A Novel Phospholipase C Inhibitor,S-PLI Produced by Streptomyces Sp. Strain No. A-6288

Abstract

S-PLI, an inhibitor of phospholipase C (PLC) produced by Strepromyces sp. strain No. 6288, was purified from the culture filtrate by salting-out with solid ammonium sulfate, column chromatography on CM-cellulose and gel filtration on Sephadex G-75. The molecular weight of S-PLI was estimated to be 65,000 by SDS-polyacrylamide gel electrophoresis. The inhibitor was found to be a glycoprotein with a composition of 609 amino acids and 19 glucose residues having an isoelectric point at 7.8. S-PLI was stable from pH 3 to 10 at 37°C and up to 40° at pH 6.0. The inhibitory activity showed pH-and temperature-dependence with a maximum around pH 7.0 at 50°C. S-PLI inhibited phospholipase C in a competitive manner (K_i value; 9.5 × 10-⁶ mM), but did not inhibit S-Hemolysin, phospholipase A₂, phospholipase B, phospholipase D and phosphatases. S-PLI is the first reported example of a glycoproteinaceous inhibitor of microbial origin which is able to specifically inhibit phospholipase C.     

Purification and Some Properties of Amylase Inhibitor (S-AI)

Amylase inhibitor (S-AI) was purified by about 25 times from culture filtrate of Streptomyces diastaticus subsp. amylostaticus No. 2476 through the methods of adsorption on active carbon, column chromatographies on Dowex 50 W × 2 (H-form) and Dowex 50 W × 2 (NH₄-form), gel filtration on Sephadex G-25, El-complex formation with BLA, isolation of complex by gel filtration on Sephadex G-75, dissociation from complex by the method of acid denaturation, rechromatographies on Dowex 50 W × 2 (NH₄-form) and Sephadex G-25. Homogeneity of this S-AI was examined by means of TLC, where S-AI gave a single spot in various solvent systems. S-AI specially inhibited α-amylases and glucoamylase, but not β-amylases and other glucoside hydrolases.

S-AI was a very stable substance, as it retained 100% of its original activity after being kept for 30 min at 100°C in a pH range between 3.0 and 10.0. The molecular weight of S-AI was estimated to be about 1500 by gel filtration on Sephadex G-15.

S-AI was regarded to be an oligosaccharide which was mainly composed of glucose in an amount of about 85 %. S-AI was hydrolyzed by β-amylase from non-reducing terminal and released two moles of maltose succesively.     

Chromatographic characteristics and subcellular localization of synthase phosphatase,phosphorylase phosphatase and histone phosphatase in human polymorphonuclear leukocytes

Summary Synthase phosphatase, phosphorylase phosphatase and histone phosphatase activity in a leukocyte homogenate were found to have different sedimentation charcteristics: both synthase phosphatase and phosphorylase phosphatase activity are associated with the microsomal fraction, while the majority of histone phosphatase activity (75–85%) was found in the cytosol. Synthase phosphatase, phosphorylase phosphatase and histone phosphatase activities accompanying the microsomal fraction are readily solubilized by 0.3% Triton X-100.When the solubilized microsomal enzymes were chromatographed on Sephadex G-200, the majority of synthase phosphatase, phosphorylase phosphatase and histone phosphatase activity migrated in single peaks corresponding to apparent molecular weights of 380 000, 250 000 and 68 000, respectively. A minor peak of 30 000, which had phosphatase activity against all three substrates was also obtained.Ethanol treatment resulted in solubilization and dissociation of the three phosphatase activities. It was found that although ethanol treatment resulted in a 4-fold increase of phosphorylase phosphatase activity, histone phosphatase activity was decreased (by 60%), while synthase phosphatase activity remained stable. Similar results were obtained when ethanol treatment was performed on the 17 000 × g supernatant.Chromatography of the ethanol-treated microsomes (or homogenate) on Sephadex G-200 showed that the phosphatase activity towards synthase D, phosphorylase a and phosphohistone coincided a M_r 30 000 species. Heat treatment of the M_r 30 000 peak resulted in dissociation of synthase phosphatase and phosphorylase phosphatase activity.Synthase phosphatase was inhibited by phosphorylase a in a kinetically non-competitive manner while histone phosphatase activity was notinhibited by synthase D (8.5 unit/ ml) orby phosphorylase a(12 unit/ ml).     

