Identification of N- and C-terminal corticotropin peptides in the Mr 80000 form of neurophysin

The 125I-labeled Mr 80000 form of neurophysin has been purified from bovine neurohypophysi. Tryptic digests of this species were analyzed, prior to or after treatment with carboxypeptidase B, by high-pressure liquid chromatography followed by isoelectric focusing and the fragments compared with those generated by a similar treatment of reference bovine 1-39 adrenocorticotropin. The ACTH peptides 22-39 and 1-8, as well as the 1-7 derivative of the latter were identified by those two independent criteria. This provides chemical evidence supporting the hypothesis [8] that high Mr neurophysin may contain the sequence of ACTH.     

Measurement of Methionine-Enkephalin [Arg6,Phe7] in Rat Brain by Specific Radioimmunoassay Directed at Methionine Sulphoxide Enkephalin[Arg6,Phe7]

Abstract: A specific and sensitive radioimmunoassay procedure for Metenkephalin[Arg⁶,Phe⁷] which allows its measurement in regions of the rat brain is described. The antiserum was raised against the methionine sulphoxide derivative of the peptide, and all samples and standards were oxidized with hydrogen peroxide prior to use in the assay with chloramine T-oxidized ¹²⁵I-labelled Met(O)-enkephalin[Arg⁶,Phe⁷]. The only significant cross-reactivity was 30% with the reduced heptapeptide Met-enkephalin[Arg⁶,Phe⁷]. The assay showed less than 0.15% cross-reactivity with fragments of the heptapeptide and with leucine-enkephalin-containing peptides. Acid acetone extraction of rat striatum followed by Sephadex G-50 chromatography and reverse-phase high pressure liquid chromatography showed that essentially all immunoreactivity co-chromatographed with Met-enkephalin[Arg⁶,Phe⁷]. This confirmed the specificity of the assay and showed that the striatum does not contain a high concentration of larger molecular weight forms with the heptapeptide at the COOH terminus. Distribution of the heptapeptide followed that of methionine enkephalin, with highest concentrations in the globus pallidus, intermediate levels in caudate-putamen and hypothalamus, and low levels in cortex and cerebellum.     

Differentiation of Ca2+ pumps linked to plasma membrane and endoplasmic reticulum in the microsomal fraction from intestinal smooth muscle

ATP promotes ⁴⁵Ca uptake by the microsomal fraction from the longitudinal smooth muscle of guinea-pig ileum and this uptake is stimulated by oxalate. As the microsomal fraction is made up of various subcellular entities, we examined the localization of the Ca²⁺-transport activity by density gradient centrifugation, taking advantage of the selective effect of digitonin (at low concentration) on the density of plasmalemmal elements. When the ⁴⁵Ca-uptake activity was measured in the absence of oxalate, its behavior in subfractionation experiments closely paralleled that of the plasmalemmal marker 5′-nucleotidase. In contrast, the additional Ca²⁺-transport activity elicited by oxalate behaved like NADH-cytochrome $\text{c}$ reductase, a putative endoplasmic reticulum marker. The endoplasmic reticulum vesicles constituted only a small part of the membranes in the microsomal fraction, which explains that their Ca²⁺-storage capacity was not detectable in the absence of Ca²⁺-trapping agent. Low digitonin concentrations selectively increased the Ca²⁺ permeability of the plasmalemmal vesicles. The two Ca²⁺-transport activities were further differentiated by their distinct sensitivities to K⁺, vanadate and calmodulin. In this respect, the oxalte-insensitive and oxalate-stimulated Ca²⁺-transport systems resembled, respectively, the sarcolemmal and sarcoplasmic reticulum Ca²⁺ pumps in cardiac and skeletal muscle, in accordance with the subcellular locations established by density gradient centrifugation.     

