Biomedical subjects

C Chapus

Publications and source records attributed to C Chapus.

At least 37 records · Page 2Linked to original sources

PubMed1991

The activation peptide of pancreatic procarboxypeptidase A is the keystone of the bovine procarboxypeptidase A-S6 ternary complex.

In some ruminant species, pancreatic procarboxypeptidase A is the central element of a ternary complex involving two other components, a C-type chymotrypsinogen and an inactive protease E. Although the complex is devoted to protein digestion, the fate of this system upon activation of its constituent subunits has, as yet, not been clearly established. In this paper, the activation peptide of procarboxypeptidase A is shown to play a key role in the association of the three subunits and a model is proposed for the in vivo function of the complex.

Animals↗

PubMed1991

Microcalorimetric investigation of the interactions between the subunits of the bovine pancreatic procarboxypeptidase A-S6 complex.

A complete microcalorimetric investigation of the interactions between the native subunits of the bovine pancreatic procarboxypeptidase A-S6 ternary complex has been performed. All the association constants and thermodynamic parameters associated with the reactions forming the various complexes have been determined. The influence of pH and ionic strength on the binding reactions has been investigated. Interestingly, the affinity between the subunits is not significantly modified by varying the ionic strength. In this respect, an enthalpy/entropy compensatory effect is observed for the binding of subunit III to subunit I when the ionic strength is increased, suggesting a physiological function for the association. The various pathways for formation of the ternary complex have been studied. Binding of subunit II (or III) to subunit I, the central element of the ternary complex, does not significantly modify the affinity of the other subunit for subunit I. From a thermodynamic point of view, the same final state is obtained whatever the pathway of ternary complex formation. This study is the first step of a kinetic investigation of the associated subunits.

Animals↗

PubMed1990

Spectrofluorimetric investigation of the interactions between the subunits of bovine pancreatic procarboxypeptidase A-S6.

A spectrofluorimetric investigation of the interactions between the subunits of the pancreatic bovine procarboxypeptidase A ternary complex was carried out after covalent insertion of a fluorescent probe at the active center of one of the constituent subunits. The specific insertion of an anthraniloyl group at the active center of subunit II free or bound to subunit I, after its conversion into chymotrypsin II, allowed us to determine the value of the dissociation constant between subunit I and anthraniloyl-chymotrypsin II (Kd = 0.7 +/- 0.1 x 10(-7) M) and between subunit III and the binary complex subunit I-anthraniloyl-chymotrypsin II (Kd = 1.6 +/- 0.3 x 10(-7) M). Moreover, the influence of the association on the flexibility of the active center of chymotrypsin II was deduced from fluorescence polarization measurements and rotational correlation time determination of anthraniloyl-chymotrypsin II free or bound to subunit I. The anthraniloyl group has no motion independently of the whole chymotrypsin II molecule and the binding of subunit I to anthraniloyl-chymotrypsin II results in an increase of the rigidity of the active site in the latter protein.

Animals↗

PubMed1990

Binding of terbium and of an elastase inhibitor to bovine pancreatic subunit III, an inactive protease E.

Using the Tb3+ luminescence technique, we showed that bovine subunit III, a defective pancreatic serine endopeptidase-like protease, possessed a single metal ion binding site able to bind Tb3+ with a high affinity comparable to that of porcine elastase. The topology of the metal ion binding site in subunit III is predicted from sequence homologies and modeling experiments based on the known crystallographic three-dimensional structures of the equivalent sites in porcine elastase 1 and bovine beta-trypsin. Moreover, the Tb3+ luminescence technique in parallel to a 19F NMR investigation, allowed us to measure the binding of a very potent specific inhibitor of porcine elastase (trifluoroacetyl-L-lysyl-alanyl p-trifluoromethylphenylanilide) to bovine subunit III. These results confirm that, although devoid of any specific activity, subunit III might possess a conformation close to that of an active enzyme and further support the analogy between subunit III and an elastase-like family.

Amino Acid Sequence↗

PubMed1990

Further studies on the human pancreatic binary complexes involving procarboxypeptidase A.

