Summary
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.
Key words
Human platelet - Subcellular fractionation - Adenylate cyclase - Prostaglandins -
GTP
