Reaction of the main proteolytic fraction of Schistosoma mansoni cercarial enzymes with synthetic substrates and inhibitors of proteolytic enzymes

The purified proteolytic fraction of Schistosoma mansoni cercarial enzymes (PCF) was inhibited by Diisopropylphosphofluoridate (DFP). Its esterolytic activity was not affected by either of two specific active center reagents of proteolytic enzymes: (1) 1-chloro-3-tosylamido-7-ammo-2-heptanone (trypsin) and (2) l-1-tosylamido-2-phenylethyl chloromethyl ketone (chymotrypsin). Furthermore, PCF did not destroy the biological activity of bradykinin on the isolated guinea pig ileum, nor did it release bradykinin from purified dog plasma bradykininogen.     

Demonstration of protease activity for epidermal growth factor-binding protein

The epidermal growth factor can be isolated from the male mouse submaxillary gland as part of a high molecular weight complex. The complex is composed of two molecules of epidermal growth factor and two molecules of epidermal growth-factor binding protein (J.M. Taylor, W.M. Mitchell, and S. Cohen, 1974, J. Biol. Chem.249, 3198–3203). The proteolytic activity of epidermal growth-factor binding protein was demonstrated by its self-proteolysis in moderate (3–7 m) concentrations of urea, and, its inhibition by formation of a complex with pancreatic trypsin inhibitor. This complex was characterized by its pI and by its ability to yield pancreatic trypsin inhibitor and epidermal growth factor-binding protein in sodium dodecyl sulfate-urea gel electrophoresis. The association equilibrium constant was determined to be 3.6 × 10⁷m^?1 by inhibition studies of the esteropeptidase. These results, which indicate that epidermal growth factor-binding protein is capable of autodigestion and of forming a stable complex with a macromolecular inhibitor of trypsin, lend strong support to the hypothesis that epidermal growth factor-binding protein is capable of cleaving a larger precursor by its proteolytic action.     

A trypsin and chymotrypsin inhibitor from seeds of Bauhenia purpurea

A trypsin and chymotrypsin inhibitor was partially purified from Bauhenia purpurea seeds and separated from a second inhibitor by Ecteola cellulose chromatography. The factor inhibited bovine trypsin and chymotrypsin as well as pronase trypsin and elastase. It formed a complex with trypsin and with chymotrypsin, but a ternary complex could not be detected. Differences were detected in the effect on trypsin and on chymotrypsin, although one enzyme interfered with the inhibition of the other. The results obtained point to two active centers on the inhibitor for the trypsin and chymotrypsin inhibition such that the one cannot complex with the inhibitor after this inhibitor had complexed with the other.     

Characterization of proteolytic enzymes of the midgut and excreta of the biting fly Stomoxys calcitrans

Proteolytic enzymes were characterized in the midgut and the excreta of the stable fly Stomoxys calcitrans (L) with proteins, synthetic substrates, and inhibitors. Inhibition studies suggested trypsinlike activity in sugar-fed fly midguts, whereas excreta and blood-fed fly guts exhibited other proteases. Trypsinlike activity in midguts removed 20 and 30 h after a blood meal increased from 20% to 50% of the total proteolytic enzymes present. Trypsinlike activity was inhibited with human sera, trypsin-specific inhibitors, and a protein isolated from the stable fly thorax. When human albumin and globulin fractions were incubated with trypsinlike enzymes isolated from the midgut and excreta, the albumin fraction was less inhibitory than the globulin fractions and was readily hydrolyzed by the proteolytic enzymes. These results may indicate that the proteolytic enzymes produce an abortive complex with the globulin fractions of the sera. Such a complex may explain the temporary inhibition of proteolysis by the blood meal. Soybean trypsin inhibitor fed to stable flies caused 50% inhibition in proteolytic activity in the midguts of sugar-fed stable flies and 25% inhibition in the midguts of blood-fed stable flies. Complete inhibition of proteolytic enzyme activity was achieved only in vitro. pH profiles of proteolytic enzyme activity isolated from the excreta of blood-fed stable flies indicated that several proteolytic enzymes were excreted.     

