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Citrate Metabolism by Pediococcus halophilus

Several strains of non-citrate-metabolizing Pediococcus halophilus have previously been isolated from soy sauce mash or moromi. The factors controlling the metabolism of citrate in soy pediococci were studied. All the soy pediococcal strains tested which failed to decompose citrate did not possess citrate lyase [citrate (pro-3S)-lyase; EC 4.1.3.6] activity. In P. halophilus, citrate lyase was an inducible enzyme, and the optimum pH for activity was 7.0. The metabolism of citrate in P. halophilus was different from that observed in lactic streptococci. The main products from citrate were acetate and formate, and this bacterium produced no acetoin or diacetyl. Formate production from citrate was greatly reduced in the presence of glucose. P. halophilus 7117 (Cit) was proved to contain citrate lyase, pyruvate formate-lyase (EC 2.3.1.54) phosphotransacetylase (phosphate acetyltransferase; EC 2.3.1.8), and acetate kinase (EC 2.7.2.1), i.e., all the enzymes necessary to convert citrate to acetate and formate.

EVIDENCE FOR THE PRESENCE OF MALTASE AND α-GLUCOSIDASE ISOENZYMES IN BARLEY

An examination of the development of α-glucosidase and maltase activities (as measured by the hydrolysis of p-nitrophenyl α-D-glucoside and maltose respectively) indicated that two genotypes of Glacier barley had the same general pattern of enzyme development. However, the development of α-glucosidase activity followed a different course from that of maltase activity suggesting that separate enzyme proteins are involved in hydrolysing these substrates. Further evidence that separate enzyme proteins were responsible for hydrolysis of maltose and p-nitrophenyl α-glucoside was obtained by column chromatography of extracts of germinated barley which indicated the presence of two maltases and two α-glucosidases. The maltases and the α-glucosidases differed in molecular weight, pH of optimum activity and in thermostability. When isomaltose was used as a substrate the optimum pH and behaviour on gel chromatography were coincident with that of maltase activity but different from the α-glucosidases.

Purification and Properties of an Antimicrobial Substance Produced by Lacfobacillus bulgaricus

ABSTRACTMedia taken from cultures of Lactobacillus bulgaricus were found to contain an antibiotic substance different from lactic acid. This antibiotic substance was active against Pseudomonas frugi and, to a lesser extent, Staphyloccus aureus. The optimum pH for activity was approximately 4.0. At this pH the activity was stable at 6°C for 18 days and for at least 1 hr at 100°C. Active substance was isolated from the growth medium and purified by reverse phase high performance liquid chromatography procedures after removal of proteins by ethanol precipitation. Chromatography was performed first on an uncapped C‐8 column, then on a fully capped C‐18 column. A negative Biuret test indicated that the purified substance was not a polypeptide. UV absorption and mass spectra were consistent with a structure having an aromatic moiety and a molecular weight ≤ 700.

Phosphorylated fructose-1,6-bisphosphatase dephosphorylating protein phosphatase from Saccharomyces cerevisiae.

Phosphorylation of fructose-1,6-bisphosphatase with cyclic AMP-dependent protein kinase from yeast is accompanied by a 50% decrease in the catalytic activity (Pohlig, G. and Holzer, H. (1985) J. Biol. Chem. 260, 13818-13823). Using reactivation of phoshorylated fructose-1,6-bisphosphatase as assay, a protein phosphatase was about 2,000-fold purified to electrophoretic homogeneity from Saccharomyces cerevisiae. Upon incubation with phosphorylated fructose-1,6-bisphosphatase the purified protein phosphatase not only reverses the 50% inactivation caused by phosphorylation, but also the previously observed change in the pH optimum and in the ratio of activity with Mg2+ or Mn2+. The phosphatase is strongly inhibited by heparin and fluoride. L-Carnitine, orthophosphate, pyrophosphate, and succinate inhibit to 50% at concentrations from 1 to 10 mM. The molecular mass of the native phosphatase was found to be 180,000 Da. Sodium dodecyl sulfate-gel electrophoresis suggested four subunits with a molecular mass of 45,000 Da each. Half-maximal activity was observed with 5 mM Mg2+ or Mn2+, the pH optimum of activity was found at pH 7. Using polyclonal antibodies, disappearance of 32P-labeled fructose-1,6-bisphosphatase and concomitant liberation of the expected amount of inorganic [32P] phosphate was demonstrated.

Catalytic-regulatory subunit interactions and allosteric effects in aspartate transcarbamylase.

