Purification and properties of DL-lactate dehydrogenase from Leuconostoc mesenteroides

A dl-lactate dehydrogenase from the bacterium, Leuconostoc mesenteroides, has been purified and characterized with respect to amino acid composition, molecular weight, and kinetic properties. The turnover number of the enzyme was 1.7 × 10⁵ moles DPNH/mole enzyme/min for the most active of three preparations. On the basis of a sedimentation constant of 3.52 S and a diffusion constant of 5.0 × 10^?7 cm²/ ml, the molecular weight of the enzyme was determined to be approximately 64,000. Similar values were derived from sedimentation equilibrium data. The enzyme exhibits typical Michaelis-Menten kinetics except when lactate is the variable substrate. In this case, double reciprocal plots of activity versus substrate concentration are curved upward, suggesting that lactate either activates or stabilizes a more active form of the enzyme.     

Purification and properties of the extracellular dextransucrase from Leuconostoc mesenteroides NRRL B-1299.

Dextransucrase [EC 2.4.1.5] activity from cell-free culture supernatant of Leuconostoc mesenteroides NRRL B-1299 was purified by (NH4)2SO4 fractionation, adsorption on hydroxyapatite, chromatography on DEAE-cellulose and gel filtration on Sephadex G-75. The extracellular enzyme was separated into two principal forms, enzymes I and N, and the latter was shown to be an aggregated form of the protomer, enzyme I. Enzymes I and N were both electrophoretically homogeneous and their relative activities reached 820 and 647 times that of the culture supernatant, respectively. On sodium dodecylsulfate (SDS)-polyacrylamide gel electrophoresis, enzyme N dissociated into the protomer enzyme I, with a molecular weight of 48,000. Enzyme I was gradually converted into enzyme N upon aging, and this conversion was stimulated in the presence of NaCl. The optimum pH and temperature of enzyme I activity were pH 6.0 and 40 degrees, respectively, while those of enzyme N were pH 5.5 and 35 degrees. The Km values of enzymes I and N were 13.9 and 13.1 mM, respectively. Ca2+, Mg2+, Fe2+, and Co2+ stimulated the activity of enzyme N, and EDTA showed a potent inhibitory effect on this enzyme. Moreover, the activity of enzyme N was more effectively stimulated by exogenous dextrans as compared with enzyme I.     

Purification and characterization of NADH oxidase from a strain of Leuconostoc mesenteroides

An NADH oxidase was purified to homogeneity from Leuconostoc mesenteroides with a specific activity 100-fold higher than that of the crude extract. The purified NADH oxidase was an acidic protein having an S0 20,W of 5.49S and a molecular weight of 104,000, consisting of a dimer with 53,000 subunit size. The enzyme could use O2, dichlorophenolindophenol and methylene blue as oxidants, but not H2O2, cytochrome c, or ferricyanide. The physiological substrate was beta-NADH (Km = 0.12 mM) with O2 as the oxidant, probably forming H2O, rather than H2O2. Activity toward alpha-NADH was observed (Km = 0.14 mM), but the maximum velocity was 3 orders of magnitude lower than that with beta-NADH. alpha-NADPH and beta-NADPH were inert for the reaction. The enzyme showed a flavoprotein absorption spectrum with maxima at 273, 379, and 450 nm with a shoulder at 465 nm: the absorption at 450-465 nm disappeared on adding excess NADH or hydrosulfite. One mol of the holoenzyme contained approximately 2 mol of FAD. The apoenzyme was obtained by treatment with EDTA-KBr solution and could be reconstituted partially by adding FAD, but not riboflavin or FMN. The maximum activity of the reaction was observed at pH 6.5 in a temperature range of 35-45 degrees C. The activation energy was estimated to be 3.77 kcal/mol. The enzyme was inhibited by SH reagents, quinacrine, quinine, and Cu2+, but not by EDTA. Adenine and its nucleoside 5'-di- and triphosphates showed competitive inhibitions, while various metabolites, such as H2O2, FDP, acetyl phosphate, lactate, ethanol, and acetate, did not affect the reaction.     

