Purification and amino acid sequence of a bitter gourd inhibitor against an acidic amino acid-specific endopeptidase of Streptomyces griseus.

An inhibitor (BGIA) against an acidic amino acid-specific endopeptidase of Streptomyces griseus (Glu S. griseus protease) was isolated from seeds of the bitter gourd Momordica charantia L., and its amino acid sequence was determined. The molecular weight of BGIA based on the amino acid sequence was calculated to be 7419. BGIA competitively inhibited Glu S. griseus protease with an inhibition constant (Ki) of 70 nM, and gel filtration analyses suggested that BGIA forms a 1:1 complex with this protease. However, two other acidic amino acid-specific endopeptidases, protease V8 from Staphylococcus aureus and Bacillus subtilis proteinase (Glu B. subtilis protease), were not inhibited by BGIA. BGIA had no inhibitory activity against chymotrypsin, trypsin, porcine pancreatic elastase, and papain, although subtilisin Carlsberg was strongly inhibited. The amino acid sequence of BGIA shows similarity to potato chymotrypsin inhibitor, barley subtilisin-chymotrypsin inhibitor CI-1 and CI-2, and leech eglin C, especially around the reactive site. Although the residue at the putative reactive site of these inhibitors is leucine or methionine, the corresponding amino acid in BGIA is alanine.     

Characterization and partial purification of the digestive proteases of the black field cricket,Teleogryllus commodus (Walker): Elastase is a major component

The major proteases of the black field cricket, Telleogryllus commodus, digestive system have been identified, partially purified and characterized. Classification of proteases into different classes of endo- and exopeptidases was made on the ability to hydrolyse specific synthetic substrates, pH optima and their interaction with a range of specific chemical and proteinaceous inhibitors. The major activities detected were trypsin, elastase, an uncharacterized proteinase (proteinase Tc), leucine aminopeptidase and carboxypeptidases A and B. Chymotrypsin activity was very low and neither cysteine endopeptidase nor metalloendopepitidase activities were found. Elastase is a newly discovered protease activity for insects.Trypsin, elastase and proteinase Tc have molecular weights of 24,300, 19,500 and 23,600, respectively; show alkaline pH optima and chemical inhibition indicative of serine endopeptidases; and interact most strongly with their characteristic class of proteinaceous inhibitors. Elastase and proteinase Tc are inhibited by a very similar spectrum of specific inhibitors, but the latter lacks activity against all specific synthetic substrates tested. Leucine aminopeptidase and carboxypeptidase A have molecular weights of 94,000 and 39,700, respectively, and show optimum activity at pH 8 and pH 9, respectively.The equilibrium dissociation constants for trypsin, elastase and proteinase Tc with 25 serine proteinase inhibitors were measured. Values spanning a 1000-fold range were obtained in each case.     

Leukocyte chemoattractant peptides from the serpin heparin cofactor II

Heparin cofactor II (HC) is a plasma serine proteinase inhibitor (serpin) that inhibits the coagulant proteinase alpha-thrombin. We have recently demonstrated that proteolysis of HC by catalytic amounts of polymorphonuclear leukocyte proteinases (elastase or cathepsin G) generates leukocyte chemotaxins (Hoffman, M., Pratt, C. W., Brown, R. L., and Church, F. C. (1989) Blood 73, 1682-1685). One of four peptides produced when HC is degraded by neutrophil elastase has chemotactic activity for both monocytes and neutrophils with maximal migration comparable to formyl-Met-Leu-Phe, the "gold standard" bacterially derived chemotaxin. The amino-terminal sequence of this HC peptide is Asp-Phe-His-Lys-Glu-Asn-Thr-Val-... and the peptide corresponds to Asp-39 to Ile-66 of HC. A variety of synthetic peptides derived from this sequence were evaluated for leukocyte migration activity, and a dodecapeptide from Asp-49 to Tyr-60 (Asp-Trp-Ile-Pro-Glu-Gly-Glu-Glu-Asp-Asp-Asp-Tyr) was identified as the active site for leukocyte chemotactic action. The 12-mer synthetic peptide possesses significant neutrophil chemotactic action at 1 nM (60% of the maximal activity of formyl-Met-Leu-Phe), while a peptide with the reverse sequence has essentially no chemotactic activity. Cross-desensitization experiments also show that pretreatment of neutrophils with a 19-mer peptide (Asn-48 to Ile-66) greatly reduces subsequent chemotaxis to HC-neutrophil elastase proteolysis reaction products. When injected intraperitoneally in mice, the HC-neutrophil elastase digest elicits neutrophil migration. Our results demonstrate that not only does HC function as a thrombin inhibitor, but that limited proteolysis of HC near the amino terminus yields biologically active peptide(s) which might participate in inflammation and in wound healing and tissue repair processes.     

