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P Fromageot

Publications and source records attributed to P Fromageot.

At least 37 records · Page 2Linked to original sources
PubMed1984

On the molecular mechanisms of neutralization of a cobra neurotoxin by specific antibodies.

Examination of 76 homologous neurotoxin sequences suggested that the "toxic" domain of these compounds consists of twelve highly conserved residues. Five of these, namely Lys-27, Trp-29, Asp-31, Arg-33 and Glu-38, together with a variant residue at position 36 are organized into a pattern which resembles that of d-tubocurarine. Two lines of experimental evidence are in agreement with the proposed topology of the "toxic" site in Naja nigricollis toxin alpha--Three highly conserved residues (Lys-27, Trp-29 and Lys-47) have been modified individually in toxin alpha. These modifications induce a decrease in binding affinity of toxin alpha for its target, the nicotinic acetylcholine receptor. In contrast, modifications of three residues (Leu-1, Lys-15 and Lys-51) excluded from the "toxic" domain, do not alter the binding properties of toxin alpha.--Five toxin derivatives carrying a nitroxide group at residues 1, 15, 27, 47 or 51 have been prepared. ESR spectra have been recorded for each derivative in both the free state and bound to the receptor. Mobility of the probes of the residues excluded from the "toxic" site is not altered upon receptor binding. In contrast mobility of the nitroxide of the presumed "toxic" Lys-47 becomes markedly reduced after toxin receptor complex formation. Lys-27 nitroxide is immobilized in both the free and bound state. The antigenic structure of N. nigricollis toxin alpha has been partially clarified using two different approaches. --Fifteen antigenically important residues of toxin alpha have been identified by analyzing cross-reactions between toxin alpha and eleven homologous neurotoxins, using polyclonal antibodies.--- One monoclonal antibody (M alpha 1) specific for toxin alpha has been prepared. Competition experiments, made with (3H) toxin alpha, six mono modified toxin derivatives or alpha three homologous neurotoxins, showed that the binding site of (M alpha 1) comprises the N-terminal group, Lys-15, Pro-18 and probably Thr-16. This site is topographically different from the "toxic" domain. (M alpha 1) inhibits the toxicity of toxin alpha under both in vivo and in vitro conditions. In addition, (M alpha 1) is capable of "removing" toxin molecules bound to the receptor, allowing a rapid recovery of the functional properties of the receptor.

Amino Acid Sequence↗
PubMed1983

Analysis of yeast RNA polymerases with subunit-specific antibodies.

Specific antibodies directed against each polypeptide component of yeast RNA polymerases A or B were prepared and their affinity spectrum determined by protein blot immunodetection. The majority of enzyme A or B subunits were specifically recognized by their respective antiserum. A direct correspondence was established between the polypeptides immunologically related in the three forms of RNA polymerases A, B, and C by reacting the different antibodies with enzymes subunits transferred to a nitrocellulose membrane. Subunit-specific antibodies and antibodies to native enzymes A and B were used to probe the activity of RNA polymerases A, B, and C. Based on DNA protection experiments, the largest subunit of enzymes A and B as well as the common subunit ABC23 appear to be involved in DNA binding.

Antibodies↗
PubMed1983

On the phosphorylation of yeast RNA polymerases A and B.

In exponentially growing cells, RNA polymerase B is exclusively form BI enzyme with several phosphorylated subunits: B220, B23 and possibly B44.5. In RNA polymerase A an average of fifteen phosphate groups are distributed on the five phosphorylated subunits: A190 (6), A43 (4), A34.5 (2), A23 (1-2) and A19 (1-2). Phosphorylation of enzyme A by a yeast protein kinase in vitro adds less than 1 mol phosphate/mol enzyme but occurs essentially at the physiological sites, as shown by a comparison of the peptide patterns obtained by limited proteolysis of subunits 32P-labelled in vivo and in vitro. No evidence was found in favor of a modulation of RNA polymerase activity in vitro or in vivo via phosphorylation.

Chemical Phenomena↗
PubMed1982

Activation of a latent RNAase from yeast by nucleoside triphosphates.

