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Query: UNIPROT:O95477 (membrane-bound)
 29,236 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Previous reports from this laboratory and others have established that both the rabbit and human erythrocyte membranes contain multiple protein kinase and phosphate acceptor activities. We now report that these membranes also contain phosphoryl acceptor sites for the soluble cyclic AMP-dependent and -independent protein kinases from rabbit erythrocytes. The rabbit erythrocyte membrane, which does not contain a cyclic AMP-dependent protein kinase, has at least four polypeptides (Bands 2.1, 2.3, 4.5, and 4.8) which are phosphorylated in the presence of the soluble cyclic AMP-dependent protein kinases I, IIa, and IIb isolated from rabbit erythrocyte lysates. The resulting phosphoprotein profile is very similar to that obtained for the cyclic AMP-mediated autophosphorylation of human erythrocyte membranes. The activities of the soluble cyclic AMP-dependent protein kinases toward the membranes have been studied at several pH values. Although the substrate specificity of the three kinases is similar, polypeptide 2.3 appears to be phosphorylated to a greater extent by kinase IIa than by I or IIb. This occurs at all pH values studied. Also apparent is that the pH profile for membrane phosphorylation is different from that of histone phosphorylation. The phosphorylation of membrane proteins can also be catalyzed by the soluble erythrocyte casein kinases. These enzymes are not regulated by cyclic nucleotides and can use either ATP or GTP as their phosphoryl donor. Polypeptides 2.1, 2.9, 4.1, 4.5, 4.8, and 5 of both human and rabbit erythrocyte membranes are phosphorylated in the presence of GTP and the casein kinases. This reaction is optimal at pH 7.5. Experiments were performed to determine whether the phosphorylation of the membranes by the soluble and membrane-bound kinases is additive or exclusive. Our results indicate that after maximal autophosphorylation of the erythrocyte membranes, phosphoryl acceptor sites are available to the soluble cyclic AMP-dependent and -independent protein kinases. Furthermore, after maximal phosphorylation of the membranes with one type of soluble kinase, further 32P incorporation can occur as a result of exposure to the other type of soluble kinase.
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PMID:Phosphorylation of rabbit and human erythrocyte membranes by soluble adenosine 3':5'-monophosphate-dependent and -independent protein kinases. 18 4

In the preceding paper (Sheetz, M. and S.J. Singer. 1977. J Cell Biol. 73:638-646) it was shown that erythrocyte ghosts undergo pronounced shape changes in the presence of mg-ATP. The biochemical effects of the action of ATP are herein examined. The biochemical effects of the action of ATP are herein examined. Phosphorylation by ATP of spectrin component 2 of the erythrocyte membrane is known to occur. We have shown that it is only membrane protein that is significantly phosphorylated under the conditions where the shape changes are produced. The extent of this phosphorylation rises with increasing ATP concentration, reaching nearly 1 mol phosphoryle group per mole of component 2 at 8mM ATP. Most of this phosphorylation appears to occur at a single site on the protein molecule, according to cyanogen bromide peptide cleavage experiments. The degree of phosphorylation of component 2 is apparently also regulated by a membrane-bound protein phosphatase. This activity can be demonstrated in erythrocyte ghosts prepared from intact cells prelabeled with [(32)P]phosphate. In addition to the phosphorylation of component 2, some phosphorylation of lipids, mainly of phosphatidylinositol, is also known to occur. The ghost shape changes are, however, shown to be correlated with the degree of phosphorylation of component 2. In such experiment, the incorporation of exogenous phosphatases into ghosts reversed the shape changes produced by ATP, or by the membrane-intercalating drug chlorpromazine. The results obtained in this and the preceding paper are consistent with the proposal that the erythrocyte membrane possesses kinase and phosphates activities which produce phosphorylation and dephosphorylation of a specific site on spectrin component 2 molecules; the steady-state level of this phosphorylation regulates the structural state of the spectrin complex on the cytoplasmic surface of the membrane, which in turn exerts an important control on the shape of the cell.
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PMID:On the mechanism of ATP-induced shape changes in human erythrocyte membranes. II. The role of ATP. 19 4

By an improved isolation procedure chloroplasts could be obtained from the alga Bumilleriopsis filiformis (Xanthophyceae) which exhibited high electron transport rates tightly coupled to ATP formation. Uncouplers both stimulate electron transport and inhibit photophosphorylation. These chloroplasts retain almost all soluble cytochrome c-553 besides a membrane-bound cytochrome c-554.5 (=f-554.5). Sonification or iron deficiency removed the soluble cytochrome only with a concurrent decrease of electron transport from water to methyl viologen or to NADP and decreased non-cyclic and cyclic photophosphorylation. However, photosynthetic control and the P/2e ratios remain unaltered. In Bumilleriopsis, which apparently has no plastocyanin, the soluble cytochrome c-553 seemingly links electron transport between the bound cytochrome c and P-700.
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PMID:The role of plastidic cytochrome c in algal electron transport and photophosphorylation. 20 17

