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Purification of an AlF4- and G-protein beta gamma-subunit-regulated phospholipase C-activating protein
G L Waldo1, J L Boyer, A J Morris
1Department of Pharmacology, University of North Carolina School of Medicine, Chapel Hill 27599.
Abstract:
A 150-kDa phospholipase C has previously been purified from turkey erythrocytes and has been shown by reconstitution with turkey erythrocyte membranes to be a receptor- and G-protein-regulated enzyme (Morris, A. J., Waldo, G. L., Downes, C.P., and Harden, T. K. (1990) J. Biol. Chem. 265, 13501-13507; Morris, A.J., Waldo, G.L., Downes, C.P., and Harden, T.K. (1990) J. Biol. Chem. 265, 13508-13514). Combination of this 150-kDa protein with phosphoinositide substrate-containing phospholipid vesicles prepared with a cholate extract from purified turkey erythrocyte plasma membranes resulted in conferrence of AlF4- sensitivity to the purified phospholipase C. Guanosine 5'-3-O-(thio)triphosphate also activated the reconstituted phospholipase C in a manner that was inhibited by guanosine 5'-2-O-(thio)-diphosphate. The magnitude of the AlF4- stimulation was increased with increasing amounts of plasma membrane extract, and was also dependent on the concentration of purified phospholipase C. Using reconstitution of AlF4- sensitivity as an assay, the putative G-protein conferring regulation to the 150-kDa phospholipase C was purified to near homogeneity by sequential chromatography over Q-Sepharose, Sephacryl S-300, octyl-Sepharose, hydroxylapatite, and Mono-Q. Reconstituting activity co-purified with an approximately 43-kDa protein identified by silver staining; lesser amounts of a 35-kDa protein was present in the final purified fractions, as was a minor 40-kDa protein. The 43-kDa protein strongly reacted with antiserum against a 12-amino acid sequence found at the carboxyl terminus of Gq and G11, the 35-kDa protein strongly reacted with G-protein beta-subunit antiserum, and the 40-kDa protein reacted with antiserum that recognizes Gi3. Immunoprecipitation of the 43-kDa protein resulted in loss of phospholipase C-stimulating activity of the purified fraction. The idea that this is a phospholipase C-regulating G-protein is further supported by the observation that co-reconstitution of G-protein beta gamma-subunit with the purified phospholipase C-activating fraction resulted in a beta gamma-subunit-dependent inhibition of AlF(4-)-stimulated phospholipase C activity in the reconstituted preparation.
Insights
Researchers purified a G-protein that regulates a 150-kDa phospholipase C enzyme in turkey red blood cells. This G-protein, identified as a 43-kDa protein, activates phospholipase C and is sensitive to aluminum fluoride.
Area of Science:
- Biochemistry
- Cell Signaling
- Molecular Biology
Background:
- A 150-kDa phospholipase C (PLC) from turkey erythrocytes is known to be regulated by receptors and G-proteins.
- Previous studies demonstrated its regulation by G-proteins through reconstitution experiments.
Purpose of the Study:
- To purify and identify the specific G-protein responsible for regulating the 150-kDa turkey erythrocyte phospholipase C.
- To characterize the regulatory interaction between the purified G-protein and phospholipase C.
Main Methods:
- Purification of a G-protein using sequential chromatography (Q-Sepharose, Sephacryl S-300, octyl-Sepharose, hydroxylapatite, Mono-Q).
- Reconstitution assays to assess G-protein mediated activation of purified phospholipase C using AlF4- and GTPγS.
- Western blot analysis using specific antisera against G-protein subunits (Gq/G11, Gβ, Gi3).
- Immunoprecipitation to confirm the role of the 43-kDa protein in phospholipase C activation.
Main Results:
- A 43-kDa protein, identified as a Gq/G11 family member, was purified and co-purified with phospholipase C-stimulating activity.
- The reconstituted phospholipase C was activated by AlF4- and GTPγS, and this activation was dependent on the purified 43-kDa protein.
- Immunoprecipitation of the 43-kDa protein abolished the phospholipase C-stimulating activity.
- G-protein beta gamma-subunits inhibited AlF4-stimulated activity, further supporting the G-protein's role.
Conclusions:
- The 43-kDa protein is a G-protein, likely a member of the Gq/G11 family, that directly regulates the 150-kDa turkey erythrocyte phospholipase C.
- This study elucidates a specific G-protein-phospholipase C signaling pathway in turkey erythrocytes.