Isolation of a crotalase-like protease with alpha-fibrinogenase activity from the western diamondback rattlesnake, Crotalus atrox.

Venom toxins were isolated from rattlesnake (Crotalus atrox) venom by cation-exchange chromatography. Seven major fractions could be obtained by single-step ion-exchange chromatography with two fractions showing essentially apparent homogeneity by SDS-gel electrophoresis. All fractions showed various extents of specific proteolytic activity against alpha- or beta-chains of fibrinogen molecules. Further characterization of one of the purified fractions with alpha-fribrinogenase activity indicated that it is a single-chain thrombin-like protease with a molecular mass of about 30 kDa. It is relatively heat stable, inhibited by phenylmethanesulfonyl fluoride, N alpha-p-tosyl-L-phenylalanine chloromethyl ketone and N alpha-p-tosyl-L-lysine chloromethyl ketone but not by soybean trypsin inhibitor and beta-mercaptoethanol. Amino acid analysis showed that the enzyme possesses an amino acid composition very similar to thrombin and crotalase characterized before from the closely related snake venoms. N-Terminal sequence analysis of the enzyme corroborated the close similarity between this enzyme and those sequences of crotalase and kallikrein-like enzymes characterized from the same Crotalidae snake family. This study is in contrast to the previous reports which indicated a lack of thrombin- and crotalase-like enzyme in the venom of Western diamondback rattlesnake.     

Isolation and characterization of fibrinogenase from western diamondback rattlesnake venom and its comparison to the thrombin-like enzyme, crotalase

A new type of fibrinogenase was isolated from the venom of the western diamondback rattlesnake (Crotalus atrox). Unlike thrombin, the newly isolated fibrinogenase did not cause formation of a fibrin clot. Various properties of the fibrinogenase we isolated were compared with crotalase isolated from the venom of C. adamanteus. It was found that fibrinogenase has considerable similarity to crotalase isolated by Markland and Damus in 1971. Crotalase is a thrombin-like enzyme and produces a fibrin clot from fibrinogen. The A alpha chain of fibrinogen was first split and the B beta chain was cleaved later. The fact that no fibrin clot forms indicates that the cleavage sites in A alpha and B beta chains of fibrinogen must be different from thrombin sites. The fibrinogenase also released bradykinin by interacting with plasma proteins. It hydrolyzed TAME (p-toluenesulfonyl-L-arginine methyl ester), BAEE (N-benzoyl-L-arginine ethyl ester). TLME (N-tosyllysine methyl ester) but not BAA (N-benzoylarginine amide), TAA (N-tosylarginineamide) or ATEE (N-acetyltyrosine ethyl ester). The enzyme is an acidic protein with pI of 4.6 and a mol. wt of 31,000. It consists of 272 total amino acid residues, 21% of which are acidic amino acids. Fibrinogenase is a specific form of protease. A newly liberated amino group after hydrolysis of dimethyl-casein can be detected by the reagent trinitrobenzenesulfonic acid (TNBS). Fibrinogenase differs from trypsin as the soybean trypsin inhibitor does not inhibit the enzyme's action.(ABSTRACT TRUNCATED AT 250 WORDS)     