Recognition of Human Anti-DNA Autoantibodies by Secretory Antigen 85 Complex of Mycobacterium Tuberculosis H37Rv

Secreted mycobacterial protein antigens were isolated from the culture filtrate of Mycobacterium tuberculosis H₃₇R_v strain grown in Sauton's medium. These secretory proteins of Mycobacterium tuberculosis culture filtrate (MTCF) were separated by SDS-PAGE. High titre anti-DNA autoantibodies from the sera of systemic lupus erythematosus (SLE) patients showed remarkable binding and specificity towards MTCF proteins in dot blot and solid phase immunoassays. The major immunodominant secretory protein in MTCF, fractionated by Sephadex G-200 chromatography was the antigen 85 complex comprising of 30 and 31 kDa molecular weight components. Binding of SLE anti-DNA autoantibodies to the antigen 85 complex was further confirmed by Western blotting. The results suggest the possible involvement of secretory mycobacterial protein antigens in anti-DNA antibody induction in SLE.     

Electrophoretic Studies of Alkaline Proteinases from Strains of Aspergillus flavus Group

Entevobacter sp. G-1 which produces chitinolytic and chitosanolytic enzymes, was previously isolated in our laboratory. One major chitinase, designated ChiA, was purified 42.9-fold from a culture filtrate of Entevobacter sp. G-L To purify the chitinase, ammonium sulfate fractionation, DEAE-Sephadex A-50 column chromatography, and gel filtration on Sephadex G-100 column chromatography were used. The ChiA protein had a molecular weight of 60,000 estimated by SDS polyacrylamide gel electrophoresis and an isoelectric point of 6.6. The optimal pH and optimal temperature of ChiA against colloidal chitin were pH 7.0, and 40°C, respectively. The purified ChiA degraded colloidal chitin mainly to GlcNAc₂ with a small amount of GlcNAc₃ and GlcNAc₄. ChiA hydrolyzed flaked chitin, colloidal chitin, and ethylenglycol chitin, but did not hydrolyze carboxymethyl cellulose (CMC), nor >90% deacetylated flaked chitosan. The chitinase activity was 42% inhibited by 10mm EDTA, but was not inhibited by Ca²⁺ (<50 mm) or NaCl (<400 mm). The purified ChiA hydrolyzed colloidal chitin and chitin-related compounds in an endo splitting manner.     

Serratia marcescens-Lytic Enzyme Produced by Micromonospora sp. Strain No. 152

A strain of Micromonospora sp. producing a lytic enzyme toward Serratia marcescens was isolated from soil. The lytic enzyme, called 152-enzyme, was purified from the culture filtrate by salting-out with ammonium sulfate, DEAE-cellulose column chromatography, and gel filtration on Sephadex G-75. The molecular weight of 152-enzyme was 17,000 and the isoelectric point was pH 7.3. The 152-enzyme showed lytic activity toward S. marcescens, Pseudomonas aeruginosa, Proteus vulgaris, Escherichia coli, and Bacillus subtilis, but was completely intert toward Staphylococcus aureus. The enzyme also showed caseinolytic activity. The lytic and caseinolytic activities of 152-enzyme were maximum around pH 11.0 and at 60°C. Both activities were inhibited by DFP and API-2c. Liberation of amino groups from cell walls of P. aeruginosa by incubation with 152-enzyme suggested that the enzyme was a kind of cell wall-lytic peptidase.     

