Ostrich (Struthio camelus) carboxypeptidase B: Purification, kinetic properties and characterization of the pancreatic enzyme

Abstract

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.

Molecular Characterization of the Carboxypeptidase B1 of Anopheles stephensi and Its Evaluation as a Target for Transmission-Blocking Vaccines

“…5). These results confirmed that cpbAs1 encodes an active CPB, and they are consistent with other CPB reports (13,(40)(41)(42). The CPBAs1 kinetic parameters were determined by the progress of an enzyme-catalyzed reaction using fixed amounts of CPBAs1 and a series of different concentrations of Hip-Arg as the substrate according to the standard Michaelis-Menten method.…”

“…Moreover, its specificity for Arg is higher than that for Lys residues as reported for other CPBs (13,45). The recombinant CPBAs1 K m is about 8.5-fold greater than the amounts which have been reported for the CPB enzyme of vertebrates by using the same substrates (13,(40)(41)(42). Furthermore, the CPBAs1 alignment with CPB enzymes of other organisms (Fig.…”

Elucidating the effects of arginine and lysine on a monoclonal antibody C-terminal lysine variation in CHO cell cultures

“…This decrease in pHi might be caused by the increased accumulation of acidic metabolite (e.g., lactic acid), which might outweigh the opposite effect caused by the higher levels of Arg and Lys (Jeong et al 2006). Nevertheless, such a difference in pHi was believed not to significantly affect the activity of basic Cps (Bradley et al 1996;Wolff et al 1962). …”

“…A lower pHi value was observed throughout the culture process under higher Arg and Lys concentrations (HH) when compared with the other conditions. However, this difference in pHi (no more than 0.05 unit) was believed not to significantly affect the activity of basic Cps (Bradley et al 1996;Wolff et al 1962). Additionally, extracellular pH (pHe) was also measured during the batch cultures, and no significant difference was observed among the four conditions (data not shown), thus indicating that the different lysine variant levels should not be attributed to pH condition.…”

Characteristics of carboxypeptidase B from pyloric ceca of the starfish Asterina pectinifera

“…However, lower molecular weights of CPB were reported for cod (26,000) (Overnell, 1973). The optimum pH of the A. pectinifera CPB (pH 7.5) was similar to those of other animal species (pH7-8.2) (Marinkovicet al, 1977;Ajlan & Bailey, 1999;Bradley et al, 1996;Hajjou et al, 1995;Yoshinaka et al, 1984b).…”