A Simplified Colorimetry without Incineration of Phosphorus in Phosphatides

An NAD linked formate dehydrogenating enzyme which catalyzed the last step of methanol oxidation system was extracted from the methanol-grown Kloeckera sp. No. 2201. The specific activity of the enzyme in the extract of methanol-grown cells was found to be considerably higher than that of the glucose-grown cells. The enzyme was purified about 35-fold from the extract of methanol-grown cells by heat treatment, column chromatographies on DEAE-cellulose and on hydroxylapatite, and Sephadex G-200 gel filtration. The purified enzyme was shown to be homogeneous by analyses with electrophoresis and ultracentrifuga-tion. The purified enzyme was a kind of NAD: formate oxidoreductase (EC, 1.2.1.2) which catalyzed specifically the oxidation of formate to carbon dioxide. The Km values were 22 mm for formate and 0.1 mm for NAD. The enzyme was inactivated by potassium cyanide, sodium azide, and p-chloromercuribenzoate but not by any metal-chelating reagents tested. Other general properties of the enzyme were also investigated.     

Regioselective synthesis of kojic acid esters by Bacillus subtilis protease

The lipophilicity of kojic acid [5-hydroxy-2-(hydroxymethyl)-4H-pyran-4-one] was improved by esterifying kojic acid with either divinyl adipate, vinyl hexanoate, vinyl octanoate or vinyl decanoate using protease from Bacillus subtilis for 7 d. ¹H-NMR and ¹³C-NMR showed that the primary hydroxyl group at the C-7 position of kojic acid was regioselectively esterified to afford 7-O-vinyl adipoyl kojic acid, 7-O-hexanoyl kojic acid, 7-O-octanoyl kojic acid and 7-O-decanoyl kojic acid (13–27% yield). The kojic acid esters had radical scavenging activities, inhibited tyrosinase activity and was biodegradable.     

Studies on Metabolic Pathway of NAD in Yeast

The enzyme system of NAD degradation was extracted from autolysate of Saccharomyces oviformis.

The enzymes were partially separated by ammonium sulfate fractionation and DEAE-cellulose column chromatography, and then the metabolic pathway of NAD in yeast was presented, in which four enzymes were contained. It has been found that, among them, the 5?nucleotidase has more affinity for AMP and the nucleosidase has strict affinity for nicotinamide riboside.

In the degradation of NAD in the yeast, nucleotide pyrophosphatase was main enzyme, but NADase, nucleotide pyrophosphorylase and adenosine deaminase seemed not to play an important role.     

Purification and properties of a nicotinamide adenine dinucleotide-linked cyclohexanol dehydrogenase from aNocardia species

An inducible pyridine nucleotide-linked cyclohexanol dehydrogenase activity was present in crude extracts from aNocardia species following growth on cyclohexane. The enzyme was purified 126-fold by affinity chromatography and has an oligomeric molecular weight of 145,000 ±5,000. There was an absolute requirement for NAD for activity and the products of the dehydrogenase reaction were stoichiometric amounts of NADH and cyclohexanone. The enzyme had a broad specificity for secondary alcohols including straight-chain secondary alcohols, cyclic and substituted cyclic alcohols, and cyclohexane diols. The apparentK _m values for cyclohexanol and NAD were 3.7×10⁻⁵ M and 2.4×10⁻⁵ M, respectively, and the optimal pH for cyclohexanol oxidation was 10.5. The enzyme was heat sensitive, losing about 50% activity after a 1-min incubation at 45°C. Enzyme activity was completely inhibited by the thiol agent,p-chloromercuribenzoate but not by metal chelating agents.     

Isolation and Sequence Analysis of Bovine αsl-Casein cDNA Clone

Nucleoside oxidase, a novel nucleoside oxidizing enzyme has been purified from a crude extract of Pseudomonas maltophilia LB-86 by a ammonium sulfate fractionation, heat treatment, column chromatography on DEAE-Toyopearl, and gel filtration twice on Sephacryl S-200. The overall purification was approximately 60-fold with a yield of 35 %. The purified enzyme gave a single protein band on acrylamide gel electrophoresis.

