Redox regulation of yeast flavin-containing monooxygenase

The flavin-dependent monooxygenase from yeast (yFMO) oxidizes biological thiols such as cysteine, cysteamine, and glutathione. The enzyme makes a major contribution to the pools of oxidized thiols that, together with reduced glutathione from glutathione reductase, create the optimum cellular redox environment. We show that the activity of yFMO, as a soluble enzyme or in association with the ER membrane of microsomal fractions, is correlated with the redox potential. The enzyme is active under conditions normally found in the cytoplasm, but is inhibited as GSSG accumulates to give a redox potential similar to that found in the lumen of the ER. Site-directed mutations show that Cys 353 and Cys 339 participate in the redox regulation. Cys 353 is the principal residue in the redox-sensitive switch. We hypothesize that it may initiate formation of a mixed disulfide that is partially inhibitory to yFMO. The mixed disulfide may exchange with Cys 339 to form an intramolecular disulfide bond that is fully inhibitory.     

Interactions of iron-thiol-nitrosyl compounds with the phosphoroclastic system of Clostridium sporogenes

Certain reagents, such as ascorbate or iron salts and thiols, enhance the bacteriostatic action of nitrite on food-spoilage bacteria. This may be due to the formation of nitric oxide and iron-thiol-nitrosyl [( Fe-S-NO]) complexes. The minimum concentrations of these reagents required to inhibit growth of Clostridium sporogenes were investigated. A mixture of nitrite (0.72 mM) with iron (1.44 mM) and cysteine (2.16 mM) was found to be extremely inhibitory when autoclaved and diluted into the culture medium. This mixture caused rapid cessation of growth and loss of cell viability at a final concentration corresponding to 40 microM-nitrite. If added to the initial culture medium, it prevented growth at 5 microM-nitrite. The mixture was more inhibitory, on the basis of the nitrite concentration used, than the 'Perigo factor', obtained by autoclaving nitrite in growth medium. [Fe-S-NO] compounds of known chemical structure were tested to determine if they were responsible for this effect. Total inhibition of cell growth was observed with the tetranuclear clusters [Fe4S3(NO)7] (Roussin's black salt), [Fe4S4(NO)4] or [Fe4Se3(NO)7], added at concentrations equivalent to 10 microM-nitrite, or with [Fe2(SMe)2(NO)4] (methyl ester of Roussin's red salt), equivalent to 200 microM-nitrite. The rate of hydrogen production in growing cell cultures was inhibited by [Fe4S3(NO)7] at levels equivalent to 2.5 microM-nitrite. EPR spectra of the inhibited cells showed features with g-values of 2.03, characteristic of mononuclear iron-nitrosyl species, and, under non-reducing conditions, an unusual signal at g = 1.65. There was no correlation between growth inhibition and the g = 2.03 signal, though there was a better correlation between inhibition and the g = 1.65 signal. The direct effects of the compounds were tested on the iron-sulphur proteins of the phosphoroclastic system, namely ferredoxin, pyruvate-ferredoxin oxidoreductase and hydrogenase. EPR spectra and enzyme assays showed that these proteins were not destroyed by [Fe4S3(NO)7], [Fe4S4(NO)4], [Fe2(SMe)2(NO)4], [Fe(SPh)2(NO)2], or M2 (an autoclaved mixture of 66 mM-cysteine, 3.6 mM-FeSO4 and 0.72 mM-NaNO2) at concentrations higher than those that caused total inhibition of cell growth. Inhibition of cells by [Fe-S-NO] compounds is unlikely to be due to interaction with the preformed enzymes. The possible formation of iron-nitrosyl complexes in vivo, and their inhibitory actions, are discussed.     

