THE IMPORTANT DISCREPANCY BETWEEN THE APPARENT AFFINITY OBSERVED IN CA2-D MEASURED IN BINDING-STUDIES IS A CONSEQUENCE OF THE QUANTAL PROCESS BY WHICH INOSITOL 1,4,5-TRISPHOSPHATE RELEASES CA2+ FROM BOVINE ADRENAL-CORTEX MICROSOMES( MOBILIZATION STUDIES AND THE K)

Inositol 1,4,5-trisphosphate (InsP(3)) is a second messenger responsib le for Ca2+ release from a non-mitochondrial intracellular store. An i mportant discrepancy has been observed between the affinity measured i n binding studies (K-d) and the apparent affinity obtained in Ca2+ mob ilization studies (EC(50)). It has been proposed that this discrepancy could be due to different experimental conditions used for Ca2+ mobil ization studies and for InsP(3) binding studies. With the fluorescent indicator Fura-2, we studied InsP(3)-induced Ca2+ release activity at 7 degrees C and at 37 degrees C, in bovine adrenal cortex microsomes. Under both conditions, the Ca2+ releasing effect of InsP(3) (1 mu M) w as completed within about 2 s, as a result of the quantal process of I nsP(3) receptor action. The apparent affinity (EC(50)) observed for In sP(3)-induced Ca2+ release at 7 degrees C and at 37 degrees C were 0.6 4 +/- 0.2 mu M and 0.9 +/- 0.2 mu M respectively. InsP(3) degradation studies, at 37 degrees C, indicated that less than 10% of [H-3]-InsP(3 ) was degraded within the first 10 s of incubation. InsP(3) associatio n rates were evaluated, at low temperature, with increasing concentrat ions of [H-3]-InsP(3). These kinetic studies revealed a direct relatio nship between the initial rate of association (V-i) and InsP(3) concen tration. From this relationship, we evaluated that the concentration o f InsP(3) needed to occupy half of the binding sites within the first second of incubation was 271 nM. We conclude that the discrepancy betw een K-d and EC(50) is related to a kinetic constraint dictated by the quantal process by which InsP(3) releases Ca2+.