Inhibition Of NADH Oxidase Research Articles

The effect of an NADH oxidase inhibitor (hydrocortisone) on polymorphonuclear leukocyte bactericidal activity.

Polymorphonuclear neutrophils (PMN) from patients with chronic granulomatous disease of childhood have impaired bactericidal activity and are deficient in diphosphopyridine nucleotide, reduced form of, (NADH) oxidase. Since hydrocortisone had been shown to inhibit NADH oxidation, experiments were undertaken to determine the effect of hydrocortisone on several parameters of human PMN function. The phagocytic and bactericidal capacity of PMN with or without hydrocortisone (2.1 mM) was determined by quantitation of cell-free, cell-associated, and total bacteria. Phagocytosis of Staphylococcus aureus and several gram-negative rods was unimpaired by the presence of hydrocortisone in the media. In contrast, killing of bacteria was markedly impaired by hydrocortisone. After 30 min of incubation, there were 20-400 times as many bacteria surviving in hydrocortisone-treated PMN as in simultaneously run controls without hydrocortisone. The defect of intracellular killing noted in the presence of hydrocortisone was not related to impaired degranulation. Quantitative kinetic studies of degranulation revealed no difference in the release of granule associated acid phosphatase in hydrocortisone-treated and control PMN after phagocytosis. Electron microscopy of PMN also indicated that the presence of hydrocortisone had no effect on the extent of degranulation after phagocytosis. These observations were confirmed by studies using histochemical techniques to detect lysosomal enzymes. After phagocytosis, hydrocortisone-treated PMN demonstrated less NADH oxidase activity, oxygen consumption, and hydrogen peroxide production than postphagocytic control PMN. In addition, Nitro blue tetrazolium dye reduction was diminished in hydrocortisone-treated PMN.Thus, impairment of NADH oxidase activity in normal human PMN by hydrocortisone results in reduced intracellular killing of bacteria, diminished postphagocytic oxygen consumption, decreased ability to reduce Nitro blue tetrazolium, and decreased hydrogen peroxide production. These abnormalities are similar to those seen in the PMN of patients with chronic granulomatous disease of childhood.

Effects of chloramphenicol and its isomers and analogues on the mitochondrial respiratory chain

The nature of the inhibition of mitochondrial respiration by chloramphenicol isomers and analogues has been examined further to explain differences in their action in several systems. With coupled rat-liver mitochondria, D-threo-chloramphenicol inhibited β-hydroxybutyrate oxidase 50% at 1.3 mM and succinoxidase at 3.5 mM. L-threo-Chloramphenicol inhibited 50% at 3.6 mM and 3.7 mM, respectively. The sensitivity of succinoxidase to D-chloramphenicol was lost on freezing the mitochondria. Chloramphenicol inhibited dinitrophenol-activated ATPase with 50% inhibition at 6 mM for the D-isomer and at 2.2 mM for the L isomer. The inhibition of NADH oxidase of frozen–thawed beef-heart mitochondria by D-chloramphenicol analogues was dependent on the analogues' solubility properties. A relationship which approximates a straight line was obtained by plotting log (1/concentration50% inhibition) against log (solubilityethanol/solubilitywater) indicating that the inhibition was greater for analogues which would partition into an organic phase. The inhibition did not depend on the orientation of the mitochondrial membrane; the NADH oxidase of submitochondrial particles prepared either by sonication or with digitonin had the same sensitivity to the analogues. The inhibition of NADH oxidase by rotenone and amytal fitted into the straight line relationship obtained with the chloramphenicol analogues. Beef-liver glutamic dehydrogenase was inhibited by some of the analogues of chloramphenicol with the same relationship as for the inhibition of NADH oxidase. It was suggested that the inhibition of NADH oxidase and glutamic dehydrogenase depends on the inhibitors reacting with a hydrophobic site on the enzyme.

Studies of the energy-transfer system of submitochondrial particles. I. Competition between oxidative phosphorylation and the energy-linked nicotinamide-adenine dinucleotide transhydrogenase reaction.

The relationship between oxidative phosphorylation and the energy‐linked nicotinamide‐adenine dinucleotide transhydrogenase reaction catalyzed by phosphorylating submitochondrial particles from beef heart has been investigated. The efficiency of the particles in exhibiting oxidative phosphorylation (P/O ratio) and NADH‐linked NADP+ reduction (NADP+/O ratio) was measured, using succinate as oxidizable substrate and rotenone as inhibitor of NADH oxidase, under conditions where the phosphorylating and transhydrogenating systems were operating one by one or in parallel. When succinate oxidation was limited by increasing concentrations of malonate, the P/O ratio remained unchanged in the absence of an active phosphorylating system, and decreased in its presence. The NADP+/O ratio increased with increasing concentrations of malonate, both in the absence and presence of an active phosphorylating system. The (P + NADP+)/O ratio in the combined system approached 2, i.e., the generally accepted maximal ∼/O ratio for succinate oxidase. Active transhydrogenation caused an increase in the apparent Km for Pi in the phosphorylating system from 1.7 to 4.5 mM, and active phosphorylation caused an increase in the apparent Km for NADH in the transhydrogenating system from 21 to 40 μM. It is concluded that oxidative phosphorylation and the energy‐linked transhydrogenase reaction are competing for a common nonphosphorylated high‐energy intermediate generated by the respiratory chain. The kinetic evidence presented further suggests that the two systems interact with two different moieties of this high‐energy intermediate.

