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)

The biologic activity of substance P has been demonstrated to be limited both in in vivo and in vitro by a membrane-bound protease, neutral endopeptidase (EC 3.4.24.11). The interaction of substance P with its receptor on guinea pig lung tissues was studied in the presence of an inhibitor of neutral endopeptidase under conditions that protect the peptide from degradation. Uptake of 0.1 nM [125I]-BH-substance P in lung membrane preparations was rapid at 4 degrees C, reaching equilibrium in 30 to 40 min, and binding was stable for at least 30 min thereafter. Binding was reversible and saturable. Scatchard analyses of saturation binding data are consistent with a single class of receptor molecules in both lung parenchymal and airway membranes, with a Kd of 2 to 3 nM and a receptor density of 4,000 to 5,000 fmol/g wet wt of tissue. In competitive binding experiments, neurokinin A and substance P methyl ester were equipotent and required approximately 100-fold higher concentrations to effect equivalent displacement than unlabeled substance P. Eledoisin also competed for [125I]-BH-substance P binding, but was less effective than the other analogs. The spasmogenically inactive derivative, substance P 1-9, did not compete for substance P binding at concentrations as high as 1 microM. Binding of [125I]-BH-substance P was rapidly and completely reversed by addition of 0.1 mM GTP, suggesting that association with a GTP binding protein is required for high affinity binding of substance P to its receptor in lung. The substance P receptor molecule was further characterized by covalently crosslinking [125I]-BH-substance P to membrane preparations followed by SDS-PAGE of the solubilized material.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Characterization of the substance P receptor in guinea pig lung tissues. 248 20

Adrenal medullary homogenates and chromaffin granule membranes were separated by SDS-polyacrylamide gel electrophoresis and GTP-binding proteins detected using [alpha-32P]GTP binding to nitrocellulose blots. Four GTP-binding polypeptides of 24, 22, 20 and 18 kDa were routinely found in medullary homogenates and all were also found in isolated chromaffin granule membranes. The GTP-binding polypeptides co-sedimented with granule membrane markers following separation on sucrose gradients. On the basis of trypsin sensitivity and resistance to extraction, the GTP-binding proteins appeared to be tightly bound to the cytoplasmic surface of the granules. One or more of the secretory granule GTP-binding proteins could be involved in exocytosis in adrenal chromaffin cells.
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PMID:Low molecular mass GTP-binding proteins of adrenal chromaffin cells are present on the secretory granule. 249 70

Isolated uncoupling protein (UCP) can be cross-linked, by various disulfide-forming reagents, to dimers. The best cross-linking is achieved with Cu2+-phenanthroline oxidation. Because cross-linking is independent of UCP concentration and prevented by SDS addition, a disulfide bridge must be formed between the two subunits of the native dimer. Cross-linking is prevented by SH reagent and reversed by SH-reducing reagents. In mitochondria, cross-linking of UCP with disulfide-forming agents is even more efficient than in isolated state. It proves that UCP is a dimer in mitochondria, before isolation. Disulfide-bridge formation does not inhibit GTP-binding to UCP. Cross-linked UCP re-incorporated in proteoliposomes either before or after cross-linking fully retains the H1-transport function. Rapid cross-linking by membrane impermeant reagents indicates a surface localization of the C-terminus in soluble UCP and projection to the outer surface in mitochondria. Intermolecular disulfide-bridge formation in a dimer requires juxtaposition of identical cysteines at the twofold symmetry axis. A rigid juxtaposition of cysteines is unlikely, unless intended for a native disulfide bridge. The absence of such a bridge in UCP suggests that juxtaposition of cysteines is generated by high mobility. In order to localize the cysteine involved, cross-linked UCP was cleaved by BrCN. The CB-7 C-terminal peptide, which contains cysteines at positions 287 and 304, disappears. Limited trypsinolytic cleavage, previously shown to occur at Lys-292, removed cross-linking in UCP both in the solubilized and mitochondrially bound state. The cleaved C-terminal peptide of 11 residues contains only cystein-304 which, thus, should be the only one (out of 7 cysteines in UCP) involved in the S-S bridge formation. Obviously, the C-terminal location of the cysteine, because of its high mobility, permits juxtapositioning for cross-linking. This agrees with predictions from hydrophobicity analysis that the last 14 residues in UCP protrude from the membrane.
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PMID:The uncoupling protein dimer can form a disulfide cross-link between the mobile C-terminal SH groups. 249 40