In contrast to procarboxypeptidase B which has always been reported to be secreted by the pancreas as a monomer, procarboxypeptidase A occurs as a monomer and/or associated to one or two functionally different proteins, depending on the species. Recent studies showed that, in the human pancreatic secretion, procarboxypeptidase A is mainly secreted as a 44 kDa protein involved in at least three different binary complexes. As previously reported, two of these complexes associated procarboxypeptidase A to either a glycosylated truncated protease E or zymogen E. In this paper, we identified proelastase 2 as the partner of procarboxypeptidase A in the third complex, thus reporting for the first time the occurrence of a proelastase 2/procarboxypeptidase A binary complex in vertebrates. Moreover, from N-terminal sequence analyses, the 44 kDa procarboxypeptidase A involved in these complexes was identified as being of the A1 type. Only one type of procarboxypeptidase B, the B1 type, has been detected in the analyzed pancreatic juices, thus emphasizing the previously observed genetic differences between individuals.

Amino Acid Sequence↗

PubMed1989

A cross-linked complex between horse pancreatic lipase and colipase.

The water soluble carbodiimide N-cyclohexyl-N'-2-morpholinoethyl-carbodiimide-methyl-p-toluolsulfona te was found to effectively covalently cross-link pancreatic colipase to lipase as evidenced by Western blotting experiments using antibodies directed either against lipase or colipase. Moreover the resulting covalent complex has a Mr consistent with a stoichiometry of 1 mol colipase per mol lipase. Cross-linked lipase and colipase retain their activity implying a correct covalent binding between the two proteins. The specificity of the lipase-colipase binding was further supported by the very low amount of cross-linked products when lipase or colipase alone were incubated in the presence of carbodiimide. The formation of a covalent lipase-colipase complex in the presence of carbodiimide clearly demonstrates that the binding between both proteins involves ion pairing. Furthermore, the formation of an active covalent complex strongly suggests that the lipase-colipase binding site is distinct from the colipase interfacial recognition site as well as from the lipase catalytic site.

Animals↗

PubMed1989

Identification of a procarboxypeptidase A-truncated protease E binary complex in human pancreatic juice.

The characterization, in human pancreatic juice, of a binary complex associating procarboxypeptidase A with a 32 kDa inactive glycoprotein (G32) is reported in this paper. Free G32 was isolated after dissociation of the binary complex. N-terminal sequence analysis revealed a complete homology between this protein and human protease E (HPE 1), except for the two strongly hydrophobic N-terminal residues (Val-Val) which are missing in G32. This protein might be a truncated protease E highly analogous to the subunit III of the ruminant procarboxypeptidase A-S6 ternary complex. The analogy with bovine subunit III is further supported by interspecies reassociation experiments showing that bovine procarboxypeptidase A can specifically bind human G32.

Carbohydrates↗

PubMed1989

Crystallization and preliminary X-ray study of horse pancreatic lipase.

Horse (Equus caballus) pancreatic lipase (EC 3.1.1.3) has been crystallized using the hanging drop method of vapour diffusion at 20 degrees C. The best crystals were grown from an 8 mg/ml solution in 10 to 20% (w/v) polyethylene glycol 8000, 10 mM-MgCl2, 0.1 M-NaCl, 0.1 M-Mes buffer (pH 5.6). They reach dimensions of 0.8 mm x 0.4 mm x 0.6 mm. X-ray examination of the lipase crystals shows that they are orthorombic with a space group P2(1)2(1)2(1). Their cell dimensions are a = 79.8 A, b = 97.2 A c = 145.3 A. Two molecules per asymmetric unit give a Vm value of 2.82 A3/dalton (56% water content). Lipase crystals strongly diffract to at least 1.8 A resolution. Some molecular properties of horse lipase compared to those of the better-known porcine enzyme are also presented.

Animals↗

PubMed1988

Primary structure of the activation peptide from bovine pancreatic procarboxypeptidase A.

The complete sequence of the 94 residues composing the activation peptide of bovine procarboxypeptidase A has been determined by automated analysis of the intact activation segment and of three peptides resulting from enzymatic cleavages of the isolated peptide. The sequencing of a CNBr peptide isolated from procarboxypeptidase A allowed to connect the activation peptide with alpha-carboxypeptidase A (peptidylprolyl-L-amino-acid hydrolase, EC 3.4.17.1). The activation segment has a high content of acidic residues and a proline-rich region. Conformational prediction studies show that the bovine peptide, as the porcine and rat peptides, contains a high proportion of secondary structure and that the structural disposition of the regions in secondary structure is similar in the three peptides. The comparison of the sequence of the bovine, porcine and rat peptides, although exhibiting a striking homology, clearly shows that 40% of the substitutions have led to a charge change.

