Phospholipase C in rabbit thymocytes: subcellular distribution and influences of calcium and GTP gamma S on the substrate dependence of cytosolic and plasma membrane-associated phospholipase C.
The subcellular distribution of phospholipase C (PLC) activity in rabbit thymocytes was examined by measuring the enzyme's activity in different subcellular fractions. PLC activity was determined using exogenously added [3H]PIP2 as substrate. Approx. 80% of the activity of the cell homogenate was found in the cytosolic fraction. A minor portion of PLC activity was attached to the particulate fraction. This membrane-associated PLC activity was found to be predominantly bound to the plasma membrane. Both PIP2-cleaving PLCs (the PLC associated with the plasma membrane and the PLC in the cytosol) exhibited maximum activity at pH 5. GTP gamma S stimulated the cytosolic and the membrane-bound PLC. As revealed by computer analysis of the substrate dependence of both basal and GTP gamma S-stimulated PLC activity, GTP gamma S enhanced the Vmax of the enzymes. Calcium, at a concentration of 1 mM, decreased PLC activity, as compared to a calcium concentration of 100 nM. The characteristic increase in Vmax induced by GTP gamma S was observed at a concentration of 1 mM calcium and was similar to that at 100 nM. These data suggest that the stimulatory effect of GTP gamma S is not due to an increased affinity of PLCs to calcium.
D000818:Animals; D002118:Calcium / Q000494:pharmacology*; D002462:Cell Membrane / Q000201:enzymology*; D003600:Cytosol / Q000201:enzymology*; D004789:Enzyme Activation / Q000187:drug effects; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D006160:Guanosine Triphosphate / Q000031:analogs & derivatives* / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007294:Inositol / Q000378:metabolism; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D019269:Phosphatidylinositol 4,5-Diphosphate; D010716:Phosphatidylinositols / Q000378:metabolism; D011817:Rabbits; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism; D013873:Thionucleotides / Q000494:pharmacology*; D013950:Thymus Gland / Q000201:enzymology* / Q000648:ultrastructure; D010738:Type C Phospholipases / Q000378:metabolism*; D005723:gamma-Glutamyltransferase / Q000378:metabolism
D019269:Phosphatidylinositol 4,5-Diphosphate; D010716:Phosphatidylinositols; D013873:Thionucleotides; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D007294:Inositol; D006160:Guanosine Triphosphate; D005723:gamma-Glutamyltransferase; D010738:Type C Phospholipases; D000254:Sodium-Potassium-Exchanging ATPase; D008274:Magnesium; D002118:Calcium