G-protein linked polyphosphoinositide phospholipase C activity in cell membranes from clonally derived and ras-transformed Madin-Darby canine kidney cells.
Membranes prepared from clone D1 of Madin-Darby canine kidney (MDCK) cells contain activity that can be attributed to Gp, a guanine nucleotide binding protein linked to phosphatidylinositol 4,5-bisphosphate dependent phospholipase C. Polyphosphoinositides are produced by addition of GTP, nonhydrolyzable GTP analogs, or fluoroaluminate. This production is inhibited by guanosine 5'-(beta-thiodiphosphate). While Ca2+ at 1 microM or more can generate high yields of inositol phosphates, guanine nucleotide activation of Gp can potentiate this Ca2(+)-dependent yield at resting levels of the cation. Membranes from cells expressing large amounts of ras-p21 exhibit small differences in guanine nucleotide induced polyphosphoinositide quantities. The greatest difference between normal and ras membranes was seen with AlF4- incubation. Of the three inositol phosphates measured, only the inositol bisphosphate yield was greatly increased in ras membranes compared with membranes from both parental and the D-1 clone of MDCK cells. From these data, we conclude that the presence of ras-p21 may affect production of polyphosphoinositides in MDCK cell membranes by some means other than direct participation in phospholipase C activation.
Waite
J J
JJ
Department of Pharmacology, University of California, San Diego, La Jolla, CA 92093.
Insel
P A
PA
D000818:Animals; D002118:Calcium / Q000502:physiology; D002461:Cell Line, Transformed; D002462:Cell Membrane / Q000737:chemistry; D004285:Dogs; D004848:Epithelium / Q000737:chemistry; D019204:GTP-Binding Proteins / Q000502:physiology*; D006150:Guanine Nucleotides / Q000502:physiology; D007668:Kidney / Q000737:chemistry*; D015689:Oncogene Protein p21(ras) / Q000235:genetics / Q000502:physiology*; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D010716:Phosphatidylinositols / Q000096:biosynthesis*; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism*
D006150:Guanine Nucleotides; D010716:Phosphatidylinositols; D010727:Phosphoric Diester Hydrolases; D019204:GTP-Binding Proteins; D015689:Oncogene Protein p21(ras); D043265:Phosphatidylinositol Diacylglycerol-Lyase; D002118:Calcium