Biomedical subjects

J M Yon

Publications and source records attributed to J M Yon.

At least 19 recordsLinked to original sources

PubMed1992

First determination of the secondary structure of purified factor VIII light chain.

The first analysis of the secondary structure of human factor VIII light chain was performed by c.d. spectroscopy. The purification process described in this paper allowed us to obtain the large amounts of purified factor VIII light chains required for c.d. experiments. Since this 80 kDa protein is non-covalently associated with a heavy chain to form the active molecule, isolated factor VIII light chains were obtained after immunoadsorption and dissociation of the immobilized active complexes by EDTA. Furthermore, factor VIII light chains were discriminated from the residual active complexes and the free heavy chains by a final ion-exchange-chromatography step. This f.p.l.c. analysis showed that factor VIII light chains were less electronegative than the active complexes. The results of conformational analysis by c.d. show that the protein possesses a high degree of regular secondary structure (58%) with approx. 22% of alpha-helix and 36% of beta-strand structures. The protein was completely unfolded by 3 M-guanidine hydrochloride. The results obtained from the analysis of c.d. spectra were compared with those predicted from three different statistical methods based on amino-acid sequence. The secondary structure information obtained from these methods was in good agreement with the c.d. results. These results were comparable with the secondary structure prediction of ceruloplasmin, a protein known to show sequence identity to factor VIII.

Chromatography, High Pressure Liquid↗

PubMed1992

Kinetic studies of the refolding of yeast phosphoglycerate kinase: comparison with the isolated engineered domains.

Unfolding and refolding kinetics of yeast phosphoglycerate kinase were studied by following the time-dependent changes of two signals: the ellipticity at 218 nm and 222 nm, and the fluorescence emission at 330 nm (following excitation at 295 nm). The protein is composed of two similar-sized structural domains. Each domain has been produced by recombinant DNA techniques. It has been previously demonstrated that the engineered isolated domains are able to fold into a quasinative structure (Minard, P., et al., 1989b, Protein Eng. 3, 55-60; Missiakas, D., Betton, J.M., Minard, P., & Yon, J.M., 1990, Biochemistry 29, 8683-8689). The behavior of the isolated domains was studied using the same two conformational probes as for the whole enzyme. We found that the refolding kinetics of each domain are multiphasic. In the whole protein, domain folding and pairing appeared to be simultaneous events. However, it was found that some refolding steps occurring during the refolding of the isolated C-domain are masked during the refolding of yeast phosphoglycerate kinase. The N-domain was also found to refold faster when it was isolated than when integrated.

Guanidine↗

PubMed1992

The slow-refolding step of phosphoglycerate kinase as monitored by pulse proteolysis.

The kinetics of refolding of yeast phosphoglycerate kinase were studied by following the variation in circular dichroism at 218 nm, the recovery of enzyme activity, and the susceptibility to proteolysis by trypsin and V8-protease. A very rapid phase followed by a slower one was detected by circular dichroism, which revealed the formation of secondary structures. The slower phase, with a macroscopic rate constant of 0.35 min-1, was also detected by the susceptibility of the enzyme to both proteases. It was shown that cleavage sites located in the hinge region, in a part of the C-domain and, to a lesser extent, in a region of the N-domain, which are accessible in the intermediate state, became inaccessible during the slow-refolding step of the molecule. These results demonstrate, on the one hand, the role of domains as folding intermediates, and, on the other hand, the locking of the domain structure and the domain pairing that occurs during the slow-refolding step with a rate constant of 0.35 min-1. The return of the enzyme activity occurred in a slower last step upon conformational readjustments induced by domain interactions.

Circular Dichroism↗

PubMed1992

Comparison of muscle phosphofructokinase from euthermic and hibernating Jaculus orientalis. Purification and determination of the quaternary structure.

1. The structural properties of skeletal muscle phosphofructokinase from euthermic and hibernating jerboa were compared. 2. The enzyme was purified by a rapid procedure; suspended in ammonium sulfate in the presence of ATP, it was found to be stable for three weeks. 3. A specific activity of 76 U/mg and at most 65 U/mg was obtained for the enzyme from the euthermic and hibernating jerboa, respectively. 4. The molecular weight was estimated to be 320 kDa for the oligomer and 80 kDa for the subunit. 5. A unique alanine residue was found at the C-terminal end, suggesting that the enzyme is a tetramer made of four identical subunits. 6. The tetrameric structure of phosphofructokinase was confirmed by using crosslinking with disuccinimidyl esters. 7. The kinetics of formation of the different crosslinked species were found to be in agreement with a model of the tetramer corresponding to a dihedral symmetry with isologuous contacts between protomers. 8. The same molecular characteristics and immunochemical properties were found for the enzyme extracted from the euthermic and hibernating animals.

