Purification and characterization of two novel arginine aminopeptidases from Streptococcus mitis ATCC 9811

Two novel aminopeptidases (I and II) which have specificity for amino-terminal arginine residues and strong sensitivity to divalent cations were purified from Streptococcus mitis ATCC 9811 by a procedure that involved treatment with a lytic enzyme for bacterial cell walls, followed by a series of chromatographies. Enzyme I was obtained as a homogeneous protein as judged by polyacrylamide gel electrophoresis and had a specific activity of 484.8 units per mg protein using L-arginine-2-naphthylamide as substrate; its Km value was 2.6 X 10(-5) M. The molecular weight was estimated to be 62,000, and its isoelectric point was pH 4.4. Enzyme II was purified to a specific activity of 128.0 units per mg protein and had a Km value of 3.8 X 10(-5) M. The molecular weight was estimated to be 360,000, and its isoelectric point was pH 5.7. The pH optima of enzymes I and II were 8.6 and 7.6, respectively. Both enzymes were inactivated by sulfhydryl reagents and metal ions but were markedly activated by EDTA. The chloride ion had an inhibitory rather than a stimulatory effect on the activity of both enzymes. Substrate specificity studies indicated that both the enzymes specifically hydrolyze N-terminal arginine residues from a-aminoacyl 2-naphthylamides and peptides, but they could not attack the L-arginyl-L-prolyl-peptide.     

Isolation and characterization of N-long chain acyl aminoacylase from Pseudomonas diminuta

N-Long chain acyl aminoacylase II (Enzyme II) catalyzing the hydrolysis of N-long chain acyl amino acids was purified about 2,000-fold from the cell extracts of Pseudomonas diminuta with 1.8% of activity yield. The purified enzyme was homogeneous on polyacrylamide gel electrophoresis and the molecular weight was 220,000. Enzyme II differed from N-long chain acyl aminoacylase I (Enzyme I) in molecular weight, in substrate specificity, and in behavior toward temperature and pH. Enzyme II showed broader substrate specificity than Enzyme I and catalyzed the hydrolysis of lipoamino acids containing various amino acid residues, although Enzyme I was almost specific to the lipoamino acids containing L-glutamate. The extent of hydrolysis by Enzyme II reaction varied depending on the kinds of lipoamino acids and were: 100% for palmitoyl-L-glutamate, 91% for myristoyl-L-glutamate, 85% for lauroyl-L-glutamate, 54% for lauroyl-L-aspartate, 28% for stearoyl-L-glutamate and 17.5% for lauroyl-glycine.     

Purification and characterization of beta-N-acetylhexosaminidase from the ascidian, Halocynthia roretzi

beta-N-Acetylhexosaminidase [EC 3.2.1.30] was purified 820-fold from the viscera of Halocynthia roretzi by Sephadex G-200 gel filtration and chromatography on columns of DEAE-Sephadex and CM-Sephadex. The final preparation was sufficiently free from alpha-N-acetylglucosaminidase, alpha-N-acetylgalactosaminidase, alpha- and beta-glucosidases, alpha- and beta-galactosidases, alpha- and beta-mannosidases, and alpha-L-fucosidase, and gave one protein band on disc gel electrophoresis. Two different molecular weight forms which depended upon the pH were observed on Sephadex gel filtration. At pH 7.0, a species with a molecular weight of 170,000 was observed, whereas at pH 4.5, an enzyme of 330,000 daltons was seen. The enzyme was active at pH 4.5 but inactive at pH 7.0. The optimum pH and the Km were pH 4.2 and 1.9 mM for p-nitrophenyl beta-N-acetylglucosaminide and pH 4.0 and 0.9 mM for p-nitrophenyl beta-N-acetylgalactosaminide. The terminal beta-N-acetylhexosamine of glycolipids such as globoside I, GM2, and asialo GM2 was cleaved by the ascidian beta-N-acetylhexosaminidase though GM2 was less susceptible to the enzyme.     

