Reconstitution of bovine procarboxypeptidase A-S6 from the free subunits

Puigserver, Antoine; Desnuelle, P.

doi:10.1021/bi00630a028

Cited by 25 publications

(20 citation statements)

References 26 publications

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“…However, this activation segment does not bind to the complementary CPB, as deduced by the observed independent eiect'rophoresis of both proteins along the whole urea-gradient when they are run as a 1: 1 molar ratio mixture ( fig.2c). The inability of asB to bind to its active enzyme, in contrast to the strong binding of asA to its enzyme, could help to explain the great differences in the proteolytic activation process of both proenzymes, which requires a few minutes in the former case and several hours in the latter [3,5,8,9]. Thus, during the action of trypsin asB would be quickly released from CPB, the activity of which would be expressed very early.…”

Section: Resultsmentioning

confidence: 99%

Urea‐gradient gel electrophoresis studies on the association of procarboxypeptidases A and B, proproteinase E, and their tryptic activation products

Vilanova¹,

Burgos²,

Cuchillo³

et al. 1985

FEBS Letters

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Monomeric procarboxypeptidase A (PCPA) and isolated proproteinase E (PPE), both from pig pancreas, were shown by means of electrophoresis on transverse urea gradients (0–9 M) to form a very stable complex, identical to their natural binary complex. Although the complex is maintained by the interaction of both active regions, the activation segment of PCPA participates directly in the binding. Procarboxypeptidase B (PCPB) also associates with PPE, but in this case the complex shows low stability. In contrast with carb‐oxypeptidases A that strongly bind to their corresponding severed activation segments, no interaction was observed between carboxypeptidase B and its severed activation segment. The above results give some insight into several characteristics of the structure and activation properties of pancreatic PCPA and PCPB.

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Section: Resultsmentioning

confidence: 99%

Urea‐gradient gel electrophoresis studies on the association of procarboxypeptidases A and B, proproteinase E, and their tryptic activation products

Vilanova¹,

Burgos²,

Cuchillo³

et al. 1985

FEBS Letters

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“…The oligomeric association is the most common occurrence of pro-CPAs (Yamasaki et al, 1963;Uren & Neurath, 1972;Kobayashi et al, 1978;Oppezzo et al, 1994), and it has been observed that the quaternary structure affects their activation behavior (Puigserver & Desnuelle, 1977;Kobayashi et al, 1981; Puigserver et al, 1986;Chapus et al, 1987). No complex with proteinase precursors has yet been described for the B form.…”

Section: Introductionmentioning

confidence: 99%

The activation pathway of procarboxypeptidase B from porcine pancreas: Participation of the active enzyme in the proteolytic processing

1995

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The activation process of porcine pancreatic procarboxypeptidase B (pro-CPB) has been studied in detail by a number of complementary methodologies, and a description of the molecular events that lead to the generation of active carboxypeptidase B (CPB) has been deduced. The generated CPB participates in the degradation of its own activation segment by excising C-terminal residues from fragments produced by tryptic proteolysis. The trimming action of CPB is, however, not essential for the release of a fully functional enzyme, in contrast to what was previously reported for porcine procarboxypeptidase A (pro-CPA). In the model presented here, the activation process is solely dependent on the first tryptic cleavage, at the limit between the activation segment and the enzyme region, and the former piece loses all of its inhibitory capacity once severed from the proenzyme. The use of heterologous inhibitors of CPB activity during the study of the tryptic activation process of pro-CPB has been required for the capture of short-lived, otherwise nondetectable, intermediates. This has allowed a complete description of the process and shown that the first proteolytic action of trypsin can also take place on a second target bond. Structural considerations that take into account the three-dimensional structures of the A and B forms of the proenzymes lead us to propose that the differences in conformation at the region that connects the globular activation domain to the enzyme are the main responsible elements for the differences observed in the activation processes of both proenzymes.Keywords: activation domain; activation segment; inhibition mechanism; limited proteolysis; procarboxypeptidase; zymogen Porcine pancreatic secretion contains considerable amounts of the precursor forms of the two major pancreatic carboxypeptidases: AI and B. Procarboxypeptidase A1 (pro-CPA1) is found in the monomeric state and as a member of a binary complex with proproteinase E, whereas procarboxypeptidase B (pro-CPB) is found only in the monomeric state (Aviles et al., 1993). The oligomeric association is the most common occurrence of pro-CPAs (Yamasaki et al., 1963;Uren & Neurath, 1972;Kobayashi et al., 1978;Oppezzo et al., 1994), and it has been observed that the quaternary structure affects their activation behavior (Puigserver & Desnuelle, 1977;Kobayashi et al., 1981; Puigserver et al., 1986;Chapus et al., 1987). No complex with proteinase precursors has yet been described for the B form. The Reprint requests to: Francesc X. Aviles, Departament de Bioquimica i Biologia Molecular, Unitat de Ciencies and lnstitut de Biologia Fonamental, Universitat Autonoma de Barcelona, 08193 Bellaterra, Spain.Abbreviations: BZS, benzylsuccinic acid; CP, carboxypeptidase; PCI, potato carboxypeptidase inhibitor; MMGETP, 2-mercaptomethyl-3-guanidinethylthiopropanoic acid.-~ ~-~-~-presence of the two monomeric zymogens in porcine pancreas makes this system suitable for comparison studies on their activation processes. The activation process of pancr...