A chymotrypsin-like proteinase from the midgut of Tenebrio molitor larvae

A chymotrypsin-like proteinase was isolated from the posterior midgut of larvae of the yellow mealworm, Tenebrio molitor, by ion-exchange and gel filtration chromatography. The enzyme, TmC1, was purified to homogeneity as determined by SDS-PAGE and postelectrophoretic activity detection. TmC1 had a molecular mass of 23.0 kDa, pI of 8.4, a pH optimum of 9.5, and the optimal temperature for activity was 51 degrees C. The proteinase displayed high stability at temperatures below 43 degrees C and in the pH range 6.5-11.2, which is inclusive of the pH of the posterior and middle midgut. The enzyme hydrolyzed long chymotrypsin peptide substrates SucAAPFpNA, SucAAPLpNA and GlpAALpNA and did not hydrolyze short chymotrypsin substrates. Kinetic parameters of the enzymatic reaction demonstrated that the best substrate was SucAAPFpNA, with k(cat app) 36.5 s(-1) and K(m) 1.59 mM. However, the enzyme had a lower K(m) for SucAAPLpNA, 0.5 mM. Phenylmethylsulfonyl fluoride (PMSF) was an effective inhibitor of TmC1, and the proteinase was not inhibited by either tosyl-l-phenylalanine chloromethyl ketone (TPCK) or N(alpha)-tosyl-l-lysine chloromethyl ketone (TLCK). However, the activity of TmC1 was reduced with sulfhydryl reagents. Several plant and insect proteinaceous proteinase inhibitors were active against the purified enzyme, the most effective being Kunitz soybean trypsin inhibitor (STI). The N-terminal sequence of the enzyme was IISGSAASKGQFPWQ, which was up to 67% similar to other insect chymotrypsin-like proteinases and 47% similar to mammalian chymotrypsin A. The amino acid composition of TmC1 differed significantly from previously isolated T. molitor enzymes.     

Neutral elastolytic proteinase from canine leucocytes. Purification and characterization.

1. A neutral proteinase (EC 3.4.-.-) with elastolytic activity was isolated from canine bloodstream leucocytes, and purified to apparent homogeneity by a two-step procedure consisting of DEAE-Sephadex chromatography and molecular sieving on Sephadex G-75. 2. The molecular weight of the enzyme was 23 500, and the absorbance (A1%1cm) at 282 nm was 6.1. Amino acid analysis showed high content of glycine, aspartic acid, and valine, and low proportion of methionine, lysine and histidine as well as the absence of tyrosine in the enzyme molecule. 3. The proteinase was active against several protein substrates as well as towards N-t-butyloxycarbonyl-L-alanine p-nitrophenyl ester, N-acetyl-L-alanyl-tyrosine ethyl ester. 4. The enzyme was inactivated by diisopropylfluorophosphate, N-acetyl-L-alanyl-L-alanyl-L-alanine chloromethyl ketone, and N-p-tosyl-L-phenylalanine chloromethyl ketone. Inhibition by some natural proteinase inhibitors was also noted.     

Purification of a toxic metalloprotease produced by the pathogenic Photobacterium damselae subsp. piscicida isolated from cobia (Rachycentron canadum)

The aim of the present study was to purify and characterize a toxic protease secreted by the pathogenic Photobacterium damselae subsp. piscicida strain CP1 originally isolated from diseased cobia (Rachycentron canadum). The toxin isolated by anion exchange chromatography, was a metalloprotease, inhibited by L-cysteine, ethylenediaminetetraacetic acid (EDTA), ethylene glycol-bis(beta-aminoethyl ether)N,N,N',N'-tetraacetic acid (EGTA), 1,10-phenanthroline, N-tosyl-L-phenylalanine-chloromethyl ketone (TPCK), and N-alpha-p-tosyl-L-lysine-chloromethyl ketone (TLCK), and showed maximal activity at pH 6.0-8.0 and an apparent molecular mass of about 34.3 kDa. The toxin was also completely inhibited by HgCl2, and partially by sodium dodecyl sulfate (SDS) and CuCl2. The extracellular products and the partially purified protease were lethal to cobia with LD50 values of 1.26 and 6.8 microg protein/g body weight, respectively. The addition of EDTA completely inhibited the lethal toxicity of the purified protease, indicating that this metalloprotease was a lethal toxin produced by the bacterium.     

Cloning, expression, and characterization of a cDNA encoding snake venom metalloprotease

A cDNA clone, MT-c, encoding metalloprotease was isolated from snake (Agkistrodon halys brevicadus) venom gland cDNA library. Deduced amino acid sequence indicated that MT-c is composed of a signal sequence, amino-terminal propeptide, a central metalloprotease domain, and a Lys-Gly-Asp (KGD) disintegrin domain. The partial cDNA encoding metalloprotease and disintegrin domain was subcloned and expressed in E. coli. The expressed MT-c protein was purified and successfully refolded into functional form retaining the enzyme activity. Analyses of the purified recombinant protease activity revealed that the enzyme hydrolyzes extracellular matrix proteins including type I gelatin, type IV and type V collagen, while type I, II, III collagens and fibronectin were insensitive to the proteolytic digestion. The recombinant enzyme was also able to degrade fibrinogen by specifically cleaving A alpha chain of the protein.     