Animals↗

PubMed1991

Conformational changes in yeast phosphoglycerate kinase upon ligand binding: fluorescence of a linked probe and chemical reactivity of genetically introduced cysteinyl residues.

The effects of ligands on the conformation of yeast phosphoglycerate kinase were explored by introducing cysteinyl residues at different positions in the molecule by site-directed mutagenesis. Thus several mutants were constructed, each containing a unique cysteinyl residue. Neither the conformation nor the enzyme activity was affected by the substitutions. The reactivity of the thiol groups and the fluorescence of N-acetyl-N'-(5-sulfo-1-naphthyl)ethylene-diamine covalently linked to these thiols were used to monitor the conformational changes induced upon ligand binding. It was found that the observed changes mainly involve the part of the protein located in the cleft, particularly the environment of residues 35 and 183. No alteration was observed on the external side of the protein. Only 3-Phosphoglycerate induced these conformational changes. However, when the fluorescent probe was attached to residue 377, the binding of the two substrates was required to induce a modification in the fluorescence of the probe. These results indicate that the substrates separately or together induce discrete molecular motions in phosphoglycerate kinase.

Adenosine Triphosphate↗

PubMed1990

Unfolding-refolding of the domains in yeast phosphoglycerate kinase: comparison with the isolated engineered domains.

The role of domains as folding units was investigated with a two-domain protein, yeast phosphoglycerate kinase. Each of the domains was produced independently by site-directed mutagenesis. It has been previously demonstrated by several criteria that these domains are able to fold in vivo into a quasi-native structure [Minard et al. (1989a) Protein Eng. 3, 55-60; Fairbrother et al. (1989) Protein Eng. 3, 5-11]. In the present study, the reversibility of the unfolding-refolding process induced by guanidine hydrochloride was investigated for the intact protein and the isolated domains. The transitions were followed by circular dichroism for both domains and the intact protein and by the variations in enzyme activity for the intact protein. Tryptophan residues were used as intrinsic conformational probes of the C-domain. An extrinsic fluorescent probe, N-[[(iodoacetyl)amino]ethyl]-8-naphthylamine-1-sulfonic acid (IAEDANS), was bound to the unique cysteinyl residue Cys97 to observe the conformational events in the N-domain. The unfolding-refolding transitions of each domain in the intact protein and in the isolated domains prepared by site-directed mutagenesis were compared. It was shown that the two domains are able to refold in a fully reversible process. A hyperfluorescent intermediate was detected during the folding of both the isolated C-domain and the intact yeast phosphoglycerate kinase. The stability of each isolated domain was found to be similar, the free energy of unfolding being approximately half that of the intact molecule.

Fungal Proteins↗

PubMed1990

The effects of ligands on the conformation of phosphoglycerate kinase: fluorescence anisotropy decay and theoretical interpretation.

Horse muscle phosphoglycerate kinase (PGK) is a monomer folded into two widely distant domains. In the glycolytic pathway, this enzyme catalyzes the first reaction that produces ATP. It was suggested, by analogy with yeast hexokinase, that a hinge-bending motion may be induced by the binding of specific substrates to the protein. To analyze such a motion, or any structural changes induced by ligand binding, fluorescence anisotropy decay of tryptophan residues in free and liganded PGK was studied. At 293 K, for the free protein and the binary complex with 3-phosphoglycerate, a single correlation time of 26 ns was observed, corresponding to the rotation of the overall protein, whereas upon addition of MgADP, this correlation time decreased to 10 ns. Such a decrease cannot be merely due to a change of the protein's shape and volume. To explain this, it was suggested that the fluorescence anisotropy decay of the PGK-MgADP complex corresponded to the rotation of the only buried tryptophan (Trp 335). The rotational paths of this tryptophan, in the presence and absence of the nucleotide, were established by potential energy minimization calculations. The results indicated that MgADP induces a displacement of helix alpha-13 that decreases the rotational energy barrier of Trp 335 from 16 kcal/mol in the free protein to 8 kcal/mol in the complex.

Adenosine Diphosphate↗

PubMed1990

Regulation of the skeletal muscle metabolism during hibernation of Jaculus orientalis.

1. The glycolytic flow in the skeletal muscle was considerably depressed during hibernation of Jaculus orientalis. 2. Although the ATP content was not modified, the ATP/AMP ratio was twice as large than under homeothermic conditions. 3. Furthermore, the hexose phosphates were markedly depressed. 4. The total activities of the key enzymes, hexokinase, phosphofructokinase and pyruvate kinase, which are regulated through the adenylates, decreased. 5. Under in vivo conditions, taking into account the small amount of fructose-6-phosphate and the ATP/AMP ratio, it is likely that phosphofructokinase was totally inhibited, explaining the undetectable amount of fructose 1.6 bisphosphate.

