Dehydrogenases for the synthesis of chiral compounds.
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Chemical Phenomena↗
Biomedical subjects
W Hummel
Publications and source records attributed to W Hummel.
A convenient enzymatic method for the determination of 4-methyl-2-oxopentanoate in plasma: comparison with high performance liquid chromatographic analysis.
A simple and rapid spectrophotometric method for the estimation of 4-methyl-2-oxopentanoate in plasma samples by use of NAD+-dependent D-2-hydroxyisocaproate dehydrogenase from Lactobacillus casei ssp. pseudoplantarum is described. It is based on the kinetic measurement of the decrease of NADH absorbance at 334 nm. Applicability is demonstrated by comparative measurement of 4-methyl-2-oxopentanoate content in plasma of patients with maple syrup urine disease by the enzymatic and a reversed phase high performance liquid chromatographic method.
Alcohol Oxidoreductases↗
Monitoring of phenylketonuria: a colorimetric method for the determination of plasma phenylalanine using L-phenylalanine dehydrogenase.
A simple, rapid, accurate, and precise colorimetric assay for the determination of L-phenylalanine in plasma samples using L-phenylalanine dehydrogenase [L-phenylalanine:NAD+-oxidoreductase (deaminating)] from Rhodococcus sp. M 4 is described. The enzyme catalyzes the NAD-dependent oxidative deamination of L-phenylalanine. However, the equilibrium of reaction favors L-phenylalanine formation. By stoichiometric coupling of this reaction with diaphorase/iodonitro tetrazolium chloride (INT) the formed NADH converts INT to a formazan whereby the reaction is displaced in favor of phenylpyruvate. Using a kinetic approach the increase in absorbance at 492 nm shows linearity over more than 30 min. Deproteinized standard solutions of L-phenylalanine in the range from 30 to 1200 mumol/liter show a linearity between the dAformazan/30 min and the substrate concentration. In phenylketonuria (PKU) plasma samples no interferences caused by L-tyrosine or phenylpyruvic acid are seen. Applicability is demonstrated by comparative determination of plasma L-phenylalanine of treated PKU patients by the colorimetric method and automated amino acid analysis.
Amino Acid Oxidoreductases↗
Enzymatic determination of L-phenylalanine and phenylpyruvate with L-phenylalanine dehydrogenase.
An enzymatic method is described for the determination of L-phenylalanine or phenylpyruvate using L-phenylalanine dehydrogenase. The enzyme catalyzes the NAD-dependent oxidative deamination of L-phenylalanine or the reductive amination of the 2-oxoacid, respectively. The stoichiometric coupling of the coenzyme allows a direct spectrophotometric assay of the substrate concentration. The equilibrium of the reaction favors L-phenylalanine formation; however, by measuring initial reaction velocities, the enzyme can be used for L-phenylalanine determination, too. Standard solutions of L-phenylalanine in the range of 10-300 microM and of phenylpyruvate (5-100 microM) show a linearity between the value for dENADH/min and the substrate concentration. Besides phenylalanine, the enzyme can convert tyrosine and methionine, and their oxoacids, respectively. The Km values of these substrates are higher. The influence of tyrosine on the determination of phenylalanine was studied and appeared tolerable for certain applications.
Amino Acid Oxidoreductases↗
[A computer program for registering all daily accumulated data at a school for vision disorders].
Despite increasing medical use of the computer, many clinics have experienced only limited success (or none at all) in computerized registration of patient data. One important reason for this lies in the complicated user interfaces of most programs. The clinician has no time to familiarize himself with the complicated command sequences involved, while necessary acceptance of the new technology by administrative staff is often lacking. This paper shows that computerized registration and interpretation of patient data are well within the bounds of possibility, given the use of newly-developed user-friendly operating systems and suitable personal computers. A concrete example of computerized registration of data in a remedial center is given: a case with squint symptoms for which 176 parameters can be entered.
Documentation↗
Characterization of hydantoinase from Pseudomonas fluorescens strain DSM 84.
