An alkaline proteinase of an alkalophilicBacillus sp.

An alkaline serine proteinase was purfied from the culture broth of an alkalophilicBacillus sp. NKS-21. The molecular weight was estimated to be 22,000 by a gel filtration method and 31,000 by SDS-polyacrylamide gel electrophoresis. The isoelectric point was determined to be 8.2. The amino acid composition and CD spectrum were determined. The alkaline proteinase had a pH optimum at 10–11 for milk casein digestion. The specific activity of the alkaline proteinase was 0.35 katal/kg of protein at pH 10.0 for milk casein hydrolysis.The substrate specificity of the alkaline proteinase was studied by using the oxidized, insulin B-chain and angiotensin. An initial cleavage site was observed at Leu¹⁵-Tyr¹⁶, secondary site at Leu¹¹-Val¹², and additional sites at Gln⁴-His⁵, Tyr²⁶-Thr²⁷, and Asn³-Gln⁴ in the oxidized insulin B-chain at pH 10.0. In comparison with the subtilisins Carlsberg and Novo, the alkaline proteinase fromBacillus sp. showed a unique specificity toward the oxidized insulin B-chain. Hydrolysis of angiotensin at pH 10.0 with the alkaline proteinase was observed at Tyr⁴-Ile⁵. The proteinase has aK _m of 0.1 mM andk _cat of 3.3 s^–1 with angiotensin as substrate.     

Macluralisin — a serine proteinase from fruits of Maclura pomifera (Raf.) Schneid.

A serine proteinase was isolated from fruits of Maclura pomifera (Raf.) Schneid. by affinity chromatography on bacitracin-containing sorbents and gel-filtration. The enzyme, named macluralisin, is a glycoprotein with a molecular mass of 65 kDa; its protein moiety corresponds to a molecular mass of 50 kDa. The substrate specificity of macluralisin towards synthetic peptides and insulin B-chain is similar to that of cucumisin, a subtilisin-like proteinase from melon fruit. The enzyme is completely inhibited by diisopropylfluorophosphate. Its amino-acid composition resembles that of a serine proteinase isolated from the Cucurbitaceae. The N-terminal sequence has 33% of its residues identical to those of the sequence of fungal subtilisin-like proteinase K. Hence, Maclura pomifera serine proteinase belongs to the subtilisin family, which seems to be broadly distributed in the plant kingdom.Abbreviations DFP diisopropylfluorophosphate - PMSF phenylmethylsulfonylfluoride - Glp pyroglutamyl - NHC₆H₄NO₂ p-nitroanilide This work was supported in part by a grant from the Russian Foundation for Basic Research.     

Substrate specificity of carboxyl proteinase fromPycnoporus coccineus,a wood-deteriorating fungus

A carboxyl proteinase was purified from submerged-culture filtrate of a wood-deteriorating basidiomycete,Pycnoporus coccineus. The purified enzyme was found to be essentially homogeneous in disc gel electrophoresis tests at pH 9.4 and 2.3. The specificity and mode of action ofP. coccineus carboxyl proteinase I_a were investigated with the oxidized B-chain of insulinP. coccineus carboxyl proteinase I_a hydrolyzed primarily three peptide bonds, Ala¹⁴-Leu¹⁵, Tyr¹⁶-Leu¹⁷, and Phe²⁴-Phe²⁵ bonds, in the oxidized B-chain of insulin.     

Some newly characterized collagenases from procaryotes and lower eucaryotes

Summary Chemical and enzymatic properties of four collagenases newly isolated from anaerobic Clostridium histolyticum, aerobic Achromobacter iophagus, and from two lower eucaryotes, the fungus Entomophthora coronata and the insect Hypoderma lineatum are reviewed.The problems of their biosynthesis and precursors, namely the effect of induction of collagenase and neutral proteinase in Achromobacter by their macromolecular substrates are discussed.The two bacterial collagenases are Zn-metallo-enzymes; the highly purified Clostridium collagenase contains cyst(e)ine, serine phosphate and tryptophan additionally to amino acids reported previously. Achromobacter collagenase has the highest specific activity of all collagenases; it yields by autolysis enzymatically active degraded forms. The active dimer is composed of two identical subunits of molecular weight 35,000. Similarities between Achromobacter collagenase, thermolysin and Bacillus subtilis neutral proteinase in molecular weight, amino acid composition, and amino acids important for the active sites are discussed.The two collagenases from low eucaryotes are serine proteinases; Hypoderma collagenase is homologous to the trypsin family in the amino terminal sequence.The initial cleavage of native collagen by highly purified bacterial collagenases occurs in the central helical part of the a chains and not progressively from the amino terminal end. One of the two initial cleavages produced by Achromobacter collagenase is situated in the region cleaved specifically by vertebrate collagenases, but with different bond specificity. The same is true for the insect collagenase. Entomophthora collagenase is a proteinase of broad specificity which also cleaves collagen in its helical parts. All four collagenases also degrade other proteins according to their bond specificity.     