CO2.- radical induced cleavage of disulfide bonds in proteins. A gamma-ray and pulse radiolysis mechanistic investigation

Disulfide bond reduction by the CO2.- radical was investigated in aponeocarzinostatin, aporiboflavin-binding protein, and bovine immunoglobulin. Protein-bound cysteine free thiols were formed under gamma-ray irradiation in the course of a pH-dependent and protein concentration dependent chain reaction. The chain efficiency increased upon acidification of the medium, with an apparent pKa around 5, and decreased abruptly below pH 3.6. It decreased also at neutral pH as cysteine accumulated. From pulse radiolysis analysis, CO2.- proved able to induce rapid one-electron oxidation of thiols and of tyrosine phenolic groups in addition to one-electron donation to exposed disulfide bonds. The bulk rate constant of CO2.- uptake by the native proteins was 5- to 10-fold faster at pH 3 than at pH 8, and the protonated form of the disulfide radical anion, [symbol: see text], appeared to be the major protein radical species formed under acidic conditions. The main decay path of [symbol: see text] consisted of the rapid formation of a thiyl radical intermediate [symbol: see text] in equilibrium with the closed, cyclic form. The thiyl radical was subsequently reduced to the sulfhydryl level [symbol: see text] on reaction with formate, generating 1 mol of the CO2.- radical, thus propagating the chain reaction. The disulfide radical anion [symbol: see text] at pH 8 decayed through competing intramolecular and/or intermolecular routes including disproportionation, protein-protein cross-linking, electron transfer with tyrosine residues, and reaction with sulfhydryl groups in prereduced systems. Disproportionation and cross-linking were observed with the riboflavin-binding protein solely. Formation of the disulfide radical cation [symbol: see text], phenoxyl radical Tyr-O. disproportionation, and phenoxyl radical induced oxidation of preformed thiol groups should also be taken into consideration to explain the fate of the oxygen-centered phenoxyl radical.     

Radical reactions in aqueous disulphide-thiol systems

Absolute rate constants have been measured for the reaction of (CH3SSCH3)+. and sulphur centred radical cations of lipoic acid, lip (SS)+., with various thiols including penicillamine, cysteamine and cysteine. Under pulse radiolysis conditions no reactions was observed between the disulphide radical cations and the neutral thiols, RSH, i.e. kappa less than or equal to 10(7) M-1 s-1. Rate constants in the order of 10(9) M-1 s-1, i.e. close to the diffusion controlled limit, were, however, found for the corresponding reactions with the thiolates, RS-. In systems containing lipoate and cysteamine the lip (S therefore S)+. induced oxidation of CyaS- proceeds via CyaS., (CyaS therefore SCya)- and lip (S S)- as intermediates, i.e. results in a cysteamine mediated conversion of an oxidizing lip (S therefore S)+. radical cation to a reducing lip (S S)- radical anion along the reaction route. In other cases the reaction of disulphide radical cations with thiolate anions was found to proceed via an optically absorbing transient (lambda max approximately 380 nm) which is suggested to be an adduct radical. The mechanism of the (RSSR)+. induced oxidation of thiolate appears to depend on the stability of the 3-electron bonded disulphide radical anion.     

Oxidized LDL activates phospholipase A2 to supply fatty acids required for cholesterol esterification

We examined the roles of phospholipase A2 (PLA2) in oxidized LDL (oxLDL)-induced cholesteryl ester formation in macrophages. In [3H]oleic acid-labeled RAW264.7 cells and mouse peritoneal macrophages, oxLDL induced [3H]cholesteryl oleate formation with an increase in free [3H]oleic acid and a decrease in [3H]phosphatidylcholine. The changes in these lipids were suppressed by methyl arachidonyl fluorophosphonate (MAFP), a cytosolic PLA2 (cPLA2) inhibitor. However, MAFP had no effect on the ACAT activity or the binding and/or uptake of oxLDL. Stimulation with oxLDL in the presence of [3H]cholesterol increased [3H]cholesteryl ester bearing fatty acyl chains derived from cellular and/or exogenous (oxLDL) lipids. The formation of cholesteryl ester under this condition was also inhibited by MAFP, and the inhibitory effect was reversed by adding oleic acid. While oxLDL did not affect the activity or amounts of cPLA2, preincubation with oxLDL enhanced the release of oleic acid and arachidonic acid induced by ionomycin in RAW264.7 cells. 13(S)-hydroxyoctadecadienoic acid, but not 7-ketocholesterol, also enhanced ionomycin-induced oleic acid release. These results suggest that oxLDL induces cPLA2 activation, which contributes, at least in part, to the supply of fatty acids required for the cholesteryl esterification, probably through the acceleration by oxidized lipids of the catalytic action of cPLA2 in macrophages.     