A latent RNAase activity stimulated by nucleoside triphosphates has been isolated from a yeast chromatin extract, by filtration on Sepharose 6B and hydroxyapatite chromatography. The RNAase was separated from a thermolabile proteic inhibitor on phosphocellulose. When separated from the inhibitor, the RNAase hydrolyses RNA to 5'-mononucleotides. Its activity is retained in the presence of EDTA, and 50% inhibited by 1 mM ATP or CTP. The RNAase is inhibited by the thermolabile component only in the presence of divalent cations. The activity is recovered upon addition of 0.01 mM ATP to the mixture. The Km for ATP is 10 microM. ATP can be replaced by other ribo- or deoxyribonucleoside triphosphates with varying efficiency but not by ADP, AMP or cAMP. These results suggest multiple interactions between the RNAase, a regulatory component, divalent cations and nucleoside triphosphates.

Adenosine Triphosphate↗
PubMed1982

Neutralizing monoclonal antibody specific for Naja nigricollis toxin alpha: preparation, characterization, and localization of the antigenic binding site.

One homogeneous population of high-affinity monoclonal antibodies (KD = 0.35 nM) specific for Naja nigricollis toxin alpha has been produced. It neutralizes the biological activity of the toxin under both the vivo and in vitro conditions. The molecular zone of the toxin to which the antibody binds has been precisely defined on the basis of cross-reaction experiments using five derivative of toxin alpha monomodified at a single amino group and two naturally occurring homologous toxins. The epitope is located at the base of the first beta-sheet loop of the toxin, involving the two positive charges at the N-terminal position and lysine-15 proline-18, and probably threonine-16. It is shown that this region is topographically distinct from the "toxic" site of toxin alpha. Several possibilities are offered to explain the mechanisms(s) of specific neutralization.

Animals↗
PubMed1982

Natural variation in yeast RNA polymerase A. Formation of a mosaic RNA polymerase A in a meiotic segregant from an interspecific hybrid.

There is a natural variation in the molecular structure of RNA polymerase A isolated from several genetically distant yeast species, Saccharomyces cerevisiae, Saccharomyces douglasii, Schizosaccharomyces pombe, and Candida tropicalis. Several biochemical criteria were used to identify their homologous polypeptide components. Based on these correlations, the minimal subunit composition of S. cerevisiae (and Saccharomyces carlsbergensis) RNA polymerase A was tentatively defined as A190, A135, A40, A27, A23, A19, and A14.5. Without the two Saccharomyces species, S. cerevisiae and S. douglasii, 7 of 13 polypeptides of enzyme A(A49, A43, A40, A34.5, A19, A14.5, and A14) differ slightly in molecular weight and can be resolved by electrophoresis on polyacrylamide gel. The RNA polymerase A isolated from the diploid interspecific hybrid contains all the polypeptides characteristic of the two parents. One meiotic segregant had a hybrid RNA polymerase A with five of the polymorphic polypeptides (A49, A43, A19, A14.5, and A14) coming from S. douglasii and two (A40 and A34.5) from S. cerevisiae. In three successive backcrosses with S. cerevisiae, all the genes for S. douglasii polypeptides were shown to recombine although parental ditype tetrads predominated in the four four-spored asci examined. Thus, the genes for the seven polymorphic polypeptides are not clustered: they lie on at least three different chromosomes.

Ascomycota↗
PubMed1982

Separation of intermediates in the refolding of reduced erabutoxin b by analytical isoelectric focusing in layers of polyacrylamide gel.

Isoelectric focusing in a thin layer of polyacrylamide gel is shown to be a suitable method for the resolution of intermediates trapped during the refolding process of reduced cystine-containing proteins. The method has been applied to the well-characterized snake neurotoxin erabutoxin b. An explanation is offered for the relatively low rate of refolding of this polypeptide.

Animals↗
PubMed1982

Probing yeast RNA polymerase A subunits with monospecific antibodies.

Monoclonal antibodies were raised in mouse against native RNA polymerase A from Saccharomyces cerevisiae. After screening with the spot-immunodetection technique, 14 hybridomas were selected and the antibodies produced in mice. Their specificity, analyzed by blot-immunodetection, was found to be markedly biased towards a few RNA polymerase subunits: A135 , A49 , A43 , and A14.5. A different monoclonal antibody directed against the largest subunit, A190 , was obtained by immunizing a mouse with RNA polymerase A dissociated into its subunits with SDS. Two antibodies, which probably recognized the same antigenic determinant on subunit A135 , inhibited in vitro RNA synthesis. Inhibition was prevented by preincubation of the enzyme with DNA, suggesting a role for the A135 subunit in template binding. The antibody directed against A14.5 interacted with the A14.5 kd subunit present in all three forms of the yeast nuclear RNA polymerases but did not interfere with RNA polymerase activity. These antibody probes will be useful to study subunit function in reconstituted transcription systems.

