Purification and properties of an intracellular leucine aminopeptidase from the fungus,Penicillium citrinum strain IFO 6352

An intracellular leucine aminopeptidase (LAP) fromPenicillium citrinum (IFO 6352) was purified to homogeneity using three successive purification steps. The enzyme has a native molecular mass of 63 kDa using HPLC gel filtration analysis and a molecular mass of 65 kDa when using SDS-polyacrylamide gel electrophoresis. This monomeric aminopeptidase showed maximum enzyme activity at pH 8.5. An optimum temperature was 45–50°C whenl-Leu-p-nitroanilide (pNA) was the substrate, and enzyme activity drastically decreased above 60°C. The Michaelis-Menten constants forl-Leu-pNA andl-Met-pNA were 2.7 mM and 1.8 mM, respectively. When the enzyme reacted with biosynthetic methionyl human growth hormone, it showed high specificity for N-terminal methionine residue and recognized a stop sequence (Xaa-Pro). The aminopeptidase was inactivated by EDTA or 1,10-phenanthroline, indicating that it is a metallo-exoprotease. Enzyme activity was restored to 90% of maximal activity by addition of Co²⁺ ions. The activity of EDTA-treated enzyme was restored by addition of Zn²⁺, but reconstitution with Ca²⁺, Mg²⁺ or Mn²⁺ restored some enzyme activity. It is likely that Co²⁺ ions play an important role in the catalysis or stability of thePenicillium citrinum aminopeptidase, as zinc plays a similar function in other leucine aminopeptidases.     

Synthesis of 1-(2,6,6-Trimethyl-4-hydroxy-1-cyclohexen-1-yl)-1,3-butanedione

Streptococcus dysgalactiae IID 678, belonging to group C of the streptococci, secreted a large amount of hyaluronidase (hyaluronate lyase, EC 4.2.2.1) into a culture medium containing hyaluronic acid. The purification procedures of hyaluronidase were 70% ammonium sulfate precipitation, ECTEOLA-cellulose chromatography, phospho-cellulose chromatography, and gel filtration on Sephacryl S-300. The hyaluronidase was purified approximately 27,000-fold from the culture filtrate. The purified enzyme was homogeneous by SDS-poIyacrylamide gel electrophoresis. The enzyme degradated only hyaluronic acid and chondroitin to zl 4,5-unsaturated disaccharides and did not act on other glycosaminoglycans containing sulfate groups, while the degradation rate of chondroitin was about 1/60 of that of hyaluronic acid. The optimum pH was wide, from pH 5.8 to pH 6.6, and the optimum temperature was 37°C. Fe^{2 +}, Cu^{2 +}, Pb^{2 +}, and Hg^{2 +} ions inhibited the activity strongly and Zn²⁺ inhibited it by half. The molecular weight of the enzyme was estimated to be 125,000 by gel filtration and 117,000 by SDS-polyacrylamide gel electrophoresis. The enzyme was different immunochemically from the hyaluronidase from Streptococcus pyogenes belonging to group A.     

Selection of High Ubiquinone 10-Producing Strain of Tobacco Cultured Cells by Cell Cloning Technique

A novel enzyme, which was named N^α-benzyloxycarbonyl amino acid urethane hydrolase, was purified from a cell-free extract of Streptococcus faecalis R ATCC 8043, using N^α-benzyloxycarbonyl glycine as substrate. The enzyme was purified 1300-fold with an activity yield of 8%. The purified enzyme was homogeneous by disc electrophoresis. The molecular weight of the native enzyme is about 220,000 by gel filtration, and a molecular weight of 32,000 was determined for the reduced and denatured enzyme by gel electrophoresis in sodium dodecyl sulfate. The isoelectric point was 4.48. The enzyme was inhibited by p-chloromercuribenzoate. The presence of divalent cations (i.e., Co²⁺ or Zn²⁺) is essential for its activity.     

