A study on the identities of the three species of chromatin-associated proteinases in a mutant of Saccharomyces cerevisiae which lacks four major vacuolar proteinases

Using mutant strain ABYS1 of Saccharomyces cerevisiae lacking four main vacuolar proteinases, proteinase A, proteinase B, carboxypeptidase Y, and carboxypeptidase S, we examined the identities of chromatin-associated proteinases, ruling out possible contamination of the chromatin fraction by them. The chromatin of strain ABYS1 showed three peaks of proteolytic activity at pH 4, 7, and 11, and these activities were found to be derived from three species of proteinases, the aspartic, serine neutral, and serine alkaline ones. As these chromatin-associated proteinases of strain ABYS1 were identical in both quality and quantity to those of wild-type strain of yeast, we suggest that the yeast chromatin contains three species of specific proteinases as essential components.     

Phylogenetic distribution of cysteine proteinases in beetles: evidence for an evolutionary shift to an alkaline digestive strategy in Cerambycidae

We characterized the digestive proteinases of eight species of beetles to improve our understanding of the phylogenetic distribution of serine and cysteine proteinases. Serine proteinases function optimally under alkaline pH conditions, whereas cysteine proteinases require acidic pH. The phylogenetic distribution of cysteine proteinases suggests that they first appeared in an early cucujiform ancestor, however, data for some groups is patchy, and there has been speculation that they have been lost in at least one group, the long-horned beetles (Cerambycidae). The pattern we found supports the hypothesized origin of the proteinases and extends their distribution to an additional superfamily. In addition, we confirmed the presence of cysteine proteinases in some Curculionoidea. Cysteine proteinases were absent, however, from all three species of cerambycids surveyed, supporting the hypothesis that this group has reverted to the more ancestral serine (alkaline) digestive strategy. In four species we compared the pH optima for total proteolytic activity to the actual pH of the midgut and found the match between optimal and actual pH to be weaker in the cerambycids. These findings suggest that either a close correlation between midgut pH and the proteolytic pH optimum is not needed for adequate digestive efficiency, or that midgut pH is a more constrained digestive feature and there has been insufficient time for it to shift upwards to maximize serine proteinase activity.     

Aspartic proteinases in the digestive tract of marine decapod crustaceans

Decapod crustaceans synthesize highly active proteolytic enzymes in the midgut gland and release at least a part of them into the stomach where they facilitate the first step in peptide hydrolysis. The most common proteinases in the gastric fluid characterized so far are serine proteinases, that is, trypsin and chymotrypsin. These enzymes show highest activities at neutral or slightly alkaline conditions. The presence of acid proteinases, as they prevail in vertebrates, has been discussed contradictorily yet in invertebrates. In this study, we show that acid aspartic proteinases appear in the gastric fluid of several decapods. Lobsters Homarus gammarus showed the highest activity with a maximum at pH 3. These activities were almost entirely inhibited by pepstatin A, which indicates a high share of aspartic proteinases. In other species (Panulirus interruptus, Cancer pagurus, Callinectes arcuatus and Callinectes bellicosus), proteolytic activities were present at acid conditions but were distinctly lower than in H. gammarus. Zymograms at pH 3 showed in each of the studied species at least one, but mostly two-four bands of activity. The apparent molecular weight of the enzymes ranged from 17.8 to 38.6 kDa. Two distinct bands were identified which were inhibited by pepstatin A. Acid aspartic proteinases may play an important role in the process of extracellular digestion in decapod crustaceans. Activities were significantly higher in clawed lobster than in spiny lobster and three species of brachyurans. Therefore, it may be suggested that the expression of acid proteinases is favored in certain groups and reduced in others.     

