Regulation of calcium accumulation and efflux from platelet vesicles: Possible role for cyclic-AMP-dependent phosphorylation and calmodulin |
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Authors: | Christian J. Le Peuch Danielle A.M. Le Peuch Sidney Katz Jacques G. Demaille Maxwell T. Hincke R. Bredoux J. Enouf S. Levy-Toledano Jacques Caen |
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Affiliation: | 1. U-249 de l''Institut National de la Santé et de la Recherche Médicale, Centre de Recherches de Biochimie Macromoléculaire du Centre National de la Recherche Scientifique, and Faculté de Médecine, BP 5015, 34033 - Montpellier, France;2. U - 150 of Institut National de la Santé et de la Recherche Médicale, 6 rue Guy - Patin, 75010 Paris France |
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Abstract: | Calcium-accumulating vesicles were isolated by differential centrifugation of sonicated platelets. Such vesicles exhibit a activity of about 10 nmol (min·mg)?1 and an ATP-dependent Ca2+ uptake of about 10 nmol (min·mg)?1. When incubated in the presence of Mg[γ-32P]ATP, the pump is phosphorylated and the acyl phosphate bond is sensitive to hydroxylamine. The [32P]phosphate-labeled Ca2+ pump exhibits a subunit molecular weight of 120 000 when analyzed by lithium dodecyl sulfate-polyacrylamide gel electrophoresis. Platelet calcium-accumulating vesicles contain a 23 kDa membrane protein that is phosphorylatable by the catalytic subunit of cAMP-dependent protein kinase but not by protein kinase C. This phosphate acceptor is not phosphorylated when the vesicles are incubated in the presence of either Ca2+ or Ca2+ plus calmodulin. The latter protein is bound to the vesicles and represents 0.5% of the proteins present in the membrane fraction. Binding of 125I-labeled calmodulin to this membrane fraction was of high affinity (16 nM), and the use of an overlay technique revealed four major calmodulin-binding proteins in the platelet cytosol (). Some minor calmodulin-binding proteins were enriched in the membrane fractions (). When the vesicles are phosphorylated in the presence of MgATP and of the catalytic subunit of cAMP-dependent protein kinase, the rate of Ca2+ uptake is essentially unaltered, while the Ca2+ capacity is diminished as a consequence of a doubling in the rate of Ca2+ efflux. Therefore, the inhibitory effect of cAMP on platelet function cannot be explained in such simple terms as an increased rate of Ca2+ removal from the cytosol. Calmodulin, on the other hand, was observed to have no effect on the initial rate of calcium efflux when added either in the absence or in the presence of the catalytic subunit of the cyclic AMP-dependent protein kinase, nor did the addition of 0.5 μM calmodulin result in increased levels of vesicle phosphorylation. |
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Keywords: | Phosphorylation cyclic AMP Calmodulin (Platelet vesicle) cAMP cyclic adenosine 3′5′-monophosphate PMSF phenylmethylsulfonyl fluoride Hepes SDS sodium dodecyl sulfate LDS lithium dodecyl sulfate EGTA EDTA ethylenediaminetetraacetic acid |
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