Involvement of SH-groups in the regulation of adenylate cyclase signal system by isoproterenol and serotonin in cultured murine fibroblasts L (subline LSM)

The adenylyl cyclase system (ACS) plays a key role in transduction of a hormonal signal into eukaryotic cells. The functional activity of the system depends on SH-groups of proteins involved in the ACS: receptor, G-protein, and enzyme adenylyl cyclase (AC). We studied the influence of thiols and SH-blockers on the regulation of AC activity by nonhormonal (NaF and Gpp[NH]p) and hormonal (biogenic amines isoproterenol and serotonin) agents in homogenates of cultured murine fibroblasts of line L (subline LSM). In the presence of thiols 2-mercaptoethanol (5 mM) and dithiothreitol (1 mM) the basal AC activity somewhat increased, whereas the stimulating effects of NaF, Gpp[NH]p, and hormones decreased. No potentiating action of Gpp[NH]p on hormonal effect in this case was found. The SH-blockers 25 mkM p-chloromercuribenzoic acid (CMBA) and 0.2 mM N-ethylmaleimide significantly inhibited both the basal AC activity and that stimulated by different agents. Thiols partially restored CMBA inhibited AC activity (in the case of N-ethylmaleimide restoring effects of thiols were insignificant). This, the ACS of murine fibroblasts of subline LSM is SH-sensitive. The forms of SH-groups in proteins involved in the ACS determine their functional activities and a possibility of transduction of the hormonal signal on the effector systems.     

Regulation of binding of subfragment 1 in isolated rigor myofibrils

A steric-hindrance model has been used to explain the regulation of muscle contraction by tropomyosin-troponin complex. The regulation of binding was studied by microscopic observation of mixtures of fluorescent subfragment 1 (S1) with rigor myofibrils at different actin- to-S1 ratios and in the presence and absence of calcium. Procedures were adapted to protect the critical thiols of S1 before conjugation to thiol-specific fluorochromes, this giving fluorescent S1 with unaltered enzyme activity. S1 binding was greatest in the I band (except at the Z- lines) in the presence of calcium regardless of the [S1]. The patterns in the absence of calcium depended on the actin-to-S1 ratios: low [S1], binding in the myosin-actin overlap region; intermediate [S1], highest binding at the A-I junction; high [S1], greatest binding in the I-band. The two distinct binding patterns observed at low [S1] were demonstrated by dual-channel fluorescence microscopy when myofibrils were sequentially incubated with fluorescent S1 without calcium followed by a different fluorescent S1 with calcium. These observations support the concept of rigor activation of actin sites. The change in the pattern upon increasing [S1] without calcium demonstrate cooperative interactions along the thin filament. However, these interactions (under the conditions used without calcium) do not appear to extend over greater than 2-3 tropomyosin-troponin-7 actin functional units.     

Regulation of the chromaffin granule aggregating activity of annexin I by phosphorylation.

Annexin I (lipocortin I) binds to secretory granule membranes and promotes their aggregation in a Ca(2+)-dependent manner [Creutz, C. E., et al. (1987) J. Biol. Chem. 262, 1860-1868; Drust, D. S., & Creutz, C. E. (1988) Nature 331, 88-91]. It is also phosphorylated on serine residues when bovine chromaffin cells are stimulated to secrete [Michener, M. L., et al. (1986) J. Biol. Chem. 261, 6548-6555], suggesting phosphorylation may be involved in modulating the function of annexin I. We report here that phosphorylation of the N-terminal tail by protein kinase C strongly inhibits the ability of annexin I to aggregate chromaffin granules by increasing the calcium requirement 4-fold. This inhibition was readily reversed when the protein was dephosphorylated by protein phosphatase 2A. The inhibition was not due to inability of phosphorylated annexin I to bind to chromaffin granules, since the phosphorylated form bound to the granule membrane at slightly lower levels of calcium than the native form. The phosphorylated annexin I also bound to 20% phosphatidylserine/80% phosphatidylcholine vesicles at lower Ca2+ levels than the native form. The inhibitory effect of phosphorylation on the granule aggregating activity of annexin I was found to be amplified by an unusual mechanism: The phosphorylated form inhibited the activity of the unphosphorylated form. The possible importance of the regulation of annexin I activity by phosphorylation in exocytosis is discussed.     

Purification and properties of a recombinant sulfur analog of murine selenium-glutathione peroxidase.