Acid proteinases in various species of cellular slime mold

The proteolytic activities of the cellular slime moldsDictyostelium mucoroides, Dictyostelium purpureum, Polysphondylium pallidum, andPolysphondylium violaceum have been examined. Myxamoebae possessed activity against Hide Powder Azure at pH 2–5 which was enhanced by dithiothreitol: this enhancement was small inDictyostelium species but three- to four-fold in thePolysphondylium species. Following electrophoresis on polyacrylamide gels containing denatured haemoglobin five or more proteinases could be detected in each species. Activity against Hide Powder Azure was inhibited severely by HgCl₂ and to a lesser extent by other thiol proteinase inhibitors such asN-α-p-tosyl-l-lysine chloromethyl ketone-HCl, antipain, and leupeptin. Inhibitors of aspartyl and serine proteinases had no effect. All proteinases visualized on gels were inhibited by HgCl₂, and some, but not the major one of each species, were sensitive to the other thiol proteinase inhibitors. Extracts of fruiting bodies retained acid proteolytic activity. New proteinases were detected inD. mucoroides; there was a relative increase in one proteinase inP. violaceum but three proteinases were lost during fruiting body formation inP. pallidum. During microcyst formation inP. pallidum there was a decrease in proteolytic activity but most of the myxamoebal proteinases could be detected. Overall the results demonstrate that the cellular slime molds possess similar types of proteinase although there were significant differences between the actual proteinases observed in individual species.     

Analysis of Leishmania proteinases reveals developmental changes in species-specific forms and a common 68-kDa activity

Abstract The proteinases of three species of Leishmania have been analysed by electrophoresis. Amastigotes of L. mexicana mexicana have several high-activity, low- M _r cysteine proteinases which are absent from log-phase promastigotes of L. m. mexicana and from all developmental stages of the other species analysed ( L. donovani and L. major ). Low-activity, low- M _r proteinases were present in populations of stationary-phase promastigotes of L. m. mexicana . All three species of Leishmania had higher M _r proteinases, a number of which showed developmental regulation, some of them being stage-specific. Significantly, at all stages of the life cycle in all three species a 68-kDa proteinase was apparent. In its size, sensitivity to inhibitors and ability to bind concanavalin A-agarose, this resembles the major surface protein thought to be present in all Leishmania species and which has recently been reported to possess proteinase activity in L. major promastigotes.     

MECHANISM OF ACTIVATION AND POSSIBLE ROLES OF THE CATHEPSIN B-LIKE AND D-LIKE PROTEINASES IN THE EGGS OF PHILOSAMIA CYNTHIA RICINI*

Abstract The mechanism of activation and possible roles in the yolk protein's degradation of the cathepsin B-like and cathepsin D-like proteinases in eggs of Philosamia cynthia ricini were studied. The results showed that acidification could not obviously change the molecular masses of the proteinases. The two proteinases were not likely to modify with each other. The results suggested that the two proteinases might hydrolyze different yolk proteins, or hydrolyze the yolk proteins at different stages during embryogenesis. The results of double immunodiffusion showed that the cathepsin B-like and cathepsin D-like proteinases might exist in the eggs of some different species in Lepidoptera.     

蓖麻蚕卵组织蛋白酶B和组织蛋白酶D活化机制及作用(英)

本文研究了蓖麻蚕卵内组织蛋白酶B和组织蛋白酶D在酸性条件下的活化机理和在卵黄蛋白水解中的作用。活性检测结果表明,酸处理用以使蛋白酶活化,但SDS-聚丙烯酰胺凝胶电泳实验表明酸化没有使蛋白酶分子量降低。推测酸化可能通过改变蛋白酶构象从而使蛋白酶活化。两种蛋白酶之间未表现出相互修饰导致分子量变化,也未表现出明显的相互促进活性的作用。蛋白酶水解产物分子量的不同,说明两种蛋白酶作用位点不同。组织蛋白酶B的水解产物分子量较小而组织蛋白酶D的水解产物分子量大,说明两种蛋白酶在体内可能作用于蛋白质水解的不同阶段或作用于不同的蛋白质。免疫扩散结果显示,抗蓖麻蚕卵蛋白酶血清可以识别柞蚕和棉铃虫卵内的成分,表明其它一些鳞翅目昆虫卵内可能存在相似的蛋白酶。     

Purification and characterization of the cysteine proteinases in the latex of Vasconcellea spp

