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Thromb Haemost 1986; 55(02): 178-183
DOI: 10.1055/s-0038-1661517
Original Article
Schattauer GmbH Stuttgart

Isolation and Partial Characterization of Adenylate Cyclase-Enriched Membranes from Human Platelets

N Shimada
1   The Department of Biochemistry, Tokyo Metropolitan Institute of Gerontology, Tokyo, Japan
,
M Tsubokura
2   The Tokyo Red Cross Blood Center, Tokyo, Japan
,
N Kimura
1   The Department of Biochemistry, Tokyo Metropolitan Institute of Gerontology, Tokyo, Japan
› Author Affiliations
Further Information

Publication History

Received 05 August 1985

Accepted 03 January 1986

Publication Date:
18 July 2018 (online)

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Summary

Isolation of adenylate cyclase-enriched membranes from human platelets was attempted using glycerol lysis technique followed by ultracentrifugation on discontinuous sucrose gradients composed of 24, 30, 34, 37, and 41% (w/w). Adenylate cyclase activity was enriched 4-fold in sample/24% sucrose interface, 7-fold in 24%/30% sucrose interface, and 4-fold in 30%/ 34% sucrose interface fractions with the recovery of 15-20% of the total activity. The enrichment and subcellular distribution of adenylate cyclase resembled in general those of phosphodiesterase and acid phosphatase with slight differences in each other. Protein profiles from SDS-polyacrylamide gel electrophoresis showed that the heavy chain of myosin (Mr = 200,000) was enriched in sample/24% sucrose interface and lower molecular weight proteins in 34%/37% sucrose interface and pellet. The interface fractions between 24 and 34% sucrose were, therefore, collected as adenylate cyclase-enriched membranes.

Adenylate cyclase associated with the membranes displayed high specific activity (0.1 and 1-2 nmol/min/mg protein in the absence and presence of stimulants, respectively), and possessed sensitivities to prostaglandins (E1, I2, and D2) as well as cholera toxin. Activation of adenylate cyclase by these compounds required added GTP, indicating that the contamination of the membrane preparations with GTP-like substance (s) was minimal, if at all present.

Key words

Human platelet - Subcellular fractionation - Adenylate cyclase - Prostaglandins - GTP
 