We previously constructed plasmids for synthesis of glutathione-peroxidase (GPx) mutants in an Escherichia coli expression system. In these recombinant proteins either cysteine ([Cys]GPx mutant) or serine ([Ser]GPx mutant) were present in place of the active-site selenocysteine (SeCys) of the natural enzyme. We have now investigated GPx activity of [Cys]GPx and [Ser]GPx mutants. Enzyme assays performed on preparations of these partially purified proteins demonstrated that the [Cys]GPx mutant exhibited a significant GPx activity, unlike the [Ser]GPx mutant. Purification of [Cys]GPx was performed in two steps of ion-exchange chromatography giving a 98% homogenous protein in 50% yield. The purified [Cys]GPx protein was shown to be a symmetrical tetramer by the means of gel-filtration HPLC and SDS/PAGE. Two isoelectric points were found (6.8 and 7.2) which may reflect two different oxidation states of the mutant protein. The GPx activity of the [Cys]GPx mutant was optimal at pH 8.5. The [Cys]GPx mutant had a specific activity approximately 1000-fold smaller than that of the natural enzyme, and was very easily inactivated by hydroperoxides. Inhibition of the activity with iodoacetate determined a pKa of 8.3, presumably that of the active-site cysteine. Unlike that of SeGPx, the GPx activity of [Cys]GPx was only slightly inhibited by mercaptosuccinate. We discuss hypothetical mechanistic constraints of either catalytic cycle, which may explain such results.     

Cholesteryl ester hydrolysis in rat liver lysosomes: different response to female sex hormones

The regulation of the hydrolysis of cholesteryl oleate by female sex hormones was studied in the lysosomal fraction of rat liver. Cholesterol ester hydrolase activity was determined at pH 5.0 with an acetone-dissolved cholesteryl [1-14C]oleate substrate preparation. The administration of a single dose of progesterone decreased the enzyme activity during a 3- to 24-hr period following hormone injection. This effect was not correlated to changes in the lysosomal protein synthesis rate. The lysosomal hydrolysis of cholesteryl esters was also inhibited in a noncompetitive manner by the addition of progesterone at concentrations higher than 100 microM. The esterase failed to respond to the estradiol in vivo as well as in vitro. The findings of the present paper suggest that the lysosomal breakdown of cholesteryl esters in rat liver may be under selective hormonal regulation and that the inhibitory effect of progesterone on the enzyme activity might be, at least in part, responsible for the liver cholesterol ester accumulus produced by the administration of the hormone.     

Dual role of cysteine 172 in redox regulation of ribulose 1,5-bisphosphate carboxylase/oxygenase activity and degradation

Alkylation and oxidation of cysteine residues significantly decrease the catalytic activity and stimulate the degradation of ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO). We analyzed the role of vicinal cysteine residues in redox regulation of RuBisCO from Synechocystis sp. strain PCC 6803. Cys172 and Cys192, which are adjacent to the catalytic site, and Cys247, which cross-links two large subunits, were replaced by alanine. Whereas all mutant cells (C172A, C192A, C172A-C192A, and C247A) and the wild type grew photoautotrophically at similar rates, the maximal photosynthesis rates of C172A mutants decreased 10 to 20% as a result of 40 to 60% declines in RuBisCO turnover number. Replacement of Cys172, but not replacement of Cys192, prominently decreased the effect of cysteine alkylation or oxidation on RuBisCO. Oxidants that react with vicinal thiols had a less inhibitory effect on the activity of either the C172A or C192A enzyme variants, suggesting that a disulfide bond was formed upon oxidation. Thiol oxidation induced RuBisCO dissociation into subunits. This effect was either reduced in the C172A and C192A mutant enzymes or eliminated by carboxypentitol bisphosphate (CPBP) binding to the activated enzyme form. The CPBP effect presumably resulted from a conformational change in the carbamylated CPBP-bound enzyme, as implied from an alteration in the electrophoretic mobility. Stress conditions, provoked by nitrate deprivation, decreased the RuBisCO contents and activities in the wild type and in the C192A and C247A mutants but not in the C172A and C172A-C192A mutants. These results suggest that although Cys172 does not participate in catalysis, it plays a role in redox regulation of RuBisCO activity and degradation.     

