Human placental 3 beta-hydroxy-5-ene-steroid dehydrogenase and steroid 5----4-ene-isomerase: purification from microsomes, substrate kinetics, and inhibition by product steroids

In human pregnancy, placental 3 beta-hydroxy-5-ene-steroid dehydrogenase and steroid 5----4-ene-isomerase produce progesterone from pregnenolone and metabolize fetal dehydroepiandrosterone sulfate to androstenedione, an estrogen precursor. The enzyme complex was solubilized from human placental microsomes using the anionic detergent, sodium cholate. Purification (500-fold, 3.9% yield) was achieved by ion exchange chromatography (Fractogel-TSK DEAE 650-S) followed by hydroxylapatite chromatography (Bio-Gel HT). The purified enzyme was detected as a single protein band in sodium dodecylsulfate-polyacrylamide gel electrophoresis (monomeric Mr = 19,000). Fractionation by gel filtration chromatography at constant specific enzyme activity supported enzyme homogeneity and determined the molecular mass (Mr = 76,000). The dehydrogenase and isomerase activities copurified. Kinetic constants were determined at pH 7.4, 37 degrees C for the oxidation of pregnenolone (Km = 1.9 microM, Vmax = 32.6 nmol/min/mg) and dehydroepiandrosterone (Km = 2.8 microM, Vmax = 32.0 nmol/min/mg) and for the isomerization of 5-pregnene-3,20-dione (Km = 9.7 microM, Vmax = 618.3 nmol/min/mg) and 5-androstene-3,17-dione (Km = 23.7 microM, Vmax = 625.7 nmol/min/mg). Mixed substrate analyses showed that the dehydrogenase and isomerase reactions use the appropriate pregnene and androstene steroids as alternative, competitive substrates. Dixon analyses demonstrated competitive inhibition of the oxidation of pregnenolone and dehydroepiandrosterone by both product steroids, progesterone and androstenedione. The enzyme has a 3-fold higher affinity for androstenedione than for progesterone as an inhibitor of dehydrogenase activity. Based on these competitive patterns of substrate utilization and product inhibition, the pregnene and androstene activities of 3 beta-hydroxy-5-ene-steroid dehydrogenase and steroid 5----4-ene-isomerase may be expressed at a single catalytic site on one protein in human placenta.     

Effect of verapamil on the aldosterone-stimulating properties of metoclopramide: in vitro and in vivo studies

The role of calcium in the regulation of aldosterone secretion has been recently clarified. Angiotensin II and potassium stimulate aldosterone secretion through a calcium-entry dependent mechanism, while ACTH action is both calcium and cyclic AMP dependent. To establish whether also the so-called aldosterone dopaminergic regulatory system is calcium-dependent we have studied, in vitro and in vivo, the effect of verapamil, a calcium entry blocker agent, on the aldosterone-stimulating properties of the antidopaminergic drug, metoclopramide. In the rat adrenal cells perfusion system, verapamil blocked both angiotensin II and metoclopramide-stimulated aldosterone. This effect on metoclopramide action seems to be present also in vivo in normal subjects: in fact aldosterone response was slightly but significantly reduced after pretreatment with verapamil. In conclusion the results suggest that also the dopaminergic system could regulate aldosterone secretion through calcium-mediated mechanisms.     

Topography of the subunits of Micrococcus lysodeikticus F1-ATPase

Summary The combined use of proteolytic digestion and lactoperoxidase catalyzed labelling with [¹²⁵I] applied to membrane-bound or soluble pure F₁-ATPase from Micrococcus lysodeikticus has allowed us to establish the topography of its , , and subunits within the protein molecule and with respect to the plane of the membrane.The subunit is most externally located to the membrane bilayer looking towards the cytoplasmic face, a position consistent with its proposed catalytic role. The and subunits lie in an intermediate layer between the subunits and the membrane, in which the subunit occupies a central position within the F₁-ATPase molecule in contact with the subunit. The subunit appears to be tightly bound to the F₀ component of the ATPase complex, probably buried in the membrane bilayer. A molecular arrangement of M. lysodeikticus ATPase is proposed that, taking into account the subunit stoichiometry _{3 3 2 2} (MW 420 000), accommodates the role assigned to each subunit and most, if not all, the known properties of this bacterial energy-transducing protein.     

Existence and uniqueness of a sharp travelling wave in degenerate non-linear diffusion Fisher-KPP equations

In this paper we use a dynamical systems approach to prove the existence of a unique critical value c ^* of the speed c for which the degenerate density-dependent diffusion equation u _ct = [D(u)u _x ] _x + g(u) has: 1. no travelling wave solutions for 0 < c < c ^*, 2. a travelling wave solution u(x, t) = (x - c ^* t) of sharp type satisfying (– ) = 1, () = 0 ^*; '(^*–) = – c ^*/D'(0), '(^*+) = 0 and 3. a continuum of travelling wave solutions of monotone decreasing front type for each c > c ^*. These fronts satisfy the boundary conditions (– ) = 1, '(– ) = (+ ) = '(+ ) = 0. We illustrate our analytical results with some numerical solutions.     

