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. 1987 Jul 15;245(2):617–620. doi: 10.1042/bj2450617

Detection of 23-27 kDa GTP-binding proteins in platelets and other cells.

R P Bhullar 1, R J Haslam 1
PMCID: PMC1148167  PMID: 3117049

Abstract

Membrane proteins from rabbit and human platelets were separated by SDS/polyacrylamide-gel electrophoresis and the resolved polypeptides blotted on nitrocellulose. A family of GTP-binding proteins, termed Gn proteins, was detected by incubation of these blots with [alpha-32P]GTP in the presence of Mg2+. A major Gn protein with a molecular mass of 27 kDa (Gn27) and lesser amounts of 23, 24 and 25 kDa Gn proteins were observed in platelet membranes; much smaller amounts were in the platelet soluble fraction. Binding of [alpha-32P]GTP by platelet Gn proteins was blocked by GDP, GTP or guanosine 5'-[gamma-thio]triphosphate, but not by GMP or adenosine 5'-[beta gamma-imido]triphosphate. Rabbit and human red-cell membranes contained only Gn27. When rat tissues were analysed for Gn proteins, the largest amounts were found in brain, which contained two membrane-bound forms (Gn27 and Gn26) and a soluble form (Gn26).

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Brass L. F., Laposata M., Banga H. S., Rittenhouse S. E. Regulation of the phosphoinositide hydrolysis pathway in thrombin-stimulated platelets by a pertussis toxin-sensitive guanine nucleotide-binding protein. Evaluation of its contribution to platelet activation and comparisons with the adenylate cyclase inhibitory protein, Gi. J Biol Chem. 1986 Dec 25;261(36):16838–16847. [PubMed] [Google Scholar]
  2. Downes C. P., Mussat M. C., Michell R. H. The inositol trisphosphate phosphomonoesterase of the human erythrocyte membrane. Biochem J. 1982 Apr 1;203(1):169–177. doi: 10.1042/bj2030169. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Evans T., Brown M. L., Fraser E. D., Northup J. K. Purification of the major GTP-binding proteins from human placental membranes. J Biol Chem. 1986 May 25;261(15):7052–7059. [PubMed] [Google Scholar]
  4. Fleischman L. F., Chahwala S. B., Cantley L. ras-transformed cells: altered levels of phosphatidylinositol-4,5-bisphosphate and catabolites. Science. 1986 Jan 24;231(4736):407–410. doi: 10.1126/science.3001936. [DOI] [PubMed] [Google Scholar]
  5. Fox J. E., Say A. K., Haslam R. J. Subcellular distribution of the different platelet proteins phosphorylated on exposure of intact platelets to ionophore A23187 or to prostaglandin E1. Possible role of a membrane phosphopolypeptide in the regulation of calcium-ion transport. Biochem J. 1979 Dec 15;184(3):651–661. doi: 10.1042/bj1840651. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Gierschik P., Falloon J., Milligan G., Pines M., Gallin J. I., Spiegel A. Immunochemical evidence for a novel pertussis toxin substrate in human neutrophils. J Biol Chem. 1986 Jun 15;261(17):8058–8062. [PubMed] [Google Scholar]
  7. Gilman A. G. G proteins and dual control of adenylate cyclase. Cell. 1984 Mar;36(3):577–579. doi: 10.1016/0092-8674(84)90336-2. [DOI] [PubMed] [Google Scholar]
  8. Grandt R., Aktories K., Jakobs K. H. Evidence for two GTPases activated by thrombin in membranes of human platelets. Biochem J. 1986 Aug 1;237(3):669–674. doi: 10.1042/bj2370669. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Hallam T. J., Rink T. J. Agonists stimulate divalent cation channels in the plasma membrane of human platelets. FEBS Lett. 1985 Jul 8;186(2):175–179. doi: 10.1016/0014-5793(85)80703-1. [DOI] [PubMed] [Google Scholar]
  10. Haslam R. J., Lynham J. A. Activation and inhibition of blood platelet adenylate cyclase by adenosine or by 2-chloroadenosine. Life Sci II. 1972 Dec 8;11(23):1143–1154. doi: 10.1016/0024-3205(72)90269-x. [DOI] [PubMed] [Google Scholar]
  11. Heyworth C. M., Whetton A. D., Wong S., Martin B. R., Houslay M. D. Insulin inhibits the cholera-toxin-catalysed ribosylation of a Mr-25000 protein in rat liver plasma membranes. Biochem J. 1985 Jun 15;228(3):593–603. doi: 10.1042/bj2280593. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Holz G. G., 4th, Rane S. G., Dunlap K. GTP-binding proteins mediate transmitter inhibition of voltage-dependent calcium channels. Nature. 1986 Feb 20;319(6055):670–672. doi: 10.1038/319670a0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Houslay M. D., Bojanic D., Wilson A. Platelet activating factor and U44069 stimulate a GTPase activity in human platelets which is distinct from the guanine nucleotide regulatory proteins, Ns and Ni. Biochem J. 1986 Mar 15;234(3):737–740. doi: 10.1042/bj2340737. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Imaoka T., Lynham J. A., Haslam R. J. Purification and characterization of the 47,000-dalton protein phosphorylated during degranulation of human platelets. J Biol Chem. 1983 Sep 25;258(18):11404–11414. [PubMed] [Google Scholar]