Sexual agglutinin of mating-type minus gametes inChlamydomonas reinhardtii

The sexual agglutinin from the mating-type minus gametes ofChlamydomonas reinhardtii was purified by gel filtration and hydroxyapatite chromotography. The minus agglutinin was identified as a single glycopolypeptide termed Agg(-) of very high molecular weight by SDS-poly-acrylamide gel electrophoresis. It was also observed as a glycoprotein with agglutinin activity on non-SDS polyacrylamide gels. The native agglutinin appeared to be composed of a complex of Agg(-) subunits. It consisted of about 60% protein and 40% carbohydrate and the activity was diminished by a mild periodate oxidation. When the plus agglutinin was also purified and compared with the minus agglutinin, it was found that both agglutinins migrate in the same position by SDS-polyacrylamide gel electrophoresis, whereas their behaviors on gel filtration and hydroxyapatite chromatography are different.Abbreviations mt ^+/– mating-tape plus or minus - SDS sodium dodecyl sulfate - V_e elution volume - V_o void volume - kDa kilodalton     

Isolation and partial characterization of carbonic anhydrase from erythrocytes of Meleagris gallopavo

Summary A soluble enzyme with carbonic anhydrase activity has been isolated from domestic turkey(Meleagris gallopavo) erythrocytes and purified by chloroformethanol precipitation, ammonium sulfate fractionation and gel filtration on a Sephadex G-75 column. Analytical polyacrylamide gel disc electrophoresis showed one major and two minor bands. The specific activity for the CO₂ hydration reaction was approximately 2000 Wilbur-Anderson units/mg protein at 0°C. The presence of the reducing agents 2-mercaptoethanol or dithioerythritol was required throughout the procedure. Upon removal of the 2-mercaptoethanol by dialysis the activity was lost but could be restored by addition of the reducing agent. The enzymatic activity was inhibited by acetazolamide,p-chloromercuribenzoate ando-iodosobenzoate. Esterase activity was detected withp-nitrophenylacetate as the substrate. The molecular weight of the enzyme was determined as 31,000 by gel filtration and 34,000 ± 2000 with analytical ultracentrifugation. Atomic absorption spectroscopy indicated the presence of zinc in the ratio of one mole of zinc per one mole of enzyme.     

Properties of polyphosphatase ofAcinetobacter johnsonii 210A

Polyphosphatase, an enzyme which hydrolyses highly polymeric polyphosphates to P_i, was purified 77-fold fromAcinetobacter johnsonii 210A by Q-Sepharose, hydroxylapatite and Mono-Q column chromatography. The native molecular mass estimated by gel filtration and native gel electrophoresis was 55 kDa. SDS-polyacrylamide gel electrophoresis indicated that polyphosphatase ofAcinetobacter johnsonii 210A is a monomer. The enzyme was specific for highly polymeric polyphosphates and showed no activity towards pyrophosphate and organic phosphate esters. The enzyme was inhibited by iodoacetamide and in the presence of 10 mM Mg²⁺ by pyro- and triphosphate. The apparent K_m-value for polyphosphate with an average chain length of 64 residues was 5.9 µM and for tetraphosphate 1.2 mM. Polyphosphate chains were degraded to short chain polymers by a processive mechanism. Polyphosphatase activity was maximal in the presence of Mg²⁺ and K⁺.     

Purification and properties of extracellular phytase from Bacillus sp. KHU-10

Bacillus species producing a thermostable phytase was isolated from soil, boiled rice, and mezu (Korean traditinal koji). The activity of phytase increased markedly at the late stationary phase. An extracellular phytase from Bacillus sp. KHU-10 was purified to homogeneity by acetone precipitation and DEAE-Sepharose and phenyl-Sepharose column chromatographies. Its molecular weight was estimated to be 46 kDa on gel filtration and 44 kDa on SDS-polyacrylamide gel elctrophoresis. Its optimum pH and temperature for phytase activity were pH 6.5-8.5 and 40°C without 10 mM CaCl₂ and pH 6.0-9.5 and 60°C with 10 mM CaCl₂. About 50% of its original activity remained after incubation at 80°C or 10 min in the presence of 10 mM CaCl₂. The enzyme activity was fairly stable from pH 6.5 to 10.0. The enzyme had an isoelectric point of 6.8. As for substrate specificity, it was very specific for sodium phytate and showed no activity on other phosphate esters. The K _m value for sodium phytate was 50 M. Its activity was inhibited by EDTA and metal ions such as Ba²⁺, Cd²⁺, Co²⁺, Cr³⁺, Cu²⁺, Hg²⁺, and Mn²⁺ ions.     

