of human platelets. Of the proteinases examined, trypsin, acrosin and a recently described trypsin-like proteinase from bovine sperm, but not chymotrypsin, increased GTP hydrolysis. Similar to what was described previously for hormone-like agents, the stimulation of GTP hydrolysis by the proteinases was only observed at low GTP concentrations, with apparent Km values of 0.2-0.3 microM-GTP. Stimulation of the high-affinity GTPase by the proteinases occurred without apparent lag phase and was constant over a long period of incubation. The proteinase inhibitors leupeptin and soya-bean trypsin inhibitor blocked the stimulation of GTP hydrolysis, but did not reverse the effect of the proteinases. Treatment of platelet membranes with N-ethylmaleimide, which eliminates Gi-protein (inhibitory guanine-nucleotide-binding protein)-related GTPase stimulation by adrenaline, decreased stimulation of GTP hydrolysis by the proteinases only partially. Activation of GTP hydrolysis by the proteinases was partially additive with that caused by adrenaline, whereas thrombin stimulation was not increased further. The data indicate that, similarly to the proteinase thrombin, trypsin and trypsin-like proteinases can activate GTP-hydrolysing protein(s) that exhibit high affinity for GTP in platelet membranes. It is suggested that the proteinases interact in platelet membranes with a receptor site similar to that used by thrombin and that the observed GTPase stimulation is a reflection of a proteinase-receptor interaction with a guanine-nucleotide-binding regulatory protein.
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Dubin, A., J. Potempa, and J. Travis. "Structural and functional characterization of elastases from horse neutrophils." Biochemical Journal 300, no. 2 (June 1, 1994): 401–6. http://dx.doi.org/10.1042/bj3000401.
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In order better to understand the pathophysiology of the equine form of emphysema, two elastinolytic enzymes from horse neutrophils, referred to as proteinases 2A and 2B, have been extensively characterized and compared with the human neutrophil proteinases, proteinase-3 and elastase. Specificity studies using both the oxidized insulin B-chain and synthetic peptides revealed that cleavage of peptide bonds with P1 alanine or valine residues was preferred. Further characterization of the two horse elastases by N-terminal sequence and reactive-site analyses indicated that proteinases 2A and 2B have considerable sequence similarity to each other, to proteinase-3 from human neutrophils (proteinase 2A), to human neutrophil elastase (proteinase 2B) and to a lesser extent to pig pancreatic elastase. Horse and human elastases differed somewhat in their interaction with some natural protein proteinase inhibitors. For example, in contrast with its action on human neutrophil elastase, aprotinin did not inhibit either of the horse proteinases. However, the Val15, alpha-aminobutyric acid-15 (Abu15), alpha-aminovaleric acid-15 (Nva15) and Ala15 reactive-site variants of aprotinin were good inhibitors of proteinase 2B (Ki < 10(-9) M) but only weak inhibitors of proteinase 2A (Ki > 10(-7) M). In summary, despite these differences, the horse neutrophil elastases were found to resemble closely their human counterparts, thus implicating them in the pathological degradation of connective tissue in chronic lung diseases in the equine species.
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Coppedge, B. R., J. M. Jones, G. W. Felton, and F. M. Stephen. "Examination of Midgut Proteinases of the Adult Southern Pine Beetle (Coleoptera: Scolytidae)." Journal of Entomological Science 29, no. 4 (October 1, 1994): 457–65. http://dx.doi.org/10.18474/0749-8004-29.4.457.
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The midgut of adult southern pine beetles, Dendroctonus frontalis Zimmermann (Coleoptera: Scolytidae), contains digestive enzymes with optimal proteolytic activity in vitro near pH 7. General proteinase activity was significantly inhibited by serine and cysteine proteinase class inhibitors, while limited activation by cysteine proteinase class activators was apparent. These results indicate that both cysteine and serine proteinases are present in the adult midgut. The presence of both proteinase classes in adult southern pine beetles coincides with previous studies showing widespread occurrence of these two classes of proteinases as digestive enzymes in midguts of other coleopteran species, but represents one of few beetle species known to possess both proteinase classes simultaneously.
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FERNÁNDEZ, J., A. F. MOHEDANO, P. GAYA, M. MEDINA, and M. NUÑEZ. "Purification and Characterization of Three Extracellular Proteinases Produced by Pseudomonas fluorescens INIA 745, an Isolate from Ewe's Milk." Journal of Food Protection 62, no. 5 (May 1, 1999): 543–46. http://dx.doi.org/10.4315/0362-028x-62.5.543.
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Three proteinases were isolated from culture medium of Pseudomonas fluorescens INIA 745 and purified to homogeneity by a combination of Phenyl-Sepharose, DEAE-Sepharose, and Sephadex G-100 chromatography. Optimal temperature for enzymatic activity was 45°C for all three proteinases. The pH optimum of proteinases I and II was found to be 7.0, while that of proteinase III was 8.0. Divalent metal ions like Cu2+, Co2+, Zn2+, Fe2+, and Hg2+ were inhibitory to proteinase activity while Ca2+, Mg2+, and Mn2+ had little or no inhibitory effect. The three enzymes were strongly inhibited by EDTA and 1,10-phenantroline and partially by cysteine. The three enzymes are metalloproteinases since they were inhibited by chelators and reactivated by Co2+, Mn2+, Cu2+, and Zn2+. The Km values of proteinases I, II, and III for casein were calculated to be 3.2, 2.6, and 5.2 mg/ml, respectively. Proteinases II and III rapidly degraded β-casein, with preference to αs1-casein, whereas proteinase I hydrolyzed both casein fractions at a slow rate.
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Todorova, V. K., D. P. Knox, and M. W. Kennedy. "Proteinases in the excretory/secretory products (ES) of adult Trichinella spiralis." Parasitology 111, no. 2 (August 1995): 201–8. http://dx.doi.org/10.1017/s0031182000064957.
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SUMMARYAdult Trichinella spiralis were maintained in vitro using defined media and the material excreted/secreted (ES) during this time examined for proteolytic enzyme (proteinase) activity using an azocasein assay and gelatin-substrate gels. Several discrete proteinases in the size range 14–100 kDa were observed with optimal activity at pH 7·5. The use of a class-differentiating panel of proteinase inhibitors indicated that serine proteinases were predominant although some inhibition was evident in the presence of cysteine and metalloproteinase inhibitors. Of a panel of potential natural protein substrates tested, ES proteinases only degraded fibrinogen and plasminogen and degradation was, in part, susceptible to the action of serine, cysteine and aspartyl proteinase inhibitors. In addition, antibody harvested from immune but not normal mice inhibited ES proteinase activity, an observation of relevance to the immunobiology of Trichinosis.
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St. Leger, Raymond J. "The role of cuticle-degrading proteases in fungal pathogenesis of insects." Canadian Journal of Botany 73, S1 (December 31, 1995): 1119–25. http://dx.doi.org/10.1139/b95-367.
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The proteinaceous outer integument of insects forms an effective barrier against most microbes. Only the 700 known species of entomopathogenic fungi effect entry into their hosts by breaching the cuticle. There is accumulating evidence that the ability of fungi to degrade protein may aid their invasion of and growth in this orderly complex structure. Evidence for the particular importance of proteinases derives largely from studies of their production in infected cuticles associated with cuticle degradation, the effects of proteinase inhibitors on pathogen behavior, and by the analysis of protease-deficient mutants. More recently, studies have included the cloning, identification, and manipulation of specific protease genes of Metarhizium anisopliae, particularly those of the subtilisin (chymoelastase) type (designated Pr1) also produced by many other entomopathogenic fungi. Following solubilization of cuticle proteins by Pr1-type endoproteases, complete degradation of the cuticle involves a number of interacting enzyme species including a family of trypsin-like proteinases, metalloproteinases, several aminopeptidases, and a carboxypeptidase. Testing genetically engineered M. anisopliae null mutants of Pr1 indicated that the other endopeptidases can partially substitute for Pr1. The exopeptidases further degrade peptides released by the endopeptidases producing free amino acids for uptake and metabolism. Utilization of these enzymes has assisted investigators in understanding cuticle structure and how the cuticle is degraded naturally, and could lead to improved strain selection of entomopathogenic fungi or the introduction of their genes into other microbes and plants for the purpose of insect control. Key words: proteinaceous insect cuticle, pathogen endopeptidases, exopeptidases, multiple isozymes, enzyme regulation.