The predominant guanine nucleotide-binding protein (G-protein) of bovine lung membranes, termed GL, has been purified and compared biochemically, immunochemically and functionally with Gi and Go purified from rabbit brain. The purified GL appeared to have a similar subunit structure to Gi and Go, being composed of alpha, beta and possibly gamma subunits. On Coomassie Blue-stained SDS/polyacrylamide gels and immunoblots, the alpha subunit of GL (GL alpha) displayed an intermediate mobility (40 kDa) between those of Gi and Go (Gi alpha and Go alpha). GL alpha was [32P]ADP-ribosylated in the presence of pertussis toxin and [32P]NAD+. Analysis of [32P]ADP-ribosylated alpha subunits by SDS/polyacrylamide-gel electrophoresis and isoelectric focusing showed that GL alpha was distinct from Gi alpha and Go alpha, but very similar to the predominant G-protein in neutrophil membranes. Immunochemical characterization also revealed that GL was distinct from Gi and Go, but was indistinguishable from the G-protein of neutrophils, which has been tentatively identified as Gi2 [Goldsmith, Gierschik, Milligan, Unson, Vinitsky, Maleck & Spiegel (1987) J. Biol. Chem. 262, 14683-14688]. In functional studies, higher Mg2+ concentrations were required for guanosine 5'-[gamma-[35S]thio]triphosphate (GTP[35S]) binding to GL than were required for nucleotide binding to Go, whereas Gi showed a Mg2+-dependence similar to that of GL. The kinetics of GTP[35S] binding to GL was quite different from those of Gi and Go; t1/2 values of maximal binding were 30, 15 and 5 min respectively. In contrast, the rate of hydrolysis of [gamma-32P]GTP by GL (t1/2 approximately 1 min) was approx. 4 times faster than that by Gi or Go. These results indicated that the predominant G-protein purified from lung is structurally and functionally distinct from Gi and Go of brain, but structurally indistinguishable from Gi2 of neutrophils.
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PMID:Purification and characterization of predominant G-protein from bovine lung membranes. Biochemical and immunochemical comparison with Gi1 and Go purified from brain. 249 37

We recently identified a phosphoinositide-specific phospholipase C (PI-PLC)-stimulating GTP-binding protein (G protein) in calf thymocyte cytosol (Wang, P., Toyoshima, S., & Osawa, T. (1987) J. Biochem. 102, 1275-1287; and (1988) 103, 137-142). In this study we completely purified a G protein whose properties are quite similar to the G protein mentioned above from the calf thymocyte membrane and determined partial amino acid sequences of it. The purification was achieved by first treating the membrane with GTP gamma S, followed by sequential column chromatographies on DEAE-Sepharose CL-6B, Sephacryl S-200, Mono Q, and Mono S. The G protein was purified in a GTP gamma S-binding form and assayed as to the radioactivity of the [35S]GTP gamma S-bound PI-PLC-associated G protein standard obtained from calf thymocyte cytosol. The purified G protein could stimulate the activity of a partially purified PI-PLC for phosphatidylinositol 4,5-bisphosphate hydrolysis. From approximately 5.6 g of membrane protein we obtained about 5 micrograms of a purified sample. The purified G protein showed a molecular weight of 21 kDa on SDS-PAGE and one of 25 kDa on gel filtration. The partial amino acid sequences were determined by treating the purified sample with lysylendopeptidase, purifying the resultant peptide fragments on a HPLC-reverse phase column and then sequencing the peptide fragments with a sequencer. Comparison of the obtained sequences with those of known lower molecular weight GTP-binding proteins suggested that, although structurally similar to rho gene products, this is a novel G protein.
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PMID:Purification and partial amino acid sequences of a phospholipase C-associated GTP-binding protein from calf thymocytes. 249 75