Substrate specificity of carboxyl proteinase ofAspergillus sojae

The specificity and mode of action ofAspergillus sojae carboxyl proteinase I were investigated with the oxidized B-chain of insulin.A. sojae carboxyl proteinase I hydrolyzed primarily two peptide bonds in the oxidized B-chain of insulin, the Leu¹⁵-Tyr¹⁶ bond and the Phe²⁴-Phe²⁵ bond. Additional cleavage of the bond Tyr¹⁶-Leu¹⁷ was also noted.     

Miltpain, a cysteine proteinase, from milt of Pacific cod (Gadus macrocephalus): purification and characterization

Miltpain (EC.3.4.22.-) is a cysteine proteinase that preferentially hydrolyzes basic proteins, previously found in the milt of chum salmon. Here we report a similar cysteine proteinase in the milt of the marine Pacific cod. The enzyme was isolated and purified 6900-fold and with an estimated mass of 63 kDa by gel filtration chromatography and 72 kDa by SDS/PAGE. Cod miltpain has an optimum pH of 6.0 for Z-Arg-Arg-MCA hydrolysis, and K_m of 11.5 μM and k_cat of 19.0 s⁻¹ with Z-Arg-Arg-MCA. It requires a thiol-inducing reagent for activation and is inhibited by E-64, iodoacetamide, CA-074, PCMB, NEM, TLCK, TPCK, ZPCK and o-phenanthroline. This proteinase strongly hydrolyzes basic proteins such as salmine, clupeine and histone, and exhibits unique substrate specificity toward paired basic residues such as Lys-Arg, Arg-Arg on the substrates of P2-P1. The isoelectric point is 5.2 by isoelectric focusing. N-Terminal sequencing gave a sequence of <EVPVEVVRXYVTSAPEK. The cysteine proteinase from Pacific cod very closely matches the previously reported miltpain from chum salmon.     

Modulation of the Cell-Surface Proteinase Activity of Thermophilic Lactobacilli by the Peptide Supply

The proteolytic system of thermophilic lactobacilli is considered important for bacterial nutrition as well as for the formation of flavor and texture in fermented products. We investigated the influence of peptide content on the cell surface proteinase and intracellular aminopeptidase activities from seven thermophilic lactobacilli strains. The proteinase activities were remarkably reduced in cells grown in the peptide-rich medium MRS or in a chemically defined medium supplemented with Casitone compared with those found in a synthetic medium. The degree of inhibition observed was strain dependent. When proteinase activities were analyzed by their hydrolytic patterns of α- and β-casein degradation, four types of P_III-caseinolytic cleavage specificity were distinguished. Lactobacillus helveticus strains possessed aminopeptidase activities with broader specificity than those found in L. delbrueckii subsp. lactis strains. However, the aminopeptidase activities were not influenced by the peptide content of the medium. Received: 1 February 2002 / Accepted: 27 February 2002     

Characterization of cathepsin B proteinase (AcCP-2) in eggs and larvae stages of hookworm Ancylostoma caninum

Cathepsin B proteinase constitutes a large multigenes family in parasitic and non-parasitic nematodes. The localization of cathepsin B proteinases (AcCP-1 and AcCP-2) in adult worm of Ancylostoma caninum has been characterized (Harrop et al., 1995), but the localization and function in eggs and larval stages remained undiscovered. Here we described the expressing of cathepsin B proteinase (AcCP-2) in Escherichia coli, and immuno-localization of cathepsin B proteinase in eggs and larvae stages of A. caninum. A cDNA fragment encoding a cathepsin B proteinase (AcCP-2) was cloned from A. caninum and expressed in E. coli. Gelatin digestion showed that recombinant cathepsin B proteinase (AcCP-2) has protease activity. The protein level of cathepsin B proteinase in larval and adult worm was detected by western blot. The immuno-localization of cathepsin B proteinase in eggs and larval stages was characterized. The expression of cathepsin B proteinase was more abundant in eggs and larvae stages of A. caninum. It implied that cathepsin B proteinase might play roles in the early development of A. caninum.     