Secretion of a trypsin-like thiol protease by a new keratinolytic strain of Bacillus licheniformis

When cultured in feather-containing broth with a growth optimum of pH 7.0 and 47 degrees C, a Bacillus licheniformis strain exhibited a high chicken feather-degrading activity. A trypsin-like protease was isolated from its ferment broth and was partially characterized. The enzyme was constitutively secreted and was highly active towards N-benzoyl-Phe-Val-Arg-p-nitroanilide as chromogenic substrate. Its pH optimum was 8.5 and it exhibited the highest activity at 52 degrees C. Fractionation on Sephadex G-100 column revealed that its molecular mass was about 42 kDa. The enzyme, which is new for the genus Bacillus, is a thiol protease, as tosyl-L-phenylalanine chloromethyl ketone, tosyl-L-lysine chloromethyl ketone, phenylmethylsulfonyl fluoride and ethylenediamine tetraacetate did not inhibit it, while HgCl2 and para-chloromercuribenzoate lowered its activity.     

Isolation and characterization of a proteinase from the sarcocarp of melon fruit.

A proteinase from the sarcocarp of melon (Cucumis Melo L. var. Prince) was purified by a three-step procedure involving batch-wise treatment with CM-cellulose fibers, column chromatography on CM-cellulose powder and gel filtration on Sephadex G-75. The final enzyme preparation was homogeneous on acrylamide gel electrophoresis. Its molecular weight was estimated by two different methods to be about 50,000. Anlayses indicated tha presence of 475 amino acid residues and at least 7 moles of hexose. The maximum activity was found in the alkaline pH region against casein as a substrate. The optimum temperature against casein was 70 degrees at pH 7.1. The enzyme was strongly inhibited by diisopropyl fluorophosphate, partly inhibited by HgCl2 and not inhibited by EDTA, p-chloromercuribenzoic acid, N-tosyl-L-lysine chloromethyl ketone, N-tosyl-L-phenylalanine chloromethyl ketone, and soybean trypsin inhibitor. The reduced and carboxymethylated insulin B-chain was cleaved at the peptide bonds of Asn3-Gln4, Cm-Cys7-Gly8, Glu13-Ala14, Leu15-Tyr16, Cm-Cys19-Gly20, Phe25-Tyr26, Pro28-Lys29, and Lys29-Ala30 by the enzyme.     

Enzymatic characterization of arginine esterase from dog seminal plasma

Previously purified arginine esterase from dog seminal plasma was characterized enzymatically. The enzyme was found to have a rather narrow specificity for arginine esters, much less for lysine esters and was practically devoid of activity towards tyrosine esters, casein, albumin and azocoll. It had a broad optimum pH between 8 and 9. It presented no kallikrein-like activities either in the blood pressure test in dog or in the rat uterus contraction test. It was inhibited by bovine pancreas trypsin inhibitor, aprotinin, phenylalanylprolyl arginine chloromethyl ketone, diisopropylfluorophosphate, phenylmethylsulfonyl fluoride, sodium dodecyl sulfate and leupeptin, but not by soybean trypsin inhibitor, tosyllysine chloromethyl ketone, tosylamide-2-phenylethyl chloromethyl ketone, iodoacetamide, Triton X-100 and EDTA. Experiments involving incubation of prostatic cytosol with purified arginine esterase showed that actin was the only important prostatic protein that was extensively hydrolyzed by this enzyme. It is not known presently whether the hydrolysis of actin is related to a true physiological function of the enzyme and whether actin and arginine esterase ever come into contact with each other in vivo. These properties indicate that arginine esterase from dog seminal plasma is different from other known proteinases including classical kallikreins, although it presents many similarities with this class of enzyme.     