Purification and some properties of NAD(P)H dehydrogenase from Saccharomyces cerevisiae: Contribution to glycolytic methylglyoxal pathway

NAD(P)H dehydrogenase was purified approximately 480-fold from Saccharomyces cerevisiae with 6.5% activity yield. The enzyme was homogeneous on polyacrylamide gel electrophoresis. The molecular weight of the enzyme was estimated to be 40,000–44,000 by gel filtration on Sephadex G-150 column chromatography and SDS-polyacrylamide gel electrophoresis. The K_m values for NADPH and NADH were 7.3 μM and 0.1 mM, respectively. The activity of the enzyme increased approximately 4-fold with Cu²⁺. FAD, FMN and cytochrome c were not effective as electron acceptors, although Fe(CN)₆³⁻ was slightly effective. NADH generated by the reaction of lactaldehyde dehydrogenase in the glycolytic methylglyoxal pathway will be reoxidized by NAD(P)H dehydrogenase. NAD(P)H dehydrogenase thus may contribute to the reduction/oxidation system in the glycolytic methylglyoxal pathway to maintain the flux of methylglyoxal to lactic acid via lactaldehyde.     

A Crystalline Aminopeptidase from Streptomyces peptidofaciens: Physico-chemical Properties and Characteristics as a Ca-Metalloprotease

A crystalline aminopeptidase obtained from the culture filtrate of Streptomyces peptidofaciens KY 2389 appeared to be homogeneous on ultracentrifugation and acrylamide gel electrophoresis. The sedimentation coefficient, s_{20, w}., was determined to be 2.6 S. The molecular weight was estimated to be approximately 19,000 by sedimentation equilibrium studies. The amino acid analyses indicated that the enzyme was composed of 147 amino acid residues and contained no sulfhydryl group. The isoelectric point was found to be around pH 7.4 by isoelectric focusing on ampholites.

The enzyme required Ca²⁺ for its maximal activity and was strongly inhibited by some metal-chelating agents such as ethylenediaminetetraacetic acid (EDTA) and o-phenanthroline. The EDTA-inactivated enzyme restored its activity almost completely by the addition of Ca²⁺ The crystalline preparation of aminopeptidase contained 1 g-atom of calcium and about 2 g-atoms of magnesium per mole of enzyme protein, and the calcium was essential for the activity of the enzyme.     

Purification and Characterization of Phospholipase C Preferentially Hydrolysing Phosphatidylcholine in Tetrahymena Membranes

A phospholipase C (PLC) activity that preferentially hydrolyses phosphatidylcholine to diacylglycerol and phosphorylcholine was found to be present in Tetrahymena pyriformis, strain W and most of its activity was recovered in the membrane fraction. This enzyme was extracted with 1% Triton X-100 from the membrane fraction and purified to apparent homogeneity by sequential chromatographies on Fast Q-Sepharose, hydroxyapatite HCA-100S, Mono Q and Superose 12 gel filtration columns. The purified enzyme had specific activity of 2083 nmol of diacylglycerol released/mg of protein/min for dipalmitoylphosphatidylcholine hydrolysis. Its apparent molecular mass was 128 kDa as determined by SDS-polyacrylamide gel electrophoresis and was 127 kDa by gel filtration chromatography, indicating that the enzyme is present in a monomeric form. The enzyme exhibited an optimum pH 7.0 and the apparent K_m value was determined to be 166 μM for dipalmitoylphosphatidylcholine. A marked increase was observed in phosphatidylcholine hydrolytic activity in the presence of 0.05% (1.2 mM) deoxycholate. Ca²⁺ but not Mg²⁺ enhanced the activity at a concentration of 2 mM. This purified phospholipase C exhibited a preferential hydrolytic activity for phosphatidylcholine but much less activity was observed for phosphatidylinositol (～ 9%) and phosphatidylethanolamine (～ 2%).     

