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Affinity purification of pancreastatin receptor-Gq/11 protein complex from rat liver membranes
J Santos-Alvarez1, V Sánchez-Margalet
1Department of Medical Biochemistry and Molecular Biology, School of Medicine, Virgen Macarena University Hospital, Seville, Spain.
Abstract:
Pancreastatin, a chromogranin A derived peptide, exerts a glycogenolytic effect on the hepatocyte. This effect is initiated by binding to membrane receptors which are coupled to pertussis toxin insensitive G proteins belonging to the Gq/11 family. We have recently solubilized active pancreastatin receptors from rat liver membranes still functionally coupled to G proteins. Here, we have purified pancreastatin receptors by a two-step procedure. First, pancreastatin receptors with their associated Gq/11 regulatory proteins were purified from liver membranes by lectin absorption chromatography on wheat germ agglutinin immobilized on agarose. A biotinylated rat pancreastatin analog was tested for binding to liver membranes before using it for affinity purification. Unlabeled biotinylated rat pancreastatin competed for 125I-labeled [Tyr0]PST binding to solubilized receptors with a Kd = 0.27 nM, comparable to that of native pancreastatin. The biotinylated analog was immobilized on streptavidin-coated Sepharose beads and used to further affinity purify wheat germ agglutinin eluted receptor material. Specific elution at low pH showed that the receptor protein was purified as an 80-kDa protein in association with a G protein of the q/11 family, as demonstrated by specific immunoblot analysis. The specificity of the receptor band was assessed by chemical cross-linking of the purified material followed by SDS-PAGE and autoradiography. In conclusion, we have purified pancreastatin receptor as a glycoprotein of 80 kDa physically associated with a Gq/11 protein.
Insights
Researchers purified the pancreastatin receptor, a key protein involved in glycogen breakdown in liver cells. This 80-kDa glycoprotein is physically linked to Gq/11 proteins, advancing our understanding of cellular signaling pathways.
Area of Science:
- Biochemistry
- Cellular signaling
- Molecular endocrinology
Background:
- Pancreastatin, a peptide derived from chromogranin A, influences hepatocyte glycogenolysis.
- This effect is mediated by membrane receptors coupled to pertussis toxin-insensitive Gq/11 family G proteins.
- Active pancreastatin receptors, coupled to G proteins, have been previously solubilized from rat liver membranes.
Purpose of the Study:
- To purify the pancreastatin receptor.
- To characterize the purified receptor and its associated proteins.
- To confirm the receptor's interaction with Gq/11 proteins.
Main Methods:
- A two-step purification procedure involving wheat germ agglutinin affinity chromatography and streptavidin-based affinity purification.
- Utilized a biotinylated pancreastatin analog for receptor binding assays and affinity purification.
- Analyzed purified material using immunoblot analysis, SDS-PAGE, and autoradiography.
Main Results:
- Purified pancreastatin receptors associated with Gq/11 proteins.
- Identified the receptor as an 80-kDa glycoprotein.
- Confirmed the specificity of the receptor band through cross-linking and SDS-PAGE.
Conclusions:
- Successfully purified the pancreastatin receptor.
- The purified receptor is an 80-kDa glycoprotein physically associated with a Gq/11 protein.
- This purification provides a tool for further investigation into pancreastatin signaling.