The hydantoinase (EC 3.5.2.2) from Pseudomonas fluorescens strain DSM 84 was purified either by hydrophobic interaction chromatography on phenyl-Sepharose or by salting out chromatography on Sepharose 4B, gel filtration on Sephacryl S-400, and preparative electrophoresis. Molecular weight values of 230,000 and 60,000 for the native enzyme and each of the four subunits were estimated for the hydantoin hydrolysing activity. The hydantoinase was stable at temperatures up to 40 degrees C but showed an optimal activity at 55 degrees C. The enzyme was markedly inhibited by copper, para-hydroxymercuribenzoate, 8-hydroxyquinoline, and 2,2'-dipyridyl but not by zinc, and poorly by EDTA and o-phenanthroline. The hydantoin-hydrolyzing activity could be reactivated by ferrous ions. Dihydrouracil was the most readily hydrolyzed substrate. The dihydropyrimidinase produced by strain DSM 84 could also hydrolyze 5-substituted hydantions such as isopropylhydantoin (valine derivative) continuously for 10 days in a membrane reactor at a conversion rate of 30%. The only identified end product was N-carbamyl-D-valine.
Amidohydrolases↗
Improved enzyme screening by automated fast protein liquid chromatography.
The assay for NADH-dependent dehydrogenases in crude extracts is often interfered with non-specific reactions. Therefore a screening for such enzymes is hampered by high blank values. To overcome such problems we chromatographed crude extracts on a fast protein liquid chromatography system during part of an enzyme screening for 2-hydroxyisocaproate dehydrogenases and lactate dehydrogenases. The automated chromatography procedure presented consists of a combination of gel filtration and ion-exchange chromatography. The total time needed to perform one cycle of the two-column purification, including the equilibration and regeneration steps, is about 35 min. The procedure described separates the desired enzyme, 2-hydroxyisocaproate dehydrogenase, totally from any interfering activity such as NADH-oxidase and also from the second enzyme of interest, the lactate dehydrogenase. Besides the elimination of the side reactions the desired enzymes are purified up to 20-fold.
Alcohol Oxidoreductases↗
Recent developments in separation and purification of biomolecules.
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Amino Acid Oxidoreductases↗
Purification and characterization of a nicotinamide adenine dinucleotide-dependent secondary alcohol dehydrogenase from Candida boidinii.
From the yeast Candida boidinii grown on glucose a new secondary alcohol dehydrogenase was purified 426-fold by heat treatment, column chromatography on DEAE-Sephacel, affinity chromatography on Blue Sepharose Cl-6b, and gel filtration on Sephacryl S-300. The purified enzyme was homogeneous as judged by analytical polyacrylamide gel electrophoresis. The molecular weight was found to be 150000 by sedimentation equilibrium as well as by gel filtration. The enzyme appears to be composed of four identical subunits (Mr=38000) as determined by SDS-gel electrophoresis. The enzyme catalyzes the oxidation of isopropanol to acetone in the presence of NAD+ as an electron acceptor. The Km values were found to be 0.099 mM for isopropanol and 0.14 mM for NAD+. Besides isopropanol also other secondary alcohols like butan-2-ol, pentan-2-ol, pentan-3-ol, hexan-2-ol, cyclobutanol, cyclopentanol, and cyclohexanol served as a substrate and were oxidized to the corresponding ketones. Isopropanol seems to be the best substrate for this enzyme which we therefore call isopropanol dehydrogenase. Primary alcohols are not oxidized by the enzyme. The optimum pH for enzymatic activity in the oxidation reaction was found to be 9.0, the optimal temperature is 45 degrees C. The isoelectric point of the isopropanol dehydrogenase was found to be pH 4.9. The enzyme is inactivated by mercaptide-forming reagents and chelating agents, 2-mercaptoethanol is an inhibitor. Zinc ions appear necessary for enzyme production.
Alcohol Oxidoreductases↗
Preliminary characterization of ferrichrome synthetase from Aspergillus quadricinctus.
