Activation of phospholipase C in rabbit brain membranes by carbachol in the presence of GTP gamma S; effects of biological detergents.
Rabbit brain cortical membranes incubated with carbachol in the presence of GTP gamma S show a marked increase in the degradation of exogenous phosphatidylinositol 4,5-bisphosphate. This activation of phospholipase C is dependent on the presence of deoxycholate and maximal at 0.8-1 mM deoxycholate. There is negligible activation by carbachol alone but in the presence of GTP gamma S a carbachol effect can be readily demonstrated. Optimal activation of phospholipase C by carbachol was seen at 10 to 100 nM free Ca2+. Washing cortical membranes with hypertonic buffer extracted 60% of the membrane protein yet the carbachol and GTP gamma S coupling remained intact. Incubation of the membranes with lysophosphatidylcholine, Nonidet P-40, sodium deoxycholate or digitonin at concentrations considerably less than those frequently used to solubilize membrane proteins abolished the carbachol response. Octyl glucoside and sodium cholate also uncoupled receptor regulation of phospholipase C but only at concentrations where solubilization of membrane proteins occurred. Prior exposure of membranes to carbachol did not prevent the uncoupling observed as a result of detergent treatment. Incubation of the membranes with carbachol and GTP gamma S did not appear to be accompanied by specific release of either active phospholipase C or inhibitors of phospholipase C activity.
Carter
H R
HR
Department of Biochemistry, University of Tennessee, Memphis 38163.
Wallace
M A
MA
Fain
J N
JN
D000818:Animals; D001921:Brain / Q000201:enzymology*; D002217:Carbachol / Q000494:pharmacology*; D002462:Cell Membrane / Q000201:enzymology; D003902:Detergents / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D006160:Guanosine Triphosphate / Q000031:analogs & derivatives* / Q000494:pharmacology; D019269:Phosphatidylinositol 4,5-Diphosphate; D010716:Phosphatidylinositols / Q000378:metabolism; D011817:Rabbits; D013873:Thionucleotides / Q000494:pharmacology*; D010738:Type C Phospholipases / Q000378:metabolism*; D014475:Uncoupling Agents / Q000494:pharmacology
D003902:Detergents; D019269:Phosphatidylinositol 4,5-Diphosphate; D010716:Phosphatidylinositols; D013873:Thionucleotides; D014475:Uncoupling Agents; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D006160:Guanosine Triphosphate; D002217:Carbachol; D010738:Type C Phospholipases