Reaction products from inosine were identified as inosine-5′-aldehyde and inosine-5′-carboxylic acid. The enzyme catalyzed the oxidation of inosine to inosine-5′-carboxylic acid via inosine-5′-aldehyde using molecular oxygen as a primary electron acceptor with no formation of hydrogen peroxide.     

Phosphodiesterase from the venom of Crotalus ruber ruber

Phosphodiesterase was isolated from the venom of Crotalus ruber ruber from the U.S.A. using the gel filtration on a Sephadex G-75 column, followed by anion or cation exchange chromatography. Phosphodiesterase was homogeneous as established by a single band on acrylamide gel electrophoresis and isoelectric focusing electrophoresis. Phosphodiesterase activity was inhibited by ethylenediamine tetraacetic acid (EDTA), o-phenanthroline, thioglycolic acid or p-chloromercuribenzoate (PCMB), but not by soybean trypsin inhibitor (SBTI) or benzamidine. The molecular weight of this enzyme was determined to be approx. 98,000 and the isoelectric point was found to be pH 10.5 by isoelectric focusing with carrier ampholyte. This enzyme contained 1.04 mol zinc per mol. The Michaelis constant (Km) of this enzyme for p-nitrophenyl thymidine-5'-phosphate and inhibition constant (Ki) for PCMB were found to be 8.3 X 10(-3) and 1.2 X 10(-2) M, respectively.     

Purification and Some Properties of Two Types of Penicillium Lipase,I and II,and Conversion of Types I and II under Various Modification Conditions

The lipase produced by a strain of Penicillium crustosum Thom was fractionated into three lipase components, I～III by DEAE cellulose column chromatography, and two of them, I and II were purified and obtained in crystalline form respectively, which proved homogeneous by electrophoresis and ultracentrifugal analysis. Lipase I was an ordinary lipase with molecular weight about 29,000 hydrolyzing olive oil and tributyrin favourably in almost the same degree, while II, rather, a so-called tributyrinase with M. W. about 32,000 hydrolyzing tributyrin more efficiently than olive oil. The site of the activity on olive oil in these lipase was generally sensitive to sodium desoxycholate, ethylenediamine-tetraacetate (EDTA), and p-chloromercuribenzoate (PCMB), and lipase I was converted to a lipase II by a treatment with these reagents. Also, partial degradation of I by proteinase (‘pronase’) yielded the enzyme fragment of type II. On the other hand, treatment of the enzymes with hydrogen peroxide or sodium borohydride caused the conversion of type II into I. From the observation of UV difference spectrum during incubation with sodium desoxycholate it was indicated that the situation of tryptophane residue in enzyme molecule may have a significance in the activity of lipase I on olive oil.     

Effect of Carbohydrates and Starvation on Nitrogen Sparing Action of Methionine and Threonine in Rats

Formaldehyde dehydrogenase from Pseudomonas putida C-83 was found to contain 7 halfcystine residues per subunit monomer, as checked by the method of performic acid oxidation. Approximately 7 sulfhydryl groups per subunit monomer were titrated with 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB) after denaturation with 8 m urea. In the native enzyme, modification of three sulfhydryl groups per subunit with p-chloromercuribenzoate (PCMB) led to the complete loss of enzyme actiyities for both formaldehyde and n-butanol. Hydrogen-peroxide competitively inhibited the enzyme activity for formaldehyde, while it was only slightly inhibitory to the activity for n-butanol. Both formaldehyde and hydrogen-peroxide protected one sulfhydryl group per subunit monomer from modification with PCMB. Moreover, hydrogen-peroxide was hardly reactive to the enzyme which was preincubated with formaldehyde.

From these observations, we conclude that one of three PCMB-reactive sulfhydryl groups is essential for the binding of formaldehyde, and hydrogen-peroxide modifies this sulfhydryl group.     

Inhibitory activity of novel kojic acid derivative containing trolox moiety on melanogenesis

A novel kojic acid derivative containing a trolox moiety, (±)-5-hydroxy-4-oxo-4H-pyran-2-yl methyl 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylate (3a), was synthesized. The two biologically active compounds, namely, kojic acid and trolox, were conjugated via an ester bond as they are expected to behave synergistically. The antioxidant activity and the tyrosinase inhibitory activity of this novel kojic acid derivative on melanogenesis were evaluated. Compound 3a exhibited potent tyrosinase inhibitory activity and radical scavenging activity. Limited structure–activity relationship (SAR) investigations indicated that the tyrosinase inhibitory activities may originate from the kojic acid moiety, and the radical scavenging activity may be due to the phenolic hydroxyl group of trolox. Compound 3a also exhibited potent depigmenting activity in a cell-based assay. The limited SAR investigations revealed that the depigmenting activity of 3a may be due to the synergistic activities of kojic acid and its trolox moiety.     