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Talbot, James A., Klaus Nielsen, and Lynette B. Corbeil. "Cleavage of proteins of reproductive secretions by extracellular proteinases of Tritrichomonas foetus." Canadian Journal of Microbiology 37, no. 5 (May 1, 1991): 384–90. http://dx.doi.org/10.1139/m91-062.
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Cleavage of host defense proteins from reproductive secretions was investigated as a potential virulence mechanism for Tritrichomonas foetus extracellular proteinases. Three categories of susceptibility to digestion were found among the defense proteins tested. Cleavage of fibrinogen, fibronectin, and albumin occurred rapidly with more than 50% of these digested within 30 min. Lactoferrin, immunoglobulin G1, and immunoglobulin G2 were more than 50% digested after 4 h. Transferrin, immunoglobulin M, and immunoglobulin A were the most resistant to the Tritrichomonas foetus extracellular proteinases, since 50% or more of the parent molecule remained after 24 h. The responsible proteinases were classified as cysteine (thiol) proteinases because cleavage was inhibited by the cysteine proteinase specific inhibitor, trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane and not by the serine proteinase specific inhibitor, phenylmethylsulfonyl fluoride. In addition, α2-macro-globulin, but not α1-antitrypsin, inhibits the action of the proteinases. The ratio of this naturally occurring inhibitor to the quantity of proteinases released may determine whether the above substrates are cleaved in vivo. Since these substrates are implicated in iron acquisition, cell adherence, and acquired immunity, Tritrichomonas foetus proteinases are likely to play a role in host–parasite interactions. Key words: cysteine proteinase, Tritrichomonas foetus, host defense proteins, reproductive secretions.
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D'AVILA-LEVY, C. M., F. M. ARAUJO, A. B. VERMELHO, R. M. A. SOARES, A. L. S. SANTOS, and M. H. BRANQUINHA. "Proteolytic expression inBlastocrithidia culicis: influence of the endosymbiont and similarities with virulence factors of pathogenic trypanosomatids." Parasitology 130, no. 4 (November 2, 2004): 413–20. http://dx.doi.org/10.1017/s0031182004006705.
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Blastocrithidia culicisis an insect trypanosomatid that presents bacterial endosymbionts. The cell-associated and secreted proteinases of the endosymbiont-bearing and aposymbiotic strains were compared through the incorporation of proteinaceous substrates into sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Few qualitative changes could be detected in the proteolytic zymograms in the 2 strains studied when gelatin, casein, haemoglobin or bovine serum albumin (BSA) were tested. However, the level of proteolytic activities was significantly higher in the aposymbiotic strain. Some of theB. culicisproteins reacted in Western blots with antibodies raised against gp63, a zinc-metalloproteinase, and cruzipain, a cysteinyl-proteinase, which are virulence factors of the human pathogenic trypanosomatids,Leishmaniaspp. andTrypanosoma cruzi, respectively. The anti-cross-reacting determinant (CRD) antibody recognized 2 polypeptides (50 and 58 kDa) in the spent culture media and in the supernatant from glycosylphosphatidylinositol-phospholipase C (GPI-PLC)-treated cells, suggesting that these proteins are GPI-anchored to the plasma membrane. In addition, the anti-gp63 reacted with the 50 kDa protein. The identification of protein homologues in trypanosomatids with distinct life-cycles may help to determine the importance of proteinases in trypanosomatids.
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Mills, J. S. "Virus Proteinase Inhibitors — What Next after HIV?" Antiviral Chemistry and Chemotherapy 7, no. 6 (December 1996): 281–93. http://dx.doi.org/10.1177/095632029600700601.
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The recent approval by the regulatory authorities in the United States of several HIV proteinase inhibitors as therapeutics for the treatment of AIDS confirms that virus proteinases are valid molecular targets in the search for new antiviral drugs. This review summarizes the available approaches that can be taken to discover virus proteinase inhibitors and reviews the current status of our knowledge with respect to virus proteinases in viruses of clinical significance other than HIV. The major focus is on proteinases identified in the viruses that cause the common cold, hepatitis C virus and the herpesviruses.
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Hortin, Glen L., Ilka Warshawsky, and Maryline Laude-Sharp. "Macromolecular Chromogenic Substrates for Measuring Proteinase Activity." Clinical Chemistry 47, no. 2 (February 1, 2001): 215–22. http://dx.doi.org/10.1093/clinchem/47.2.215.
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Abstract Background: Proteinase activities are often measured using chromogenic substrates that are much smaller than physiological substrates. Methods: The hydrodynamic size of macromolecular substrates (macrosubstrates) prepared by linking small chromogenic substrates to polyethylene glycol was determined by gel filtration. Efficiency of macrosubstrate cleavage by proteinases and α2-macroglobulin-proteinase complexes was monitored spectrophotometrically. Results: Macrosubstrates had hydrodynamic radii of ∼20 Å, similar to proteins with a molecular weight of 18 000. Different macrosubstrates served as efficient substrates for chymotrypsin, trypsin, and thrombin. Linking small substrates to a polymer variably affected substrate efficiency, with the impact on activity ranging from a 60-fold decrease to a 30-fold increase. Proteinases complexed with α2-macroglobulin had ∼10-fold lower activity vs macrosubstrates than small substrates. Conclusions: Macrosubstrates are efficient substrates that allow decreased measurement of sterically hindered proteinase molecules such as α2-macroglobulin-proteinase complexes. Thus, macrosubstrates may provide more accurate functional assays of proteinases such as coagulation factors.
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Jean, D., J. Hermann, F. Rodrigues-Lima, M. Barel, M. Balbo, and R. Frade. "Identification on melanoma cells of p39, a cysteine proteinase that cleaves C3, the third component of complement: amino-acid-sequence identities with procathepsin L." Biochemical Journal 312, no. 3 (December 15, 1995): 961–69. http://dx.doi.org/10.1042/bj3120961.
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We previously identified, on normal or tumour cells, two membrane proteinases, p57 and p65, that cleave human C3, the third component of complement, thus regulating C3′s biological properties. Whereas p57 was purified from human erythrocytes, p65 was identified using polyclonal anti-p57 antibodies on a human melanoma cell line resistant to complement lysis. Analysis of cell distribution of C3-cleaving proteinases established that DSm, a murine melanoma cell line, expressed a C3-cleaving proteinase distinct from p57 and p65 proteinases. Thus we purified the C3-cleaving proteinase solubilized from membranes of DSm cells. The purified proteinase, termed ‘p39’ on the basis of its molecular mass of 39 kDa, was identified, using specific proteinase inhibitors, as a cysteine proteinase. Anti-p39 antibodies, prepared against highly purified p39, localized the p39 C3-cleaving proteinase mainly at the cell surface and demonstrated that p39 is also secreted. Anti-p39 antibodies inhibited solubilized C3-cleaving activity. Preincubation of DSm cells with anti-p39 F(ab′)2 fragments increased up to 60% complement cell susceptibility. Amino acid analysis of N-terminal and three other regions of p39 demonstrated that this C3-cleaving proteinase carries 100% identity within four regions of procathepsin L. This is the first demonstration that a melanoma cell line expresses on its surface and secretes a p39 C3-cleaving cysteine proteinase that shares sequence identities with procathepsin L. Thus the p39 cysteine proteinase represents a new member of the C3-cleaving proteinase family associated with, and/or expressed on, the cell surface.
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Kang, Yoon-Suk, Young-Bae Chun, and Moon-Jae Cho. "Purification and Characterization of proteinase from Ruditapes philippinarum infected with Perkinsus sp." Journal of Medicine and Life Science 1, no. 1 (December 1, 2003): 53–56. http://dx.doi.org/10.22730/jmls.2003.1.1.53.