  15. Kahn R. A., Gilman A. G. The protein cofactor necessary for ADP-ribosylation of Gs by cholera toxin is itself a GTP binding protein. J Biol Chem. 1986 Jun 15;261(17):7906–7911. [PubMed] [Google Scholar]
  16. Katada T., Bokoch G. M., Smigel M. D., Ui M., Gilman A. G. The inhibitory guanine nucleotide-binding regulatory component of adenylate cyclase. Subunit dissociation and the inhibition of adenylate cyclase in S49 lymphoma cyc- and wild type membranes. J Biol Chem. 1984 Mar 25;259(6):3586–3595. [PubMed] [Google Scholar]
  17. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  18. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  19. Lapetina E. G. Effect of pertussis toxin on the phosphodiesteratic cleavage of the polyphosphoinositides by guanosine 5'-O-thiotriphosphate and thrombin in permeabilized human platelets. Biochim Biophys Acta. 1986 Nov 19;884(2):219–224. doi: 10.1016/0304-4165(86)90166-2. [DOI] [PubMed] [Google Scholar]
  20. Litosch I., Fain J. N. Regulation of phosphoinositide breakdown by guanine nucleotides. Life Sci. 1986 Jul 21;39(3):187–194. doi: 10.1016/0024-3205(86)90529-1. [DOI] [PubMed] [Google Scholar]
  21. Logothetis D. E., Kurachi Y., Galper J., Neer E. J., Clapham D. E. The beta gamma subunits of GTP-binding proteins activate the muscarinic K+ channel in heart. Nature. 1987 Jan 22;325(6102):321–326. doi: 10.1038/325321a0. [DOI] [PubMed] [Google Scholar]
  22. McGrath J. P., Capon D. J., Goeddel D. V., Levinson A. D. Comparative biochemical properties of normal and activated human ras p21 protein. Nature. 1984 Aug 23;310(5979):644–649. doi: 10.1038/310644a0. [DOI] [PubMed] [Google Scholar]
  23. Michel T., Winslow J. W., Smith J. A., Seidman J. G., Neer E. J. Molecular cloning and characterization of cDNA encoding the GTP-binding protein alpha i and identification of a related protein, alpha h. Proc Natl Acad Sci U S A. 1986 Oct;83(20):7663–7667. doi: 10.1073/pnas.83.20.7663. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Pfaffinger P. J., Martin J. M., Hunter D. D., Nathanson N. M., Hille B. GTP-binding proteins couple cardiac muscarinic receptors to a K channel. Nature. 1985 Oct 10;317(6037):536–538. doi: 10.1038/317536a0. [DOI] [PubMed] [Google Scholar]
  25. Pollock W. K., Rink T. J., Irvine R. F. Liberation of [3H]arachidonic acid and changes in cytosolic free calcium in fura-2-loaded human platelets stimulated by ionomycin and collagen. Biochem J. 1986 May 1;235(3):869–877. doi: 10.1042/bj2350869. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Schaffner W., Weissmann C. A rapid, sensitive, and specific method for the determination of protein in dilute solution. Anal Biochem. 1973 Dec;56(2):502–514. doi: 10.1016/0003-2697(73)90217-0. [DOI] [PubMed] [Google Scholar]
  27. Spiegel A. M. Signal transduction by guanine nucleotide binding proteins. Mol Cell Endocrinol. 1987 Jan;49(1):1–16. doi: 10.1016/0303-7207(87)90058-x. [DOI] [PubMed] [Google Scholar]
  28. Stein R. B., Robinson P. S., Scolnick E. M. Photoaffinity labeling with GTP of viral p21 ras protein expressed in Escherichia coli. J Virol. 1984 May;50(2):343–351. doi: 10.1128/jvi.50.2.343-351.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Sternweis P. C., Robishaw J. D. Isolation of two proteins with high affinity for guanine nucleotides from membranes of bovine brain. J Biol Chem. 1984 Nov 25;259(22):13806–13813. [PubMed] [Google Scholar]
  30. Sweatt J. D., Blair I. A., Cragoe E. J., Limbird L. E. Inhibitors of Na+/H+ exchange block epinephrine- and ADP-induced stimulation of human platelet phospholipase C by blockade of arachidonic acid release at a prior step. J Biol Chem. 1986 Jul 5;261(19):8660–8666. [PubMed] [Google Scholar]
  31. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Wakelam M. J., Davies S. A., Houslay M. D., McKay I., Marshall C. J., Hall A. Normal p21N-ras couples bombesin and other growth factor receptors to inositol phosphate production. Nature. 1986 Sep 11;323(6084):173–176. doi: 10.1038/323173a0. [DOI] [PubMed] [Google Scholar]

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