Increased proteolytic resistance of ribonuclease A by protein engineering.

Although highly stable toward unfolding, native ribonuclease A is known to be cleaved by unspecific proteases in the flexible loop region near Ala20. With the aim to create a protease-resistant ribonuclease A, Ala20 was substituted for Pro by site-directed mutagenesis. The resulting mutant enzyme was nearly identical to the wild-type enzyme in the near-UV and far-UV circular dichroism spectra, in its activity to 2',3'-cCMP and in its thermodynamic stability. However, the proteolytic resistance to proteinase K and subtilisin Carlsberg was extremely increased. Pseudo-first-order rate constants of proteolysis, determined by densitometric analysis of the bands of intact protein in SDS-PAGE, decreased by two orders of magnitude. In contrast, the rate constant of proteolysis with elastase was similar to that of the wild-type enzyme. These differences can be explained by the analysis of the fragments occurring in proteolysis with elastase. Ser21-Ser22 was identified as the main primary cleavage site in the degradation of the mutant enzyme by elastase. Obviously, this bond is not cleavable by proteinase K or subtilisin Carlsberg. The results demonstrate the high potential of a single mutation in protein stabilization to proteolytic degradation.     

Identification of a soluble enzyme from C3H/10T1/2 cells which is inhibited by the Bowman-Birk proteinase inhibitor

The anticarcinogenic Bowman-Birk proteinase inhibitor (BBI) inhibits a 70-kDa serine proteinase in C3H/10T1/2 transformed fibroblasts. Two serine proteinases, the proline endopeptidase and a novel neutral proteolytic activity, both having a mass of approximately 70-kDa, were isolated from the cytoplasm of C3H/10T1/2 cells. BBI did not inhibit diisopropylfluorophosphate binding to the proline endopeptidase or its ability to hydrolyze peptides. However, BBI blocked the binding of diisopropylfluorophosphate and inhibited the cleavage of peptides by the novel cytoplasmic enzyme. Thus BBI does not inhibit the proline endopeptidase but another soluble 70-kDa serine proteinase from C3H/10T1/2 cells.     

Human lysosomal elastase. Catalytic and immunological properties.

1. The elastase of human spleen was shown to exhibit endopeptidase activity against azo-casein and elastin. 2. Activity against several synthetic substrates was detected, and benzyloxycarbonyl-L-alanine 2-naphthyl ester was found to be a good substrate for routine use. 3. The enzyme showed a broad pH optimum in the range of 8.2-9.2 against azo-casein and the synthetic substrate. 4. The effect of inhibitors on the spleen elastase showed it to be a serine proteinase with a specificity similar to that of porcine pancreatic elastase. 5. Specific antisera were raised against the enzyme, and it was shown to be immunologically identical with the lysosomal elastase of human neutrophil leucocytes.     

The dual nature of the reaction between porcine elastase and human plasma alpha1 proteinase inhibitor.

Only a portion of human plasma α₁ proteinase inhibitor (α₁PI) forms a 1:1 complex with porcine elastase; the other portion is inactivated via proteolysis. High temperature (37°) and high salt (2 M) enhance complex formation. The complex is unstable, but no significant liberation of active elastase could be demonstrated. Probably the same two major products of ～50,000 and ～4,000 daltons are formed from α₁PI via proteolysis and via disintegration of the complex. Iodination of α₁PI or oxidation with chloramine-T prevents complex formation with elastase but not with trypsin. Iodinated elastase, however, forms a complex with α₁PI.     

Carboxylmethylation affects the proteolysis of myelin basic protein by Staphylococcus aureus V8 proteinase.