Purification of proteins similar to HPr and enzyme I from the oral bacterium Streptococcus salivarius. Biochemical and immunochemical properties

The phosphoenolpyruvate:sugar phosphotransferase system (PTS) is made of several proteins. Two of them are designated general proteins because they are required for the transport and phosphorylation of all sugars of the PTS. These two proteins are found in the soluble fraction of cellular extracts and are termed HPr and enzyme I (EI). We reported in this work the purification and the characterization of these two proteins from Streptococcus salivarius ATCC 25975. HPr was purified by DEAE-cellulose chromatography, molecular sieving on Ultrogel AcA44, and carboxymethylcellulose chromatography. Sodium dodecyl sulfate electrophoresis in the presence of urea revealed a single band with a molecular weight of 6700. The protein contained no tryptophan and had a pI of 4.8. The purification scheme of EI was as follows: DEAE-cellulose chromatography, hydroxylapatite chromatography, DEAE-Sephadex A-50 chromatography, preparative electrophoresis, and molecular sieving on Ultrogel AcA34. The five-step purification for EI produced a 199-fold purified preparation with a specific activity of 530 mumol of HPr phosphorylated per minute per milligram of protein at 37 degrees C. The fraction obtained after filtration on Ultrogel AcA34 gave one band (68 000) on sodium dodecyl sulfate - polyacrylamide gel electrophoresis. The molecular weight of the native enzyme determined by gel filtration at 4 degrees C was 135 000, suggesting that it was a dimer. Enzyme I had a pI of 4.2, a pH optimum of 6.7, a Km for HPr of about 27 microM, a Km for phosphoenolpyruvate of 0.48 mM, and kinetics that were consistent with a Ping-Pong mechanism. Evidence had been obtained which indicated that S. salivarius enzyme I was antigenically very similar to enzyme I from various strains of Streptococcus mutans, but not to the enzyme from Bacillus subtilis, Staphylococcus aureus, Streptococcus faecalis, and Escherichia coli.     

Glutamate synthase from rice leaves

Ferredoxin-dependent glutamate synthase (EC 1.4.7.1) from rice leaves (Oryza sativa L. cv Delta) was purified 206-fold with a final specific activity of 35.9 mumoles glutamate formed per min per milligram protein by a procedure including ammonium sulfate fractionation, DEAE-cellulose chromatography, Sephacryl S-300 gel filtration, and ferredoxin-Sepharose affinity chromatography. The purified enzyme yielded a single protein band on polyacrylamide gel electrophoresis. Molecular weight of the native enzyme was estimated to be 224,000 daltons by Sepharose 6B gel filtration. Electrophoresis of the dissociated enzyme in sodium dodecyl sulfate-polyacrylamide gel gave a single protein band which corresponds to the subunit molecular weight of 115,000 daltons. Thus, it is concluded that the glutamate synthase is composed of two polypeptidic chains exhibiting the same molecular weight. Spectrophotometric analysis indicated that the enzyme is free of iron-sulfide and flavin. The pH optimum was 7.3. The enzyme had a negative cooperativity (Hill number of 0.70) for glutamine, and its K(m) value increased from 270 to 570 mum at a glutamine concentration higher than 800 mum. K(m) values for alpha-ketoglutarate and ferredoxin were 330 and 5.5 mum, respectively. Asparagine and oxaloacetate could not be substituted for glutamine and alpha-ketoglutarate, respectively. Enzyme activity was not detected with pyridine nucleotides as electron donors. Azaserine and several divalent cations were potent inhibitors. The purified enzyme was stabilized by dithiothreitol.     

Purification and some properties of alkaline pullulanase from a strain of bacillus no. 202-1, an alkalophilic microorganism.

Pullulanase (pullulan 6-glucanohydrolase EC 3.2.1.41) was purified about 290-fold from the culture fluid of Bacillus No. 202-1 by DEAE-cellulose adsorption, acetone fractionation, (NH4) 2SO4 precipitation and DEAE--cellulose column chromatography followed by Sephadex G-200 molecular sieve chromatography. The enzyme gave a single band of protein by disc polyacrylamide gel electrophoresis. The molecular weight was estimated as 92 000 by sodium dodecyl sulfate gel electrophoresis. The isolectric point was lower than pH 2.5. The optimum pH for enzyme action was about 8.5-9.0. The action of the enzyme on amylopectin and glycogen resulted in increase in the iodine coloration of 85% and 70%, respectively. The enzyme completely hydrolyzed 1,6-alpha-glucosidic linkages in amylopectin, glycogen and pullulan.     

Flavor Specificities of Satsuma Mandarin Juice Extracted by a New-type Screw Press Extraction System

Dextransucrases from Leuconostoc mesenteroides NRRL B-1416 and B-1375 strains were purified to electrophoretically homogeneous preparations. After successive column chromatographies, the enzyme fractions were treated with endodextranase, then subjected to preparative polyacrylamide gel electrophoresis. The purified dextransucrase from each strain had a dimeric structure of molecular weight 130,000~133,000. Alkaline treatment (pH 10.5) dissociated these dimer forms into the respective monomer forms having molecular weight of 64,000~68,000. The two enzymes were closely similar to each other in optimum conditions and thermal and pH stabilities. The purified B-1416 enzyme was activated 4.35-fold by the addition of exogenous dextran (0.5%), while the B-1375 enzyme was activated 2.76-fold. In the absence of exogenous dextran, both enzymes gave 5~10 min lag periods for reaction, which were abolished by the clinical dextran.     