The x-ray structure of the unliganded aspartate transcarbamylase reveals that Arg-113 of the catalytic chain is involved in an important set of interactions at the interface between the catalytic and regulatory subunits (Honzatko, R.B., Crawford, J.L., Monaco, H.L., Ladner, J.E., Edwards, B.F.P., Evans, D.R., Warren, S.G., Wiley, D.C., Ladner, R.C., and Lipscomb, W. N. (1982) J. Mol. Biol. 160, 219-263). In order to disturb this interaction, site-directed mutagenesis has been used to replace Arg-113 with glycine. This modification results in a substantial weakening of the interface between the catalytic and regulatory subunits leading to a high tendency for dissociation. The unliganded mutant enzyme exhibits a pH dependence and a sensitivity toward mercurials analogous to that obtained for the relaxed conformation of the wild-type enzyme. Moreover, the presence of saturating concentrations of aspartate is accompanied by only a slight shift in the optimal pH for activity. The bisubstrate analog N-(phosphonacetyl)-L-aspartate induces a 2-fold increase in the sulfhydryl reactivity as compared to the 4-fold increase observed for the wild-type enzyme. Despite this change in the interactions at the interface between the catalytic and regulatory subunits, the mutant enzyme still retains homotropic and heterotropic effects and exhibits a normal affinity for aspartate. Together these data show that a substantial weakening of the catalytic-regulatory interface can occur without altering the allosteric properties of the enzyme. These results also indicate that the intersubunit interactions involving Arg-113, between the polar domain of the catalytic chain and the zinc domain of the regulatory chain, do not participate in the homotropic cooperativity of the enzyme.

Isolation and partial characterization of two alkaline proteases of the greater wax moth Galleria mellonella (L.)

Two alkaline proteases were isolated from whole-body extracts of Galleria mellonella larvae. The two proteases were separated by cation-exchange chromatography on CM-Sepharose CL6B and further purified by gel filtration on Ultrogel ACA 54. The optimal pH of activity using Azocoll as substrate was 10.5 for protease P-1 and 11.2 for protease P-2. The molecular weights of the two enzymes determined by gel filtration were respectively 12,500 and 10,500. Protease P-1 was inhibited by soybean trypsin inhibitor, TPCK, TLCK and activated by non-ionic detergents. Protease P-2 was inhibited by soybean trypsin inhibitor, 4-aminobenzamidine, ovomucoid and activated by dithiothreitol. Both enzymes were partially inhibited by PMSF. Distribution studies suggested that the two proteases were digestive enzymes.

The Partial Purification and Characterization of Nuclear and Mitochondrial Uracil-DNA Glycosylase Activities from Zea mays Seedlings

Uracil-DNA glycosylase activities from etiolated Zea mays seedling nuclei and mitochondria were partially purified and characterized. Nuclei and mitochondria were separated using sucrose differential and step gradient centrifugation. Experiments with osmotically shocked organelles indicated that enzyme activity from mitochondria was soluble, whereas nuclear enzyme activity was only partially soluble under the conditions tested. Purification using DEAE-cellulose and Affigel Blue column chromatography yielded distinct elution profiles from both columns for each of the organellar enzyme activities. Final purification was 490- and 850- fold for the nuclear and mitochondrial uracil-DNA glycosylase, respectively. Characterization studies demonstrated significant differences between the nuclear and mitochondrial uracil-DNA glycosylase with respect to K(m), temperature, and pH activity optimum, the effect of salts, and substrate preference. Molecular weight as determined by gel filtration was 18,000 for enzymes from both sources. Both were also sensitive to the sulfhydryl group-blocking agent N-ethylmaleimide. A number of uracil analogs were tested for their ability to inhibit nuclear and mitochondrial uracil-DNA glycosylase activities. 5-Azauracil, uracil, 6-aminouracil, 6-azauracil, 5-aminouracil, and 5-fluorouracil all inhibited both activities to variable degrees.

Purification and properties of β-galactosidase from chicken seminal plasma

1. 1. β-Galactosidase (EC 3.2.1.23) from chicken seminal plasma was purified approx. 111-fold to homogeneity. 2. 2. pH optimum of the enzyme ranged from 3.6 to 4.0 and its K m was 0.65 mM with p- nitrophenyl-β- d- galactoside as substrate. 3. 3. The enzyme was unstable at its optimal activity pH and was activated by Cl − ions. 4. 4. The enzyme had pI value of 4.0. 5. 5. The active enzyme had M r approx. 100,000 by Sephacryl S-300 chromatography. SDS electrophoresis in the presence of β-mercaptoethanol showed four bands corresponding to M r of approx. 90,000, 75,000, 65,000 and 13,000.

