Skip to main content
PMC home page
Biochemical Journal logoLink to Biochemical Journal
. 1988 Mar 1;250(2):355–361. doi: 10.1042/bj2500355

A monoclonal antibody (PL/IM 430) to human platelet intracellular membranes which inhibits the uptake of Ca2+ without affecting the Ca2+ +Mg2+-ATPase.

N Hack 1, J M Wilkinson 1, N Crawford 1
PMCID: PMC1148863  PMID: 2965578

Abstract

To probe the structure-function relationships of proteins present in the endoplasmic reticulum-like intracellular membranes of human blood platelets a panel of monoclonal antibodies have been raised, using as immunogen highly purified platelet intracellular membrane vesicles isolated by continuous flow electrophoresis [Menashi, Weintroub & Crawford (1981) J. Biol. Chem. 256, 4095-4101]. Four of these antibodies recognize a single 100 kDa polypeptide in the platelet membrane by immunoblotting. One antibody PL/IM 430 (of IgG1 subclass) inhibited (approximately 70%) the energy-dependent uptake of Ca2+ into the vesicles without affecting the Ca2+ +Mg2+-ATPase activity or the protein phosphorylation previously shown to proceed concomitantly with Ca2+ sequestration [Hack, Croset & Crawford (1986) Biochem. J. 233, 661-668]. The inhibition is independent of ATP concentration over a range 0-2 mM-ATP but shows dose-dependency for external [Ca2+] with maximum inhibition of Ca2+ translocation at concentrations of Ca2+ greater than 500 nM. This capacity of the antibody PL/IM 430 functionally to dislocate components of the intracellular membrane Ca2+ pump complex may have value in structural studies.

