Ca2+ transport in human platelet membranes. Kinetics of active transport and passive release.
Active Ca2+ transport and passive release were characterized in crude and purified human platelet membranes to facilitate comparison with skeletal muscle sarcoplasmic reticulum. Endoplasmic reticulum markers were enriched from 3- to 14-fold in the purified membranes, while surface membrane antigens were reduced 4-fold and mitochondrial contamination was completely eliminated. The pH optimum for active Ca2+ transport in platelet membranes was 7.6, and the optimum for Ca2+-ATPase activity ranged from 7.6 to 8.0. Upon addition of MgATP there was a burst in active Ca2+ transport activity. In the absence of phosphate, steady state was reached within 20 s; added phosphate promoted continued uptake for greater than 1 h. The maximum pump stoichiometry was 2.0 Ca2+/ATP. The Ca2+ ionophore A23187 caused rapid release of 90% of the sequestered Ca2+ in the presence of phosphate. The dependence of Ca2+ transport on MgATP was biphasic with apparent Km values of 0.6 mM and 9.5 microM. Kinetic measurements with varied external Ca2+ yielded a single Km of 0.1 microM. Mg2+ stimulated Ca2+ transport and Ca2+-ATPase activities. Results with crude and purified membranes were similar, and comparison with the Ca2+ pump from sarcoplasmic reticulum revealed nearly identical enzymatic properties. In contrast to the results of comparing active Ca2+ transport, the characteristics of Ca2+ release from platelet membranes were quite different from those of sarcoplasmic reticulum. External Ca2+ did not promote release of sequestered Ca2+ from platelet membranes in contrast to sarcoplasmic reticulum. In addition, spontaneous release of Ca2+ from platelet membranes did not occur after ATP depletion. Inositol trisphosphate induced rapid partial release of Ca2+ from platelet membranes but had no effect on sarcoplasmic reticulum under identical conditions. Thus active Ca2+ transport is quite similar in internal membranes of platelet and skeletal muscle, but the mechanism of Ca2+ release appears to be entirely different.
D000255:Adenosine Triphosphate / Q000378:metabolism; D001692:Biological Transport; D001693:Biological Transport, Active; D001792:Blood Platelets / Q000166:cytology* / Q000378:metabolism; D002118:Calcium / Q000097:blood*; D000252:Calcium-Transporting ATPases / Q000097:blood; D002462:Cell Membrane / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D015544:Inositol 1,4,5-Trisphosphate; D007295:Inositol Phosphates / Q000494:pharmacology; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D015636:Magnesium Chloride; D010710:Phosphates / Q000494:pharmacology; D011188:Potassium / Q000494:pharmacology; D017680:Potassium Compounds; D012519:Sarcoplasmic Reticulum / Q000378:metabolism
D007295:Inositol Phosphates; D010710:Phosphates; D017680:Potassium Compounds; D015636:Magnesium Chloride; D015544:Inositol 1,4,5-Trisphosphate; D000255:Adenosine Triphosphate; C013216:potassium phosphate; D000252:Calcium-Transporting ATPases; D008274:Magnesium; D011188:Potassium; D002118:Calcium