Tunicamycin Inhibits Biosynthesis of Acid Mucopolysaccharides in Cultures of Chick Embryo Fibroblasts

Enterobacter cloacae KY 3074 grown in a medium containing xanthine, hypoxanthine, guanine, or their nucleosides and nucleotides produced xanthine oxidase. The purified enzyme preparation showed a major protein band and a few minor bands in acrylamide gel electrophoresis. Molecular oxygen was the most effective electron acceptor. Ferricyanide and 2,6-dichlorophenolindophenol also served as electron acceptors, but NAD and NADP did not. Xanthine and hypoxanthine were good substrates, and guanine was also an effective substrate. The activity was inhibited by Ag²⁺, Cu²⁺, PCMB, and ascorbate. The spectrum of the Enterobacter enzyme resembled that of some known xanthine oxidizing enzymes, and this suggests a similarity in the prosthetic groups of these enzymes. The molecular weight of the native enzyme and subunit was 128,000 and 69,000, respectively.     

Studies on Chrysanthemic Acid

Ribose-5-phosphate ketol-isomerase, an enzyme isomerizing ribose-5-phosphate to ribulose-5-phosphate, is isolated from Candida utilis which is grown in a medium containing xylose. The enzyme is also purified by means of fractionation with ammonium sulfate, acetone, and by DEAE-cellulose column chromatography.

The enzyme has its optimum pH at 7.5 and optimum temperature at 50°C.

Michaelis-Menten constant for d-ribose-5-phosphate is 7.38 × 10^?4 M and activation energy of the enzyme reaction is 10,525 calories.

The enzyme activity is inhibited by p-CMB, EDTA and sodium pyrophosphate, and activated by the addition of magnesium ion.

Extract of Candida utilis contains polyol: NAD oxidoreductase which catalyzes the conversion of polyols to the corresponding ketoses.

By fractionation with ammonium sulfate and on DEAE-cellulose column chromatography, the purity of enzyme has been increased about 14-fold.

The relatively high activity with both xylitol and sorbitol suggests that they may be the natural substances for the enzyme.

Evidence suggests that this enzyme relates to the metabolism of d-xylose in Candida utilis.     

Deacetylcephalosporin C Formation by Cephalosporin C Acetylhydrolase Induced in a Cephalosporium acermonium Mutant

Cephalosporin C acetyl-hydrolase, which had not yet been found in Cephalosporium acremonium cultures, was partially purified from the culture fluid of the mutant No. 81 by ammonium sulfate fractionation, dialysis and DEAE-cellulose column chromatography. The optimum pH and temperature of the enzyme reaction were found to be about 8.0 and 50°C, respectively. The enzyme activity was hardly affected by Mg²⁺, Mn²⁺, Zn²⁺, Co²⁺, Ni²⁺, Na⁺, K⁺, EDTA, PCMB and 2,4-dinitrophenol, but markedly inhibited by diisopropylfluoro-phosphate at 1 mm. The product formed from cephalosporin C by the enzyme reaction was proved to be deacetylcephalosporin C by physical and chemical analyses and chromatographic behaviors.     

Milk Clotting Enzyme from Microorganisms

The purification of the milk clotting enzyme from Mucor pusillus Lindt could be achieved by column chromatography on Amberlite IRC-50 by raising pH from 3.5 to 4.5 and about 70% of activity was recovered after this treatment. After the treatment through the column of DEAE-Sephadex A-25, the trace cellulase activity could be eliminated.

The homogeneity of the purified preparation was proved by ultracentrifugal analysis and electrophoretic patterns at various pH values.

Isoelectric point of this enzyme is considered to lie between pH 3.5 and 3.8.

The enzyme activity was inhibited by Hg⁺⁺ or Fe⁺⁺⁺.

Trypsinogenkinase activity was not contained in this enzyme.