Hypodermin B, a trypsin-related enzyme from the insect Hypoderma lineatum. Comparison with hypodermin A and Hypoderma collagenase, two serine proteinases from the same source

Hypodermin B, a serine proteinase with a molecular weight of 23000, was purified to homogeneity from the larvae Hypoderma lineatum. It is stoichiometrically inhibited by diisopropylfluorophosphate and fully inactivated by N-tosyllysine chloromethyl ketone and soya bean and bovine pancreatic trypsin inhibitors. N-Tosylphenylalanine chloromethyl ketone and ovomucoid are without effect on its activity. Hypodermin B hydrolyses both amide and ester substrates of trypsin but does not display any chymotryptic activity on synthetic substrates. Its specificity on the B chain of insulin is slightly broader than that of bovine trypsin. Its amino acid composition and N-terminal sequence suggest structural homology with serine proteinases of the trypsin family and with two other serine proteinases, hypodermin A and Hypoderma collagenase, previously isolated from the same larvae. Hypodermins A and B are very similar with respect to their inhibition and specificity, they differ however strongly from Hypoderma collagenase.     

Purification and characterization of a rat liver cytosol neutral thiol peptidase that degrades glucagon, insulin, and isolated insulin A and B chains

A thiol peptidase that catalyzes at near neutral pH the hydrolysis of insulin, the isolated A and B chains of insulin, and glucagon was purified from rat liver cytosol by fractionation on Sephadex G-200, Affi-Gel Blue, and Spherogel TSK-G 3000 SW. The purified enzyme showed a single component by chromatography on a Spherogel TSK column and by gel filtration on a Sephadex G-200 column. The native enzyme has a molecular weight of approximately 180,000 and consists of two subunits having pI's of 5.9 and 6.3. Studies on its substrate specificity showed that the purified enzyme degrades glucagon, insulin, insulin B chain, and insulin A chain, but it does not degrade proinsulin, ACTH, or denatured hemoglobin. Kinetic analyses were performed on three substrates. The Km values were: 34 nM for insulin, 276 nM for insulin B chain, and 3.5 microM for glucagon. The kcat and Vm/Km values were glucagon greater than B chain greater than insulin. Thus, the enzyme has the highest affinity/lowest efficiency for insulin, an intermediate affinity/intermediate efficiency for B chain of insulin and the lowest affinity/highest efficiency for glucagon. The effect of several potential activators and inhibitors on the enzyme's activity was investigated. The enzyme activity was markedly inhibited by N-ethylmaleimide, p-chloromercuribenzoic acid, iodoacetamide, and Np-tosyl-L-phenylalanine chloromethyl ketone (TPCK), and was partially inhibited by dithiothreitol, by the chelating agents EDTA and EGTA, and by phenylmethylsulfonyl fluoride (PMSF). Bacitracin inhibited the activity of the enzyme, but the protease inhibitors aprotinin, leupeptin, pepstatin, and phosphoramidon had little or no effect. Reduced glutathione, iodoacetate, and N alpha,p-tosyl-L-lysine chloromethyl ketone (TLCK) also had little or no effect on the enzyme activity.     

Characterization of tumor cell membrane serine proteases by polyacrylamide gel electrophoresis

Studies in our laboratory have indicated that tumor cell membrane-bound proteases are responsible for the ability of tumor cells to lyse normal cells in vitro. In order to evaluate the tumor cell membrane enzymes, a purified tumor cell membrane preparation was prepared and the nonionic detergent Triton X-100 was used to extract active enzymes from the cell membranes. The solubilized membrane enzymes were then studied by Triton X-100 polyacrylamide gel electrophoresis under non-denaturing conditions. Using this technique the tumor cell membranes were shown to contain esterproteases that reacted with the substrates alpha-naphthyl acetate and naphthol-AS-aminocaproate. These esterproteases were inhibited by diisopropyl fluorphosphate and tosyl lysine chloromethyl ketone but not by tosylamide phenylethyl chloromethyl ketone, soybean trypsin inhibitor p-chloromercuribenzene sulfonic acid; N-ethylmaleimide choline iodide, alpha-1-anti-trypsin. NaF, epsilon-aminocaproic acid, ethylenediamine tetraacetic acid, or eserine. SBTI affinity chromatography of the tumor cell membrane extract revealed that some of the serine esterproteases bound to the SBTI column. The proteolytic activity of the tumor cell membrane extract and a fraction eluted from the SBTI affinity column was demonstrated using casein. We conclude that the tumor cell membranes contain previously undescribed serine proteases that are identifiable by their esterase activity and inhibitor profiles in polyacrylamide gels.     