Amino Acid Sequence↗

PubMed1988

Subunit III of ruminant procarboxypeptidase A-S6 complexes and pancreatic proteases E. A new family of pancreatic serine endopeptidases?

Subunit III (BSIII) of the bovine ternary complex of procarboxypeptidase A-S6 (PCPA-S6), a defective serine endopeptidase-like protein, actively synthesized by the pancreas of some ruminant species, is highly homologous to human protease E (HPE). Both proteins possess the same atypical disulfide bridge in position 98-99b. They are structurally related to porcine elastase 1 and human elastase 2 (about 56% identity). However, in contrast to those two enzymes which have an overall positive net charge, BSIII and HPE are negatively charged. Three-dimensional models of BSIII and HPE have been constructed from the crystallographic structure of porcine pancreatic elastase 1. The inhibitor-binding site for TFAI in these three proteins seems to be very similar; the atypical disulfide bridge does not seem to be involved in this binding site. The specific structural features of BSIII and HPE strongly support the assumption that BSIII is a truncated protease E and that both proteins belong to a separate serine endopeptidase family.

Amino Acid Sequence↗

PubMed1988

Morphology of the procarboxypeptidase A-S6 complex. A solution X-ray scattering study.

Bovine pancreatic procarboxypeptidase A is secreted as a non-covalent association of three different proteins (pro CPA-S6). The free native subunits can be obtained by dissociation of the complex by dimethylmaleylation. Moreover, two specific binary complexes resulting from the high affinity of procarboxypeptidase A (subunit I) for its other two partners (subunits II and III) can also be obtained. In order to better understand the function of the association, an investigation of the morphology of the ternary complex by solution X-ray scattering has been carried out. The radii of gyration of all the molecular species have been obtained and the experimental results have been interpreted in terms of compact objects of simple shape. The various components correspond to globular particles as shown by the value of the ratio Rg/M1/3. This is confirmed by the moderate anisotropy of the simple geometric shapes determined using an assumed value of 0.3 g H2O/g protein for the hydration. The distances between the centres of gravity of pairs of species strongly suggest that the components are in the closest distance configuration or close to it. However, the binary complex I-III appears to be more open than the complex I-II. Finally, a model of the interaction between carboxpeptidase A and its activation peptide has been constructed by comparing the hypothetical geometric model of subunit I to the crystallographically determined structure of carboxypeptidase A.

Animals↗

PubMed1988

The bovine pro-carboxypeptidase A-S6 ternary complex: a rare case of a secreted protein complex.

Up to now, a non-covalent ternary complex in which the pro-carboxypeptidase A (subunit I) is associated to two functionally different proteins (subunits II and III) has only been found in the pancreas of ruminant species. In the other species studied so far, the pro-carboxypeptidase A is secreted either as a monomer or as a binary association with a functionally different protein. Subunit I is the immediate precursor of carboxypeptidase A. Subunit II is a chymotrypsinogen of the C-type, involved, like subunit I, in the degradation of proteins and peptides. Although closely related to the pancreatic serine endopeptidases, subunit III appears to be devoid of any specific enzymatic activity. Information about the spatial organization of the subunits in the ternary complex has been deduced from the sequential dissociation of the complex. In contrast to the mechanism of activation of subunits I and II, which is independent of their aggregation state, the catalytic properties of the resulting enzymes are sensitive to their aggregation state. Moreover, the structural basis of inactivity of subunit III as well as the physiological role of the ternary complex are also discussed in this review.

Amino Acid Sequence↗

PubMed1988

Minireview on pancreatic lipase and colipase.

By hydrolyzing the dietary triacylglycerols, pancreatic lipase causes catalysis in heterogeneous medium. In vivo, lipase action cannot take place without colipase due to the presence of bile salts. The cofactor enables lipase anchoring to the water-lipid interface. The lipase-colipase system furnishes an excellent example of specific interactions (protein-protein and protein-lipid). The studies of lipase catalytic properties brought to light the importance of certain parameters related to the 'quality of the interface'. The structure-function relationship analyses revealed a certain number of functional amino acid residues in lipase and colipase involved either in the catalytic site of the enzyme or in the recognition sites (lipase-colipase and protein-interface). Comparisons of the sequences of lipases derived from different sources display interesting similarities in certain cases.

Amino Acid Sequence↗

PubMed1987

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

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. alpha-Carboxypeptidase A was mainly formed under the relatively mild conditions used, indicating that the Arg-1-Ala+1 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.