Latex of all Vasconcellea species analyzed to date exhibits higher proteolytic amidase activities, generally attributed to cysteine proteinases, than the latex of Carica papaya. In the present study, we show that this higher activity is correlated with a higher concentration of enzymes in the latex of Vasconcellea fruits, but in addition also results from the presence of other cysteine proteinases or isoforms. In contrast to the cysteine proteinases present in papaya latex, which have been extensively studied, very little is known about the cysteine proteinases of Vasconcellea spp. In this investigation, several cDNA sequences coding for cysteine proteinases in Vasconcellea x heilbornii and Vasconcellea stipulata were determined using primers based on conserved sequences. In silico translation showed that they hold the characteristic features of all known papain-class cysteine proteinases, and a phylogenetic analysis revealed the existence of several papain and chymopapain homologues in these species. Ion-exchange chromatography and gel filtration procedures were applied on latex of V. x heilbornii in order to characterize its cysteine proteinases at the protein level. Five major protein fractions (VXH-I-VXH-V) revealing very high amidase activities (between 7.5 and 23.3 nkat x mg protein(-1)) were isolated. After further purification, three of them were N-terminally sequenced. The observed microheterogeneity in the N-terminal and cDNA sequences reveals the presence of several distinct cysteine proteinase isoforms in the latex of Vasconcellea spp.     

Extracellular proteinases from the phytopathogenic fungus Fusarium culmorum

The growth of Fusarium culmorum fungus on a medium containing thermostable proteins from potato tubers was accompanied by the production of proteinases, exhibiting activity over a broad pH range (from 6.0-10.0). When studied by SDS-PAGE in the presence of beta-mercaptoethanol, extracellular proteinases were represented by at least five species with a molecular weight of 30-60 kDa. Inhibitor analysis and studies of enzyme activities with synthetic substrates demonstrated that the culture liquid of Fusarium culmorum contained serine proteinases of various classes. The amount of subtilisin-like proteinases was the highest. A near-complete inhibition of the enzymes was caused by proteinaceous proteinase inhibitors from potato tubers. These data suggest that proteinases of the phytopathogen Fusarium culmorum serve as a metabolic target for natural inhibitors of potato proteinases.     

Aspartic proteinases in Antarctic fish

The present review surveys several recent studies of the aspartic proteinases from Antarctic Notothenioidei, a dominating fish group that has developed a number of adjustments at the molecular level to maintain metabolic function at low temperatures. Given the unique peculiarities of the Antarctic environment, studying the features of Antarctic aspartic proteinases could provide new insights into the role of these proteins in fish physiology. We describe here: (1) the biochemical properties of a cathepsin D purified from the liver of the hemoglobinless icefish Chionodraco hamatus; (2) the biochemical characterization of Trematomus bernacchii pepsins variants A1 and A2 obtained by heterologous expression in bacteria; and (3) the identification of two closely related, novel aspartic proteinases from the liver of the two Antarctic fish species mentioned above. Overall, the results show that Notothenioidei aspartic proteinases display a number of characteristics that are remarkably different from those of mammalian aspartic proteinases, including high turnover number or high catalytic efficiency. We have named the newly identified aspartic proteinases "Nothepsins" and classified them relative to aspartic proteinases from other species.     

Extracellular proteinases from the phytopathogenic fungus Fusarium culmorum

The growth of Fusarium culmorum fungus on a medium containing thermostable proteins from potato tubers was accompanied by the production of proteinases, exhibiting activity over a broad pH range (from 6.0–10.0). When studied by SDS-PAGE in the presence of β-mercaptoethanol, extracellular proteinases were represented by at least five species with a molecular weight of 30–60 kDa. Inhibitor analysis and studies of enzyme activities with synthetic substrates demonstrated that the culture liquid of Fusarium culmorum contained serine proteinases of various classes. The amount of subtilisin-like proteinases was the highest. A near-complete inhibition of the enzymes was caused by proteinaceous proteinase inhibitors from potato tubers. These data suggest that proteinases of the phytopathogen Fusarium culmorum serve as a metabolic target for natural inhibitors of potato proteinases.     

Effect of pH on the proteinase activities in the intestine mucosa,chyme, and enteral microbiota in the piscivorous fish differing in their ecological traits

The effect of pH on the activity of proteinases of intestine mucosa, chyme, and enteral microbiota was studied in three piscivorous fish species of the Rybinsk Reservoir differing in their ecological traits: pike Esox lucius, burbot Lota lota, and zander Sander lucioperca. In all fish species, studied pH of ten is optimal for the functioning of proteinases of intestine mucosa; in chyme proteinases, optimal pH values vary from six to ten. Optimal pH for functioning of proteinases of enteral microbiota is seven for zander and pike; pH of nine, for burbot. High activity of chyme and microbiota proteinases within the diapason of pH values of six to nine is a characteristic of burbot. Relative activity of proteinases of intestine mucosa in all fish species is not more than 15% of the maximal activity; that of chyme and enteral microbiota is lower than 40% in zander, close to 50% in burbot, and 80 and 50%, respectively, in pike. It is suggested that diversity of the patterns of pH-dependence of enteral microbiota proteinases relates to the specificity of microbiota in various ecological zones of the reservoir (littoral, sublittoral, and bathyal).     

