D-Aspartate stimulation of testosterone synthesis in rat Leydig cells. (9/10540)

D-Aspartate increases human chorionic gonadotropin-induced testosterone production in purified rat Leydig cells. L-Aspartate, D-,L-glutamate or D-,L-asparagine could not substitute for D-aspartate and this effect was independent of glutamate receptor activation. Testosterone production was enhanced only in cells cultured with D-aspartate for more than 3 h. The increased production of testosterone was well correlated with the amounts of D-aspartate incorporated into the Leydig cells, and L-cysteine sulfinic acid, an inhibitor of D-aspartate uptake, suppressed both testosterone production and intracellular D-aspartate levels. D-Aspartate therefore is presumably taken up into cells to increase steroidogenesis. Intracellular D-aspartate probably acts on cholesterol translocation into the inner mitochondrial membrane, the rate-limiting process in steroidogenesis.  (+info)

The internal Cys-207 of sorghum leaf NADP-malate dehydrogenase can form mixed disulphides with thioredoxin. (10/10540)

The role of the internal Cys-207 of sorghum NADP-malate dehydrogenase (NADP-MDH) in the activation of the enzyme has been investigated through the examination of the ability of this residue to form mixed disulphides with thioredoxin mutated at either of its two active-site cysteines. The h-type Chlamydomonas thioredoxin was used, because it has no additional cysteines in the primary sequence besides the active-site cysteines. Both thioredoxin mutants proved equally efficient in forming mixed disulphides with an NADP-MDH devoid of its N-terminal bridge either by truncation, or by mutation of its N-terminal cysteines. They were poorly efficient with the more compact WT oxidised NADP-MDH. Upon mutation of Cys-207, no mixed disulphide could be formed, showing that this cysteine is the only one, among the four internal cysteines, which can form mixed disulphides with thioredoxin. These experiments confirm that the opening of the N-terminal disulphide loosens the interaction between subunits, making Cys-207, located at the dimer contact area, more accessible.  (+info)

Thiol-dependent degradation of protoporphyrin IX by plant peroxidases. (11/10540)

Protoporphyrin IX (PP) is the last porphyrin intermediate in common between heme and chlorophyll biosynthesis. This pigment normally does not accumulate in plants because its highly photodynamic nature makes it toxic. While the steps leading to heme and chlorophylls are well characterized, relatively little is known of the metabolic fate of excess PP in plants. We have discovered that plant peroxidases can rapidly degrade this pigment in the presence of thiol-containing substrates such as glutathione and cysteine. This thiol-dependent degradation of PP by horseradish peroxidase consumes oxygen and is inhibited by ascorbic acid.  (+info)

Hairpin-shaped DNA duplexes with disulfide bonds in sugar-phosphate backbone as potential DNA reagents for crosslinking with proteins. (12/10540)

Convenient approaches were described to incorporate -OP(=O)O(-)-SS-O(-)(O=)PO- bridges in hairpin-shaped DNA duplexes instead of regular phosphodiester linkages: (i) H2O2- or 2,2'-dipyridyldisulfide-mediated coupling of 3'- and 5'-thiophosphorylated oligonucleotides on complementary template and (ii) more selective template-guided autoligation of a preactivated oligonucleotide derivative with an oligomer carrying a terminal thiophosphoryl group. Dithiothreitol was found to cleave completely modified internucleotide linkage releasing starting oligonucleotides. The presence of complementary template as an intrinsic element of the molecule protects the hairpin DNA analog from spontaneous exchange of disulfide-linked oligomer fragments and makes it a good candidate for auto-crosslinking with cysteine-containing proteins.  (+info)

Structure and function in rhodopsin: further elucidation of the role of the intradiscal cysteines, Cys-110, -185, and -187, in rhodopsin folding and function. (13/10540)

The disulfide bond between Cys-110 and Cys-187 in the intradiscal domain is required for correct folding in vivo and function of mammalian rhodopsin. Misfolding in rhodopsin, characterized by the loss of ability to bind 11-cis-retinal, has been shown to be caused by an intradiscal disulfide bond different from the above native disulfide bond. Further, naturally occurring single mutations of the intradiscal cysteines (C110F, C110Y, and C187Y) are associated with retinitis pigmentosa (RP). To elucidate further the role of every one of the three intradiscal cysteines, mutants containing single-cysteine replacements by alanine residues and the above three RP mutants have been studied. We find that C110A, C110F, and C110Y all form a disulfide bond between C185 and C187 and cause loss of retinal binding. C185A allows the formation of a C110-C187 disulfide bond, with wild-type-like rhodopsin phenotype. C187A forms a disulfide bond between C110 and C185 and binds retinal, and the pigment formed has markedly altered bleaching behavior. However, the opsin from the RP mutant C187Y forms no rhodopsin chromophore.  (+info)

