Solubilization and reconstitution of hepatic system A-mediated amino acid transport. Preparation of proteoliposomes containing glucagon-stimulated transport activity

• Published in: Biochimica et biophysica acta Vol. 899; no. 1; pp. 51 - 58
• Main Authors: BRACY, D. S, SCHENERMAN, M. A, KILBERG, M. S
• Format: Journal Article
• Language: English
• Published: Amsterdam Elsevier 12.05.1987; North-Holland
• Subjects: Amino Acids - metabolism; Animals; Biological and medical sciences; Biological Transport - drug effects; Carrier Proteins - metabolism; Cell Membrane - drug effects; Cell Membrane - metabolism; Cell physiology; Fundamental and applied biological sciences. Psychology; Glucagon - pharmacology; Kinetics; Liposomes; Liver - metabolism; Male; Membrane and intracellular transports; Membrane Proteins - metabolism; Molecular and cellular biology; Proteolipids - isolation & purification; Proteolipids - metabolism; Rats; Rats, Inbred Strains; Glucagon; Transportation system; Aminoacid; Liver; Hormonal regulation; Liposome; Artificial membrane; Reconstitution
• ISSN: 0006-3002; 1878-2434
• DOI: 10.1016/0005-2736(87)90238-0

System A-mediated amino acid transport activity from rat liver plasma membrane vesicles has been solubilized and reconstituted into proteoliposomes using a freeze-thaw-dilution technique. The presence of cholate, at a cholate to protein ratio of 1:1, during the freeze-thaw step resulted in an enhancement in recoverable transport activity. The carrier required both phosphatidylcholine and phosphatidylethanolamine for optimal activity, but the addition of cholesterol to the reconstitution procedure appeared to have no significant effect on the resulting activity. A lipid to protein ratio of 20:1 yielded maximal transport activity. Sonication of the proteoliposomes provided some improvement in the accuracy of replicate assays for a given proteoliposome preparation. Isolated liver plasma membrane vesicles prepared from rats treated in vivo with glucagon in combination with dexamethasone contained stimulated System A activity. This enhanced transport activity could be solubilized and recovered in proteoliposomes generated from these plasma membranes. The data support the proposal that hormone regulation of the hepatic System A gene results in the de novo synthesis and plasma membrane insertion of the carrier protein itself.