摘要:

The oxidative inactivation of rabbit skeletal muscle Ca2+-ATPase in sarcoplasmic reticulum (SR) vesicles by peroxynitrite (ONOO−) was investigated. The exposure of SR vesicles (10 mg/ml protein) to low peroxynitrite concentrations (≤0.2 mM) resulted in a decrease of Ca2+-ATPase activity primarily through oxidation of sulf-hydryl groups. Most of this deactivation (ca. 70%) could be chemically reversed by subsequent reduction of the enzyme with either dithiothreitol (DTT) or sodium borohydride (NaBH4), indicating that free cysteine groups were oxidized to disulfides. The initial presence of 5 mM glutathione failed to protect the SR Ca2+-ATPase activity. However, as long as peroxynitrite concentrations were kept≤0.45 mM, the efficacy of DTT to reverse Ca2+-ATPase inactivation was enhanced for re action mixtures which initially contained 5 mM gluta thione. At least part of the disulfides were formed intermolecularly since gel electrophoresis revealed protein aggregation which could be reduced under reducing conditions. The application of higher Peroxnitrite concentrations (≥0.45 mM) resulted in Ca2+-ATPase in activation which could not be restored by exposure of the modified protein to reducing agents. On the other hand, treatment of modified protein with NaBH4recovered all SR protein thiols. This result indicates that possibly the oxidation of other amino acids contributes to enzyme inactivation, corroborated by amino acid analysis which revealed some additional targets for peroxynitrite or peroxynitrite-induced processes such as Met, Lys, Phe, Thr, Ser, Leu and Tyr. Tyr oxidation was confirmed by a significant lower sensitivity of oxidized SR proteins to the Lowry assay. However, neither bityrosine nor nitrotyrosine were formed in significant yields, as monitored by fluorescence spectroscopy and immunodetection, respectively. The Ca2+-ATPase of SR is involved in cellular Ca2+-homeostasis. Thus, peroxynitrite mediated oxidation of the Ca2+-ATPase might significantly contribute to the loss of Ca2+-homeostasis observed under biological conditions of oxidative stress.

ISSN:1071-5762    

DOI:10.3109/10715769609088022

出版商:Taylor&Francis    

年代:1996

数据来源: Taylor

摘要:

Capacitation of spermatozoa is an essential procedure for fertilization. Capacitated spermatozoa have an in crease in the intracellular cAMP and acrosome reaction (AR) occurs immediately. The effect of exogenous superoxide anion (O−2) on the level of intracellular c AMP and the percentages of both spontaneous AR and lysophosphatidylcholine-induced AR (LPC-AR) were studied using semen samples collected from 10 healthy and fertile volunteers working or studying in Lanzhou Medical College. Spermatozoa were separated by Percoll and incubated at 37°C in Ham's F-10 medium with O−2generation system: xanthine + xanthine oxidase + catalase + diethylenetriaminepentaacetic acid + sodium formate. The intracellular cAMP was deter mined by (3H)—cAMP radioimmunoassay at 3 h of incubation, and the percentages of AR and LPC-AR were evaluated by the triple-stain technique at 3.5 h of incubation. The effects of SOD with different concentration were also determined. The results showed: the level of intracellular CAMP (pmol/108spermatozoa) of spermatozoa increased from 14.0±1.3 to 23.2±2.5 (P<0.01), and the percentages of AR and LPC-AR increased from 4.5±1.1% and 14±1.9% to 16±2.0% and 32.5±1.7%, respectively (P<0.01 in both comparisons). SOD inhibited these processes concentration dependently. To investigate the source of O−2duringin vivosperm capacitation, female genital tract fluids collected from 6 healthy nonpregnant donors of reproductive age, and seminal plasma, capacitated and noncapacitated spermatozoa from 10 fertile volunteers were investigated by spin trapping method. The results showed: A typical electron paramagnetic resonance spectrum for O−2spin adduct was exhibited only in capacitated spermatozoa but not in vaginal or cervical secretions, uterine and fallopian tubalfluids, nor in seminal plasma and noncapacitated spermatozoa. These results suggested that only capacitated spermatozoa themselves are able to generate O−2which stimulated their capacitation in turn. Furthermore, on the basis of these data, we pro pose that it may be possible to utilize the inhibitory effect of SOD on sperm capacitation so as to regulate fertilization.