Purification and characterization of an extracellular poly(L-lactic acid) depolymerase from a soil isolate, Amycolatopsis sp. strain K104-1

Poly(L-lactic acid) (PLA)-degrading Amycolatopsis sp. strains K104-1 and K104-2 were isolated by screening 300 soil samples for the ability to form clear zones on the PLA-emulsified mineral agar plates. Both of the strains assimilated >90% of emulsified 0.1% (wt/vol) PLA within 8 days under aerobic conditions. A novel PLA depolymerase with a molecular weight of 24,000 was purified to homogeneity from the culture supernatant of strain K104-1. The purified enzyme degraded high-molecular-weight PLA in emulsion and in solid film, ultimately forming lactic acid. The optimum pH for the enzyme activity was 9.5, and the optimum temperature was 55 to 60 degrees C. The PLA depolymerase also degraded casein and fibrin but did not hydrolyze collagen type I, triolein, tributyrin, poly(beta-hydroxybutyrate), or poly(epsilon-caprolactone). The PLA-degrading and caseinolytic activities of the enzyme were inhibited by diisopropyl fluorophosphate and phenylmethylsulfonyl fluoride but were not significantly affected by soybean trypsin inhibitor, N-tosyl-L-lysyl chloromethyl ketone, N-tosyl-L-phenylalanyl chloromethyl ketone, and Streptomyces subtilisin inhibitor. Thus, Amycolatopsis sp. strain K104-1 excretes the unique PLA-degrading and fibrinolytic serine enzyme, utilizing extracellular polylactide as a sole carbon source.     

Characterization of soybean trypsin inhibitor sensitive protease from unfertilized sea urchin eggs

A serine protease from sea urchin eggs has been isolated by affinity chromatography on soybean trypsin inhibitor-agarose. Benzamidine hydrochloride was included to minimize autodegradation. We present data on the properties of the protease with respect to molecular weight and its interaction with trypsin inhibitors and substrates. The molecular weight of the enzyme is 47 000 by gel filtration under nonreducing conditions and 35 000 by electrophoresis in the presence of sodium dodecyl sulfate and dithiothreitol. The pH optimum and Km with N alpha-benzoyl-L-arginine ethyl ester (BAEE) are 8.0 and 75 microM, respectively. The specific activity is comparable to that of bovine pancreatic trypsin. Proteolytic activity was measured by beta-casein hydrolysis. The caseinolytic activity is completely inhibited by 1 mumol of soybean trypsin inhibitor (SBTI) per micromole of enzyme. BAEE esterase activity is inhibited competitively by SBTI (Ki = 1.6 nM), lima bean trypsin inhibitor (150 nM), chicken ovomucoid (100 nM), and leupeptin (130 nM). Bowman-Birk inhibitor, benzamidine hydrochloride, and antipain are also inhibitors of the purified enzyme. Inhibition by phenylmethanesulfonyl fluoride and N alpha-p-tosyl-L-lysine chloromethyl ketone indicates the presence of serine and histidine residues in the active center, respectively. The chymotrypsin inhibitor L-1-(tosylamido)-2-phenylethyl chloromethyl ketone is ineffective. The protease is susceptible to autodegradation which can result in the appearance of a minor 23-kilodalton component. The egg protease appears to be similar in many respects to trypsins and trypsin-like enzymes isolated from a wide variety of sources, including sea urchin and mammalian sperm.     