Proteolytic Activation of the Human Epithelial Sodium Channel by Trypsin IV and Trypsin I Involves Distinct Cleavage Sites

Proteolytic activation is a unique feature of the epithelial sodium channel (ENaC). However, the underlying molecular mechanisms and the physiologically relevant proteases remain to be identified. The serine protease trypsin I can activate ENaC in vitro but is unlikely to be the physiologically relevant activating protease in ENaC-expressing tissues in vivo. Herein, we investigated whether human trypsin IV, a form of trypsin that is co-expressed in several extrapancreatic epithelial cells with ENaC, can activate human ENaC. In Xenopus laevis oocytes, we monitored proteolytic activation of ENaC currents and the appearance of γENaC cleavage products at the cell surface. We demonstrated that trypsin IV and trypsin I can stimulate ENaC heterologously expressed in oocytes. ENaC cleavage and activation by trypsin IV but not by trypsin I required a critical cleavage site (Lys-189) in the extracellular domain of the γ-subunit. In contrast, channel activation by trypsin I was prevented by mutating three putative cleavage sites (Lys-168, Lys-170, and Arg-172) in addition to mutating previously described prostasin (RKRK¹⁷⁸), plasmin (Lys-189), and neutrophil elastase (Val-182 and Val-193) sites. Moreover, we found that trypsin IV is expressed in human renal epithelial cells and can increase ENaC-mediated sodium transport in cultured human airway epithelial cells. Thus, trypsin IV may regulate ENaC function in epithelial tissues. Our results show, for the first time, that trypsin IV can stimulate ENaC and that trypsin IV and trypsin I activate ENaC by cleavage at distinct sites. The presence of distinct cleavage sites may be important for ENaC regulation by tissue-specific proteases.     

Ligula intestinalis (Cestoda: Pseudophyllidae): Studies on the properties of proteolytic and protease inhibitor activities of plerocercoid larvae

The somatic extract of L. intestinalis plerocercoids reveals hydrolytic activity against N-Benzoyl-l-tyrosine ethyl ester (BTEE) and Azocoll, and inactivates the esterolysis by mammalian trypsin and chymotripsin. The proteolytic enzyme activity and the inhibitory effect were completely separated by Sephadex G-100 column chromatography. Gel chromatography of the somatic extract revealed two peaks of proteolytic activity : one is bound to macromolecular substances, the other appears to be in free form and has a molecular weight of approx 60,000–65,000. The proteolytic activity showed the following characteristics : Tris-HCl buffer provided the highest activity against BTEE, the pH optimum was 7·4–7·8; the enzyme was activated by 10^?5m-Ca²⁺, Mg²⁺ or Mn²⁺, it was inhibited by 10^?5m-Cu²⁺, but not by 10^?5m-Zn²⁺. 0.001% soybean trypsin inhibitor, 2 × 10^?3m-EDTA, 1 mm-tosyl-l-phenylalanyl chloromethane, 1000 KIU/ml Trasylol did not inhibit the proteolytic activity, but it was inhibited by 1 mm-phenylmethyl-sulphonyl fluoride. The enzyme activity completely ceased upon 5 % TCA treatment or incubation at 56°C for 30 min. The trypsin and chyrnotrypsin inhibitor activities were eluted from the Sephadex G-100 column in a single peak with an estimated molecular weight of 6700–7200. The inhibitory effect was not sensitive to pH changes, and treatment by 5% TCA or incubation at 80°C for 15 min was ineffective. The proteolytic activity of plerocercoid extract was not effected ‘in vitro’ by the inhibitors isolated from this parasite.     

Proglobulin processing enzyme in vacuoles isolated from developing pumpkin cotyledons

