Pierre Fromageot

Tritium labelling of 8-lysine vasopressin and its purification by affinity chromatography on sepharose bound neurophysins.

The antidiuretic hormone vasopressin is a nonapeptide containing one disulfide bridge and tyrosyl and phenylalanyl residues in position -2 and -3, respectively. 8-Lysine vasopressin (LVP) is found in pig and hippopotamus while 8-arginine vasopressin (AVP) occurs in several other mammals (man, ox, rat...). These pituitary polypeptides exert selective effects on the permeability of the distal part of the mammalian nephron and on the active sodium transport and passive permeability to water and other low molecular weight molecules of the amphibian skin and bladder epithelial cells. These effects have been shown to result from a specific activation of an adenyl cyclase located within the membrane of the target cells (for review, see for instance [ 1 ] ). Either LVP or AVP is associated with oxytocin, a related peptide, and carried in the post-hypophysis by a family of relatively small proteins, the neurophysins [2], with which they form non covalent complexes [3]. The isotopic labelling of polypeptidi¢ hormones has been successfully applied, in this laboratory, to oxytocin [4], angiotensine II [5] and other compounds [6]. The principle is an iodination of the tyrosyl (or histidyl) residue(s) followed by catalytic substitution of the peptide bound iodine with tritium. In this report we describe a similar procedure which produces, after purification by affinity chromatography on a column of bovine neurophysins immobilized on Agarose, a highly radioactive compound (10 Ci/mmole) which exhibits all the biological and biochemical properties of the native hormone. This, obviously, provides a very useful tool for the examination of the molecular mechanisms by which this important biological compound interacts with neurophysins [7] and biological membranes.

Proton NMR studies on thyrotropin releasing factor

The synthesis of the tripeptide pyroglutamylhistidyl-prolineamide (TRF), the thyreotropin releasing factor [ 1,2], and its labelling [3] have promoted studies of its mode of action at the molecular level [4]. One important step towards this aim is to define the shape of the molecule and the relationships between biological activity and conformations. For this purpose ‘H-NMR investigations of TRF and related peptides have been carried out. The conformation of the histidyl residue was deduced from the relationships between the vicinal coupling constant J (NH-CctH) and the dihedral angle (C&H-NH). @ was derived from the equation 8 = (O-60) [5-S]. The behaviour of the C&H-CPH, histidyl protons and the non equivalence of the two prolyl C6 protons, on one hand, the differences between the cis and the trans-protons of the terminal amide group on the other hand suggested additional ~onformational restrictions. On this basis a model of TRF conformation is presented.

Hormonal interactions at the molecular level: A high resolution proton magnetic resonance study of bovine neurophysins and their interactions with oxytocin.

The neurohypophyseal nonapeptides oxytocin and 8-arginine (or 8-lysine) vasopressin are biologically important hormones known to associate non-covalently with the proteins, neurophysins I and II [l-3]. The physical and chemical properties of the neurophysins, whose polypeptides have molecular weight near 10,000, have been investigated by several authors [4,5], and some amino acid sequences for bovine neurophysins I and II have been proposed [6,7]. The study of the molecular interactions between neurophysins and peptide hormones has been approached using classical equilibrium methods [8-121 and optical techniques [13,14]. High resolution proton magnetic resonance (PMR) has proved useful in providing information about selected residues in proteins during the processes of denaturation [15-l 71 and inhibitor-enzyme interactions [18,19]. A few studies of protein-polypeptide or protein-protein interactions using high resolution magnetic resonance techniques have been reported [20-221. We present here PMR observations on highly purified bovine neurophysins and the complex formed with oxytocin. In particular, high resolution PMR methods enable us to study the possible involvement of the lone histidine residue of neurophysin I or of the single tyrosyl residue of oxytocin in the hormonal complexes.

Inhibition of viral RNA synthesis by levallorphan

Studies of the actions of narcotic alkaloids on bacterial and mammalian cells have shown that levallorphan and related compounds are potent inhibitors of RNA synthesis. At low concentrations, these drugs appear to inhibit RNA synthesis rather selectively [ 1, 21 and to induce in Escherichia coli a loss of cellular polyamines [3]. Higher concentrations lead to the inhibition of protein synthesis [4,5] and produce a decrease in the intracellular levels of ATP [4]. The inhibition of RNA synthesis in E. coli has been re ported to involve chiefly the various species of ribosomal RNA, including the 5 S component [6] ; the formation of sRNA and the rapidly labeled, presumably mRNA are more resistant to the effect of the drugs. These’observations all suggested the some aspect of nuclear functions or of genetic transcription, i.e. DNAdirected RNA synthesis, might be the primary site of action of levallorphan. Since the formation of RNA of some RNA viruses differs from that of the host with respect to the template, polymerizing enzyme and cellular localization, we have studied the effect of levallorphan on the RNA synthesis of an RNA virus, Mengovirus. The results show that the synthesis of Mengoviral RNA is at least as sensitive to levallorphan as host cell RNA synthesis. p IOoz f P 9Y .

Interaction entre pigments et acides nucleiques Etude sur l'interaction de l'Actinomycine D avec les nucléosides par Dichroïsme Circulaire

The interaction of Actinomycin C1 (AM) with purine nucleosides was investigated by circular dichroism. While dG and dGMP show identical interactions, rG, rGMP and 8‐aza‐GMP show lower tendency for complex formation. ara‐G shows no complex formation. 6‐Thio‐GMP shows the strongest affinity for AM in accordance with previous results. Substitution on N‐7 (methylation, protonation) or C‐8 (8‐Br‐rG, 8‐SH‐rG) eliminate completely the complexing capacity. Another type of weaker complexes is formed by dA, dAMP and rIMP, indicated by the appearance of a positive band at 440 nm. It is concluded that in these complexes both the electron donor capacity of the purine base as well as the steric flexibility of the sugar around the glycosidic linkage determine their complexing capacity.