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)

Human platelets were disrupted using nitrogen cavitation and fractionated on sucrose density gradients to permit isolation of alpha-granules, the major secretory granule of platelets. Membrane proteins prepared from intact alpha-granules by alkali extraction were separated by SDS-polyacrylamide gel electrophoresis, transferred to nitrocellulose and the blot probed for the presence of GTP-binding proteins using [alpha-32P]GTP. Two low molecular mass GTP-binding proteins with molecular mass of 27 and 24 kDa, respectively, were identified on the alpha-granule membrane. In contrast to the 27-kDa protein which was present in significant amounts in the plasma membrane-enriched fraction, the 24-kDa protein showed a preferential association with the alpha-granule membrane. On immunoblotting with specific antiserum, the 24-kDa GTP-binding protein was found to be distinct from rab3A. To the best of our knowledge, the present report represents the first identification of low molecular mass GTP-binding proteins associated with a platelet secretory granule.
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PMID:Association of a 24-kDa GTP-binding protein, Gn24, with human platelet alpha-granule membranes. 193 39

When frozen plasma membranes isolated from maize seedling roots are thawed, a significant portion of GTP-binding activity goes into solution. The GTP-binding protein was purified by ion exchange chromatography on Mono-Q and gel filtration on Superose 6. Its molecular weight was estimated at 61 kDa by gel filtration. The same molecular weight was obtained upon solubilization of the GTP-binding protein with cholic acid followed by gel filtration in the presence of this detergent. SDS-PAGE demonstrated that the isolated GTP-binding protein consists of two types of subunit of molecular weights 27 kDa and 34 kDa.
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PMID:Purification and subunit composition of a GTP-binding protein from maize root plasma membranes. 193 67

Bovine liver mitochondrial translational initiation factor 2 (IF-2mt) has been purified to near homogeneity. The scheme developed results in a 24,000-fold purification of the factor with about 26% recovery of activity. SDS-polyacrylamide gel electrophoresis indicates that IF-2mt has a subunit molecular mass of 85 kDa. IF-2mt promotes the binding of formyl(f)Met-tRNA to mitochondrial ribosomes but is inactive with the nonformylated derivative. IF-2mt is active on chloroplast 30 S ribosomal subunits, but IF-2chl has no activity in promoting fMet-tRNA binding to animal mitochondrial ribosomes. IF-2mt is sensitive to elevated temperatures and is inactivated by treatment with N-ethylmaleimide. It is partially protected from heat and N-ethylmaleimide inactivation by the presence of either GTP or GDP suggesting that guanine nucleotides may bind to this factor directly. The binding of fMet-tRNA to mitochondrial ribosomes requires the presence of GTP and is inhibited by GDP. DeoxyGTP is very effective in replacing GTP in promoting fMet-tRNA binding to ribosomes and some activity is also observed with ITP. No activity is observed with ATP, CTP, or UTP. Nonhydrolyzable analogs of GTP can promote formation of both 28 S and 55 S initiation complexes indicating that GTP hydrolysis is not required for subunit joining in the animal mitochondrial system.
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PMID:Initiation of protein synthesis in animal mitochondria. Purification and characterization of translational initiation factor 2. 193 22

We found small GTP-binding proteins in the outer envelope membrane of pea chloroplasts. The proteins in this membrane were separated by SDS-PAGE, transferred to a nitrocellulose filter, and incubated with [alpha-32P]GTP. Three GTP-binding proteins with the molecular weight of 24,000 were found. Binding was prevented by 10(-8)-10(-7) M GTP or by 10(-7) M guanosine 5'-[gamma-thio]triphosphate or GDP; binding was unaffected by 10(-8)-10(-6) M ATP. Thermolysin treatment of intact chloroplasts resulted in the loss of GTP-binding activity, suggesting that these proteins were in the cytosolic side of the outer envelope membrane.
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PMID:Detection of small GTP-binding proteins in the outer envelope membrane of pea chloroplasts. 195 43