Characterization of midgut proteinase activities of white grubs: Lepidiota noxia,Lepidiota negatoria,and Antitrogus consanguineus (scarabaeidae,melolonthini)

The proteinases in the midguts of three scarab white grub species, Lepidiota noxia, L. negatoria, and Antitrogus consanguineus, were investigated to classify the proteinases present and to determine the most effective proteinase inhibitor for potential use as an insect control agent. pH activity profiles indicated the presence of serine proteinases and the absence of cysteine proteinases. This was confirmed by the lack of inhibition by specific cysteine proteinase inhibitors. Trypsin, chymotrypsin, elastase, and leucine aminopeptidase activities were detected by using specific synthetic substrates. A screen of 32 proteinase inhibitors produced 9 inhibitors of trypsin, chymotrypsin, and elastase which reduced proteolytic activity by greater than 75%. © 1995 Wiley-Liss, Inc.     

Comparison of secretory acid proteinases from Candida tropicalis, C. parapsilosis and C. albicans.

Acid proteinases secreted by Candida tropicalis and C. parapsilosis were newly isolated. Their physico-chemical and enzymatic properties of molecular weight, pH stability, isoelectric points, specific activity, and N-terminal amino acid sequences were determined and compared with those of a C. albicans acid proteinase. The two acid proteinases secreted by C. parapsilosis were found to be new enzymes in their molecular weights. The acid proteinases from C. tropicalis and C. parapsilosis showed lower activity at neutral pH, less resistance to neutral and alkaline pH than that from C. albicans, and a half or a third of the specific activity of the C. albicans enzyme. These differences seemed to be associated with the difference of pathogenesis between Candida species. Of the 31 N-terminal amino acids, the enzymes of these three Candida species revealed 12 homologous amino acids.     

Overexpression of cysteine proteinase 2 in Entamoeba histolytica or Entamoeba dispar increases amoeba-induced monolayer destruction in vitro but does not augment amoebic liver abscess formation in gerbils

To study the role of cysteine proteinases in the pathogenicity of Entamoeba histolytica , we have attempted to overexpress the three main cysteine proteinases (EhCP1, EhCP2, EhCP5) of this parasite in trophozoites of E. histolytica as well as in non-pathogenic Entamoeba dispar by episomal transfection. Although each of the corresponding coding sequences were cloned in identical expression plasmids, we were unable to overexpress EhCP1 and EhCP5, respectively, but could substantially induce expression of EhCP2 in both amoeba species by sevenfold, leading to a threefold increase in total cysteine proteinase activity. Overexpression of EhCP2 did not influence expression of other cysteine proteinases and could be attributed to an increase of a single 35 kDa activity band in substrate gel electrophoresis. In contrast to previous findings, which indicated that amoeba cysteine proteinases are involved in erythrophagocytosis and liver abscess formation, cells overexpressing EhCP2 showed no difference in erythrophagocytosis or liver abscess formation compared with respective controls. However, overexpression of EhCP2 in both amoeba species resulted in a marked increase of in vitro monolayer destruction.     

The basic difference in catalyses by serine and cysteine proteinases resides in charge stabilization in the transition state

Besides the mechanistic similarities, in particular acylenzyme formation, kinetic investigations and X-ray diffraction studies have revealed some differences between the mechanisms of serine and cysteine proteinases: general base-catalysis in acylation, catalytic contribution by oxyanion binding, and a negatively charged catalytic triad in serine proteinases, but not in cysteine proteinases. In this paper we point out that all these differences are related and connected with the mode of stabilization of the zwitterionic species developing in the transition state of the reactions. In the case of serine proteinases this charge separation requires facilitation by the oxyanion binding and the negative charge of the catalytic triad. On the other hand cysteine proteinases do not require such contributions as they are capable of stabilizing the ion-pair even in the ground state of the reaction. Therefore, cysteine proteinases, in contrast to serine proteinases, may be regarded as "activated" enzymes.     

