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Query: UMLS:C0272170 (SDS)
 50,377 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Actinoplanes missouriensis utilizes arogenate as an intermediate in L-tyrosine biosynthesis, while no evidence of prephenate dehydrogenase was observed. Arogenate dehydrogenase has been partially purified by a five-step procedure. The enzyme requires NAD as cofactor. The Km values for NAD and arogenate are 0.2 mM and 0.15 mM, respectively. The molecular weight of arogenate dehydrogenase is about 68,000, and SDS gel electrophoresis indicates a composition of two identical subunits. The enzyme is not feedback inhibited by L-tyrosine and unaffected by L-phenylalanine, prephenate, phenylpyruvate, p-hydroxyphenylpyruvate or L-tryptophan. Arogenate dehydrogenase is quite sensitive to p-hydroxymercuribenzoate with 50% inhibition at 12.5 microM of the SH-specific reagent. The presence of malate in usually applied arogenate preparations is demonstrated and the consequence of an impure substrate on arogenate dehydrogenase studies is discussed.
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PMID:Purification and properties of arogenate dehydrogenase from Actinoplanes missouriensis. 259 Mar 41

The high heterogeneity of native rat liver EF-2 prepared from either 105000 x g supernatant or microsome high-salt extract was detected by two-dimensional equilibrium isoelectric focusing-SDS-polyacrylamide gel electrophoresis in the presence of 9.5 M urea. Five spots were always detected, all of Mr 95,000, which were not artefactual for their amount varied when EF-2 was specifically ADP-ribosylated by diphtheria toxin in the presence of NAD+, and/or phosphorylated on a threonine residue by a Ca2+/calmodulin-dependent protein kinase (most likely Ca2+/calmodulin-dependent protein kinase III described by others [(1987) J. Biol. Chem. 262, 17299-17303; (1988) Nature 334, 170-173]). Results of ADP-ribosylation and/or phosphorylation experiments with either unlabeled or labeled reagents ([14C]NAD and [32P]ATP) strongly suggest that our preparation contained native ADP-ribosylated and native phosphorylated forms which could be estimated at about 20% and 40% of the whole EF-2. Phosphorylated and ADP-ribosylated forms of EF-2 could be ADP-ribosylated and phosphorylated, respectively, but a native form both ADP-ribosylated and phosphorylated was not detected. Our results also suggest the existence of a minor native form of EF-2 and of its phosphorylated and ADP-ribosylated derivatives.
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PMID:Heterogeneity of native rat liver elongation factor 2. 279 73

NADP-dependent glutamate dehydrogenase (NADP-GDH) was purified to homogeneity from Pseudomonas aeruginosa strain 8602 (PAC 1). The Mr determined by Sephadex gel filtration was 280,000; the subunit Mr determined by SDS-PAGE was 45,000. Mutant strains lacking NADP-GDH and glutamate synthase (Gdh-Glt-) required glutamate for growth. Transductants that lacked only NADP-GDH were indistinguishable from the wild-type strain in growth properties. It was concluded that NADP-GDH is not essential for growth of the wild-type organism and that glutamate formation via NAD-dependent glutamate dehydrogenase does not occur to a significant extent. A mutant strain, 39, producing high NADP-GDH activity, synthesized normal NADP-GDH and had the same intracellular glutamate concentrations as its parent. The mutation responsible for the synthesis of high levels of NADP-GDH was shown, by transduction, to be closely linked to the NADP-GDH structural gene (gdhA).
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PMID:Mutations affecting the synthesis of NADP-dependent glutamate dehydrogenase in Pseudomonas aeruginosa. 284 62

Highly active superoxide (O2-)-forming NADPH oxidase was extracted from plasmamembranes of phorbol-12-myristate-13-acetate-activated pig neutrophils and was partially purified by gel filtration chromatography. Oxidase activity copurified with cytochrome b-245 in an aggregate containing phospholipids and was almost completely separated from FAD and NAD(P)H-cytochrome c reductase. A polypeptide with molecular weight of 31,500 strictly paralleled the purification of NADPH oxidase, suggesting that it is a major component of the enzyme. The enzyme complex was then dissociated by high detergent and salt concentration and cytochrome b-245 was isolated by a further gel filtration chromatography, with a 147 fold purification with respect to the initial preparation. The cytochrome b-245 showed a 31,500 molecular weight by SDS electrophoresis, indicating that it is actually the component previously identified in the partially purified enzyme. The 31,500 protein was phosphorylated in enzyme preparations from activated but not from resting neutrophils, suggesting that phosphorylation of cytochrome b-245 is involved in the activation mechanism of the O2(-) -forming enzyme responsible for the respiratory burst in phagocytes.
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PMID:Studies on the nature and activation of O2(-)-forming NADPH oxidase of leukocytes. Identification of a phosphorylated component of the active enzyme. 285 Feb 66

