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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.

Due to their conjugated double bonds the leukotrienes themselves are photolabile compounds and may therefore be used directly for photoaffinity labeling of leukotriene binding sites. Cryofixation eliminates unspecific labeling taking place in solution by photoisomers and photodegradation products of leukotrienes. After fixation of receptor ligand interactions by shock-freezing of the samples, irradiation-induced highly reactive excited states and/or intermediates can form covalent bonds with the respective binding site in the frozen state. After cryofixation of a solution of albumin incubated with [3H8]leukotriene E4, irradiation at 300 nm resulted in time-dependent incorporation of radioactivity into the protein. Photoaffinity labeling of rat as well as of human blood serum with [3H8]leukotriene E4 after cryofixation revealed that only one polypeptide with an Mr of 67,000 was labeled. This polypeptide was identified as albumin. Photoaffinity labeling of rat liver membrane subfractions enriched with sinusoidal membranes resulted in the labeling of a polypeptide with an apparent Mr of 48,000, whereas no polypeptide was predominantly labeled in the subfraction enriched with canalicular membranes. Photoaffinity labeling of isolated hepatocytes disclosed different leukotriene E4 binding polypeptides. In the particulate fraction of hepatocytes a polypeptide with an apparent Mr of 48,000 was labeled predominantly, whereas in the soluble fraction several polypeptides were labeled to a similar extent. One of these, with an apparent Mr of 25,000, was identified as subunit 1 of glutathione transferases by immunoprecipitation. The method of direct photoaffinity labeling in the frozen state after cryofixation using leukotrienes as photoactivatable compounds, as exemplified by leukotriene E4, may be most useful for the identification and characterization of various leukotriene binding sites, including receptors, leukotriene-metabolizing enzymes, and transport systems.

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.

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.

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.

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.