五种黄精属植物的蛋白水解酶谱研究

用蛋白水解酶复性电泳方法 (G- PAGE)分析了 5种黄精属植物根状茎和叶的蛋白水解酶的种类和活性。结果表明 :(1)它们的根状茎均含有 85k D和 55k D的蛋白水解酶 ;叶均含有 82 k D的蛋白水解酶 ;(2 )根状茎和叶的蛋白水解酶种类和活性有很大差异 ,叶的蛋白水解酶活性为根状茎的 10倍 ,它们的活性均受 p H影响 ,其最适 p H为 7;(3)每种植物都含有自己特有蛋白水解酶 ;(4 )蛋白水解酶在植物鉴定中有参考价值     

Comparative biochemistry of acid proteinase from animal origins

Acid proteinases from 17 tissues of 12 animal species were compared with respect to molecular weight, inhibition by pepstatin and activation by tripolyphosphate. Gel filtration of acid proteinases from protochordates and vertebrates showed a common elution profile and three peaks with mol. wts of -20,000, -45,000 and above 150,000 were detected with acid-denatured hemoglobin as substrate at pH 3.6. The main component of vertebrate acid proteinases was identified as cathepsin D. In the invertebrate acid proteinases, the elution profiles through gel filtration were characteristic to the tissues examined, and were not so distinct as those of vertebrates. Through a biochemical survey, the animal acid proteinase was discussed from a comparative point of view.     

Proteinase activity in rumen ciliate protozoa

Azocasein-degrading proteinase activity was detected in all rumen ciliate protozoa that were examined from four entodiniomorphid and two holotrich genera. All of the activities were optimal in the range pH 4.0-5.0 and were inhibited by cysteine proteinase inhibitors, notably leupeptin. The inhibition profiles and extent of inhibition observed with the different groups of inhibitors were organism-specific. Gelatin-SDS-polyacrylamide gel electrophoresis of protozoal lysates revealed multiple forms of the proteinases in the species examined. The number of enzymes detected, their molecular masses, the level of activity and inhibitor susceptibility was genus-dependent. The proteinase profiles of the two holotrich species differed and inter-species differences were also apparent among species of the genus Entodinium. The characteristics and molecular size distribution of rumen bacterial proteinases were different to the protozoal proteinases. Low levels of proteinase activity, of apparently bacterial origin, were detected by gelatin-SDS-PAGE analysis of cell-free rumen liquor.     

Shewasin A, an active pepsin homolog from the bacterium Shewanella amazonensis

The view has been widely held that pepsin-like aspartic proteinases are found only in eukaryotes, and not in bacteria. However, a recent bioinformatics search [Rawlings ND & Bateman A (2009) BMC Genomics10, 437] revealed that, in seven of ～ 1000 completely sequenced bacterial genomes, genes were present encoding polypeptides that displayed the requisite hallmark sequence motifs of pepsin-like aspartic proteinases. The implications of this theoretical observation prompted us to generate biochemical data to validate this finding experimentally. The aspartic proteinase gene from one of the seven identified bacterial species, Shewanella amazonensis, was expressed in Escherichia coli. The recombinant protein, termed shewasin A, was produced in soluble form, purified to homogeneity, and shown to display properties remarkably similar to those of pepsin-like aspartic proteinases. Shewasin A was maximally active at acidic pH values, cleaving a substrate that has been widely used for assessment of the proteolytic activity of other aspartic proteinases, and displayed a clear preference for cleaving peptide bonds between hydrophobic residues in the P1*P1' positions of the substrate. It was completely inhibited by the general inhibitor of aspartic proteinases, pepstatin, and mutation of one of the catalytic Asp residues (in the Asp-Thr-Gly motif of the N-terminal domain) resulted in complete loss of enzymatic activity. It can thus be concluded unequivocally that this Shewanella gene encodes an active pepsin-like aspartic proteinase. It is now beyond doubt that pepsin-like aspartic proteinases are not confined to eukaryotes, but are encoded within some species of bacteria. The distinctions between the bacterial and eukaryotic polypeptides are discussed and their evolutionary relationships are outlined.