The mechanism of heterologous desensitization of adenylate cyclase stimulation was studied in cultured neonatal rat heart muscle cells. After culturing of the cells for 3 days in the presence of 1 microM noradrenaline there was in addition to a 52% decrease in isoproterenol-stimulated adenylate cyclase activity, a lessening of the stimulation of beta-adrenoceptor-independent adenylate cyclase by guanosine-5'-O-(thiotriphosphate) and forskolin by 24 and 34%, respectively. The decrease in receptor-independent adenylate cyclase stimulation by forskolin, but not the attenuation of isoproterenol-stimulated adenylate cyclase activity, was abolished by pertussis toxin (PTX) pretreatment of the cells. Gi, the inhibitory G-protein of adenylate cyclase was therefore quantitated. Labelling of the Mr approximately 40 kDa PTX substrates in membranes of noradrenaline-treated cells was increased by 70% as shown by pertussis toxin-catalyzed ADP ribosylation of heart cell membranes. This increase was also seen in the presence of an excess of purified beta gamma-subunits of transducin and of GTP, suggesting that the increased labelling was not due to elevation of the level of beta gamma-subunits or increase in the concentration of GTP in the membranes of noradrenaline-treated cells. Analysis of the PTX substrates on high resolution urea/SDS-polyacrylamide gels revealed that at least two distinct PTX substrates (40 and 41 kDa) were present in rat heart cell membranes. The labelling of both substrates was increased in membranes of desensitized cells. Immunoblotting of heart cell membranes with anti-Gi alpha-antibodies demonstrated a marked increase in the amount of Gi alpha in membranes of noradrenaline-treated cells. In contrast, immunoblotting with anti-beta-antibodies showed that the level of the beta-subunit of G-proteins (36 kDa) was unchanged after noradrenaline exposure. The data indicate that prolonged treatment of rat heart muscle cells with noradrenaline leads to an increase in the level of alpha-subunits of Gi-proteins. This suggests that this increase is responsible for the observed heterologous desensitization of adenylate cyclase stimulation.
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PMID:Mechanism of noradrenaline-induced heterologous desensitization of adenylate cyclase stimulation in rat heart muscle cells: increase in the level of inhibitory G-protein alpha-subunits. 250 67

As a first step in determining the molecular mechanism of membrane fusion stimulated by GTP in rough endoplasmic reticulum (RER), we have looked for GTP-binding proteins. Rough microsomes from rat liver were treated for the release of ribosomes, and the membrane proteins were separated by SDS/polyacrylamide-gel electrophoresis. The polypeptides were then blotted on to nitrocellulose sheets and incubated with [alpha-32P]GTP [Bhullar & Haslam (1987) Biochem. J. 245, 617-620]. A doublet of polypeptides (23 and 24 kDa) was detected in the presence of 2 microM-MgCl2. Binding of [alpha-32P]GTP was blocked by 1-5 mM-EDTA, 10-10,000 nM-GTP or 10 microM-GDP. Either guanosine 5'-[gamma-thio]triphosphate or guanosine 5'-[beta gamma-imido]triphosphate at 100 nM completely inhibited binding, but ATP, CTP or UTP at 10 mciroM did not. Pretreatment of microsomes by mild trypsin treatment (0.5-10 micrograms of trypsin/ml, concentrations known not to affect microsomal permeability) led to inhibition of [alpha-32P]GTP binding, suggesting a cytosolic membrane orientation for the GTP-binding proteins. Two-dimensional gel-electrophoretic analysis revealed the 23 and 24 kDa [alpha-32P]GTP-binding proteins to have similar acid isoelectric points. [alpha-32P]GTP binding occurred to similar proteins of rough microsomes from rat liver, rat prostate and dog pancreas, as well as to a 23 kDa protein of rough microsomes from frog liver, but occurred to distinctly different proteins in a rat liver plasma-membrane-enriched fraction. Thus [alpha-32P]GTP binding has been demonstrated to two low-molecular-mass (approx. 21 kDa) proteins in the rough endoplasmic reticulum of several varied cell types.
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PMID:Detection of GTP-binding proteins in purified derivatives of rough endoplasmic reticulum. 250 29

The distribution of the alpha- and beta-subunits of G-proteins and their disposition in rat liver plasma and intracellular membranes was investigated. Western blotting, using antibodies that recognised the alpha-subunit of the inhibitory and the beta-subunits of most G-proteins, identified 41 and 36 kDa polypeptides respectively in all plasma membrane functional domains, in endosomes as well as in Golgi membranes. Lysosomes were devoid of these subunits. The highest levels of G-protein subunits were found in bile canalicular plasma membranes prepared by density gradient centrifugation followed by free-flow electrophoresis. Separation of membrane proteins into extrinsic and intrinsic components was carried out by extraction of the membranes at pH 11.0 and by partitioning the membranes in Triton X-114/aqueous phases. The results demonstrated that the alpha- and beta-subunits were tightly associated with the hepatic membranes but they could be solubilised by extraction with detergent, e.g. SDS. Prolonged incubation in the presence of GTP analogues also released up to approximately 50% of the alpha-subunit of inhibitory G-proteins from membranes. The beta-subunit was still associated with membranes after alkaline extraction. The results emphasise the strong association of G-protein subunits with liver membranes, and show that these proteins are distributed widely in the plasma membrane and along the endocytic pathways of hepatocytes.
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PMID:G-proteins of rat liver membranes. Subcellular compartmentation and disposition in the plasma membrane. 251 42

