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G Kurz

Publications and source records attributed to G Kurz.

At least 55 records · Page 3Linked to original sources
PubMed1983

6-phospho-D-gluconate dehydrogenase from Pseudomonas fluorescens. Properties and subunit structure.

1. The 6-phospho-D-gluconate dehydrogenase (decarboxylating) (EC 1.1.1.44) from Pseudomonas fluorescens, a B-side stereospecific enzyme, is active with both NAD+ and NADP+, having a specific activity of the homogeneous enzyme of 121 mumols NADH and 23 mumols NADPH, respectively, formed min-1 mg protein-1. The pI of the native enzyme is 4.62, the pH optimum is about 8.2. 2. The molecular weight of the native enzyme has been determined to be 126000 by sedimentation equilibrium studies. The molecular weight of the polypeptide chains composing the enzyme has been found to be 32000 by dodecylsulfate/polyacrylamide gel electrophoresis and 31000 by sedimentation equilibrium studies in presence of 6 M guanidine hydrochloride. The native enzyme is composed of four polypeptide chains. 3. Reacting enzyme centrifugation studies gave at pH 8.2 a sedimentation coefficient s20, w of 8.04 S and a diffusion coefficient D20, w of 6.56 F, resulting in a molecular weight of 115000 for the catalytically active form. Thus, the enzyme is active as the tetramer. So far the enzyme from P. fluorescens is the sole 6-phospho-D-gluconate dehydrogenase (decarboxylating) composed of four polypeptide chains.

Amino Acids↗
PubMed1983

Photolabile derivatives of bile salts. Synthesis and suitability for photoaffinity labeling.

In an approach to the identification of bile salt-binding carriers, the photoactivable bile acid derivatives A) 3 beta-azido, 7 alpha,12 alpha-dihydroxy-5 beta-cholan-24-oic acid, B) 7,7-azo-3 alpha,12 alpha-dihydroxy-5 beta-cholan-24-oic acid, and C) 11 xi-azido-12-oxo-3 alpha,7 alpha-dihydroxy-5 beta-cholan-24-oic acid were synthesized in unconjugated and taurine-conjugated form. Photolysis of the 3 beta-azido derivatives was studied using a light source with a maximum emission at 300 nm and established a half-life time of 18.5 min. The photochemistry of the 7,7-azo derivatives was investigated using light with a maximum at 350 nm and had a half-life time of 2.2 min. The 11 xi-azido-12-oxo derivatives were photolyzed with light having a maximum at 300 nm resulting in a half-life time of 8.5 min. The suitability of the 7,7-azo derivatives for photoaffinity labeling was demonstrated by photolyses in 14C-labeled methanol and acetonitrile. The generated carbene reacted with the solvents under covalent bond formation of 6 to 12%. The efficiency of all synthesized photolabile derivatives for photoaffinity labeling of bile salt binding proteins was demonstrated.

Affinity Labels↗
PubMed1982

Bile-salt-binding polypeptides in plasma membranes of hepatocytes revealed by photoaffinity labelling.

1. Photoaffinity labelling of a subfraction of plasma membranes of rat liver, enriched with sinusoidal surfaces, with the sodium salts of (3 beta-azido-7 alpha,12 alpha-dihydroxy-5 beta-cholan-24-oyl)-2-amino[2-3H(N)]ethanesulfonic acid, (7,7-azo-3 alpha,12 alpha-dihydroxy-5 beta-cholan-24-oyl)-2-amino[2-3H(N)]ethanesulfonic acid and (11 xi-azido-12-oxo-3 alpha,7 alpha-dihydroxy- 5 beta-cholan-24-oyl)-2-amino[2-3H(N)]ethanesulfonic acid resulted with each derivative in a clear covalent incorporation of radioactivity into polypeptides with the apparent molecular weights of 67,000, 52,000, 48,000, 43,000 and about 20,000. 2. Photoaffinity labelling of a membrane subfraction predominantly composed of bile canalicular membranes by the photolabile derivatives of the conjugated bile salts also showed covalent incorporation of radioactivity into polypeptides of the same apparent molecular weights as with the subfraction enriched with the sinusoidal membranes. 3. The extent of photoaffinity labelling of the different membrane polypeptides is dependent upon the photolabile bile-salt derivative used. However, with each of the photolabile derivatives the relative ratio of the labelling of the different membrane polypeptides was similar for both membrane subfractions. Provided that the uptake as well as the secretion of bile salts by hepatocytes are carrier-mediated processes, this suggests the participation of the same polypeptides in both processes.