Adenosine Monophosphate↗

PubMed1990

Introduction of internal cysteines as conformational probes in yeast phosphoglycerate kinase.

Several mutants of yeast phosphoglycerate kinase, each containing only one internal cysteine residue, were constructed from a single mutant devoid of cysteine. These cysteines were introduced as local conformational probes in selected buried positions. The enzyme activity, conformational characteristics and stability indicated that the mutations introduced only small perturbations in the molecule. The folding-unfolding process mediated by guanidine hydrochloride under equilibrium conditions was studied by following the variations in ellipticity and the reactivity of the cysteine residue towards 5,5'-dithiobis(nitrobenzoate). The process was found to be reversible except for mutant C97A, V49C, suggesting that this region located in helix I might be crucial in determining an intermediate on the folding pathway. The transitions obtained by the two signals did not coincide, indicating that the local structures, in several parts inside the molecule, are more sensitive to the denaturant than the overall conformation.

Catalysis↗

PubMed1989

Study of the fast-reacting cysteines in phosphoglycerate kinase using chemical modification and site-directed mutagenesis.

Horse muscle phosphoglycerate kinase, like other mammalian phosphoglycerate kinases, contains seven cysteine residues of which two react rapidly with 5,5'-dithio-bis(2-nitrobenzoate) (Nbs2) following second-order kinetics (k = 640 M-1.s-1). Selective cyanylation of the fast-reacting cysteines, followed by chemical cleavage and subsequent sodium dodecyl sulfate/polyacrylamide gel electrophoresis analysis of the resulting polypeptides, suggested that these cysteines are at positions 378 and 379. Cysteine residues were introduced into yeast phosphoglycerate kinase by site-directed mutagenesis. Mutant enzymes, each containing only one cysteine residue at position 364, 376, or 377, were constructed from a mutant devoid of cysteine (Cys97----Ala). In the last two mutants, the cysteines were at positions corresponding to Cys378 and Cys379, respectively, in the horse muscle enzyme. The chemical reactivity of the cysteine groups in these latter two yeast mutant enzymes was similar to that of the fast-reacting cysteines in the horse muscle enzyme. Furthermore, they were similarly modified upon substrate binding. All these data demonstrate unambiguously that the fast-reacting cysteines in the horse muscle enzyme are Cys378 and Cys379.

Animals↗

PubMed1989

Isolation and characterization of different activated forms of factor VIII, the human antihemophilic A factor.

Factor VIII was purified 1200-fold from commercial concentrates (Centre National de Transfusion Sanguine) by immunoaffinity chromatography using an anti-(80-kDa light chain) monoclonal antibody. The different molecular forms isolated were subsequently separated and analyzed using Fast Protein Liquid Chromatography and sodium dodecyl sulphate polyacrylamide gel electrophoresis analysis. The different factor-VIII forms obtained, consisted of 80-kDa light chains, each being associated with one more-or-less fragmented heavy chain ranging over 90-210 kDa. The specific activity of these different forms was 7000 U/mg. At different stages of activation of factor VIII by thrombin, various forms were separated and identified. Activated complexes were found to result from the association of the 70-kDa light chain (generated from the 80-kDa light chain) with heavy chains ranging over 90-210 kDa. Two different thrombin activation steps were characterized. The first step corresponding to the cleavage of the 80-kDa light chain led to a sixfold increase in the procoagulant activity, and yielded a stable activated intermediate form. Compared with normal factor VIII, the ratio of von Willebrand activity to factor-VIII activity, measured in the activated fractions, decreased, indicating that von Willebrand factor dissociates from factor VIII after proteolysis of the light chain by thrombin. In the second step, the 90-kDa heavy chain was cleaved into two polypeptides of 45 kDa and 50 kDa, which were associated with the 70-kDa proteolyzed light chain, generating the final activated complex (45-50-70 kDa). The new intermediate forms we described imply a new scheme for the multistep activation process of factor VIII.

Adsorption↗

PubMed1989

Detection of intermediates in the unfolding transition of phosphoglycerate kinase using limited proteolysis.

The accessibility of peptide bonds to cleavage by Staphylococcus aureus V8 protease bound on a Sepharose matrix was used as a conformational probe in the study of the unfolding-folding transition of phosphoglycerate kinase induced by guanidine hydrochloride. It was shown that the protein is resistant to proteolysis below a denaturant concentration of 0.4 M. The transition curve, determined by susceptibility toward proteolysis, was similar to that obtained following the enzyme activity [Betton et al. (1984) Biochemistry 23, 6654-6661]. Proteolysis under conditions where the folding intermediates are more populated, i.e., 0.7 M Gdn.HCl, gave two major fragments of Mr 25K and 11K, respectively. The 25K polypeptide fragment was identified as the carboxy-terminal domain. Its conformation was similar to that of a folding intermediate trapped at a critical concentration of denaturant, and in this form, it was not able to bind nucleotide substrates [Mitraki et al. (1987) Eur. J. Biochem. 163, 29-34]. From the present data and those previously reported, we concluded that the intermediate detected on the folding pathway of phosphoglycerate kinase has a partially folded carboxy-terminal domain and an unfolded amino-terminal domain.

