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PMID: 8280097 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't

Phospholipase C in Dictyostelium discoideum. Cyclic AMP surface receptor and G-protein-regulated activity in vitro.

The Biochemical journal ·Vol. 297 ( Pt 1) ·1994-01-01 ·Pages 181-7

Bominaar AA, Kesbeke F, Van Haastert PJ

Abstract

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.

MeSH Terms
Animals Calcium/pharmacology Cyclic AMP/pharmacology Dictyostelium/enzymology Egtazic Acid/pharmacology Enzyme Activation/drug effects GTP-Binding Proteins/physiology Guanine Nucleotides/pharmacology Guanosine 5'-O-(3-Thiotriphosphate)/pharmacology Guanosine Diphosphate/analogs & derivatives,pharmacology Inositol 1,4,5-Trisphosphate/metabolism Kinetics Phosphatidylinositols/metabolism Receptors, Cyclic AMP/metabolism Thionucleotides/pharmacology Tritium Type C Phospholipases/metabolism
Chemicals
Guanine Nucleotides Phosphatidylinositols Receptors, Cyclic AMP Thionucleotides Tritium Guanosine Diphosphate 6-thioguanosine 5'-diphosphate Guanosine 5'-O-(3-Thiotriphosphate) Egtazic Acid Inositol 1,4,5-Trisphosphate Cyclic AMP Type C Phospholipases GTP-Binding Proteins Calcium
Authors & Affiliations
3 authors, click to expand affiliations / ORCID
Bominaar A A
Department of Biochemistry, University of Groningen, The Netherlands.
Kesbeke F
Van Haastert P J
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Article Info
Journal
The Biochemical journal
Abbr.
Biochem J
ISSN
0264-6021
DOI
10.1042/bj2970181
Published
1994-01-01
Pages
181-7
Language
English
Region
England
NLM ID
2984726R
PMCID
PMC1137808
Subset
IM
External Links
PubMed source DOI: 10.1042/bj2970181 PMC Full Text View journal details
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