Characterization of a proteolytic enzyme from Lachesis muta venom (Serpentes: Viperidae).

A proteolytic enzyme from L. muta stenophrys was isolated by gel filtration on Bio Gel P-100 followed by FPLC on MONO S column. The enzyme exhibited proteolytic activity toward casein, hemoglobin and fibrinogen with a pH optimum around 10. The activity was inhibited by EDTA while trypsin inhibitors were not inhibitory. It is a glycoprotein, Mr 14 kDa with a high content of Asp, Glu, and Leu residues and a low content of Cys and Trp. The protease is devoid of myotoxic, hemorrhagic, esterolytic and amidolytic activities. It lyses the alfa and beta chains of human fibrinogen and releases kinin from L.M.W. kininogen. No release of histamine was observed upon incubation with mast cells.     

Peptide bond cleavage site determination of novel proteolytic enzymes found in ROS 17/2.8 cell lysates

We have identified proteolytic activities in the rat osteoblastic osteosarcoma cell line ROS 17/2.8 which are capable of cleaving a peptide substrate for protein kinase C-mediated phosphorylation (PSPKC, Pro-Leu-Ser-Arg-Thr-Leu-Ser-Val-Ala-Ala-Lys). Using polyacrylamide gel electrophoresis conditions similar to those used to resolve small molecular weight proteins, the peptide bonds of PSPKC which are cleaved by the proteolytic activities present in ROS 17/2.8 cell lysates have been determined. These activities cleave the Ser-Arg, Thr-Leu, and Ser-Val peptide bonds. To date, no proteolytic activities present in osteoblast cell lysates have been described with the aforementioned peptide bond specificities, suggesting that these activities are novel. The PSPKC-cleaved peptide fragment pattern generated was similar for several different osteoblast cell lysates. Lysates generated from different rat tissues were also able to cleave PSPKC, but the peptide fragment pattern generated by ROS 17/2.8 cell lysates appeared to be unique amongst these tissues. © 1996 Wiley-Liss, Inc.     

Taxonomic Characters and Culture Conditions of a Bacterium which Produces a Lytic Enzyme on Rhizopus Cell Wall

A bacterium R–4 which produces a novel type of lytic enzyme which lyses fungal and yeast cell walls was isolated from the air and was identified to belong to the genus Bacillus.

Production of the enzyme appeared to require a high concentration of nitrogen source in medium. No inducing substance was needed for the enzyme production.

A crude preparation of the enzyme was used to characterize the lytic activity. From the lytic spectrum, the enzyme seemed to have the highest activity toward the cell walls of species in the genus Rhizopus among various fungi and yeasts tested, A proteolytic activity was shown to be parallel with the lytic activity. The lytic activity was also accompanied with the liberation of reducing sugars from Rhizopus cell wall, but no activity on some known carbohydrates tested was detected in the preparation.     

Proteolytic Activity of the MMGP1 Antifungal Peptide Derived from Marine Metagenome

An antifungal peptide, MMGP1 with direct cell penetrating property was recently reported from marine metagenome. The peptide showed efficient in vitro proteolytic activity, which could be associated with its antifungal activity. The proteolytic activity of MMGP1 was confirmed by tricine SDS-PAGE and gel filtration chromatography. Liquid chromatography-mass spectrometry analysis of MMGP1 treated bovine serum albumin (BSA), RNaseA and casein substrates revealed that the peptide does not have common cleavage position and it cleaves the substrates non-specifically at all peptide bonds. The proteolytic activity of MMGP1 was enhanced in the presence of Mn²⁺. Molecular docking studies revealed that the predicted active site residues of MMGP1 could interact with BSA, RNaseA and casein.     