Filamin is a high molecular weight actin-binding protein found in large quantities in smooth muscle and other non-muscle cells. We have studied the phosphorylation of filamin in a mammalian smooth muscle, the guinea pig vas deferens. Intact vas deferens incorporated [32P]orthophosphate into filamin. Incubation of particulate fractions of vas deferens with [gamma-32P]ATP resulted in 32P-labeling of filamin. Cyclic AMP stimulated this phosphorylation, whereas cyclic GMP and Ca2+ had no effect. Purified vas deferens filamin can be phosphorylated by purified cyclic AMP-dependent protein kinase. We have compared cyclic AMP and cyclic GMP effects on phosphorylation in smooth muscle. Cyclic GMP stimulated phosphorylation of two particulate proteins, G-I (Mr = 130,000) a protein previously described by Casnellie, J. E., and Greengard, P. (1974) Proc. Natl. Acad, Sci. U.S.A. 71, 1891-1895 and G-III (Mr = 240,000). Both proteins and the kinase responsible for their phosphorylation appear to be membrane-bound. Phosphorylation of both proteins is stimulated by cyclic GMP (Ka = 3 x 10(-8) M), cyclic AMP (Ka = 3 x 10(-7) M), and to a lesser degree by Ca2+. In contrast, filamin phosphorylation is due to a soluble kinase stimulated only by cyclic AMP (Ka = 3 x 10(-7) M) and not by cyclic GMP or Ca2+.
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PMID:Cyclic AMP-dependent phosphorylation of filamin in mammalian smooth muscle. 20 8

In the presence of ATP and a cytosolic factor, cholera toxin fragment A1 catalyzes the transfer of ADP-ribose from NAD to a number of soluble and membrane-bound proteins of the pigeon erythrocyte. Evidence is presented that suggests that the most readily modified membrane protein (Mr 42,000) is the adenylate cyclase-associated GTP-binding protein. Its modification by toxin is stimulated by guanine nucleotides. Adenylate cyclase activity increases in parallel with the addition of ADP-ribose to this protein and decreases in parallel with the subsequent reversal of ADP-ribosylation by toxin and nicotinamide. The protein is only accessible to toxin A subunits if the erythrocytes are lysed. When adenylate cyclase activity reaches a maximum, the number of ADP-ribose residues bound to this protein (about 1500 per cell) is similar to the reported number of beta-adrenergic receptors.
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PMID:ADP-ribosylation of membrane proteins catalyzed by cholera toxin: basis of the activation of adenylate cyclase. 21 Apr 49

Antibodies which were raised against highly purified membrane-bound (Na+ -K+)-ATPase from the outer medulla of rat kidneys inhibit the (Na+-K+)-ATPase activity up to 95%. The antibody inhibition is reversible. The time course of enzyme inhibition and reactivation is biphasic in semilogarithmic plots. In the purified membrane-bound (Na+-K+)-ATPase negative cooperativity was observed (a) for the ATP dependence of the (Na+ -K+)-ATPase activity (n = 0.86), (b) for the ATP binding to the enzyme (n = 0.58), and (c) for the ouabain inhibition of the (Na+ -K+)-ATPase activity (n = 0.77). By measuring the Na+ dependence of the (Na+ -K+)-ATPase reaction, a positive homotropic cooperativity (n = 1.67) was found. As reactivation of the antibody-inhibited enzyme proceeds very slowly (t0.5 = 5.2 hr), it was possible to measure characteristics of the antibody-(Na+ -K+)-ATPase complex: The antibodies exerted similar effects on the ATP dependence of the (Na+ -K+)-ATPase reaction and on the ATP binding of the enzyme. Vmax of the (Na+ -K+)-ATPase reaction and the number of ATP binding sites were reduced while K0.5 ATP for the (Na+ -K+)-ATPase activity and for the ATP binding were increased by the antibodies. The Hill coefficients for the ATP binding and for the ATP dependence of the enzyme activity were not significantly altered by the antibodies. The antibodies increased the K0.5 value for the Na+ stimulation of the (Na+ -K+)-ATPase activity, but they did not alter the homotropic interactions between the Na+-binding sites. The negative cooperativity which was observed for the ouabain inhibition of the (Na+ -K)-ATPase activity was abolished by the antibodies. The data are tentatively explained by the following model: The antibodies bind to the (Na+ -K+)-ATPase from the inner membrane side, reduce the ATP binding symmetrically at the ATP binding sites and reduce thereby also the (Na+ -K+)-ATPase activity of the enzyme. The antibodies may inhibit the ATP binding by a direct interaction or by means of a conformational change at the ATP binding sites. This may possibly also lead to the alteration of the Na+ dependence of the (Na+ -K+)-ATPase activity and to the observed alteration of the dose response to the ouabain inhibition.
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PMID:Characteristics of antibody inhibition of rat kidney (Na+ -K+)-ATPase. 21 73

