Studies on the interaction between Streptomyces pepsin inhibitor and several acid proteinases by means of a zinc(II)-dye complex as a probe.

The zinc(II) complex of pyridine-2-azo-p-dimethylaniline is bound to several acid proteinases, at pH 5.0, accompanied by a change is the visible absorption spectrum. Streptomyces pepsin inhibitor, which was discovered by Satoi and Murao (Satoi, S. and Murao, S. (1970) Agric. Biol. Chem. 34, 1265-1267 and Satoi, S. and Murao, S. (1971) Agric. Biol. Chem. 35, 1482-1487), is also bound to acid proteinases. Spectrophotometric studies with ten acid proteinases from different sources have revealed that in several acid proteinases, zinc(II)-pyridine-2-azo-p-dimethylaniline is released from the enzyme by the inhibitor, while some acid proteinase forms a quaternary complex, zinc(II)-pyridine-2-azo-p-dimethylaniline-inhibitor-enzyme. It is speculated that zinc(II)-pyridine-2-azo-p-dimethylaniline is bound to two catalytic carboxylate groups in the active site of the acid proteinases and the inhibitor is bound mainly to the substrate-binding site of the enzymes. The binding of the inhibitor may overlap the catalytic site completely or partially. The degree of overlapping is characteristic of the kind of acid proteinases.     

Some Properties of Neutral Proteinases I and II of Aspergillus sojae as Zinc-containing Metalloenzyme

Some experiments were carried out with purified neutral proteinases I and II of Aspergillus sojae in relation to their characteristics as metalloenzyme.

The both enzymes contained one gram atom of zinc and about two gram atoms of calcium per mole (molecular weights of 41,700 for I and 19,800 for II were estimated by gel filtration) of enzyme protein, and the zinc was essential for the activity. Some metal-chelating agents, such as ethylenediaminetetraacetic acid (EDTA), o-phenanthroline, 8-hydroxyquinoline and α,α′-dipyridyl, inhibited the activity of the both enzymes. In the inactivation of neutral proteinase II by EDTA a distinct pH-dependency was observed. The EDTA-inactivated enzymes were reactivated fully or partially by the addition of some metal ions such as Zn²⁺, Co²⁺, Mn²⁺, Cu²⁺ (only neutral proteinase II) and Ni²⁺. Zinc-free apo-enzymes were prepared from the native enzymes by the dialysis against EDTA solution. The apo-enzyme of neutral proteinase I still contained calcium, while that of neutral proteinase II did not. The apo-enzymes restored their activity for the most part either by the addition of excess amount of zinc or by mixing with a stoichiometric amount of zinc in the presence of calcium at an alkaline condition.     

Purification and Characterization of Extracellular Proteinases of Aspergillus oryzae

The extracellular proteinases of Aspergillus oryzae EI 212 were separated into two active fractions by (NH4)2SO4 and ethanol fractionation followed by diethylaminoethyl-Sephadex A-50 and hydroxyapatite chromatography. The molecular weight was estimated by gel filtration to be about 70,000 and 35,000 for proteinases I and II, respectively. Optimum pH for casein and hemoglobin hydrolysis was 6.5 at 60 C for proteinase I and 10.0 at 45 C for proteinase II, and for gelatin hydrolysis it was 6.5 at 45 C for both enzymes. The enzymes were stable over the pH range 6 to 8 at 30 C for 60 min. The enzyme activity for both the proteinases was accelerated by Cu2+ and inhibited by Fe2+, Fe3+, Hg2+, and Ag+. Halogenators (e.g., N-chlorosuccinimide) and diisopropyl fluorophosphate inhibited proteinase II. Sulfhydryl reagents such as p-chloromercuribenzoate and iodoacetate inhibited proteinase I. Sulfhydryl compounds accelerated the action of both enzymes.     

Some Properties of Two Proteinases from a Luminous Bacterium,Vibrio harveyi Strain FLN-108

Two proteinases (I and II) from a marine luminous bacterium, FLN-108, were purified to homogeneity. The molecular weights of proteinases I and II were estimated to be 49,000 and 46,000, comprising a dimer of 23,000 molecular weight subunits, respectively. These enzymes were most active at from pH 8.0 to pH 9.0 and 50°C, and stable below 45°C. These enzyme activities were inhibited by EDTA and orthophenanthrolin. Phosphoramidon inhibited the activity of proteinase II, but not that of proteinase I. Metal ions such as Cu²⁺ , Hg²⁺ , and Ni²⁺ strongly inhibited these activities. These results indicate that the proteinases I and II are metal-chelater-sensitive, alkaline proteinases.     

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.     