Bovine myelin basic protein (MBP), charge isoform 1 (C1) was carboxylmethylated by the enzyme D-aspartyl/L-isoaspartyl protein methyltransferase (EC. 2.1.1.77) and the carboxylmethylated protein was subjected to proteolysis by sequencing grade staphylococcal V8 proteinase at pH 4.0 to identify its carboxylmethylated modified aspartate and/or asparagine residues which are recognized by this methyltransferase. Native MBP, C1 was treated similarly and the proteolysis products were compared, using electrophoretic, chromatographic and amino acid sequencing techniques. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) revealed differences in the kinetics of proteolysis between the native and the carboxylmethylated MBP, C1 which were confirmed using HPLC. Partial sequencing of the native and carboxylmethylated fragments eluting at about 29 min (P29) revealed cleavage of native MBP, C1 at Gly-127-Gly-128 and of the carboxylmethylated MBP, C1 at Phe-124-Gly-125. Additional evidence including tryptic subdigestion of carboxylmethylated P29 disclosed the following partial sequence for this peptide: Gly-Tyr-Gly-Gly-Arg-Ala-Ser-Asp-Tyr-Lys-Ser-Ala-His-Lys-Gly-Leu-Lys- Gly-His-Asp-Ala-Gln-Gly-Thr-Leu-Ser-Lys-Ileu-Phe-Lys-. This sequence matches MBP residues 125-154. As a result of these findings, Asp-132 and Asp-144 were identified as two of the modified (isomerized or racemized) methyl-accepting L-aspartates in MBP. The results of the proteolysis experiments wherein the sequencing grade staphylococcal V8 proteinase was used at the rarely tested pH of 4.0, rather than at its commonly tested pH of 7.8, also disclose that the proteinase totally failed to recognize and hence cleave the two Glu-X bonds (Glu-82-Asn-83 and Glu-118-Gly-119) of MBP, preferring to cleave the protein at a number of hitherto unreported sites.     

Limited proteolysis of prolactin in secretory granules from the pituitary gland of estrogenized rats

The LTH-converting proteolytic activity in LTH granules isolated from estrogenized rat hypophysis was studied. Suspensions of granules were incubated at different values of pH for 4 hours at 37 degrees C. The reaction was controlled by SDS electrophoresis. Intensive proteolysis of LTH was observed at pH 6.0 and 3.9, which was accompanied by the formation of fragments with Mr 10, 12 and 17 kD and probably of smaller peptides. An inhibitory analysis revealed that the formation of the 17 kD fragment at pH 3.9 was partly and selectively inhibited by chloroquine, phenanthroline and phenylmethylsulfonyl fluoride. Pepstatin A fully inhibited the proteolysis, whereas leupeptin had no inhibiting influence. The data obtained testify to the presence in the granular fraction of the endopeptidase LTH-converting activity which is sensitive to pepstatin A, an aspartyl proteinase inhibitor as well as to chelators and a serine proteinase inhibitor.     

Organization of a multifunctional protein in pyrimidine biosynthesis. A domain hypersensitive to proteolysis.

When the multifunctional protein that catalyses the first three steps of pyrimidine biosynthesis in hamster cells is treated with staphylococcal V8 proteinase, a single cleavage takes place. The activities of carbamoyl-phosphate synthetase (EC 6.3.5.5), aspartate carbamoyltransferase (EC 2.1.3.2) and dihydro-orotase (EC 3.5.2.3) and the allosteric inhibition by UTP are unaffected. One fragment, of Mr 182000, has the first and third enzyme activities, whereas the other fragment, of Mr 42000, has aspartate carbamoyltransferase activity and an aggregation site. A similar small fragment is observed in protein digested with low concentrations of trypsin. A similar large fragment is seen after digestion with trypsin and as the predominating form of this protein in certain mutants defective in pyrimidine biosynthesis. These results indicate that a region located adjacent to the aspartate carbamoyltransferase domain is hypersensitive to proteinase action in vitro and may also be sensitive to proteolysis in vivo.     

Midgut proteinase activities of three keratinolytic larvae,Hofmannophila pseudospretella,Tineola bisselliella,and Anthrenocerus australis,and the effect of proteinase inhibitors on proteolysis

The midgut proteinase activities were characterized from the keratinolytic larvae of two lepidopterans, Hofmannophila pseudospretella (Stainton) (Oecophoridae) and Tineola bisselliella (Hummel) (Tineidae), and one coleopteran, Anthrenocerus australis (Hope) (Dermestidae). The major endopeptidase activities, characterized using specific enzyme inhibitors, were serine proteinases with hydrolytic activity against N-benzoyl-DL-arginine-p-nitroanilide and against N-succinyl-L-alanyl-L-alanyl-L-prolyl-L-leucine-p-nitroanilide. No significant levels of metalloendopeptidase or cysteine endopeptidase activities were detected. Aminopeptidase activity was present in all larvae. The enzyme levels and properties of the two moth larvae were similar to each other and to those of phytophagous lepidopteran larvae but different from those of the beetle larva. Whereas only a limited number of serine proteinase inhibitors inhibited the midgut proteolysis of the lepidopteran larvae, most inhibitors inhibited the midgut proteolysis of the beetle larva. © 1994 Wiley-Liss, Inc.     

