The role of glutathione in the isomerization of delta 5-androstene-3,17-dione catalyzed by human glutathione transferase A1-1

Human glutathione transferase (GST) A1-1 efficiently catalyzes the isomerization of Delta(5)-androstene-3,17-dione (AD) into Delta(4)-androstene-3,17-dione. High activity requires glutathione, but enzymatic catalysis occurs also in the absence of this cofactor. Glutathione alone shows a limited catalytic effect. S-Alkylglutathione derivatives do not promote the reaction, and the pH dependence of the isomerization indicates that the glutathione thiolate serves as a base in the catalytic mechanism. Mutation of the active-site Tyr(9) into Phe significantly decreases the steady-state kinetic parameters, alters their pH dependence, and increases the pK(a) value of the enzyme-bound glutathione thiol. Thus, Tyr(9) promotes the reaction via its phenolic hydroxyl group in protonated form. GST A2-2 has a catalytic efficiency with AD 100-fold lower than the homologous GST A1-1. Another Alpha class enzyme, GST A4-4, is 1000-fold less active than GST A1-1. The Y9F mutant of GST A1-1 is more efficient than GST A2-2 and GST A4-4, both having a glutathione cofactor and an active-site Tyr(9) residue. The active sites of GST A2-2 and GST A1-1 differ by only four amino acid residues, suggesting that proper orientation of AD in relation to the thiolate of glutathione is crucial for high catalytic efficiency in the isomerization reaction. The GST A1-1-catalyzed steroid isomerization provides a complement to the previously described isomerase activity of 3beta-hydroxysteroid dehydrogenase.     

Conversion of 5 alpha-pregnane-3,20-dione, 3 alpha-hydroxy-5 alpha-pregnan-20-one and 3 beta-hydroxy-5 alpha-pregnan-20-one to delta 16-C19 steroids by the reconstituted delta 16-C19-steroid synthetase system

The substrate specificity of the reconstituted delta 16-C19-steroid synthetase system, which catalyzes the formation of 5,16-androstadien-3 beta-ol or 4,16-androstadien-3-one from pregnenolone or progesterone, respectively, was studied. The reconstituted system consisted of a partially purified cytochrome P-450, NADPH-cytochrome P-450 reductase, cytochrome b5 and NADH-cytochrome b5 reductase all from pig testicular microsomes. It was found that 5 alpha-reduced C21 steroids such as 5 alpha-pregnane-3,20-dione, 3 alpha-hydroxy-5 alpha-pregnan-20-one and 3 beta-hydroxy-5 alpha-pregnan-20-one can be substrates for the enzyme system, resulting in the formation of 5 alpha-androst-16-en-3-one, 5 alpha-androst-16-en-3 alpha-ol and 5 alpha-androst-16-en-3 beta-ol, respectively. The results suggest that 5 alpha-reduced delta 16-C19 steroids might be synthesized from pregnenolone and progesterone via 5 alpha-reduced C21 steroids as intermediates. The pathways would bypass 5,16-androstadien-3 beta-ol and 4,16-androstadien-3-one which have been assumed as obligatory intermediates in the formation of 5 alpha-reduced delta 16-C19 steroids from pregnenolone and progesterone.     

Contacts between gamma delta resolvase and the gamma delta res site.

We have investigated the interaction between resolvase and the res site of the transposon gamma delta by methylation and ethylation interference experiments. We have examined the effect of these DNA modifications both on binding and resolution in vitro. Major groove methylations within a 9 bp sequence that borders each site inhibit binding of resolvase to that site. Ethylation of certain phosphates within, and adjacent to, this border sequence inhibits binding. Together, these interference points define a contact region, present at all three res sites. In vitro resolution is inhibited only by modifications within site I. Inhibition of resolution by methylation of adenines at the center of site I suggests that minor groove contacts near the crossover may be required for resolution activity.     

The delta nifB (or delta nifE) FeMo cofactor-deficient MoFe protein is different from the delta nifH protein

We have examined three strains of Azotobacter vinelandii, which contain defined deletions within the nifH, nifB, or nifE genes. All three strains accumulate inactive FeMo cofactor-deficient forms of the MoFe protein of nitrogenase. These forms can be activated in vitro by addition of isolated FeMo cofactor in N-methylformamide. Although the phenotypes of these strains are superficially the same, our characterizations demonstrate that the FeMo cofactor-deficient MoFe protein synthesized by the delta nifH strain is quite different from that synthesized by either the delta nifB or delta nifE strains. These differences include the following: 1) the activation of the delta nifH protein requires MgATP, whereas the activation of the delta nifB and delta nifE proteins does not; 2) the delta nifH extracts can be activated with FeMo cofactor to wild-type levels of activity, whereas delta nifB and delta nifE extracts cannot; 3) the delta nifH protein is markedly less heat stable than the delta nifB and delta nifE proteins; and 4) the migration of the delta nifH protein on native gels is very different when compared with delta nifB and delta nifE, which look like each other. These data can be explained if the nifB and nifE gene products are only involved in FeMo cofactor biosynthesis, whereas the nifH gene product is involved in both the initial synthesis of FeMo cofactor and in the insertion of preformed FeMo cofactor into the MoFe protein. A model is presented that suggests that the FeMo cofactor-deficient MoFe protein synthesized by the delta nifH strain is the one that normally participates in MoFe protein assembly in wild-type cells.     