Proteinase activities in resting and germinating seeds of Scots pine, Pinus sylvestris

Resting seeds of Scots pine contained a moderate amount of acid proteinase activity, about 90% of which was inhibited by pepstatin A and about 10% by p-hydroxymer-curibenzoate. In gel chromatography on Sephacryl S-200 the proteinase activity showed a complex elution pattern with poorly separated peaks at positions corresponding to mol. wts. 100,000 and 30,000 and several shoulders. The results suggested that pine proteinases I and II, which are the main proteinases in the endosperms of germinating seeds (Salmia 1981: Physiol. Plant. 51: 253–258), were not present in the resting seeds.—Seedling extracts showed a low level of acid proteinase activity, which separated into several peaks in chromatography on Sephacryl S-200. As none of the peaks had the catalytic properties of proteinase I or II, it seems that these endospermal enzymes are also lacking in the seedling tissues.—In the endosperms of germinating seeds the activity of the pepstatin-sensitive acid proteinase(s) remained at a constant level throughout the period of reserve protein mobilization (lasting up to the stage when the length of dark-grown seedlings was 60 mm). Proteinases I and II were absent from resting seeds, showed a small increase up to the 20-mm stage, and then increased rapidly up to the 60-mm stage.—Resting embryos contained relatively higher acid proteinase activity than resting endosperms, and again about 90% of it was inhibited by pepstatin A and about 10% by p-hy-droxymercuribenzoate. During germination the former activity decreased, the latter activity remained at approximately the same level, and the activity of the other acid proteinases increased continuously with the growth of the seedling.—It is concluded that the pepstatin-sensitive proteinase(s), which is not affected by endogenous proteinase inhibitors, plays a central role in the initiation of reserve protein mobilization in both the embryo and the endosperm. Proteinases I and II, on the other hand, seem to account for the greater part of reserve protein breakdown in the main protein storage tissue, the endosperm.     

Extracellular proteinase spectrum ofAspergillus terreus grown on various substrates

The level of proteinase activity and the ratio of proteinases I and II, secreted byAspergillus terreus, a cellulase producer, was followed during its growth on media containing various carbon sources. Correlation was found between the level of proteinase secretion and the rate of change of the cellulase complex spectrum. The extracellular proteolytic system ofA. terreus was presented mainly by proteinase II (metalloproteinase) during cultivation under conditions favoring fast accumulation of low-molar mass cellulases. The results indicate that proteinase II could be responsible for the limited proteolysis of high-molar mass cellulases ofA. terreus into smaller enzymes of the cellulolytic complex, thus changing their substrate specificity.     

Isolation and immunological characterization of three highly purified serine proteinases from Antarctic krill (Euphausia superba)

Summary Three individual serine proteinases (I, II, III) originating from Antarctic krill (E. superba) were separated and highly purified using a combination of affinity and high resolution ion exchange chromatography. Each enzyme showed a single protein band (30 000 Daltons) in sodium dodecyl sulphate polyacrylamide gradient gel electrophoresis indicating high purity and identical molecular weights. Moreover, each enzyme demonstrated one immunoprecipitate on crossed immunoelectrophoresis (two-dimensional agarose gel electrophoresis) using polyclonal rabbit antibodies which confirmed the high purity of the individual enzymes. A mixture of the three enzymes (I, II, III) revealed two immunoprecipitates, not one or three which should have been the case for identical or non-identical immunological properties. Double immunodiffusion test according to Ouchterlony exhibited immunological identity between enzyme II and III. Enzyme I showed only partial identity with II/III. These findings correlated well with biochemical data on the three serine proteinases. Enzyme I is able to liberate free amino acids from polypeptides in comparison with enzyme II and III (classical true endopeptidases), which do not. We suggest that these unique biochemical properties also have their immunological counterpart expressed as other antigenic determinants of the molecular structure.     

Phage display of a double-headed proteinase inhibitor: Analysis of the binding domains of potato proteinase inhibitor II

Potato proteinase inhibitor II (PI2) is a serine proteinase inhibitor composed of two domains that are thought to bind independently to proteinases. To determine the activities of each domain separately, various inactive and active domain combinations were constructed by substituting amino acid residues in the active domains by alanines. These derivatives were expressed as soluble protein inEscherichia coli and exposed on M13 phage as fusions to gene 3 in a phagemid system for monovalent phage display. Inactivation of both active domains by Ala residues reduced binding of phage to trypsin and chymotrypsin by 95%. Ten times more phage were bound to proteinases by domain II compared to domain I, while a point mutation (Leu⁵ Arg) altered the binding specificity of domain I of PI2 phage from chymotrypsin to trypsin. The mutants were used to show that functional PI2 phage mixed with nonfunctional PI2 phage could be enriched 323 000-fold after three rounds of panning. Thus, these results open up the possibility to use phage display for the selection of engineered PI2 derivatives with improved binding characteristics towards digestive proteinases of plants pests.The nucleotide sequence data reported will appear in the EMBL, GenBank and DDBJ Nucleotide Sequence Databases under the accession number L37519 (p303.51).     

