Ostrich (Struthio camelus) carboxypeptidase B: purification, kinetic properties and characterization of the pancreatic enzyme.
Carboxypeptidase B has been isolated from numerous mammalian and invertebrate species. In contrast, very little is known about carboxypeptidases of avian origin. To provide information for a comparative study, we have undertaken an investigation of the kinetic and physical properties of ostrich carboxypeptidase B. Carboxypeptidase B from the pancreas of the ostrich was purified by water extraction of acetone powder and aminobenzylsuccinic acid affinity and hydroxylapatite chromatography. The effects of pH and temperature on CPB activity were examined. K(i)-values for numerous inhibitors (PCI, ABSA, hipp-D-lys, epsilon-aminocaproic acid, D-arg and 3-phenylproprionic acid) and kinetic parameters (K(m), k(cat) and k(cat)/K(m)) for several substrates (hipp-arg, hipp-lys, FAAA, FAAL and hipp-AA) were determined. N-terminal sequencing and amino acid analysis were also performed. Purified ostrich carboxypeptidase B was assessed to be homogeneous by SDS-PAGE with a M(r) value of approx. 35,000. For ostrich carboxypeptidase B the K(m) values for the different substrates were of the same order as those reported for other species, whereas the k(cat) values were 8- to 21-fold lower than the reported values. FAAA and hipp-AA were the preferred substrates. PCI was the most effective inhibitor, with a K(i) in the nM region, and no inhibition was shown with 3-phenylpropionic acid. The N-terminal sequence showed a high degree of homology when aligned with CPB from other species. Amino acid analysis showed significantly lower levels of Asx and Cyh and higher levels of Trp and Leu when compared with other species. Ostrich carboxypeptidase B would appear to show many physical, chemical and kinetic properties similar to those of other known carboxypeptidases.
Bradley
G
G
Department of Biochemistry, University of Port Elizabeth, South Africa.
Naudé
R J
RJ
Muramoto
K
K
Yamauchi
F
F
Oelofsen
W
W
D000595:Amino Acid Sequence; D000818:Animals; D001717:Birds / Q000378:metabolism*; D043424:Carboxypeptidase B; D002268:Carboxypeptidases / Q000737:chemistry / Q000302:isolation & purification*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D010179:Pancreas / Q000201:enzymology*; D017386:Sequence Homology, Amino Acid
D002268:Carboxypeptidases; D043424:Carboxypeptidase B