Characterization of an alkaline protease from Bacillus sp. no. AH-101

Summary The Bacillus sp. no. AH-101 alkaline protease showed higher hydrolysing activity against insoluble fibrous natural proteins such as elastin and keratin in comparison with subtilisins and Proteinase K. The optimum pH of the enzyme toward elastin and keratin was pH 10.5 and pH 11.0–12.0 respectively. The specific activity toward elastin and keratin was 10 600 units/mg protein and 3970 units/mg protein, respectively. The enzymatic activity was not inhibited by p-chloromercuribenzoic acid and iodoacetic acid. Carbobenzoxy-glycyl-glycyl-L-phenylalanyl chloromethyl ketone completely inhibited the caseinolytic activity, but 36% elastolytic activity remained. No inhibitory effect on caseinolytic and elastolytic activity was shown by tosyl-L-phenylalanyl-chloromethyl ketone, tosyl-L-lysine chloromethyl ketone, carbobenzoxy-L-phenylalanyl chloromethyl ketone, and elastatinal. The amino acid composition and amino terminal sequence of the enzyme were determined. The no. AH-101 alkaline protease was compared with subtilisin BPN', subtilisin Carlsberg, no. 221, and Ya-B alkaline proteases. Extensive sequence homology existed among these enzymes. Offprint requests to: H. Takami     

Novel Ca2+-activated neutral protease from an aquatic fungus, Allomyces arbuscula.

A Ca2+-activated neutral protease was purified to homogeneity from an aquatic Phycomycete fungus, Allomyces arbuscula. It requires millimolar concentrations of Ca2+ for activation (1.8 to 2 mM for 50% activation). Sr2+ can replace Ca2+ but at higher concentrations (4 mM for 50% activation). The enzyme is a dimer of 40-kilodalton subunits and contains six cysteine residues, three of which are revealed only after the addition of micromolar concentrations of Ca2+; the other three are free. Enzyme activity is strongly inhibited by SH-group inhibitors and some trypsin inhibitors (leupeptin and alpha-N-tosyl-L-lysine chloromethyl ketone). The enzyme lacks general trypsinlike specificity, since substrates containing tryptic cleavage sites are not cleaved nor is enzyme activity inhibited by other trypsin inhibitors. The enzyme has many functional similarities to the extensively characterized mammalian and avian Ca2+-activated neutral proteases but differs in its substrate specificity, inhibition by alpha-N-tosyl-L-phenylalanine chloromethyl ketone, and subunit structure. It is, nevertheless, presumed that this enzyme has a similar high order of specificity and is involved in the regulation of a specific growth function.     

Primary structure of rat brain prostaglandin D synthetase deduced from cDNA sequence

The amino acid sequence of rat brain prostaglandin D synthetase (Urade, Y., Fujimoto, N., and Hayaishi, O. (1985) J. Biol. Chem. 260, 12410-12415) was determined by a combination of cDNA and protein sequencing. cDNA clones specific for this enzyme were isolated from a lambda gt11 rat brain cDNA expression library. Nucleotide sequence analyses of cloned cDNA inserts revealed that this enzyme consisted of a 564- or 549-base pair open reading frame coding for a 188- or 183-amino acid polypeptide with a Mr of 21,232 or 20,749 starting at the first or second ATG. About 60% of the deduced amino acid sequence was confirmed by partial amino acid sequencing of tryptic peptides of the purified enzyme. The recognition sequence for N-glycosylation was seen at two positions of amino acid residues 51-53 (-Asn-Ser-Ser-) and 78-80 (-Asn-Leu-Thr-) counted from the first Met. Both sites were considered to be glycosylated with carbohydrate chains of Mr 3,000, since two smaller proteins with Mr 23,000 and 20,000 were found during deglycosylation of the purified enzyme (Mr 26,000) with N-glycanase. The prostaglandin D synthetase activity was detected in fusion proteins obtained from lysogens with recombinants coding from 34 and 19 nucleotides upstream and 47 and 77 downstream from the first ATG, indicating that the glycosyl chain and about 20 amino acid residues of N terminus were not essential for the enzyme activity. The amino acid composition of the purified enzyme indicated that about 20 residues of hydrophobic amino acids of the N terminus are post-translationally deleted, probably as a signal peptide. These results, together with the immunocytochemical localization of this enzyme to rough-surfaced endoplasmic reticulum and other nuclear membrane of oligodendrocytes (Urade, Y., Fujimoto, N., Kaneko, T., Konishi, A., Mizuno, N., and Hayaishi, O. (1987) J. Biol. Chem. 262, 15132-15136) suggest that this enzyme is a membrane-associated protein.     

