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1.
Presence of Active and Inactive Molecules of a Cell Wall-Associated Proteinase in Lactobacillus helveticus CP790
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Monoclonal antibodies against a cell wall-associated 45-kDa proteinase from Lactobacillus helveticus CP790 were prepared and used for an immunoblotting analysis of the cell wall extract of CP790. They were found to react with an unidentified 46-kDa protein as well as the 45-kDa proteinase. The 46-kDa protein was copurified with the 45-kDa proteinase by affinity column chromatography using antibody-fixed Sepharose and sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then extracted from the gels. An elution profile of the cyanogen bromide digest of the purified 46-kDa protein obtained by reversed-phase high-performance liquid chromatography was identical to that of the 45-kDa proteinase except for one peak. An analysis of the N-terminal 21-amino-acid sequence revealed that the 46-kDa protein possesses an extra 7 amino acids at the N terminus of the 45-kDa proteinase. The 46-kDa protein was produced at constant levels during fermentation in a skim milk medium, while the 45-kDa protein was mainly observed in the middle of the exponential phase of growth and was produced in proportion to the proteinase activity. Moreover, only the 46-kDa protein was detected in the crude extract of L. helveticus CP791, a variant strain of CP790 defective in proteinase activity. These data strongly suggest that the 46-kDa protein is a precursor, inactive form of the 45-kDa proteinase. 相似文献
2.
《Bioscience, biotechnology, and biochemistry》2013,77(6):1228-1230
Polyclonal antibodies against an extracellular proteinase of Lactobacillus helveticus CP53 were raised. The antibodies reacted with a 170-kDa enzyme with activity and a 53-kDa protein that seemed to be a degradation product of the 170-kDa proteinase from results of immunoblotting. The antibodies reacted also with a 45-kDa extracellular proteinase of L. helveticus CP790. However, monoclonal antibodies to the CP790 proteinase did not react with the proteinase of L. helveticus CP53. Seventeen strains of L. helveticus were tested for immunological reactivity with the two kinds of antibodies. The strains all had the same reactivity as either strain CP53 or strain CP790. Eleven strains with the 45-kDa proteinase were identified as L. helveticus biovar jugurti because they did not ferment maltose, four other strains with the 170- and 53-kDa proteins were identified as L. helveticus biovar helveticus because they fermented maltose. The remaining two strains dit not fit this pattern; they had both the 170- and 53-kDa proteins, but classification by their sugar utilization showed them to be L. helveticus biovar jugurti. 相似文献
3.
Characterization of a human coronavirus (strain 229E) 3C-like proteinase activity. 总被引:15,自引:8,他引:7
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The RNA polymerase gene of human coronavirus (HCV) 229E encodes a large polyprotein that contains domains with motifs characteristic of both papain-like cysteine proteinases and proteinases with homology to the 3C proteinase of picornaviruses. In this study, we have, first, expressed the putative HCV 229E 3C-like proteinase domain as part of a beta-galactosidase fusion protein in Escherichia coli and have shown that the expressed protein has proteolytic activity. The substitution of one amino acid within the predicted proteinase domain (His-3006-->Asp-3006) abolishes, or at least significantly reduces, this activity. Amino-terminal sequence analysis of a purified, 34-kDa cleavage product shows that the bacterial fusion protein is cleaved at the dipeptide Gln-2965-Ala-2966, which is the predicted amino-terminal end of the putative 3C-like proteinase domain. Second, we have confirmed the proteolytic activity of a bacterially expressed polypeptide with the amino acid sequence of the predicted HCV 229E 3C-like proteinase by trans cleavage of an in vitro translated polypeptide encoded within open reading frame 1b of the RNA polymerase gene. Finally, using fusion protein-specific antiserum, we have identified a 34-kDa, 3C-like proteinase polypeptide in HCV 229E-infected MRC-5 cells. This polypeptide can be detected as early as 3 to 5 h postinfection but is present in the infected cell in very low amounts. These data contribute to the characterization of the 3C-like proteinase activity of HCV 229E. 相似文献
4.