Changes in the plasma membrane ATPase activity in relationship to cell proliferation in Dictyostelium

The plasma membrane ATPase activity of Dictyostelium amoebae increases ca 2.5 fold from non dividing stationary phase cells to synchronously growing cells. This increase in ATPase activity takes place during the three hours lag period that precede the cell division after diluting stationary cells into fresh medium and is prevented by cycloheximide. No differences in the Km for ATP or in the optimal pH for activity were observed in kinetic studies carried out with purified plasma membranes from stationary and proliferating cells.

Characterization of an alkaline elastase from alkalophilic Bacillus Ya-B

The alkaline elastase produced by alkalophilic Bacillus Ya-B was a new type of proteinase which had a very high optimum pH and high elastolytic activity. It also had a high hydrolyzing activity against keratin and collagen. The molecular weight was determined to be 23 700 and 25 000 by ultracentrifugation analysis and SDS-polycrylamide gel electrophoresis, respectively. The isoelectric point was 10.6. The optimum reaction temperature was 60°C. Like many alkaline proteinases, this enzyme required Ca 2+ for stability. The optimum reaction pH was 11.75 toward casein and elastin-orcein. The K cat/ K m values of the enzyme to synthetic substrates were constant from pH 8.5 up to 12.75. The enzyme was stable in the pH range 5.0–10.0. The enzyme was inhibited by alkaline proteinase inhibitors Streptomyces subtilisin inhibitor and microbial alkaline proteinase inhibitor, but not by elastatinal or the metalloproteinase inhibitor metalloproteinase inhibitor. Sodium chloride inhibited the elastolytic activity but not the caseinolytic activity at a concentration below 0.2 M. The inhibitory effect of sodium chloride to elastolytic activity was much more prominent at pH 9.0 than at pH 11.5. More than 50% of the enzyme bound onto elastin in the pH range below the isoelectric point of this enzyme. The amino-terminal sequence of the enzyme was determined, and compared with those of subtilisin BPN′ and subtilisin Carlsberg. Extensive sequence homology was noted among these three enzymes.

Characterization of Seed Lipoxygenase of Phaseolus vulgaris cv, Haricot

ABSTRACT Phaseolus vulgaris cv, haricot seed lipoxygenase was extracted and partially purified. The precipitation of an active lipoxygenase fraction with solid ammonium sulfate (at 20–50% of saturation) increased its activity by a factor of 3. The pH for optimum activity was 7.3. The addition of 40 mM potassium cyanide resulted in a doubling of the enzyme activity. Enzyme activity was completely lost on storage at 4°C for 48 hr. The activity of haricot lipoxygenase extract was considerably greater on linoleic acid than on its esters, and decreased in the order: mono‐ > di > trilinolein. The haricot lipoxygenase shares many of the characteristics of the corresponding enzyme found in several seed sources.

Purification and Characterization of Human Platelet Glutathione-S-Transferase

A glutathione-S-transferase was isolated and purified to homogeneity from human platelets. With a combination of ammonium sulfate fractionation and chromatographic methods, 0.2 mg of pure enzyme was obtained from 9 X 10(11) platelets with a 12% recovery. The purified enzyme had a specific activity of 7.5 U per milligram, representing an approximately 1,100-fold purification. The enzyme was found to be anionic, with an isoelectric point of 4.6. With reduced glutathione as a co-substrate, platelet glutathione-S-transferase was most active with the synthetic substrate, 1-chloro-2,4-dinitrobenzene, less active with 1,2-dichloro-4-nitrobenzene, and essentially inactive with nitroglycerin and 1,2-epoxy-3-(p-nitrophenoxy)-propane. The pH optimum for activity with glutathione and 1-chloro-2,4-dinitrobenzene was 7.0. Indomethacin (1-(p-chlorobenzoyl)-5-methoxy-2-methyindole-3-acetic acid), a chlorobenzene derivative, noncompetitively inhibited human platelet glutathione-S-transferase with an apparent KI of 0.23 mmol/L. This study represents the first complete purification and characterization of a glutathione-S-transferase from platelets. The presence of this enzyme in the platelet, within which high concentrations of reduced glutathione coexist, suggests the potential importance of the platelet in detoxification reactions and in the synthesis of the glutathione adducts of leukotriene metabolism.

