Solubilization, purification, and characterization of an inositol trisphosphate receptor.
Inositol 1,4,5-trisphosphate is a second messenger of the phosphoinositide system which can mobilize calcium from intracellular stores. Rat cerebellum is an abundant source of a receptor for inositol 1,4,5-trisphosphate (Worley, P. F., Baraban, J. M., Supattapone, S., Wilson, V. S., and Snyder, S. H. (1987) J. Biol. Chem. 262, 12132-12136). In this study we have solubilized and purified this receptor to apparent homogeneity from rat cerebellum. Crude membrane, detergent-solubilized, and purified receptor preparations display similar selectivity for inositol 1,4,5-trisphosphate over other inositol phosphates. The purified receptor is globular with a Stokes' radius of approximately 10 nm. Electrophoretic analysis reveals one protein band with an Mr of 260,000. While binding is reversibly inhibited by 300 nM calcium in particulate fractions and detergent-solubilized membranes, the purified protein is not inhibited by calcium concentrations up to 1.5 mM. Inhibition by calcium is reconstituted by addition of detergent-solubilized cerebellar membranes, but not by the cytosolic fraction of cerebellum.
Supattapone
S
S
Department of Neuroscience, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205.
Worley
P F
PF
Baraban
J M
JM
Snyder
S H
SH
D000818:Animals; D002118:Calcium / Q000378:metabolism; D015220:Calcium Channels; D002531:Cerebellum / Q000032:analysis; D015544:Inositol 1,4,5-Trisphosphate; D053496:Inositol 1,4,5-Trisphosphate Receptors; D007295:Inositol Phosphates / Q000378:metabolism; D008297:Male; D008970:Molecular Weight; D051381:Rats; D011919:Rats, Inbred Strains; D011956:Receptors, Cell Surface / Q000302:isolation & purification*; D018160:Receptors, Cytoplasmic and Nuclear; D012995:Solubility
D015220:Calcium Channels; D053496:Inositol 1,4,5-Trisphosphate Receptors; D007295:Inositol Phosphates; D011956:Receptors, Cell Surface; D018160:Receptors, Cytoplasmic and Nuclear; D015544:Inositol 1,4,5-Trisphosphate; D002118:Calcium