Purification and Properties of a Malolactic Enzyme from a Strain of Leuconostoc mesenteroides Isolated from Grapes

An enzymatic complex able to transform l-malate to l-lactate was obtained from a Leuconostoc mesenteroides strain isolated from grapes. The molecular weight was about 235,000, the isoelectric point was at pH 4.35, and the optimal pH for activity was 5.75. The malolactic activity followed a sequential pattern concerning the involved substrates. At pH values substantially different from the optimum, a positive cooperativity between malate molecules was observed. Oxamate, fructose-1, 6-diphosphate, and l-lactate acted as noncompetitive inhibitors, whereas succinate, citrate, and tartrate isomers produced a competitive inhibition.     

Characterization of a bifunctional HPr kinase/phosphorylase from Leuconostoc mesenteroides SY1

The hprK gene encoding bifunctional HPrK/P (kinase/ phosphorylase) was cloned from L. mesenteroides SY1, a strain isolated from kimchi. hprK was transcribed as a monocistronic gene. His-tagged HPrH16A and HPrK/P were produced in E. coli BL21(DE3) using pET26b(+) and purified. HPrK/P phosphorylation assay with purified proteins showed that the kinase activity of HPrK/P increased at slightly acidic pHs. Divalent cations such as Mg2+ and Mn2+ and glycolytic intermediates such as fructose-1, 6-bisphosphate (FBP) and phosphoenolpyruvate (PEP) increased the kinase activity of HPrK/P, but inorganic phosphate strongly inhibited it. Kinetic studies for the kinase activity of HPrK/P showed that the apparent Km values were 0.18 and 14.57 microM for ATP and HPr, respectively. The Km value for the phosphorylase activity of HPrK/P was 14.16 microM for P-Ser-HPr (HPr phosphorylated at the serine residue).     

Purification and some physico-chemical properties of myocardial adenylate deaminase

A procedure for isolation of adenylate deaminase from duck heart muscle has been developed. The method includes extraction of enzyme, chromatography on cellulose phosphate, fractionation by ammonium sulfate, chromatography on Sephadex G-25 and ion-exchange chromatography on DEAE-cellulose. The enzyme was purified approximately 4000-fold with a yield of 25%. Electrophoresis in polyacrylamide gel revealed that the enzyme contains no proteins other than adenylate deaminase. The enzyme has a UV absorption spectrum typical for proteins which contain no nucleic acid impurities. Using sievorptive chromatography, it was shown that the myocardial extract contains two adenylate deaminase forms, which are tetramers with mol. weights of 190 000 and 240 000. The molecular weights of the subunits are 47 000 and 63 000, respectively. In the oligomeric form the enzyme is only detected at high enzyme concentrations and in the presence of large amounts of substrate.     

Purification and some properties of protamine kinase from rabbit brain

A protein kinase of high specificity towards protamine and ATP was isolated from rabbit brain and purified about 200-fold. The enzyme does not catalyse phosphorylation of acidic proteins, e.g. casein and phosvitin, nor is it susceptible to cyclic AMP or protein inhibitors of the cAMP-dependent kinase. The enzyme shows the highest activity in the presence of 10 mM-Mg2+ and 2 mM-dithiothreitol at pH 8. The only phosphorylated amino acid found in protamine phosphorylated by the enzyme was phosphoserine.     