Alvarez-Sánchez ME Carvajal-Gamez BI Solano-González E Martínez-Benitez M Garcia AF Alderete JF Arroyo R 《The international journal of biochemistry & cell biology》2008,40(11):2442-2451
Recently, we found that inhibition of putrescine synthesis by ornithine decarboxylase (ODC) significantly increased Trichomonas vaginalis adherence mediated by protein adhesins. Surprisingly and unexpectedly, trichomonal contact-dependent cytotoxicity was absent. Therefore, a role for polyamine depletion on regulation of T. vaginalis cytotoxicity mediated by the cysteine proteinase (CP) of 65-kDa, CP65, was investigated. We performed cytotoxicity and cell-binding assays followed by zymograms, as well as Western blot and indirect immunofluorescence assays using specific anti-CP65 antibodies to detect CP65. Trichomonads grown in the presence of the ODC inhibitor, 1-4-diamino-2-butanone (DAB) had lower levels of cytotoxicity that corresponded with diminished CP65 proteolytic activity when compared to untreated organisms handled identically. Likewise, semiquantitative and qRT-PCR as well as Western blot and immunofluorescence assays showed decreased amounts of tvcp65 mRNA and CP65 protein in DAB-treated parasites. These effects were reversed by addition of exogenous putrescine. These data show a direct link between polyamine metabolism and expression of the cytotoxic CP65 proteinase involved in trichomonal host cellular damage. 相似文献
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6.
Identification of a novel cleavage activity of the first papain-like proteinase domain encoded by open reading frame 1a of the coronavirus Avian infectious bronchitis virus and characterization of the cleavage products
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The coronavirus Avian infectious bronchitis virus (IBV) employs polyprotein processing as a strategy to express its gene products. Previously we identified the first cleavage event as proteolysis at the Gly(673)-Gly(674) dipeptide bond mediated by the first papain-like proteinase domain (PLPD-1) to release an 87-kDa mature protein. In this report, we demonstrate a novel cleavage activity of PLPD-1. Expression, deletion, and mutagenesis studies showed that the product encoded between nucleotides 2548 and 8865 was further cleaved by PLPD-1 at the Gly(2265)-Gly(2266) dipeptide bond to release an N-terminal 195-kDa and a C-terminal 41-kDa cleavage product. Characterization of the cleavage activity revealed that the proteinase is active on this scissile bond when expressed in vitro in rabbit reticulocyte lysates and can act on the same substrate in trans when expressed in intact cells. Both the N- and C-terminal cleavage products were detected in virus-infected cells and were found to be physically associated. Glycosidase digestion and site-directed mutagenesis studies of the 41-kDa protein demonstrated that it is modified by N-linked glycosylation at the Asn(2313) residue encoded by nucleotides 7465 to 7467. By using a region-specific antiserum raised against the IBV sequence encoded by nucleotides 8865 to 9786, we also demonstrated that a 33-kDa protein, representing the 3C-like proteinase (3CLP), was specifically immunoprecipitated from the virus-infected cells. Site-directed mutagenesis and expression studies showed that a previously predicted cleavage site (Q(2583)-G(2584)) located within the 41-kDa protein-encoding region was not utilized by 3CLP, supporting the conclusion that the 41-kDa protein is a mature viral product. 相似文献
7.
Human cytomegalovirus maturational proteinase: expression in Escherichia coli, purification, and enzymatic characterization by using peptide substrate mimics of natural cleavage sites. 总被引:8,自引:8,他引:0
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P J Burck D H Berg T P Luk L M Sassmannshausen M Wakulchik D P Smith H M Hsiung G W Becker W Gibson E C Villarreal 《Journal of virology》1994,68(5):2937-2946
The proteolytic processing of the human cytomegalovirus (HCMV) assembly protein, resulting in truncation of its C terminus, is an essential step in virion maturation. The proteinase responsible for this cleavage is the amino-terminal half of the protein encoded by the UL80a open reading fame. We have obtained high expression levels of this 256-amino-acid HCMV proteinase, assemblin, in Escherichia coli. In addition to the 28-kDa proteinase, a 15-kDa protein comprising the first 143 amino acids and a 13-kDa protein comprising the last 113 amino acids of the 28-kDa HCMV proteinase were present. Both the 28-kDa proteinase and the 15-kDa protein were purified by a two-step chromatographic procedure utilizing anion exchange in urea and dithiothreitol and size exclusion in NaSCN and dithiothreitol. Activation of the purified 28-kDa proteinase required denaturation in urea as well as complete reduction of all five cysteine residues in the molecule. Removal of the urea by dialysis with retention of the reducing agent yielded an active proteinase. Addition of glycerol to 50% enhanced the activity. The HCMV proteinase cleaved the peptides RGVVNASSRLAK and SYVKASVSPE, which are mimics of the maturational (M)- and release (R)-site sequences, respectively, in the UL80a-encoded protein. The cleavage site in the peptides was at the same Ala-Ser scissile bond as observed in the UL80a protein. The Km value for the cleavage of RGVVNASSRLAK (M-site mimic) by the proteinase was similar to that for SYVKASVSPE (R-site mimic), but the turnover (kcat) of the M-site peptide mimic substrate by the proteinase was six to eight times faster. The peptide homologs of the herpes simplex virus type 1 M- and R-site sequences in the UL26-encoded protein were also cleaved by the HCMV proteinase, although at rates slower than those for the HCMV substrates. The HCMV proteinase was inhibited by Zn2+ and by alkylating agents, but only at very high inhibitor concentrations. The purified 15-kDa protein, subjected to the same activation conditions as the 28-kDa proteinase, had no enzymatic activity against the HCMV M- and R-site peptide substrates. 相似文献
8.