The enzymic conversion of proglobulin to globulin catalyzed by the extracts of vacuoles isolated from developing pumpkin (Cucurbita sp. cv Kurokawa Amakuri Nankin) cotyledons was investigated. The endoplasmic reticulum fraction isolated from the developing cotyledons pulselabeled with [³⁵S]methionine was shown to contain mainly the radiolabeled proglobulin, which was used as a substrate for assaying the proteolytic processing in vitro. The vacuolar extracts catalyzed the proteolytic processing of the proglobulin molecule to produce globulin containing two kinds of polypeptide chains, γ and δ. The pH optimum for the vacuole-mediated conversion was at pH 5.0. The proteolytic processing of proglobulin by the vacuolar extracts was inhibited in the presence of various thiol reagents, e.g. p-chloromercuribenzoate, N-ethylmaleimide, iodoacetic acid, Hg²⁺, and Cu²⁺, but not phenylmethylsulfonyl fluoride, EDTA, o-phenanthroline, leupeptin, antipain, pepstatin, chymostatin, or pumpkin trypsin inhibitor, and was activated in the presence of dithiothreitol and cysteine, indicating that the processing enzyme is a thiol protease. The suborganellar fractionation of the vacuoles showed that the processing activity was localized in the matrix fraction, but not in the membrane or crystalloid fractions. During the seed development, the enzyme was shown to increase, exhibiting the maximal activity at the late developmental stage. The matrix fraction of the protein bodies isolated from the dry castor bean (Ricinus communis) exhibited the processing activity toward the pumpkin proglobulin molecules in the same manner as that by the matrix fraction of pumpkin vacuoles.     

Digestive enzymes associated with the glycocalyx,microvillar membranes and secretory vesicles from midgut cells of Tenebrio molitor larvae

Acetylglucosaminidase, amylase, cellobiase and maltase are more active in anterior midgut cells, whereas aminopeptidase, carboxypeptidase and trypsin are more active in posterior midgut cells of Tenebrio molitor larvae. Differential centrifugation of midgut homogenates prepared in saline (or mannitol) isotonic buffered solutions revealed that aminopeptidase is associated with membranes, which occur in subcellular fractions displaying many microvilli. Carboxypeptidase, trypsin and the carbohydrases are mostly found in the soluble fraction, although significant amounts sediment together with cell vesicles. Data on differential calcium precipitation of midgut homogenates and on partial ultrasound disruption of midgut tissue suggest that aminopeptidase is a microvillar enzyme and that the digestive enzymes recovered in the soluble fraction of cells are loosely bound to the cell glycocalyx. About 5% of the non-absorbable dye amaranth fed to T. molitor larvae remains in the midgut tissue after rinsing. Most dye was recovered in the soluble fraction of midgut cells. This provided further support for the hypothesis that the digestive enzymes found in the soluble fraction are actually extracellular and that the true intracellular enzymes are those associated with cell vesicles. The results suggest that the carbohydrases are secreted by exocytosis from the anterior midgut and carboxypeptidase and trypsin from the posterior midgut.     

Differential interactions of rabbit plasma alpha-1-antiproteinases S and F with porcine trypsin

Two major forms of rabbit plasma alpha-1-antiproteinase, S and F, were separated by affinity chromatography on Red Sepharose, and their modes of interaction with porcine trypsin were studied. The S form interacted with trypsin much more slowly than the F form, and the resulting complex partially retained the amidolytic and proteolytic activities towards benzoyl-L-arginine p-nitroanilide and remazol brilliant blue hide powder, respectively. This S form-trypsin complex also prevented the inactivation of bound trypsin by soybean trypsin inhibitor. In marked contrast, an equimolar complex of trypsin and the F form retained neither amidolytic nor proteolytic activity. These results suggest that the F form blocks the active site of trypsin while the S form does not bind directly to the active site, thereby preserving the catalytic potential of trypsin. No similar interaction was observed, however, between the S form and either bovine chymotrypsin or porcine pancreatic elastase. Both the S and F forms inactivated these proteinases in a stoichiometric manner with differing inhibitor/proteinase binding ratios. The S form showed about twofold greater capacity to inhibit elastase than the F form, whereas the reverse was the case for chymotrypsin.     

The secretory nature of the excretory gland cells of Strephanurus dentatus. 3. Proteinase inhibitors

A proteinase inhibitor(s) was found in extracts of the excretory gland cells, intestines, esophagi, reproductive organs, and body walls from Stephanurus dentatus adults. The specific activity of the inhibitor(s) in the excretory gland cell extract was 45–175 times greater than in the other tissues. It is heat stable at pH 5.0 and inhibits the esterolytic activity of trypsin and chymotrypsin using p-toluenesulfonyl-l-arginine methyl ester hydrochloride (TAME) and benzoyl-l-tyrosine ethyl ester (BTEE) as the substrates, respectively, and also the proteolytic activity of both chymotrypsin and trypsin using casein as the substrate. S. dentatus adults maintained in NCTC 109 medium, secreted a trypsin inhibitor.     