Initial site of cleavage towards oxidized B-chain of insulin by thermophilicStreptomyces alkaline proteinase

The initial site of cleavage towards the oxidized B-chain of insulin by thermophilicStreptomyces alkaline proteinase (EC 3.4.21.14) was investigated. The most susceptible bond was between Leu¹⁵ and Tyr¹⁶. Additional cleavage of the bonds Leu¹¹-Val¹², Leu¹⁷-Val¹⁸, and Gln⁴-His⁵ were noted. The results suggested that the specificity of bond, cleavage of thermophilicStreptomyces alkaline proteinase is close to that of subtilisin Carlsberg.     

On the Mode of Action of Scopulariopsis brevicaulis Proteinase (Prot. II) on the Oxidized Insulin

The proteinase (Prot. II) from Scopulariopsis brevicaulis has rather a broader specificity in its action on oxidized A- and B-chain of bovine insulin, than that of trypsin or chymotrypsin.

The cleavage in the above peptides occurred rapidly at such bonds, where leucine, valine or glutamic acid is linked by its respective carboxyl group, and slowly at the carboxyl side of cysteic acid or alanine.     

Specificity of induced resistance in the tomato, Lycopersicon esculentum

Specificity in the induced responses of tomato foliage to arthropod herbivores was investigated. We distinguished between two aspects of specificity: specificity of effect (the range of organisms affected by a given induced response), and specificity of elicitation (ability of the plant to generate distinct chemical responses to different damage types). Specificity of effect was investigated by examining the effect of restricted feeding by Helicoverpa zea on the resistance of tomato plants to an aphid species (Macrosiphum euphorbiae), a mite species (Tetranychus urticae), a noctuid species (Spodoptera exigua), and to a phytopathogen, Pseudomonas syringae pv. tomato. Prior H. zea feeding was found to increase the resistance of tomato plants to all four organisms. Specificity in elicitation was investigated by examining the effect of aphid feeding on the activities of four defense-related proteins and on the suitability of foliage for S. exigua. Aphid feeding was found to induce peroxidase and lipoxygenase activities but not polyphenol oxidase and proteinase inhibitor activities; this response is distinct from the response to H. zea feeding, which induces polyphenol oxidase and proteinase inhibitors but not peroxidase. Leaflets which had been fed upon by aphids were better sources of food for S. exigua than were leaflets which had not been fed upon by aphids. Studies of both these aspects of specificity are needed to understand the way in which plants coordinate and integrate induced responses against insects with other physiological processes. Received: 20 December 1996 / Accepted: 2 July 1997     

Crystal structure of the bovine α-chymotrypsin:kunitz inhibitor complex. An example of multiple protein:protein recognition sites

The crystal structure of bovine α-chymotrypsin (α-CHT) in complex with the bovine basic pancreatic trypsin inhibitor (BPTI) has been solved and refined at 2.8 Å resolution (R-factor=0.18). The proteinase:inhibitor complex forms a compact dimer (two α-CHT and two BPTI molecules), which may be stabilized by surface-bound sulphate ions, in the crystalline state. Each BPTI molecule, at opposite ends, is contacting both proteinase molecules in the dimer, through the reactive site loop and through residues next to the inhibitor's C-terminal region. Specific recognition between α-CHT and BPTI occurs at the (re)active site interface according to structural rules inferred from the analysis of homologous serine proteinase:inhibitor complexes. Lys15, the P₁ residue of BPTI, however, does not occupy the α-CHT S₁ specificity pocket, being hydrogen bonded to backbone atoms of the enzyme surface residues Gly216 and Ser217. © 1997 John Wiley & Sons, Ltd.     

A new alkaline proteinase with pI 2.8 from alkalophilicBacillus sp.