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In this study, we characterized a proteinase from marine bivalve Ruditapes philippinarum (Manila clam) infected with the protozoan parasite Perkinsus sp. When infected with Perkinsus sp., Manila clam produced a proteinase, which was not appeared in non-infected manila clam and Perkinsus hypnospore. A clear baiid of 45 kDa was detected on 0.2% gelatin containing gel SDS-PAGE. The proteinase was inhibited by phenylmethyl sulfonyl fluoride (PMSF) known as a serine proteinase inhibitor. Using Mucin-Sepharose CL-6B affinity chromatography, proteinases were eluted by Tris-HCl buffer (pH 7.4) containing 20 mM EDTA. Like lectins, N-acetyl-D-galactosamine also could elute proteinases. Based on these results, isolated Ruditapes philippinarum proteinase belonged to the class of serine protease and may have lectin activities or may be associated with lectin
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Takeuchi, K., H. Wood, and R. T. Swank. "Lysosomal elastase and cathepsin G in beige mice. Neutrophils of beige (Chediak-Higashi) mice selectively lack lysosomal elastase and cathepsin G." Journal of Experimental Medicine 163, no. 3 (March 1, 1986): 665–77. http://dx.doi.org/10.1084/jem.163.3.665.
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A profound decrease in activities of the two lysosomal serine proteinases, elastase, and cathepsin G, was found in neutrophils of four independent beige mutants. Elastase and cathepsin G activities were assayed with the specific synthetic substrates MeO-Suc-Ala-Ala-Pro-Val-MCA and Suc-Ala-Ala-Pro-Phe-pNA, respectively. The defect is intrinsic to cells of beige mice, since transplantation of bone marrow from normal to mutant mice restored normal proteinase activity, and normal mice transplanted with beige marrow produced neutrophils with a deficiency of proteinase activity. The loss of elastase and cathepsin G activity was confirmed by separation of [3H]diisopropylfluorophosphate-labeled proteins on denaturing gels, which also revealed that other serine proteinases are at normal levels in beige neutrophil extracts. The deficiency of lysosomal proteinase activity appears specific, in that four other common neutrophil lysosomal enzymes, plus the spectrum of major neutrophil proteins are not affected by the beige mutation. The deficiency of proteinase activity is likely not the primary genetic alteration of the beige mutation, since more than one proteinase is affected, and heterozygous F1 mice have normal rather than intermediate levels of both proteinases. The lowered proteinase activity may contribute to the high susceptibility of beige mice and Chediak-Higashi patients to infection.
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Manocha, M. S., and R. Balasubramanian. "In vitro regulation of chitinase and chitin synthase activity of two mucoraceous hosts of a mycoparasite." Canadian Journal of Microbiology 34, no. 10 (October 1, 1988): 1116–21. http://dx.doi.org/10.1139/m88-197.
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Effects of partially purified preparations of proteinases extracted from Choanephora cucurbitarum and Phascolomyces articulosus, susceptible and resistant hosts of Piptocephalis virginiana, respectively, were studied in vitro on the activities of chitinase and chitin synthase of the same hosts. Both chitinase and chitin synthase are membrane bound, zymogenic, and activated by partial proteolysis. Host proteinases of acid and neutral type stimulated chitinase activity, but only the acid proteinase stimulated chitin synthase activity of the two hosts. Neutral proteinase of C. cucurbitarum inhibited its chitin synthase, whereas that of Ph. articulosus increased its chitin synthase activity. Partially purified preparations of neutral proteinase from the mycoparasite, Piptocephalis virginiana, however, enhanced chitinase and suppressed chitin synthase activity of both the hosts alike. The role of host proteinases in regulating the activity of chitinase and chitin synthase of the two host species is suggested.
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Peng, Chih-Wen, Valera V. Peremyslov, Arcady R. Mushegian, William O. Dawson, and Valerian V. Dolja. "Functional Specialization and Evolution of Leader Proteinases in the Family Closteroviridae." Journal of Virology 75, no. 24 (December 15, 2001): 12153–60. http://dx.doi.org/10.1128/jvi.75.24.12153-12160.2001.
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ABSTRACT Members of the Closteroviridae andPotyviridae families of the plant positive-strand RNA viruses encode one or two papain-like leader proteinases. In addition to a C-terminal proteolytic domain, each of these proteinases possesses a nonproteolytic N-terminal domain. We compared functions of the several leader proteinases using a gene swapping approach. The leader proteinase (L-Pro) of Beet yellows virus (BYV; a closterovirus) was replaced with L1 or L2 proteinases of Citrus tristeza virus (CTV; another closterovirus), P-Pro proteinase of Lettuce infectious yellows virus (LIYV; a crinivirus), and HC-Pro proteinase of Tobacco etch virus(a potyvirus). Each foreign proteinase efficiently processed the chimeric BYV polyprotein in vitro. However, only L1 and P-Pro, not L2 and HC-Pro, were able to rescue the amplification of the chimeric BYV variants. The combined expression of L1 and L2 resulted in an increased RNA accumulation compared to that of the parental BYV. Remarkably, this L1-L2 chimera exhibited reduced invasiveness and inability to move from cell to cell. Similar analyses of the BYV hybrids, in which only the papain-like domain of L-Pro was replaced with those derived from L1, L2, P-Pro, and HC-Pro, also revealed functional specialization of these domains. In subcellular-localization experiments, distinct patterns were observed for the leader proteinases of BYV, CTV, and LIYV. Taken together, these results demonstrated that, in addition to a common proteolytic activity, the leader proteinases of closteroviruses possess specialized functions in virus RNA amplification, virus invasion, and cell-to-cell movement. The phylogenetic analysis suggested that functionally distinct L1 and L2 of CTV originated by a gene duplication event.
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MORADIAN-OLDAK, Janet, Wendy LEUNG, James P. SIMMER, Margarita ZEICHNER-DAVID, and Alan G. FINCHAM. "Identification of a novel proteinase (ameloprotease-I) responsible for the complete degradation of amelogenin during enamel maturation." Biochemical Journal 318, no. 3 (September 15, 1996): 1015–21. http://dx.doi.org/10.1042/bj3181015.
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During enamel formation the proteins of the extracellular matrix, particularly amelogenins, are removed prior to maturation. In order to investigate this process and to improve our understanding of the function of proteinases during enamel maturation, proteinase fractions were isolated from developing pig enamel and assayed for proteolytic activity in vitro. A recombinant murine amelogenin, M179, was used as a substrate. Two major groups of enamel proteinases were defined as high-molecular-mass [‘high-molecular-weight’ in Moradian-Oldak, Simmer, Sarte, Zeichner-David and Fincham (1994) Arch. Oral Biol. 39, 647–656] and low-molecular-mass proteinases. Here we report the characterization of one of the proteinases present in the low-molecular-mass group. We demonstrate that this proteinase is a serine proteinase capable of degradation of M179 following cleavage of the tyrosine-rich amelogenin polypeptide from the N-terminal region. A partial N-terminal sequence of the proteinase was obtained (LPHVPHRIPPGYGRPXTXNEEGXNPYFXFFXXHG). An anti-peptide antibody directed against a synthetic peptide corresponding to the first 14 amino acids of the above sequence was produced. The presence of the proteinase in the acetic acid extract was confirmed by Western blotting. Searching using the amino acid sequence determined in this study showed it to be also present in the 32 kDa and 89 kDa enamelin proteins reported by Fukae, Tanabe, Murakami and Tohi [(1996) Adv. Dent. Res., in the press]. We therefore identify the 32 kDa enamelin as an enamel proteinase (‘ameloprotease-I’) which is responsible for amelogenin degradation in maturing enamel. We propose that the 89 kDa enamelin is a precursor of ameloprotease-I, the first enamel protein for which a function has been defined.
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Rymerson, Robert T., and Robert P. Bodnaryk. "GUT PROTEINASE ACTIVITY IN INSECT PESTS OF CANOLA." Canadian Entomologist 127, no. 1 (February 1995): 41–48. http://dx.doi.org/10.4039/ent12741-1.