Isolation and characterization of trypsin inhibitors from cowpea (Vigna unguiculata)

The trypsin inhibitor fraction from cowpea (Vigna unguiculata) has been purified and characterized. Although the total trypsin inhibitor as purified by affinity chromatography on immobilised trypsin was shown to be heterogeneous by gel electrophoresis and isoelectric focusing as well as by function, it was relatively homogeneous in MW (ca 17 000) on gel filtration. The total trypsin inhibitor was divided into inhibitors active against trypsin only and active against trypsin and chymotrypsin by affinity chromatography on immobilised chymotrypsin. The ‘trypsin-only’ inhibitor was the major component of the total trypsin inhibitor. It was shown by isoelectric focusing and gel electrophoresis to contain several isoinhibitors. Determination of the combining weight of this inhibitor and investigation of the complexes formed with trypsin by gel filtration indicated the presence of two protease binding sites per inhibitor molecule. The chymotrypsin/trypsin inhibitor was also shown to be composed of several isoinhibitors. On the basis of gel electrophoresis and gel filtration in dissociating and non-dissociating media both inhibitors were considered to be dimeric molecules with the subunits linked by disulphide bonds; this implies that the ‘trypsin-only’ inhibitor has one binding site per subunit.     

cDNA cloning and phylogenetic analysis of pancreatic serine proteases from Japanese flounder,Paralichthys olivaceus

To better understand the digestive physiology and phylogeny of the pancreatic serine proteases of teleosts, we cloned trypsin, chymotrypsin and elastase from flounder (Paralichthys olivaceus). Fifty phage plaques randomly chosen from a flounder pancreatic cDNA library were found to contain three species of trypsin, two species of chymotrypsin and four species of elastase. cDNAs of two species of carboxypeptidase A, one carboxypeptidase B and lipase were also obtained. In total, 23 out of 24 digestive enzyme cDNAs were those of proteolytic enzymes. Such a high ratio of proteolytic enzyme cDNA in the pancreas may reflect the carnivorous feeding habits of flounder. A phylogenetic comparison of the peptide sequences of flounder enzymes with those of other teleosts and mammals suggested that duplication of trypsin, chymotrypsin and elastase occurred before the divergence of the ray finned fish. It is also hypothesized that functional descendants of both duplicated genes of elastase exist in the teleosts and mammals, whereas only one of the genes of trypsin and chymotrypsin gave rise to the functional descendants in the teleosts but not in the mammals.     

The interaction of a trypsin-dependent neutral protease and its inhibitor found in tumour cells. Analysis of complex kinetic data involved in a thiol-disulphide exchange mechanism

Ehrlich ascites tumour cells contain a granule-derived zymogen which on trypsin activation yields a collegenolytic neutral protease. The preparation of the granule fraction by subcellular fractionation procedure results in the preparation of a second fraction referred to as the post-granule supernatant fraction. The post-granule supernatant fraction contains a latent form of the granule-derived neutral protease and an excess of cytoplasmic inhibitor for this enzyme. The inhibitor of neutral protease is also capable of inhibiting trypsin and in each case the chemical mechanism of enzyme.inhibitor complex formation has been shown to be a reversible thiol-disulphide exchange. The post-granule supernatant fraction exhibited complex kinetic data when the interactions between the inhibitor, the latent enzymes and trypsin were examined simultaneously by incremental analysis. The data were interpreted and quantitatively analysed by computer analysis. It was demonstrated that the conventional types of analysis could not have provided meaningful interpretations of the experimental data provided by these complex-interacting systems.     

Proteolytic processing of Bacillus thuringiensis toxin Cry1Ab in rice brown planthopper, Nilaparvata lugens (Stål)

To understand the low toxicity of Cry toxins in planthoppers, proteolytic activation of Cry1Ab in Nilaparvata lugens was studied. The proteolytic processing of Cry1Ab protoxin by N. lugens midgut proteases was similar to that by trypsin activated Cry1Ab. The Cry1Ab processed with N. lugens midgut proteases was highly insecticidal against Plutella xylostella. However, Cry1Ab activated either by trypsin or the gut proteases of the brown planthopper showed low toxicity in N. lugens. Binding analysis showed that activated Cry1Ab bound to brush border membrane vesicles (BBMV) from N. lugens at a significantly lower level than to BBMV from P. xylostella.     

