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

Vilanova, María; Burgos, Francisco Javier Antón; Cuchillo, Claudi M.; Avilés, Francesc X.

doi:10.1016/0014-5793(85)80023-5

Cited by 26 publications

(17 citation statements)

References 13 publications

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“…The peptide was found to exert a very strong inhibitory effect on the CPA activity with a Ki in the nanomolar range [24]. Like other large peptides, it was found to possess a secondary structure including in the present case 50% a-helix and 10% fl-sheet [25]. Therefore it may be expected to be rather compact and resistant to proteolysis.…”

mentioning

confidence: 69%

“…Thus prior dissocia-tion was assumed to occur in vivo to allow a fast conversion of pro-CPA into CPA [33]. Yet CPA activity is known to be expressed during trypsin treatment of the binary and ternary complexes of porcine and bovine pro-CPA without any detectable dissociation of the subunits [6, 13,25]. Furthermore the present work shows that the susceptible bond was cleaved at a similar rate in the free or complex forms of bovine pro-CPA, thus implying that the accessibility to trypsin of the bond is not hindered in the complexes.…”

Section: Amino Acid Composition and N-terminal Sequence Of The Bovinementioning

confidence: 99%

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Further studies on the activation of bovine pancreatic procarboxypeptidase A by trypsin

et al. 1987

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Unlike the pancreatic endopeptidase zymogens, procarboxypeptidase A is activated very slowly in vitro. The activation proceeds through the removal of about 100 amino acids away from the N-terminus of the chain. The cleavage of the susceptible bond(s) in monomeric and aggregated forms of bovine procarboxypeptidase A by catalytic amounts of trypsin was found to be very fast. However, as in the case of the porcine zymogen, the expression of the carboxypeptidase activity was considerably delayed by the inhibitory effect of the activation peptide which remains bound to the enzyme molecule after the trypsin treatment of the zymogen. a-Carboxypeptidase A was mainly formed under the relatively mild conditions used, indicating that the Arg-' -Ala" bond is probably the first to be cleaved during in vitro activation.The bovine carboxypeptidase activity was immediately and reversibly expressed upon dimethylmaleylation of the activation mixtures. This expression does not require full dissociation of the enzyme-peptide complex but merely a suitable change in its quaternary structure resulting from a modification of some electrostatic interactions upon dimethylmaleylation.Separation of bovine carboxypeptidase A from its activation peptide was only achieved upon filtration of the dimethylmaleylated mixtures in the presence of 6 M urea. The bovine activation peptide contains at least 93 amino acids compared to the 94 amino acids found by other authors for the rat and porcine peptides and sequencing of the first 53 amino acids showed a 75 -85% homology with the latter two peptides.Pancreatic carboxypeptidase A (CPA) is a zinc exopeptidase that sequentially degrades proteins and peptides by cleavage of certain C-terminal bonds. Its chemical properties and mechanism of action have been extensively studied (for recent reviews see [l, 21). The enzyme is synthesized in the form of the inactive zymogen procarboxypeptidase A (pro-CPA) which, depending on the species, is present in pancreatic juice either as a monomer [3 -61 or an aggregate with one [7, 81 or two [6, 9 -111 other proteins. A ternary complex with a sedimentation coefficient of 6s in which pro-CPA is associated with a chymotrypsinogen of the C-type [I21 and an inactive protein (subunit 111) related to an elastase [13, 141 has so far been described only in three ruminant species: ox, goat and sheep [6]. In the pig, pro-CPA is partly monomeric and We wish to dedicate this paper to the memory of Professor Pierre Desnuelle (Centre National de la Recherche Scientifique, Marseille) for his kind interest during the course of this work and for his helpful criticisms during the preparation of this manuscript.Abbreviations. CPA, carboxypeptidase A; pro-CPA, procarboxypeptidase A.Enzynies. Carboxypeptidase A (EC 3.4.17.1); trypsin (EC 3.4.21.4; chymotrypsin (EC 3.4.21 .1).Note. The numbering system used for amino acid residues corresponds to the following scheme for the conversion of the bovine zymogen into the various forms of CPA porcine CPAs resulting from both tryptic and chym...