Purification and Properties of Pectinesterase from Acrocylindrium

The crude enzyme fraction of precipitates resulting from the addition of 70% alcohol to the culture filtrate of A. lunatus was separated by CM-Sephadex and Sephadex G-75 chromatography into 13 fractions having lytic activity for M. radiodurans, M. lysodeikticus and P. radiora. Five of the fractions showed similar lytic activity spectra, but the other fractions were separated by the specificities of their lytic activities. This result indicates that the wide lytic spectrum of the crude enzyme against microorganisms is attributable to the action of many lytic enzymes. All fractions, except for P2-2 fraction (designated as the P2-2. enzyme), contained at least two proteins as determined by disc gel electrophoresis. The P2-2 enzyme was purified 34-fold by rechromatography on Sephadex G-75, and appeared to be homogeneous on disc gel electrophoresis. The enzyme was able to lyse intact cells of M. radiodurans and M. lysodeikticus without detergent, and those of P. radiora with detergent, but was not able to digest casein.     

Isolation and Purification of Ascorbate Oxidase from Acremonium sp. HI-25

A screening test was undertaken to isolate microorganisms that produced ascorbate oxidase. The enzyme activity was found in a culture filtrate of a fungal strain (HI-25), newly isolated from a soil sample. Based on the morphological characteristics, this isolate was identified as Acremonium sp. From the examinations of cultural conditions, optimum conditions for enzyme production were found; strain HI-25 was aerobically cultured by a jar fermenter at 25°C in a medium containing 5% glycerol, 2% defatted soybeans, 0.1% monosodium L-glutamate, 0.1% KH₂PO₄, 0.02% MgSO₄ ·7H₂O, and 0.01% KCl, pH 6.0. After cultivation, an ascorbate oxidase was purified from the culture filtrate by an ammonium sulfate fractionation, column chromatographies on DEAE-cellulose and Butyl-Toyopearl, and gel filtration twice on Sephadex G-100. The purification was 850-fold with an activity yield of 8.8%. The purified enzyme gave a single band on SDS polyacrylamide gel electrophoresis, and had a molecular weight of 80,000 by SDS polyacrylamide gel electrophoresis and 76,000 by native gel filtration. This enzyme was most active at pH 4.0, 45°C, and was most stable between pH 6.0–10.0 and at temperatures below 60°C.     

Purification and Properties of Lipase Activator Produced by Streptomyces sp. Strain No. BR-1381

To obtain actinomycetes capable of producing new enzyme affectors such as enzyme inhibitors or activators, a screening test was carried out. Streptomyces sp. strain No. BR-1381 isolated in our laboratory produced a proteinous lipase activator abbreviated as LAV. LAV was purified from the culture filtrate by salting-out with ammonium sulfate, DEAE-cellulose column chromatography and gel filtration on Sephadex G-100. LAV was stable in the pH range from 3 to 7 at 37°c for 20 hr and in a wider range of pH at 4°C for 5 days. LAV itself was very stable against heat treatment, but LAV did not have any effect on the thermal stability of Phycomyces nitens lipase. LAV activated several microbial lipases, but did not activate pancreatic or rice bran lipases. LAV particularly showed strong activation for Phycomyces nitens lipase.     

Partial Hydrolysis of a Synthetic Heptapeptide Related to Gramicidin S

Galactomyces reessii L, isolated as a protopectin-solubilizing enzyme-producing strain, produced protopectin-solubilizing enzyme in the culture filtrate. The enzyme was purified by repeated CM-Sephadex C-50 column chromatography, and isolated as a crystalline form with a yield of 16% of the initial activity. The enzyme was a glycoprotein containing about 2.6% carbohydrate (as pentose). Its isoelectric point was around pH 8.4, and the sedimentation coefficient (s_20,w) was determined to be 3.83 S. The molecular weight was determined to be 30,000 by gel filtration on Sephadex G-75 and 29,300 by ultracentrifugal analysis. The enzyme catalyzed the release of highly polymerized pectin from various protopectins. The enzyme also catalyzed the depolymerization of pectic acid or galacturonic acid oligomers, and was confirmed to be an endo- polygalacturonase.     