Using a radioactively tagged, photoaffinity analog of cAMP, 8-azidoadenosine-3',5'-cyclic monophosphate (8-N3 cAMP) and [gamma32P]ATP, the membrane-binding properties of both the regulatory and catalytic subunits of the cAMP-activated protein kinase of human erythrocyte membranes were investigated. [32P]8-N3 cAMP was used to locate and quantify regulatory subunits. Increased phosphorylation of specific membrane proteins by [gamma32P]ATP was used to determine the presence of the catalytic subunit. The data support a mechanism which operates through a tight membrane-bound regulatory subunit and a catalytic subunit that is released from the membrane when cAMP is present and the Mg.ATP concentration is below approximately 10 micrometer. The catalytic subunit is not required for the Mg.ATP inhibition of 8-N3 cAMP binding. Experiments with a photoaffinity analog of ATP, 8-azidoadenosine triphosphate (8-N3ATP), support the hypothesis that ATP hydrolysis and phosphorylation are not involved in the regulation. The data indicate that the regulatory subunit contains an ATP regulatory site which inhibits 8-N3 cAMP binding and the release of the catalytic subunit. These results indicate that the membrane-bound type I enzyme (type IM) differs significantly from the soluble (type IS) enzyme studied on other tissues. These enzymes are compartmentalized by being in different cellular locations and are regulated differently by Mg.ATP.
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PMID:Use of photoaffinity nucleotide analogs to determine the mechanism of ATP regulation of a membrane-bound, cAMP-activated protein kinase. 21 38

The endogenous phosphorylation of specific proteins was studied in subcellular fractions from proliferating and cAMP-induced differentiated neuroblastoma cells. Fractions containing nuclear, membrane-bound, and cytosolic proteins were incubated with [gamma-32P]ATP, in the presence and absence of added cyclic nucleotides. Phosphate incorporation into specific proteins was determined by slab-gel electrophoresis of sodium dodecyl sulfate-solubilized reaction products. Cytosol fractions from differentiated cells demonstrated a twofold increase in cAMP-dependent phosphorylation of a specific protein with apparent mol wt of 59,000 daltons and a comparable decrease in cAMP-independent phosphorylation of another protein (97,000). The nuclear fraction of differentiated cells showed an increase in the cAMP-independent phosphorylation of two nonhistone proteins (110,000 and 102,000). Membrane fractions from differentiated cells exhibited a differential decrease in endogenous phosphorylation of specific proteins. Selective alterations in the phosphorylation of specific proteins in various subcellular components may be important biochemical events associated with the increased levels of differentiated functions in neuroblastoma cells in culture.
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PMID:Selective changes in the phosphorylation of endogenous proteins in subcellular fractions from cyclic AMP-induced differentiated neuroblastoma cells. 21 47

The membrane-bound adenylase cyclase (ATP pyrosphosphate-lyase (cyclizing), EC 4.6.1.1) of isolated rat adrenal cortex cells can be rendered soluble using 0.02 M Lubrol 12A9. The solubilized enzyme can be filtered through Milipore filters with pores 0.22 micron in diameter. Using gel filtration, on Sephadex G-200, adenylate cyclase activity was eluted with a distribution coefficient of 0.139, whereas on Sephadex G-100 the activity was eluted in the excluded volume. Half-maximum activation of the postulated guanyl nucleotide regulator site of adenylate was achieved with 5'-guanylyl-imidodiphosphate at a concentration of 1 . 10(-6)M. In contrast, however, using intact isolated rat adrenal cortex cells the guanyl nucleotide regulator site could not be stimulated by 5'-guanylyl-imidodiphosphate.
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PMID:Solubilization of membrane-bound adenylate cyclase of isolated rat adrenal cortex cells. 21 37

Aging has recently been shown to promote lipid peroxidation of mitochondrial membranes by a mechanism involving chaotropic oxidants (5). The present paper reports on the relationship between these membrane alterations and the activities of lipid-dependent enzymes of isolated heart mitochondria from 3 month and 24 month old rats. 1. Temperature breaks of Arrhenius plots reveal age-dependent shifts to higher temperatures for the succinate oxidase system (delta t = 1,7 degrees C), the beta-hydroxybutyrate dehydrogenase, the succinate dehydrogenase and the ATP-ase (delta t = 3,0 degrees C). 2. Specific activities of the above enzymes are distinctly lowered in preparation from aged rats. 3. Thermotropic differences of the particular enzyme activities completely disappeared after solubilization of the lipid components by Triton X-100. 4. ESR studies exhibit a clear decrease in the fluidity of membrane lipids as a function of age. 5. Analysis of the membrane lipids by gas-liquid chromatography reveals a distinct age-dependent fall in the content of polyunsaturated fatty acids accompanied by a slow increase in the degree of fatty acid saturation. 6. It is concluded from the results that aging influences enzyme-protein-lipid interactions by radical-induced peroxidation of the surrounding membrane lipids, but does not affect the intrinsic properties of the membrane-bound enzymes themselves.
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PMID:Influence of age on thermotropic kinetics of enzymes involved in mitochondrial energy-metabolism. 21 32


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