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confidence: 69%

Section: Amino Acid Composition and N-terminal Sequence Of The Bovinementioning

confidence: 99%

Further studies on the activation of bovine pancreatic procarboxypeptidase A by trypsin

et al. 1987

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“…This would be a highly unstable complex because of autolysis of the propancreatopeptidase E component, as shown in the human species [14]. Or, alternatively, because of easy activation by traces of trypsin in the preparation, since tryptic activation of such binary complexes decreases their stability [23] or even promotes dissociation [9]. The bovine ternary complex is the only quaternary association that has been shown to be stable upon tryptic activation [13].…”

Section: Discussionmentioning

confidence: 99%

“…The structural determinants responsible, respectively, for the null and great inhibitory power of porcine pro-CPB and pro-CPAl activation segments [23,241, probably also play a functional role in the corresponding rat proenzymes. The lack of evidence for trypsin cleavages at Arg85-Glu86 in rat pro-CPA2 and Lys85-Gln86 in rat pro-CPAl are in favour of the occurrence of these residues in a region with a stable a-helix.…”

Section: Discussionmentioning

confidence: 99%

Procarboxypeptidase in rat pancreas Overall characterization and comparison of the activation processes

Oppezzo

Ventura

Bergman

et al. 1994

European Journal of Biochemistry

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Three monomeric procarboxypeptidases and a binary complex consisting of a procarboxypeptidase and a chymotrypsinogen have been isolated from rat pancreas by HPLC. N-terminal sequence determination, substrate-specificity analysis and physico-chemical characterization showed that the carboxypeptidase precursors were the Al, A2 and B forms. No isomorphism could be detected for any of these proenzymes and no clear evidence was obtained for the presence of procarboxypeptidase-containing quaternary complexes of the types previously described for other species. Instead, we observed the presence of a binary complex between procarboxypeptidase A2 and chymotrypsinogen B. Among the major pancreatic endoproteinases, only trypsin was found to be a general activator of rat procarboxypeptidases in vitro. Time-course analysis of the products generated after trypsin addition confirmed that full activation of procarboxypeptidase A1 requires several cleavages in the C-terminal region (residues 87 -94) of the activation segment, while procarboxypeptidases A2 and B require a single cleavage each. The carboxypeptidases released participate in the trimming of the activation segment in A1 and B, but not in A2, probably because of the high specificity of the latter in the active form.The present knowledge about the precursor forms of pancreatic metallo-pro-carboxypeptidases (Metallo-pro-CP) has been obtained from studies on many different species [l]. A significant point in this progress was the gene cloning and sequence analysis of pro-CP Al, A2 and B from rat pancreas [2, 31. Together with these studies, the functional mapping of the active site of the rat carboxypeptidase A1 isoform (CPA1) by site-directed mutagenesis [4, 51, and the X-ray crystallographic analysis of rat carboxypeptidase A2 (CPA2) [6], made the rat system suitable to serve as one of the reference models in the field. However, the properties of the pro-CP complement in rat pancreas have not been evaluated. These characteristics are now determined.Pro-CP occur in a diversity of isoforms and alleloforms in the pancreas of vertebrates [7-91. Two isoforms of pro-CPB and pro-CPA (Bl, B2, A1 and A2) are found in certain species, and pro-CPA is found in at least two alleloforms in bovine pancreas. We now show that three of the isoforms mentioned, Al, A2 and B, are found and expressed at similar levels in rat pancreas. In addition to the monomeric forms [9-111, pro-CPA also occur in different oligomeric complexes with other pancreatic zymogens [9,11-151. A question remains open about the potential role of these complexes and the reason for the species dependence of their occurrence, as ternary complexes in ruminants and as binary complexes in non-ruminants [13]. In rat pancreas we find that a fraction of pro-CPA2 is complexed with a chymotrypsinogen-like zymogen of the B form. Such a binary complex has no counterpart in other species, where only binary complexes between pro-CPAl and either propancreatopeptidase E [9, 11, 161 or chymotrypsinogen C [17] have been described.R...

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“…The sample was immediately mixed with electrophoretic loading buffer (containing 7 M urea), heated at 100 "C for 1 min, and analyzed in a polyacrylamide gel with a transverse gradient of urea (0-9 M), as described previously (Vilanova et al, 1985a).…”

Section: Binding Studiesmentioning

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

Cited by 26 publications

References 13 publications

Further studies on the activation of bovine pancreatic procarboxypeptidase A by trypsin

Further studies on the activation of bovine pancreatic procarboxypeptidase A by trypsin

Procarboxypeptidase in rat pancreas Overall characterization and comparison of the activation processes

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

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