Purification and Some Enzymatic Properties of Acid Protease A and B of Scytalidium lignicolum ATCC 24568

Three kinds of acid proteases were purified from the culture filtrate of Scytalidium lignicolum ATCC 24568. About 3 mg of A–1, 6 mg of A–2 and 60 mg of B were obtained from one liter of culture broth. These purified enzymes were monodisperse by physicochemical criteria such as ultracentrifugal analysis and disc electrophoresis.

A–1 and A–2 were very similar to each other on their enzymatic properties except the small difference of isoelectric point. A–1 and A–2 were active between pH 3.0~3.5 toward casein, and stable between pH 2.5 and 5.5 for 20 hr at 37°C. Both enzymes were strongly inhibited by NBS, but not by EDTA, DFP and sulfhydryl reagents.

B was most active at pH 2.0, and stable at pH values between 1.5 and 5.0. This enzyme was also inhibited by NBS and KMnO₄, but not by EDTA, DFP and sulfhydryl reagents.

The molecular weights and isoelectric points of A–1, A–2 and B were 43,000, pH 3.6; 43,000, pH 3.8 and 22,000, pH 3.2, respectively.

A–1 and A–2 were not inhibited by S–PI and synthetic pepsin inhibitor such as diazoacetyl-dl-norleucine methylester (DAN) and 1,2-epoxy-3-(p-nitrophenoxy)-propane (EPNP). B was inhibited by EPNP, but not by S–PI and DAN.     

Two Alkaline Phosphatases from a Butirosin A Producer Bacillus vitellinus

In low-phosphate medium, a butirosin A producer B. vitellinus produced two alkaline phosphatases. These enzymes were fractionated by DEAE-cellulose column chromatography. One phosphatase (Pho I) was eluted with the lower concentration of NaCl compared with the other phosphatase (Pho II). In the wild type strain, Pho I was completely repressed in the high-phosphate medium, but 30% of the fully-derepressed level of Pho II was still produced.

The phosphatase-negative mutant, P-15, that was shown to accumulate butirosin A-6′-N-diphosphate in our previous study, produced only one phosphatase (Pho I) under the low-phosphate condition. Therefore, P-15 was characteristic of the deficiency in Pho II synthesis.

The partially purified preparations of Pho I and II were characterized. Although both enzymes had a similar molecular weight, they could be differentiated in control of synthesis, heat stability, substrate specificity and other properties. Kinetic properties showed that Pho-II was more specific than Pho I to aminoglycoside-phosphates; butirosin A-3′-phosphate, butirosin A-6′-N-diphosphate and 6′-deamino-6′-hydroxybutirosin A-6′-O-diphosphate. The roles of the two phosphatases in butirosin A biosynthesis were discussed.     

Regulation of the formation of proteinases inBacillus megaterium

The exocellular proteinases of asporogenic and sporogenicBacillus megaterium KM (megaterioproteinase A and S) were found to be active enzymes of the monomer type. The electrophoretic mobility of megaterioproteinase A is higher than that of S on acrylamide gel. After mixing, the enzymes could be separated again. The molecular weight of megaterioproteinase A was found to be 20,000–23,500, that of megaterioproteinase S 16,500–20,000 daltons, according to the “molecular sieving” method. The electrophoretic mobility of both proteinases was determined at different pH and the graphically calculated isoelectric point (pI) was found to be 7.3–7.4. The pK values of the ES complex estimated by plotting the logarithm of the maximum velocity of the enzymic reaction against pH were 6.0–6.1 and 7.8–8.0 for both megaterioproteinases. The activation energy was 13,500–13,600 for both enzymes. It is concluded that the above two enzymes resemble each other in enzymic properties but differ in electrophoretic mobility and probably also in molecular weight.     

Reevaluation of the role of auxin binding site II

Binding of 1-naphthylacetic acid (1-NAA) was assayed in microsomal membranes from Zea mays coleoptiles and from hypocotyls of Cucurbita pepo. Auxin binding site II was differentiated from site I binding by using phenylacetic acid (PAA) to saturate site I binding capacity. The amount of type-II binding sites, per gram original fresh weight, was 34 pmol with Zea and 6.4 pmol with Cucurbita. When maize membranes were separated by dextran gradient centrifugation, auxin binding site II migrated coincident with tonoplast marker enzymes. The physiologically active auxin 4-chloroindoleacetic acid (4-Cl-IAA) competed very poorly with 1-NAA binding to both site I and site II. This result suggests that sites I and II are not involved in the regulation of growth. When comparing isolated outer epidermis with intact coleoptile of Zea, similar amounts and ratios of site I and site II binding activities were observed.     

