Activation and membrane binding of carboxypeptidase E.
Carboxypeptidase E (CPE) is a carboxypeptidase B-like enzyme that is thought to be involved in the processing of peptide hormones and neurotransmitters. Soluble and membrane-associated forms of CPE have been observed in purified secretory granules from various hormone-producing tissues. In this report, the influence of membrane association on CPE activity has been examined. A substantial amount of the membrane-associated CPE activity is solubilized upon extraction of bovine pituitary membranes with either 100 mM sodium acetate buffer (pH 5.6) containing 0.5% Triton X-100 and 1 M NaCl, or by extraction with high pH buffers (pH greater than 8). These treatments also lead to a two- to threefold increase in CPE activity. CPE extracted from membranes with either NaCl/Triton X-100 or high pH buffers hydrolyzes the dansyl-Phe-Ala-Arg substrate with a lower Km than the membrane-associated CPE. The Vmax of CPE present in extracts and membrane fractions after the NaCl/Triton X-100 treatment is twofold higher than in untreated membranes. Treatment of membranes with high pH buffers does not affect the Vmax of CPE in the soluble and particulate fractions. Pretreatment of membranes with bromoacetyl-D-arginine, an active site-directed irreversible inhibitor of CPE, blocks the activation by NaCl/Triton X-100 treatment. Thus the increase in CPE activity upon extraction from membranes is probably not because of the conversion of an inactive form to an active one, but is the result of changes in the conformation of the enzyme that effect the catalytic activity.
Fricker
L D
LD
Department of Molecular Pharmacology, Albert Einstein College of Medicine, Bronx, New York 10461.
D000818:Animals; D043423:Carboxypeptidase H; D002268:Carboxypeptidases / Q000378:metabolism*; D002417:Cattle; D002462:Cell Membrane / Q000201:enzymology; D003902:Detergents; D004789:Enzyme Activation; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D017830:Octoxynol; D010902:Pituitary Gland / Q000201:enzymology*; D011092:Polyethylene Glycols; D012965:Sodium Chloride
D003902:Detergents; D011092:Polyethylene Glycols; D012965:Sodium Chloride; D017830:Octoxynol; D002268:Carboxypeptidases; D043423:Carboxypeptidase H