The Putative Helicase of the Coronavirus Mouse Hepatitis Virus Is Processed from the Replicase Gene Polyprotein and Localizes in Complexes That Are Active in Viral RNA Synthesis 总被引:7,自引:0,他引:7
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Mark R. Denison Willy J. M. Spaan Yvonne van der Meer C. Anne Gibson Amy C. Sims Erik Prentice Xiao Tao Lu 《Journal of virology》1999,73(8):6862-6871
The coronavirus mouse hepatitis virus (MHV) translates its replicase gene (gene 1) into two co-amino-terminal polyproteins, polyprotein 1a and polyprotein 1ab. The gene 1 polyproteins are processed by viral proteinases to yield at least 15 mature products, including a putative RNA helicase from polyprotein 1ab that is presumed to be involved in viral RNA synthesis. Antibodies directed against polypeptides encoded by open reading frame 1b were used to characterize the expression and processing of the MHV helicase and to define the relationship of helicase to the viral nucleocapsid protein (N) and to sites of viral RNA synthesis in MHV-infected cells. The antihelicase antibodies detected a 67-kDa protein in MHV-infected cells that was translated and processed throughout the virus life cycle. Processing of the 67-kDa helicase from polyprotein 1ab was abolished by E64d, a known inhibitor of the MHV 3C-like proteinase. When infected cells were probed for helicase by immunofluorescence laser confocal microscopy, the protein was detected in patterns that varied from punctate perinuclear complexes to large structures that occupied much of the cell cytoplasm. Dual-labeling studies of infected cells for helicase and bromo-UTP-labeled RNA demonstrated that the vast majority of helicase-containing complexes were active in viral RNA synthesis. Dual-labeling studies for helicase and the MHV N protein showed that the two proteins almost completely colocalized, indicating that N was associated with the helicase-containing complexes. This study demonstrates that the putative RNA helicase is closely associated with MHV RNA synthesis and suggests that complexes containing helicase, N, and new viral RNA are the viral replication complexes. 相似文献
9.
A cell adhesion protein from the crayfish Pacifastacus leniusculus, a serine proteinase homologue similar to Drosophila masquerade 总被引:1,自引:0,他引:1
Huang TS Wang H Lee SY Johansson MW Söderhäll K Cerenius L 《The Journal of biological chemistry》2000,275(14):9996-10001
A cDNA encoding a protein resembling masquerade, a serine proteinase homologue expressed during embryogenesis, larval, and pupal development in Drosophila melanogaster, was identified in hemocytes of the adult freshwater crayfish, Pacifastacus leniusculus. The crayfish protein is similar to Drosophila masquerade in the following aspects: (a) overall sequence of the serine proteinase domain, such as the position of three putative disulfide bridges, glycine in the place of the catalytic serine residue, and the presence of a substrate-lining pocket typical for trypsins; (b) the presence of several copies of a disulfide-knotted motif in the putative propeptide. This masquerade-like protein is cleaved into a 27-kDa fragment, which could be detected by immunoblot analysis using an affinity-purified antibody against a synthetic peptide in the C-terminal domain of the protein. The 27-kDa protein could be immunoaffinity-purified from hemocyte lysate supernatant and exhibited cell adhesion activity in vitro, indicating that the C-terminal domain of the crayfish masquerade-like protein mediates cell adhesion. 相似文献
10.