Acetyl-CoA synthetase was purified 800-fold from Bradyrhizobium japonicum bacteroids. A specific activity of 16 mumol/min per mg of protein was achieved, with a 30-40% yield. The purification scheme consisted of only three consecutive chromatography steps. The enzyme has a native Mr of 150,000, estimated by gel-permeation chromatography, and a subunit Mr of 72,000, determined by SDS/polyacrylamide-gel electrophoresis. The optimum pH and temperature are 8.5 and 50 degrees C respectively. The Km values for acetate, CoA and ATP were 146, 202 and 275 microM respectively. The reaction was specific for acetate, as propionate and oleate were used very poorly. Likewise, the enzyme used only ATP, ADP or dATP; AMP, GTP, XTP and UTP could not replace ATP. Acetyl-CoA synthetase showed a broad specificity for metals; MnCl2 could replace MgCl2. In addition, CaCl2 and CoCl2 were approx. 50% as effective as MgCl2, but FeCl3, NiCl2 or ZnCl2 could not effectively substitute for MgCl2. The enzyme may be regulated by NADP+ and pyruvate; no effect was seen of amino acids, glucose catabolites, reduced nicotinamide nucleotides or acetyl-CoA. Inhibition was seen with AMP, PPi, FMN and pyridoxal phosphate, with Ki values of 720, 222, 397 and 1050 microM respectively.
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PMID:Purification and properties of acetyl-CoA synthetase from Bradyrhizobium japonicum bacteroids. 197 Feb 39

Pertussis toxin (PT), preactivated with 20 mm dithiothreitol (DTT), was incubated with different serum dilutions (1/10-1/200) before addition to the reaction mixture. Final concentrations of the reagents were: PT (50 ng/ml), dithiothreitol (DTT)(less than or equal to 0.3 mM), bovine transducin (2 micrograms/ml), ATP (1 mM). GTP (1 mM), lysophosphatidylcholine (LTC) (0.1 mg/ml), sodium acetate (NaAc) (0.1 M), Tris-HCl, pH 7.1 (0.06 M) and 32P-NAD+ (10 microCi 28 Ci/mM). The reaction was stopped by precipitation with 10% TCA (w/v), the pellet was collected and the samples were submitted to SDS-PAGE followed by autoradiography. ADP-ribosylation was detected as the radiolabelling of a protein band (m.w. approximately 40 kD) corresponding to the alpha-subunit of transducin. 32P-ADP incorporation was a linear function of PT concentration. By this assay quantitative differences in PT neutralizing antibodies were found between sera which were not revealed by measuring PT neutralizing antibodies by a Chinese hamster ovary (CHO) cell culture test or by an enzyme-linked immunosorbent assay (ELISA). The conditions of the ADP-ribosylation of bovine transducin have been optimized to permit detection of the enzymic activity of as low amounts of PT as 0.5 ng. This opens the possibility of the study of the presence and avidity of neutralizing antibodies to PT in post-vaccination sera without preceding purification and concentration of the antibodies and thus may provide a useful tool for evaluation of whooping cough vaccines.
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PMID:A sensitive method for measuring neutralizing antibodies to Bordetella pertussis toxin: optimized ADP-ribosylation of transducin. 204 77

Polyclonal antibodies raised against specific recombinant low molecular mass GTP-binding proteins were tested for their ability to recognize partially purified human platelet membrane Gn-proteins (i.e. proteins that bind [alpha-32P]GTP on nitrocellulose blots of SDS/polyacrylamide gels). An antiserum against simian ralA protein recognized a 27 kDa human platelet protein with the same apparent molecular mass as the major platelet Gn-protein (Gn27). In further analysis by two-dimensional polyacrylamide gel electrophoresis, the isoelectric focusing step permitted resolution of 12 major Gn-protein forms, seven of 27 kDa (Gn27a-g), one of 26 kDa (Gn26) and four of 24 kDa (Gn24a-d). The ralA antibody reacted strongly with the five most basic Gn27 species (a-e), weakly with Gn26 and not at all with Gn27f, Gn27g or Gn24a-d. We conclude that ral gene products account for some but probably not for all of the platelet Gn-proteins.
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PMID:Identification of multiple ral gene products in human platelets that account for some but not all of the platelet Gn-proteins. 210 35