Amino Acid Sequence↗

PubMed1986

Crystallization and preliminary X-ray study of subunit III of the bovine pancreatic procarboxypeptidase A-S6 ternary complex.

Subunit III of the bovine pancreatic procarboxypeptidase A-S6 ternary complex was dissociated from the complex, purified and crystallized using the hanging- or sitting-drop method of vapour diffusion, with ammonium sulphate as the precipitant. The assays were carried out at pH 4.2 (20 mM-acetate buffer). An X-ray examination of the crystals shows that they are monoclinic, with a space group P21 and cell dimensions a = 47.9 A, b = 61.3 A, c = 39.0 A and beta = 95.0 degrees. The asymmetric unit contains one molecule of 25,800 Mr. The crystals are suitable for structure determination to at least 2.8 A resolution.

Animals↗

PubMed1986

The inactive subunit of ruminant procarboxypeptidase A-S6 complexes. Structural basis of inactivity and physiological role.

Subunit III has so far been found only in the pancreas of ruminants in a non-covalent association (procarboxypeptidase A-S6) with two different proteins: the procarboxypeptidase A itself (subunit I) and a C-type chymotrypsinogen (subunit II). In contrast with these latter two proteins, which are zymogens of pancreatic proteases, subunit III seems to be devoid of any activity towards specific substrates of pancreatic proteases. However, it possesses a weakly functional active site which allows it to hydrolyze a non-specific ester, p-nitrophenyl acetate, and to react with several active-site titrants. The binding of proflavin to subunit III shows that this protein owns a non-polar binding site with a very high Kd compared to that of chymotrypsin. The comparison of the amino acid sequences of subunit III and some serine proteases showed that subunit III is closely related to an elastase. Models of the tertiary structure of subunit III suggest a conformational modification that affects the substrate binding and could explain the lack of specific enzymatic activity. The presence of subunit III in the ternary complex is not related to an enzymatic function. This protein does not participate in the activation process of subunit I but prevents the denaturation of this subunit at low pH. This may represent its biological role in the acidic environment of the duodenum in ruminants.

Amino Acid Sequence↗

PubMed1985

Existence of ternary complexes of procarboxypeptidase A in the pancreas of some ruminant species.

The existence of procarboxypeptidase A, in the form of a non-covalent ternary complex containing the apparently inactive serine protease (subunit III), has so far been observed only in the ox pancreas. Evidence, obtained in the present study, shows that a ternary complex of procarboxypeptidase A, with a subunit III highly homologous with that of the bovine complex, is also present in two other ruminant species, sheep and goat. The biological significance of these complex forms of procarboxypeptidase A and the consistently high biosynthesis level of the apparently inactive subunit III in all three ruminant species is still unknown. Yet the synthesis of subunit III is not related to the animal diet since in the horse, which is a non-ruminant herbivorous animal, the procarboxypeptidase A is monomeric. Reassociation assays between either bovine subunits II or III and monomeric as well as binary forms of procarboxypeptidase A from various species show that, unlike subunit II, the recognition site for subunit III is highly conserved in all the procarboxypeptidases A and that bovine subunit II is different from porcine chymotrypsinogen C with regard to association.

Amino Acid Sequence↗

PubMed1984

Two-step dissociation of bovine 6S procarboxypeptidase A by dimethylmaleylation.

Reversible condensation of the ternary complex form of bovine pancreatic procarboxypeptidase A with 2,3-dimethyl maleic anhydride was investigated at pH 9.0 and low concentration of reagent over the acylable amino groups. After subsequent modification of only a few lysyl residues, subunit III was found to have been released from the quaternary structure leading to the separation of an apparently native protein devoid of any contaminating subunit II, while dissociation of the remaining binary complex occurred upon further addition of the anhydride. This observation suggests that the electrostatic interactions existing between subunits I and III are more rapidly weakened than those between subunits I and II, probably because fewer lysyl residues are involved and/or there is greater accessibility to the chemical reagent . Although completely inactive on the specific substrates of trypsin, chymotrypsin and elastase, subunit III hydrolyzed p-nitrophenyl acetate at a rate similar to that of chymotrypsin but without any burst of p-nitrophenol, which indicates that the weakly functional active site of the subunit is not quite comparable to that of serine protease zymogens. Subunit III already has some of the functional characteristics of the corresponding active enzymes.

Animals↗