The antiserum against the milk clotting enzyme from Mucor pusillus reacted with the purified and crude enzyme preparations in precipitin test and inhibited their enzyme activities, but did not react with other enzymes such as rennin, pepsin, acid proteases from Aspergillus saitoi and Aspergillus oryzae, or the culture filtrates of some strains of Mucor and Rhizopus.

The antigen-antibody reaction was so specific that it might be possible with this antibody to identify this enzyme and also the strain itself.

Normal sera from some mammals inhibited this enzyme activity too, but the degree was less than that with rennin.     

Calmodulin-dependent and independent NAD kinase activities from cytoplasmic and chloroplastic fractions of spinach (Spinacia oleracea L.)

NAD kinase activity has been found in a soluble, cytoplasmic fraction and in the chloroplasts prepared from green spinach leaves. A small amount of both the cytoplasmic and the chloroplastic NAD kinase activities was retained on a calmodulin-Sepharose affinity column. The cytoplasmic NAD kinase eluted from the affinity column was found to be enhanced by calmodulin in a Ca²⁺-dependent manner. The chloroplastic enzyme which is located exclusively in the stroma and not in the envelope and thylakoid fractions was not affected by Ca²⁺ and calmodulin. The stromal fraction of purified chloroplasts contained only a negligible amount of calmodulin, most probably due to cytoplasmic contamination. Based on these data, two different mechanisms for the light-dependent modulation of spinach NAD kinase activity are suggested.     

Localization and stability of hydrogenases from aerobic hydrogen bacteria

Alcaligenes eutrophus strains H 16, B 19, G 27 and N9A contained two different hydrogenases. One enzyme catalyzed the reduction of NAD by hydrogen and was strictly localized in the soluble cell fraction, while the second enzyme was found to be particulate and unable to react with NAD.All other tested strains, Alcaligenes paradoxus SA 29, Pseudomonas facilis, P. palleronii RH 2, Pseudomonas sp. strain GA 3, Paracoccus denitrificans, Aquaspirillum autotrophicum SA 32, and Corynebacterium autotrophicum 14g and 7C contained only a single enzyme exclusively bound to membranes. This was established using fractional centrifugation, indicator enzyme systems, gentle methods of cell disintegration and discontinuous sucrose density gradient centrifugation. In cell-free extracts obtained by rough disruption (sonication) of cells, hydrogenase was associated to particles of different size and sedimentation velocity. A partial solubilization of hydrogenase caused by sonication was observed with P. facilis.Without exception, the particulate hydrogenases were found (1) to be unable to reduce pyridine nucleotides, and (2) to reduce methylene blue at an extremely high activity. The eminent reaction rate of 34 moles H₂ oxidized per min and mg protein has been determined in particle suspensions of Pseudomonas sp. strain GA 3. All hydrogenases were stable during storage under hydrogen atmosphere, except the soluble enzyme from A. eutrophus H 16 which was shown to be more stable under aerobic conditions.     

Reconstitution of the Z-Disk

Methanol-utilizing bacteria, Klebsiella sp. No. 101 and Microcyclus eburneus could grow aerobically and statically on 1,2-propanediol. The authors examined the presence of enzyme activity of adenosyl-B₁₂ dependent diol dehydratase as well as NAD dependent diol dehydroagenase. Adenosyl-B₁₂ dependent diol dehydratase activity was not detected in these organisms, even if these grown statically.

The dehydrogenase activity was found in the extract from these methanol-utilizing bacteria cells grown on various carbon sources. The partially purified enzyme preparation from the cells of Mic. eburneus grown aerobically on 1,2-propanediol dehydrogenated 1,2-propanediol, 1,2-butanediol and 2,3-butanediol. The enzyme activity was separated into two fractions, namely enzyme I and II on DEAE-Sephadex A-25 column chromatography. The enzyme I was different from the enzyme II in the ratio of enzyme activity to 1,2-propanediol and 2,3-butanediol, heat stability, pH stability and pH optimum, and effect of 2-mercaptoethanol.     

Characterization of the adenosine triphosphatase of avian myeloblastosis virus.