An enzyme synthesizing the cyclic hexapeptide, ferrichrome, was partially purified from extracts of Aspergillus quadricinctus by fractional (NH4)2SO4 precipitation and Bio-Gel A 1.5 m filtration. About a 20-fold purification was achieved. The enzyme system incorporated delta-N-acetyl-delta-N-hydroxyornithine into ferrichrome and catalyzed ATP-PP1 exchange reactions, dependent on the constituent amino acids, glycine and delta-N-acetyl-delta-N-hydroxyornithine, in the presence of Mg2+. The optimal temperature was 27 degrees C. Km values were 3.1 . 10(-4) M for glycine and 5.3 . 10(-6) M for delta-N-acetyl-delta-N-hydroxyornithine. Both Km values were significantly lowered in the presence of 1 . 10(-6) M Fe3+. From the inhibition experiments it is concluded that sulfhydryl groups of the enzyme are involved. Both monomers are covalently bound to the enzyme in the course of the reaction. A molecular weight of 1.1 . 10(6) was determined by gel filtration. As the partially purified protein fraction also catalyzed transacetylation of hydroxyornithine from acetyl CoA, the peptide synthesizing activity may be part of a multienzyme complex. No ferrichrome synthetase activity can be found when the fungus is grown in the presence of 1. 10(-5) M Fe3+.
Amino Acids↗
A double-labelling radioassay for the determination of 5'-nucleotidase activity.
For the determination of 5'-ribonucleotide phosphohydrolase (EC 3.1.3.5;5'-Nase) in rat liver, a radiochemical double-labelling assay was developed. [14C]-labelled AMP which is hydrolyzed to [14C]-adenosine by 5'-Nase activity is added to crude liver homogenates. After 30 min, the process is stopped and [2-3H]-adenosine added to estimate the loss of [14C]-adenosine during separation by ion exchange column chromatography. The enzymatic reaction was found to be linear in correlation with the enzyme content and the incubation time. The specificity of the reaction was evaluated by addition of beta-glycerophosphate which acts as a competitive inhibitor to eliminate the catalytic effect of non-specific phosphatases, and addition of alpha, beta-methylene adenosine 5'-diphosphate, a specific inhibitor of 5'-Nase; both cause an almost complete suppression of enzyme activity.
Adenosine↗
[A new procedure for the direct demonstration of microbial aminoacylase on agar plates with o-phthalaldehyde].
Not less than 1 nmol of methionine and phenylalanine were detected as fluorescent spots on agar plates with o-phthalaldehyde reagent under UV light. Microorganisms were grown on thin-agar-layer coated filter papers placed on nutrient agar plates, and then transferred onto new plates lacking nutrients by moving the papers. After the background amino compounds were removed by the diffusion to the bottom plates, the paper cultures were moved and incubated on assay plates containing N-acetylmethionine or N-acetylphenylalanine. The amino acids formed around the colonies were visualized by the o-phthaladehyde procedure to indicate aminoacylase activity of microorganisms. The substrate-and stereospecificity of the enzyme was shown for some strains on the agar plates by this procedure.
Agar↗
The effect of clotrimazole and triadimefon on 3-hydroxy-3-methyl-glutaryl-CoA-reductase-[EC 1.1.1.34]-activity in Saccharomyces cerevisiae.
Clotrimazole and triadimefon are known as potent inhibitors of ergosterol synthesis in pathogenic yeast and fungi, respectively. As their mode of action generally the inhibition of sterol desmethylation reactions is accepted. We report about a second effect, a "feed-back" inhibition of 3-hydroxy-3-methyl-glutaryl (HMG)-CoA-reductase by accumulation of ergosterol precursors. Addition of lanosterol to intact cells leads to an inhibition of HMG-CoA-reductase as well, but not to fungistatic effects. From the reported data the influences of clotrimazole and triadimefon have to be considered as an inhibition of desmethylation reactions involved in ergosterol synthesis of yeast and fungi with a concomitant decreased production of mevalonate.
Cell Fractionation↗