Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0272170 (SDS)
 50,377 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We have found in water-soluble extracts of rat liver (and RL-PR-C cloned rat hepatocytes), prepared in the absence of detergent, a factor that markedly enhances basal, isoproterenol and cholera toxin activation of adenylate cyclase of rigorously washed hepatocyte membranes, in the absence of added GTP. The factor, which has characteristics of a protein with an Mr of approx. 35000, has been fractionated from crude cytosol by gel filtration, and then further purified over 50-fold by sequential ion-exchange chromatography. The site of action of the protein appears to be at the level of the guanine nucleotide regulatory (G) protein of the plasma membrane adenylate cyclase complex, as the factor, cooperatively with GTP, also permitted cholera toxin to ADP-ribosylate (from 32P-labeled NAD) two integral membrane proteins that migrated on SDS-polyacrylamide gel electrophoresis gels with the mobilities (Mr approx. 46 000 and 48 000) generally observed for the guanine nucleotide regulator protein subunits. In this system, isoproterenol did not stimulate ADP-ribosylation, in either the presence or absence of the liver protein factor.
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PMID:Partial purification of a water-soluble liver protein that regulates adenylate cyclase activity (basal, hormone- and cholera-toxin-activated) and cholera-toxin-catalyzed ADP-ribosylation of the membrane G protein. 643 82

An NADP-dependent 7 beta-hydroxysteroid dehydrogenase was purified 11.5-fold over the activity in crude cell extracts prepared from Peptostreptococcus productus strain b-52, by using Sephadex G-200 and DEAE-cellulose column chromatography. 7 beta-Dehydrogenation was the sole transformation of bile acids catalyzed by the partially purified enzyme. The enzyme preparation (spec. act. 2.781 IU per mg protein) had an optimum pH of 9.8. Lineweaver-Burk plots showed a Michaelis constant (Km) value of 0.05 mM for 3 alpha, 7 beta-dihydroxy-5 beta-cholanoic acid whereas higher values were obtained with 3 alpha,7 beta-dihydroxy-5 beta-cholanoyl glycine (0.20 mM), and 3 alpha,7 beta-dihydroxy-5 beta-cholanoyl taurine (0.26 mM). NADP but not NAD could function as an electron acceptor, and had a Km value of 0.30 mM. A molecular weight of 64000 was determined by SDS-polyacrylamide gel electrophoresis. The addition of 0.4 mM of either bile acid to the growth medium suppressed not only cell growth, but also the enzyme yield.
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PMID:Purification and characterization of NADP-dependent 7 beta-hydroxysteroid dehydrogenase from Peptostreptococcus productus strain b-52. 657 75

The effects of intravesicular NAD on Na+-dependent 32Pi uptake were investigated in isolated rat kidney brush border membrane vesicles (BBMV). NAD was introduced into the vesicles by osmotic shock, and extravesicular NAD was removed by passing the vesicles through a anion exchange column. The effectiveness of the osmotic shock procedure and the hydrolysis of extra- and intravesicular NAD were controlled by enzymatic analysis and thin layer chromatography. ADP-ribosylation of the membrane proteins was analyzed in vesicles osmotically shocked in the presence of either [adenylate-32P]-NAD or [adenine-2,8-3H]-NAD by SDS-polyacrylamide gel electrophoresis. It was found that the Na+-dependent Pi uptake was inhibited when the BBMV were incubated with NAD at alkaline pH, which resulted in rapid NAD hydrolysis. When NAD was present in the intravesicular space only, the Na+-dependent Pi uptake was not inhibited. 32P from NAD was rapidly incorporated into a number of brush border membrane proteins, but no incorporation of 3H-adenine could be detected. The results provide evidence that NAD does not inhibit Pi transport by a direct interaction with the cytoplasmic side of the brush border membrane. No evidence of ADP-ribosylation of the brush border membrane protein(s) was found.
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PMID:Intravesicular NAD has no effect on sodium-dependent phosphate transport in isolated renal brush border membrane vesicles. 670 90