Purification and some properties of ornithine decarboxylase from rat liver

Ornithine decarboxylase (EC 4.1.1.17) was purified to near homogeniety from livers of thioacetamide- and dl-α-hydrazino-δ-aminovaleric acid-treated rats by using three types of affinity chromatography with pyridoxamine phosphate-Sepharose, pyridoxamine phosphate-dipropylenetriamine-Sepharose and heparin-Sepharose. This procedure gave a purification of about 3.5·10⁵-fold with an 8% yield; the specific activity of the final enzyme preparation was 1,1·10⁶ nmol CO₂/h per mg protein. The purified enzyme gave a single band of protein which coincided with activity peak on polyacrylamide gel electrophoresis and also gave a single major band on SDS-polyacrylamide gel electrophoresis. A single precipitin line was formed between the purified enzyme and an antiserum raised against a partially purified enzyme, on Ouchterlony immunodiffusion. The molecular weight of the enzyme was estimated to be 105 000 by polyacrylamide gel electrophoresis at several different gel concentrations; the dissociated subunits had molecular weights of 50 000 on SDS-polyacrylmide gels. The isoelectric point of the enzyme was pH 4.1.     

Purification and properties of human placental acid lipase

Two peaks of lysosomal acid lipase activity were purified from normal human placenta. Acid lipase I, with an estimated molecular weight of 102 500, was purified 1016-fold while acid lipase II, with an estimated molecular weight of 30 600, was purified 3031-fold. The final yields of enzyme activity for acid lipase I and II were 0.9% and 2.2% respectively. The purity of the final preparations was documented by demonstration of a single protein band on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Both preparations of the purified enzyme demonstrated activity towards triolein, cholesteryl oleate and the artificial substrate 4-methylumbelliferyl oleate. Examination of Km values, thermal stability, pH optima, and electrophoretic mobility revealed similar properties for the two enzyme peaks. The response of the two enzyme preparations to inhibitors was similar with both being significantly inhibited by 0.2 M NaCl, 0.2 M KCl, 5 mM HgCl2 and 5 mM p-chloromercuribenzoate. The activity of the two preparations as assayed with either triolein or cholesterol oleate was not significantly affected by the addition of bovine serum albumin. In contrast, the 4-methylumbelliferyl oleate activity of both preparations was significantly inhibitred by albumin. These findings support the hypothesis that the same enzyme or enzymes are responsible for the intralysosomal hydrolysis of triacylglycerols and cholesterol esters in human tissues.     

Purification and properties of two ribonucleases and a nuclease from barley seeds

Three enzymes possessing RNAase activity were isolated from barley seeds. These enzymes were further purified by ammonium sulphate precipitation DEAE-cellulose chromatography, gel filtration on Sephadex G-75 and DEAE-Sephadex A-50 chromatography. These enzymes have been characterized and classified as: 1. Plant RNAase I (EC 3.1.27.1). It has a pH optimum at 5.7 and molecular weight of 19 000. 2. Plant RNAase II (EC 3.1.27.1). It has a pH optimum at 6.35 and molecular weight of 19 000. 3. Plant nuclease I (EC 3.1.30.2). It has a pH optimum at 6.8 and molecular weight of 37 000. Two RNAases were purified to homogeneity by means of affinity chromatography on poly(G)-Sepharose 4B, as shown by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate.     

Purification and properties of an acidic β-glucosidase from Acidobacterium capsulatum

Acidobacterium capsulatum, an acidophilic, mesophilic and chemoorganotrophic bacterium, produced an inducible, acidic β-glucosidase in the cellobiose medium. The enzyme was successively purified 109 times by CM-Sepharose, Sephacryl S-200 chromatography and preparative discontinuous polyacrylamide gel electrophoresis. Polyacrylamide gel electrophoresis of the purified enzyme gave a single band at pH 4.3. The enzyme had an optimum pH of 3.0 and optimum reaction temperature of 55°C, being stable from pH 1.5 to 6.0 and at temperatures from 20 to 45°C. No activity was detected above pH 6.5 or above 65°C. The molecular weight of 90,000 was estimated by gel filtration and the enzyme had an isoelectric point of 7.0. The enzyme hydrolyzed aryl-β-glycosides and β-linked disaccharides.     