Native membranes from human erythrocytes contain the following G proteins which are ADP-ribosylated by a number of bacterial toxins: Gi alpha and Go alpha (pertussis toxin), Gs alpha (cholera toxin), and three proteins of 27, 26 and 22 kDa (exoenzyme C3 from Clostridium botulinum). Three additional C3 substrates (18.5, 16.5 and 14.5 kDa) appeared in conditions of unrestrained proteolysis during hemolysis. SDS-PAGE separation of erythrocyte membrane proteins followed by electroblotting and incubation of nitrocellulose sheets with radiolabeled GTP revealed consistently four GTP-binding proteins with Mr values of 27, 26, 22 and 21 kDa. Although a 22 kDa protein was immunochemically identified as ras p21, the C3 substrate of 22 kDa is a different protein probably identifiable with a rho gene product. Accordingly, at least five distinct small molecular weight guanine nucleotide-binding proteins, whose functions are so far undetermined, are present in native human erythrocyte membranes.
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PMID:Multiple small molecular weight guanine nucleotide-binding proteins in human erythrocyte membranes. 210 17

Activation of adenylyl cyclase by cholera toxin A subunit (CT-A) results from the ADP-ribosylation of the stimulatory guanine nucleotide binding protein (GS alpha). This process requires GTP and an endogenous guanine nucleotide binding protein known as ADP-ribosylation factor (ARF). One membrane (mARF) and two soluble forms (sARF I and sARF II) of ARF have been purified from bovine brain. Because the conditions reported to enhance the binding of guanine nucleotides by ARF differ from those observed to promote optimal activity, we sought to characterize the determinants influencing the functional interaction of guanine nucleotides with ARF. High-affinity GTP binding by sARF II (apparent KD of approximately 70 nM) required Mg2+, DMPC, and sodium cholate. sARF II, in DMPC/cholate, also enhanced CT-A ADP-ribosyltransferase activity (apparent EC50 for GTP of approximately 50 nM), although there was a delay before achievement of a maximal rate of sARF II stimulated toxin activity. The delay was abolished by incubation of sARF II with GTP at 30 degrees C before initiation of the assay. In contrast, a maximal rate of activation of toxin by sARF II, in 0.003% SDS, occurred without delay (apparent EC50 for GTP of approximately 5 microM). High-affinity GTP binding by sARF II was not detectable in SDS. Enhancement of CT-A ADP-ribosyltransferase activity by sARF II, therefore, can occur under conditions in which sARF II exhibits either a relatively low affinity or a relatively high affinity for GTP. The interaction of GTP with ARF under these conditions may reflect ways in which intracellular membrane and cytosolic environments modulate GTP-mediated activation of ARF.
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PMID:Mechanism of activation of cholera toxin by ADP-ribosylation factor (ARF): both low- and high-affinity interactions of ARF with guanine nucleotides promote toxin activation. 211 Nov 67

From porcine thyroid cell membranes, we purified five GTP-binding proteins (G-proteins); Nos. 1 to 3 have 41-kDa alpha-subunits, and Nos. 4 and 5 have 40-kDa alpha-subunits. They were chromatographically (Mono Q) separable and served as specific substrates for islet-activating protein (pertussis toxin). G-proteins 1 and 2 were indistinguishable from porcine brain Gi1 with respect to three criteria, i.e., mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), pI of the ADP-ribosylated alpha-subunit, and immunoreactivity. G-protein 3 was identified as Gi3 by immunoreactivity. The SDS-PAGE and isoelectric focusing (IEF) analyses identified G-proteins 4 and 5 as being chromatographically heterogeneous subtypes of Gi2 in comparison with a pure porcine brain preparation. The IEF analysis also disclosed that each of the Gi1, Gi2, and Gi3 subspecies isolated in the present study has a minor component characterized by a slightly lower pI of its alpha-subunit. We conclude that porcine thyroid tissue contains at least Gi1, Gi2, and Gi3, and that each is made up of heterogeneous populations.
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PMID:Molecular heterogeneity of the subclasses of islet-activating protein (pertussis toxin)-sensitive GTP-binding proteins in porcine thyroid tissue. 211 33


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