The reactive site of human alpha 2-antiplasmin

Human alpha 2-antiplasmin rapidly forms a stable, equimolar complex with either its target enzyme, plasmin, or with trypsin. Perturbation of the inhibitor-trypsin complex results in peptide bond cleavage at the reactive site of the inhibitor with the concomitant release of a small peptide fragment which apparently represents the carboxyl-terminal segment of the inhibitor. Sequence analysis of this fragment, together with that of an overlapping peptide obtained by treatment of native inhibitor with either Staphylococcus aureus V8 proteinase or human neutrophil elastase, yields data which indicate that the reactive site of alpha 2-antiplasmin encompasses a P1-P'1 Arg-Met sequence. However, unlike alpha 1-1-proteinase inhibitor which has a Met residue in the P1-position, oxidation of alpha 2-antiplasmin has no effect on its inhibitory activity toward either plasmin, trypsin, or chymotrypsin, indicating the lesser mechanistic importance of the P'1-residue during enzyme inactivation by this inhibitor.     

Inter-alpha-trypsin inhibitor. Inhibition spectrum of native and derived forms

The conversion of inter-alpha-trypsin inhibitor (I alpha I) into active, acid-stable derivatives by proteolytic degradation has been tested with 10 different proteinases. Of these, only plasma kallikrein, cathepsin G, neutrophil elastase, and the Staphylococcus aureus V-8 proteinase were found to be effective, each releasing more than 50% of this activity. However, a strong correlation between inhibitor degradation and significant release of acid-stable activity could only be found with the V-8 enzyme. Inhibition kinetics for the interaction of native I alpha I, the inhibitory fragment released by digestion with S. aureus V-8 proteinase, or the related urinary trypsin inhibitor, with seven different proteinases indicated that all had essentially identical Ki values with an individual enzyme and, where measurements were possible, nearly identical second order association rate constants. Significantly, none of the five human proteinases tested, including trypsin, chymotrypsin, plasmin, neutrophil elastase, and cathepsin G, would appear to have low enough Ki values to be physiologically relevant. Thus, the role of native I alpha I or its degradation products in controlling a specific proteolytic activity is still unknown.     

Characterization of a maize root proteinase.

The major proteinase in maize (Zea mays) roots behaves as a serine endopeptidase. A possible physiological role of this enzyme could be in the turnover of nitrate reductase (NR) and, as such, it could be of great importance in regulating the assimilation of nitrate. The objective of this research was to elucidate the specificity and uniqueness of maize root proteinase. When bovine serum albumin and an NR purified from Chlorella vulgaris were used as substrates, the maize root proteinase exhibited a preference for cleavages such that the amino acid on the amino side of the scissile bond was alanine. This information was established by microsequence analysis of the N termini of proteolytic fragments, and carboxypeptidase Y analysis of the C termini of proteolytic fragments of substrates hydrolyzed by the proteinase. Cleavage occurred at the sequence Ala/Ala-Ala-Ala-Pro-Glu in Chlorella NR, and at the sequence Ala-Asp-Glu-Ser-His-Ala-Gln in bovine serum albumin. When bovine serum albumin was the substrate, the maize root proteinase yielded a peptide map that is unique relative to those created with the other serine endopeptidases elastase, trypsin, or chymotrypsin. Based on our data, the maize root proteinase appears to cleave peptide bonds at the carboxy side of alanine. Because of its specificity, it should have useful applications in protein chemistry.     

Proteolytic cleavage of the puromycin-sensitive aminopeptidase generates a substrate binding domain

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.     

Purification, characterization and gene cloning of a novel glutamic acid-specific endopeptidase from Staphylococcus aureus ATCC 12600.