Vasopressin V1 receptors were solubilized from rat liver plasma membranes with the detergent lysophosphatidylcholine. [[3H]Arginine]vasopressin (AVP) binding to the solubilized preparations was specific and saturable, with a dissociation constant of 0.6 nM. Cross-linking of [125I]vasopressin to the solubilized fraction, studied by SDS/polyacrylamide-gel-electrophoretic analysis, demonstrated the presence of a 65 kDa band which was specifically labelled with [125I]vasopressin. Specific binding of [3H]AVP to these solubilized receptors was decreased by guanine nucleotides, but not by adenosine 5'-[beta gamma-imido]triphosphate. Addition of vasopressin increased specific binding of 35S-labelled guanosine 5'-[gamma-thio]triphosphate (GTP[35S]) to the solubilized fractions, indicating co-solubilization of GTP-binding protein(s) [G-protein(s)] and vasopressin receptors. The solubilized fraction was insensitive to both cholera- and pertussistoxin treatment. Immunoblotting of the solubilized fraction with antibodies specific for a phosphoinositide-specific phospholipase C (PI-PLC I) demonstrated the presence of a 60 kDa protein. Anti-PI-PLC I antiserum immunoprecipitated solubilized vasopressin-binding sites from rat liver (V1), but not solubilized vasopressin-binding sites from hog kidney (V2). Similar results were obtained with an anti-PI-PLC I IgG affinity column. The solubilized (V1) receptors were enriched by ion-exchange and high-performance gel-filtration liquid chromatography. Vasopressin-binding activity was co-eluted with PI-PLC I and GTP[S]-binding activity on a DEAE-Sepharose column. The major vasopressin- and GTP[35S]-binding activities were co-eluted with PI-PLC I activity at approx. 240 kDa suggesting that vasopressin receptors from rat liver membranes can be solubilized as a complex of receptor-coupler-effector by using the detergent lysophosphatidycholine.
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PMID:Solubilization of rat liver vasopressin receptors as a complex with a guanine-nucleotide-binding protein and phosphoinositide-specific phospholipase C. 254 66

Somatostatin receptors of plasma membranes from beta cells of hamster insulinoma were covalently labelled with 125I-[Leu8,D-Trp22,Tyr25]somatostatin-28 (125I-somatostatin-28) and solubilized with the non-denaturing detergent Triton X-100. Analysis by SDS/PAGE and autoradiography revealed three specific 125I-somatostatin-28 receptor complexes with similar molecular masses (228 kDa, 128 kDa and 45 kDa) to those previously identified [Cotroneo, P., Marie, J.-C. & Rosselin, G. (1988) Eur. J. Biochem. 174, 219-224]. The major labelled complex (128 kDa) was adsorbed to a wheat-germ-agglutinin agarose column and eluted by N-acetylglucosamine. Also, the binding of 125I-somatostatin-28 to plasma membranes was specifically inhibited by the GTP analog, guanosine-5'-O-(3-thiotriphosphate) (GTP[S]) in a dose-dependent manner. Furthermore, when somatostatin-28 receptors were solubilized by Triton X-100 as a reversible complex with 125I-somatostatin-28, GTP[S] specifically dissociated the bound ligand to a larger extent from the soluble receptors than from the plasma-membrane-embedded receptors, the radioactivity remaining bound after 15 min at 37 degrees C being 30% and 83% respectively. After pertussis-toxin-induced [32P]ADP-ribosylation of pancreatic membranes, a 41-kDa [32P]ADP-ribose-labelled inhibitory guanine nucleotide binding protein coeluted with the 128-kDa and 45-kDa receptor complexes. The labelling of both receptor proteins was sensitive to GTP[S]. The labelling of the 228-kDa band was inconsistent. These results support the conclusion that beta cell somatostatin receptors can be solubilized as proteins of 128 kDa and 45 kDa. The major labeled species corresponds to the 128-kDa band and is a glycoprotein. The pancreatic membrane contains a 41-kDa GTP-binding protein that can complex with somatostatin receptors.
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PMID:Solubilization of somatostatin receptors in hamster pancreatic beta cells. Characterization as a glycoprotein interacting with a GTP-binding protein. 257 68


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