Three different procarboxypeptidases A and two different procarboxypeptidases B have been isolated for the first time, in a pure and native state, from human pancreatic extracts. These proteins were purified in one or two quick steps by anion-exchange HPLC. All these forms have been biochemically characterized. Two of the procarboxypeptidases A, the A1 and A2 forms, are obtained in a monomeric state while the other, the A3 form, is obtained as a binary complex of a procarboxypeptidase A with a proproteinase E. This complex is stable in aqueous buffers at various ionic strengths and develops carboxypeptidase A and proteinase E activities in the presence of trypsin. The A3 and A2 forms show clear differences in electrophoretic mobility in SDS/polyacrylamide gels, isoelectric point, proteolytic activation process with trypsin and susceptibility to thermal denaturation. In contrast, these properties are similar in the A1 and A3 (binary complex) forms. On the other hand, with respect to the properties listed above, the B1 and B2 forms differ from each other mainly in isoelectric point. An overall comparison of the above properties reveals the unusual character of the A2 form, midway between the other A and B forms. N-terminal extended sequence analysis carried out on these proenzymes confirm that they constitute different isologous forms.The existence of procarboxypeptidases in two forms which generate active enzymes of different specificity, the A and the B forms, is a widely recognized fact [l]. However, the presence of further isology in these forms has been studied in depth in only a few species. Thus, Neurath et al. [2, 31 have isolated and characterized two alellomorphic forms of bovine carboxypeptidase A. Both forms were isolated under autolytic conditions and no information was obtained on the possible existence of further heterogeneity in the precursor molecules. Scheele et al. [4-81 have also obtained evidence, using electrophoretic techniques, for the existence of two or three forms of procarboxypeptidase A protomer and one to three forms of procarboxypeptidase B in different mammals (guinea pig, rat, dog and human). Recently, Rutter and coworkers [9, 101 have reported the probable existence of a single procarboxypeptidase A gene in the rat genome and have already located the chromosome bands that contain the gene(s) for the same protein in humans and mice.This work reports, for the first time, the rapid and complete isolation of different homologous procarboxypeptidases, three A forms and two B forms, in a native state from human pancreas, using HPLC. The existence of a stable binary complex of a procarboxypeptidase A and a proproteinase E is also reported. Several fundamental properties of these proteins have been characterized and compared with those of the equivalent proenzymes from porcine pancreas, which have been previously studied by our group [11 -181. In addition, the chromatographic behaviour of these human procarboxypeptidases has been analyzed under different conditions and using different HPL...

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The molecular events which lead to the proteolytic transformation of porcine procarboxypeptidase B (PCPB) in carboxypeptidase B (CPB) have been determined. Among pancreatic and other tested proteinases, trypsin is the only one capable of generating carboxypeptidase B activity from the zymogen, in vitro. In the first step of this process, trypsin produces cleavage at the boundary between the activation region and the CPB region. Subsequently, a definite sequence of cleavages occurs at the C-terminal end of the released activation segment of 95 residues, giving rise to characteristic intermediates and to a proteolytically resistant activation fragment of 81 residues. In this process, the newly formed CPB participates in the quick-trimming of the released activation peptides. Only a single CPB species is formed in the activation process. This fact and the inability of the released activation peptides to inhibit CPB--and, therefore, their inability to slow down the kinetics of appearance of CPB activity--are two important characteristics differentiating between the activation processes of procarboxypeptidases A and B. The sequence of the 95 residues (MW = 12,835) of the activation region of porcine PCPB has also been deduced, largely from the information obtained by Edman degradation of its fragments and in part by considerations of homology with the rat precursor. The porcine PCPB activation region contains a high percentage of acidic residues, lacks cysteines, methionines, and side-chain posttranslational modifications, and presents a low but significant homology (31%) with the corresponding sequence of porcine procarboxypeptidase A.

This article is available online at http://dmd.aspetjournals.org ABSTRACT:Almotriptan is a novel highly selective 5-hydroxytryptamine 1B/1D agonist developed for the acute oral treatment of migraine. The in vitro metabolism of almotriptan has been investigated using human liver subcellular fractions and cDNA-expressed human enzymes, to study the metabolic pathways and identify the enzymes responsible for the formation of the major metabolites.