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“…Neurath and coworkers isolated the first pancreatic procarboxypeptidases from cattle [7-91, dogfish [lo], and lungfish [Ill, amongst others. Also, Puigserver and coworkers [12, 131, Hirs and coworkers [14, IS] and Avilts and coworkers [16, 171, made significant contributions to the characterization of procarboxypeptidases, in particular those from cattle, pig and human.According to two-dimensional electrophoresis studies [ 18, 191, both the A and B proenzymes are secreted in the pancreatic juice as isoforms or alleloforms of similar molecular mass, within the range 45 -47 kDa, in different mammalian species (human, cattle, dog, pig, rat, etc.). The PI range of these proenzymes varies little in the A forms, from 4.3 to 5.2 (5.7…”

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confidence: 99%

Advances in metallo‐procarboxypeptidases

Avilés

Vendrell

Guasch

et al. 1993

European Journal of Biochemistry

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Our knowledge on the structure and functionality of pancreatic carboxypeptidases is rapidly expanding to include that of their zymogen forms. The recent application of fast and mild isolation procedures, together with modern molecular genetic and biochemical-biophysical characterization approaches, has provided a clearer view of the basic structures and functional states in which these zymogens occur, and their evolutionary relationships. The same holds for related metallocarboxypeptidases, either in the pro or active forms, that have been isolated and characterized in non-digestive fluids and tissues, where they probably play an important role in protein and peptide processing. The determination of the three-dimensional structure of the A and B pancreatic zymogens has revealed the molecular determinants of their inactivity and proteolytic activation. The folding of their 95-residue activation segment in a globular N-terminal domain (74-81 residues) and in a connecting region (20-14 residues), and the specific contacts of these pieces with the substrate binding sites of the enzyme, are important factors in zymogen inhibition. On the other hand, the different length of the a-helical connecting region and the stability of its contacts with the enzyme account for the different activation properties of A and B zymogens.The hydrolysis of the peptide bond at the C-terminus of peptides and proteins in living organisms is performed by enzymes belonging either to the metallo-carboxypeptidase family or to the serine-carboxypeptidase family. The latter contain a reactive Ser residue at the active center and other catalytic residues which resemble and have a similar role to those of the Ser-endopeptidases superfamily [I -31. The residues and conformation of the active center of metallocarboxypeptidases are different, having one atom of Zn2 + directly involved in the catalytic mechanism. The present review refers to this latter class of carboxypeptidases and to their precursors.Pancreatic carboxypeptidases, the digestive enzymes involved in the hydrolysis of alimentary proteins from their Cterminal, are the best known enzymes among metallocarboxypeptidases and their occurrence and fundamental properties are very well documented [4 - (EC 3.4.17.10); CPM, membrane-bound carboxypeptidase M (EC 3.4.17.12); CPN, plasma carboxypeptidase N or kininase I (EC 3.4.17.3). specificity between the A form (CPA, with preference for aliphatic C-terminal residues) and the B form (CPB, with preference for basic C-terminal residues), as well as the tertiary structure and mechanism of action of carboxypeptidase A are well known. A number of non-digestive pancreatic-like carboxypeptidases have also been reported in the recent literature, expanding the field of interest in metallo-carboxypeptidases. In contrast to the active enzymes, and for many years, information on the inactive precursor forms of synthesis and storage, procarboxypeptidases A and B (pro-CPA and pro-CPB), has been limited.The lability of these precursors and their occurrence...

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Reconstitution of bovine procarboxypeptidase A-S6 from the free subunits

Cited by 25 publications

References 26 publications

Urea‐gradient gel electrophoresis studies on the association of procarboxypeptidases A and B, proproteinase E, and their tryptic activation products

Urea‐gradient gel electrophoresis studies on the association of procarboxypeptidases A and B, proproteinase E, and their tryptic activation products

The activation pathway of procarboxypeptidase B from porcine pancreas: Participation of the active enzyme in the proteolytic processing

Advances in metallo‐procarboxypeptidases

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