Metallo-protease activity increases prior to ovulation in brook trout (Salvelinus fontinalis) and yellow perch (Perca flavescens) follicle walls

Proteolytic activity was measured in the follicle wall surrounding oocytes from brook trout (Salvelinus fontinalis) and yellow perch (Perca flavescens) by use of two different protease assays: sodium dodecyl sulfate-polyacrylamide gel electrophoresis (substrate-SDS-PAGE) and a chromogenic synthetic peptide for type I collagen. In brook trout follicle walls, substrate-SDS-PAGE studies demonstrated that the activity of two proteolytic enzymes (80 kDa and 66 kDa) increased significantly before ovulation. The 80 kDa enzyme decreased significantly after ovulation whereas the 66 kDa enzyme remained elevated following ovulation. In yellow perch follicle walls, substrate-SDS-PAGE studies demonstrated that the activity of the major protease (66 kDa) increased before ovulation and remained elevated after ovulation. A chromogenic synthetic peptide was used to assay collagenolytic activity in follicle walls of brook trout and yellow perch. This assay revealed that collagenolytic activity increased significantly in both species before ovulation and remained elevated after ovulation. These findings suggest that metallo-proteases are involved in digesting the follicle wall in teleosts before and after ovulation.     

Various physico-chemical properties of lytic proteinase L2 of the enzyme preparation lysoamidase isolated from bacteria of Pseudomonadaceae family

Some physico-chemical properties of lytic proteinase L2 isolated from the enzymatic microbial preparation of lysoamidase were studied. The molecular mass of the enzyme is 15 000 Da, pI is 5.3. The enzyme hydrolyzes casein as well as the cells and cell walls of Staphylococcus aureus 209-P. The pH optimum of casein hydrolysis lies at 9.5; that for cell wall hydrolysis at 8.0. The temperature optimum for casein hydrolysis and cell lysis lies at 55 degrees C and 65 degrees C, respectively. The enzyme proteolytic activity is inhibited by serine proteinase inhibitors in a greater degree than the lytic activity. 50% of the proteolytic and lytic activities is lost upon enzyme heating for 15 min at 65 degrees C.     

Trypanosoma cruzi: Isolation and characterization of a protease

Ion-exchange chromatography and Sephadex gel filtration were used to isolate a soluble proteolytic enzyme from culture (epimastigote) forms of Trypanosoma cruzi. The enzyme had a molecular weight of ~200,000 and an isoelectric point of pH 5.5. The enzyme exhibited protease, esterase, and transamidase activity, with a Michaelis constant of 0.122 mmole/liter [substrate: α-N-benzoyl-dl-arginine-p-nitroanilide (BAPA)]. The enzyme was specific for peptide bonds, involving the carboxyl groups of arginine, tryptophan, or α-N-substituted lysine. Two percent of the enzyme molecule was carbohydrate; glucose, mannose, xylose, galactose, and glucosamine were detected. The enzyme was inhibited by several sulfhydryl inhibitors, and was highly susceptible to oxidation. We concluded that the enzyme possesses active sulfhydryl groups.     

A Protease that Participates in Yeast Cell Wall Lysis during Zymolyase Digestion

Zymolyase B decreased the turbidity of a yeast cell wall suspension by about 50% and caused release of peptide-mannan from the cell walls. However cell walls treated with the enzyme still maintained the cell shape. The effect of the enzyme on the cell walls was inhibited by yeast mannan and completely counteracted by treatment of the enzyme with DFP. The activity was not affected by pH, but was considerably reduced by incubation of the enzyme at 55°C for 15 min, a treatment that did not affect the proteolytic activity. Heat-treatment decreased the molecular weight of the enzyme from 29,000 to 22,500 and its sensitivity to yeast mannan. Yeast mannan caused noncompetitive inhibition of the proteolytic activity of the native enzyme and competitive inhibition of that of the heat-treated enzyme. Modification of tryptophan residues of Zymolyase B resulted in decreased sensitivity to yeast mannan and a decrease in the activity of the enzyme on yeast cell walls as well as heat-treatment. On the basis of these results, it is hypothesized that Zymolyase B binds to the cell wall mannans and changes their conformation, making the attached proteins susceptible to proteolysis, and then releases peptide-mannan from the cell walls.     