Purification and characterization of human platelet glutathione-S- transferase

A glutathione-S-transferase was isolated and purified to homogeneity from human platelets. With a combination of ammonium sulfate fractionation and chromatographic methods, 0.2 mg of pure enzyme was obtained from 9 X 10(11) platelets with a 12% recovery. The purified enzyme had a specific activity of 7.5 U per milligram, representing an approximately 1,100-fold purification. The enzyme was found to be anionic, with an isoelectric point of 4.6. With reduced glutathione as a co-substrate, platelet glutathione-S-transferase was most active with the synthetic substrate, 1-chloro-2,4-dinitrobenzene, less active with 1,2-dichloro-4-nitrobenzene, and essentially inactive with nitroglycerin and 1,2-epoxy-3-(p-nitrophenoxy)-propane. The pH optimum for activity with glutathione and 1-chloro-2,4-dinitrobenzene was 7.0. Indomethacin (1-(p-chlorobenzoyl)-5-methoxy-2-methyindole-3-acetic acid), a chlorobenzene derivative, noncompetitively inhibited human platelet glutathione-S-transferase with an apparent KI of 0.23 mmol/L. This study represents the first complete purification and characterization of a glutathione-S-transferase from platelets. The presence of this enzyme in the platelet, within which high concentrations of reduced glutathione coexist, suggests the potential importance of the platelet in detoxification reactions and in the synthesis of the glutathione adducts of leukotriene metabolism.

Isolation and characterization of a new aminopeptidase from bovine lens.

An aminopeptidase has been purified to homogeneity from bovine lens tissue by gel filtration and DEAE-cellulose chromatography. This enzyme has a molecular weight of 96,000 under both native and denaturing conditions. The purified enzyme hydrolyzed a variety of synthetic substrates as well as di-, tri-, and higher molecular weight peptides. Significantly this enzyme is capable of hydrolyzing arginine, lysine, and proline aminoacyl bonds. The pH optimum for activity and stability was 6.0. Both a reduced sulfhydryl group and a divalent metal ion are essential for activity. The native enzyme contains 1.6 mol of zinc and 1.0 mol of copper/mol of enzyme. No activation was seen upon incubation with either magnesium or manganese; however, heavy metal ions were inhibitory. Bestatin and puromycin were effective inhibitors and no endopeptidase activity could be detected in the purified preparation. This enzyme is clearly distinct from the lens leucine aminopeptidase, but rather, is identical to a cytosolic aminopeptidase III isolated from other tissues. Evidence is presented which argues that this enzyme may be the major lens aminopeptidase under in vivo conditions.

Purification and Some Properties of the 2-Hydroxy-6-oxohepta-2,4-dienoate Hydrolase (2-Hydroxymuconic Semialdehyde Hydrolase) Encoded by the TOL Plasmid pWW0 from Pseudomonas putida mt-2

SUMMARY: The 2-hydroxy-6-oxohepta-2,4-dienoate (HOD) hydrolase encoded by the TOL plasmid pWW0 from Pseudomonas putida mt-2 (PaWl) was purified to homogeneity. It has an M r of 65000 and is dissociated by SDS into two subunits of equal size. Alanine was the only N-terminal residue detected, and each subunit contained one cysteine thiol group. The pH optimum for activity and for enzyme stability was around 7·5, whereas the isoelectric point was 4·7. Only the products of catechol 2,3-oxygenase action on linear chain alkylcatechols and 4-chlorocatechol served as substrates. Kinetic measurements showed that the higher activity against the ketone substrates (from 3-substituted catechols) over aldehyde substrates (from 4-substituted catechols) was the result of higher V max values rather than lower K m values. Antisera prepared against this purified HOD hydrolase were shown by Ouchterlony double diffusion and inhibition studies to be related to the HOD hydrolase from phenol-grown P. putida strain U (NCIB 10015) in which the enzyme is chromosomally encoded.

Purification of peroxisomal malate synthase from alkane-grown Candida tropicalis and some properties of the purified enzyme.

Malate synthase, one of the key enzymes in the glyoxylate cycle, was purified from peroxisomes of alkane-grown yeast, Candida tropicalis. The enzyme was mainly localized in the matrix of peroxisomes, judging from subcellular fractionation followed by exposure of the organelles to hypotonic conditions. The molecular mass of this peroxisomal malate synthase was determined to be 250,000 daltons by gel filtration on a Sepharose 6B column as well as by ultracentrifugation. On sodium dodecylsulfate/polyacrylamide slab-gel electrophoresis, the molecular mass of the subunit of the enzyme was demonstrated to be 61,000 daltons. These results revealed that the native form of this enzyme was homo-tetrameric. Peroxisomal malate synthase showed the optimal activity pH at 8.0 and absolutely required Mg2+ for enzymatic activity. The Km values for Mg2+, acetyl-CoA and glyoxylate were 4.7 mM, 80 microM and 1.0 mM, respectively.