Purification of apo-3-D-(-) hydroxybutyrate dehydrogenase from rat liver mitochondria

Summary 3-D-(-) hydroxybutyrate dehydrogenase (EC 1.1.1.30) from rat-liver mitochondria was purified in the form of the soluble, phospholipid-free apoenzyme by a procedure involving: (1) solubilization of the membrane bound enzyme by controlled digestion of membrane phospholipids with porcine pancreas phospholipase A₂; (2) stabilization and separation of the released apoenzyme as a complex with egg-lecithin by gel filtration on Sephadex G-100; and (3) specific displacement of the apoenzyme from the enzyme-lecithin complex by treatment withBothrops atrox venom phospholipase A₂ (in the absence of Ca²⁺ ions) and subsequent separation of the displaced apoenzyme by gel filtration on Sephadex G-100. The method described is adequate for samples containing about 40 mg of mitochondrial protein. The yield in activity is 42% of that present in mitochondria and the degree of purification of the apodehydrogenase is about 170 fold. The purified apodehydrogenase shows one single sharp band when submitted to SDS polyacrylamide gel electrophoresis, with a mobility corresponding to a molecular weight of 38000 daltons. Gel filtration of the apoenzyme on Sephadex G-100 shows two active peaks with molecular weights of 76000 and 38500 daltons, indicating two different states of aggregation, namely, monomer and dimer. The corresponding diffusion coefficients are 7.73 (monomer) and 5.70 (dimer) × 10^–7. The apodehydrogenase preparation is devoid of phospholipids and is catalytically inactive. It can be reactivated by addition of egg lecithin or phospholipid mixtures containing lecithin in a suitable physical state. Reactivation occurs after formation of an active apodehydrogenase phospholipid complex.Abbreviations HBD 3-D-(-) hydroxybutyrate dehydrogenase - apoHBD 3-D-(-) hydroxybutyrate dehydrogenase apoenzyme - SMP submitochondrial particles - DFP diisopropylfluorophosphate - BSA bovine serum albumin - MPL mitochondrial phospholipids - L-diC₁₄ 1,2-dimyristoyl-sn-glycero-3-phosphorylcholine - lysoC₁₄ 1-myristoyl-sn, glycero-3-phosphorylcholine - D-diC₁₀ 2.3-didecanoyl-sn-glycero-1-phosphorylcholine - tlc thin layer chromatography - SDS sodium dodecylsulfate Dedicated to ProfessorLuis F. Leloir on the occasion of his 70th birthday.     

Purification and Characterization of Phosphatidylinositol-specific Phospholipase C in Suspension-cultured Cells of Rice (Oryza sativa L.)

Phosphatidylinositol-specific phospholipase C was purified from the soluble fraction of suspension-cultured rice cells. The apparent molecular weight of rice enzyme was estimated to be 50,000 by both Sephadex G-100 gel filtration and SDS–polyacrylamide gel electrophoresis, indicating that the enzyme is composed of a single polypeptide. The enzyme had an isoelectric point of 6.3. The soluble phospholipase C had a high degree of specificity toward phosphatidylinositol and a weak activity toward phosphatidyl-inositol monophosphate, while the enzyme did not hydrolyze the other phospholipids or p-nitrophenylphosphorylcholine. V_max and K_m values were 5.0, μmol/min/mg protein and 0.3 mM, respectively. The pH dependency of the enzyme activity was sharp with an optimum of 5.2. In addition, the phospholipase C was a Ca²⁺ -dependent enzyme. The marked activation of enzyme was observed in the presence of 10 to 250, μM Ca²⁺ and higher Ca ²⁺ concentrations than 1 mM had a strong inhibitory effect. A possible regulation of the phospholipase C activity by pH and Ca²⁺ concentrations in the rice cells is discussed.     