A novel endopeptidase with a strict specificity for threonine residues at the P1' position

An endopeptidase was purified from Archachatina ventricosa by chromatography on columns of gel filtration, DEAE-Sepharose and phenyl-Sepharose. The preparation was shown to be homogeneous by polyacrylamide gel electrophoresis and capillary electrophoresis. The purified enzyme displayed two protein bands on SDS-polyacrylamide gel electrophoresis with estimated molecular weights of 90,000 and 121,000. The protease exhibited maximum proteolytic activity at 55 degrees C and at pH 8.0, but it retained more than 85% of its activity in the pH range 7.5 to 8.5. It was completely inactivated by the chelating agents EDTA and 1,10-phenanthroline which are metalloprotease inhibitors. Studies on substrate specificity showed that only the amide bonds of peptide substrates having a threonine residue at the P1' position were hydrolyzed by the purified protease. This endopeptidase constitutes a novel tool for the study of proteins in view of its narrow and unique substrate specificity.     

Sphere-rod morphogenesis in Arthrobacter crystallopoietes. II. Peptides of the cell wall peptidoglycan

Cell walls of Arthrobacter crystallopoietes grown as spheres and as rods were solubilized by treatment with the B enzyme from Chalaropsis, an N-acetylmuramidase. The neutral glycopeptides were then isolated by chromatography on ECTEOLA cellulose. The glycopeptides, consisting of disaccharide-peptide units interlinked by peptide cross-bridges, were fractionated by gel filtration on Sephadex columns into oligomers of various sizes. The size distribution ranged from monomers with no cross-bridges to polymers with a high degree of polymerization, but did not differ significantly between cell walls from cells grown as spheres or rods. Some small differences in the distribution of C- and N-terminal amino acids were found. Analyses revealed that all the peptide bridges in the glycopeptide fractions from rod cell walls were formed by one l-alanine residue. In sphere cell walls, l-alanine was also found, but, in addition, higher oligomers of the glycopeptide contained glycine in their cross-bridges. These results were confirmed by determinations of C- and N-terminal amino acids released after lysostaphin and AL-1 enzyme digestions and by Edman degradations. Models representing the structures of the sphere and rod cell walls are presented. These structures indicate that the sphere cell wall is probably a more loosely knit macromolecule than is the rod cell wall.     

Proteolysis of spectrin by trypsin and pronase in the presence of phospholipid suspensions

The effect of phospholipid suspensions on the proteolysis of isolated spectrin was examined by SDS-polyacrylamide gradient gel electrophoresis. Proteolysis of spectrin in the membranes by trypsin and pronase was also studied. It was found that electrophoretic patterns of spectrin fragments were influenced by the presence of the suspension prepared from phosphatidylethanolamine:phosphatidylserine (60:40) mixture and of phosphatidylcholine. Qualitative changes in the proteolytic patterns obtained after proteolysis of spectrin by pronase in the presence of phosphatidylcholine suspension were observed. The changes in the sensitivity of spectrin towards proteases result probably from changes in the accessibility of some peptide bonds upon the interaction of this extrinsic protein with phospholipids.     

Heterotropic interactions of AMP and glucose binding sites in phosphorylase a are destroyed by limited proteolysis

Subtilisin BPN' hydrolyses a single peptide bond in phosphorylase a. The two proteolytic fragments are attached to each other by noncovalent bonds in solution as shown by gel filtration and ultracentrifugation studies. The subtilisin nicked phosphorylase a is inactive, however, still binds AMP and glucose as judged by equilibrium dialysis and fluorescence experiments. The modified enzyme can be dephosphorylated by protein phosphatase and AMP is an effective inhibitor of the dephosphorylation reaction. Glucose cannot cancel the AMP inhibition as well as cannot expel AMP from the nucleotide binding site. Thus a single nick in the polypeptide chain breaks the "communication" between the two ligand binding domains.     

Purification and properties of an extracellular protease from Myxococcus virescens.