Respiratory chain strongly oxidizes the CXXC motif of DsbB in the Escherichia coli disulfide bond formation pathway. (14/10540)

Escherichia coli DsbB has four essential cysteine residues, among which Cys41 and Cys44 form a CXXC redox active site motif and the Cys104-Cys130 disulfide bond oxidizes the active site cysteines of DsbA, the disulfide bond formation factor in the periplasm. Functional respiratory chain is required for the cell to keep DsbA oxidized. In this study, we characterized the roles of essential cysteines of DsbB in the coupling with the respiratory chain. Cys104 was found to form the inactive complex with DsbA under respiration-defective conditions. While DsbB, under normal aerobic conditions, is in the oxidized state, having two intramolecular disulfide bonds, oxidation of Cys104 and Cys130 requires the presence of Cys41-Cys44. Remarkably, the Cys41-Cys44 disulfide bond is refractory to reduction by a high concentration of dithiothreitol, unless the membrane is solubilized with a detergent. This reductant resistance requires both the respiratory function and oxygen, since Cys41-Cys44 became sensitive to the reducing agent when membrane was prepared from quinone- or heme-depleted cells or when a membrane sample was deaerated. Thus, the Cys41-Val-Leu-Cys44 motif of DsbB is kept both strongly oxidized and strongly oxidizing when DsbB is integrated into the membrane with the normal set of respiratory components.  (+info)

Nitric oxide inhibits caspase-3 by S-nitrosation in vivo. (15/10540)

In cultured human endothelial cells, physiological levels of NO prevent apoptosis and interfere with the activation of the caspase cascade. In vitro data have demonstrated that NO inhibits the activity of caspase-3 by S-nitrosation of the enzyme. Here we present evidence for the in vivo occurrence and functional relevance of this novel antiapoptotic mechanism. To demonstrate that the cysteine residue Cys-163 of caspase-3 is S-nitrosated, cells were transfected with the Myc-tagged p17 subunit of caspase-3. After incubation of the transfected cells with different NO donors, Myc-tagged p17 was immunoprecipitated with anti-Myc antibody. S-Nitrosothiol was detected in the immunoprecipitate by electron spin resonance spectroscopy after liberation and spin trapping of NO by N-methyl-D-glucamine-dithiocarbamate-iron complex. Transfection of cells with a p17 mutant, where the essential Cys-163 was mutated into alanine, completely prevented S-nitrosation of the enzyme. As a functional correlate, in human umbilical vein endothelial cells the NO donors sodium nitroprusside or PAPA NONOate (50 microM) significantly reduced the increase in caspase-3-like activity induced by overexpressing caspase-3 by 75 and 70%, respectively. When human umbilical vein endothelial cells were cotransfected with beta-galactosidase, morphological analysis of stained cells revealed that cell death induction by overexpression of caspase-3 was completely suppressed in the presence of sodium nitroprusside, PAPA NONOate, or S-nitroso-L-cysteine (50 microM). Thus, NO supplied by exogenous NO donors serves in vivo as an antiapoptotic regulator of caspase activity via S-nitrosation of the Cys-163 residue of caspase-3.  (+info)

Blockade of nitric oxide-induced relaxation of rabbit aorta by cysteine and homocysteine. (16/10540)

AIM: To examine the inhibition by L-cysteine (Cys) and L-homocysteine (HoCys) of NO-induced relaxation of aorta. METHODS: The tension of rabbit aortic rings in oxygenated Krebs' solution was recorded isometrically. RESULTS: Pretreatment of endothelium-denuded rings with Cys or HoCys inhibited the NO-induced increase in cGMP. The inhibitory effects of Cys or HoCys on relaxation responses to subsequent additions of NO 75 nmol.L-1 gradually diminished with time, which was consistent with the loss of the sulfhydryl concentration of Cys and HoCys. Superoxide dismutase (SOD) 35 kU.L-1 attenuated the inhibition by Cys and HoCys of NO-induced relaxation. Neither boiled SOD nor catalase 100 kU.L-1 antagonized the inhibitory effects of Cys. Preaddition of SOD 35 kU.L-1 inhibited the reduction of cytochrome C by Cys. Increasing concentrations of SOD from 35 to 350 kU.L-1 intensified the cytochrome C reduction. Addition of xanthine 300 mumol.L-1 plus xanthine oxidase 1 U.L-1 to the mixture of cytochrome C 60 mumol.L-1 and Cys 100 mumol.L-1 produced an additional augmentation of SOD-inhibitable reduction of cytochrome C. The rate of the reduction of cytochrome C induced by HoCys 100 mumol.L-1 was much slower than with Cys. Addition of NO reduced the SH concentrations of both the supernatant of aortic homogenate and Cys in Krebs' solution. CONCLUSION: The inhibition by the SH compounds of NO is mediated partly by the superoxide generated by the auto-oxidation of these compounds, and partly by a direct reaction of SH groups with NO.  (+info)