Our previous study concerning guanine nucleotides regulation of labeled somatostatin binding has suggested that somatostatin receptors on pancreatic acinar cell membranes probably couple with the inhibitory guanine nucleotide regulatory protein (Ni). In order to clarify the possible role of Ni in mediating signal transduction of somatostatin in the pancreas, we further examined the effect of pretreatment with islet activating protein (IAP) on the inhibition of VIP-stimulated cellular cyclic AMP content by somatostatin in isolated rat pancreatic acini. Increasing concentrations of somatostatin decreased VIP-stimulated cellular content of cyclic AMP in the acini, with a maximal inhibition at 10(-8) M somatostatin. When pancreatic acini were pretreated with varying concentrations of IAP for 4 hours, the somatostatin-induced inhibition of cyclic AMP content was attenuated in a dose dependent manner by IAP pretreatment. Incubation of pancreatic acinar membrane with preactivated IAP and [32P] NAD resulted in labeling of a Mr = 41,000 protein band, consistent with alpha-subunit of Ni in many other cell types previously reported. On the other hand, a Mr = 41,000 protein band on SDS gel was reduced in a dose dependent fashion by IAP pretreatment, when acini were pretreated with increasing concentrations of IAP. These results suggest that only the Mr = 41,000 protein is a specific substrate in pancreatic acinar membranes for IAP-induced ADP-ribosylation. Furthermore, the reduction of 32P incorporation to Mr = 41,000 protein by IAP pretreatment occurred in parallel to decreases in somatostatin-induced inhibition of cellular cyclic AMP contents in pancreatic acini.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:[Effect of islet activating protein on somatostatin-induced inhibition of cellular cyclic AMP content in isolated rat pancreatic acini]. 290 81

ADP-ribosylation and phosphorylation of chromatin proteins was studied in rat uterine nuclei isolated after estrogen treatment and then incubated with [adenylate-32P]NAD or [gamma-32P]ATP. Histone acetylation was studied in uteri from immature rats treated with estradiol by incubating the whole uterus in a medium containing [14C]acetic acid. Chromatin proteins were isolated from uterine nuclei and separated by electrophoresis on SDS polyacrylamide gels followed by autoradiography or fluorography. Chromatin proteins H1, H2B, H3, HMG 14 and HMG 17 were almost exclusively ADP-ribosylated. Uterine histones H1, H3, H4, HMG 14 and HMG 17 were phosphorylated. There was a general increase in [32P]ADP-ribose uptake in chromatin proteins after estrogen stimulation, whereas [32P]phosphate incorporation into chromatin proteins showed a biphasic pattern. The [14C]acetate activity associated with all histone proteins increased gradually after estrogen treatment.
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PMID:Estrogen effects on modifications of chromatin proteins in the rat uterus. 291 95

A testosterone-binding protein (Mr = 50,500) has been isolated from the Gram-negative bacterium Pseudomonas testosteroni. The protein was partially purified by a combination of ion exchange chromatography and chromatofocusing. Final purification was achieved by electroelution of the 50 kDa protein from SDS-polyacrylamide gels. Following renaturation from a diluted solution of guanidine-HCl, specific binding of [3H]testosterone to the purified protein was observed. The native protein has a pI of 6.8. It appears to contain 428 amino acids, 39% of which are hydrophobic. There is only one cysteine residue. Both chymotrypsin and V8 protease were used to produce peptide maps of the protein for use in future identification. The first 10 amino acids situated at the N-terminal of the protein were Ser-Pro-Phe-Asp-Leu-Arg-Pro-Leu-Ser-Gly. Testosterone binding to the protein was saturable at approximately 3.8 nmol/mg protein; the binding constant was approximately 25 nM. Unlabelled testosterone, androstenedione, 5 alpha-dihydrotestosterone and 5 beta-dihydrotestosterone were able to compete for [3H]testosterone bound to the protein; 17 beta-estradiol also competed for [3H]testosterone but to a lesser degree. Neither progesterone nor desoxycorticosterone competed for the testosterone-binding site. Binding of testosterone to the protein was stable at pH's ranging from 5.5 to 9.0 and at various temperatures ranging from 4 to 30 degrees C. The protein was unable to metabolize testosterone in either the presence or absence of the cofactor NAD.
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PMID:Isolation and characterization of a 50 kDa testosterone-binding protein from Pseudomonas testosteroni. 291 97