Human somatotropin 53. Synthesis and biological activity of the amino terminal fifty-four residue fragment

The amino terminal 54 residue peptide fragment of human somatotropin [Cys(Gam)53]-HGH-(1-54), has been synthesized by the solid-phase method. The symmetrical anhydride and active ester coupling methods were used exclusively. The synthetic product was purified by gel fitration isoelectric focusing, and partition chromatography. It was found to be homogeneous by six additional criteria. In complement fixation experiments the synthetic product was immunologically active with antisera to HGH and [cys(Cam)53]-HGH-(134). Antiserum raised against the synthetic product was immunologically active in the homologous assay and with HGH,[Cys(Cam)53]-HGH-(1-134), and [Cys(Cam)53]-HGH-(15-125). The synthetic fragment exhibited 53% of the activity of [Cys(Cam)53]-HGH-(1-134) in the rat tibia assay.     

Encapsulation of platinum(II)-based DNA intercalators within cucurbit[6,7,8]urils

The partial encapsulation of platinum(II)-based DNA intercalators of the type [Pt(5-Cl-phen)(ancillary ligand)](2+), where 5-Cl-phen is 5-chloro-1,10-phenanthroline and the ancillary ligand is ethylenediamine, (1S,2S)-diaminocyclohexane (S,S-dach) or (1R,2R)-diaminocyclohexane, within cucurbit[n]uril (CB[n], where n is 6, 7 or 8) has been examined by (1)H and (195)Pt NMR and mass spectrometry. For CB[7], the molecule encapsulates over the ancillary ligand of all metal complexes, whether this is ethylenediamine or diaminocyclohexane. For CB[8], encapsulation occurs over the sides of the 5-Cl-phen ligand at low [Pt(5-Cl-phen)(S,S-dach)](2+) (5CLSS) to CB[8] ratios (i.e. 0.25:1) but over the ancillary ligand at higher ratios (i.e. 2:1). For CB[6] binding, 5CLSS exhibits both portal and cavity binding, with the ancillary ligand displaying chemical shifts consistent with fast exchange kinetics on the NMR timescale for portal binding and slow exchange kinetics for cavity binding. Binding constants could not be determined using UV-vis, circular dichroism or fluorescence spectrophotometry, but a binding constant for binding of 5CLSS to CB[6] of approximately 10(5) M(-1) was determined using (1)H NMR. Finally, the effect of CB[n] encapsulation on the cytotoxicity of the metal complexes was examined using L1210 murine leukaemia cells in vitro growth inhibition assays. The cytotoxicity is highly dependent on both the metal complex and the CB[n] size, and whilst CB[7] and CB[8] generally decreased cytotoxicity, it was found that CB[6] increased the cyotoxicity of 5CLSS up to 2.5-fold.     

Beta-endorphin. Synthesis and biological activity of analogs with disulfide bridges

Two analogs of human beta-endorphin (beta-EP) which contain cystine bridges, [Cys15-Cys26,Phe27,Gly31]-beta-EP (I) and [Cys16-Cys26,Phe27,Gly31]-beta-EP (II), were synthesized by the solid-phase method. Peptides I and II were shown to contain 2-2.5 times the opiate receptor binding activity of beta-endorphin. We also synthesized two analogs with reduced alkylated cysteine residues and these peptides, [Arg9,19,24,28,29 Cys(Cam)11,26,Phe27,Gly31] and [Arg9,19,24,28,29,Cys-(Cam)12,26,Phe27,Gly31], were shown to have approximately the same opiate receptor activity as beta-endorphin.     

Implication of the disulfide bridge in trypsin inhibitor SFTI-1 in its interaction with serine proteinases

Fourteen monocyclic analogues of trypsin inhibitor SFTI-1 isolated from sunflower seeds were synthesized by the solid-phase method. The purpose of this work was to establish the role of a disulfide bridge present in inhibitor’s side chains of Cys3 and Cys11 in association with serine proteinases. This cyclic fragment was replaced by the disulfide bridges formed by l-pencillamine (Pen), homo-l-cysteine (Hcy), N-sulfanylethylglycine (Nhcy) or combination of the three with Cys. As in the substrate specificity the P₁ position of the synthesized analogues Lys, Nlys [N-(4-aminobutyl)glycine], Phe or Nphe (N-benzylglycine) were present, and they were checked for trypsin and chymotrypsin inhibitory activity. The results clearly indicated that Pen and Nhcy were not acceptable at the position 3, yielding inactive analogues, whereas another residue (Cys11) could be substituted without any significant impact on the affinity towards proteinase. On the other hand, elongation of the Cys3 side chain by introduction of Hcy did not affect inhibitory activity, and an analogue with the Hcy–Hcy disulfide bridge was more than twice as effective as the reference compound ([Phe⁵] SFTI-1) in inhibition of bovine α-chymotrypsin.     

