title: Identification, purification and partial characterization of a carboxypeptidase from the matrix of rat liver mitochondria: a novel metalloenzyme.
abstract: A novel carboxypeptidase has been purified to apparent homogeneity from the matrix fraction of rat liver mitochondria by using a procedure mainly based on immobilized-metal-affinity chromatography (IMAC). This carboxypeptidase has been named mCP-III, since it represents the third major peak of carboxypeptidase activity after the IMAC step of purification. mCP-III hydrolyses a number of N-blocked dipeptides, with preference for Cbz-Phe-Ala, and shows no degrading activity towards 125I-casein. The optimal pH of its activity is 7.6, the apparent Km for Cbz-Phe-Ala is 0.12 mM and the specific activity is 145.5 mumol/min per mg of protein. The enzyme is a typical metalloproteinase, is inhibited by 1,10-phenanthroline and carboxypeptidase inhibitor and re-activated by added Zn2+ and Co2+. The molecular mass estimated by molecular-sieve h.p.l.c. was approx. 115 kDa with two protein bands of 61 and 50 kDa shown by SDS/PAGE analysis, indicating that the enzyme is active as a dimer. This is the first clearly identified carboxypeptidase within mitochondria.
authors.affiliation: Instituto Gulbenkian de Ciência, Oeiras, Portugal.
authors.forename: E
authors.initials: E
authors.lastname: Figueiredo
authors.forename: M C
authors.initials: MC
authors.lastname: Duque-Magalhães
mesh_terms: D000818:Animals; D002268:Carboxypeptidases / Q000032:analysis / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008297:Male; D008666:Metalloendopeptidases / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D008930:Mitochondria, Liver / Q000201:enzymology*; D008970:Molecular Weight; D051381:Rats; D017208:Rats, Wistar; D013379:Substrate Specificity
chemical_list: D002268:Carboxypeptidases; C087419:mitochondrial carboxypeptidase-III; D008666:Metalloendopeptidases