We have examined a variety of conditions for solubilizing and electrophoresing cell proteins in order to define optimum conditions for studying proteins modified by ADP-ribosylation. We have identified conditions in which proteins can be quantitatively extracted from cells in an undegraded form with the protein-ADPribose linkages intact. Effective measures include boiling cells briefly (4 min) in the presence of 2% SDS and 2 M urea at pH 6.8. Both SDS and urea were present in the 6-18% gradient polyacrylamide gel matrix used for electrophoresis. Under these conditions good resolution of proteins of a wide molecular-weight range is obtained. This system has been used to compare protein ADP-ribosylation in non-transformed and polyma virus-transformed baby hamster kidney (BHK) fibroblasts, since the latter cells have a greater NAD+ ADP-ribosyltransferase activity (measured in isolated nuclei and permeabilized cells). Addition of DNAase to permeabilized BHK cells over the range 10-150 micrograms led to a progressively greater activation of transferase compared with controls. When PyY cells were used, however, maximum activation was achieved with only 10 micrograms of DNAase, further additions producing a successively smaller activation relative to control cells without added nuclease. There were also differences between these cells in response to salt. Addition of NaCl (to about 0.3 M) to BHK cells resulted in various extents of transferase activation, whereas any addition of NaCl to the incubate of permeabilized PyY cells decreased transferase activity. These different enzyme activities between this transformed and non-transformed cell line are for the most part not reflected in the protein modification profiles seen on autoradiograms of acrylamide gels after electrophoresis 32P-labelled proteins. A variety of proteins are modified and their molecular weights depend on the NA concentration in the permeabilized cell incubation. At 0.5 microM NAD+ there were two major acceptors with Mr values of 14 kDa and 30 kDa, and at 100 microM NAD+, three major acceptors, with Mr values of 19 kDa. 45 kDa and greater than 170 kDa. NAD concentrations of between 1 microM and 100 microM had no further effect on protein ADP-ribosylation profiles, except for the protein(s) of Mr greater than 170 kDa, pointing to a critical difference around 0.5-1.0 microM substrate. In some experiments, however, a difference was observed in the intensity of radioactivity in two bands. This may represent two different proteins, or a single protein modified to different extents.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:A gel-electrophoretic analysis of protein ADP-ribosylation in polyoma virus-transformed and non-transformed BHK-21/C13 fibroblasts. 300 86

The cytoplasmic, NAD-linked hydrogenase of Alcaligenes eutrophus H16 consists of four non-identical subunits. From the mutant strain HF14, defective in this enzyme, a protein was isolated that reacted with specific antibodies raised against the wild-type hydrogenase; the reaction type was of partial identity. The same protein was also tested with specific antibodies raised against each of the four denatured subunits of the wild-type hydrogenase and was found to be completely identical with the second largest subunit; it reacted weakly with the antibody against the largest subunit and not at all with the antibody against the small subunits. In SDS-polyacrylamide gel electrophoresis the protein of the mutant migrated as a single polypeptide and corresponded to the second largest subunit of soluble hydrogenase with Mr = 56,000. The mutant enzyme strongly differed from the wild-type hydrogenase in its binding behaviour to chromatographic gels. It had pronounced hydrophobic properties and bound strongly to phenyl-Sepharose; it had high affinity to triazin dye gels. Enzymatically the polypeptide was totally inactive with NAD as electron acceptor, but showed weak residual activities with methylene blue, ferricyanide and cytochrome c. The protein also contained nickel; however, because of the instability of this polypeptide the amount varied between 0.2-1.4 nickel per enzyme molecule. As shown by ESR studies, the iron is organized in a [4Fe-4S] cluster but is partially present also in the 3Fe-form. No nickel signal could be detected. The role of the polypeptide subunit for hydrogen activation in the intact hydrogenase is discussed.
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PMID:Characterization of a native subunit of the NAD-linked hydrogenase isolated from a mutant of Alcaligenes eutrophus H16. 308 7

Poly(ADP-ribose)synthetase has been purified over 600-fold from HeLa cell nuclei. Upon electrophoresis on 9% polyacrylamide slab-gel in the presence of SDS, the purified enzyme resolved in two bands that showed similar apparent Mr values, 110,000 and 118,000. The aminoacid composition of the two components differed from compositions reported for the same enzyme from other sources. In accordance with other reports, the enzyme purified from HeLa cell nuclei exhibited an absolute dependence on DNA and its activity was modulated by changes in the histone concentration. Incubation of intact nuclei from HeLa cells with [14C]NAD resulted in the isolation of a poly(ADP-ribose)synthetase to which a definite radioactivity is bound. These results are taken as a further demonstration that the enzyme can be auto-ADP-ribosylated.
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PMID:Poly(ADP-ribose)synthetase from HeLa cell nuclei: purification and properties. 311 36


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