Native and Acid-denatured L-Lactate Dehydrogenase Are Different in the Lysosomal Proteinases Involving in Their Overall Degradation

When native and acid-denatured lactate dehydrogenase (LDH) were incubated with total lysosomal enzymes in vitro, amino acids from their degradation were produced at various acidic pH. The pH profile in the overall degradation of native LDH was markedly different from that of acid-denatured LDH. Disappearance of the 35-kDa subunit of native LDH was markedly suppressed by a low level of cystatin α as well as by a general cysteine proteinase inhibitor, N-(L-3-trans-carboxyoxirane-2-carbonyl)-L-leucine-3-methylbutylamide (E-64-c). On the other hand, the degradation of acid-denatured LDH was only slightly suppressed by these inhibitors. It was concluded that at least a part of the proteinases involved in the overall degradation of native LDH is different from the proteinases involved in the degradation of acid-denatured form and a role of a cystatin α-sensitive cysteine proteinase is critical in the lysosomal degradation of native LDH, but not in that of acid-denatured form.     

Evidence for the presence of proteolytically active secreted aspartic proteinase 1 of Candida parapsilosis in the cell wall

Pathogenic yeasts of the genus Candida produce secreted aspartic proteinases, which are known to enhance virulence. We focused on Sapp1p proteinase secreted by Candida parapsilosis and studied the final stage of its passage through the cell wall and release into the extracellular environment. We found that Sapp1p displays enzyme activity prior to secretion, and therefore, it is probably fully folded within the upper layer of the cell wall. The positioning of cell surface-associated Sapp1p was detected by cell wall protein labeling using biotinylation agents, extraction of cell wall proteins by β-mercaptoethanol, immunochemical detection, and mass spectrometry analysis. All lysine residues present in the structure of soluble, purified Sapp1p were labeled with biotin. In contrast, the accessibility of individual lysines in cell wall-associated Sapp1p varied with the exception of four lysine residues that were biotinylated in all experiments performed, suggesting that Sapp1p has a preferred orientation in the cell wall. As the molecular weight of this partially labeled Sapp1p did not differ among the experiments, we can assume that the retaining of Sapp1p in the cell wall is not a totally random process and that pathogenic yeasts might use this cell-associated proteinase activity to enhance degradation of appropriate substrates.     

Peculiarities of digestive function of proteinases in invertebrates—Inhabitants of cold seas

Digestive proteinases of various taxa of invertebrates of the boreal seas have been studied: crustaceans Paralithodes camtchaticus, Pandalus borealis; molluscs Chlamys islandicus, Buccinum undatum, Serripes groenlandicus, and echinoderms Strongylocentrotus droebachiensis, Cucumaria frondosa, Asterias rubens, and Crossaster papposus. The presence of two proteolytic activity peaks in the acidic (pH 2.5–3.5) and lower alkaline ranges (pH 7.5–8.5) and a similar proteinase spectrum have been revealed in digestive organs of the studied animals. The proteolytic activity in digestive organs of the Barents Sea invertebrates exceeds significantly that of terrestrial homoiothermal animals, which seems to be an extensive compensation for poor differentiation of the digestive system and for low substrate specificity of the enzymes as well as for cold conditions of the habitat. The principal qualitative difference between vertebrates and invertebrates consists in that the latter have no pepsin activity, but do have the cathepsin activity that is absent in vertebrate digestive organs. Contribution to the acid proteolysis is made by lysosomal cathepsins, rather than by pepsins. Activity in the alkaline and neutral pH ranges is provided by serine proteinases. In digestive cavities of invertebrates, hydrolysis of proteins and mechanical processing of food occur only in the lower alkaline pH range, whereas acid proteolysis has intracellular lysosomal localization.     