Biochemical and genetic characterization of arazyme, an extracellular metalloprotease produced from Serratia proteamaculans HY-3

Serratia proteamaculans HY-3 isolated from the digestive tract of a spider produces an extracellular protease named arazyme, with an estimated molecular mass of 51.5 kDa. The purified enzyme was characterized as having high activities at wide pH and temperature ranges. We further characterized biochemical features of the enzymatic reactions under various reaction conditions. The protease efficiently hydrolyzed a broad range of protein substrates including albumin, keratin, and collagen. The dependence of enzymatic activities on the presence of metal ions such as calcium and zinc indicated that the enzyme is a metalloprotease, together with the previous observation that the proteolytic activity of the enzyme was not inhibited by aspartate, cysteine, or serine protease inhibitors, but strongly inhibited by 1,10-phenanthroline and EDTA. The araA gene encoding the exoprotease was isolated as a 5.6 kb BamHl fragment after PCR amplification using degenerate primers and subsequent Southern hybridization. The nucleotide sequence revealed that the deduced amino acid sequences shared extensive similarity with those of the serralysin family of metalloproteases from other enteric bacteria. A gene (inh) encoding a putative protease inhibitor was also identified immediately adjacent to the araA structural gene.     

Purification of an acid protease from Mucor rouxii that inactivates chitin synthetase

An acid protease was purified from the mycelial form of Mucor rouxii by a method which involved salt and acid precipitation, gel filtration and anion-exchange chromatography. The enzyme had a molecular mass of 16,000 Da. Its optimum pH was 4.0, maximal activity was obtained at 50°C, and it was inactivated at 70°C. It was not affected by leupeptin or N -p-tosyl-L-lysine chloromethyl ketone (TLCK) but diazoacetyl-DL-norleucine methyl ester (DNME) in the presence of Cu²⁺ and more noticeably pepstatin A, strongly inhibited the activity. This acid protease did not activate zymogenic chitin synthetase from the fungus, but brought about its inactivation even at low concentrations and after short periods of incubation time.Abbreviations TLCK N -p-tosyl-L-lysine chloromethyl ketone - DNME diazoacetyl-DL-norleucine methyl ester - TCA trichloroacetic acid - SDS sodium dodecyl sulfate     

Characterization of serine proteases of Lumbriculus variegatus and their role in regeneration

Serine proteases, ubiquitous enzymes known to function in digestion and immune protection in both vertebrates and invertebrates and implicated in regeneration in some species, were investigated in the California blackworm, Lumbriculus variegatus. Several serine proteases, rather than a single enzyme with broad specificity, were present in tissue extracts from the worms. Extracts were treated with a fluorescein‐labeled peptide chloromethyl ketone that specifically binds to trypsin/thrombin‐like proteases. Denaturing gel electrophoresis of labeled extracts showed several serine proteases with their molecular weight ranging 28,000–38,000 daltons. The trypsin/thrombin‐like activity was localized, using the fluorescein‐conjugated reagent, to the pharynx and digestive tract of L. variegatus. Movement of cells labeled by the reagent into regenerating tissues suggests that some differentiated endodermal tissues were used for reformation of digestive structures during regeneration in L. variegatus. The types of serine proteases in the extracts were further characterized by inhibitor studies. Presence of plasmin‐like activity was indicated by degradation of fibrin by tissue homogenates from the worms and the inhibitory effect of aprotinin on enzymes in these extracts. The ability of L. variegatus extracts to generate clots when incubated with rabbit plasma and partial inhibition of extract activity by phenylmethylsulfonyl fluoride and hirudin indicated presence of thrombin‐like activity. Consistent with the detection of trypsin, chymotrypsin, and plasmin‐like enzymes in the extracts was partial inhibition of L. variegatus serine protease activity by aminoethyl benzenesulfonyl fluoride and soybean trypsin inhibitor. Selective inhibition of chymotrypsin‐like activity by N‐tosyl‐l ‐phenylalanine chloromethyl ketone and chymostatin as well as trypsin‐like activity by N‐tosyl‐l ‐lysine chloromethyl ketone was observed. A potential role during regeneration for serine proteases is suggested by blockage of formation of head and tail structures by aminoethyl benzenesulfonyl fluoride, an inhibitor of these proteases.