Isozymes of alpha-galactosidase from Bacillus stearothermophilus

Two molecular forms of alpha-galactosidase (EC 3.2.1.22) synthesized constitutively by Bacillus stearothermophilus, strain AT-7, have been purified. alpha-Galactosidase I (with the substrate p-nitrophenyl alpha-D-galactopyranoside (PNPG)) has a pH optimum of 6 and half-life at 65 degrees C of > 2 h at low protein concentration. alpha-Galactosidase II has a pH optimum of 7 with PNPG and a half-life at 65 degrees C of about 3 min. The isozymes also differ with respect to their Km with PNPG and melibiose. Both enzymes are inhibited competitively by D-galactose, melibiose, and Tris. With the beta-glycosides cellobiose and lactose either noncompetitive or mixed-type inhibition is observed, with the pattern dependent on both the pH and the isozyme. The two isozymes have similar Arrhenius activation energies (about 20 kcal/mol, 1 kcal = 4.184 kJ). Their molecular weights, estimated by disc gel electrophoresis, are alpha-galactosidase I, 280 000 +/- 30 000 and alpha-galactosidase II, 325 000 +/- 15 000. Dodecyl sulfate gel electrophoresis gave a single band for each enzyme. The respective molecular weights, 81 000 +/- 500 for alpha-galactosidase I and 84 000 +/- 500 for alpha-galactosidase II, suggest that both enzymes consist of four subunits.     

Heat-Moisture Treatment of Cereal Starch Observed by X-ray Diffraction

Chitinases I and II were purified from the culture supernatant of Aeromonas sp. 10S-24 by ammonium sulfate precipitation, SP-Sephadex C-50 chromatography, Sephacryl S-200 gel filtration, and chromatofocusing. Both enzymes were most active at pH 4.0 and the optimum temperature for I and II were 50°C and 60°C. Chitinase I was stable at pHs between 4 and 9 and at temperatures below 50°C and chitinase II was stable at pHs between 5 and 7 and at temperatures below 45°C. The molecular weights were estimated by 8D8 polyacrylamide gel electrophoresis to be 112,000 and 115,000 for I and II respectively, while gel filtration showed the molecular weight to be 114,000 for both types of the enzyme. The pIs for I and II were 7.9 and 8.1, respectively. The activities of both enzymes were inhibited by Ag⁺ and iodoacetic acid.     

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.     

Purification and properties of alkaline phosphatase from boar seminal plasma

An alkaline phosphatase was purified from boar seminal plasma using adsorption to calcium phosphate gel, gel filtration, and ion-exchange chromatography. The preparation gave a single band on SDS polyacrylamide electrophoresis. The enzyme was a non-specific alkaline phosphatase that hydrolysed pyrophosphate slowly and had no phosphodiesterase activity. The pH optimum was 10 and the Km was approximately 0.2 mM with p-nitrophenyl phosphate as substrate. The enzyme was a zinc metalloenzyme as indicated by the loss of activity when treated with o-phenanthroline and the restoration of activity by zinc and magnesium ions. It also lost activity when treated with thiols. Molecular weight estimates from SDS polyacrylamide gel electrophoresis and gel filtration suggest that the enzyme is a tetramer of identical subunits, each of which has a molecular weight of 68,000.     

A rapid purification method for human erythrocyte pyruvate kinase

Human erythrocyte pyruvate kinase (ATP: pyruvate phosphotransferase, E.C.2.7.1.40) is purified 30,000-fold, using a method which includes ammonium sulfate precipitation, Sephadex G-75 filtration, and Blue Dextran-Sepharose 4B chromatography. The enzyme is resolved into two peaks on Blue Dextran-Sepharose 4B. The first peak with sp act of 300 corresponds to the mature form (R4) whereas the second peak with sp act of 180 corresponds to R2R'2. Peaks I and II give one band on 10% polyacrylamide gel without SDS. Peak II gives two bands on 10% SDS gel with molecular weights 60,000 (R') and 57,500 (R). On the other hand, peak I gives only one band on 10% SDS gel having a molecular weight of 57,500. Both the R4 and R2R'2 forms of the enzyme have the same pH optimum of 7.2.     

Purification and characterization of microsomal and lysosomal beta-glucuronidase from rat liver by use of immunoaffinity chromatography.