Recombinant cold-adapted trypsin I from Atlantic cod-expression, purification, and identification

Atlantic cod trypsin I is a cold-adapted proteolytic enzyme exhibiting approximately 20 times higher catalytic efficiency (kcat/KM) than its mesophilic bovine counterpart for the simple amide substrate BAPNA. In general, cold-adapted proteolytic enzymes are sensitive to autolytic degradation, thermal inactivation as well as molecular aggregation, even at temperatures as low as 18-25 degrees C which may explain the problems observed with their expression, activation, and purification. Prior to the data presented here, there have been no reports in the literature on the expression of psychrophilic or cold-adapted proteolytic enzymes from fish. Nevertheless, numerous cold-adapted proteolytic microbial enzymes have been successfully expressed in bacteria and yeast. This report describes successful expression, activation, and purification of the recombinant cod trypsin I in the His-Patch ThioFusion Escherichia coli expression system. The E. coli pThioHis expression vector used in the study enabled the formation of a fusion protein between a highly soluble fraction of HP-thioredoxin contained in the vector and the N-terminal end of the precursor form of cod trypsin I. The HP-thioredoxin part of the fusion protein binds to a metal-chelating ProBond column, which facilitated its purification. The cod trypsin I part of the purified fusion protein was released by proteolytic cleavage, resulting in concomitant activation of the recombinant enzyme. The recombinant cod trypsin I was purified to homogeneity on a trypsin-specific benzamidine affinity column. The identity of the recombinant enzyme was demonstrated by electrophoresis and chromatography.     

Trypsin promotes efficient influenza vaccine production in MDCK cells by interfering with the antiviral host response

Trypsin is commonly used in Madin–Darby canine kidney (MDCK) cell culture-based influenza vaccine production to facilitate virus infection by proteolytic activation of viral haemagglutinin, which enables multi-cycle replication. In this study, we were able to demonstrate that trypsin also interferes with pathogen defence mechanisms of host cells. In particular, a trypsin concentration of 5 BAEE U/mL (4.5 μg/mL porcine trypsin) used in vaccine manufacturing strongly inhibited interferon (IFN) signalling by proteolytic degradation of secreted IFN. Consequently, absence of trypsin during infection resulted in a considerably stronger induction of IFN signalling and apoptosis, which significantly reduced virus yields. Under this condition, multi-cycle virus replication in MDCK cells was not prevented but clearly delayed. Therefore, incomplete infection can be ruled out as the reason for the lower virus titres. However, suppression of IFN signalling by overexpression of viral IFN antagonists (influenza virus PR8-NS1, rabies virus phosphoprotein) partially rescued virus titres in the absence of trypsin. In addition, virus yields could be almost restored by using the influenza strain A/WSN/33 in combination with fetal calf serum (FCS). For this strain, FCS enabled trypsin-independent fast propagation of virus infection, probably outrunning cellular defence mechanisms and apoptosis induction in the absence of trypsin. Overall, addition of trypsin provided optimal conditions for high yield vaccine production in MDCK cells by two means. On the one hand, proteolytic degradation of IFN keeps cellular defence at a low level. On the other hand, enhanced virus spreading enables viruses to replicate before the cellular response becomes fully activated.     

Production and properties of recombinant glutenin-hydrolyzing proteinases from Eurygaster integriceps Put.

cDNAs coding for a mature form of glutenin-hydrolyzing trypsin-like proteinase (referred to as glutenin-hydrolyzing proteinase 3 or GHP3) from the insect pest Eurygaster integriceps Put. and a zymogen of this proteinase containing a signal peptide required for protein secretion were cloned into vectors pPIC9 and pPIC3.5, respectively. The constructs were used for protein expression in cells of the methylotrophic yeast Pichia pastoris. The recombinant protein corresponding to the mature form of the proteinase was secreted into the culture medium and possessed proteolytic activity, while the zymogen acquired activity after trypsin treatment. Both recombinant enzymes hydrolyzed high-molecular weight glutenin subunits from wheat of the variety Ege-88 and a range of other soft and durum wheat varieties. Chymotrypsin inhibitor I from potatoes and related inhibitors from seeds of plants of the subclass Asteridae, the Kunitz-type trypsin inhibitor from soybeans, and bovine aprotinin had a weak inhibitory effect on the recombinant proteinases, while the Bowman-Birk trypsin and chymotrypsin inhibitor from soybeans did not interact with these enzymes.     