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AbstractThe digestive proteinases of three important pests of canola, Brassica napus L. and B. rapa L., in western Canada were characterized by assessing the proteolytic activity of homogenates of their midguts against azocasein or azoalbumin at various pH levels and in the presence of diagnostic proteinase inhibitors. The midgut of larvae of the bertha armyworm, Mamestra configurata Wlk., had maximum proteolytic activity at pH 10.5 which was inhibited 45–60% by serine proteinase inhibitors such as the soybean trypsin inhibitor. The midgut of larvae of the diamondback moth, Plutella xylostella L., had maximum proteolytic activity at pH 10 which was inhibited 56–75% by serine proteinase inhibitors. The two lepidopterans thus use a serine-like proteinase in digestion. The midgut of adults of the flea beetle, Phyllotreta cruciferae Goeze, exhibited maximum proteolytic activity at pH 5 which was inhibited 33–61% by specific cysteine proteinase inhibitors such as cystatin and trans-epoxysuccinyl-L-leucylamido (4-guanidino)-butane (E-64) and was activated strongly by L-cysteine. Aspartic proteinase inhibitors such as pepstatin A also decreased proteolytic activity by 21–50%. Serine proteinase inhibitors were without effect. Therefore, P. cruciferae appears to use both cysteine- and aspartic-like proteinases in digestion. Cotyledons and first true leaves of canola, B. napus cv. Westar, contained inhibitory activity against serine, cysteine, and aspartic proteinases when tested against bovine trypsin, papain, or porcine pepsin, but the level of antiproteinase activity is insufficient to provide significant resistance against any of these pests.
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Fang, Weihuan, and Markus Sandholm. "Inhibition of the proteinase activity in mastitic milk." Journal of Dairy Research 62, no. 1 (February 1995): 61–68. http://dx.doi.org/10.1017/s0022029900033677.
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SummaryThe antiproteolytic activity of selected proteinase inhibitors was studied in mastitic bovine milk and urokinase-activated normal milk using a caseolytic agar diffusion assay. The inhibition profiles of mastitic milk and urokinase-activated milk were compared with those of purified proteinases. The proteinase inhibition profile of mastitic milk did not resemble that of any of the pure proteinases, indicating a mixed type of proteinase system in mastitic milk. The trypanocidals diminazene (equivalent to Berenil®) and pentamidine (equivalent to Lomidine®), together with aprotinin (Trasylol®), showed most promise when considering possible applications in mastitis to break up the proteolytic cascade within the inflamed udder.
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Michaud, Dominique, Thierry C. Vrain, and Hugh A. Daubeny. "Potential of Plant Cystatins for the Production of Transgenic Strawberry Plants Resistant to the Black Vine Weevil." HortScience 30, no. 4 (July 1995): 828D—828. http://dx.doi.org/10.21273/hortsci.30.4.828d.
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Transformation of plant genomes with cysteine proteinase inhibitor (cystatin) genes represents an attractive option for the biological control of insect pests. However, this strategy must be carefully considered, because the transgenic plant endogenous proteinases may represent potential target enzymes for the exogenous inhibitors produced. For example, we are considering the transformation of strawberry (Fragaria ×ananassa) with cystatin cDNA clones, to control the Coleoptera pest black vine weevil (BVW; Otiorynchus sulcatus). Electrophoretic analyses of adult BVW proteinases have revealed the involvement of at least five proteinase forms for protein digestion, and the major form was strongly inhibited by oryzacystatins (OCI and OCII), two cystatins isolated from rice seeds. A similar analysis of proteinases showed the existence of OC-sensitive proteinase activity in the leaves of strawberry, suggesting a possible risk of interference of the inhibitors in the transformed plants. In addition, the two rice inhibitors were rapidly hydrolyzed at 25C when incubated with proteinase extracts from either young, mature or senescent leaves. An efficient control of BVW by plant cystatin-expressing transgenic strawberry plants is therefore potentially possible, but the correct targeting of the inhibitors in the plant cells using appropriate signal peptides could be necessary.
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Goodenough, Peter W., Peter J. Kilshaw, Fiona McEwan, and A. Jane Owen. "Monoclonal antibodies to the two most basic papaya proteinases." Bioscience Reports 6, no. 8 (August 1, 1986): 759–66. http://dx.doi.org/10.1007/bf01116544.
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The proteinases from Carica papaya include papain, isoenzymes of chymopapain and two proteinases A and B distinguished by their unusually high pI. The identity of one of the most basic proteinases has been questioned. The present report describes the preparation and characterisation of two monoclonal antibodies that react specifically with papaya proteinases A and B respectively and a third that identifies a common structural feature found in papain and proteinase A.
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Thøgersen, I. B., G. Salvesen, F. H. Brucato, S. V. Pizzo, and J. J. Enghild. "Purification and characterization of an α-macroglobulin proteinase inhibitor from the mollusc Octopus vulgaris." Biochemical Journal 285, no. 2 (July 15, 1992): 521–27. http://dx.doi.org/10.1042/bj2850521.
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The cell-free haemolymph of the mollusc Octopus vulgaris inhibited the proteolytic activity of the thermolysin against the high-molecular-mass substrate hide powder azure. The purified inhibitor was a glycoprotein composed of two identical 180 kDa disulphide-linked subunits. In addition to the inhibition of the metalloproteinase thermolysin, the protein inhibited the serine proteinases human neutrophil elastase, pig pancreatic elastase, bovine chymotrypsin, bovine trypsin and the cysteine proteinase papain. A fraction of the proteinase-inhibitor complex resisted dissociation after denaturation indicating that some of the proteinase molecules became covalently bound. The nucleophile beta-aminopropionitrile decreased the covalent binding of proteinases to the Octopus vulgaris protein, suggesting that this interaction is mediated by an internal thiol ester; the reactivity and the amino acid sequence flanking the reactive residues of the putative thiol ester were consistent with this hypothesis. Bound trypsin remained active against the low-molecular-mass chromatogenic substrate H-D-Pro-Phe-Arg p-nitroanilide and was protected from inhibition by active-site-directed protein inhibitors of trypsin; however, the bound trypsin was readily inhibited by small synthetic inhibitors. This indicates that the inhibition of proteinases is accomplished by steric hindrance. The proteinase-inhibitory activity of this protein is characteristic of inhibition by mammalian alpha-macroglobulins and the presence of a putative thiol ester suggests that the Octopus vulgaris proteinase inhibitor is a homologue of human alpha 2-macroglobulin.
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Sinden, Nicola J., Michael J. Baker, David J. Smith, Jan-Ulrich Kreft, Timothy R. Dafforn, and Robert A. Stockley. "α-1-Antitrypsin variants and the proteinase/antiproteinase imbalance in chronic obstructive pulmonary disease." American Journal of Physiology-Lung Cellular and Molecular Physiology 308, no. 2 (January 15, 2015): L179—L190. http://dx.doi.org/10.1152/ajplung.00179.2014.
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The excessive activities of the serine proteinases neutrophil elastase and proteinase 3 are associated with tissue damage in chronic obstructive pulmonary disease. Reduced concentrations and/or inhibitory efficiency of the main circulating serine proteinase inhibitor α-1-antitrypsin result from point mutations in its gene. In addition, α-2-macroglobulin competes with α-1-antitrypsin for proteinases, and the α-2-macroglobulin-sequestered enzyme can retain its catalytic activity. We have studied how serine proteinases partition between these inhibitors and the effects of α-1-antitrypsin mutations on this partitioning. Subsequently, we have developed a three-dimensional reaction-diffusion model to describe events occurring in the lung interstitium when serine proteinases diffuse from the neutrophil azurophil granule following degranulation and subsequently bind to either α-1-antitrypsin or α-2-macroglobulin. We found that the proteinases remained uninhibited on the order of 0.1 s after release and diffused on the order of 10 μm into the tissue before becoming sequestered. We have shown that proteinases sequestered to α-2-macroglobulin retain their proteolytic activity and that neutrophil elastase complexes with α-2-macroglobulin are able to degrade elastin. Although neutrophil elastase is implicated in the pathophysiology of emphysema, our results highlight a potentially important role for proteinase 3 because of its greater concentration in azurophil granules, its reduced association rate constant with all α-1-antitrypsin variants studied here, its greater diffusion distance, time spent uninhibited following degranulation, and its greater propensity to partition to α-2-macroglobulin where it retains proteolytic activity.