Toxic exoproducts of citrobacter freundii

A strain of Citrobacter freundii isolated from the feces of a patient with diarrhoea was examined for growth kinetics and toxic exoproduct formation using the complete (BHI) and synthetic culture media. It was found that the test organism in synthetic medium grew distinctly slower than in BHI. Fractionations on Sephadex G-100 column yielded 3 fractions from the complete medium culture filtrate and 2 fractions from the culture filtrate obtained from synthetic medium. The first culture filtrate fractions (F1) were represented by components of the molecular weight over 100,000, the respective second fractions (F2) from complete and synthetic medium were of the molecular weights of about 40,000 and 10,000. In the early skin test on rabbits the toxicity of culture filtrates and their fractions manifested itself by an increased permeability of blood vessels, in the late skin test by a hemorrhagic reaction associated with dilatation of blood vessels and induration of the skin tissue. In a test on mouse foot pad all separated filtrate fractions gave a positive edematous reaction. In cultured Vero cells samples of synthetic medium fractions gave a distinct cytotoxic reaction. Immunochemically, the presence of LPS in culture filtrates as well as some variations in the antigenicity of components from the complete and synthetic medium fractions were found. Apart from LPS some additional high-molecular-weight components were also present in the toxic complex of both first filtrate fractions (F1). Much more attention should be given to analysis of these first fraction complexes as well as to toxinogenicity of second fractions (F2) using some additional tests.     

Purification of two lipases with high phospholipase A1 activity from guinea-pig pancreas

• 1.1. Two cationic lipases (Ia and Ib) were purified from homogenates of fresh guinea-pig pancreas by ion-exchange chromatography on DEAE-Sepharose and CM-Sepharose (twice for the latter) followed by gel filtration on Sephadex G-100.
• 2.2. Both enzymes were homogeneous upon polyacrylamide gel electrophoresis. Their molecular weights are 37000 and 42000 for lipases Ia and Ib, respectively, as determined by gel filtration on Sephadex G-100. Very close values for isoelectric points were found in the pH range 9.3–9.4.
• 3.3. The cationic lipases are characterized by a high phospholipase A activity (500 IU/mg protein using a potentiometric assay with egg yolk lecithin as substrate), resulting in an unusual phospholipase/lipase activity ratio of 1.
• 4.4. Using doubly labelled phosphatidylcholine, a specificity, A₁, was described for the two enzymes, which are unaffected by N-ethylmaleimide, diisopropylfluorophosphate and p-bromophenacylbromide. The enzymes are insensitive to EDTA and slightly inhibited by CaCl₂and MgCl₂, whereas sodium deoxycholate is required for maximal activity.

     

Photoinduced Decarboxylation-dependent Transamination of Pyridoxal 5′-Phosphate-α-Amino Acid Schiff Bases

Bilirubin oxidase was purified from the culture filtrate of Myrothecium verrucaria MT-1 by a procedure involving ammonium sulfate precipitation, charcoal treatment, and QAE-Sephadex A-50 and Sephadex G-100 column chromatographies. The purified enzyme was homogeneous on disc gel electrophoresis.

Copper and carbohydrate were contained in the enzyme. The enzyme was inhibited by Fe²⁺ and compounds that complex with copper. Bilirubin, biliverdin, hemin and chlorophyllin which consist of tetrapyrrole, and substrates of laccase were oxidized by the enzyme. Bilirubin was oxidized more rapidly than other substances. Bilirubin oxidase differed from laccase in reactivity with substances consisting of tetrapyrrole. Substances consisting of tetrapyrrole were oxidized only a little by laccase but rapidly oxidized by bilirubin oxidase. The apparent Km value for bilirubin was calculated to be 190 μm.     

Purification and characterization of oxalate oxidase from wheat seedlings

Oxalate oxidase (OxO, EC 1.2.3.4.) was purified to homogeneity from wheat (Triticum aestivum) seedlings by sequential thermal treatment, ultrafiltration, Sephadex G-100 gel filtration and affinity chromatography with concanavalin A. The enzyme was purified 66.11-fold with a recovery of 21.97%. It showed a subunit molecular mass of 32.6 kDa on SDS-PAGE and a native molecular mass of 170 kDa on Sephadex G-150 filtration, suggesting that it is a pentamer. The wheat OxO had a maximum activity at pH 3.5. Its K _m for oxalate was 0.21 mM. Chemical modification revealed that cysteine, lysine and carboxylate residues were essential for OxO activity, whereas arginine, serine, threonine and tryptophane residues were not essential.     