The ATPase of avian myeloblastosis virus (AMV) is not a recognizable cellular enzyme. It hydrolyzes ATP, GTP, ITP, UTP, and dCTP at equal rates, is inhibited by high concentrations of dithiothreitol, and is partially inhibited by 1 × 10^?5mp-chloromercuribenzoic acid (PCMB) and p-chloromercuribenzene sulfonate acid (PCMBS). The inhibition by the mercurials is reversed by increasing the concentration of PCMB or PCMBS to 1 × 10^?3m. The enzyme requires phospholipid for activity. Incubation with phospholipase C inhibits activity and subsequent addition of lecithin-containing saturated fatty acids partially restores activity, whereas lecithin-containing unsaturated fatty acids further inhibit activity.     

Metabolism of Indole-3-acetaldehyde. III. Some Characteristics of the Aldehyde Oxidase of Avena Coleoptiles

Coleoptiles of Avena contain a soluble enzyme system, capable of oxidizing indoleacetaldehyde (lAAld) to indoleacetic acid (IAA). There is a gradient in the concentration of the enzyme along the length of the coleoptile and the first inter-node. The top 5 mm segment of each organ is relatively richer in this enzyme than the rest of the tissue. The enzyme was purified 17.7-fold by fractional precipitation with ammonium sulphate followed by gel filtration on Sephadex. Optimal pH for lAAld oxidation is ca. 4.4. Activity of the enzyme is normally oxygen obligatory. But, in the absence of oxygen, phenazine methosulphate (PMS) serves as hydrogen acceptor for aldehyde oxidation, but not some other dyes tried. Approximately one mole of oxygen was consumed for each mole of IAA formed. Formation of H₂O₂ could not be detected. Added H₂O₂ inhibited the reaction. Prolonged dialysis progressively inactivated the enzyme. Added NAD, NADP, FMN, FAD, cytochrome c, cyanoco-balamin, folic acid and ascorbic acid did not restore the lost activity. But 10^?3M cysteine restored about 60 % of the lost activity. The enzyme is an acidic protein, isoelectric at pH 4.05. For lAAld, under the conditions of experimentation, a Km of 3.45 × 10^?4M was calculated. Besides lAAld, indole-3-aldehyde and phenylacetaldehyde served as substrates, but not acetaldehyde, propionaldehyde, salicylaldehyde, xanthine, hypoxanthine or catechol. Cyanide, dithionite and mercapto-ethanol totally inactivated the enzyme depending upon the concentration and duration of treatment. X-ray irradiation up to a dosage of 2900 r promoted the lAAld oxidizing activity of cell-free preparations made from irradiated coleoptiles. As yet, no cofactor requirements have been found for the activity. The enzyme is unlikely to be a pyridino- or a flavoprotein.     

Inhibition of Hog Liver Crystalline Quinolinate Phosphoribosyltransferase by Nucleotides,Quinolinate Analogues and Sulfhydryl Reagents

Effects of the precursors and intermediates of the NAD biosynthetic pathway, and of quinolinate analogues etc. on hog liver crystalline quinolinate phosphoribosyltransferase (an intermediary enzyme in the de novo NAD biosynthetic pathway) activity were investigated. The enzyme activity was inhibited by many kinds of nucleotides, phthalic acid and SH reagents. But amino acids, nicotinic acid and nicotinamide had practically no effect. The apparent inhibition by ATP removed by raising Mg²⁺ concentration. Phthalic acid was proved to be a competitive inhibitor to quinolinic acid. The Ki value for phthalic acid was calculated at 1.7 × 10^?4 m by a Dixon plot.     

Some Characteristics of Formyltetrahydrofolate Synthetase from Pea Seedlings

It has been found, that ammonium sulfate is effective not only in stabilizing, but also in stimulating the activity of formyltetrahydrofolate synthetase (E. C. 6, 3. 4. 3) purified approximately 500-fold from pea seedlings. Kinetic studies have indicated that the stimulation by ammonium sulfate is due to the enhancement of the binding of the substrate, formate, with the enzyme. The binding of the another substrate, FAH₄, with the enzyme was not affected by the addition of ammonium sulfate. The enzyme activity was inhibited by various sulfhydryl reagents, and the inhibition by PCMB was overcome by the addition of l-cysteine. The inhibition by PCMB was competitive with FAH₄, and the K_i value for PCMB was 0.8 × 10?⁶m.