Distinct ryanodine- and inositol 1,4,5-triphosphate-binding sites in hepatic microsomes

A light hepatic microsomal preparation was fractionated by sucrose-density centrifugation into one rough, one intermediate and two smooth fractions. The four fractions were characterized with respect to parameters relevant to Ca²⁺ sequestration. Ca²⁺-ATPase activity was similar in the rough, intermediate and smooth I fractions, but lower in the smooth II fraction. Ca²⁺ accumulation was the highest in the smooth I and intermediate fractions. On the other hand, Ca²⁺ efflux from the rough fraction was several-fold faster than from the smooth I fraction. All four subfractions exhibited specific binding sites for inositol 1,4,5-triphosphate (IP₃) and ryanodine; however, the receptors were especially enriched in the smooth I fraction. The total binding sites for ryanodine in that fraction exceeded the number of binding sites for IP₃ by about 10-fold. The two receptors responded differently to pharmacological agents; caffeine and dantrolene strongly inhibited ryanodine binding but not IP₃ binding, whereas heparin inhibited IP₃ binding only. Thus the two receptors are distinct entities. The four fractions also showed distinct gel electrophoretic patterns. The use of two different SDS/polyacrylamide-gel gradients and two protein-staining methods revealed major differences in the distribution of the bands corresponding to M(r) values of (x10^-3) 380, 320, 260, 170, 90, 29 and 21. These proteins were enriched in the smooth fraction. The results indicate that the smooth I fraction might have special importance in stimulus-evoked Ca²⁺-release processes.