Incubation of purified rat brain tubulin with cholera toxin and radiolabeled [32P] or [8-3H]NAD results in the labeling of both alpha and beta subunits as revealed on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Treatment of these protein bands with snake venom phosphodiesterase resulted in quantitative release of labeled 5'-AMP, respectively labeled with the corresponding isotope. Two-dimensional separation by isoelectric focusing and SDS-PAGE of labeled and native tubulin revealed that labeling occurs at least in four different isotubulins. The isoelectric point of the labeled isotubulins was slightly lower than that of native purified tubulin. This shift in mobility is probably due to additional negative charges involved with the incorporation of ADP-ribosyl residues into the tubulin subunits. SDS-PAGE of peptides derived from [32P]ADP-ribosylated alpha and beta tubulin subunits by Staphylococcus aureus protease cleavage showed a peptide pattern identical with that of native tubulin. Microtubule-associated proteins (MAP1 and MAP2) of high molecular weight were also shown to undergo ADP-ribosylation. Incubation of permeated rat neuroblastoma cells in the presence of [32P]NAD and cholera toxin results in the labeling of only a few cell proteins of which tubulin is one of the major substrates.
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PMID:ADP-ribosylation of microtubule proteins as catalyzed by cholera toxin. 676 71

Acceptors of poly(ADP-ribosylation) were identified and compared between inducer-treated and untreated Friend erythroleukemia cells. When permeabilized Friend cells were pulse labeled with 0.6 microM [32P]NAD for 1 min and labeled proteins analyzed by SDS-polyacrylamide gel electrophoresis, nucleosome core histones were found to be the primary acceptors, with an additional minor radioactive peak at a position corresponding to Mr = 170 000. Friend cells induced to differentiate by DMSO treatment showed a similar distribution of radioactivity, but with a 60% reduction in the overall level of poly(ADP-ribosylation) under identical labeling conditions. When isolated nuclei were pulse labeled with 0.6 microM [32P]NAD, radioactive peaks were not restricted mainly at the positions of core histones but widely dispersed in the area from 10 to 50 kDa with another peak at 170 kDa. Increase of NAD concentration resulted in the overall shift of peaks to higher molecular weight positions. When pulse-labeled nuclei or permeable cells were chased with 1 mM NAD, radioactive peaks migrated to positions of very high molecular weight (greater than Mr = 180 000). Remarkable suppression of poly(ADP-ribose) synthesis was observed when DMSO, hexamethylene bisacetamide, butyric acid, or hemin were used as the inducers.
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PMID:Acceptors of poly(ADP-ribosylation) in differentiation inducer-treated and untreated Friend erythroleukemia cells. 715 93

The influence of the pH of the medium on growth and production of extracellular proteins was examined in a group C streptococcus (SA. equisimilis strain H 46 A). The pH values employed ranged from 5.0 to 8.5 and the analytical methods used included the determination of enzyme activities, isoelectric focusing, crossed-immunoelectrophoresis, and SDS electrophoresis. The strain is able to grow in the entire pH range used, with maximum growth occurring at pH 6.5. The amount of extracellular proteins elaborated depended on both the pH of the medium and the biomass produced. A specific pH range was defined for optimum production of all enzymes examined. With the exception of streptodornase and NAD-glycohydrolase, the pH optimum for product formation was generally in the neutral and weakly alkaline range. Active proteinase was formed only a pH 6.0, leading to gross degradation of most of the other proteins in the culture medium. At pH values ranging from 6.5 to 7.5, isoelectric focusing revealed the production of 28 different extracellular proteins, and crossed immunoelectrophoresis demonstrated 30 to 32 antigens. The number of extracellular products produced greatly declined at pH values above and below this range. Under optimum conditions, the main extracellular product at H 46 A was streptokinase, followed by deoxyribonuclease A.
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PMID:[Influence of physical parameters on the production of streptococcal extracellular proteins in cultures with stabilized pH. I. Communication: pH-dependence of extracellular protein production (author's transl)]. 719 49

