Isolation and partial characterization of adenylate cyclase-enriched membranes from human platelets.
Isolation of adenylate cyclase-enriched membranes from human platelets was attempted using glycerol lysis technique followed by ultracentrifugation on discontinuous sucrose gradients composed of 24, 30, 34, 37, and 41% (w/w). Adenylate cyclase activity was enriched 4-fold in sample/24% sucrose interface, 7-fold in 24%/30% sucrose interface, and 4-fold in 30%/34% sucrose interface fractions with the recovery of 15-20% of the total activity. The enrichment and subcellular distribution of adenylate cyclase resembled in general those of phosphodiesterase and acid phosphatase with slight differences in each other. Protein profiles from SDS-polyacrylamide gel electrophoresis showed that the heavy chain of myosin (Mr = 200,000) was enriched in sample/24% sucrose interface and lower molecular weight proteins in 34%/37% sucrose interface and pellet. The interface fractions between 24 and 34% sucrose were, therefore, collected as adenylate cyclase-enriched membranes. Adenylate cyclase associated with the membranes displayed high specific activity (0.1 and 1-2 nmol/min/mg protein in the absence and presence of stimulants, respectively), and possessed sensitivities to prostaglandins (E1, I2, and D2) as well as cholera toxin. Activation of adenylate cyclase by these compounds required added GTP, indicating that the contamination of the membrane preparations with GTP-like substance (s) was minimal, if at all present.
D000262:Adenylyl Cyclases / Q000302:isolation & purification*; D001792:Blood Platelets / Q000166:cytology / Q000201:enzymology* / Q000648:ultrastructure; D002458:Cell Fractionation; D002462:Cell Membrane / Q000201:enzymology; D002499:Centrifugation, Density Gradient; D002772:Cholera Toxin / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D006160:Guanosine Triphosphate / Q000494:pharmacology; D006801:Humans; D007425:Intracellular Membranes / Q000201:enzymology; D008970:Molecular Weight; D011453:Prostaglandins / Q000494:pharmacology; D013347:Subcellular Fractions / Q000201:enzymology
D011453:Prostaglandins; D006160:Guanosine Triphosphate; D002772:Cholera Toxin; D000262:Adenylyl Cyclases