ISSN:1071-5762    

DOI:10.3109/10715769609088023

出版商:Taylor&Francis    

年代:1996

数据来源: Taylor

摘要:

The method of Electron Paramagnetic Resonance (EPR) spectroscopy was used to study the reaction of human methaemoglabin (metHb) with hydrogen peroxide. The samples for EPR measurements were rapidly frozen in liquid nitrogen at different times after H2O2was added at 3- and 10-fold molar excess to 100μM metHb in 50 mM phosphate buffer, pH 7.4, 37°C. Precautions were taken to remove all catalase from the haemoglobin preparation and no molecular oxygen evolution was detected during the reaction. On addition of H2O2the EPR signals (- 196°C) of both high spin and low spin metHb rapidly decreased and free radicals were formed. The low temperature (- 196°C) EPR spectrum of the free radicals formed in the reaction has been deconvoluted into two individual EPR signals, one being an anisotropic signal (g°= 2.035 and g°= 2.0053), and the other an isotropic singlet (g = 2.0042, AH = 20 G). The former signal was assigned to peroxyl radicals. As the kinetic Pehaviour of both peroxyl (ROO*) and nonperoxyl (P*) free radicals were similar, we concluded that ROO*radicals are not formed from P*radicals by addition of O2. The time courses for both radicals showed a steady state during the time required for H2O2to decompose. Once all peroxide was consumed, the radical decayed with a first order rate constant of 1.42±10-3s-1(1:3 molar ratio). The level of the steady state was higher and its duration shorter at lower initial concentration of H2O2. The formation of the rhombic Fe(III) non-haemcentres with g = 4.35 was found. Their yield was proportional to the H2O2concentration used and the centers were ascribed to haem degradation products. The reaction was also monitored by EPR spectroscopy at room temperature. The kinetics of the free radicals measured in the reaction mixture at room temperature was similar to that observed when the fast freezing method and EPR measurement at—196°C were used.

ISSN:1071-5762    

DOI:10.3109/10715769609088024

出版商:Taylor&Francis    

年代:1996

数据来源: Taylor

摘要:

Effects of various derivatives ofα-tocopherol (VE) and coenzyme Q (CoQ) on superoxide (O2*-) generation of neutrophils and protein kinase C (PKC) activity were examined. VE and CoQ8inhibited O2*-generation of neutrophils stimulated by a protein kinase C mediated process monitored by cytochrome c reduction and spin trapping methods. The inhibitory action was observed not only withα-tocopherol, but also withβ-,γ-, dL-tocopherols and with tocol which is a chemical similar to VE but lacking methyl groups on the chromanol ring structure and which is not a radical scavenger. By con trast, no inhibition was observed with 2-carboxy-2,5,7,8-tetramethyl-6-chromanol (CTMC, trolox) or 2,2,5,7,8,-pentamethyl-6-chromanol (PMC) which are water soluble VE derivatives having radical scavenging activity. Compounds having a similar isoprenoid chain, such as CoQ, also have inhibitory activity on PKC-dependent O2*-generation of neutrophils. The inhibitory activity of CoQ derivatives is dependent on the length of the unsaturated isoprenoid chain. CoQ derivatives having 16,24 and 32 carbon isoprenoid chains corresponding to CoQ4,6, and 8 inhibited O2*-generation but 4 and 40 carbon isoprenoid chains corresponding to CoQ2 and 10 had no inhibitory activity on O2*-generation. Alpha-tocopherol and CoQ inhibited PKC activity but the ID50for O2*-generation and PKC activity was different for each compound. However, no direct relationship between VE content and O2*-generation of neutrophils was observed. These results suggest that isoprenoids of VE and CoQ participate in the inhibition of the NADPH oxidase activation system through modulation of the neutrophil membrane probably by the inhibition of PKC.

ISSN:1071-5762    

DOI:10.3109/10715769609088025

出版商:Taylor&Francis    

年代:1996

数据来源: Taylor

摘要:

