Met-enkephalin immunoreactivity in blood platelets

Picogram amounts (50–150 pg/mg protein) of immunoreactive met-enkephalin material (met-enkephalin in IR) were detected by radioimmunoassay in human, rat and rabbit platelets. Characterization of this material by thin-layer chromatography, gel filtration chromatography and high-pressure liquid chromatography indicated that it behaves identically with synthetic met-enkephalin. No high molecular weight met-enkephalin IR could be detected in the platelet extracts, even after trypsin hydrolysis, using two antisera which are able to recognize some of the putative met-enkephalin precursors present in the adrenal gland or striatum. $\text{In vitro}$, thrombin released platelet met-enkephalin in IR concomitantly with 5-hydroxytryptamine (5-HT), suggesting a common subcellular localization, i.e. the 5-HT storing organelles, for met-enkephalin IR and the amine. $\text{In vivo}$, platelet met-enkephalin IR in the Sprague-Dawley rat was affected neither by adrenalectomy nor by hypophysectomy. Thirteen- and 18-week-old spontaneous hypertensive rats (SHR) had lower platelet concentrations of met-enkephalin in IR than age matched normotensive Wistar-Kyoto rats.     

The stereochemistry of matricin and 4-epimatricin,proazulene sesquiterpene lactones from Artemisia arborescens

An investigation of Artemisia arborescens afforded, in addition to the known compounds matricin, artabsin and artemetin, the new guaianolide 4-epimatricin. The stereostructures of 4-epimatricin and matricin were assigned on the basis of spectroscopic evidence.     

Effect of temperature on the stability and activity of crystalline ox liver nuclear and mitochondrial glutamate dehydrogenases

A study on the response of the stability and activity of crystalline ox liver nuclear and mitochondrial glutamate dehydrogenases to temperature variations has been carried out. The thermodynamic properties of the heat inactivation process and of the reaction with the substrates glutamate and α-ketoglutarate have been investigated. The heat inactivation of nuclear glutamate dehydrogenase proceeds at a faster rate than that of the mitochondrial enzyme in the temperature range 40–51 °C; the enthalpy of activation of the inactivation process is higher and the entropy is almost double, compared to the values of mitochondrial glutamate dehydrogenase. The effect of temperature on the maximal velocity shows that, with both glutamate and α-ketoglutarate, the enthalpy of activation with nuclear glutamate dehydrogenase is double and the decrease in entropy almost half of the values of the mitochondrial enzyme. The variation of the apparent K_m with temperature shows a decrease of the affinity of both enzymes for glutamate, with no major difference in the thermodynamic properties of the reaction. With α-ketoglutarate, on the other hand, the affinity of nuclear glutamate dehydrogenase decreased, whereas that of the mitochondrial enzyme increased with temperature. The process is therefore exothermic with the former enzyme, endothermic with the latter; furthermore, it occurs with a decrease in enthropy with nuclear glutamate dehydrogenase, but with a large increase with the mitochondrial enzyme. The studies on the effect of temperature on the activity were carried out in the range 20–44 °C.     

Time-dependent phosphorescence anisotropy measurements of the slow rotational motions of proteins in viscous solution

A method of monitoring slow rotational motions of proteins from the decay of the intrinsic phosphorescence is described. The phosphorescence is excited with a 10-μsec pulse of vertically polarized light from an air gap lamp, and the anisotropy was computed as a function of time from the simultaneously detected vertically and horizontally polarized components of the emission. The approach is illustrated with time-dependent measurements of the anisotropy of the tryptophan phosphorescence of Staphylococcus aureus nuclease, bovine carbonic anhydrase, and liver alcohol dehydrogenase in glycerol-phosphate buffer between ?90 and ?70°C. The temperature- and molecular-weight dependence of the exponential decays in the anisotropy indicate that overall rotation of the proteins is at the origin of the depolarization. The potential of the approach as a probe of the slow rotational motions of proteins in membranes and other macromolecular complexes is stressed.     

Determination of cytoplasmic receptors for specific steroid hormones. II) Androgen receptor

Cytoplasmic receptors for 5 alpha-dihydrotestosterone (3H-DHT) were determined in normal and hypertrophic human prostate using the slightly modified DCC method we previously standardized for 17beta-estradiol-receptor. Incubations were always performed at 0 degree C for 1 hr. Discrimination between 3H-DHT binding to cytoplasmic receptor and to Sex Hormone Binding Globulin (SHBG) was achieved on the basis of binding affinity, thermolability and pattern of specificity by various steroid hormones. In particular, 5 beta-DHT did not bind to cytoplasmic receptor, while it did to SHBG.     

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     

Effect of mutation in the aromatic amino acid pathway on sporulation of Saccharomyces cerevisiae.

Mutations in ARO1 and ARO2 genes coding for enzymes involved in the common part of the aromatic amino acid pathway completely block the sporulation of Saccharomyces cerevisiae when in a homozygous state, whereas mutations in all the other genes of the same pathway do not. This effect is not due to the lack of any intermediate metabolite but rather to the accumulation of a metabolite preceding chorismic acid. Shikimic acid or one of its precursors was identified as the possible inhibitor. The presence of the three aromatic amino acids in the sporulation medium restores the ability to undergo meiosis. This seems not to be due to a feedback inhibition of the first enzymes of the pathway but rather to a competition between aromatic amino acids and the inhibitor on a site specific for the meiotic process. The inhibition of sporulation seems to occur at a very early step in meiosis, as indicated by the lack of premeiotic DNA synthesis in aro1 and aro2 mutants.