1 '-(6-Hydroxyoctanoyl) nornicotine and 1 '-(7-Hydroxyoctanoyl) nornico-tine,Two New Alkaloids from Japanese Domestic Tobacco

A Streptomyces-pepsin inhibitor (S-PI or pepstatin Ac)-insensitive carboxyl proteinase was found in a still culture filtrate of Ganoderma lucidum (Mannen-take). The new carboxyl proteinase was purified, and about 15 mg of the purified enzyme was obtained from 15 liters of culture filtrate, with 13% recovery. The enzyme showed a single protein band on Polyacrylamide gel electrophoresis.

The enzyme was most active at pH 3.2 toward hemoglobin, and at pH 2.0 toward casein, and stable only in the narrow pH range of 3.5 to 5.2 even under mild treatment (37°C for 3hr). The molecular weight and isoelectric point were 36,000 and pH 5.3, respectively. The enzyme did not contain methionine.

The enzyme was characterized by the following points: (1) the proteolytic activity was not inhibited by carboxyl proteinase inhibitors such as S-PI, DAN, and EPNP; (2) the enzyme was very unstable; (3) the caseinolytic activity was very low compared with the hydrolysis of hemoglobin (about 15%); (4) the enzyme split preferentially the Phe(24)–Phe(25) bond of oxidized insulin B-chain at the rate of 50% for total hydrolysis. These characteristics were compared with the carboxyl proteinases isolated from Scytalidium lignicolum and Lentinus edodes, which were reported to be SPI- and DAN-insensitive carboxyl proteinases.     

Involvement of Microbial Alkyl Hydroxybenzenes in the Regulation of Autolytic Degradation of Yeast Cells

A comparative study was performed of the processes of autolytic degradation of the cells of Saccharomyces cerevisiae and Schizosaccharomyces pombe under conditions simulating the phase of cell death in microbial cultures: (1) during autolysis induced by oleic acid, which is the chemical analogue of factors d₂ (autolysis autoinducer), (2) under the effect of extracellular microbial proteinases (enzymatic lysis), and (3) under the concomitant effect of the enzymes of the endogenous autolytic complex and exogenous proteinases (heterolysis). Regulatory mechanisms controlling the rate and profundity of autolysis were elucidated, relying on the stabilization of hydrolytic enzymes and enhancement of their activity in their complexes with a chemical analogue of microbial autoregulatory factors d₁, which belong to alkylhydroxybenzenes and fulfill functions of chemical chaperones. The changes in the activity of proteinases and enzymes of the autolytic complex were shown to be dependent on the concentration of the analogue at the moment of complex formation.     

Cysteinyl proteinases of Schistosoma mansoni eggs: purification and partial characterization

The egg stage of Schistosoma mansoni, a trematode blood fluke, is known to be responsible for an immunologically mediated granuloma formation. Proteolytic enzymes of S. mansoni eggs may be involved in the penetration of host tissue by eggs and/or may act as antigens to cause a humoral as well as a cell-mediated response leading to granuloma formation. Three acidic, thiol-dependent proteinases from the eggs of S. mansoni were isolated, and 2 major proteinases (I and II) were purified to homogeneity using chromatofocusing, AcA54 ultrogel chromatography, and thiopropyl-Sepharose 6B affinity chromatography. Proteinases I and II have molecular weights of 25,400 and 30,500, and isoelectric points of 6.0 and 5.6, respectively. These enzymes were found to be cathespin B-like cysteinyl proteinases based on similarities in molecular weight, isoelectric point, optimal assay pH, instability to neutral pH, substrate specificity, and inhibitor sensitivity. A monoclonal antibody, specific to S. mansoni egg proteinases was used in immunoblotting studies. Under native, but not under denaturing, conditions for gel electrophoresis, this monoclonal antibody reacted with egg proteinases. This antibody had previously been shown to recognize an antigen in the miracidial penetration glands of schistosome eggs.     

Isolation and Identification of a Microorganism which Produces Non Streptomyces Pepsin Inhibitor and N-Diazoacetyl-dl-norleucine Methylester Sensitive Acid Proteases

Non pepsin inhibitor (S–PI) and diazoacetyl-dl-norleucine methylester (DAN) sensitive acid proteases producing microorganism was isolated from farm soil of Osaka Prefecture.