Twenty strains of Staphylococcus aureus from ATCC type cultures and strains found in clinical studies were cultivated, and their endopeptidase activity specific for glutamic acid was surveyed using benzyloxycarbonyl-Phe-Leu-Glu-p-nitroanilide (Z-Phe-Leu-Glu-pNA) as a substrate. The activity was found in two of the strains, ATCC 12600 and ATCC 25923. A glutamic acid-specific proteinase, which we propose to call SPase, was purified from the culture filtrate of S. aureus strain ATCC 12600 by a series of column chromatographies on DEAE-Sepharose twice and on Sephacryl S-200. A single band was observed on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of the purified SPase. The molecular weight of the proteinase was estimated to be 34000 by SDS-PAGE. When synthetic peptides and oxidized insulin B-chain were used as substrates, SPase showed the same substrate specificity as V8 proteinase, EC 3.4.21.9, which specifically cleaves peptide bonds on the C-terminal side of glutamic acid and aspartic acid. Examination with p-nitroanilides of glutamic acid and aspartic acid as substrates, however, revealed that both proteinases are highly specific for a glutamyl bond in comparison with an aspartyl bond. To elucidate the complete primary structure of SPase, its gene was cloned from genomic DNA of S. aureus ATCC 12600, and the nucleotide sequence was determined. Taking the amino acid sequence of SPase from the NH2-terminus to the 27th residue into consideration, the clones encode a mature peptide of 289 amino acids, which follows a prepropeptide of 68 residues. SPase was confirmed to be a novel endopeptidase specific for glutamic acid, being different from V8 proteinase which consists of 268 amino acids.     

Immunochemical characterization of a proline endopeptidase from rat brain. Its relationship to proline endopeptidase from other tissues and from other species

Monospecific antiserum raised against rat brain proline endopeptidase is used to demonstrate the ubiquity of the enzyme and its unique role in the degradation of proline-containing peptides. All endoproteolytic activity directed toward proline residues in several rat tissues is shown to share one or more common antigenic determinants with rat brain proline endopeptidase. Similar activity from tissue of other species crossreacts with rat proline endopeptidase. The data presented suggest that proline endopeptidase is the sole cytoplasmic enzyme capable of degrading proline-containing peptides in every tissue examined and that previously reported proline-specific endoproteolytic activities observed in a variety of systems may be ascribed to proline endopeptidase. The putative role of proline endopeptidase in protein degradation is discussed.     

Generation of peptides suitable for sequence analysis by proteolytic cleavage in reversed-phase high-performance liquid chromatography solvents

A methodological study of practical importance to protein sequencing has been carried out. Peptide mapping and sequence analysis of the cleavage products of reduced and carboxymethylated ribonuclease have been applied to the study of the activity and specificity of trypsin, chymotrypsin, elastase, lysyl endopeptidase (Achromobacter protease I), endoproteinase Arg-C (from mouse submaxillary gland), Staphylococcus aureus V8 protease, pepsin, and thermolysin in the presence of 20% methanol, ethanol, 2-propanol, and acetonitrile at 22 and 37 degrees C. The peptide bond specificities were retained, and the activities were generally unaffected or moderately reduced at 22 degrees C and pH 8. At 37 degrees C the activity of chymotrypsin, endoproteinase Arg-C, V8 protease at pH 4, and pepsin was substantially reduced and decreased in the order methanol, ethanol, 2-propanol, and acetonitrile. The activity of thermolysin at 55 degrees C was reduced very little in the presence of 20% organic solvent and 50 mM Ca2+. In low calcium and 20% 2-propanol at 22 degrees C the activity of thermolysin was restricted to the complete and specific cleavage of peptide bonds N-terminally of Phe, Ile, and Leu. The experiments suggest that secondary proteolytic digestions can be carried out directly in reversed-phase-HPLC fractions, and that organic cosolvents can be applied to control the degree of proteolysis. Moreover, the denaturing potential of these solvents might be useful in the degradation of proteins resistant to proteolysis, for example, in studies aimed at identification of disulfide bridges.     

Mouse macrophage elastase. Purification and characterization as a metalloproteinase.

Macrophage elastase was purified from tissue-culture medium conditioned by inflammatory mouse peritoneal macrophages. Characterized as a secreted neutral metalloproteinase, this enzyme was shown to be catalytically and immunochemically distinct from the mouse pancreatic and mouse granulocyte elastases, both of which are serine proteinases. Inhibition profiles, production of nascent N-terminal leucine residues and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of degraded elastin indicated that macrophage elastase is an endopeptidase, with properties of a metalloproteinase, rather than a serine proteinase. Macrophage elastase was inhibited by alpha 2-macroglobulin, but not by alpha 1-proteinase inhibitor. Macrophage elastase was resolved into three chromatographically distinct forms. The predominant form had mol.wt. 22 000 and was purified 4100-fold. Purification of biosynthetically radiolabelled elastase indicated that this form represented less than 0.5% of the secreted protein of macrophages. Approx. 800% of the starting activity was recovered after purification. Evidence was obtained for an excess of an endogenous inhibitor masking more than 80% of the secreted activity.