Characterization of a glucagon receptor-linked protease from canine hepatic plasma membranes. Partial purification, kinetic analysis, and determination of sites for hormone processing.
Previous studies have identified the association of the hepatic glucagon receptor with a membrane-bound protease which cleaves [[125I]iodo-Tyr10]glucagon in a hormone- and GTP-sensitive manner (Sheetz, M. J., and Tager, H. S. (1988) J. Biol. Chem. 263, 8509-8514). The current investigations were undertaken to characterize the receptor-linked protease. Treatment of canine hepatic membranes with buffers containing Lubrol and NaCl, followed by gel filtration of soluble material on Sepharose CL-6B and subsequent fractionation of proteins by use of (NH4)2SO4, resulted in a 50-fold purification of the enzyme. Kinetic analysis of the solubilized protease under conditions of linear initial rate (by use of glucagon as substrate and high performance liquid chromatography to separate and quantitate the products) revealed its minimal dependency on pH between values of 7 and 9, its relative preference for glucagon as substrate, and its sensitivity to the presence of salt. Initial rates and the ratio Vmax/Km typically increased 5- to 7-fold and 10-fold, respectively, in the presence of 1 M NaCl. Identification by amino acid analysis of peptide fragments resulting from the incubation of glucagon with the partially purified enzyme and analysis of related time courses for hormone processing demonstrated that the glucagon Tyr13-Leu14 peptide bond is the primary site for proteolytic cleavage by the receptor-linked protease. The implications of these and related findings are discussed.
Sheetz
M J
MJ
Department of Biochemistry and Molecular Biology, University of Chicago, Illinois 60637.
Tager
H S
HS
D000818:Animals; D002462:Cell Membrane / Q000201:enzymology; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D004285:Dogs; D005934:Glucagon / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D010447:Peptide Hydrolases / Q000378:metabolism*; D011964:Receptors, Gastrointestinal Hormone / Q000378:metabolism*; D018027:Receptors, Glucagon
D011964:Receptors, Gastrointestinal Hormone; D018027:Receptors, Glucagon; D005934:Glucagon; D010447:Peptide Hydrolases