Distinct ryanodine- and inositol 1,4,5-trisphosphate-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-trisphosphate (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 Mr values of (x 10(-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.
Shoshan-Barmatz
V
V
Department of Biology, Ben-Gurion University of Negev, Beer-Sheva, Israel.
Zhang
G H
GH
Garretson
L
L
Kraus-Friedmann
N
N
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels; D000252:Calcium-Transporting ATPases / Q000378:metabolism; D015544:Inositol 1,4,5-Trisphosphate / Q000037:antagonists & inhibitors / Q000378:metabolism*; D053496:Inositol 1,4,5-Trisphosphate Receptors; D008297:Male; D008862:Microsomes, Liver / Q000201:enzymology / Q000378:metabolism* / Q000648:ultrastructure; D051381:Rats; D011919:Rats, Inbred Strains; D011956:Receptors, Cell Surface / Q000187:drug effects / Q000378:metabolism*; D011950:Receptors, Cholinergic / Q000187:drug effects / Q000378:metabolism*; D018160:Receptors, Cytoplasmic and Nuclear; D012433:Ryanodine / Q000037:antagonists & inhibitors / Q000378:metabolism; D019837:Ryanodine Receptor Calcium Release Channel; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism
D015220:Calcium Channels; D053496:Inositol 1,4,5-Trisphosphate Receptors; D011956:Receptors, Cell Surface; D011950:Receptors, Cholinergic; D018160:Receptors, Cytoplasmic and Nuclear; D019837:Ryanodine Receptor Calcium Release Channel; D012433:Ryanodine; D015544:Inositol 1,4,5-Trisphosphate; D000252:Calcium-Transporting ATPases; D000254:Sodium-Potassium-Exchanging ATPase; D002118:Calcium