Purification and Properties of Three Types of Xylanases Induced by Methyl β-Xyloside from Streptomyces sp.

When mycelia of Streptomyces sp. No. 3137 were cultivated in a medium containing methyl β-xyloside, xylanases (EC 3.2.1.8) were inductively produced into the medium. Three types of enzyme from the culture filtrate have been purified by ultrafiltration with DIAFLO UM-10, chromatography on DEAE-Sephadex A-25, gel filtration on Bio Gel P-100, and isoelectric focusing with Servalyt 6~8 or 9~11. The three purified enzymes, tentatively named X-I, X-II-A, and X-II-B, were homogeneous by Polyacrylamide gel electrophoresis at pH 4.3. The molecular weight of X-I was about 50,000 by SDS-polyacrylamide gel electrophoresis or gel filtration on Bio Gel P-100. The molecular weight of X-II-A and X-II-B were both approximately 25,000 by SDS-polyacrylamide gel electrophoresis and that of X-II-B was 25,680 by the sedimentation-equilibrium method. X-I had an isoelectric point at 7.10, and X-II-A and X-II-B had different isoelectric points, 10.06 and 10.26, respectively. The three enzymes were optimally active at 60~65°C and stable to 55°C. The optimal pH of X-I, X-II-A, and X-II-B were pH 5.5~6.5, 5.0~6.0, and 5.0~6.0, respectively. The ranges of two X-I’s pH stability (pH 1.5 ~ 11.5) were wider than that of X-I’s (pH 3.0 ~ 10.5). These purified preparations hydrolyzed xylotriose, xylotetraose, and xylan but not xylobiose, cellobiose, maltose, carboxymethyl cellulose, or soluble starch. Their actions were inhibited by Hg²⁺ and Fe³⁺ ions, sodium dodecyl sulfate, and N-bromosuccinimide.     

The Structures of Myocotrienins I. and II,a Novel Class of Ansamycin Antibiotic

Endopolygalacturonase I [EC 3.2.1.15], the major component of endopolygalacturonases causing silver-leaf symptoms, was purified from culture liquids of Stereum purpureum by column chromatographies on CM-52 and Sephadex G-100. The purified enzyme was homogeneous on Polyacrylamide gel electrophoresis and ultracentrifugation. The sedimentation coefficient (S_20,W) was determined to be 3.21 S, and the molecular weight was estimated to be 40,000 by gel filtration, 41,000 by SDS-polyacrylamide gel electrophoresis and 44,000 by sedimentation equilibrium. The enzyme had an isoelectric point of pH 8.5. The optimal pH of the enzyme was 3.5 for trigalacturonic acid, 4.0 for tetragalacturonic acid, and 4.5 for pectic acid. The enzyme was stable in the range of pH 4.0 to 9.0 and up to 70%C for 30 min. The amount of the enzyme which was required to induce silver-leaf symptoms on apple trees was 20 μg/tree.     

trans-2, cis-4-Heptadienoic Acid,One of Metabolites of Sporobolomyces odorus

Water-soluble phospholipase B was purified to homogeneity from Torulaspora delbrueckii cell washings. The washings were concentrated by ultrafiltration, and then a fraction with phospholipase B activity was precipitated with ammonium sulfate, and purified by sequential column chromatographies on Octyl-Sepharose CL-4B, DEAE-Sephacel, and Sepharose 6B. The molecular weight of the enzyme was estimated to be 170,000~200,000 by SDS-polyacrylamide gel electrophoresis and by gel filtration with a Sephadex G-200 column. The isoelectric point of the enzyme was 4.0. The purified enzyme had two pH optima at pH 2.5 and pH 7.5. The activity at acidic pH was greatly stimulated by the divalent metal ions tested, but the activity at alkaline pH was stimulated mainly by Ca²⁺ and Fe²⁺. The purified enzyme had both lysophospholipase activity and phospholipase B activity in a ratio of 37:1 at acidic pH and 73:1 at alkaline pH. The amino acid composition of the enzyme was characterized by high contents of Asp, Ser, Leu, and Gly.     