Mapping of the mastoparan-binding site on G proteins. Cross-linking of [125I-Tyr3,Cys11]mastoparan to Go

Mastoparan (MP) activates GTP-binding regulatory proteins (G proteins) by promoting GDP/GTP exchange through a mechanism similar to that of G protein-coupled receptors (Higashijima, T., Burnier, J., and Ross, E. M. (1990) J. Biol. Chem. 265, 14176-14186). [Tyr3, Cys11]MP was synthesized and shown to have regulatory activity similar to that of mastoparan when assayed in the presence of dithiothreitol (DTT). Activation by [Tyr3,Cys11]MP in the absence of DTT was complex in its kinetics, concentration dependence, and dependence on detergents. [125I-Tyr3,Cys11]MP bound covalently to the alpha subunit of G proteins. Cross-linking was blocked by mastoparan or [Tyr3,Cys11]MP. Cross-linking was enhanced by the addition of beta gamma subunits, but no cross-linking to beta gamma subunits was observed. Cross-linking was inhibited by incubation of Go with guanosine 5'-O-(thiotriphosphate) and Mg2+ and was reversed by incubation with DTT or 2-mercaptoethanol. Stoichiometry of labeling was consistent with the cross-linking of one molecule of [125I-Tyr3,Cys11]MP/alpha subunit, and CNBr hydrolysis of the [125I-Tyr3,Cys11]MP-alpha o adduct yielded one major labeled peptide fragment of approximately 6 kDa. Amino acid sequencing of this CNBr fragment prepared from recombinant alpha o showed that cross-linking occurred at Cys3. No alpha o sequence was obtained from the same fragment prepared from bovine brain alpha o, which is blocked by a myristoyl group at Gly2. Regulation of Go by MP was eliminated by tryptic proteolysis of the amino-terminal region. These observations suggest that the amino-terminal region of G protein alpha subunits contributes to the mastoparan-binding site, which may also be the receptor-binding site, and is involved in regulation of nucleotide exchange.     

Effects of novel 17alpha-hydroxylase/C17, 20-lyase (P450 17, CYP 17) inhibitors on androgen biosynthesis in vitro and in vivo

Aiming at the development of new drugs for the treatment of prostate cancer, the effects of steroidal compounds and one non-steroidal substance on androgen biosynthesis were evaluated in vitro and in vivo. Sa 40 [17-(5-pyrimidyl)androsta-5,16-diene-3beta-ol], its 3-acetyl derivate Sa 41 and BW 19 [3,4-dihydro-2-(4-imidazolylmethyl)-6-methoxy-1-methyl-naphthalene] are compounds from our group, which have been developed as inhibitors of CYP 17 (17alpha-hydroxylase-C17, 20-lyase, the key enzyme in androgen biosynthesis). They have been compared with CB 7598 [abiraterone: 17-(3-pyridyl)androsta-5,16-diene-3beta-ol], its 3-acetyl compound CB 7630 and ketoconazole, compounds which already have been used clinically. The most potent compound toward human CYP 17 (testicular microsomes) was Sa 40 (IC(50) value of 24 nM), followed by Sa 41, CB 7598, BW 19, CB 7630 and ketoconazole. Sa 40 shows a type II difference spectrum and a non-competitive type of inhibition (K(i) value of 16 nM). No recovery of enzyme activity was observed after preincubation of CYP 17 with Sa 40 and subsequent charcoal treatment. In Escherichia coli cells coexpressing human CYP 17 and NADPH-P450 reductase, Sa 40 was more active than CB 7598 and BW 19, whereas the acetyl compounds were not active. The latter three compounds were equally active towards rat CYP 17. Male Sprague-Dawley (SD) rats were administered daily for 14 days BW 19 and the acetyl derivatives Sa 41 and CB 7630 as prodrugs (0.1 mmol/kg intraperitoneally). The test compounds strongly reduced plasma testosterone concentration, as well as prostate and seminal vesicles weights. They showed moderate inhibitory effects on the weights of levator ani, bulbocavernosus and testes, whereas they led to an increase in adrenal and pituitary weights. The only exception was BW 19 which did not change pituitary weights. Based on its superiority on the human enzyme, it was concluded that Sa 40 in its 3beta-acetate form (Sa 41) could be a promising candidate for clinical evaluation.     