Effect of environmental pH on the fermentation balance of Lactobacillus reuteri

The influence of environmental pH on the regulation of glucose catabolism by Lactobacillus reuteri was examined in anaerobic batch cultures. Under acidic conditions both glucose consumption and end-products formation were low. Maximum biomass was reached at pH 5·0, with a specific growth rate of µ= 0·78 h^-1. The shift in pH values from 4.3 to 6.5 reflected an increase in glucose uptake as well as in the yield ( Y _p/x) of acetate, lactate and ethanol after 12 h of incubation. Ethanol was the major metabolite produced at all pH values assayed.     

Effects of oxygen level on metabolism and development of seedlings of Trapa natans and two ecologically related species

Seeds of the water plant Trapa natans L. (water chestnut) can germinate in strict anoxia. The seedlings show seminal roots growing upwards while shoot buds remain quiescent until O₂ becomes available. Trapa seedlings are highly tolerant to anoxia. The rate of ethanol fermentation was 21.2 μmol (g FW)⁻¹ h⁻¹, while production of lactate was negligible and lower than that of succinate. The seminal root of Trapa compares better to the rice coleoptile rather than to the rice root, both functionally and as to the metabolic response to anoxia. The anaerobic germination of Nuphar luteum L. and Scirpus mucronatus L. was also characterized by a limited developmental program.     

Immunochemistry of membrane F1-Adenosine triphosphatase fromMicrococcus lysodeikticus. Role of the alpha subunit

The membrane-bound ATPase activity from two substrains ofMicrococcus lysodeikticus, designated as A and B, was inhibited by antibodies raised against the two forms of purified F₁-ATPase. Form B of the enzyme, which behaved as a poorer immunogen than form A, also showed less reactivity as an antigen, independent of the physical state of the F₁-ATPase form. Antibodies were raised against the two major subunits ( and ) isolated fromM. lysodeikticus F₁-ATPase form A, which was the most stable form of the enzyme. Anti-(-subunit) serum strongly inhibited the ATPase activity of membrane-bound ATPase but showed little inhibition of the purified, soluble F₁-ATPase. The anti-(-subunit) serum inhibited the soluble F₁-ATPase, but to a lesser extent than the membrane-bound enzyme. In any event, the effect of anti- antibodies on the membrane-bound ATPase was smaller than that of anti- antibodies. It was postulated that the subunit ofM. lysodeikticus F₁-ATPase plays an essential and regulatory role in the expression of the immunochemical properties of the protein.     

Purification and some properties of NAD-glycohydrolase from conidia of Neurospora crassa

NAD-glycohydrolase from conidia of Neurospora crassa was purified by affinity chromatography, using 4-methylnicotinamide adenine dinucleotide as ligand immobilized onto Sepharose through a hydrophilic spacer arm. The pure enzyme is a glycoprotein with an isoelectric point of 5.5 and a molecular weight of 33 000 as determined by sodium dodecylsulphate gel electrophoresis. The specific activity is the highest so far found for NAD-glycohydrolases and in various aspects the enzyme is different from that isolated from mycelia of N. crassa grown in a 'zinc-deficient' medium.     

Luminescence properties of the Ln–EDTA complexes covalently linked to the chitosan biopolymers containing β‐diketonate as antenna ligands

This paper reports on the preparation, characterization, and photoluminescence properties of novel hybrid materials, in which the EDTA–Ln–L complexes (where L: H₂O, acac, bzac, dbm, and tta ligands, and Ln: Eu, Gd, and Tb) were covalently linked to the precursor medium molecular weight chitosan surface (CS) matrices or on the chitosan surfaces previously crosslinked with epichlorohydrin (CSech). The emission spectra of these materials were characterized by intraconfigurational‐4fN transitions centred on the Eu³⁺ and Tb³⁺ ions. Some broad bands from the polymeric matrix were also observed in the emission spectra, however the relative intensities of the intraconfigurational bands increased significantly for systems containing diketonate ligands when the antenna effect became more efficient. The values of the radiative rates (A_rad) were higher for crosslinked hybrid systems with epichlorohydrin, while nonradiative rates (A_nrad) presented the opposite behaviour. These data contributed to an increase in the values of emission quantum efficiency (η) for crosslinked materials. The effect of the modification process and antenna ligand on the values of intensities, intensity parameters Ω₂ e Ω₄ of the Eu³⁺ complexes were also investigated. The results showed that the crosslinked biopolymer surfaces have great potential for applications in molecular devices light converters.