Phospholipase C in Dictyostelium discoideum. Cyclic AMP surface receptor and G-protein-regulated activity in vitro.
The cellular slime mould Dictyostelium discoideum shows several responses after stimulation with the chemoattractant cAMP, including a transient rise in cyclic AMP (cAMP), cGMP and Ins(1,4,5)P3. In this paper the regulation of phospholipase C in vitro is described. Under our experimental conditions commercial PtdIns(4,5)P2 cannot be used to analyse phospholipase C activity in Dictyostelium lysates, because it is hydrolysed mainly to glycerophosphoinositol instead of Ins(1,4,5)P3. Enzyme activity was determined with endogenous unlabelled PtdInsP2 as a substrate. The product was measured by isotope-dilution assay and identified as authentic Ins(1,4,5)P3. Since phospholipase C is strictly Ca(2+)-dependent, with an optimal concentration range of 1-100 microM, cell lysates were prepared in EGTA and the enzyme reaction was started by adding 10 microM free Ca2+. Phospholipase C activity increased 2-fold during Dictyostelium development up to 8 h of starvation, after which the activity declined to less than 10% of the vegetative level. Enzyme activity in vitro increased up to 2-fold after stimulation of cells with the agonist cAMP in vivo. Addition of 10 microM guanosine 5'-[gamma-thio]triphosphate during lysis activated the enzyme to the same extent, and this effect was antagonized by guanosine 5'-[beta-thio]diphosphate. These results strongly suggest that surface cAMP receptors and G-proteins regulate phospholipase C during Dictyostelium development.
Bominaar
A A
AA
Department of Biochemistry, University of Groningen, The Netherlands.
Kesbeke
F
F
Van Haastert
P J
PJ
D000818:Animals; D002118:Calcium / Q000494:pharmacology; D000242:Cyclic AMP / Q000494:pharmacology; D004023:Dictyostelium / Q000201:enzymology*; D004533:Egtazic Acid / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D019204:GTP-Binding Proteins / Q000502:physiology*; D006150:Guanine Nucleotides / Q000494:pharmacology; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D006153:Guanosine Diphosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism; D007700:Kinetics; D010716:Phosphatidylinositols / Q000378:metabolism; D011953:Receptors, Cyclic AMP / Q000378:metabolism*; D013873:Thionucleotides / Q000494:pharmacology; D014316:Tritium; D010738:Type C Phospholipases / Q000378:metabolism*
D006150:Guanine Nucleotides; D010716:Phosphatidylinositols; D011953:Receptors, Cyclic AMP; D013873:Thionucleotides; D014316:Tritium; D006153:Guanosine Diphosphate; C032829:6-thioguanosine 5'-diphosphate; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D004533:Egtazic Acid; D015544:Inositol 1,4,5-Trisphosphate; D000242:Cyclic AMP; D010738:Type C Phospholipases; D019204:GTP-Binding Proteins; D002118:Calcium