Amino Acids↗

PubMed1989

Conformational modification of muscle phosphofructokinase from Jaculus orientalis upon ligand binding.

Phosphofructokinase from Jaculus orientalis muscle is an allosteric enzyme regulated by substrates and nucleotide effectors. The conformational modifications upon ligand binding were probed by UV difference spectra and reactivities of thiol groups towards dithiobisnitrobenzoate and N-ethylmaleimide. The binding of Fru-6-P induced significant perturbations in the environment of the aromatic residues and buried the most reactive on the three accessible cysteines per protomer. The same effect on thiol reactivity was observed upon binding of the activator AMP. Various perturbations of both difference spectra and thiol reactivity were detected in the presence of either Mg-ATP, an allosteric inhibitor, or Mg-ITP which is not an effector.

Adenosine Monophosphate↗

PubMed1989

Nuclear magnetic resonance studies of isolated structural domains of yeast phosphoglycerate kinase.

The structural integrity and substrate binding properties of the two genetically engineered domains of yeast phosphoglycerate kinase were investigated using one- and two-dimensional nuclear magnetic resonance techniques. Both domains were found to fold with regions of native-like structure, with the N-domain showing greater conformational flexibility than the C-domain. The 'basic patch' region of the N-domain is, however, clearly perturbed by removal of the C-domain. This is most likely due to the absence of stabilizing interactions between the C-terminal peptide (including alpha-helices XIII and XIV) and the N-domain. The C-domain is able to bind nucleotide with an affinity only three times less than that of the native protein.

Hydrogen-Ion Concentration↗

PubMed1989

Efficient expression and characterization of isolated structural domains of yeast phosphoglycerate kinase generated by site-directed mutagenesis.

The two domains of yeast phosphoglycerate kinase were produced by recombinant techniques. The N-domain was obtained by the introduction of a termination codon at the position coding for Phe185, and the C-domain by a deletion in the gene of the coding sequence between Ser1 and Leu186. Both domains were efficiently expressed in yeast, the level for the C-domain being greater than that for the N-domain. Both domains were found to have a quasi-native structure; the C-domain retained its ability to bind nucleotides. Small local differences were detected in domain structure compared to that in the whole enzyme, probably due to the lack of interdomain stabilizing interactions. Nevertheless, such an approach provides direct evidence for independent folding of domains in a two-domain protein.

Cloning, Molecular↗

PubMed1988

Survey of the folding pathway of a two-domain protein phosphoglycerate kinase.

The role of structural domains as folding units in the folding process which generates an active enzyme, is considered through several studies on phosphoglycerate kinase, a two-domain enzyme which catalyzes the first step of ATP production in glycolysis. The folding pathway was found to be a complex multi-step process, the C-terminal domain being more stable folding first. Inactive species originating from an intermediate in the folding pathway have been identified. Isolated domains recently obtained using genetic engineering are under investigation in our laboratory; this might probably allow to understand the way by which the N-terminal domain reaches its final native conformation and interacts with the other domain.

Animals↗

PubMed1988

The effect of phosphate on the unfolding-refolding of phosphoglycerate kinase induced by guanidine hydrochloride.

Phosphate ions were found to stabilize the native structure of phosphoglycerate kinase without modifying the folding pathway. The transition curves obtained from different signals: enzyme activity, ellipticity at 220 nm and fluorescence intensity at 336 nm (excitation at 292 nm) are shifted to smaller guanidine hydrochloride cm values in the absence of phosphate. The kinetic characteristics are qualitatively similar, unfolding rate constants being slightly smaller in the presence of phosphate. The mechanism by which the native structure of phosphoglycerate kinase is stabilized by phosphate probably occurs upon specific phosphate binding to the nucleotide beta- or gamma-phosphate binding site of nucleotides.

Animals↗

PubMed1979

[Conformational coupling between structural units. A decisive step in the functional structure formation].

Proteins built up of several structural domains are compared with oligomeric proteins made up of several subunits. On this basis the functional consequences of a conformational coupling between domains are analyzed. From the data obtained on the elastase molecule it is proposed that the last step in protein folding insures the optimal coupling between domains and the energy of interaction is utilized for catalysis.

Catalysis↗