Structural requirements for the action of steroids as inhibitors of electron transfer

A series of C 21, C 19, and C 18-steroids (including hormonal steroids and derivatives) have been tested as inhibitors of NADH-oxidase and succinate oxidase activities of heart-muscle sarcosomal fragments (Keilin-Hartree preparation). NADH oxidation is much more sensitive to steroids than succinate oxidation, in accordance with electron transfer inhibition in the vicinity of the NADH-flavoprotein site. Steroid activity on NADH-oxidase has been titrated and the 50% inhibitory concentration ( I 50) of each steroid has been determined. Among the steroids tested, the following were the more effective inhibitors, having I 50 values ≦ 1.0 μM 19-norethynyltestosterone acetate, 5-pregnen-3β,20α-diol, 5α-pregnan-3β,20α-diol, 5-pregnen-3β,20β-diol, 5-pregnen-3β,17α,20α-triol, testosterone acetate, testosterone enol diacetate, testosterone benzoate, androstanolone acetate, and 17β-ethynylestradiol benzoate. Comparison I 50 values for steroids differing in a single group (or structure) shows that the following structural features promote the inhibitory potency: (a) a trans A B junction; (b) an alcohol or ketone group at C-3 and/or C-17; (c) an acetyl chain at C-17; (d) 17α-methyl or ethynyl groups at C-17 of 17β-hydroxylated steroids; (e) acetylated or benzoylated hydroxyl groups at C-3 or C-17; (f) α configuration of 3-OH groups in 5α-androstane derivatives and β-OH configuration in 5β-pregnane derivatives; (g) α configuration of hydroxyl groups at C-17; (h) an angular β-methyl group at C-10; (i) a hydroxyl group at C-20 of 5α-pregnane and 5-pregnene derivatives. Decreased inhibitory potency is determined by the following structural features: (j) a cis A B junction; (k) Δ 1 and Δ 4 double bonds; (l) ionizable (hydrophylic) groups at C-3 or C-17; (m) hydroxyl groups at C-17 or C-21 of C 21-steroids; (n) relatively large substituents at C-3 or C-17 (hexahydrobenzoate and propionate derivatives); (o) substituents at the skeleton of the steroid molecule (C-2, C-4, C-6, C-11, C-16) irrespective of group-polarity and configuration. Accordingly, the most effective steroid structure to inhibit electron transfer is a planar and narrow hydrocarbon framework with relatively small, adequately orientated polar end-groups and a large intermediate hydrophobic area. These structural characteristics are consistent with the interaction of steroids with the phospholipid components of the electron transfer chain at the NADH-flavoprotein site. Many of the structural requirements for inhibition of electron transfer resemble those for androgenicity.

Inhibition of electron transport in Prototheca zopfii

The effects of a number of inhibitors on the respiration of the colourless alga, Prototheca zopfii, have been studied, and twenty-six compounds related to rotenone tested as inhibitors of NADH oxidase. The results are compared with those obtained with other algal systems and the mammalian respiratory chain.

Diphenylamine inhibition of electron transport in plant mitochondria

Diphenylamine (DPA) at concentrations of 10 −4 M and above inhibits NADH oxidase and succinoxidase activities of plant mitochondria. At the lower concentrations (≦2.7 × 10 −4 M) the inhibitory effects result primarily from DPA acting in the region between succinic and NADH dehydrogenases, and the first cytochrome component of the electron transport chain(s). This conclusion was based on the following results: (1) DPA delayed the reduction of mitochondrial cytochromes in the sweet potato succinoxidase system; no reduced-oxidized cytochrome pairs were observed; (2) NADH- and succinic-cytochrome c reductase activities of the mitochondria were severely inhibited by 2.7 × 10 −4 M DPA; (3) succinic dehydrogenase and NADH diaphorase activities of the mitochondria were not inhibited by that concentration of DPA; (4) menadione effects an antimycin A-insensitive shunt from NADH to cytochrome c which is not inhibited by DPA; and (5) much smaller effects were exerted by DPA on mitochondrial cytochrome c oxidase activity. The antimycin A-insensitive respiration in sweet potato mitochondria was also inhibited by DPA, suggesting that DPA may be useful in testing alternate pathway theories.