Malic enzyme has been purified from Ascaris suum by polyethylene glycol precipitation, ion-exchange chromatography, ammonium sulfate precipitation, and NAD-agarose affinity chromatography to a specific activity of 80 units/mg (V/[E]t = 350 s-1). The preparation was shown to be homogeneous by SDS polyacrylamide disc gel electrophoresis. The procedure can be accomplished in a maximum of four days with a 74% yield.
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PMID:Purification of malic enzyme from Ascaris suum using NAD+-agarose. 724 72

The particulate fraction, heat-labile factor, heat-stable factor, and NADPH are essential for the conversion of lignoceric acid (tetracosanoic acid) to cerebronic acid (alpha-hydroxylignoceric acid). The heat-labile factor was extracted from calf cerebellum and partially purified in four steps: ammonium sulfate precipitation, hydroxylapatite chromatography, isoelectric focusing, and NAD-Agarose affinity chromatography. The specific activity of the heat-labile factor was increased 105-fold during the last three steps, with a yield of 37% of the activity. One major and several minor bands were visible when the preparation was examined by SDS-polyacrylamide gel electrophoresis with Coomassie blue staining. The major band corresponded to a protein of molecular weight 32,700, and the minor bands corresponded to proteins of molecular weights 62,000 and 67,000. The activity was lost when the heat-labile factor was incubated with 1 mM-N-ethylmaleimide. This inhibition was prevented by preincubating the heat-labile factor with 1 mM-NADH. These observations indicate that the heat-labile factor contains a sulfhydryl group which is essential for activity, and that it is located at or near the binding site for the pyridine nucleotide.
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PMID:alpha-Hydroxylation of fatty acids in brain: characterization of heat-labile factor. 726 65

D-Erythrulose reductase from chicken liver has been purified to homogeneity as judged by acrylamide gel electrophoresis and ultracentrifugation. The overall purification of the enzyme was 164-fold from a crude extract. The enzyme was crystallized from ammonium sulfate solution at pH 7.0 to give hexagonal plates. The molecular weight determined by sedimentation equilibrium analysis was 94,600 and that by SDS-polyacrylamide gel electrophoresis was 22,400, which suggests a tetrameric structure for the native enzyme. The enzyme was found to contain up to 3 molecules of NADP+ per enzyme; this high amount of NADP+ resulted in a higher absorption at 260 nm than at 280 nm. The extinction coefficient of the enzyme at 290 nm was found to be 4.0. The contents of various amino acids were very similar to those of the beef liver enzyme formerly crystallized in our laboratory. The isoelectric point of the enzyme determined by Ampholine isoelectric focusing was pH 6.43. The enzyme was shown to catalyze the reduction of D-erythrulose to D-threitol with the concomitant oxidation of NAD(P)H to NAD(P)+, and was highly specific to D-erythrulose with an apparent Km of 0.38 mM. NADH was less effective than NADPH and the Km's for NADH and NADPH were 67 micrometers and 7.9 micrometers, respectively. D-Threitol was slightly oxidized by the enzyme with either NADP+ or NAD+ as a cofactor at pH's 7.5 and 9.0.
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PMID:Studies on D-tetrose metabolism. Crystallization and properties of D-erythrulose reductase from chicken liver. 735 41

The locations of ATP- and NAD-binding sites on diphtheria toxin were investigated by ultraviolet irradiation of ligand . toxin complexes. Illumination of ATP with ultraviolet light (253.7 nm) in the presence of various proteins resulted in photoinduced cross-linking only with Fraction II of diphtheria toxin. Under the same conditions, NAD was cross-linked most effectively to Fragment A, followed by Fraction II and CRM 45. For both ATP and NAD, the degree of protein labelling correlated well with binding data, suggesting that photoinduced cross-linking ocurred only at the high affinity binding sites for these ligands. Nonspecific labeling of unrelated proteins was consistently less than 5% of that observed for Fraction II. Analysis of nicked and reduced Fraction II . ligand complexes on SDS polyacrylamide gels demonstrated that essentially all of the cross-linked label migrated with the A fragment, whether photolysis was performed with ATP or NAD.
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PMID:Ligand interactions of diphtheria toxin. III. Direct photochemical cross-linking of ATP and NAD to toxin. 744 May 83


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