Evidence for the participation of Cys558 and Cys559 at the active site of mercuric reductase

Mercuric reductase, with FAD and a reducible disulfide at the active site, catalyzes the two-electron reduction of Hg(II) by NADPH. Addition of reducing equivalents rapidly produces a spectrally distinct EH2 form of the enzyme containing oxidized FAD and reduced active site thiols. Formation of EH2 has previously been reported to require only 2 electrons for reduction of the active site disulfide. We present results of anaerobic titrations of mercuric reductase with NADPH and dithionite showing that the equilibrium conversion of oxidized enzyme to EH2 actually requires 2 equiv of reducing agent or 4 electrons. Kinetic studies conducted both at 4 degrees C and at 25 degrees C indicate that reduction of the active site occurs rapidly, as previously reported [Sahlman, L., & Lindskog, S. (1983) Biochem. Biophys. Res. Commun. 117, 231-237]; this is followed by a slower reduction of another redox group via reaction with the active site. Thiol titrations of denatured Eox and EH2 enzyme forms show that an additional disulfide is the group in communication with the active site. [14C]Iodoacetamide labeling experiments demonstrate that the C-terminal residues, Cys558 and Cys559, are involved in this disulfide. The fluorescence, but not the absorbance, of the enzyme-bound FAD was found to be highly dependent on the redox state of the C-terminal thiols. Thus, Eox with Cys558 and Cys559 as thiols exhibits less than 50% of the fluorescence of Eox where these residues are present as a disulfide, indicating that the thiols remain intimately associated with the active site.(ABSTRACT TRUNCATED AT 250 WORDS)     

Chemical modification of thiol groups of mitochondrial F1-ATPase from the yeast Schizosaccharomyces pombe. Involvement of alpha- and gamma-subunits in the enzyme activity

Mitochondrial F1-ATPase from the yeast Schizosaccharomyces pombe has been prepared under a stable form and in relatively high amounts by an improved purification procedure. Specific chemical modification of the enzyme by the thiol reagent N-ethylmaleimide (NEM) at pH 6.8 leads to complete inactivation characterized by complex kinetics and pH dependence, indicating that several thiols are related to the enzyme activity. A complete protection against NEM effect is afforded by low concentrations of nucleotides in the presence of Mg2+, with ADP and ATP being more efficient than GTP. A total binding of 5 mol of [14C]NEM/mol of F1-ATPase is obtained when the enzyme is 85% inactivated: 3 mol of the label are located on the alpha-subunits and 2 on the gamma-subunit. Two out of the 3 mol on the alpha-subunits bind very rapidly before any inactivation occurs, indicating that the two thiols modified are unrelated to the inactivation process. Complete protection by ATP against inactivation by NEM prevents the modification of three essential thiols out of the group of five thiols labeled in the absence of ATP: one is located on a alpha-subunit and two on the gamma-subunit. These two essential thiols of the gamma-subunit can be differentiated by modification with 6,6'-dithiodinicotinic acid (CPDS), another specific thiol reagent. A maximal binding of 4 mol of [14C]CPDS/mol of enzyme is obtained, concomitant to a 25% inhibition. Sequential modification of the enzyme by CPDS and [14C]NEM leads to the same final deep inactivation as that obtained with [14C]NEM alone. One out of the two thiols of the gamma-subunit is no longer accessible to [14C]NEM after CPDS treatment. When incubated at pH 6.8 with [3H]ATP in the presence of Mg2+, F1-ATPase is able to bind 3, largely exchangeable, mol of nucleotide/mol of enzyme. Modification of the three essential thiols by NEM dramatically decreases the binding of 3H-nucleotide down to about 1 mol/mol of enzyme. Partial modification modifies the cooperative properties, the enzyme being no longer sensitive to anion activation.