A new serine alkaline proteinase (ALPase II) was purified from the culture broth of an alkalophilicBacillus sp. NKS-21. The molecular weight of ALPase II was estimated to be 32,000 by SDS-polyacrylamide gel electrophoresis. The enzyme had a very low isoelectric point (pI), which was determined to be 2.8. An optimum pH of this enzyme was 10.2. The specific activity was 0.28 katal/kg of protein for milk casein, 0.34 katal/kg for succinyl-l-alanyl-l-alanyl-l-prolyl-l-phenylalanyl-4-methyl-coumaryl-7-amide (Suc-Ala-Ala-Pro-Phe-MCA) and 8.5 katal/kg for succinyl-l-alanyl-l-alanyl-l-prolyl-l-phenylalanyl-p-nitroanilide (Suc-Ala-Ala-Pro-Phe-pNA).The substrate specificity of the alkaline proteinase was studied with the synthetic fluorogenic and chromogenic substrates. It was most favorable for the enzyme that the P₁ site of the substrate might be hydrophobic and bulky amino residue (Phe or Tyr). When the substrate contained four amino residues, the proteinase efficiently expressed its activity. The alkaline proteinase had higher specificity than those of the bacterial serine proteinases, subtilisins Carlsberg and BPN, and lower specificity than that of serine alkaline proteinase with pI 8.2 (ALPase I) obtained from the same bacteria NKS-21. ALPase II did not react with the anti-ALPase I antiserum.     

Alkaline serine proteinase and lectin isolation from the culture fluid of Bacillus subtilis

Bacillus subtilis strain 316 M was found to produce extracellular alkaline serine proteinase and lectin. The characteristics of proteinase and lectin accumulation during the growth of the producer organism were found to be similar. The maxima of proteolytic and lectin activities were close and observed at 16 h and 14 h of B. subtilis 316 M batch cultivation, respectively. Alkaline serine proteinase was purified by ion exchange chromatography directly from the culture fluid. Proteinase (eluate) purified 40-fold possessed 60–90 units/ml of caseinolytic activity and 240–320 units/ml of elastolytic activity. Eluate obtained after enzyme sorption on the ion exchanger was used for lectin isolation followed by ammonium sulphate precipiration. Lectin purified 12.3-fold was shown to have a high carbohydrate specificity to N-glycolylneuraminic, N-acetylneuraminic, N-acetylmuramic and d-galacturonic acids with minimal inhibiting concentrations of 2.5–7.5 mm. *** DIRECT SUPPORT *** AG903053 00002     

Purification and characterization of the mature,membrane-associated cell-envelope proteinase of Lactobacillus helveticus L89

Lactobacillus helveticus L89 possesses a cell-envelope proteinase (Lb-CEP) that is biochemically and genetically related to that of the lactococci (Lc-CEP). The in-situ proteinase is resistant to autoproteolysis and remains associated with the membrane during lysozyme treatment of cells and subsequent mechanical disruption of the treated cells. The proteinase was purified from isolated membranes by a procedure that preserves the complete in-situ proteinase (mature proteinase) assumed to be the N-terminally processed translation product including the membrane anchor: its monomer molecular mass is approximately 180 kDa. The purified enzyme appeared to be more stable towards heat than hitherto known related, but C-terminally truncated cell-envelope proteinases of lactobacilli and lactococci, which were released from the cells by autoproteolysis. On the basis of its specificity towards caseins, towards the _sl-casein-(1-23)-fragment and towards two differently charged chromophoric peptides, the proteinase was recognized as an (Lb-)CEP_I/III mixed-type variant different from those identified so far among the lactococcal proteinases. Correspondence to: F. A. Exterkate     

Investigating human immunodeficiency virus-1 proteinase specificity at positions P4 to P2 using a bacterial screening system

Inhibitors of human immunodeficiency virus-1(HIV-1) proteinase have been used for several years to treat acquired immunodeficiency syndrome patients. Despite intensive research, however, the substrate specificity of this enzyme is not completely elucidated. Here, we assessed the HIV-1 proteinase P₄ to P₂ substrate specificity using a bacterial screening system. In this system, the bacterial enzyme β-galactosidase has been transformed into an HIV-1 proteinase substrate by insertion of the p6/PR cleavage site. Consequently, HIV-1 processing can be determined by measuring the β-galactosidase activity on X-gal plates and by examination of the extent of cleavage of the β-galactosidase protein itself. We screened a library containing randomized sequences at the P₄ to P₂ positions and found strong preferences for Thr, Ser, and Pro at P₄, for Leu, Met, and Phe at P₃, and for Ser, Met, and Leu at P₂. The frequent observations of Thr at P₄ and Ser at P₂ extend previous findings and offer the possibility of producing inhibitors with different properties. These new data on HIV proteinase specificity illustrate the usefulness of random libraries in the genetic screening system. This approach can be applied to examine any proteinase that has a recognition site extending across several amino acids.     