Purification and characterization of endo-.BETA.-N-acetylglucosaminidase from a Flavobacterium sp.

A gram negative bacterium isolated from soil was found to produce a high level of endo-β-N-acetylglucosaminidase in the culture medium. The organism was identified as a Flavobacterium sp. from various bacteriological characteristics. The enzyme from the Flavobacterium sp. was purified to homogeneity from culture broth by fractionation with ammonium sulfate and column chromatographies on DEAE-cellulose, hydroxylapatite, and Sephadex G-150 and G-100. The molecular weight of the enzyme was estimated to be 27,000 and 30,000 by gel filtration and SDS-polyacrylamide gel electrophoresis, respectively, and it appeared to consist of a single polypeptide chain. The optimal pH for activity was 5.0 to 6.0 and the stable pH range was 5~7. The Michaelis constant was 0.30 mm with dansyl-Asn-(GlcNAc)2(Man)6 as the substrate. The enzyme hydrolyzed oligosaccharides of native ovalbumin, bovine pancreatic ribonuclease B and a yeast invertase.

A human lung mast cell chymotrypsin-like enzyme. Identification and partial characterization.

We have used a high performance liquid chromatography assay, which detects chymotryptic cleavage of the phe8-his9 bond of angiotensin I to yield angiotensin II, in order to examine human lung mast cells for the presence of chymotryptic activity. Mast cells, purified from human lung by enzymatic dispersion, countercurrent elutriation, and Percoll gradient centrifugation, were lysed or challenged with goat anti-human IgE. In multiple experiments angiotensin II-converting activity was detected in lysates of 10-99% pure mast cell preparations. Regression analysis of net percent release values of histamine and the angiotensin I-converting activity from dose-response experiments demonstrated a correlation between the two parameters, indicating that the chymotrypsin-like enzyme is a constituent of the mast cell secretory granule. The chymotryptic activity was completely inhibited by 10(-3) M phenylmethylsulfonylfluoride but not by 10(-3) M Captopril, and the pH optimum of activity was 7.5-9.5. Gel filtration of released material separated the activity from tryptase and demonstrated an approximate molecular weight of 30-35,000. The mast cell enzyme, like a human skin chymotrypsin-like proteinase, can be distinguished from leukocyte cathepsin G by lack of susceptibility to inhibition by bovine pancreatic trypsin inhibitor. Thus, an enzyme with limited chymotryptic specificity is present in human lung mast cells. The Michaelis constant of the enzyme for angiotensin I of 6.0 X 10(-5) M is similar to that of endothelial cell angiotensin-converting enzyme and is consistent with a reaction of physiologic importance.

Immobilization of exo-D-galacturonanase by coupling to a polyacrylamide type support

Exo-D-galacturonanase (E.C. 3.2.1.67) from carrot was immobilized by covalent bonding to a polyacrylamide type support (with free carboxyl groups) activated by water-soluble carbodiimides. The activity of the immobilized enzyme (under optimal reaction conditions of the immobilization) was around 43% of the activity of the free enzyme. The pH-optimum of activity was shifted from 5.1 to 5.3. The immobilization of the enzyme did not change its temperature optimum and the thermal stability of the enzyme was slightly increased after its immobilization. No change in the mode of action of the immobilized enzyme on a polymeric substrate or digalacturonic acid was observed. When sodium pectate was digested with the immobilized enzyme the value of Kmapp was substantially increased and the Vapp-value dropped to 40% of that observed with the free enzyme.

Further characterization of the two catalases inEscherichia coli

Crude extracts of wild-typeEscherichia coli contain two catalase species that separate on native polyacrylamide gels. The slow-migrating enzyme (HPII) has two pH optima of activity (at pH 6.8 and 10.5), is activated at 70°C, is sensitive to inhibitory by 3-amino-1,2,4-triazole and has Km values of 18.2 mM at pH 6.8 and of 10 mM at pH 10.5. The fast-migrating enzyme has a single pH optimum of 6.8 and is composed of two isozymes (HPI-A and HPI-B). Its activity is labile at 70°C, it is relatively resistant to inhibition by 3-amino-1,2,4-triazole and has a Km value of 3.7 mM.