Abstract
Bovine liver cytosol contains a phosphoinositide phospholipase C (PLCcyt) that is activated by guanosine 5'-O-(3-thio)triphosphate (GTP gamma S)-activated G-proteins from liver plasma membranes. Heparin-Sepharose chromatography indicated that PLCcyt was immunologically distinct from PLC-beta 1, PLC-gamma 1, or PLC-delta 1 from brain. Initial purification of the GTP gamma S-activated G-proteins that stimulated PLCcyt indicated that the beta gamma complex was responsible. G-proteins were subsequently extracted from liver membranes as heterotrimers and purified in the presence of AlCl3, MgCl2, and NaF to allow reversible activation. Immunoblot analysis with an antiserum selective for the beta subunit showed that the stimulatory activity corresponded with the presence of this protein at every chromatographic step. When liver beta gamma complex was purified and separated from all detectable alpha subunits, as shown by immunoblotting and silver staining, it strongly stimulated PLCcyt after removal of the activating ligand [AlF4]- by gel filtration. beta gamma prepared from brain was approximately equipotent with that from liver. beta gamma was half-maximally effective at 33 nM and produced a maximal 50-fold activation of the PLC. Under identical conditions, beta gamma had no effect on brain PLC-gamma 1 or PLC-delta 1 and produced a 2-fold stimulation of PLC-beta 1 activity. Addition of purified GDP-bound alpha o, which had no effect by itself, completely reversed the beta gamma activation of PLCcyt, confirming that beta gamma was the active species. These data provide evidence for a novel mechanism by which beta gamma subunits of pertussis toxin-sensitive or -insensitive G-proteins activate phospholipase C.
Highlights
Bovine liver cytosol contains a phosphoinositide bers of a class that includes aq,all, a 1 4 9 a15) and a16 [15, 16]
Recognition of a Cytosolic Phospholipase C Responsive to G- indicted that theactive species was the Py complex and not proteins-Bovine liver cytosol waschromatographed on hep- an a subunit
It was recogarin-Sepharose and fractions containing pholipase C (PLC) were reconsti- nized that if an a subunit contaminated the /3y fractions, it tuted with a preparation of GTPyS-liganded G-proteins (Fig. would have been activated by the pretreatment of membranes lA).Immunoblot analysis showed that theG-protein prepa- with GTPyS.,the G-proteins were purified without ration included a subunits of Gi, G, and G, as well as Py prior activation with GTPyS
Summary
PLCC* weretherefore initially purified from GTPyS-treated liver plasma membranes. Chromatography of the G-proteins. Recognition of a Cytosolic Phospholipase C Responsive to G- indicted that theactive species was the Py complex and not proteins-Bovine liver cytosol waschromatographed on hep- an a subunit (see Fig. 4, and below) It was recogarin-Sepharose and fractions containing PLC were reconsti- nized that if an a subunit contaminated the /3y fractions, it tuted with a preparation of GTPyS-liganded G-proteins (Fig. would have been activated by the pretreatment of membranes lA).Immunoblot analysis showed that theG-protein prepa- with GTPyS.,the G-proteins were purified without ration included a subunits of Gi, G,, and G,, as well as Py prior activation with GTPyS. No clear peak of PLCcs stimulatory activity was observed in these fractions, and a complex pattern of G-protein subunits was shown by Western blotting (datanot shown). Using an assay for GdIlwhich measures the activation of PLC-81 in the presence of GTPyS [25], flowthrough and wash fractions were not active, whereas the a, and allthat bound to octyl-Sepharosewere active
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