Ma Z Daquin A Yao J Rodgers D Thompson MW Hersh LB 《Archives of biochemistry and biophysics》2003,415(1):80-86
The puromycin-sensitive aminopeptidase was found to be resistant to proteolysis by trypsin, chymotrypsin, and protease V8 but was cleaved into an N-terminal 60-kDa fragment and a C-terminal 33-kDa fragment by proteinase K. The two proteinase K fragments remain associated and retained enzymatic activity. Attempts to express the 60-kDa N-terminal fragment in Escherichia coli produced inclusion bodies. A hexa-histidine fusion protein of the 60-kDa N-terminal fragment was solubilized from inclusion bodies with urea and refolded by removal of the urea through dialysis. The refolded protein was devoid of aminopeptidase activity as assayed with arginine-beta-naphthylamide. However, the refolded protein bound the substrate dynorphin A(1-9) with a stoichiometry of 0.5 mol/mol and a K(0.5) value of 50 microM. Dynorphin A(1-9) binding was competitively inhibited by the substrate dynorphin B(1-9), but not by des-Tyr(1)-leucine-enkephalin, a poor substrate for the enzyme. 相似文献
11.
We have previously shown that 57-kDa protein in royal jelly (RJ) was specifically degraded in proportion to both storage temperature and storage period, and we suggested that it could be useful as a marker of freshness of RJ (Kamakura, M., Fukuda, T., Fukushima, M. and Yonekura, M., Biosci. Biotechnol. Biochem., 65, 277-284 (2001).). Here, we investigated the effects of various proteinase inhibitors on proteinase activity in RJ and on the specific degradation of 57-kDa protein during storage. Ethylenediaminetetraacetic acid (EDTA), but not other inhibitors, inhibited the proteinase activity in RJ, and dose-dependently suppressed storage-dependent degradation of 57-kDa protein. These results suggest that EDTA inhibits a specific proteinase activity in RJ, thereby suppressing the degradation of 57-kDa protein during storage at high temperature. 相似文献
12.
Zamora-Veyl FB Guedes HL Giovanni-De-Simone S 《Zeitschrift für Naturforschung. C, Journal of biosciences》2002,57(5-6):541-547
A proteolytic activity was identified in Dugesia tigrina planaria using the chromogenic substrate Phe-Ala-Ala-Phe (4-NO2)-Phe-Val-Leu-O4MP. The activity of the enzyme increased four times during the regeneration and presented a maximum at 120 hr being higher in tail than head regenerating segments. The protease that displays this activity was purified from worms by a single step on pepstatin-agarose followed by gel-filtration high performance liquid chromatography. The purification resulted in a 34-fold increase in specific activity and the final yield was 10%. The active D. tigrina hydrolase appears to be a dimeric protein composed of identical subunits with 34 kDa associated by disulphide bridges similar to vertebrate cathepsin D. By SDS-PAGE several bands were detected but upon gel filtration HPLC one proteolytically active component, termed Asp-68, was detected and isolated. The maximal activity was observed in a range between pH 3.5-5.0 and the enzyme became inactivated at a pH value above 7.2. The purified enzyme was not inhibited by inhibitors from serine (aprotinin, TPCK, PMSF and TLCK), metallo (EDTA) and cysteine proteinase (E-64) classes. In contrast, inhibitors such as pepstatin, EPNP, and 4-beta-PMA efficiently inhibited the activity of the 68-kDa protease. 相似文献
13.
A 2.6-kilobase HaeIII DNA fragment corresponding to an extracellular proteinase gene (prtY) was cloned from chromosomal DNA of Lactobacillus helveticus CP790 in Escherichia coli using a pKK223-3 vector. The transformant expressed a 48-kDa protein that reacts with monoclonal antibodies specific to the proteinase and seemed to be a pre-proproteinase, but had no proteolytic activity. About 1.6 kilobases of the 2.6-kilobase DNA fragment, which contained the complete gene for the proteinase was sequenced. Sequence analysis found an open reading frame with a capacity to encode a protein of 449 amino acids. The coding region contained a Gram-positive-type signal peptide of 30 amino acids. The N-terminal sequences of the proproteinase and the mature proteinase have been observed in the polypeptide at position + 31 and + 38. The putative amino acid sequence showed a significant similarity to a surface layer protein of L. helveticus and Lactobacillus acidophilus in the amino terminal signal sequence and carboxyl terminus. 相似文献
14.