Ultrastructural evidence of oestradiol receptor by immunochemistry

Antiserum against calf uterus oestradiol receptor has been used for detecting oestradiol receptor in rat pituitary cells at the ultrastructural level after immunochemical reaction according to Sternberger. The gonadotropic, lactotropic and somatotropic cells were positive, but not the thyrotropic and corticotropic cells. In peripubertal and adult rats, both cytoplasmic and nuclear receptors were seen, but in a long-term castrated rat, the receptor was found only in the cytoplasm. After oestradiol administration to 21-day-old animals, the cytoplasmic receptor decreased and the nuclear receptor increased in gonadotropic cells, supporting the concept of hormone-receptor complex translocation. Antibodies against α1-foetoprotein demonstrated the presence of this oestrogen-binding plasma protein in all pituitary cells, but only in the cytoplasmic area. These results and the immunological controls related to antibody specificity give the first evidence of steroid receptor at the ultrastructural level.     

Galactosyltransferase activities in subcellular fractions of two YC8 lymphoma cell variants: does a relationship exist between antigenic determinants and acceptor sites for galactose?

Experiments have been undertaken to correlate physiological changes, observed in two YC8 cells variants (P and L) and some of their immunological and enzymatic properties. These cell lines show different responses towards antilymphocyte and anti-Moloney sera. Subcellular fractionations have been made. The A fractions (d: 1.14/1.16) have the highest ouabain-inhibited Mg²⁺-stimulated (Na⁺-K⁺)-dependent ATPase and galactosyltransferase activities. Some properties of the latter enzyme have been studied: whereas optima pH and requirements for Mn²⁺ ions have been found to be the same for both cell line enzymes, on the contrary, different kinetic parameters have been shown with respect to sugar donor (UDP-galactose) on endogeneous or exogeneous (ovomucoid) acceptors. Apparent Km for UDP-galactose is 1.7 × 10⁻⁶ M (P-cells) and 3.3 × 10⁻⁶ M (L-cells), on endogeneous acceptors, and P-cell V max < L-cell V max; on ovomucoid it is 0.61 × 10⁻⁶ M, for both cell lines. These results suggest the presence on L-cells of more endogeneous acceptor sites, the higher affinity of P-cells for UDP-galactose being balanced by less endogeneous acceptor sites for galactose. When ovomucoid is added, galactose transfer on endogeneous acceptor sites of both cells is negligible. Apparent Km for ovomucoid is 8.6 × 10⁻⁵ M (P-cells) and 4.3 × 10⁻⁵ M (L-cells). These data support the above-mentioned hypothesis: L-cell enzymes would be more rapidly saturated than P-cell enzymes because of the higher number of endogeneous sites on L-cells.This supposed acquired character of L-cells as well as their immunological behaviour could explain the modified properties of L-cells as compared to P-cells.     

Studies on Porochlamydia buthi g. n., sp. n., an intracellular pathogen of the scorpion Buthus occitanus

An endemic disease caused by an intracellular microorganism related to the Chlamydiales, Porochlamydia buthi g. n., sp. n., exists in populations of the scorpion Buthus occitanus of Languedoc in southern France. The pathogen's developmental cycle occurs in the hepatopancreatic cells and progressively brings about the destruction of this organ. The microorganism is spread mostly through dejecta which contain the infectious forms during nearly the entire period of illness.     

Transfer RNA and protein synthesis

The three-dimensional structure of yeast phenylalanine transfert RNA has been determined in orthorhombic crystals. The current status of this work is reviewed together with the relationship of the transfer RNA structure in the crystal to its biologically active form. In addition some speculations are put forward regarding the mode of interaction of tRNA molecules in the ribosome and the manner in which tRNA interacts with aminoacyl synthetase.     

Binding of a Glycera convoluta neurotoxin to cholinergic nerve terminals triggers a Ca-dependent acetylcholine release

The venom glands of the annelid Glycera convoluta contain a neurotoxin which triggers ACh release from frog motor terminals and Torpedo synaptosomes. This neurotoxin binds to presynaptic, but not postsynaptic plasma membranes prepared from Torpedo electric organ. The binding site is an ectocellularly oriented protein. The binding does not require Ca. It is inhibited by pretreatment of the membrane by Concanavalin A. The toxin induced ACh release is Ca-dependent and inhibited by D 600.