Specific DNA binding by c-Myb: evidence for a double helix-turn-helix-related motif.

The c-Myb protein is a sequence-specific DNA binding protein that activates transcription in hematopoietic cells. Three imperfect repeats (R1, R2, and R3) that contain regularly spaced tryptophan residues form the DNA binding domain of c-Myb. A fragment of c-Myb that contained the R2 and R3 regions bound specifically to a DNA sequence recognized by c-Myb plus ten additional base pairs at the 3' end of the element. The R2R3 fragment was predicted to contain two consecutive helix-turn-helix (HTH) motifs with unconventional turns. Mutagenesis of amino acids in R2R3 at positions that correspond to DNA-contacting amino acids in other HTH-containing proteins abolished specific DNA binding without affecting nonspecific DNA interactions.

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.

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.

Conformational changes in two neurotoxic proteins from snake venoms.

alpha-Neurotoxin from Naja nigricollis and erabutoxin b from Laticauda semifasciata, two homologous neurotoxic proteins, are studied by circular dichroism, ultraviolet spectroscopy and fluorescence in various water/trifluoroethanol mixtures. The data obtained show that the beta structure of alpha-neurotoxin is conserved in water as well as in the organic solvent. By contrast, erabutoxin b changes from the beta-structure in water to the helix type in trifluoroethanol. The latter induces similarly for both toxins a structural modification around tryptophan 29, a residue common to all neurotoxins known to date. The vicinity of tyrosine 25, another common amino acid, is also altered by the presence of the organic solvent as demonstrated by the sudden increase of reactivity of the phenolic ring towards iodine. The present work affords some evidence for the presence of a particular structure located around the two aromatic residues, which is common to all neurotoxins and able to rearrange independently from the rest of the molecule. Biological importance of this peculiar region is highly probable.

13C-nuclear magnetic resonance studies of 85% 13C-enriched amino acids and small peptides. pH effects on the chemical shifts, coupling constants, kinetics of cis-trans isomerisation and conformation aspects.

The 13C chemical shifts of several 85% 13C-enriched amino acids and small peptides were studied as a function of pH. The results show that the chemical shifts of carbon atoms of ionizable groups vary significantly within the zone of their pK. Generally with the pH GOING FROM 7 to 1 all the deltaC are shifted more or less upfield with the exception of the carbonyl group carbon of the second last residue which is shifted slightly downfield. This suggests the formation of an hydrogen bond at acid pH involving in a seven-membered ring the C=O in question and the COOH terminal. The percentage of cis and trans conformers of glycyl-L-proline and glycyl-L-prolylglycine were studied as a function of pH. The trans form is always preponderant whatever the pH. The accessibility of the carbonyl group to protonation of the proline residue strongly influences the cis-trans equilibrium. Thus, with the pH varying from 7 to 1, the trans isomer changes from 61 to 85% for glycyl-L-proline and only from 77 to 80% for glycyl-L-prolylglycine. The proton NMR studies underline the important differences existing between the two molecular forms of glycyl-L-proline. The cis conformation is characterized with regard to the trans form by the non-equivalence of the alpha-protons of the glycine residue, by a lower pK(1) and by a larger deltadeltaHalpha of the proline residue as a function of pH. These results could suggest an end-to-end interaction in the cis form of the glycyl-L-proline molecule. The 13C-13C coupling constants were also studied as a function of pH. The results show that J(Co-Calpha) of a C-terminal residue, varying from 5 to 6 Hz and reflecting thhe pK of the carboxylate group, is a linear function of delta(Co) and delta(Calpha) as in the case of the amino acids. The total variation of the electron density of those two carbons in an amino acid is approximately 40% weaker than in a C-terminal residue. The charge distribution along the Calpha-C(o) bond, however, is practically the same in both cases. Finally the ratios of the conversion rate constants of the two isomers cis-trans of glycyl-proline were calculated at different pH values; the relations between the isomer percentages and delta(Co), delta(Calpha) on the one hand and the J(Co-Calpha) on the other were established.

Further evidence showing that neurotoxin-acetylcholine receptor dissociation is accelerated by monoclonal neurotoxin-specific immunoglobulin.

We have demonstrated that the dissociation of Naja nigricollis alpha-toxin from the two acetylcholine receptor sites [Weber and Changeux, Molec. Pharmac. 10, 1-14 (1974); Rousselet et al., Eur. J. Biochem. 140, 31-37 (1984)], is markedly accelerated by a monoclonal neurotoxin-specific antibody. The dissociation of the toxin occurs in a biphasic manner in the presence of a 900 molar excess of immunoglobulin (with respect to toxin concn). The progress curves are characterized by first-order kinetics. Under these conditions the maximal dissociation rate is achieved as further rate enhancement cannot be induced by exposure to an increased immunoglobulin level. In contrast when a toxin-immunoglobulin complex is incubated with a large excess of receptor, the dissociation kinetics of the complex are not enhanced. The data fit a kinetic model which implicates the existence of a transient ternary complex involving the receptor, the toxin and the antibody.

Two neutralizing monoclonal antibodies specific for Naja nigricollis cardiotoxin: preparation, characterization and localization of the epitopes.