A removable spacer peptide in an α-factor-leader/insulin precursor fusion protein improves processing and concomitant yield of the insulin precursor in Saccharomyces cerevisiae

An α-factor leader/insulin precursor fusion protein was produced in Saccharomyces cerevisiae and metabolically labeled in order to analyse the efficiency of maturation and secretion. A substantial fraction of the secreted material was found in a hyperglycosylated unprocessed form, indicating incomplete Kex2p endopeptidase maturation. Introduction of a spacer peptide (EAEAEAK) after the dibasic Kex2p site, creating a N-terminal extension of the insulin precursor, greatly increased the Kex2p catalytic efficiency and the fermentation yield of insulin precursor. The N-terminal extension features a Lys to allow subsequent proteolytic removal by trypsin or the Achromobacter lyticus Lys-specific protease. Dipeptidyl aminopeptidase A (DPAPA) activity removing Glu-Ala dipeptides from the extension was inhibited by adding a Glu N-terminally to the extension. Unexpectedly, this modified N-terminal extension (EEAEAEAK) was partially cleaved after the Lys during fermentation. This monobasic proteolytic activity was demonstrated to be associated with Yap3p. Yap3p cleavage could be prevented by insertion of a Pro before the Lys (EEAEAEAPK)     

Related antipodes: a comparative study on digestive endopeptidases from Northern krill and Antarctic krill (Euphausiacea)

The Antarctic krill, Euphausia superba, and the Northern krill, Meganyctiphanes norvegica, are closely related species but occupy significantly different trophic and climatic environments. E. superba holds a key position as a phytoplankton grazer in the Southern Ocean. The omnivorous M. norvegica is an important member of plankton communities in the Northeast Atlantic. Both species expressed high proteolytic activities which were dominated by serine proteinases. In the stomachs of Antarctic krill, activities of total proteinase, trypsin, and chymotrypsin were significantly higher than in Northern krill. In the midgut glands, however, total proteinase and trypsin activities were similar in both species, but chymotrypsin activity was significantly higher in Antarctic krill. Moreover, Antarctic krill expressed four trypsin isoforms while only one isoform appeared in Northern krill. Chymotrypsin was present in either species as one single isoform. Antarctic krill adapted to the low and patchy distribution of food by elevated enzyme activities and the expression of trypsin isoforms with slightly different catalytic properties. Presumably, these enzymes facilitate in concerted action the efficient utilization of proteins from phytoplankton, the major food. Northern krill, in contrast, seems not to be equipped to face food limitation. It expresses a “simple” or “basic” set of digestive enzymes for utilizing abundant and easily digestible prey.     

Oligomerization and the sensitivity of phosphoenolpyruvate carboxylase to inactivation by proteinases

Phosphenolpyruvate (PEP) carboxylase from leaves of Crassula argentea displays varying levels of sensitivity to inactivation by various proteolytic enzymes. In general, the native enzyme is sensitive to proteinases known to attack at the carbonyl end of lysine or arginine (trypsin, papain, or bromelain). The ineffective proteolytic enzymes are those which have low specificity or which attack at the N-terminal end of hydrophobic amino acids, or which cannot attack lysine. The lack of an effect of endoproteinase arginine C, which is specific for arginine, probably indicates that lysine is the critical residue. When the native enzyme, which is comprised of an equilibrium of dimers with tetramers in approximately equal quantities, is treated by preincubation with 5 millimolar PEP, the enzyme becomes much more resistant to proteolytic inactivation. When the preincubation is with 5 millimolar malate rather than buffer alone, the effect is to slightly increase (ca. 15%) the sensitivity of the enzyme to inactivation by trypsin as measured by estimates of the pseudo-first order rate constant for inactivation. PEP carboxylase from corn leaves appears to be relatively susceptible to inactivation by trypsin, but is unaffected by preincubation with malate or PEP. The sensitivity of this C₄ enzyme to inhibition by malate is also unaffected by preincubation with these ligands.