Characterization of the phosphorylated forms and the phosphorylated residues of hepatitis delta virus delta antigens

Hepatitis delta virus (HDV) replication requires both the cellular RNA polymerase and one virus-encoded protein, small delta antigen (S-HDAg). S-HDAg has been shown to be a phosphoprotein, but its phosphorylation status is not yet clear. In this study, we employed three methods to address this question. A special two-dimensional gel electrophoresis, namely, nonequilibrium pH gradient electrophoresis, was used to separate the very basic S-HDAg. By carefully adjusting the pH of solubilization solution, the ampholyte composition, and the appropriate electrophoresis time periods, we were able to clearly resolve S-HDAg into two phosphorylated isoforms and one unphosphorylated form. In contrast, the viral large delta antigen (L-HDAg) can only be separated into one phosphorylated and one unphosphorylated form. By metabolic (32)P labeling, both immunoprecipitated S-HDAg and L-HDAg were found to incorporate radioactive phosphate. The extent of S-HDAg phosphorylation was increased upon 12-O-tetradecanoylphorbol-13-acetate treatment, while that of L-HDAg was not affected. Finally, phosphoamino acid analysis identified serine and threonine as the phospho residues in the labeled S-HDAg and only serine in the L-HDAg. Therefore, HDV S- and L-HDAgs differ in their phosphorylation patterns, which may account for their distinct biological functions.     

Significance of the delta 5 and delta 4 steroidogenic pathways in the hamster preovulatory follicle

Isolated hamster granulosa cells and theca from preovulatory follicles were incubated in vitro for 2 and 6 h in the absence/or presence of LH and steroid substrates. The purpose of the experiments was to determine, in theca, the relative activities of the delta 5 and delta 4 pathways under controlled conditions, and to compare the ability of granulosa cells and theca to form progesterone from exogenous pregnenolone. The results of the experiments show that the delta 5 pathway in theca predominates before and up to 2 h after LH stimulation. The delayed effect of LH after 2 h is a switch from delta 5 to delta 4 as the major metabolic pathway. Progesterone formation from exogenous pregnenolone is 7 to 10 times greater in unstimulated granulosa cells than in theca. Acute effects of LH lead to increased conversion of exogenous pregnenolone to progesterone in granulosa cells but not theca. LH does, however, acutely stimulate the thecal conversion of DHEA to androstenedione. The longer term effect of LH in both cell types is to increase pregnenolone conversion to progesterone.     

A single antigenomic open reading frame of the hepatitis delta virus encodes the epitope(s) of both hepatitis delta antigen polypeptides p24 delta and p27 delta.

On the basis of the complete nucleotide sequence of the single-stranded, covalently closed circular hepatitis delta virus RNA genome (K.-S. Wang, Q.-L. Choo, A. J. Weiner, J.-H. Ou, R. C. Najarian, R. M. Thayer, G. T. Mullenbach, K. J. Denniston, J. L. Gerin, and M. Houghton, Nature [London] 323:508-514, 1986 [Author's correction, 328:456, 1987]), five long open reading frames (ORFs) encoding polypeptides containing a methionine proximal to the amino terminus were expressed in bacteria. Only polypeptides encoded by the antigenomic ORF5 cross-reacted with antisera obtained from patients with hepatitis delta virus infections. Immunological analysis of viral extracts and the recombinant ORF5 polypeptides synthesized in bacteria and yeast cells revealed that ORF5 encodes the immunogenic epitope(s) shared by both hepatitis delta viral polypeptides p27 delta and p24 delta and probably represents the complete structural gene for p27 delta and p24 delta. We also present evidence that ORF5 encodes the hepatitis delta antigen, an antigen originally found in the nuclei of hepatocytes of infected individuals (M. Rizzetto, M. G. Canese, S. Arico, O. Crivelli, F. Bonino, C. G. Trepo, and G. Verme, Gut 18:997-1003, 1977). A comparison of the primary structure of the predicted hepatitis delta antigen polypeptides with that of the core antigen of the hepatitis B virus shows that these polypeptides are very dissimilar.     