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Lecaille, F., D. Muno, E. Kominami, and K. Ishidoh. "Proteinases participating in the processing and activation of prolegumain in primary cultured rat macrophages." Biological Chemistry 385, no. 6 (June 7, 2004): 511–16. http://dx.doi.org/10.1515/bc.2004.060.
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Abstract The mammalian legumain is a recently identified lysosomal cysteine proteinase belonging to the clan CD and homologous to plant legumain. This enzyme has the characteristic of specifically hydrolyzing peptide bonds after asparagine residues. As in the case of papain-type cysteine proteinases, legumain is synthesized as an inactive zymogen, and processed into a mature form localized in lysosomes. However, the mechanism of its activation remains unclear. In this study, we analyze which types of proteinases may participate in the processing of legumain in rat primary cultured macrophages using various proteinase inhibitors after 24 h treatment with Bafilomycin A1, a vacuolar ATPase inhibitor. The processing of legumain in macrophages was accomplished by papain-type cysteine proteinases other than cathepsin B.
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Petersen, C. M., E. Ejlersen, S. K. Moestrup, P. H. Jensen, O. Sand, and L. Sottrup-Jensen. "Immunosuppressive properties of electrophoretically "slow" and "fast" form alpha 2-macroglobulin. Effects on cell-mediated cytotoxicity and (allo-) antigen-induced T cell proliferation." Journal of Immunology 142, no. 2 (January 15, 1989): 629–35. http://dx.doi.org/10.4049/jimmunol.142.2.629.
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Abstract Lymphokine activated killer cell lysis of K562 cells was inhibited by alpha 2-macroglobulin (alpha 2M), soybean trypsin inhibitor, and alpha 1-proteinase inhibitor. In serum free medium 2 mg/ml alpha 2M suppressed target cell lysis in a 4-h cytotoxic assay with about 40%. Suppression was dose and time dependent. Cytotoxicity was unaffected by alpha 2M concentrations less than 0.25 mg/ml, and by alpha 2M added later than 1.5 h from start of assay. Pre-treatment of effector (but not of target) cells with alpha 2M was even more suppressive than the presence of alpha 2M during assay. Cell-mediated cytotoxicity was not inhibited by alpha 2M treated with methylamine or by various alpha 2M-proteinase complexes. In contrast, alpha 2M-proteinase complex as well as native alpha 2M suppressed the proliferation of Ag-activated T cells. However, methylamine-treated alpha 2M did not inhibit T cell proliferation, and suppression by alpha 2M-proteinase complex was significantly reduced after inhibition of the alpha 2M-bound proteinase. On incubation at 4 degrees C with lymphokine-activated killer cells, alpha 2M reacted with cell associated proteinases and changed from electrophoretically "slow" to "fast" form. Cell associated proteinases bound by alpha 2M showed chymotrypsin- and trypsin-like specificities and their activity surpassed activity caused by cellular leakage and secretion. The present results strongly indicate that alpha 2M mediates immunosuppression in its capacity as a proteinase inhibitor and suggest inhibition of (T)cell surface-associated proteinases as a possible mode of suppression.
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Knight, Kenneth R., Rakesh K. Khazanchi, W. Christopher Pederson, John J. McCann, Serena A. Coe, and Bernard McC O'Brien. "Coumarin and 7-Hydroxycoumarin Treatment of Canine Obstructive Lymphoedema." Clinical Science 77, no. 1 (July 1, 1989): 69–76. http://dx.doi.org/10.1042/cs0770069.
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1. Canine obstructive lymphoedema was created in one hind leg of 30 dogs by irradiation of the groin and surgical removal of surviving lymph glands and lymphatics. The opposite leg served as a control. Once the oedema had stabilized, groups of 10 dogs were treated orally with 12.5 mg day−1 kg−1 for 8 months with either one of the benzopyrones coumarin (2H-1-benzopyran-2-one) and 7-hydroxycoumarin (7-hydroxy-2H-1-benzopyran-2-one), or placebo. 2. The two benzopyrones significantly (P < 0.01) but gradually reduced the oedema by 20–30% over 8 months, as judged by circumferential measurements of the oedematous and control limbs. There was no change with the placebo. 3. In the oedematous fluids (lymph and interstitial fluid), benzopyrone treatment reduced the protein content and increased acid and neutral proteinase activity compared with the control limbs, while the levels of the proteinase inhibitor α2-macroglobulin remained unchanged. Furthermore, these active drugs reduced the excess water content, thickness and hydroxyproline content of skin biopsies from oedematous limbs compared with those from control limbs. No changes were observed for the placebo group. 4. These biochemical changes suggest that benzopyrones can reduce the excess proteinaceous fluid in lymphoedema by increasing the levels of proteinase activity relative to the number of proteinase inhibitors. As a secondary event the amount of fibrosis in the skin is also reduced, presumably by an increase in collagenase activity from the mononuclear phagocytes. 5. These results support the hypothesis that benzopyrones activate the production of proteinases by mononuclear phagocytes. The resultant small peptides can drain locally into the bloodstream, thus bypassing the blocked lymphatics and reducing the protein concentration and its associated oedema fluid.
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Nagy, L., B. R. Johnson, P. Hauschka, and S. Szabo. "Characterization of proteases and protease inhibitors in the rat stomach." American Journal of Physiology-Gastrointestinal and Liver Physiology 272, no. 5 (May 1, 1997): G1151—G1158. http://dx.doi.org/10.1152/ajpgi.1997.272.5.g1151.
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Previously our laboratory reported increased activity of the thiol proteinase cathepsin B in gastric juice after ethanol-induced mucosal injury. In this study we measured proteinase activity (PA) and proteinase inhibitory activity (PIA) with the general substrates hemoglobin, azocasein, and bovine serum albumin (BSA) at optimal pH (2.0, 5.6, and 7.4) of aspartic, cysteine, and serine proteinases. Homogenates of glandular stomach mucosa and gastric juice from fasted rats were incubated in the presence or absence of specific inhibitors and sulfhydryl (SH) alkylators N-ethylmaleimide and iodoacetate. PIA was measured after acid and heat inactivation of endogenous proteinases and addition of 20 micrograms/ml pepsin, 20 or 100 micrograms/ml thiol proteinase papain, or 20 micrograms/ml trypsin for 5 min before digestion at 37 degrees C. The highest proteolytic activity was found at pH 2.0 (pepsin) in juice and mucosal homogenate, but proteases were also found at pH 5.6 and 7.4, where pepsin was inactive. Pepstatin inhibited most proteolytic activity at pH 2.0. The SH protease inhibitor leupeptin diminished PA mainly at pH 5.6. N-ethylmaleimide or iodoacetate substantially reduced the PA in acidic milieu, with maximum effect at pH 5.6. Endogenous PIA, expressed as inhibition of the effect of 1 microgram of pepsin, papain, and trypsin on BSA, was 13.1, 1.4, and 9.2% in gastric mucosa and 15.3, 22.5, and 6.2% in gastric juice at pH 2.0, 5.6, and 7.4, respectively. We have concluded that 1) endogenous proteinases and inhibitors in rat stomach can be measured using BSA and hemoglobin as substrates, 2) of the proteinases found in the stomach, 98% was pepsin at pH 2.0 and up to 27% or 17% was SH sensitive at pH 5.6 or 7.4, respectively, and 3) proteinases and their specific endogenous inhibitors may play a role in gastric mucosal injury and protection.
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Koritsas, V. M., and H. J. Atkinson. "Proteinases of females of the phytoparasite Globodera pallida (potato cyst nematode)." Parasitology 109, no. 3 (September 1994): 357–65. http://dx.doi.org/10.1017/s0031182000078392.
