Characterization of the calcium-sequestering process associated with human platelet intracellular membranes isolated by free-flow electrophoresis.
By using density-gradient fractionation and high-voltage free-flow electrophoresis, human platelet membranes were separated into highly purified subfractions of surface (SM) and intracellular (IM) origin. Associated exclusively with the IM fraction is an ATP-dependent Ca2+ uptake that, in the absence of oxalate, reaches steady-state levels in 5-10 min. When Ca2+-EGTA buffers were used to control the external Ca2+ concentrations (range 0.1-50 microM) there was an increase in the intravesicle steady-state level of Ca2+ up to 10 microM external Ca2+ concentration. Above this level the intravesicle space becomes saturated at a concentration between 10 and 20 nmol of Ca2+ X (mg of protein)-1. The ionophore A23187 promotes a rapid and almost total release of the sequestered Ca2+ (greater than 90%, t1/2 1-2 min). The presence of oxalate in the external medium greatly enhances the Ca2+ accumulation to levels as high as 200 nmol X (mg of protein)-1, but the uptake process is more variable and rarely reaches steady-state level even after 2 h incubation. Moreover, accumulation in the presence of oxalate effects ionophore release with less than 80% depletion in 45-60 min. These findings, taken together with the known presence in the platelet of a wide variety of functional and metabolic processes triggered by this cation, suggest that the platelet IM has a key role in controlling cytosolic Ca2+ concentrations.
D000255:Adenosine Triphosphate / Q000097:blood; D001792:Blood Platelets / Q000187:drug effects / Q000378:metabolism*; D000001:Calcimycin / Q000494:pharmacology; D002118:Calcium / Q000097:blood*; D002458:Cell Fractionation; D002462:Cell Membrane / Q000378:metabolism; D002499:Centrifugation, Density Gradient; D004586:Electrophoresis; D006801:Humans; D007425:Intracellular Membranes / Q000378:metabolism; D010070:Oxalates / Q000494:pharmacology; D019815:Oxalic Acid
D010070:Oxalates; D000001:Calcimycin; D000255:Adenosine Triphosphate; D019815:Oxalic Acid; D002118:Calcium