Activities of proteinases in invertebrate animals—Potential objects of fish nutrition. Effects of temperature,pH, and heavy metals

Differences in the degree of separate and combined effects of temperature, pH, and heavy metals (zinc, copper) on the trypsin-and chymotrypsin-like proteinase activities have been established in the whole body of some invertebrate animals—potential objects of fish nutrition: pond snail Lymnaeae stagnalis, orb snail Planorbarius purpura, zebra mussel Dreissena polymorpha, oligochaetes Tubifex sp. and Lumbriculus sp. in total, chironomid larvae Chironimus sp. and Ch. riparus, as well as crustacean zooplankton. It has been shown that enzymes of the potential prey at low temperature can compensate the low activity of intestinal proteinases of fish bentho- and planktophages.     

Digestive enzyme compartmentalization and recycling and sites of absorption and secretion along the midgut of Dermestes maculatus (Coleoptera) larvae

Bostrichiformia is the less known major series of Coleoptera regarding digestive physiology. The midgut of Dermestes maculatus has a cylindrical ventriculus with anterior caeca. There is no cell differentiation along the ventriculus, except for the predominance of cells undergoing apocrine secretion in the anterior region. Apocrine secretion affects a larger extension and a greater number of cells in caeca than in ventriculus. Ventricular cells putatively secrete digestive enzymes, whereas caecal cells are supposed to secrete peritrophic gel (PG) glycoproteins. Feeding larvae with dyes showed that caeca are water-absorbing, whereas the posterior ventriculus is water-secreting. Midgut dissection revealed a PG and a peritrophic membrane (PM) covering the contents in anterior and posterior ventriculus, respectively. This was confirmed by in situ chitin detection with FITC-WGA conjugates. Ion-exchange chromatography of midgut homogenates, associated with enzymatic assays with natural and synthetic substrates and specific inhibitors, showed that trypsin and chymotrypsin are the major proteinases, cysteine proteinase is absent, and aspartic proteinase probably is negligible. Amylase and trypsin occur in contents and decrease along the ventriculus; the contrary is true for cell-membrane-bound aminopeptidase. Maltase is cell-membrane-bound and predominates in anterior and middle midgut. Digestive enzyme activities in hindgut are negligible. This, together with dye data, indicates that enzymes are recovered from inside PM by a posterior-anterior flux of fluid outside PM before being excreted. The combined results suggest that protein digestion starts in anterior midgut and ends in the surface of posterior midgut cells. All glycogen digestion takes place in anterior midgut.     

Effect of seed extracts of <Emphasis Type="Italic">Withania somnifera,Croton tiglium</Emphasis> and <Emphasis Type="Italic">Hygrophila auriculata</Emphasis> on behavior and physiology of <Emphasis Type="Italic">Odontotermes obesus</Emphasis> (Isoptera,Termitidae)

In addition to antibiotic properties, medicinal plants are important sources of chemicals with potential application as pesticides. The present study deals with antitermitic potential of seed extracts of Withania somnifera (Indian ginseng), Croton tiglium (jamalgoota) and Hygrophila auriculata (talimkhana). The seed extracts caused changes in tunneling behaviour, number of bacterial colonies in hindgut and activities of enzymes in midgut of Odontotermes obesus. C. tiglium showed the lowest LT₅₀ (12.85 and 2.65 h) among the three seed extracts at concentrations of 50% (half dilution of the extract) and 100% (extract without dilution), respectively. There was no tunneling in soil treated with 100% concentration of seed extracts of W. somnifera and C. tiglium. Numbers of bacterial colonies in the gut of termites from soils treated with 50% and 100% concentrations of the three plants did not differ significantly, but they differed from those in termites from untreated soil. At 50% concentrations of seed extracts of the tested plants, the difference in hindgut enzyme activities was not obvious, however, at 100% concentrations the enzyme activities in the termites from soils treated with seed extracts significantly differed from controls and differences were also recorded between the plants.     

