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P Fromageot

Publications and source records attributed to P Fromageot.

At least 55 records · Page 3Linked to original sources
PubMed1980

Conformation of two homologous neurotoxins. Fluorescence and circular dichroism studies.

Two homologous short neurotoxins isolated from snake venoms (Laticauda semifasciata erabutoxin b and Naja nigricollis toxin alpha) have been studied by means of fluorescence spectroscopy and in aqueous solution at various pH values. In parallel experiments, the stability of toxin conformations was analyzed on the basis of ultraviolet circular dichroism. Total luminescence spectra (77 K) were recorded for both toxins in neutral and alkaline solutions. The data obtained indicate that, at neutral pH, the fluorescence emission is only due to the single and invariant tryptophan (29). From a comparative study with erabutoxin a, which differs from erabutoxin b by a single residue, it is unambiguously shown that the protonation of His-26 of erabutoxin b is responsible for a decrease of Trp-29 fluorescence. Also, on the basis of available X-ray data it is proposed that the protonation or deprotonation of the following titrable groups is responsible for an alteration of Trp-29 fluorescence. These are Asp-31 (pK congruent to 4) and Lys-27 (pK = 9.6) for both toxins and Lys-26 (pK congruent to 9.6) for toxin alpha. No tyrosinate emission can be observed at neutral pH and 77 K. Excitation spectra of toxin alpha revealed that 50% of the light absorbed by Tyr-25 in water is transferred to Trp-29. From the energy transfer measurements, the distance separating these two aromatic chromophores in the native toxin was estimated to be 13 A. A similar experiment was made for toxin alpha dissolved in trifluorethanol. The data indicate that the distance separating the two aromatic side chains does not depend greatly on the nature of the solvent.

Animals↗
PubMed1980

Immunological studies of yeast nuclear RNA polymerases at the subunit level.

Antisera were raised against native RNA polymerases A or B, as well as against each individual subunit of RNA polymerase A from the yeast Saccharmoyces cerevisiae. The affinity spectrum of antibodies was evaluated by reacting electrophoretically separated enzyme subunits, transferred to a membrane, with 125I-labeled immunoglobulins. Alternatively, the subunit . immunoglobulin complex was revealed by 125I-labeled Protein A. Antibodies directed against native RNA polymerase A recognized the majority of the polypeptides forming the enzyme. When challenged with RNA polymerases B or C, this antibody preparation demonstrated the presence of polypeptides common to the three enzymes. A small cross-reaction was also found at the level of the large subunits of Enzyme B as well as some additional polypeptides of Enzyme C. Similar experiments with antibodies directed against native RNA polymerase B confirmed the presence of common subunits and also showed that the large polypeptides of the three enzymes share a few immunological determinants. Common subunits are AC40, ABC27, ABC23, AC19, and ABC14.5. Immunologically related sites were conserved in the large subunits of RNA polymerase A from remote yeast species. Similarly, yeast and wheat germ RNA polymerase B share immunological determinants on the large subunit as well as on a small peptide. On the other hand, there was no significant cross-reaction between yeast and mammalian Enzyme B or Escherichia coli RNA polymerase. Antibodies raised against the different polypeptide components of RNA polymerase A reacted specifically with the corresponding subunits. Inhibition studies with these subunit-specific antibodies showed that the common subunits are not always similarly exposed to antibody attack within the three enzymes. The data are discussed in terms of the structural similarity, organization and evolution of eukaryotic RNA polymerases.

Antigen-Antibody Complex↗
PubMed1980

Refolding of reduced short neurotoxins: circular dichroism analysis.

The four disulfide bonds of nine homologous short curare-like polypeptides are cleaved by reduced dithiothreitol. Air oxidation renaturations of the reduced compounds are followed by far-ultraviolet circular dichroism analysis, and the kinetics of refolding thus determined are compared. They indicate that three toxins refold 4--10 times more slowly than the six others. It is shown that a significant difference between the refolding kinetics still subsists when renaturations are made in the presence of various concentrations of thiol-disulfide exchange reagents or at various pH values. From an examination of the toxin sequences, it is proposed that a single additional amino acid insertion is responsible for the difference in the observed kinetics. This proposal is supported by temperature studies of renaturation kinetics.

Animals↗
PubMed1980

A mutation of the B220 subunit gene affects the structural and functional properties of yeast RNA polymerase B in vitro.

The Saccharomyces cerevisiae mutant rpo B1 produces a DNA-dependent RNA polymerase B defective in RNA synthesis in vitro. RNA polymerase B purified from the mutant is altered both structurally and functionally. The enzyme is defective in the RNA chain initiation and elongation reactions. Enzyme-DNA binding is comparatively much less affected. These enzymological defects in the mutant enzyme are enhanced at elevated ionic strengths. Purified rpo B1 RNA polymerase B is lacking B32 and B16.5 subunits. However, the low activity of the mutant enzyme cannot be accounted for only by the loss of these two polypeptides. Wild type enzyme devoid of B32 and B16.5 subunits can be obtained after a mild urea treatment. This enzyme variant, called RNA polymerase B, does not share the enzymological properties of the mutant RNA polymerase. Immunoprecipitation of the enzyme from crude extracts shows that, in the rpo B1 mutant, a normal amount of RNA polymerase B is synthesized which contains the full complement of subunits. The polypeptide chain altered by the rpo B1 mutation was identified by partial proteolysis with proteinase K in the presence of sodium dodecyl sulfate. The 35S-labeled peptide pattern generated from the B220 subunit of the mutant enzyme differs markedly from the peptide pattern of the wild type subunit. The rpo B1 mutation therefore alters the B220 subunit, suggesting a role for this subunit in RNA chain elongation and in the association of the B32 and B16.5 subunits to the RNA polymerase molecule.