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SummarySensitive assays capable of detecting proteinases in single females of the phytoparasite Globodera pallida have been developed and used to define the proteinase activity of young adult females. Digestion of the large subunit of the plant protein Rubisco established a pH optimum for the proteinase activity at pH 5·7. The activity was inhibited by the cysteine proteinase inhibitors p-chloromercuribenzoic acid (PMBA) and p-chloromercurisulphonic acid (PMSA) and stimulated by both cysteine and dithiothreitol (DTT). It was moderately reduced by L-trans-epoxysuccinyl-leucylamido-(4- guanidino) butane (E64) but not by specific inhibitors of serine, aspartate or metallo-proteinases. The activity separated into 3 bands on a non-denaturing gel but only I proteinase of 62 kDa was recovered following a combination of anion-exchange chromatography and affinity chromatography using PMBA. The effect of inhibitors was similar to that reported previously for some of the cysteine proteinase activity recovered from Caenorhabditis elegans but is apparently not that for which the corresponding gene has been cloned in this nematode and Haemonchus contortus. The proteinase may have a major role in digestion of dietary protein and so offers an exciting target for future control of this important plant-parasitic nematode.
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GARCÍA-CARREÑO, F. L., M. A. NAVARRETE DEL TORO, M. DÍAZ-LÓPEZ, M. P. HERNÁNDEZ-CORTÉS, and J. M. EZQUERRA. "Proteinase Inhibition of Fish Muscle Enzymes Using Legume Seed Extracts." Journal of Food Protection 59, no. 3 (March 1, 1996): 312–18. http://dx.doi.org/10.4315/0362-028x-59.3.312.
Full textAbstract:
Seed extracts from indigenous and introduced legumes were prepared and used to search for inhibitors of fish muscle proteinases. Fish flesh extracts were prepared from samples of Merluccius productus (Pacific whiting or merluza) obtained off the Oregon coast and in the Gulf of California, respectively. The proteinase activity in the fish muscle for the Pacific whiting was the highest, followed by parasitized merluza. The lowest proteinase activity was for the nonparasitized merluza. Six out of 12 seed extracts reduced the proteinase activity from the fish flesh by more than 50%. The reduction of enzyme activity was higher for samples of fish flesh extracts from the Gulf of California than for the Oregon samples. Seed extracts also reduced the proteinase activity of commercial serine and cysteine proteinases such as trypsin, chymotrypsin, and papain. This inhibitory capacity was maintained even after heating the seed extracts to 90°C for 15 min. Several seed extracts show promise for use as proteinase inhibitors during production of surimi, the intended commercial product of massive fisheries such as Pacific whiting or merluza.
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Buttle, D. J., A. A. Kembhavi, S. L. Sharp, R. E. Shute, D. H. Rich, and A. J. Barrett. "Affinity purification of the novel cysteine proteinase papaya proteinase IV, and papain from papaya latex." Biochemical Journal 261, no. 2 (July 15, 1989): 469–76. http://dx.doi.org/10.1042/bj2610469.
Full textAbstract:
A procedure is described for the purification of a previously undetected cysteine proteinase, which we have called papaya proteinase IV, from spray-dried latex of the papaya (Carica papaya) plant. The purification involves affinity chromatography on Gly-Phe-aminoacetonitrile linked to CH-Sepharose 4B, with elution by 2-hydroxyethyl disulphide at pH 4.5. The product thus obtained is a mixture of almost fully active papain and papay proteinase IV, which are then separated by cation-exchange chromatography. A preliminary characterization of papaya proteinase IV showed it to be very similar to chymopapain in both molecular size and charge. However, the new enzyme is immunologically distinct from the previously characterized cysteine proteinases of papaya latex. It also differs in its lack of activity against the synthetic substrates of the other papaya proteinases, in its narrow specificity against protein substrates and its lack of inhibition by chicken cystatin. Papaya proteinase IV is abundant, contributing almost 30% of the protein in spray-dried papaya latex, and contamination of chymopapain preparations with this enzyme may account for some of the previously reported heterogeneity of chymopapain.
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Folco, E. J. E., L. Busconi, C. B. Martone, and J. J. Sánchez. "Fish skeletal muscle contains a novel serine proteinase with an unusual subunit composition." Biochemical Journal 263, no. 2 (October 15, 1989): 471–75. http://dx.doi.org/10.1042/bj2630471.
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Proteinase I, an enzyme previously shown to be able to degrade contractile and cytoskeletal elements of white-croaker (Micropogon opercularis) myofibrils, was purified to apparent homogeneity by chromatography on DEAE-Sephacel, octyl-Sepharose CL 4B and arginine-Sepharose 4B. Its Mr was determined to be 269,000 by Sephacryl S-300 gel filtration. Under denaturing conditions, the enzyme dissociated into two subunits with Mr 20,000 and 15,500, in a molar ratio of 1.8:1. Proteinase I showed a pH optimum of 8.5. The enzyme was strongly inhibited by several serine proteinase inhibitors, whereas inhibitors of the other types of proteinases did not affect, or only scarcely affected, its activity. Several N-terminal-blocked 4-methyl-7-coumarylamide substrates having either arginine or lysine residues adjacent to the fluorogenic group were efficiently hydrolysed by the enzyme. These results indicate that proteinase I is a trypsin-like serine proteinase. The enzyme appears to be distinct from other proteinases previously described in skeletal muscle, and might be involved in the catabolism of myofibrillar proteins.
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Buist, Girbe, Gerard Venema, and Jan Kok. "Autolysis of Lactococcus lactis Is Influenced by Proteolysis." Journal of Bacteriology 180, no. 22 (November 15, 1998): 5947–53. http://dx.doi.org/10.1128/jb.180.22.5947-5953.1998.
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ABSTRACT The autolysin AcmA of Lactococcus lactis was shown to be degraded by the extracellular lactococcal proteinase PrtP. Autolysis, as evidenced by reduction in optical density of a stationary-phase culture and concomitant release of intracellular proteins, was greatly reduced when L. lactis MG1363 cells expressed the cell wall-anchored lactococcal proteinase PrtP of the PI-type caseinolytic specificity (PI). On the other hand, lactococcal strains that did not produce the proteinase showed a high level of autolysis, which was also observed when the cells produced the secreted form of PI or a cell wall-anchored proteinase with PIII-type specificity. Autolysis was also increased when MG1363 expressed the cell wall-anchored hybrid PI/PIII-type proteinase PIac. Zymographic analysis of AcmA activity during stationary phase showed that AcmA was quickly degraded by PI and much more slowly by PrtP proteinases with PIII-type and intermediate specificities. Autolysis of L. lactis by AcmA was influenced by the specificity, amount, and location of the lactococcal proteinase. No autolysis was observed when the various proteinases were expressed in an L. lactis acmAdeletion mutant, indicating that PrtP itself did not cause lysis of cells. The chain length of a strain was significantly shortened when the strain expressed a cell wall-anchored active proteinase.
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Stepanov, V. M., G. N. Rudenskaya, L. P. Revina, Y. B. Gryaznova, E. N. Lysogorskaya, Filippova IYu, and I. I. Ivanova. "A serine proteinase of an archaebacterium, Halobacterium mediterranei. A homologue of eubacterial subtilisins." Biochemical Journal 285, no. 1 (July 1, 1992): 281–86. http://dx.doi.org/10.1042/bj2850281.
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A homogeneous serine proteinase secreted by the extreme halophilic bacterium Halobacterium mediterranei 1538 was isolated by affinity chromatography on bacitracin-Sepharose with a yield of 48% (260-fold purification). The enzyme reveals an optimum for pyroglutamyl-Ala-Ala-Leu p-nitroanilide hydrolysis at pH 8.0-8.5 (Km 0.14 mM; k(cat). 36.9 s-1). Its activity increases linearly with NaCl concentration over the range 2-5 M. The substrate specificity of the enzyme is comparable with that of secretory subtilisins, the extent of protein degradation approaching that attained with proteinase K. The enzyme has a molecular mass of 41 kDa and a pI of 7.5. The N-terminal sequence of H. mediterranei serine proteinase reveals a 50% identity with that of Thermoactinomyces vulgaris serine proteinases, indicating that the enzyme belongs to the subtilisin family. Hence the serine proteinase secreted by the halophilic bacterium should be considered as a functional analogue, and a structural homologue, of eubacterial serine proteinases (subtilisins).