Phosphofructokinases from Lactobacteriaceae. II. Purification and properties of phosphofructokinase from Streptococcus thermophilus

Phosphofructokinase (ATP : D-fructose-6-phosphate 1-phosphotransferase, EC 2.7.1.11) from Streptococcus thermophilus has been purified. It is a tetramer composed of identical subunits of molecular weight 36 000 and exhibits Michaelis-Menten kinetics. Compared to the phosphofructokinases from taxonomically related bacteria, the enzyme from S. thermophilus is more stable at high temperatures. In addition, it has been demonstrated that the phosphofructokinases from lactobacteria and also from Bacillus stearothermophilus show immunologic cross-reaction. In spite of the significantly different kinetic properties and the different thermostability of these enzymes, this finding indicates great structural resemblance.     

Purification and some properties of chlorogenic acid oxidase from apple (Malus pumila).

Chlorogenic acid oxidase was extensively purified to homogeneity from apple flesh (Malus pumila cv. Fuji). The enzyme was purified 470-fold, with a total yield close to 70% from the plastid fraction by ammonium sulfate precipitation, gel filtration and ion-exchange chromatography. The molecular weight was determined to be 65,000 by both SDS-PAGE and gel filtration chromatography. The optimum pH for the enzyme activity was around 4.0, and the enzyme was stable in the range of pH 6-8. The pI obtained by isoelectrofocusing was 5.4, and the N-terminal amino acid sequence was N-Asp-Pro-Leu-Ala-Pro-Pro-. The reaction rate of the purified enzyme was much larger for chlorogenic acid than for other o-diphenols such as (+)-catechin, (-)-epicatechin and 4-methylcatechol, and the enzyme lacked both cresolase activity and p-diphenol oxidase activity. The Km value for the enzyme was found to be 122 microM toward chlorogenic acid. The purified enzyme had far less thermal stability than the enzyme of the plastid fraction. Diethyl-dithiocarbamate, sodium azide, o-phenanthroline and sodium fluoride markedly inhibited the enzyme activity.     

Cell wall constituents of Leuconostoc citrovorum and Leuconostoc mesenteroides

The cell wall constituents of Leuconostoc citrovorum 8082, L. mesenteroides 10830a, and L. mesenteroides 11449 have been ascertained. All three strains contained glycerol. Glucose and rhamnose were the major reducing sugar constituents. Alanine, glutamic acid, lysine, glucosamine, and muramic acid were the principal amino acids and amino sugars in all three strains. In addition, strain 10830a contained l-serine as a major cell wall component. Quantitative amino acid analyses indicate that glutamic acid, lysine, glucosamine, muramic acid, and serine may be present in the cell walls in equimolar amounts and that alanine is present in three to four times these quantities. The similarities and differences between the cell wall constituents of the leuconostocs and those of the lactobacilli and streptococci are discussed.     

Purification and some properties of inducible N-acetylglucosamine kinase from Candida albicans

N-Acetylglucosamine kinase (ATP:2-acetamido-2-deoxy-D-glucose 6-phosphotransferase, EC 2.7.1.59) catalyzes the first reaction in the inducible N-acetylglucosamine catabolic pathway of Candida albicans, an obligatory aerobic yeast. As a part of continuing biochemical studies concerning the regulation of gene expression in a simple eukaryote, N-acetylglucosamine kinase has been purified and characterized biochemically. The enzyme has been purified about 300-fold from the crude extract and its molecular weight of 75 000 has been determined by Sephadex G-100 gel filtration. Isolation and analysis procedures are described. The kinase reaction is optimal within a pH range of 7--8. The enzyme is strictly specific for GlcNAc as phosphate acceptor; ATP is the phosphoryl group donor for the kinase reaction and to a lesser extent dATP and CTP. Km values for GlcNAc and ATP are 1.33 mM and 1.82 mM, respectively. The enzyme required Mg2+, which may be replaced by other bivalent metal ions such as Mn2+, Ca2+, Ba2+ and Co2+ for a lesser degree of effectiveness. The purified enzyme is extremely sensitive to thermal denaturation and becomes completely inactive by heating at 65% C for 2 min. The enzyme is also inactivated by sulphydryl reagents such as p-chloromercuribenzene sulfonic acid and N-ethylmaleimide.