Animals↗
PubMed1981

Single-stranded DNA transcription by yeast RNA polymerase B.

Single-stranded DNA is not transcribed randomly by yeast RNA polymerase B. A denatured yeast DNA fragment, containing the gene for yeast alcohol dehydrogenase I, directs the transcription of defined RNA products visualized as discrete RNA . DNA hybrid bands following S1 nuclease treatment and agarose gel electrophoresis. Blocking the 3' end of the template by 3' deoxyadenosine did not change the band pattern but reduced the proportion of RNA covalently bound to the DNA from 20 to 4%. On the other hand, the band pattern was affected by the salt concentration, the nature of the divalent cation and the nucleoside triphosphate concentration. The four major RNA bands, found at low substrate concentration, hybridized to the same region of the template. This observation suggests the potential requirement for DNA destabilization in gene activation.

Alcohol Dehydrogenase↗
PubMed1981

Native deoxyribonucleic acid transcription by yeast RNA polymerase--P37 complex.

The specific activity of yeast RNA polymerases A or B, when complexed with P37 cofactor, compares favorably with that of E. coli RNA polymerase. The stimulation is observed only with double-stranded DNA but does not result from DNase action. The Km for nucleotide substrates and the optimal conditions of transcription are not modified. P37 stimulates RNA synthesis by ternary transcription complexes in the presence of poly(rI) which prevents reinitiations. The RNA chain length, estimated by 5' end labeling or sedimentation, is increased in the presence of P37. On the other hand, the trinucleotide synthesis, which reflects the chain initiation reaction, is not affected. Therefore, the cofactor appears to act at the elongation step of RNA synthesis.

DNA-Binding Proteins↗
PubMed1981

[Binding of a tritiated enkephalinergic analog (FK 33-824) to a mitochondrial fraction of rat brain].

FK-33-824 (Try-D-Ala-Gly-MePhe-Met(O)ol) is a potent enkephalin analog which has been tritium labelled with a high specific radioactivity (41 Ci/mmole). The labelled drug exhibits specific and saturable binding to rat brain crude mitochondrial fraction. Specific binding is inhibited by low concentrations of morphine, levallorphan and beta-endorphin, suggesting that FK 33-824 [3H] binds preferentially to mu opiate sites. Binding studies at equilibrium and kinetics of formation and dissociation of the labelled ligand-receptor complex indicate that FK 33-824 [3H] binds to two classes of specific sites. Their affinities are distinguishable at 0 degree (KD = 1.3 and 5.8 nM) and very close to each other at 37 degree (KD = 1.9 nM).

Animals↗
PubMed1981

Conformational calculations on gastrin C-terminal tetrapeptide.

Energy optimizations were performed on some typical conformations of the gastrin C-terminal peptide amide NAc-Trp-Met-Asp-Phe-NH2. Two families of lowest energy conformations were found corresponding to: (a) alpha-helical structures; (b) conformations having beta-structure at the level of Trp residue, and C7-structure at the level of Asp residue. The two aromatic rings were folded on the peptide backbone and ca. 5 A distant from each other (centre to centre). The last family, favoured by energy and population probability, can better account for conformational experimental results and biological activity observations.

Amino Acid Sequence↗
PubMed1980

RNA-dependent ATPase from Saccharomyces cerevisiae.

A new RNA-dependent ATPase has been isolated from yeast chromatin extracts and partially characterized. The protein has a sedimentation coefficient of about 7 S. The enzyme hydrolyzes specifically ATP (or dATP) to ADP (or dADP) and Pi in the presence of Mg2+ or Mn2+ ions and requires a single-stranded polynucleotide as cofactor. The order of efficiency of synthetic polymers is poly(rU) > poly(rI) greater than or equal to poly(dU) > poly(rA) greater than or equal to poly(rC). Among natural polymers, single-stranded DNA and poly(rA)-containing mRNA from yeast are also active but less so than poly(rU). The enzyme exhibits a pH optimum of 8 and is fully inhibited by 0.25 M NaCl. The Km for ATP is0.2 mM. The resemblance between this ATPase and DNA-dependent ATPases from other sources, as well as the termination factor rho, is discussed.

Adenosine Triphosphatases↗