The genome of tobacco etch virus contains a single open reading frame with the potential to encode a 346-kilodalton (kDa) polyprotein. The large polyprotein is cleaved at several positions by a tobacco etch virus genome-encoded, 49-kDa proteinase. The locations of the 49-kDa proteinase-mediated cleavage sites flanking the 71-kDa cytoplasmic pinwheel inclusion protein, 6-kDa protein, 49-kDa proteinase, and 58-kDa putative polymerase have been determined by using cell-free expression, proteolytic processing, and site-directed mutagenesis systems. Each of these sites is characterized by the conserved sequence motif Glu-Xaa-Xaa-Tyr-Xaa-Gln-Ser or Gly (in which cleavage occurs after the Gln residue). The amino acid residue (Gln) predicted to occupy the -1 position relative to the scissile bond has been substituted, by mutagenesis of cloned cDNA, at each of four cleavage sites. The altered sites were not cleaved by the 49-kDa proteinase. A series of synthetic polyproteins that contained the 49-kDa proteinase linked to adjoining proteins via defective cleavage sites were expressed, and their proteolytic activities were analyzed. As part of a polyprotein, the proteinase was found to exhibit cis (intramolecular) and trans (intermolecular) activity. 相似文献
15.
Trichomonas vaginalis: characterization of a 39-kDa cysteine proteinase found in patient vaginal secretions 总被引:3,自引:0,他引:3
Hernández-Gutiérrez R Avila-González L Ortega-López J Cruz-Talonia F Gómez-Gutierrez G Arroyo R 《Experimental parasitology》2004,107(3-4):125-135
Trichomonosis, a chronic sexually transmitted disease, remains a public health problem affecting yearly over 170 million people worldwide. This disease is caused by Trichomonas vaginalis, a protozoan flagellate rich in cysteine proteinases (CPs). Although CPs are involved in trichomonal cytopathogenicity, only few of them have been defined as virulence factors. In this study, we characterize a T. vaginalis 39-kDa proteinase (CP39) found in vaginal secretions from patients with trichomonosis. The CP39 proteinase bound to HeLa epithelial cells, vaginal epithelial cells (VECs), and human prostatic cancer cells (DU-145). CP39 did not bind to a human colon cancer (CaCo) cell line, suggesting tissue-specific binding. CP39 was found in six fresh trichomonad isolates tested. In two-dimensional gels, CP39 appeared as a single spot with a pI 4.5. CP39 is inhibited by E-64, stable at 50 degrees C, and active in a wide pH range (3.6-9.0), with an optimum pH at 7.0. In addition, CP39 degraded collagens I, III, IV, and V, human fibronectin, human hemoglobin, and human immunoglobulins A and G. Indirect immunofluorescence detected CP39 on the parasite surface with specific polyclonal antibody to purified CP39. Finally, CP39 was found to be immunogenic, as evidenced by detection on immunoblots with serum of patients with trichomonosis, but not control individuals. These data suggest that CP39 may play a role during trichomonal infection. 相似文献
16.
Proteinase Activity during Tracheary Element Differentiation in Zinnia Mesophyll Cultures 总被引:7,自引:1,他引:6
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The zinnia (Zinnia elegans) mesophyll cell culture tracheary element (TE) system was used to study proteinases active during developmentally programmed cell death. Substrate-impregnated gels and single-cell assays revealed high levels of proteinase activity in differentiating TEs compared with undifferentiated cultured cells and expanding leaves. Three proteinases (145, 28, and 24 kD) were exclusive to differentiating TEs. A fourth proteinase (59 kD), although detected in extracts from all tissues examined, was most active in differentiating TEs. The 28- and 24-kD proteinases were inhibited by thiol proteinase inhibitors, leupeptin, and N-[N-(L-3-trans-carboxirane-2-carbonyl)-L-leucyl]-agmatine (E-64). The 145- and 59-kD proteinases were inhibited by the serine proteinase inhibitor phenylmethylsulfonyl fluoride (PMSF). Extracts from the TE cultures contained sodium dodecyl sulfate-stimulated proteolytic activity not detected in control cultures. Sodium dodecyl sulfate-stimulated proteolysis was inhibited by leupeptin or E-64, but not by PMSF. Other tissues, sucrose-starved cells and cotyledons, that contain high levels of proteolytic activity did not contain TE-specific proteinases, but did contain higher levels of E-64-sensitive activities migrating as 36- to 31-kD enzymes and as a PMSF-sensitive 66-kD proteinase. 相似文献
17.