An extracellular protease from Myxococcus virescens was purified by phosphate precipitation, gel exclusion, and ion-exchange chromatography. The enzyme appeared homogeneous upon disc electrophoresis. The molecular weight of the protease was estimated to be 26,000. The enzyme was rapidly inactivated by ethylenediaminetetraacetate, but the activity could be partially restored by divalent cations. Diisopropylphosphorofluoridate inhibited enzyme activity completely. Michaelis-Menten kinetics were obeyed with casein and hemoglobin as substrates. First-order kinetics were obtained with elastin as the substrate, provided trypsin was in excess. Petidolytic activity indicated that the peptide bonds hydrolyzed by the enzyme were mainly those involving amino acids with nonpolar side chains.     

Purification and partial peptide sequence analysis of the boar 32 kDa sperminogen

Boar 32 kDa sperminogen was purified from acid extracts of washed epididymal spermatozoa, and partial peptide sequence was determined. Boar sperminogen was purified from the acid extracts of boar spermatozoa by gel filtration through Sephadex G-75 column, followed by preparative SDS-PAGE. Gelatin zymographic analysis of the gel-filtered fractions showed that sperminogen was composed of three separate proteolytic bands. Among the three proteolytic bands, the 32 kDa sperminogen band which showed the strongest proteolytic activities upon activation was sliced out and eluted from the gel fragments. The eluted 32 kDa sperminogen was then subjected to peptide sequencing. Since the N-terminus of the 32 kDa sperminogen was blocked for peptide sequencing by Edman degradation method, the internal amino acid sequence of the sperminogen was obtained from the CNBr-digested peptides of sperminogen. The amino acid sequence of the analyzed peptide of the 32 kDa sperminogen showed 100% identity with that of proacrosin.     

Reevaluation of hemorrhagic toxin, HR-I, from Agkistrodon halys blomhoffii venom: proof of proteolytic enzyme

HR-I is a hemorrhagic toxin originally isolated from Agkistrodon halys blomhoffii (Mamushi) venom by Oshima et al. (1972). It was reported by the original investigators that it was nonproteolytic when casein was used as the substrate. HR-I was isolated again and proteolytic activity was tested using different substrates and assay methods. It is shown that HR-I is indeed a proteolytic enzyme hydrolyzing a number of peptide bonds. This present investigation suggests that more than one method should be used for proteolytic enzyme assay of hemorrhagic toxins. Toxicological and biochemical properties of HR-I were further investigated and are reported in this paper.     

Proteolytic inactivation of 1, 4-α-d-glucan 6-α-d-glucosyltransferase purified from Aspergillus niger R-27

An extracellular 1,4-α-d-glucan 6-α-d-glucosyltransferase [d-glucosyltransferase, 1,4-α-d-glucan:1,4-α-d-glucan(d-gluco 6-α-d-glucosyltransferase, EC 2.4.1.24] from Aspergillus niger R-27 has been purified and the kinetics of its proteolytic inactivation with subtilisin studied. The purified enzyme was shown to be homogeneous using disc polyacrylamide gel electrophoresis. It contained 16.0% mannose, 0.19% glucose and 2.95% 2-acetamido-2-deoxy-d-glucose. The characteristic feature of the proteolytic degradation of glucosyltransferase is rapid hydrolysis of ～12 peptide bonds per mol and the formation of an active intermediate product which is more resistant to further proteolysis, but is easily heat-inactivated. The isolation and some properties of glucosyltransferase are also described.     

Fractionation and partial characterization of the products of autolysis of cell walls of Bacillus subtilis

Young, Frank E. (Western Reserve University, Cleveland, Ohio). Fractionation and partial characterization of the products of autolysis of cell walls of Bacillus subtilis. J. Bacteriol. 92:839-846. 1966.-Autolysis of the cell wall of Bacillus subtilis by an indigenous autolytic enzyme results in solubilization of 90% of the cell wall. The solubilized cell wall (supernatant fraction) was fractionated by the combination of ion-exchange chromatography on diethylaminoethyl cellulose and gel filtration on Sephadex G-25 into polysaccharides (composed of N-acyl glucosamine and N-acyl muramic acid), mucopeptides, peptides, and teichoic acid. The chemical composition of the products of autolysis confirms the proposed mechanism of autolysis and establishes the autolytic enzyme as an N-acyl muramyl-l-alanine amidase. The heteropolymers in the cell wall are linked by peptide bridges. Two peptides which account for 70% of the peptides of the cell wall have a molar ratio of 1.0:0.9:1.3 for diaminopimelic acid, glutamic acid, and alanine, respectively. Other minor peptides contain diaminopimelic acid, glutamic acid, and alanine in molar ratios of 1.0:0.9:1.5, 1.0:0.5:1.0, and 1.0:1.5:1.7, respectively. The procedures employed in this study should be applicable to the fractionation of heteropolymers in cell walls of other gram-positive organisms and thereby aid in the study of the structure of antigenic determinants and endotoxins.     