Full text

PDF
360

Images in this article

357Fig. 1.
on p.357

Selected References

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

  1. Authi K. S., Lagarde M., Crawford N. Diacylglycerol lipase activity in human platelet intracellular and surface membranes. Some kinetic properties and fatty acid specificity. FEBS Lett. 1985 Jan 21;180(1):95–101. doi: 10.1016/0014-5793(85)80239-8. [DOI] [PubMed] [Google Scholar]
  2. Bird I. M., Dhoot G. K., Wilkinson J. M. Identification of multiple variants of fast muscle troponin T in the chicken using monoclonal antibodies. Eur J Biochem. 1985 Aug 1;150(3):517–525. doi: 10.1111/j.1432-1033.1985.tb09052.x. [DOI] [PubMed] [Google Scholar]
  3. Brass L. F. Ca2+ homeostasis in unstimulated platelets. J Biol Chem. 1984 Oct 25;259(20):12563–12570. [PubMed] [Google Scholar]
  4. Carey F., Menashi S., Crawford N. Localization of cyclo-oxygenase and thromboxane synthetase in human platelet intracellular membranes. Biochem J. 1982 Jun 15;204(3):847–851. doi: 10.1042/bj2040847. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Chamberlain B. K., Volpe P., Fleischer S. Inhibition of calcium-induced calcium release from purified cardiac sarcoplasmic reticulum vesicles. J Biol Chem. 1984 Jun 25;259(12):7547–7553. [PubMed] [Google Scholar]
  6. Dean W. L. Purification and reconstitution of a Ca2+ pump from human platelets. J Biol Chem. 1984 Jun 10;259(11):7343–7348. [PubMed] [Google Scholar]
  7. Enouf J., Bredoux R., Boizard B., Wautier J. L., Chap H., Thomas J., de Metz M., Levy-Toledano S. Simultaneous isolation of two platelet membrane fractions: biochemical, immunological and functional characterization. Biochem Biophys Res Commun. 1984 Aug 30;123(1):50–58. doi: 10.1016/0006-291x(84)90378-4. [DOI] [PubMed] [Google Scholar]
  8. Ey P. L., Prowse S. J., Jenkin C. R. Isolation of pure IgG1, IgG2a and IgG2b immunoglobulins from mouse serum using protein A-sepharose. Immunochemistry. 1978 Jul;15(7):429–436. doi: 10.1016/0161-5890(78)90070-6. [DOI] [PubMed] [Google Scholar]
  9. Fauvel J., Chap H., Roques V., Levy-Toledano S., Douste-Blazy L. Biochemical characterization of plasma membranes and intracellular membranes isolated from human platelets using Percoll gradients. Biochim Biophys Acta. 1986 Mar 27;856(1):155–164. doi: 10.1016/0005-2736(86)90022-2. [DOI] [PubMed] [Google Scholar]
  10. Fischer T. H., Campbell K. P., White G. C., 2nd Evidence that platelet and skeletal sarcoplasmic reticulum Ca2+-ATPase are structurally distinct. J Biol Chem. 1985 Jul 25;260(15):8996–9001. [PubMed] [Google Scholar]
  11. Galfre G., Howe S. C., Milstein C., Butcher G. W., Howard J. C. Antibodies to major histocompatibility antigens produced by hybrid cell lines. Nature. 1977 Apr 7;266(5602):550–552. doi: 10.1038/266550a0. [DOI] [PubMed] [Google Scholar]
  12. Green N. M., Taylor W. R., Brandl C., Korczak B., MacLennan D. H. Structural and mechanistic implications of the amino acid sequence of calcium-transporting ATPases. Ciba Found Symp. 1986;122:93–114. doi: 10.1002/9780470513347.ch7. [DOI] [PubMed] [Google Scholar]
  13. Hack N., Crawford N. Two-dimensional polyacrylamide-gel electrophoresis of the proteins and glycoproteins of purified human platelet surface and intracellular membranes. Biochem J. 1984 Aug 15;222(1):235–246. doi: 10.1042/bj2220235. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Hack N., Croset M., Crawford N. Studies on the bivalent-cation-activated ATPase activities of highly purified human platelet surface and intracellular membranes. Biochem J. 1986 Feb 1;233(3):661–668. doi: 10.1042/bj2330661. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Käser-Glanzmann R., Jakábová M., George J. N., Lüscher E. F. Further characterization of calcium-accumulating vesicles from human blood platelets. Biochim Biophys Acta. 1978 Sep 11;512(1):1–12. doi: 10.1016/0005-2736(78)90213-4. [DOI] [PubMed] [Google Scholar]
  16. Käser-Glanzmann R., Jakäbovä M., George J. N., Lüscher E. F. Stimulation of calcium uptake in platelet membrane vesicles by adenosine 3',5'-cyclic monophosphate and protein kinase. Biochim Biophys Acta. 1977 May 2;466(3):429–440. doi: 10.1016/0005-2736(77)90336-4. [DOI] [PubMed] [Google Scholar]
  17. 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]
  18. Lagarde M., Bryon P. A., Guichardant M., Dechavanne M. A simple and efficient method for platelet isolation from their plasma. Thromb Res. 1980 Feb 1;17(3-4):581–588. doi: 10.1016/0049-3848(80)90098-5. [DOI] [PubMed] [Google Scholar]
  19. Lagarde M., Guichardant M., Menashi S., Crawford N. The phospholipid and fatty acid composition of human platelet surface and intracellular membranes isolated by high voltage free flow electrophoresis. J Biol Chem. 1982 Mar 25;257(6):3100–3104. [PubMed] [Google Scholar]
  20. Mauco G., Fauvel J., Chap H., Douste-Blazy L. Studies on enzymes related to diacylglycerol production in activated platelets. II. Subcellular distribution, enzymatic properties and positional specificity of diacylglycerol- and monoacylglycerol-lipases. Biochim Biophys Acta. 1984 Nov 14;796(2):169–177. doi: 10.1016/0005-2760(84)90345-x. [DOI] [PubMed] [Google Scholar]
  21. Menashi S., Davis C., Crawford N. Calcium uptake associated with an intracellular membrane fraction prepared from human blood platelets by high-voltage, free-flow electrophoresis. FEBS Lett. 1982 Apr 19;140(2):298–302. doi: 10.1016/0014-5793(82)80918-6. [DOI] [PubMed] [Google Scholar]
  22. Menashi S., Weintroub H., Crawford N. Characterization of human platelet surface and intracellular membranes isolated by free flow electrophoresis. J Biol Chem. 1981 Apr 25;256(8):4095–4101. [PubMed] [Google Scholar]
  23. Mitchinson C., Wilderspin A. F., Trinnaman B. J., Green N. M. Identification of a labelled peptide after stoicheiometric reaction of fluorescein isothiocyanate with the Ca2+ -dependent adenosine triphosphatase of sarcoplasmic reticulum. FEBS Lett. 1982 Sep 6;146(1):87–92. doi: 10.1016/0014-5793(82)80710-2. [DOI] [PubMed] [Google Scholar]
  24. PORTZEHL H., CALDWELL P. C., RUEEGG J. C. THE DEPENDENCE OF CONTRACTION AND RELAXATION OF MUSCLE FIBRES FROM THE CRAB MAIA SQUINADO ON THE INTERNAL CONCENTRATION OF FREE CALCIUM IONS. Biochim Biophys Acta. 1964 May 25;79:581–591. doi: 10.1016/0926-6577(64)90224-4. [DOI] [PubMed] [Google Scholar]
  25. Pick U. Dynamic interconversions of phosphorylated and non-phosphorylated intermediates of the Ca-ATPase from sarcoplasmic reticulum followed in a fluorescein-labeled enzyme. FEBS Lett. 1981 Jan 12;123(1):131–136. doi: 10.1016/0014-5793(81)80036-1. [DOI] [PubMed] [Google Scholar]
  26. Pick U. Interaction of fluorescein isothiocyanate with nucleotide-binding sites of the Ca-ATPase from sarcoplasmic reticulum. Eur J Biochem. 1981 Dec;121(1):187–195. doi: 10.1111/j.1432-1033.1981.tb06448.x. [DOI] [PubMed] [Google Scholar]
  27. Rink T. J., Sanchez A., Hallam T. J. Diacylglycerol and phorbol ester stimulate secretion without raising cytoplasmic free calcium in human platelets. Nature. 1983 Sep 22;305(5932):317–319. doi: 10.1038/305317a0. [DOI] [PubMed] [Google Scholar]
  28. Robblee L. S., Shepro D., Belamarich F. A. Calcium uptake and associated adenosine triphosphatase activity of isolated platelet membranes. J Gen Physiol. 1973 Apr;61(4):462–481. doi: 10.1085/jgp.61.4.462. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Statland B. E., Heagan B. M., White J. G. Uptake of calcium by platelet relaxing factor. Nature. 1969 Aug 2;223(5205):521–522. doi: 10.1038/223521a0. [DOI] [PubMed] [Google Scholar]
  30. Steiner B., Lüscher E. F. Evidence that the platelet plasma membrane does not contain a (Ca2+ + Mg2+)-dependent ATPase. Biochim Biophys Acta. 1985 Sep 10;818(3):299–309. doi: 10.1016/0005-2736(85)90003-3. [DOI] [PubMed] [Google Scholar]
  31. Tanford C. Twenty questions concerning the reaction cycle of the sarcoplasmic reticulum calcium pump. CRC Crit Rev Biochem. 1984;17(2):123–151. doi: 10.3109/10409238409113603. [DOI] [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES