OSTRICH (STRUTHIO-CAMELUS) CARBOXYPEPTIDASE-B - PURIFICATION, KINETIC-PROPERTIES AND CHARACTERIZATION OF THE PANCREATIC-ENZYME

Carboxypeptidase B has been isolated from numerous mammalian and inver tebrate species, In contrast, very little is known about carboxypeptid ases of avian origin, To provide information for a comparative study, we have undertaken an investigation of the kinetic and physical proper ties of ostrich carboxypeptidase B, Carboxypeptidase B from the pancre as of the ostrich was purified by water extraction of acetone powder a nd aminobenzylsuccinic acid affinity and hydroxylapatite chromatograph y. The effects of pH and temperature on CPB activity were examined. K- i-values for numerous inhibitors (PCI, ABSA, hipp-D-lys, epsilon-amino caproic acid, D-arg and 3-phenylproprionic acid) and kinetic parameter s (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 an d amino acid analysis were also performed. Purified ostrich carboxypep tidase B wa s 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 s equence showed a high degree of homology when aligned with CPB from ot her 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 carb oxypeptidases. Copyright (C) 1996 Elsevier Science Ltd.