Two monoclonal antibodies have been raised against the native form of the potent cardiotoxin isolated from the venom of Naja nigricollis. The toxic action to mice as well as the depolarizing effect on muscle fibres in culture of the cardiotoxin are neutralized by the two immunoglobulins. Binding studies revealed that the radiolabelled toxin has a high affinity for both antibodies, the equilibrium dissociation constant values being equal to 0.2 and 0.4 nM. The epitopes that are recognized by the antibodies have been localized on the basis of competition experiments between the labelled toxin and a series of variants or a Trp-11 modified derivative, toward both antibodies. The data obtained indicate that the antibodies bind at topographically different antigenic sites. Knowing that the toxin is a single polypeptide chain folded in a structure that contains three adjacent loops emerging from a small globular region, it appears that one of the two antibodies binds on loop I, at a site which involves Trp-11 whereas the other binds at a site which involves one or both of loops II and III. Possible mechanisms of neutralization of the toxin by the antibodies are discussed.

A monoclonal antibody which recognized the functional site of snake neurotoxins and which neutralizes all short-chain variants.

We isolated a neurotoxin-specific monoclonal antibody (Mab) which is capable of recognizing and neutralizing all short-chain toxin variants that have been tested, including those with widely divergent sequences. The epitope incorporates the three invariant residues Lys-27, Trp-29 and Lys-47 which form part of the site by which the toxins bind to the nicotinic acetylcholine receptor. To our knowledge, this is the first Mab which possesses the universal capacity of neutralizing all natural variants of a toxic protein.

C-terminal amidation of neuropeptides. Gly-Lys-Arg extension an efficient precursor of C-terminal amide.

Biosynthesis of the C-terminal carboxamide group of peptide hormones was studied using comparatively pGlu-His-Pro-Gly and Glu-His-Pro-Gly-Lys-Arg as putative precursors of the tripeptide, thyroliberin (TRH). Rat hypothalamus granules were found to contain an amide group forming activity which converts both peptide substrates into TRH. Comparison of the rate of conversion of the two substrates indicated that the C-terminal dibasic extension favored a 10-fold increase in the production of amidated peptide. It is suggested that this type of structure may be present in the putative biosynthetic precursor of TRH and that it may provide a better substrate for the enzyme(s) involved in C-terminal amidation.

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.

Cloning and sequence analysis of the cDNA encoding a snake neurotoxin precursor.

A recombinant plasmid has been constructed containing a sequence of 186 nucleotides encoding a potent neurotoxin found in the venom of the sea-snake Laticauda semifasciata and designated as erabutoxin a. This sequence is flanked, in the upstream region, by a sequence of 60 nucleotides encoding a hydrophobic peptide fragment presumably involved in the secretion process of the neurotoxin. The sequence coding for the toxin ends with a termination codon which is followed by a 3'-untranslated sequence of approximately 240 nucleotides (excluding the poly(A) tract).

Further characterization of yeast RNA polymerases. Effect of subunits removal.

Two forms of yeast RNA polymerase A are resolved by phosphocellulose chromatography. One of these, called RNA polymerase A, is lacking two polypeptide chains of 48,000 and 37,000 daltons. The properties of the two enzymes are compared in the present paper. RNA polymerase A transcribes d(A-T)n with a similar efficiency as the complete enzyme, but it is comparatively much less active with native DNA. The two enzymes can also be differentiated on the basis of their ionic strength and divalent cation requirements. RNA polymerase A has a particularly low activity at high salt and low Mg2+ concentrations. Thermal inactivation curves of the two enzymes are different when residual activity is assayed with native DNA. In contrast with d(A-T)n as template the apparent inactivation curves of the two enzymes are identical. The data suggest that the two dissociable polypeptide chains play an important role in transcription. The template specificity of yeast RNA polymerase B was further investigated using SV40 DNA-FI as template. RNA polymerase B is able to retain [3H]SV40 DNA-FI on nitrocellulose filters but the enzyme-DNA complex is very unstable. The observation that RNA polymerase B can transcribe to some extent a supercoiled DNA but not a linear double stranded template supports the hypothesis that the enzyme needs some unpaired DNA structure to initiate transcription.

[Binding of a tritiated enkephalinergic analog (FK 33-824) to a mitochondrial fraction of rat brain]. / Liaison d'un analogue enképhalinergique [FK 33-824] tritié à une fraction mitochondriale de cerveau de rat.

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).

[Fate of thyrotropin releasing hormone after binding to rat prolactin secreting cells (GH3 line). Evidence for releasing of unmodified [3H]-TRH (author'r transl)]. / Devenir du trh (thyrotropin releasing hormone) après sa liaison à des cellules à prolactine de rat (lignée gh3)

It was previously shown that after a 30 min. incubation with [3H]-TRH intact GH3 cells have already performed their optimal precocious biological response to TRH (i.e increase of prolactin release) and that chemically unmodified ( greater than or equal to 93%) [3H]-TRH entered the cells and remained associated to them. The present work demonstrates that such cells spontaneously release a radioactive material ([3H]RM) when post-incubated at 37 degrees C. The binding to GH3 cells (time course and competition with unlabelled TRH) as well as the biological effect on prolactin release and the electrophoretic mobility of the [3H]-RM released in one hour were compared to those of [3H]-TRH. According to those criteria [3H]-RM is undistinguishable from [3H]-TRH at 90%. Our data, therefore, support the hypothesis of a continuous flow of TRH molecules on both sides of the plasma membrane during TRH-GH3 cells interaction.