Immunochemical evidence for the enzymatic difference of delta 6-desaturase from delta 9- and delta 5-desaturase in rat liver microsomes

The enzymatic properties of the three types of microsomal acyl-CoA desaturases, delta 6-, delta 9- and delta 5-desaturases, were immunologically compared using a monospecific antibody raised against the purified linoleoyl-CoA desaturase (delta 6-desaturase). By the double immunodiffusion technique, the anti-delta 6-desaturase antibody showed a single precipitin line to the purified delta 6-desaturase and microsomes treated with Triton X-100, but no line was observed with the partially purified delta 9-desaturase. The antibody even inhibited definitely delta 6-desaturase activity in microsomes, but neither stearoyl-CoA (delta 9-) nor eicosatrienoic acid (delta 5-) desaturations were inhibited. By these immunological investigations it was confirmed that terminal delta 6-desaturase is different enzyme from desaturases delta 9- and delta 5.     

Packaging of hepatitis delta virus RNA via the RNA-binding domain of hepatitis delta antigens: different roles for the small and large delta antigens.

Hepatitis delta virus (HDV) is composed of four specific components. The first component is envelope protein which contains hepatitis B surface antigens. The second and third components are nucleocapsid proteins, referred to as small and large hepatitis delta antigens (HDAgs). The final component is a single-stranded circular RNA molecule known as the viral genome. In order to study the mechanism of HDV RNA packaging, a four-plasmid cotransfection system in which each viral component was provided by a separate plasmid was employed. Virus-like particles released from Huh-7 cells receiving such a cotransfection were found to contain HDV RNA along with three proteins. Therefore, the four-plasmid cotransfection system could lead to successful HDV RNA packaging in vitro. The system was then used to show that the large HDAg alone was able to achieve a low level of HDV RNA packaging. Analysis of a variety of large HDAg mutants revealed that the RNA-binding domain was essential for viral RNA packaging. By increasing the incorporation of small HDAg into virus-like particles, we found a three- to fourfold enhancement of HDV RNA packaging. This effect was probably through a direct binding of HDV RNA, independent from that of large HDAg, with the small HDAg. The subsequent RNA-protein complex was packaged into particles. The results provided insight into the roles and functional domains of small and large HDAgs in HDV RNA packaging.     

Human placental 3beta-hydroxysteroid dehydrogenase: delta5-isomerase. Demonstration of an intermediate in the conversion of 3beta-hydroxypregn-5-en-20-one to pregn-4-ene-3,20-dione.

3beta-Hydroxypregn-5-en-20-one (pregnenolone) and NAD+ were incubated with a solubilized preparation of the coupled enzyme 3beta-hydroxysteroid:NAD(P) oxidoreductase-3-ketosteroid delta4,delta5-isomerase (3beta-hydroxysteroid dehydrogenase: delta5-isomerase) from the mitochondrial fraction of human placenta. Unconverted pregnenolone, pregn-4-ene-3,20-dione (rogesterone), and a small but detectable amount of pregn-5-ene-3,20-dione were isolated from the medium by Sephadex LH-20 chromomatography. The identification of pregn-5-ene-3,20-dione, confirmed by mass fragmentography, has provided the first direct evidence for the formation of the hypothetical delta5,3-ketone intermediate in the conversion of pregnenolone to progesterone. When tritium-labeled pregnenolone and [4-14C]pregnenolone were incubated simultaneously the 3H:14C ratio in isolated pregn-5-ene-3,20-dione was 4.6 times greater than in isolated progesterone and pregnenolone, indicating a kinetic isotope effect in the enzymatic isomerization of tritium-labeled pregn-5-ene-3,20-dione. Exposure of the enzyme to two steroids which inhibit the overall enzyme reaction, 2alpha-cyano-17beta-hydroxy-4,4,17alpha-trimethylandrost-5-en-3-one (cyanoketone) and 3-hydroxyestra-1,3,5(10),6,8-pentaen-17-one (equilenin), increased the relative yield of labeled pregn-5-ene-3,20-dione as well as the recovery of radioactivity remaining as unconverted pregnenolone, suggesting that both the dehydrogenase and isomerase activities were inhibited. Exposure of the enzyme to equilenin increased the ratio of isolated pregn-5-ene-3,20-dione radioactivity to progesterone radioactivity as progesterone synthesis was inhibited. Equilenin also diminished the tritium isotope effect on the isomerase reaction. Both findings suggest that it is possible to inhibit the isomerase to a greater extent than the dehydrogenase. In order to measure the rate of progesterone produced by the coupled enzymes, we have modified a radiochemical method which involves precipitation of pregnenolone by digitonin. Digitonin precipitation proved to be effective in separating unconverted pregnenolone from the steroid products of both enzyme reactions, progesterone and pregn-5-ene-3,20-dione. Neither the steroidal inhibitors nor the kinetic isotope effect altered the accuracy of the method for routine measurement of the overall rate of conversion of delta5,3beta-hydroxysteroid to delta4,3-ketosteroid.