Ammonium Sulfate↗
PubMed1980

Interaction of a new polypeptide with yeast RNA polymerase B.

A basic 37,000-dalton protein (P37), purified from yeast cells, interacts with yeast RNA polymerase B and drastically increases its specific activity. A complex of P37 and RNA polymerase can be isolated by sedimentation through a glycerol gradient. The complex is dissociated at the ionic strength of 0.9. The preferential binding of P37 with RNA polymerase form BI (with the unproteolyzed B220 subunit) was visualized by polyacrylamide gel electrophoresis under nondenaturing conditions. Kinetic analysis of the RNA polymerase cofactor interaction indicated that the dissociation constant for the complex is 5 X 10(-8) M.

DNA-Directed RNA Polymerases↗
PubMed1980

Amino acid side chain conformation in angiotensin II and analogs: correlated results of circular dichroism and 1H nuclear magnetic resonance.

[1-Sarcosine,8-isoleucine]angiotensin II (Sar-Arg-Val-Tyr-Ile-His-Pro-Ile) has been shown to be a potent antagonist of the pressor action of angiotensin II. With a view to increase half-life in vivo of this peptide, the amino acid residue at position 4 (tyrosine) or position 5 (isoleucine) was replaced with the corresponding N-methylated residue. This change drastically reduced the antagonistic properties of this analog. The present work was therefore undertaken to investigate the effect of N-methylation on overall conformation of these peptides and to determine the conformational requirements for maximum agonistic or antagonistic properties. Conformation studies were carried out by circular dichroism and proton nuclear magnetic resonance spectroscopy in aqueous solution as a function of pH. The results indicated that: (i) angiotensin II and [1-sarcosine,8-isoleucine]angiotensin II gave practically identical spectroscopic data; and (ii) N-methylation in either position 4 or position 5 resulted in remarkable changes in the peptide backbone and a severe limitation in rotational freedom of side chains in tyrosine, isoleucine, and histidine residues. However, rotational restriction of the tyrosine side chain was found to be less pronounced in [1-sarcosine,4-N-methyltyrosine,8-isoleucine]angiotensin II than in [1-sarcosine,5-N-methylisoleucine,8-isoleucine]angiotensin II. Thus, these results suggest that: (i) the backbone and side chain structure of a potent angiotensin II antagonist should resemble that of the hormone, angiotensin II, so that it can mimic the hormone in recognizing and binding with the receptor on the cell membrane; and (ii) greater impact of N-methylation in position 5 on the overall conformation of these peptides points to the controlling influence of position 5 (isoleucine) in aligning the residues in the central segment (tyrosine-isoleucine-histidine) of angiotensin II and its potent agonist and antagonist analogs in a nearly extended structure. Any change in this arrangement may lead to reduced biological activity.

Angiotensin II↗
PubMed1979

Identification of two different RNase H activities associated with yeast RNA polymerase A.

Two ribonuclease H activities have been found in yeast RNA polymerase A. The nuclease activities comigrated with subunits A49 (Mr = 49,000) and A40 (Mr = 40,000), after electrophoresis in a sodium dodecyl sulfate polyacrylamide gel containing [32P](rG)n . (dC)n as substrate. Both activities were also found, among other nucleases, in a high salt chromatin extract. Several lines of evidence suggest that the chromatin RNase H of 49,000 daltons (RNase H49) is the same protein as subunit A49. They co-migrate on sodium dodecyl sulfate-gel electrophoresis, have the same chromatographic properties, and dissociate simultaneously from RNA polymerase A. Fractions containing RNase H49 stimulate RNA synthesis by RNA polymerase A* lacking A49 and A34.5 subunits. Finally, limited proteolysis of the protein band having RNase H49 activity yields the characteristic fingerprint of the A49 subunit. This subunit, therefore, exists in two states: bound to chromatin and associated with RNA polymerase A. On the other hand, it is not yet clear whether the RNase H activity of 40,000 daltons, associated with RNA polymerase A, is due to the A40 subunit or whether it represents a trace contamination by a very active nuclease tightly bound to the enzyme.

Chromatin↗
PubMed1978

Conformation of snake toxic polypeptides studied by a method of prediction and circular dichroism.

Short and long neurotoxins as well as cardiotoxins belong to three distinct families of homologous toxic polypeptides extracted from cobra venoms. A study of their conformation was undertaken by using the method of Chou and Fasman for prediction of secondary structures of proteins. To improve the reliability of this method, an averaging scheme was developed. The data obtained showed that all toxins have a predominant trend for beta-sheet nucleation. Moreover, predicted beta-sheet strands fitted well those actually observed from X-ray data. Thus, it seems that all toxins share similarities in their secondary structure. This proposition was supported by a comparative study of the CD spectra of a set of toxins. Nevertheless, the present data suggest also that each type of toxins possesses localized structural individualities which might be responsible for the biological and/or immunological specificities.

Amino Acid Sequence↗