Determination of cytoplasmic receptors for specific steroid hormones. III) Progestin receptor

A method for determining cytoplasmic progesterone receptor was standardized in normal human endometrium comparing two different tracers, 3H-progesterone (3H-P) and 3H-medroxyprogesterone acetate (3H-MAP), a synthetic progestin which does not bind to Corticosteroid Binding Globulin (CBG). Receptor assays were performed as previously reported for 17beta-estradiol receptor, with slight modifications: incubation lasted 1 hr at 0 degree C, followed by 5 min DCC exposure under the same conditions. When 3H-P was employed as tracer, blanks performed with cold MAP gave similar results as using cortisol in incubation tubes and progesterone and cortisol in blanks. 3H-MAP was a good tracer for progesterone receptor because it neither bound to CBG nor to androgen or cortisol receptors; it had very high affinity and specificity for P-R; it was not metabolized by cytosol at 0 degree C and, finally, it detected receptor amounts quite comparable to those obtained using 3H-P.     

Reactivity of sulphydryl groups of cytosolic and mitochondrial bovine aspartate aminotransferases

Summary Reactivity of sulphydryl groups of cytosolic and mitochondrial aspartate aminotransferases from ox heart has been studied. A total of 5 and 7 cysteine residues per monomer are present in cAATo and mAATo, respectively. In native conditions only a single sulphydryl group can be titrated by Nbs₂ while the catalytic activity remains unchanged, however in the mitochondrial isozyme the reactivity depends on the functional state of the enzyme. Reactivity toward NEM reveals the existence of a syncatalytic sulphydryl group in the cytosolic isozyme. Titration of cAATo with pMB at pH 8 and pH 5 confirms the existence of two exposed sulphydryl groups with a different reactivity. The results compared with those reported on the corresponding isozymes from pig and chicken heart show that syncatalytic sulphydryl groups are of general occurrence in these enzymes.     

The generation of the proton electrochemical potential and its role in energy transduction

Summary The evidence that all energy transducing membranes can generate a proton electrochemical potential difference, _H, across the membrane and that this potential can be used to transfer energy among energy transducing units and to generate ATP, has increased the interest for the view that _H plays an obligatory role in energy transduction and ATP synthesis. In the present article we shall concentrate on two experimental questions related with the generation and role of _H: (a) the charge/site ratio; (b) the relation between the proton electrochemical potential on one side and the cation electrochemical potential, the phosphate potential and the redox potential on the other. We shall then discuss the view that energy transduction corresponds to a molecular energy machine rather than to a fuel cell.     

Analytical review enzymatic defects of hereditary porphyrias: An explanation of dominance at the molecular level

Summary In four of the five autosomal dominant porphyrias four different partial enzymatic defects of the porphyrin biosynthetic pathway have been discovered in the last few years. With the exception of protoporphyria, the residual enzymatic activity in carriers of these defects is approximately equal to 50% of that found in controls. In each case the pattern of excretion of porphyrin and/or porphyrin precursors reflects the site of the partial metabolic block. There are indications, at least in intermittent acute porphyria, that the degree of penetrance of the disorder varies according to the level of phenotypic expression, being highest for the enzyme deficiency, lower for the excretion of precursors and lowest for the clinical symptoms. It is proposed that environmental factors, and probably also gene interaction, are the cause of the different degrees of penetrance.On leave from the University of Naples, Italy     

MK-801 Prevents 1-Methyl-4-Phenyl-1,2,3,6-Tetrahydropyridine-Induced Parkinsonism in Primates

In cynomologus monkeys, systemic administration of MK-801, a noncompetitive antagonist for the N-methyl-D-aspartate receptor, prevented the development of the parkinsonian syndrome induced by the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). MK-801 also attenuated dopamine depletion in the caudate and putamen and protected dopaminergic neurons in the substantia nigra from the degeneration induced by the neurotoxin. Nevertheless, 7 days after MPTP administration in the caudate and putamen of monkeys also receiving MK-801, the levels of toxic 1-methyl-4-phenylpyridinium were even higher than those measured in monkeys receiving MPTP alone. This indicates that the protective action of MK-801 is not related to MPTP metabolism and strongly suggests that, in primates, the excitatory amino acids could play a crucial role in the mechanism of the selective neuronal death induced by MPTP.     