Antibody raised against extracellular proteinases ofSporothrix schenckii inS. schenkii inoculated hairless mice

Sporothrix schenckii produces two extracellular proteinases, namely proteinase I and II. Proteinase I is a serine proteinase, inhibited by chymostatin, while proteinase II is an aspartic proteinase, inhibited by pepstatin. Studies on substrate specificity and the effect of proteinase inhibitors on cell growth suggest an important role for these proteinases in terms of fungal invasion and growth. There has, however, been no evidence presented demonstrating thatS. schenckii produces 2 extracellular proteinases in vivo. In order to substantiate the in vivo production of proteinases and to attempt a preliminary serodiagnosis of sporotrichosis, serum antibodies against 2 proteinases were assayed usingS. schenckii inoculated hairless mice. Subsequent to an intracutaneous injection ofS. schenckii to the mouse skin, nodules spontaneously formed and disappeared for a period of 4 weeks. Histopathological examination results were in accordance with the microscopic observations. Micro-organisms disappeared during the fourth week. Serum antibody titers against purified proteinases I and II were measured weekly, using enzyme-linked immunosorbent assay (EIA). As a result, the time course of the antibody titers to both proteinases I and II were parallel to that of macroscopic and microscopic observations in an experimental mouse sporotrichosis model. These results suggest thatS. schenckii produces both proteinases I and II in vivo. Moreover, the detection of antibodies against these proteinases can contribute to a serodiagnosis of sporotrichosis.     

WIP1, a wound-inducible gene from maize with homology to Bowman-Birk proteinase inhibitors

We have cloned and sequenced a wound-inducible cDNA clone designated WIP1 (for wound-induced protein) from maize coleoptiles. It was isolated by differential screening of a cDNA library prepared from excised maize coleoptile segments. The deduced amino acid sequence predicts a secretory, cysteine-rich protein of 102 residues with a calculated molecular mass of 11 kDa and a typical N-terminal signal sequence. The protein has about 30% identity with various Bowman-Birk type proteinase inhibitors. Most interestingly, it is novel in that it is double-headed with exclusive specificity for chymotrypsin. WIP1 is strongly wound-induced in contrast to other members of the Bowman-Birk proteinase inhibitor family, which occur in seeds and are regulated during development. The response is fast, similar to defenceinduced genes, and measurable as early as 30 min after wounding. Induction can also be evoked in the intact coleoptiles and the signal is systemically transmitted in the coleoptile to adjacent regions of the wounded area. Isolation and analysis of the corresponding genomic clone reveals that WIP1 contains an intron of 90 nucleotides.     

Action of a cell wall proteinase (PI) from Streptococcus cremoris HP on bovine β-casein

Summary The specificity of a cell wall proteinase (P_I) from Streptococcus cremoris strain HP in its action on bovine -casein was determined. To this end an enzymic digest (pH 6.2; 15° C) of -casein was brought to pH 4.6 and the soluble fraction separated by semi-preparative reversed-phase HPLC. Purified peptides were analyzed by amino acid and end-group analysis. Ten chromatographic components were identified, which together accounted for at least seven cleavage sites all being located in the C-terminal fifty-residue part of -casein. In five cases it concerned a Gln-X or X-Gln peptide linkage. The specificity of this proteinase from S. cremoris HP shows similarity to that reported for a cell wall proteinase from S. lactis NCDO 763 in its action on -casein.Presented at the second FEMS Symposium on Lactic Acid Bacteria held in 1987 at Wageningen, Netherlands     

Initial cleavage site of the oxidized insulin B-chain by a ribosomal proteinase fromStreptococcus lactis,a food microorganism

The unique specificity of a ribosomal proteinase fromStreptococcus lactis, a food microorganism, was investgated with the oxidized insulin B-chain. The initial cleavage site of the enzyme was observed at Leu¹⁵-Tyr¹⁶ bond, and additional site at Arg²²-Gly²³