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H Görisch

Publications and source records attributed to H Görisch.

At least 37 records · Page 2Linked to original sources
PubMed1988

Purification and characterization of a heat-stable esterase from the thermoacidophilic archaebacterium Sulfolobus acidocaldarius.

A heat-stable esterase has been purified 1080-fold to electrophoretic homogeneity from Sulfolobus acidocaldarius, a thermoacidophilic archaebacterium; 20% of the starting activity is recovered. The purified enzyme shows a specific activity of 158 units/mg, based on the hydrolysis of p-nitrophenyl acetate. The esterase hydrolyses short-chain p-nitrophenyl esters, aliphatic esters and triacylglycerols. It is strongly inhibited by paraoxon and phenylmethanesulphonyl fluoride, but only weakly by eserine. From sedimentation-equilibrium data and molecular sieving in polyacrylamide gels, the Mr of the esterase is estimated to be 117000-128000. SDS/polyacrylamide-gel electrophoresis reveals a single band of protein, of Mr 32000. The purified esterase crystallizes in the presence of poly(ethylene glycol) in short rods. The enzyme is inactivated only on prolonged storage at temperature above 90 degrees C.

Archaea↗
PubMed1988

Drop dialysis: time course of salt and protein exchange.

By drop dialysis with membrane filters of 25 or 50 nm average pore size, salt concentrations are reduced to 15% within 25 min. During this time only 10% of ribonuclease with a Mr 13,500 will diffuse in and through the membrane. However, in the presence of 1 M NaCl about 25% of the enzyme is lost. The difference in the rate of salt removal and enzyme loss is caused by the difference in diffusion constants. Therefore with enzymes of higher molecular weights, less protein will be lost, as is shown with beta-galactose dehydrogenase. This enzyme with Mr 64,000 is lost at a lower rate than ribonuclease. The net charge of a protein apparently does not influence the rate with which it diffuses through the membrane. The time course of salt and protein exchange was studied to provide data for estimating the optimal conditions for the required reduction in salt concentration. To prepare small protein samples for electrophoresis or other analytical methods, which require low salt concentrations or a buffer change, drop dialysis is a fast and effective method with tolerable loss of protein.

Animals↗
PubMed1988

Purification, crystallisation and characterization of quinoprotein ethanol dehydrogenase from Pseudomonas aeruginosa.

Pseudomonas aeruginosa ATCC 17933 when grown on ethanol produces high levels of a quinoprotein ethanol dehydrogenase, which amounts to 7% of the soluble protein. The enzyme has been purified to homogeneity and it crystallizes readily in the presence of polyethylene glycol 1550 or 6000. The ethanol dehydrogenase (Km(ethanol) = 14 microM) resembles the dye-dependent quinoprotein methanol dehydrogenases of methylotrophic bacteria, but has a low affinity for methanol (Km (methanol) = 94mM). In addition the enzyme oxidizes secondary alcohols. With its catalytic properties the ethanol dehydrogenase is similar to the enzyme isolated from P. aeruginosa LMD 80.53 (Groen, B., Frank, J. Jzn. & Duine, J.A. (1984) Biochem. J. 223, 921-924). In contrast to this enzyme from P. aeruginosa LMD 80.53, which is a monomer, the ethanol dehydrogenase isolated from P. aeruginosa ATCC 17933 is a dimer of identical subunits of relative molecular mass 60,000. The N-terminal amino acid is lysine. Inactivation with cyclopropanone ethylhemiketal reveals one molecule of pyrroloquinoline quinone per subunit. As shown by active enzyme sedimentation, the dimer is the enzymatically active form.

Alcohol Oxidoreductases↗
PubMed1987

Enzymatic determination of pyrroloquinoline quinone using crude membranes from Escherichia coli.

Escherichia coli K-12 (ATCC 10 798) contains only the apoform of membrane-bound D-glucose dehydrogenase (pyrroloquinoline quinone-dependent). Crude membrane preparations or even crude cell extracts are suitable for a sensitive determination of pyrroloquinoline quinone under appropriate conditions. The apoform of D-glucose dehydrogenase from E. coli is reconstituted with pyrroloquinoline quinone to the holoenzyme in the presence of Mg2+ and assayed for glucose dehydrogenase activity. By increasing the time of reconstitution it is possible to detect minute amounts of about 20 pg pyrroloquinoline quinone.

Apoenzymes↗
PubMed1987

Crystalline NAD/NADP-dependent malate dehydrogenase; the enzyme from the thermoacidophilic archaebacterium Sulfolobus acidocaldarius.