Affinity Labels↗
PubMed1979

The ineffectiveness of analogs of D-galactal as competitive inhibitors of, and substrates for, beta-D-galactosidase from Escherichia coli.

2,6-Anhydro-3-deoxy-aldehydo-D-lyxo-hept-2-enose (7) and 2,6-anhydro-3-deoxy-D-lyxo-hept-2-enitol (8) were synthesized as half-chair analogs of D-galactal (1). As 1 is a strong inhibitor of, as well as a substrate for, beta-D-galactosidase from Escherichia coli, the same properties were expected for 7 and 8; however, both were ineffective. This result, together with those of other authors, allows speculative conclusions on the tight binding of 1 to the enzyme only, when water or an alcohol is bound as a co-substrate.

Escherichia coli↗
PubMed1979

Bile salt binding to serum components. Taurocholate incorporation into high-density lipoprotein revealed by photoaffinity labelling.

1. Photoaffinity labelling of human serum albumin with the sodium salts of (3 beta-azido-7 alpha,12 alpha-dihydroxy-5 beta-cholan-24-oyl)-2-amino[2(-3)H (N)]ethanesulfonic acid, (7,7-azo-3 alpha,12 alpha-dihydroxy-5 beta-cholan-24-oyl)-2-amino[2(-3)H (N)]ethanesulfonic acid and (11 zeta-azido-12-oxo-3 alpha,7 alpha-dihydroxy-5 beta-cholan-24-oyl)-2-amino[2(-3)H (N)]ethanesulfonic acid resulted, in each case, in a considerable covalent incorporation of radioactivity into the protein. 2. Photoaffinity labelling of whole serum, obtained from fasting test persons, revealed with all three photolabile derivatives of taurocholate at the physiological concentration of 2.1 microM the incorporation of radioactivity not only into albumin but also into high-density lipoprotein, as demonstrated by density gradient centrifugation and by immunological characterization. 3. The bulk of radioactivity incorporated into high-density lipoprotein by photoaffinity labelling of whole serum was found to have been associated with the lipids. Only 10-20% of the label was covalently bound to apolipoproteins, predominantly to the apolipoproteins A-I and A-II. 4. The interaction of taurocholate with high-density lipoprotein has been confirmed by density gradient centrifugation using 14C-labelled taurcholate. It is assumed that the interaction of taurocholate with high-density lipoprotein is physiologically of significance.

Adult↗
PubMed1976

1, 4-alpha-Glucan phosphorylase from Klebsiella pneumoniae purification, subunit structure and amino acid composition.

1. A 1,4-alpha-glucan phosphorylase from Klebsiella pneumoniae has been purified about 80-fold with an over-all yield greater than 35%. The purified enzyme has been shown to be homogeneous by gel electrophoresis at different pH-values, by isoelectric focusing, by dodecylsulfate electrophoresis and by ultracentrifugation. 2. The molecular weight of the native enzyme has been determined to be 180 000 by ultra-centrifugation studies, in good agreement with the value of 189 000 estimated by gel permeation chromatography. 3. The enzyme dissociates in the presence of 0.1% dodecylsulfate or 5 M guanidine hydrochloride into polypeptide chains. The molecular weight of these polypeptide chains has been found to be 88 000 by dodecylsulfate polyacrylamide gel electrophoresis and 99 000 by sedimentation equilibrium studies, indicating that the native enzyme is composed of two polypeptide chains. 4. The enzyme contains pyridoxalphosphate with a stoichiometry of two moles per 180 000 g protein, confirming that the 1,4-alpha-glucan phosphorylase from Klebsiella pneumoniae is a dimeric enzyme. 5. The amino acid composition of the enzyme has been determined, and its correspondence to that of 1,4-alpha-glucan phosphorylases from other sources is discussed. 6. The pI of the enzyme has been shown to be 5.3 and its pH-optimum to be about pH 5.9. The enzyme is stable in the range from pH 5.9 to 10.5.

Amino Acids↗
PubMed1976

A quantitation of the factors which affect the hydrolase and transgalactosylase activities of beta-galactosidase (E. coli) on lactose.

