Properties of rat erythrocyte membrane cytoskeletal structures produced by digitonin extraction: Digitonin-insoluble β-adrenergic receptor, adenylate cyclase, and cholera toxin substrate

Abstract

Rat erythrocyte plasma membranes have been extracted exhaustively with digitonin at low temperature, and the residual, detergent-extracted membrane cytoskeletal material is compared to that prepared with Triton X-100 with respect to protein, glycoprotein, phospholipid, and cholesterol content. Digitonin, a weaker detergent than Triton X-100, solubilizes only 26% of the phospholipids and none of the cholesterol. SDS-polyacrylamide gel electrophoresis reveals that differences between the proteins extracted by the two detergents are primarily quantitative. In terms of functional preservation, digitonin retains in the cytoskeleton 28% of the beta-adrenergic receptor binding activity (with the balance accounted for in the supernatant), greater than 90% of the adenylate cyclase and greater than 90% of the 45,000 mol wt polypeptide cholera toxin substrate. The cytoskeletal-associated beat-adrenergic receptor retains binding properties for antagonist and agonist which are identical to those of the native membrane receptor. The digitonin-extracted cytoskeleton containing the beta-adrenergic receptor may provide a useful vehicle for the reconstitution of a hormone-sensitive adenylate cyclase.

Cytosolic activator of adenylate cyclase: Reconstitution, characterization, and mechanism of action.

“…We had reported previously the existence of a functionally relevant interaction between membrane-associated components of the adenylate cyclase system and erythrocyte cytoskeletons prepared by detergent extraction (18,19,22). Here we show that the stimulatory effect of the cytosolic factor persisted when detergent extraction followed the activation step.…”

“…Hepatocytes were prepared by liver perfusion in situ at 37°C with Hanks' balanced salt solution containing EGTA followed by the same medium containing CaCl2 and collagenase (17) 0.1 mM EDTA and 2% Triton X-100 followed by washing the detergent shells with Triton-free buffer (18). Digitonin shells from rat erythrocytes were obtained as described (19). P-Adrenergic receptors were assayed by using the antagonist (+)-…”