  • References

  • 1 Haslam RJ, Davidson MM L, Davies T, Lynham JA, McClenaghan MD. Regulation of blood platelet function by cyclic nucleotides. Adv Cyclic Nucleotide Res 1978; 9: 533-552
    PubMedGoogle Scholar
  • 2 Feinstein MB, Rodan GA, Cutler LS. Cyclic AMP and calcium in platelet function. In Platelets in Biology and Pathology - 2 Gordon JL. (ed.). 1981: 437-472
  • 3 Rink TJ, Hallam TJ. What turns platelets on?. Trends Biochem Sci 1984; 9: 215-219
    CrossrefPubMedGoogle Scholar
  • 4 Nishizuka Y. The role of protein kinase C in cell surface signal transduction and tumor promotion. Nature 1984; 308: 693-698
    CrossrefPubMedGoogle Scholar
  • 5 Berridge MJ. Inositol triphosphate and diacylglycerol as second messengers. Biochem J 1984; 220: 345-360
    CrossrefPubMedGoogle Scholar
  • 6 Krishna G, Harwood JP, Barber AJ, Jamieson GA. Requirement for guanosine triphosphate in the prostaglandin activation of adenylate cyclase of platelet membranes. J Biol Chem 1972; 247: 2253-2254
    PubMedGoogle Scholar
  • 7 Jakobs KH, Saur W, Schultz G. Inhibition of platelet adenylate cyclase by epinephrine requires GTP. FEBS Lett 1978; 85: 167-170
    CrossrefPubMedGoogle Scholar
  • 8 Tsai BS, Lefkowits RJ. Multiple effects of guanine nucleotides on human platelet adenylate cyclase. Biochim Biophys Acta 1979; 587: 28-41
    CrossrefPubMedGoogle Scholar
  • 9 Steer ML, Wood A. Regulation of human platelet adenylate cyclase by epinephrine, prostaglandin E1 and guanine nucleotides. J Biol Chem 1979; 254: 10791-10797
    PubMedGoogle Scholar
  • 10 Insel PA, Stengel D, Ferry N, Hanoune J. Regulation of adenylate cyclase of human platelet membranes by forskolin. J Biol Chem 1982; 257: 7485-7490
    PubMedGoogle Scholar
  • 11 Stein JM, Martin BR. The role of GTP in prostaglandin E1 stimulation of adenylate cyclase in platelet membranes. Biochem J 1983; 214: 231-234
    CrossrefPubMedGoogle Scholar
  • 12 Kimura N, Nagata N. The requirement of guanine nucleotides for glucagon stimulation of adenylate cyclase in rat liver plasma membranes. J Biol Chem 1977; 252: 3829-3835
    PubMedGoogle Scholar
  • 13 Kimura N, Nagata N. Mechanism of glucagon stimulation of adenylate cyclase in the presence of GDP in rat liver plasma membranes. J Biol Chem 1979; 254: 3451-3457
    PubMedGoogle Scholar
  • 14 Kimura N, Shimada N. GDP does not mediate but rather inhibits hormonal signal to adenylate cyclase. J Biol Chem 1983; 258: 2278-2283
    PubMedGoogle Scholar
  • 15 Marcus AJ, Zucker-Franklin D, Safier LB, Ullman HL. Studies on human platelet granules and membranes. J Clin Invest 1966; 45: 14-28
    CrossrefPubMedGoogle Scholar
  • 16 Barber AJ, Jamieson GA. Isolation and characterization of plasma membranes from human blood platelets. J Biol Chem 1970; 245: 6357-6365
    PubMedGoogle Scholar
  • 17 Kaulen HD, Gross R. Metabolic properties of human platelet membranes. I. Characterization of platelet membranes prepared by sucrose and Ficoll density gradients. Thrombos Diathes Haemorrh 1973; 30: 199-214
    PubMedGoogle Scholar
  • 18 Taylor DG, Crawford N. The subfractionation of platelet membranes by zonal centrifugation: Identification of surface membranes. FEBS Lett 1974; 41: 317-322
    CrossrefPubMedGoogle Scholar
  • 19 Rittenhouse-Simmons S, Deykin D. Isolation of membranes from normal and thrombin-treated gel-filtered platelets using a lectin marker. Biochim Biophys Acta 1979; 426: 688-696
    PubMedGoogle Scholar
  • 20 Taylor DG, Crawford N. Enzymatic and chemical analyses of pig platelet membrane subfractions isolated by zonal centrifugation. Biochim Biophys Acta 1976; 436: 77-94
    CrossrefPubMedGoogle Scholar
  • 21 Kinoshita T, Nachman RL, Minick R. Isolation of human platelet membranes with polylysine beads. J Cell Biol 1979; 82: 688-696
    CrossrefPubMedGoogle 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; 256: 4095-4101
    PubMedGoogle Scholar
  • 23 Lanillo M, Cabezas JA. Isolation, characterization and chemical composition of the membrane from sheep platelets. Biochim Biophys Acta 1981; 649: 229-238
    CrossrefPubMedGoogle Scholar
  • 24 Sixma JJ, Lips JP M. Isolation of platelet membranes. A review. Thromb Haemostas 1978; 39: 328-337
    PubMedGoogle Scholar
  • 25 Crawford N. Structural and molecular properties of platelet membrane systems. In Platelet Membrane Glycoproteins George JN, Nurden AT, Phillips DR. (eds.) pp 13-49 Plenum Press; New York: 1985
  • 26 Kaulen HD, Gross R. The differentiation of acid phosphatases of human blood platelets. Thrombos Diathes Haemorrh 1971; 26: 353-361
    PubMedGoogle Scholar
  • 27 Koerner JF, Sinsheimer RL. A deoxyribonuclease from calf spleen. I. Purification and properties. J Biol Chem 1957; 228: 1039-1048
    PubMedGoogle Scholar
  • 28 King TE. Preparations of succinate-cytochrome c reductase and the cytochrome b-C1 particle, and reconstitution of succinate-cytochrome c reductase. Methods Enzymol 1967; 10: 216-225
    PubMedGoogle Scholar
  • 29 Stolzenbach F. Lactic dehydrogenases (crystalline). Methods Enzymol 1966; 9: 278-288
    PubMedGoogle Scholar
  • 30 Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970; 227d: 680-685
    PubMedGoogle Scholar
  • 31 Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem 1951; 193: 265-275
    PubMedGoogle Scholar
  • 32 Barber AJ. Cyclic nucleotides and platelet aggregation. Effect of aggregating agents on the activity of cyclic nucleotide-metabolizing enzymes. Biochim Biophys Acta 1976; 444: 579-595
    CrossrefPubMedGoogle Scholar
  • 33 Rodan GA, Feinstein MB. Interrelationship between Ca2+ and adenylate and guanylate cyclases in the control of platelet secretion and aggregation. Proc Natl Acad Sci USA 1976; 73: 1829-1833
    CrossrefPubMedGoogle Scholar
  • 34 Gorman RR, Bunting S, Miller OV. Modulation of human platelet adenylate cyclase by prostacyclin (PGX). Prostaglandins 1977; 13: 377-388
    CrossrefPubMedGoogle Scholar
  • 35 Schafer AI, Cooper B, O’Hara D, Handin RI. Identification of platelet receptors for prostaglandin I2 and D2 . J Biol Chem 1979; 254: 2914-2917
    PubMedGoogle Scholar
  • 36 Stein JM, Martin BR. The effect of carbacyclin, a prostaglandin analogue, on adenylate cyclase activity in platelet membranes. FEBS Lett 1984; 165: 290-292
    CrossrefPubMedGoogle Scholar
  • 37 Moss J, Vaughan M. Activation of adenylate cyclase by choleragen. Ann Rev Biochem 1979; 48: 581-600
    CrossrefPubMedGoogle Scholar
  • 38 Shimada N, Kimura N. GDP does not support activation of adenylate cyclase nor ADP-ribosylation of a guanine nucleotide binding protein by cholera toxin. FEBS Lett 1983; 159: 75-78
    CrossrefPubMedGoogle Scholar
  • 39 Broekman MJ, Westmoreland NP, Cohen P. An improved method for isolating alpha granules and mitochondria from human platelets. J Cell Biol 1974; 60: 507-519
    CrossrefPubMedGoogle Scholar
  • 40 Kimura N, Shimada N, Tsubokura M. Adenosine 5’-(β, γ-imino) triphosphate and guanosine 5’-o-(2-thiodiphosphate) do not necessarily provide non-phosphorylating conditions in adenylate cyclase studies. Biochem Biophys Res Commun 1985; 126: 983-991
    CrossrefPubMedGoogle Scholar