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Dreyer, T., M. J. Valler, J. Kay, P. Charlton, and B. M. Dunn. "The selectivity of action of the aspartic-proteinase inhibitor IA3 from yeast (Saccharomyces cerevisiae)." Biochemical Journal 231, no. 3 (November 1, 1985): 777–79. http://dx.doi.org/10.1042/bj2310777.
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The ability of the aspartic-proteinase inhibitor IA3 from yeast (Saccharomyces cerevisiae) to affect the activities of a range of mammalian and microbial aspartic proteinases was examined. The inhibitor appeared to be completely selective in that only the aspartic proteinase A from yeast was inhibited to any significant extent. IA3 thus represents the first example of a totally specific, naturally occurring, aspartic-proteinase inhibitor.
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Bartlett, J. D., and J. P. Simmer. "Proteinases in Developing Dental Enamel." Critical Reviews in Oral Biology & Medicine 10, no. 4 (July 1999): 425–41. http://dx.doi.org/10.1177/10454411990100040101.
Full textAbstract:
For almost three decades, proteinases have been known to reside within developing dental enamel. However, identification and characterization of these proteinases have been slow and difficult, because they are present in very small quantities and they are difficult to purify directly from the mineralizing enamel. Enamel matrix proteins such as amelogenin, ameloblastin, and enamelin are cleaved by proteinases soon after they are secreted, and their cleavage products accumulate in the deeper, more mature enamel layers, while the full-length proteins are observed only at the surface. These results suggest that proteinases are necessary for "activating" enamel proteins so the parent proteins and their cleavage products may perform different functions. A novel matrix metalloproteinase named enamelysin (MMP-20) was recently cloned from tooth tissues and was later shown to localize primarily within the most recently formed enamel. Furthermore, recombinant porcine enamelysin was demonstrated to cleave recombinant porcine amelogenin at virtually all of the sites that have previously been described in vivo. Therefore, enamelysin is at least one enzyme that may be important during early enamel development. As enamel development progresses to the later stages, a profound decrease in the enamel protein content is observed. Proteinases have traditionally been assumed to degrade the organic matrix prior to its removal from the enamel. Recently, a novel serine proteinase named enamel matrix serine proteinase-1 (EMSP1) was cloned from enamel organ epithelia. EMSP1 localizes primarily to the early maturation stage enamel and may, therefore, be involved in the degradation of proteins prior to their removal from the maturing enamel. Other, as yet unidentified, proteinases and proteinase inhibitors are almost certainly present within the forming enamel and await discovery.
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Bózner, P., A. Gombošová, M. valent, P. Demeš, and J. F. Alderete. "Proteinases ofTrichomonas vaginalis: antibody response in patients with urogenital trichomoniasis." Parasitology 105, no. 3 (December 1992): 387–91. http://dx.doi.org/10.1017/s0031182000074552.
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SUMMARYImmunoprecipitation combined with electrophoresis in gelatin-polyacrylamide gels was successfully used for detection of antibodies against numerous proteinases ofTrichomonas vaginalisin infected patients. The method proved to be highly specific as anti-proteinase antibodies were absent in women with negative cultivation ofT. vaginalisand no history of trichomoniasis. Sera of 71% and vaginal washes of 86% patients with trichomoniasis were positive for these antibodies. In vaginal washes, but not in sera, antibodies were partly complexed with proteinases, possibly of trichomonad origin. It was also shown that serum antibodies as well as local anti-proteinase antibodies persisted for weeks after patients had been cured.
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North, M. J., K. I. Scott, and B. C. Lockwood. "Multiple cysteine proteinase forms during the life cycle of Dictyostelium discoideum revealed by electrophoretic analysis." Biochemical Journal 254, no. 1 (August 15, 1988): 261–68. http://dx.doi.org/10.1042/bj2540261.
Full textAbstract:
Proteinases of the cellular slime mould Dictyostelium discoideum have been analysed using electrophoresis on polyacrylamide gels containing gelatin (gelatin/PAGE). Multiple proteinase forms were apparent in vegetative myxamoebae, but the presence of individual enzyme forms depended on the manner in which the cells were grown. Axenic cells had a characteristic A-pattern of proteinases consisting of six bands, the most active enzymes having apparent Mr values of 51,000 and 45,000 (these have been named ddCP51 and ddCP45, respectively). Some of the proteinases were also present in the medium, the major extracellular form was ddCP42, a 42,000-Mr enzyme. Cells grown in association with bacteria had a distinct B-pattern with three main enzymes that had apparent Mr values of 48,000, 43,000 and 38,000. All of the A- and B-pattern proteinases were most active at acid pH in the presence of dithiothreitol and were inhibited by various agents such as trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane (E64), leupeptin and chymostatin, which inactivate cysteine proteinases. One of the enzymes, ddCP30, was identified as cysteine proteinase B which had been purified and characterized previously [North, M.J. & Whyte, A. (1984) J. Gen. Microbiol. 130, 123-134]. During starvation of axenic cells in shaken suspensions some of the vegetative proteinases disappeared, ddCP42 was released from the cells and one new enzyme with an apparent Mr of 48,000 appeared. Addition of cyclic AMP had little effect on these changes. When the axenically grown myxamoebae underwent development on filters, similar changes in band pattern were observed and the aggregation stage was characterized by the presence of three cysteine proteinase bands (apparent Mr values of 48,000, 45,000 and 43,000). Proteinases, especially ddCP42, were released from the cells and could be collected from the buffer-saturated pads which supported the filters. The results demonstrate that cysteine proteinases are present throughout growth and development of D. discoideum and that the forms present are subject to nutritional and developmental regulation.
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Elden, T. C. "Influence of a Cysteine Proteinase Inhibitor on Alfalfa Weevil (Coleoptera: Curculionidae) Growth and Development Over Successive Generations." Journal of Entomological Science 35, no. 1 (January 1, 2000): 70–76. http://dx.doi.org/10.18474/0749-8004-35.1.70.
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The influence of leupeptin, a cysteine and serine proteinase inhibitor, on alfalfa weevil, Hypera postica (Gyllenhal), growth and development was investigated over nine successive generations. Concern that ingestion of proteinase inhibitors by phytophagous insects could induce production of inhibitor-insensitive proteinase activity initiated this investigation. The percent alfalfa weevil larval, pupal and adult survival, and defoliation was significantly lower on alfalfa foliage treated with leupeptin than on untreated foliage in all nine generations tested. Main effects for generations and treatment times generation were nonsignificant for all variables. This study demonstrates that after nine generations leupeptin, when compared to an untreated control, does not lose its ability to significantly inhibit alfalfa weevil growth and development. This suggests that the alfalfa weevil did not utilize or induce other proteinases (digestive enzymes) to compensate for inhibition of one of its major proteinases.
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LALMANACH, Gilles, Roger MAYER, Carole SERVEAU, Julio SCHARFSTEIN, and Francis GAUTHIER. "Biotin-labelled peptidyl diazomethane inhibitors derived from the substrate-like sequence of cystatin: targeting of the active site of cruzipain, the major cysteine proteinase of Trypanosoma cruzi." Biochemical Journal 318, no. 2 (September 1, 1996): 395–99. http://dx.doi.org/10.1042/bj3180395.