The purpose of this study was to determine the role of reducing agents in maintaining the integrity of vitamin E-deficient red cells. Three groups of one-month-old male Wistar rats were fed a basal vitamin E-deficient diet supplemented with either 0, 10 or 100 mg d, 1-α-tocopheryl acetate per kg diet for up to 12 weeks. Washed red blood cells (5%) were resuspended in saline-phosphate buffer, pH 7.4, and were incubated at 37°C with or without containing 12.5 mM 2,Z'-azobis (2amino- propane) dihydrochloride (AAPH), 2.8 mM glucose, 1 mM ascorbic acid, 10 mM hydrogen peroxide (H2O2), 250μM dimethylsulfoxide (DMSO) or 2.8 mM deoxyribose (DR) for up to 20 hours. Addition of either glucose, AAPH, ascorbic acid or H2O2markedly accelerated the rates of hemolysis and lipid peroxidation in the red cells of vitamin E-deficient rats. On the contrary, both glucose and ascorbic acid were protective against oxidative damage to the red cells of vitamin E-supplemented rats in a dose-dependent manner. Also, vitamin E-supplemented red cells were more resistant to AAPH and H2O2than the deficient cells. DMSO or DR had no significant effects on the rates of hemolysis or lipid peroxidation. Glucose, but not others, maintained or slowed down the loss of glutathione (GSH) during incubation. The results obtained suggest a dual role of ascorbic acid and GSH in the function of vitamin E in maintaining red cell integrity: these reducing agents may exert antioxidant function by participating in vitamin E regeneration when certain levels of vitamin E is maintained, but promote oxidative damage by enhancing free radical generation when vitamin E is low or depleted.

ISSN:1071-5762    

DOI:10.3109/10715769609088026

出版商:Taylor&Francis    

年代:1996

数据来源: Taylor

摘要:

The mechanism of H2O2-resistance of Hpr-4, a variant of Chinese hamster V79 cells, was investigated. The effect of H2O2on the mitochondria of the parental and Hpr-4 cells was compared. First, both biochemical and ultrastructural results showed that mitochondria in the parental cells were damaged by exposure to H2O2, while those in Hpr-4 cells recovered from the damage. Second, the H2O2resistance of Hpr-4 cells was reversibly reduced or recovered by the addition or removal of inhibitors of mitochondrial biosynthesis, respectively. Third, the parental cells were auxotrophic to pyruvate after exposure to H2O2. Fourth, H2O2-sensitivity of the parental cells was also enhanced by the inhibition of mitochondrial biosynthesis. From these results, it was concluded that the mitochondria of Hpr-4 cells apparently had a greater resistance to H2O2than those of the parental cells and that functional mitochondria were involved in the recovery of Chinese hamster V79 cells from H2O2-induced damage.

ISSN:1071-5762    

DOI:10.3109/10715769609088027

出版商:Taylor&Francis    

年代:1996

数据来源: Taylor

摘要:

Catecholamines (CAs: epinephrine, norepinephrine, dopamine, L-DOPA, 6-hydroxydopamine) ando-diphenols (DOPAC and catechol) enhanced dihydrolipoamide dehydrogenase (LADH) inactivation by Cu(II) /H2O2(Cu-Fenton system). The inhibition of LADH activity correlated with Cu(II), H2O2and CA concentrations. Similar inhibitions were obtained wit! the assayed CAs and o-diphenols. CAs enhanced HO radical production by Cu(II) /H2O2, as demonstrated by benzoate hydroxylation and deoxyribose oxidation; LADH counteracted the pro-oxidant effect of CAs by scavenging hydroxyl radicals. Captopril, dihydrolipo amide, dihydrolipoic acid, DL-dithiothreitol, GSSG, try-panothione and histidine effectively preserved LADH from oxidative damage, whereas N-acetylcysteine, N-(2-mercaptopropionylglycine) and lipoamide were less effective protectors. Catalase (though neither bovine serum albumin nor superoxide dismutase) protected LADH against the Cu(II)/H2O2/CAs systems. Dena tured catalase protected less than the native enzyme, its action possibly depending on Cu-binding. LADH in creased and Captopril inhibited epinephrine oxidation by Cu(II)/H2O2and Cu(II). The summarized evidence supports the following steps for LADH inactivation: (1) reduction of LADH linked-Cu(II) to Cu(I) by CAs; (2) production of HO*from H2O2by LADH-linked Cu(I) (Haber-Weiss reaction) and (3) oxidation of aminoacid residues at the: enzyme active site by site-specifically generated HO*radicals. Hydrogen peroxide formation from CAs autoxidation may contribute to LADH inactivation.

ISSN:1071-5762    

DOI:10.3109/10715769609088028

出版商:Taylor&Francis    

年代:1996

数据来源: Taylor

ISSN:1071-5762    

DOI:10.3109/10715769609088029

出版商:Taylor&Francis    

年代:1996

数据来源: Taylor