Malate dehydrogenase from Sulfolobus acidocaldarius has been purified 240-fold to apparent electrophoretic homogeneity. The enzyme shows a specific activity of 277 U/mg and crystallizes readily. The relative molecular mass of the native enzyme is estimated as 128,500 by ultracentrifugation. After cross-linking a relative molecular mass of 134,000 is found by sodium dodecyl sulfate gel electrophoresis. Malate dehydrogenase from S. acidocaldarius is composed of four subunits of identical size with a relative molecular mass of 34,000. Active-enzyme sedimentation in the analytical ultracentrifuge indicates that the tetramer is the catalytically active species. Kinetic studies in the direction of oxaloacetate reduction showed a Km for NADH of 4.1 microM and a Km for oxaloacetate of 52 microM. Oxaloacetate exhibits substrate inhibition at higher concentrations, L-malate, NAD and NADP were found to be product inhibitors. The enzymatic activity is inhibited by 2-oxoglutarate but not by the adenosine nucleotides AMP, ADP and ATP. Only low activity is detected in the direction of malate oxidation. Malate dehydrogenase from S. acidocaldarius utilizes both NADH and NADPH to reduce oxaloacetate. The enzyme shows A-side stereospecificity for both nicotinamide dinucleotides.

Amino Acids↗
PubMed1986

Purification and properties of malate dehydrogenase from the thermoacidophilic archaebacterium Thermoplasma acidophilum.

Malate dehydrogenase from the thermoacidophilic archaebacterium Thermoplasma acidophilum is purified 50-fold to electrophoretic homogeneity. The purified enzyme crystallizes readily. Native malate dehydrogenase shows a relative molecular mass of 144 000. It is a tetramer of identical subunits with a relative molecular mass of 36 600. Malate dehydrogenase from Thermoplasma uses both NADH and NADPH as coenzyme to reduce oxaloacetate. The enzyme shows A-side (pro-R) stereospecificity for both coenzymes. The pH optimum for the reduction of oxaloacetate in the presence of NADH is found to be at pH 8.1. At pH 7.4 the Km value for oxaloacetate is found to be 5.6 microM while for NADH a value of 11.7 microM is found. The homogeneous enzyme shows a turnover number of kcat = 108 s-1.

Crystallization↗
PubMed1985

Binding of histidinal to histidinol dehydrogenase.

One molecule of the enzymatic intermediate histidinal is firmly bound per subunit of histidinol dehydrogenase (EC 1.1.1.23) and protected against decomposition. The dissociation rate constant of the histidinal--histidinol dehydrogenase complex is estimated as 2.5 X 10(-5) S-1. Steady-state kinetic measurements studying the oxidation of histidinal to histidine and the reduction of histidinal to histidinol allow to calculate the association rate constants for histidinal. For both reactions the association rate constant is found as 1.9 X 10(6) M-1 S-1. Thus the dissociation constant of the histidinal--histidinol dehydrogenase complex is estimated to be of the order of 1.4 X 10(-11) M.

Alcohol Oxidoreductases↗
PubMed1985

Archaebacterial malate dehydrogenases. The enzymes from the thermoacidophilic organisms Sulfolobus acidocaldarius and Thermoplasma acidophilum show A-side stereospecificity for NAD+.

The stereoselective transfer of hydrogen from NADH to oxaloacetate catalysed by malate dehydrogenases (EC 1.1.1.37) from the thermoacidophilic archaebacteria Sulfolobus acidocaldarius and Thermoplasma acidophilum was studied by the p.m.r. method described by Zhou & Wong [(1981) J. Biochem. Biophys. Methods 4, 329-338]. Both enzymes are A-side (pro-R) stereospecific for NADH.

Archaea↗
PubMed1983

[Biosynthesis of phenylalanine and tyrosine in Streptomycetes].

The biosynthesis of phenylalanine and tyrosine from prephenic acid may proceed via phenylpyruvic acid or p-hydroxyphenylpyruvic acid or via arogenate. The enzymes necessary for these biosynthetic steps have been tested in a variety of Streptomycetes. Tyrosine is synthesized exclusively via arogenate, while phenylalanine is synthesized via phenylpyruvic acid. The feedback inhibition pattern of the corresponding enzymes has been determined. The organisms may be divided into four groups according to the properties of the phenylalanine and tyrosine biosynthetic enzymes and their regulation patterns.

Feedback↗
PubMed1981

Patterns of product inhibition of a bifunctional dehydrogenase; L-histidinol:NAD+ oxidoreductase.

The steady-state kinetic patterns of the bifunctional enzyme histidinol dehydrogenase from Salmonella typhimurium (EC 1.1.1.23) are compatible with a bi-uni uni-bi ping-pong mechanism. Studies of product inhibition make it possible to determine the sequence of substrate binding and product dissociation. Histidinol binds first to the enzyme, followed by the binding of NAD+; histidine is the last product to dissociate from histidinol dehydrogenase. Five of ten kinetic constants defined are determined from linear intercept and slope replots; Km for histidinol was found to be 16 +/- 3 microM and for NAD+ 1 +/- 0.3 mM; K2 for NAD+ was determined to be 0.8 +/- 0.4 mM and K3 for NADH to be 0.3 +/- 0.07 mM. K1 for histidine was found to be 2.1 +/- 0.5 mM.

