Preparative isolation of the two forms of pig pancreatic pro-(carboxypeptidase A) and their monomeric carboxypeptidases A

Vilanova, María; Vendrell, Josep; Lopez, Marimar; Cuchillo, Claudi M.; Aviles, Francesc X.

doi:10.1042/bj2290605

Cited by 22 publications

(7 citation statements)

References 21 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The conditions previously used to separate the activation segment from porcine CPA [36] could not be applied here because of the irreversible denaturation of bovine CPA in 7 M urea. Dissociation of the bovine-CPA .…”

Section: Mild Separation Of the Activation Segment From The Resultingmentioning

confidence: 99%

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

et al. 1987

View full text Add to dashboard Cite

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...

show abstract

Section: Mild Separation Of the Activation Segment From The Resultingmentioning

confidence: 99%

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

et al. 1987

View full text Add to dashboard Cite

show abstract

“…Porcine pro-CPB was isolated from acetone pancreatic powder by anion-exchange chromatography on DEAE sepharose according to a previously reported procedure (Vilanova et al, 1985b;Burgos et al, 1991). As a last purification step, an additional FPLC chromatography on an Ultrapac TSK-DEAE column 5PW (150 X 21.5 mm, 10 pm particle size, 0.1 pm pore, from Toyoshoda) in 20 mM Tris-acetate, p H 8.0, with an elution gradient between 0 and 0.8 M ammonium acetate, was included.…”

Section: Preparative Isolation Of Pro-cpbmentioning

confidence: 99%

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

1995

View full text Add to dashboard Cite

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...

show abstract

“…Procarboxypeptidase B was prepared from procine pancreas following the method described by Vilanova et al [19]. Carboxypeptidase B was obtained by tryptic digestion of the native proenzyme [20] followed by application to a DEAESepharose column in 40 mM Tris pH 8.0 and elution with a linear NaCl gradient (0 -0.4 M) to separate the enzyme from the severed activation domain.…”

Section: Methodsmentioning

confidence: 99%

Differential scanning calorimetric study of carboxypeptidase B, procarboxypeptidase B and its globular activation domain

Conejero‐Lara¹,

Sánchez‐Ruiz²,

Mateo³

et al. 1991

European Journal of Biochemistry

View full text Add to dashboard Cite

High-sensitivity differential scanning calorimetry has been applied to the study of porcine pancreatic carboxypeptidase B, the proenzyme and its 81-residue activation domain. The thermal study has been carried out over a range of scan rates, ionic strengths and pH values. The thermal unfolding of the isolated activation domain has been found to be reversible and corresponds to that of a typical compact globular structure, with melting temperatures higher than those of the enzyme and proenzyme. Both proteins, on the other hand, undergo an irreversible, highly scan-rate-dependent thermal denaturation under all the experimental conditions investigated. The denaturation of the enzyme at pH 7.5 and the proenzyme at pH 7.5 and 9.0 follows the two-state irreversible model [Sanchez-Ruiz, J. M., L6pez-Lacomba, J. L., Cortijo, M. & Mateo, P. L. (1988) Biochemistry 27, 1648-16521. Thus the kinetic constants and activation parameters of the denaturation process could be obtained and compared to those for other proteins, particularly those of the closely related carboxypeptidase A system.The biological function of a protein depends on the correct folding of its native structure. The loss of this folded structure, either by heat or chemicals such as detergents, leads to an unfolded, inactive state. The subject of folding/unfolding and of protein thermal stability is currently a field of increasing interest because of biotechnological implications. For example, the efficiency of bioreactors can be greatly increased by using thermostable enzymes [l]. In addition, selected amino acid residue replacement in site-directed mutagenesis is often used to enhance protein stability [2 -41, while the structural characteristics responsible for thermostability in proteins from thermophilic organisms are also the object of current attention [5, 61.Differential scanning calorimetry (DSC) is nowadays the most useful technique for characterizing the thermal stability of proteins, quantitatively defined as the Gibbs energy of unfolding [7]. Thermodynamic analysis of DSC data for simple and complex proteins, which obviously requires the denaturation to be a reversible equilibrium process, has for instance led to the structural definition of co-operative submolecular domains [8]. Nevertheless, it is well known that the thermal denaturation of most proteins is calorimetrically irreversible. Since it is also generally accepted that the folded state is thermodynamically more stable than the unfolded one at low temperatures, additional irreversible processes must occur to the unfolded state to explain this overall irreversibility. Lumry and Eyring's scheme [9] is the simplest model to describe this situation N F ? U + F where N and U stand for the native and unfolded states, and F for the final state of the protein, which has been arrived at irreversibly. When the DSC transitions corresponding to irreversible protein denaturation are found to be highly scanrate-dependent, it is clear that the overall denaturation process is kinetically controlled an...

show abstract

Preparative isolation of the two forms of pig pancreatic pro-(carboxypeptidase A) and their monomeric carboxypeptidases A

Cited by 22 publications

References 21 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

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

Differential scanning calorimetric study of carboxypeptidase B, procarboxypeptidase B and its globular activation domain

Contact Info

Product

Resources

About