Evaluation of methods for the isolation of plasma membranes displaying guanosine 5'-triphosphate-dependence for the regulation of adenylate cyclase activity: potential application to the study of other guanosine 5'-triphosphate-dependent transduction systems.
The GTP-dependence for stimulatory and inhibitory regulation of plasma membrane adenylate cyclase activity was measured in plasma membrane fractions isolated from a variety of cell types (platelets, lymphocytes, PC12 cells, GH3 cells, NBP2 cells, and hepatocytes). This report shows that the isolation of plasma membranes for the study of GTP-dependent adenylate cyclase activity was, for some cells, enhanced by the exposure of the cells to glycerol prior to cell lysis. The isolation of plasma membranes from other cells, which did not appear to be sensitive to glycerol pretreatment, was enhanced by the removal of heavy particulate matter prior to fractionation of the cell lysate. The regulation of enzyme activity by various agents was found to be dependent upon the presence of (exogenous) GTP to varying degrees, indicating variable contamination of membrane preparations with GTP. It is concluded that (i) exposure of platelets and lymphocytes to glycerol prior to cell lysis decreases subsequent contamination of the plasma membrane preparation with GTP, and (ii) although glycerol pretreatment of other cells does not ensure the subsequent isolation of plasma membrane adenylate cyclase activity displaying high requirements for (exogenous) GTP, it is a reasonable first approach to be used during the development of procedures for the isolation of plasma membranes.
Caldwell
K K
KK
Department of Pharmacology, University of Colorado Health Sciences Center, Denver 80262.
Newell
M K
MK
Cambier
J C
JC
Prasad
K N
KN
Masserano
J M
JM
Schlegel
W
W
Cooper
D M
DM
D000262:Adenylyl Cyclases / Q000378:metabolism*; D000818:Animals; D001792:Blood Platelets / Q000378:metabolism; D002458:Cell Fractionation; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism*; D005260:Female; D005990:Glycerol; D006160:Guanosine Triphosphate / Q000378:metabolism* / Q000494:pharmacology; D006801:Humans; D008099:Liver / Q000378:metabolism; D008214:Lymphocytes / Q000378:metabolism; D008297:Male; D051379:Mice; D051381:Rats; D011919:Rats, Inbred Strains
D006160:Guanosine Triphosphate; D000262:Adenylyl Cyclases; D005990:Glycerol