A study was implemented to quantitate the hydrolase and transgalactosylase activities of beta-galactosidase (E. coli) with lactose as the substrate and to investigate various factors which affect these activities. At low lactose concentrations the rate of galactose production was equal to the rate of glucose production. The rate of galactose production relative to glucose, however, dropped dramatically at lactose concentrations higher than 0.05 M and production of trisaccharides and tetrasaccharides began (galactose/glucose ratios of about 2:1 and 3:1, respectively, were found for these two types of oligosaccharides). At least five different trissacharides were formed and their patterns of formation showed that they probably utilized both lactose and allolactose as galactosyl acceptors. Allolactose was produced in amounts proportional to glucose at all lactose concentrations (ratios of allolactose/glucose were about 0.88). Analyses of various data, including a reaction analyzed at very early times, showed that the major means of production of allolactose (and the only means initially) was the direct enzymatic transfer of galactose from the 4 position to the 6 position of the glucose moiety of lactose without prior release of glucose from the enzyme. It was shown, however, that allolactose could also be formed in significant quantities by the transfer of galactose to the 6 position of free glucose, and also by hydrolysis of preformed trisaccharide. A mechanism which fits the initial velocity data was proposed in which the steps involving the formation of an enzyme-gallactose-glucose complex, the formation and breakage of allolactose on the enzyme, and the release of glucose all seem to be of roughly equal magnitude and rate determining. Various factors affected the amounts of transgalactosylase and hydrolase activities occurring. At high pH values (greater than 7.8) the transgalactosylase/hydrolyase activity ratio increased dramatically while it decreased at low pH values (less than 6.0). At mid pH values the ratio was essentially constant. The absence of Mg2+ caused a large decrease in the transgalactosylase/hydrolase activity ratio while the absence of all but traces of Na+ or K+ had no effect. The anomeric configuration of lactose altered the transgalactosylase/hydrolase activity ratios, alpha-Lactose resulted in a decrease of allolactose production (transgalactosylase activity) relative to hydrolase activities (glucose production) while beta-lactose had the opposite effect.

Chromatography, Gas↗
PubMed1975

D-glucose-6-phosphate dehydrogenase (Entner-Doudoroff enzyme) from Pseudomonas fluorescens. Purification, properties and regulation.

1. The existence of two different D-glucose-6-phosphate dehydrogenases in Pseudomonas fluorescens has been demonstrated. Based on their different specificity and their different metabolic regulation one enzyme is appointed to the Entner-Doudoroff pathway and the other to the hexose monophosphate pathway. 2. A procedure is described for the isolation of that D-glucose-6-phosphate dehydrogenase which forms part of the Entner-Doudoroff pathway (Entner-Doudoroff enzyme). A 950-fold purification was achieved with an overall yield of 44%. The final preparation, having a specific activity of about 300 mumol NADH formed per min per mg protein, was shown to be homogeneous. 3. The molecular weight of the Entner-Doudoroff enzyme has been determined to be 220000 by gel permeation chromatography, and that of the other enzyme (Zwischenferment) has been shown to be 265000. 4. The pI of the Entner-Doudoroff enzyme has been shown to be 5.24 and that of the Zwischenferment 4.27. The Entner-Doudoroff enzyme is stable in the range of pH 6 to 10.5 and shows its maximal activity at pH 8.9. 5. The Entner-Doudoroff enzyme showed specificity for NAD+ as well as for NADP+ and exhibited homotropic effects for D-glucose 6-phosphate. It is inhibited by ATP which acts as a negative allosteric effector. Other nucleoside triphosphates as well as ADP are also inhibitory. 6. The enzyme catalyzes the transfer of the axial hydrogen at carbon-1 of beta-D-glucopyranose 6-phosphate to the si face of carbon-4 of the nicotinamide ring and must be classified as B-side stereospecific dehydrogenase.

Culture Media↗
PubMed1975

Subunit structure of D-galactose dehydrogenase from Pseudomonas saccharophila.

1. D-Galactose dehydrogenase from Pseudomonas saccharophila (molecular weight 102 000) dissociates in 8 M urea into its subunits (molecular weight 25 000) which migrate in polyacrylamide gels, containing 8 M urea, as a single band. 2. The N-terminal residue determination by the dansyl method revealed only serine. 3. The C-terminal group determination with carboxypeptidase A and B indicated the sequence -Tyr-His-Leu. Leucine as the single C-terminal amino acid was confirmed by the tritiation method and by tritiation and subsequent degradation with carboxypeptidases. 4. The fragmentation of D-galactose dehydrogenase (24 mol methionine per mol enzyme) by CNBr resulted in six peptides, as detected in disc electrophoresis and substantiated by end group determination, indicating the identity of the subunits. 5. The treatment of D-galactose dehydrogenase (24 mol lysine and 52 mol arginine per mol enzyme) with trypsin and subsequent peptide mapping showed 21, perhaps 22 peptides, indicating a structure comprising four identical subunits.

Alcohol Oxidoreductases↗