The bovine plasma retinol-binding protein. Amino acid sequence, interaction with transthyretin, crystallization and preliminary X-ray data

1. The primary structure of bovine plasma retinol-binding protein (RBP) has been determined and found to be more than 90% identical to human and rabbit RBPs, and more than 80% identical to rat RBP. Main changes in amino acid sequence are observed in two regions on the surface of the protein molecule (residues 138-148 and 169-183). 2. The interactions of bovine RBP with bovine and human transthyretins were investigated using the technique of fluorescence polarization. Bovine RBP was able to form high affinity complexes (K'd = 0.34 +/- 0.02 microM) with both bovine and human transthyretins, displaying a stoichiometry of approximately 2 molecules RBP/molecule transthyretin in both cases. The sites that participate in protein-protein interactions are thus very similar, and this tends to exclude the involvement of the superficial regions more significantly substituted in mammalian RBPs (residues 138-151 and 167-183) in the protein-protein recognition. 3. Bovine RBP has been crystallized (space group P2(1)2(1)2(1), with a = 4.61 nm, b = 4.91 nm, c = 7.61 nm) and the crystals are suitable for high-resolution X-ray diffraction studies.     

Inhibition of the biosynthesis of thyroglobulin with propranolol in the rat

Thyroglobulin biosynthesis was studied in thyroid glands of rats treated during 30 days with a daily dose of propranolol, a non-selective beta-adrenergic blocking drug. Studies were carried out by extraction of soluble proteins from homogenates after incubation of the glands in presence of [3H]-4,5-L-leucine and [3H]-D-1-galactose. Thyroglobulin 19S, 12S and 4-8S soluble proteins were separated and identified by ultracentrifugation in a saccharose gradient. The glands of rats treated with propranolol showed a decreased amount of soluble proteins as well as a decreased incorporation of [3H] labeled markers. 19S thyroglobulin is poorly represented, but very large amounts of the 12S monomer are present; this suggests an impairment in the dimerization of the 12S subunit, absent in normal controls. This impairment could be due to a structure modification. Propranolol reduces the biosynthesis of thyroglobulin in thyroid gland as well as the formation of T3 from T4 in peripheral tissues; its therapeutical use against thyrotoxicosis is thus justified. The study of its action on the dimerisation of the 12S subunit could be of interest to clear the mechanism of thyroglobulin biosynthesis.     

Tunicamycin inhibition of carbohydrate incorporation into thyroglobulin

Carbohydrate chains formation into thyroglobulin (Tg) is a prerequisite for thyroid hormones formation and completeness of carbohydrates chains is necessary for secretion of Tg into the follicles. Tg biosynthesis has been investigated by in vitro experiments, incubating rat thyroid glands with labeled amino-acid and carbohydrate in the presence of tunicamycin, a specific inhibitor of protein glycosylation. Tunicamycin inhibit Tg biosynthesis which is impaired in carbohydrate chains addition but slightly in the polypeptide synthesis, as shown by inhibition of 3H-glucosamine incorporation. Thus tunicamycin inhibits carbohydrate incorporation into Tg without affecting the polypeptide chain growth and decreases its secretion into the follicles.     

Effect of change of habitat (sea and fresh water) on in vivo thyroglobulin synthesis in Atlantic glassed eels (Anguilla anguilla L.)

Thyroid biosynthesis in glassed eels (Anguilla anguilla L.) was studied to establish whether salinity changes could affect it, when they live in sea water or in fresh water containing 125I. Aqueous extrait of homogenized cephalic heads of glassed eels contains an iodinated protein 17-19 S having thyroglobulin-like properties and including iodotyrosins (MIT and DIT) and thyroid hormones (3 and T4). Biosynthesis of this proteins is roughly twice more important in fresh water than in sea water at 19-21 degrees C and its specific radioactivity (125I) is practically double in fresh water.     

High molecular weight RNA containing histone messenger in the sea urchin Paracentrotus lividus

Sea urchin RNA extracted from early and mesenchyme blastula embryos and oocytes and fractionated on denaturing sucrose density gradients, was hybridized with histone DNA recombinants of Psammechinus miliaris (clone λh22) and of Paracentrotus lividus (clone pPH70). Histone sequences are found in the 9 S and larger than 9 S regions of the formamide/sucrose density gradients. The melting of the RNA-DNA duplexes obtained by hybridization of polysomal and high molecular weight RNA of embryos of P. lividus at the stage of early blastula, suggests a degree of heterogeneity in the high M_r RNA. The high M_r RNA contains at least four of the five histone gene sequences covalently linked.