18.
《Bioscience, biotechnology, and biochemistry》2013,77(9):1536-1540
To examine, the proteolytic activities of various truncated derivatives of the potato virus Y (PVY) 50-kDa protease, the derivatives were expressed in Escherichia coli in polyprotein forms fused with coat protein (CP). For the intermolecular cleavage reaction, the truncated proteases were expressed together with the substrate protein containing the polymerase-CP junction. The activity was evaluated by the amount of the mature CP released from the precursor by the intra- and intermolecular cleavage occurring in E. coli. By this experiment, we identified the moiety responsible for the proteolytic activity of the 50-kDa protease to be a 26-kDa polypeptide mapped to the C-terminal half of the protease. Introduction of His234→Tyr, Asp269→Asn, or Cys339→Gly substitution in the putative catalytic triad of the protease abolished its activity. However, the mutated protease with Cys339→Ser replacement retained a reduced proteolytic activity. 相似文献
19.
Mathias Teschke Reinhard Saborowski 《Journal of experimental marine biology and ecology》2005,316(2):213-229
The utilization of dietary proteins in crustaceans is facilitated by a set of peptide hydrolases which are often dominated by “trypsin-like” serine proteinases. As expected, the North Sea shrimps Crangon crangon and Crangon allmani showed in their midgut glands high proteolytic activities. However, the majority of animals lacked trypsin and chymotrypsin. Conversely, a minority of about 10% of the animals had elevated trypsin activities. The appearance of trypsin was neither related to the mode of feeding nor to the nutritive state of the animals. When present, trypsin was expressed in both species as a single isoform of apparently 20 kDa. The lack of serine proteinases was also confirmed by inhibitor assays. AEBSF, a serine proteinase inhibitor, slightly reduced total proteinase activity by less than 10%. In contrast E 64, a cysteine proteinase inhibitor, caused a reduction of more than 70% of total proteinase activity, indicating that a substantial share of proteolytic activity is caused by cysteine proteinases. Cathepsin L-like proteinases were identified as major cysteine proteinases.A comparison with the eucarid crustaceans Pandalus montagui, Pagurus bernhardus, Cancer pagurus and Euphausia superba showed a similar high level of total proteinase activity in all species. Trypsin, however, varied significantly between species showing lowest activities in Caridea and the highest activity in E. superba. E 64 suppressed total proteinase activity by more than 70% in Crangon species but not in C. pagurus and E. superba. In contrast, the serine proteinase inhibitor AEBSF had only little effect in Caridea but was most effective in P. bernhardus, C. pagurus and E. superba. The results may indicate different traits of food utilization strategies in some eucarid crustaceans. Caridea may express predominantly cysteine proteinase, while in Anomura, Brachyura and Euphausiacea, serine proteinases may prevail. 相似文献
20.
Quintas-Granados LI Orozco E Brieba LG Arroyo R Ortega-López J 《Protein expression and purification》2009,63(1):26-32
The cysteine proteinase EhCP112 and the adhesin EhADH112 assemble to form the EhCPADH complex involved in Entamoeba histolytica virulence. To further characterize this cysteine proteinase, the recombinant full-length EhCP112 enzyme was expressed and purified under denaturing conditions. After a refolding step under reductive conditions, the inactive precursor (ppEhCP112) was processed to a 35.5 kDa mature and active enzyme (EhCP112). The thiol specific inhibitor E-64, but not serine or aspartic proteinase inhibitors arrested this activation process. The activation step of the proenzyme followed by the mature enzyme suggests an autocatalytic process during EhCP112 maturation. The experimentally determined processing sites observed during EhCP112 activation lie close to processing sites of other cysteine proteinases from parasites. The kinetic parameters of the mature EhCP112 were determined using hemoglobin and azocasein as substrates. The proteinase activity of EhCP112 was completely inhibited by thiol inhibitors, E-64, TLCK, and chymostatin, but not by general proteinase inhibitors. Since EhCP112 is a proteinase involved in the virulence of E. histolytica, a reliable source of active EhCP112 is a key step for its biochemical characterization and to carry out future protein structure-function studies. 相似文献