Alcohol Oxidoreductases↗
PubMed1981

Evidence for an essential lysine at the active site of L-histidinol:NAD+ oxidoreductase; a bifunctional dehydrogenase.

Histidinol dehydrogenase (EC 1.1.1.23) from Salmonella typhimurium is inhibited by formaldehyde and pyridoxal 5-phosphate (pyridoxal-P). epsilon-Pyridoxyl-lysine is isolated upon acid hydrolysis of pyridoxal-P-treated enzyme reduced by sodium borohydride. In the presence of formylhistidinol and formylhistidine (specific ligands of the enzyme) inactivation of histidinol dehydrogenase by pyridoxal-P is prevented. Extrapolation of the initial part of the inactivation curve caused by pyridoxal-P indicates that modification of two essential lysine residues results in inactivation of the dimeric enzyme. The essential lysine residues appear to participate in the reversible oxidation/reduction reaction converting histidinol to histidinal.

Alcohol Oxidoreductases↗
PubMed1979

The catalytically active form of histidinol dehydrogenase from Salmonella typhimurium.

The active-enzyme-sedimentation procedure was used to identify the catalytically competent form of histidinol dehydrogenase (EC 1.1.1.23) isolated from Salmonella typhimurium. At pH 9.4 the active species has a sedimentation coefficient S20,W of 5.4S, indicating that the dimer with a mol.wt. of approx. 83 000 is the enzymically active form.

Alcohol Oxidoreductases↗
PubMed1979

Steady-state investigations of the mechanism of histidinol dehydrogenase.

Initial velocities of the histidinol dehydrogenase reaction (EC 1.1.1.23) were measured as a function of the concentrations of the substrates histidinol and NAD+ and in the presence and absence of the product NADH. The data are consistent with a Bi Uni Uni Bi Ping Pong mechanism. The kinetic constants of this mechanism were determined; Km for histidinol was found to be 14 microM and for NAD+ 0.7 mV; Ki for NAD+ was 0.4 mM.

Alcohol Oxidoreductases↗
PubMed1976

Kinetics of ribosome dissociation and subunit association studied in a light-scattering stopped-flow apparatus.

The association-dissociation kinetics of ribosomes from Escherichia coli have been studied under various conditions in a light-scattering stopped-flow apparatus. The dissociation reaction at 2 mg/ml at 25 degrees C, induced by lowering the MgCl2 concentration from 18 to 3 mM, can best be described by three independent first-order processes with rate constants of 15 s-1, 0.9 s-u, and 3 X 10(-2) s-1, the slowest process comprising about 60% of the overall reaction. The fraction of ribosomes dissociating with the fastest rate (15 s-1) is concentration dependent and becomes negligible at 0.1 mg/ml9 Ribosomes treated with puromycin also show three dissociation rates with essentially the same rate constants as the nontreated samples. The dissociation induced by a high KCl concentration (0.85 MKCl, 18 mM MgCl) also shows three first-order phases with the same rate constants as for the dissociation induced by lowering the MgCl2 concentration. The formation of 70S ribosomes from 30S and 50S subunits, induced by increasing the MgCl2 concentration from 2 to 21 mM, follows second-order biphasic kinetics. A detailed analysis of the kinetic results shows that the two principal ribosomal forms must have one type of subunit in common. When the association data are analyzed assuming that the kinetic heterogeneity arises from two forms of only one subunit, the rate constants are found to be 6.4 X 10(6) and 1.05 X 10(6) M-1 s-1. Sequential flow experiments show that the rapid and slow association species are to be identified, respectively, with phases II (0.9 s-1) and III 0.03 s-1) of dissociation. Relaxation measurements show that these correspond to type B ("loose") and A ("tight") ribosomes, respectively. Tight and loose ribosomes were isolated by sucrose density centrifugation, and dissociation and association kinetic studies confirmed the above assignments. Furthermore, the rate constants for these ribosoems agreed within experimental error with rate constants derived from analysis of the multiphasic kinetic data. The association rate constants are for ribosomes dissociated by dilution with the appropriate buffer immediately before recording the kinetics of association. Ribosomes dissociated by dialysis overnight against 2 mM MgCl2 show an association rate constant for the slower association reaction (type-A ribosome) that is about four times smaller, whereas the rate constant for the faster process is roughly the same. The activation energies of the dissociation reactions, whether induced by lowering the MgCl2 concentration or increasing the KCl concentration, and the association raaction induced by increasing the MgCl2 concentration are less than 3.5 kcal/mol. The rate constants of the dissociation at 3 mM MgCl2 and of the association reaction at 21 mM MgCl2 do not vary between pH 7.2 and 8.4. When 30S and 50S subunits are flowed against buffer containing 20 mM spermidine, the association process is monophasic, with an association constant k - 6 X 10(6) M-1 s-1...

Binding Sites↗