Full textAbstract:
Biotin-labelled peptidyl diazomethane inhibitors of cysteine proteinases, based on the N-terminal substrate-like segment of human cystatin C, a natural inhibitor of cysteine proteinases, were synthesized. These synthetic derivatives were tested as irreversible inhibitors of cruzipain, the major cysteine proteinase of Trypanosoma cruzi, to compare the kinetics of the inhibition of the parasite proteinase with that of the mammalian cathepsins B and L. The accessibility of the active sites of these proteinases to these probes was also investigated. The inhibition of cruzipain by Biot-LVG-CHN2 (where Biot represents biotinyl and L,V and G are single-letter amino acid residue abbreviations) and Biot-Ahx-LVG-CHN2 (where Ahx represents 6-aminohexanoic acid) was similar to that of unlabelled inhibitor. Biotin labelling of the inhibitor slowed the inhibition of both cathepsin B and cathepsin L. Adding a spacer arm (Ahx) between the biotin and the peptide moiety of the derivative increased the inhibition of cathepsin B but not that of cathepsin L. The discrimination provided by this spacer is probably due to differences in the topologies of the binding sites of proteinases, a feature that can be exploited to improve targeting of individual cysteine proteinases. Analysis of the blotted proteinases revealed marked differences in the accessibility of extravidin–peroxidase conjugate to the proteinase-bound biotinylated inhibitor. Cruzipain molecules exposed to Biot-LVG-CHN2 or Biot-Ahx-LVG-CHN2 were readily identified, but the reaction was much stronger when the enzyme was treated with the spacer-containing inhibitor. In contrast with the parasite enzyme, rat cathepsin B and cathepsin L treated with either Biot-LVG-CHN2 or Biot-Ahx-LVG-CHN2 produced no detectable bands. Papain, the archetype of this family of proteinases, was poorly labelled with Biot-LVG-CHN2, but strong staining was obtained with Biot-Ahx-LVG-CHN2. These findings suggest that optimized biotinylated diazomethanes might considerably improve their selectivity for the T. cruzi target enzyme.
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Knox, D. P., D. L. Redmond, and D. G. Jones. "Characterization of proteinases in extracts of adultHaemonchus contortus, the ovine abomasal nematode." Parasitology 106, no. 4 (May 1993): 395–404. http://dx.doi.org/10.1017/s0031182000067147.
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SUMMARYThe degradation of several protein substrates, including the blood proteins haemoglobin, albumin and fibrinogen, by proteinases present in extracts of adultHaemonchus contortuswas examined over a broad pH range. These proteinases were further characterized on the basis of substrate specificity, inhibitor sensitivity and molecular size by spectrophotometric and substrate gel analysis. The majority of the proteinases capable of degrading the blood proteins tested were active at acidic pH and could be ascribed to the cysteine proteinase class. In addition, evidence is presented that these proteinases are differentially recognized and inhibited by immune sera and that parasites capable of withstanding protective host immune responses exhibit modified expression of proteinases.
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North, M. J. "A bacterial factor induces changes in cysteine proteinase forms in the cellular slime mould Dictyostelium discoideum." Biochemical Journal 254, no. 1 (August 15, 1988): 269–75. http://dx.doi.org/10.1042/bj2540269.
Full textAbstract:
The electrophoretic pattern of cysteine proteinases in axenically grown myxamoebae of Dictyostelium discoideum can be altered by the addition of either Gram-negative (Klebsiella aerogenes, Escherichia coli) or Gram-positive (Micrococcus lysodeikticus, Bacillus subtilis) bacteria to the culture. No changes occurred, however, if either yeast or latex beads were used in place of bacteria. The changes involved the simultaneous loss of proteinases characteristic of the axenic cells (the A-forms) and the acquisition of those found in cells which have been grown on bacteria (the B-forms). Using K. aerogenes the conversion was complete within 4 h. Extracellular proteinase activity was unaffected during this period. After the D. discoideum cells had been lysed, no equivalent change in proteinase band pattern could be produced either by prolonged incubation of cell extracts or by treatment with proteinases. An identical conversion could be induced in cultures of myxamoebae by a factor, cysteine proteinase converting factor (CPCF), present in the 15,000 g supernatant of a sonicated suspension of K. aerogenes. CPCF was macromolecular, as demonstrated by both ultrafiltration and gel filtration, acid-precipitable, but was soluble in ethanol or alkali. Its activity was unaffected by treatment with trypsin. The results suggested that CPCF might be a component of the bacterial cell wall, and since its activity was affected by lysozyme treatment, peptidoglycan is implicated. The results can be interpreted in terms of a novel nutrient-dependent post-translational change which affected most of the cysteine proteinases present in D. discoideum myxamoebae.
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Meléndez-López, Samuel G., Scott Herdman, Ken Hirata, Min-Ho Choi, Youngchool Choe, Charles Craik, Conor R. Caffrey, et al. "Use of Recombinant Entamoeba histolytica Cysteine Proteinase 1 To Identify a Potent Inhibitor of Amebic Invasion in a Human Colonic Model." Eukaryotic Cell 6, no. 7 (May 18, 2007): 1130–36. http://dx.doi.org/10.1128/ec.00094-07.
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ABSTRACT Cysteine proteinases are key virulence factors of the protozoan parasite Entamoeba histolytica. We have shown that cysteine proteinases play a central role in tissue invasion and disruption of host defenses by digesting components of the extracellular matrix, immunoglobulins, complement, and cytokines. Analysis of the E. histolytica genome project has revealed more than 40 genes encoding cysteine proteinases. We have focused on E. histolytica cysteine proteinase 1 (EhCP1) because it is one of two cysteine proteinases unique to invasive E. histolytica and is highly expressed and released. Recombinant EhCP1 was expressed in Escherichia coli and refolded to an active enzyme with a pH optimum of 6.0. We used positional-scanning synthetic tetrapeptide combinatorial libraries to map the specificity of the P1 to P4 subsites of the active site cleft. Arginine was strongly preferred at P2, an unusual specificity among clan CA proteinases. A new vinyl sulfone inhibitor, WRR483, was synthesized based on this specificity to target EhCP1. Recombinant EhCP1 cleaved key components of the host immune system, C3, immunoglobulin G, and pro-interleukin-18, in a time- and dose-dependent manner. EhCP1 localized to large cytoplasmic vesicles, distinct from the sites of other proteinases. To gain insight into the role of secreted cysteine proteinases in amebic invasion, we tested the effect of the vinyl sulfone cysteine proteinase inhibitors K11777 and WRR483 on invasion of human colonic xenografts. The resultant dramatic inhibition of invasion by both inhibitors in this human colonic model of amebiasis strongly suggests a significant role of secreted amebic proteinases, such as EhCP1, in the pathogenesis of amebiasis.
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Huet, G., R. M. Flipo, C. Richet, C. Thiebaut, D. Demeyer, M. Balduyck, B. Duquesnoy, and P. Degand. "Measurement of Elastase and Cysteine Proteinases in Synovial Fluid of Patients with Rheumatoid Arthritis, Sero-Negative Spondylarthropathies, and Osteoarthritis." Clinical Chemistry 38, no. 9 (September 1, 1992): 1694–97. http://dx.doi.org/10.1093/clinchem/38.9.1694.
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Abstract Synovial fluid samples were collected from 45 patients with rheumatoid arthritis, spondylarthropathy, or osteoarthritis, to study their content of elastase (EC 3.4.21.37) and of cysteine proteinases (EC 3.4.22.1, 3.4.22.15). We measured both elastase complexed with alpha 1-proteinase inhibitor and elastase activity toward the substrate L-pyroglutamyl-L-prolyl-L-valine-p-nitroanilide. Cysteine proteinase activities were measured with the substrates N-benzyloxycarbonyl-L-phenylalanyl-L-arginine-7-amido-4-methylcoumarin (Z-Phe-Arg-AMC) and Z-Arg-Arg-AMC and the inhibitor E-64 [L-trans-epoxysuccinyl-leucyl-amido-(4-guanidino)-butane]. In all these enzyme assays, higher median values were obtained in inflammatory arthropathies than in osteoarthritis. The concentration of the elastase-alpha 1-proteinase inhibitor complex and of elastase and cysteine proteinase activities were statistically higher in patients with rheumatoid arthritis than in patients with osteoarthritis. The difference in results between patients with spondylarthropathy and patients with osteoarthritis was statistically significant only for the elastase-alpha 1-proteinase inhibitor complex. The median values of the complex and of both enzyme activities were higher in patients with rheumatoid arthritis than in patients with spondylarthropathy; however, the difference was statistically significant only for the cysteine proteinase activity measured with Z-Arg-Arg-AMC substrate. These results suggest that both elastase and cysteine proteinases, which are increased in patients with inflammatory arthritis, are involved in cartilage degradation in these arthropathies.