Purification and characterization of an X-prolyl-dipeptidyl peptidase from Lactobacillus sakei

An X-prolyl-dipeptidyl peptidase has been purified from Lactobacillus sakei by ammonium sulfate fractionation and five chromatographic steps, which included hydrophobic interaction, anion-exchange chromatography, and gel filtration chromatography. This procedure resulted in a recovery yield of 7% and an increase in specificity of 737-fold. The enzyme appeared to be a dimer with a subunit molecular mass of approximately 88 kDa. Optimal activity was shown at pH 7.5 and 55 degrees C. The enzyme was inhibited by serine proteinase inhibitors and several divalent cations (Cu(2+), Hg(2+), and Zn(2+)). The enzyme almost exclusively hydrolyzed X-Pro from the N terminus of each peptide as well as fluorescent and colorimetric substrates; it also hydrolyzed X-Ala at the N terminus, albeit at lower rates. K(m) s for Gly-Pro- and Lys-Ala-7-amido-4-methylcoumarin were 29 and 88 microM, respectively; those for Gly-Pro- and Ala-Pro-p-nitroanilide were 192 and 50 microM, respectively. Among peptides, beta-casomorphin 1-3 was hydrolyzed at the highest rates, while the relative hydrolysis of the other tested peptides was only 1 to 12%. The potential role of the purified enzyme in the proteolytic pathway by catalyzing the hydrolysis of peptide bonds involving proline is discussed.     

Study of the structure of the thylakoid membrane-bound chloroplast coupling factor CF1.

The structure of thylakoid membrane-bound chloroplast coupling factor CF1 was studied by limited proteolysis followed by sodium dodecylsulfate polyacrylamide gel electrophoresis and N-terminal sequence analysis. The N-terminal fragment of the alpha-subunit was shown to have an exposed area including the peptide bond R21-E22. The cleavage of this peptide bond caused the alphaK24-V25 bond to be exposed to the outside. In the N-terminal fragment of the beta-subunit, the L14-E15 bond was identified and found to be subject to trypsinolysis. Also, the alphaR140-S141, alphaG160-R161, and betaG102-G103 bonds were accessible to the proteolytic attack. In general, the beta-subunit of membrane-bound CF1 is more sensitive to proteolysis than that of solubilized CF1. The products of proteolysis of the alpha-subunit did not contain the polypeptides typical of the reaction of cleavage of the alphaE17-G18 and alphaE22-V23 bonds in isolated CF1. These results suggest a significant structural difference between soluble and membrane-bound CF1. A number of peptide bonds, alphaG160-R161 in particular, were shown to be shielded from proteolytic attack by papain in illuminated thylakoid membranes, probably as a result of membrane energization. In contrast, the light-induced reduction of the gamma-subunit caused an increase in the accessibility of some peptide bonds to this protease, including the alphaG160-R161 bond.     

Purification and partial characterization of a lysine-specific protease of Porphyromonas gingivalis

Abstract A lysine-specific protease hydrolysing peptide bonds at the carboxyl side of lysine residues in Porphyromonas gingivalis was purified from culture supernatant by a combination of ion-exchange chromatography, gel filtration, and affinity chromatography. The molecular mass was 48 kDa and the p I value was 7.3. The enzyme hydrolysed the peptide bonds at the carboxyl side of lysine residues in synthetic substrates and natural proteins.