Isolation and Characterization of Nα-Benzyloxycarbonyl Amino Acid Urethane Hydrolase II from Lactobacillus fermenti 36 ATCC 9338

A novel enzyme, which was named N^α-benzyloxycarbonyl amino acid urethane hydrolase II, was purified from a cell-free extract of Lactobacillus fermenti 36 ATCC 9338. The enzyme catalyzed the stoichiometric hydrolysis of N^α-benzyloxycarbonyl arginine to form benzyl alcohol and arginine. The enzyme was purified 106-fold with an activity yield of 3%. The purified enzyme was homogeneous by disc gel electrophoresis. The molecular weight of the native enzyme is about 200,000 by gel filtration, and a molecular weight of 27,000 was found for the reduced and denaturated enzyme by gel electrophoresis in sodium dodecyl sulfate. The isoelectric point of the enzyme was 5.0, it was inhibited by disodium ethylenediamine tetraacetate and p-chloromercuribenzoate, and the presence of a divalent cation, i.e. Co²⁺, is essential for its activity.     

Limited Hydrolysis of Ricin D with Trypsin in the Presence of Sodium Dodecyl Sulfate

An α-amylase which produces both maltotetraose and maltopentaose from starch as the main products was found in the culture filtrate of a strain of Bacillus circulans which was newly isolated from soil. The enzyme was purified to be almost homogeneous on disc electrophoresis in polyacrylamide gel by means of ammonium sulfate fractionation, DEAE-Sepharose column chromatography and Sephadex G-200 gel filtration.

The optimum pH and temperature of the enzyme were around pH 7.0 and around 50°C, respectively. Metal ions such as Hg²⁺, Cu²⁺, Ni²⁺, Zn²⁺, Fe²⁺ and Co²⁺ strongly inhibited the enzyme activity. The molecular weight was about 45,000. The yields of maltotetraose and maltopentaose from potato starch were 30 ~ 40% and 20 ~ 30%, respectively.     

A protein modulator of erythrocyte membrane (Ca2+ + Mg2+)-ATPase inhibitor protein

A protein modulator of erythrocyte membrane (Ca²⁺ + Mg²⁺)-ATPase inhibitor protein was purified to apparent homogeneity from pig membrane-free hemolysate by a combination of carboxymethyl-Sephadex chromatography, gel filtration, chromatofocusing (pH 7-4) and subsequent removal of trace inhibitor protein by salt treatment. Gel filtration gave a molecular weight of 57 500 for the purified protein modulator, while SDS-polyacrylamide gel electrophoresis of dithiothreitol-treated modulator revealed one single band with a molecular weight of 29 000. Isoelectric focusing of the dithiothreitol-treated protein revealed one band (isoelectric pH 4.85), while untreated modulator gave an extra band (isoelectric pH 4.96). It contains no methionine and has an acidic content 73% higher than that of its basic residues. Freshly prepared or dithiothreitol-treated modulator suppressed both pig and human erythrocyte (Ca²⁺ + Mg²⁺)-ATPase inhibitor protein activity, but did not affect ATPase and calmodulin activities. Modulator-coupled Affi-Gel 15 could be employed for purification of the protein inhibitor.     

Purification and characterization of β-agarase from seaweed decomposing bacterium <Emphasis Type="Italic">Microbulbifer</Emphasis> sp. Strain CMC-5