Purification,characterization and biological activities of phospholipase a from russell's viper (Vipera russelli) venom

• 1.1. The major phospholipase A has been purified to electrophoretic homogeneity from the venom of Vipera russelli (Russell's viper).
• 2.2. The molecular weight of the purified enzyme was estimated to be 31,000 by Sephadex G-75 gel filtration chromatography and 29,000 by SDS-polyacrylamide gel electrophoresis. The enzyme exhibited an apparent K_m value of 2.3 × 10⁻² M.
• 3.3. The phospholipase A showed edema forming, indirect hemolytic and myonecrotic activities but not hemorrhagic activity.

     

Constituents of the Essential Oil of Chrysanthemum japonense var. debile

One hundred fifty strains of actinomycetes were isolated from soils on plate cultures containing beet arabinan as the sole carbon source. About one-third of the culture fluids were found to have arabinosidase activity. A wild-type strain, Streptomyces sp. No. 17-1, was selected as the best producer of arabinosidase. The highest enzymatic activity was obtained in the culture fluid when the initial pH was adjusted to 9.0. An α-l-arabinofuranosidase was highly purified from the culture filtrate of No. 17-1 by combining column chromatography on DEAE-cellulose, gel filtration on Sephadex G-100, and isoelectric focusing. The molecular weight of the purified enzyme was estimated to be about 92, 000, and its isoelectric point was pH 4.4. The enzymatic activity was maximum at pH 6.0 and was completely inhibited by Hg²⁺. The apparent Km value of the enzyme for p-nitrophenyl-α-l-arabinofuranoside was determined to be 3.6 mM.     

Purification of the cellulase complex produced byPenicillium camemberti and its partial characterization

Three cellulase components (FP-ase, CMC-ase and cellobiase) were purified by affinity binding on Avicel followed by Sephadex G-25, DEAE-Sepharose, DEAE-cellulose and Sephadex G-100 chromatography from the culture filtrate of the newly isolated strain Penicillium camemberti. The isolated enzymes had the properties of cellobiohydrolase, endo-1,4-beta-D-glucanase and cellobiase and their respective molar masses were 99, 87 and 61 kDa as determined by molecular sieve chromatography on Sephadex G-100. The amino acid composition of each fraction was also determined.     

Studies on the Autolysis of Aspergillus oryzae

An intracellular nuclease inhibitor was 1270 times purified from a heat treated cell free extract of fresh mycelia of Aspergillus oryzae, by ammonium sulfate fractionation and chromatographies using DEAE-cellulose and Sephadex G-75. The purified sample of the inhibitor showed a UV absorption curve typical for protein, and it was inactivated by proteases such as chymotrypsin. The inhibitor stoichiometrically inactivated nuclease O (an intracellular nuclease of Asp. oryzae), forming an enzyme-inhibitor complex. But, it did not affect nuclease S₁, RNase T₁, RNase T₂ or pancreatic RNase. The inhibitor was insensitive to 10^?5m p-chloromercuribenzoate or 10^?4m Pb²⁺. Molecular weights estimated by the method of Andrews were 23,000 for the inhibitor, 47,000 for nuclease O, and 82,000 for the enzyme-inhibitor complex. The nuclease activity was recovered from the inactive complex by the action of chymotrypsin.

Nuclease O of Asp. oryzae was purified and crystallized from 113.5 kg of wet mycelia and 2 kl of culture filtrate, by salting out with ammonium sulfate and by chromatographies on CM-Sephadex C-50 and Sephadex G-100. The purified nuclease showed a single peak with apparent sedimentation constant 2.9S in an ultracentrifuge. The molecular weight measured by short column method was 64,000. The nuclease was completely inhibited by the specific nuclease inhibitor obtained from Asp. oryzae. The nuclease was activated by 0.1 mm Mg²⁺ and Mn²⁺, and completely inhibited by 1 mm EDTA. Optimum pH for activity was 7.6 for RNA and 7.4 for DNA. The nuclease degraded polyadenylic acid, polyuridylic acid and polycytidylic acid without forming detectable amount of mononucleotides. And, the main product from RNA was oligonucleotides. The enzyme showed no nonspecific phosphodiesterase activity.