The isolated strain was identified as Scytalidium lignicolum M–133. When it was aerobically grown on a medium consisting of glucose 5%, meat extract 1.5%, yeast extract 0.1%, KH₂PO₄ 0.2%, MgSO₄·7H₂O 0.05% at pH 3.5 and 25°C, the strain produced two acid proteases, A and B, in the culture broth.

The acid proteases A and B were not at all inactivated by S–PI and DAN. These acid proteases were expected to be a new type of acid protease from the viewpoint of the active site.     

Purification and Characterization of Beauveria bassiana Proteinases

Two chymotrypsin‐like serine proteinases are produced by B. bassiana 278 when grown on different carbon and nitrogen sources. By employing acetone precipitation, gel filtration and ion‐exchange chromatographies, the enzymes were separated from the culture filtrate after propagation of the fungus on medium enriched either with ground larvae of Apis mellifera (Proteinase I) or porcine blood plasma (Proteinase II). The purified enzymes with a molecular mass of approximately 32 kDa hydrolyzed natural protein substrates: casein, hide powder azure (HPA), azocoll and much less elastin Congo Red and collagen. They differ from each other in the optimum pH value, amino acid composition, Michaelis constant and susceptibility to natural chymotrypsin inhibitors. Both proteinases hydrolyze suc‐Ala‐Ala‐Pro‐Phe‐p‐NA with an apparent K_m of 2.03 × 10^—3 M and 1.04 × 10^—4 M, respectively. The turkey ovomukoid (OMTKY) and cathepsin G/chymotrypsin inhibitor inhibit only Proteinase II from the larvae hemolymph of Apis mellifera (AMCI). The association constant of the interaction of this enzyme with AMCI was estimated to be very high (4.11 × 10⁹ M^—1).     

Human renin inhibition by a diazoacyl reagent: Relationship of the enzyme to other proteinases

Human renin is inactivated by a diazoacyl compound (diazoacetylglycine ethyl ester; N₂CHCO-Gly-OEt) in the presence of Cu(II). The mechanism of the inactivation is presumably identical to that which has been determined for pepsin and several other proteinases: esterification of the β-carboxyl of an aspartic acid residue at the active site of the enzyme. Renin's inhibition by the diazoacyl reagent, its specificity toward a hydrophobic sequence, and its inhibition by pepstatin, all suggest a close relationship to the acid proteinases, especially pepsin and cathepsin D. However, renin, a neutral proteinase, would be better classified together with other diazoacyl-inhibited enzymes by active site rather than pH optimum. The term “aspartic proteinase” is suggested for this group of enzymes.     

Comparative Study of Kallikrein-Like Serine Proteinases from Rat Submandibular Glands

Abstract

The present investigation describes the comparative properties, particularly the substrate specificity of three kallikrein-like serine proteinases (I, II and III) purified from rat submandibular gland extract (Bedi, G.S., Prep. Biochem. 22, 67–81. 1992). The physico-chemical and immunological properties of three proteinases were compared by Western blot analysis, immunodiffusion, immuno-electrophoresis, amino terminal sequence analysis, molecular weight determination and isoelectric focusing. Detailed substrate specificity of these proteinases was determined using chromogenic substrates, synthetic peptides and native proteins. The chromogenic substrate tosyl-gly-pro-arg-pNA was hydrolyzed preferentially by Proteinase I. The replacement of pro at the P2 position with bulky hydrophobic residues phe and leu completely abolished the hydrolysis by Proteinase I. The hydrolysis of the chromogenic substrates by Proteinase II was also affected by the amino acid residue present at the P2 position in the order of pro>gly>val>leu>phe. Neither Proteinase I nor Proteinase II hydrolyzed substrates in which arg was replaced with lys at the P1 position. Proteinase III was reactive against all the chromogenic substrates with arg or lys at the P1 position. Synthetic polypeptides T-kinin-leu and insulin B chain were resistant to cleavage by both Proteinase I and II and were cleaved specifically at arg-X peptide bond by Proteinase III. Tonin-like activity of Proteinase II was confirmed by cleavage of the angiotensin 1–14 at phe-his linkage to generate two fragments DRVYIHPF and HLLVYS respectively. All three proteinases cleaved human high molecular weight kininogen but only Proteinase III could cleave T-kininogen. Proteinase III was also reactive towards human and bovine fibronectin, fibrinogen and gelatin. Several other salivary and serum proteins were resistant to cleavage by these proteinases. Although the three enzymes are immunologically related, they differ with respect to size, isoelectric point, amino terminal sequence and inhibition profile.