Microbulbifer strain CMC-5 was isolated from decomposing seaweeds, and was found to degrade agar, alginate, carboxymethyl cellulose, carrageenan, xylan, and chitin. The extracellular agarase enzyme from strain CMC-5 was purified 103-fold by ultrafiltration, ion-exchange chromatography, using diethylaminoethyl sepharose FF, and gel filtration, using sephacryl S-300HR, with a yield of 6.7%. Zymogram and protein staining of the purified agarase on a SDS-polyacrylamide gel revealed a single band, with an apparent molecular weight of 59 kDa. The purified enzyme was endo-type β-agarase, as it was able to hydrolyze the β-1, 4 glycosidic linkages of agarose, releasing neoagarotetraose and neoagarohexaose as the end products. The optimum pH and temperature of agarase were 7 and 50°C, respectively. Thermal stability studies indicated that the agarase retained 62% of its activity after incubating at 50°C for 30 min. Treatment with EDTA reduced the agarase activity by 54%. The agarase activity was stimulated by the presence of Ca²⁺ and Mg²⁺ ions; whereas, Zn²⁺, Hg²⁺, Cu²⁺, Fe²⁺, and Co²⁺ abolished the activity. Further, the presence of NaCl at concentrations lower than 100 mM caused a decrease in the agarase activity; whereas, the activity was enhanced up to a concentration of 500 mM.     

Properties of a Zn2+-glycerophosphocholine cholinephosphodiesterase from bovine brain membranes

A Zn²⁺-glycerophosphocholine cholinephosphodiesterase was purified with a specific activity of 4.6 μmole/min·mg protein from bovine brain membranes by procedures involving PI-PLC solubilization, concanavalin A affinity chromatography, CM-sephadex chromatography and Sephadex G-150 chromatography. Based on molecular weight determination gel chromatography and SDS polyacrylamide gel electrophoresis, the phosphodiesterase activity appears to be a dimeric protein (110 kDa) composed of two subunits with a molecular weight of approximately 54 kDa. The Km value for p-nitrophenylphosphocholine and the optimum pH were found to be 16 μM and pH 10.5, respectively. The phosphodiesterase was inhibited by Cu²⁺, but not the other divalent metal ions. The activity of the apoenzyme was remarkably activated by Co²⁺ or Zn²⁺, but not Mn²⁺ or Mg²⁺. In addition, the inactivation of the enzyme in glycine buffer was prevented by Mn²⁺ or Zn²⁺, but not Co²⁺ or Mg². In a separate experiment, comparing properties of the purified and membrane-bound phosphodiesterases, the forms of two enzymes were quite similar except in stability. Both enzymes were more stable at pH 7.4 than pH 5 or 10. However, the membrane-bound enzyme was more stable than the soluble enzyme at all three pHs. These data suggest that the activity of the phosphodiesterase may be stabilized in-vivo.     

Characterization of membrane-associated arginine aminopeptidase inStreptococcus sanguis 903

Extracts of cytoplasmic membranes ofStreptococcus sanguis 903 were analyzed for aminopeptidase activity by isoelectric focusing in polyacrylamide gel and enzyme staining with 16 different aminopeptidase substrates. A single aminopeptidase with specificity for aminoterminal arginine was detected. The enzyme was stimulated by dithiothreitol and-mercaptoethanol. Urea, sodium dodecyl sulfate (SDS), andp-chloromercuribenzoate were inhibitory. Metal ions had little or no effect on activity, except that Hg²⁺, Cu²⁺, and Ni²⁺ were inhibitory. The pH optimum for activity was at 7.2. The molecular mass estimated by SDS-polyacrylamide gel electrophoresis was 170 kDa.     

Purification and Characterization of the Acidic Lectin from Winged Bean Seeds

Winged bean acidic lectin was purified by DEAE-Sephadex A-50 and affinity chromatography on N-acetylgalactosamine-agarose gel. The purified lectin was a glycoprotein homogeneous on polyacrylamide gel electrophoresis, isoelectric focusing, and gel filtration. The molecular weight of the lectin was 52,000 by gel filtration, and SDS-polyacrylamide gel electrophoresis gave a single component of molecular weight of 27,000. Its isoelectric point was 5.5. The acidic lectin was rich in acidic amino acids, and contained 2mol of methionine but no cystine. It also agglutinated both trypsinized and untreated human erythrocytes (types A, B, AB and O), but not rabbit erythrocytes. The hemagglutination was inhibited by d-galactose and related sugars. Modification of the acidic lectin with N-bromosuccinimide caused a concomitant loss of the hemagglutinating activity with oxidation of tryptophan residue. The acidic lectin was immunologically different from the purified winged bean basic lectin by double immunodiffusion using antiserum raised against the basic lectin.