The peptidases of Trypanosoma cruzi: Digestive enzymes, virulence factors, and mediators of autophagy and programmed cell death

Trypanosoma cruzi, the agent of the American Trypanosomiasis, Chagas disease, contains cysteine, serine, threonine, aspartyl and metallo peptidases. The most abundant among these enzymes is cruzipain, a cysteine proteinase expressed as a mixture of isoforms, some of them membrane-bound. The enzyme is an immunodominant antigen in human chronic Chagas disease and seems to be important in the host/parasite relationship. Inhibitors of cruzipain kill the parasite and cure infected mice, thus validating the enzyme as a very promising target for the development of new drugs against the disease. In addition, a 30 kDa cathepsin B-like enzyme, two metacaspases and two autophagins have been described. Serine peptidases described in the parasite include oligopeptidase B, a member of the prolyl oligopeptidase family involved in Ca²⁺-signaling during mammalian cell invasion; a prolyl endopeptidase (Tc80), against which inhibitors are being developed, and a lysosomal serine carboxypeptidase. Metallopeptidases homologous to the gp63 of Leishmania spp. are present, as well as two metallocarboxypeptidases belonging to the M32 family, previously found only in prokaryotes. The proteasome has properties similar to those of other eukaryotes, and its inhibition by lactacystin blocks some differentiation steps in the life cycle of the parasite. This article is part of a Special Issue entitled: Proteolysis 50 years after the discovery of lysosome.     

Differential expression of the putative Kex2 processed and secreted aspartic proteinase gene family of Cryphonectria parasitica

Kex2-silenced strains of Cryphonectria parasitica, the ascomycete causal agent of chestnut blight, show a significant reduction in virulence, reduced sexual and asexual sporulation and reductions in mating and fertility. Due to this and the known involvement of Kex2 in the processing of important proproteins in other systems, we searched the whole C. parasitica genome for putative Kex2 substrates. Out of 1299 open reading frames (ORFs) predicted to be secreted, 222 ORFs were identified as potential Kex2 substrates by this screen. Within the putative substrates we identified cell wall modifying proteins, putative proteinases, lipases, esterases, and oxidoreductases. This in silico screen also uncovered a family of nine secreted aspartic proteinases (SAPs) of C. parasitica. Northern blot analyses of this gene family showed differential expression when exposed to chestnut wood and Cryphonectria hypovirus 1 (CHV1). Due to the reduction in fungal virulence known to be caused upon hypoviral infection of C. parasitica, the differential gene expression observed, and the known involvement of SAPs in virulence in other systems, we conducted deletion analyses of four of these proteinases, representing different expression patterns. Deletion of each of the four SAPs did not affect growth rates, sporulation or virulence, suggesting that none of the considered SAPs is essential for the full development or virulence of C. parasitica under the conditions tested.     

Effect of temperature on proteinase activities in intestinal chyme and mucosa of fish of different ecological groups

Effect of temperature on activities of proteinases in intestinal chyme and mucosa was studied in three fish species (pike-perch, zope, roach) belonging to different ecological groups by their nutrition type. There was revealed a significant difference of dependence of enzyme activities in chyme on temperature in the benthophage, roach (a higher level of relative activity in the range of lower temperatures and a wider zone of temperature optimum) as well as of values of apparent energy of activation of the protein hydrolysis process as compared with that in planktoand ichtyophages, zope and pike-perch, which indicates a significant effect of the enteral microbiota proteinases and of nutrition objects on characteristics of hydrolases functioning in fish intestine.     

Extracellular proteinases of filamentous fungi as potential markers of phytopathogenesis

he presence of proteins in the culture liquid of filamentous fungi under study was found to induce the secretion of proteinases. The inhibitory analysis of the major extracellular proteinases of the saprotrophic fungus Trichoderma harzianum and the phytopathogenic fungus Alternaria alternata showed that they both belong to the group of serine proteinases. The substrate specificity of these proteinases and their sensitivity to inhibitors suggest that the enzyme of T. harzianum is a subtilisin-like proteinase and the enzyme of A. alternata is a trypsin-like proteinase. This difference between the proteinases may reflect the physiological difference between their producers (saprotroph and phytopathogen).