Rat liver beta-glucuronidase (EC 3.2.1.31), both from microsomal and lysosomal fractions, were purified about 9500-fold over the homogenate with high yield using affinity chromatography prepared by coupling purified specific immunoglobulin G against rat preputial gland beta-glucuronidase to Sepharose 2B and isoelectric focusing. The purified enzymes appeared homogeneous on electrophoresis in polyacrylamide gel and had a molecular weight of approximately 310000. In dodecylsulfate polyacrylamide gel electrophoresis, the microsomal beta-glucuronidase showed a single band corresponding to a molecular weight of 79000, while the lysosomal beta-glucuronidase had three distinct bands which consisted of one major and two minor bands corresponding to molecular weight of 79000, 74000, and 70000, respectively. A broad pH activity curve with a single optimum at pH 4.4 was observed in both the microsomal and the lysosomal beta-glucuronidases. Immunological gel diffusion technique with rabbit antiserum against rat liver lysosomal beta-glucuronidase revealed that both enzymes had the same or quite similar antigenic determinants.     

Wound-inducible pinene cyclase from grand fir: purification, characterization, and renaturation after SDS-PAGE.

The major wound-inducible monoterpene synthase (cyclase) of grand fir (Abies grandis) stems transforms geranyl pyrophosphate to both (-)-alpha-pinene (40%) and (-)-beta-pinene (60%). The enzyme was purified to apparent homogeneity by anion-exchange and hydrophobic interaction chromatography, coupled to discontinuous native polyacrylamide gel electrophoresis at neutral pH and polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (also at neutral pH) followed by renaturation in 1% Tween 20 (polyoxyethylenesorbitan monolaurate). The renatured enzyme produced a mixture of isomeric pinenes from geranyl pyrophosphate identical to that generated by the native form. The protein exhibited a molecular weight of 63,000 by gel permeation chromatography and of 62,000 by denaturing gel electrophoresis, indicating that the monomer is active. The enzyme required Mn2+ (Km = 30 microM) for activity, exhibited a Km value of 6 microM for the substrate geranyl pyrophosphate, showed a pH optimum at 7.8 and temperature optimum at 42 degrees C, and was inhibited by pyrophosphate (I50 = 0.17 mM), orthophosphate (I50 = 51 mM), and alpha-pinene, as well as by the histidine-directed reagent diethylpyrocarbonate (I50 = 0.64 mM) and the cysteine-directed reagent p-hydroxymercuribenzoate (I50 = 1.9 microM). Although similar in many respects to constitutive monoterpene cyclases of herbaceous species, this inducible cyclase, the first enzyme of this type to be purified to homogeneity from a conifer, is distinguished by the relatively high pH optimum, and the strict specificity and high affinity for the divalent metal ion cofactor.     

Purification and properties of 3-hexulosephosphate synthase from Methylomonas M 15.

3-Hexulosephosphate synthase, the first enzyme of the ribulose monophosphate cycle, was purified 15-fold from methanol-grown Methylomonas M 15. The purification procedure involved chromatography on DEAE-cellulose, Sephadex G-75, and DEAE-Sephadex A-50. The purified enzyme was more than 95% pure as judged by analytical polyacrylamide gel electrophoresis. The molecular weight was calculated to be 43000 from sedimentation equilibrium experiments. Electrophoresis in sodium dodecylsulfate gels gave a single band corresponding to a molecular weight of 22000. The enzyme catalyzes specifically the condensation formaldehyde with ribulose 5-phosphate to yield D-arabino-3-hexulose 6-phosphate. The Km values were found to be 1.1 mM for formaldehyde and 1.6 mM for ribulose 5-phosphate. A bivalent cation is essential for activity and stability of the enzyme, Mg2+ and Mn2+ serve best for this purpose. The optimum of pH for enzyme activity is 7.5--8.0.     

Spinach nitrate reductase: purification, molecular weight, and subunit composition

Nitrate reductase was purified about 3,000-fold from spinach leaves by chromatography on butyl Toyopearl 650-M, hydroxyapatite-brushite, and blue Sepharose CL-6B columns. The purified enzyme yielded a single protein band upon polyacrylamide gel electrophoresis under nondenaturing conditions. This band also gave a positive stain for reduced methylviologen-nitrate reductase activity. The specific NADH-nitrate reductase activities of the purified preparations varied from 80 to 130 units per milligram protein. Sucrose density gradient centrifugation and gel filtration experiments gave a sedimentation coefficient of 10.5 S and a Stokes radius of 6.3 nanometers, respectively. From these values, a molecular weight of 270,000 ± 40,000 was estimated for the native reductase. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the denatured enzyme yielded a subunit band having a molecular weight of 114,000 together with a very faint band possessing a somewhat smaller molecular weight. It is concluded that spinach nitrate reductase is composed of two identical subunits possessing a molecular weight of 110,000 to 120,000.