Deliberate XXXX overload as a method for in situ characterization of active calcium extrusion systems and cytoplasmic calcium binding: application to the human platelet.

@entity141 @entity1360 @entity6028
The objectives of the title were accomplished by a four-step experimental procedure followed by a simple graphical and mathematical analysis. Platelets are (i) overloaded with the indicator @entity6028 to cytoplasmic concentrations of 2.9 mM and (ii) are exposed to 2 mM external @entity141 and 1.0 microM @entity1360 to rapidly achieve cytoplasmic @entity141 ([ @entity141 ]cyt) of ca. 1.5 microM. (iii) The external @entity141 is removed by EGTA addition, and (iv) the active @entity141 extrusion process is then monitored as a function of time. Control experiments show that the ionophore shunts dense tubular uptake and does not contribute to the @entity141 efflux process during phases iii-iv and that the extrusion process is sensitive to metabolic inhibitors. The progress curves for the decline of @entity6028 fluorescence (resulting from active @entity141 extrusion) were analyzed as a function of [ @entity141 ]cyt using a mathematical model involving the probability that an exported @entity141 was removed from a @entity6028 complex (vs. a cytoplasmic binding element). The observed rates of decline of @entity6028 fluorescence at a particular [ @entity141 ]cyt are dependent upon (i) the absolute rate of the extrusion system (a function of its Km, Vm and Hill coefficient (n)), (ii) the intrinsic @entity141 buffer capacity of the cytoplasm (a function of the total site concentration ([B]T) and its Kd) and (iii) the buffer capacity of the intracytoplasmic @entity6028 (a function of its concentration and Kd). The contribution of (iii) was known and varied and was used to determine (ii) and (i) as a function of [ @entity141 ]cyt. The @entity141 binding data were verified by 45Ca2+ experimentation.(ABSTRACT TRUNCATED AT 250 WORDS)
