Hormone- and growth factor-stimulated NADH oxidase

An NADH oxidase activity of animal and plant plasma membrane is described that is stimulated by hormones and growth factors. In plasma membranes of cancer cells and tissues, the activity appears to be constitutively activated and no longer hormone responsive. With drugs that inhibit the activity, cells are unable to grow although growth inhibition may be more related to a failure of the cells to enlarge than to a direct inhibition of mitosis. The hormone-stimulated activity in plasma membranes of plants and the constitutively activated NADH oxidase in tumor cell plasma membranes is inhibited by thiol reagents whereas the basal activity is not. These findings point to a thiol involvement in the action of the activated form of the oxidase. NADH oxidase oxidation by Golgi apparatus of rat liver is inhibited by brefeldin A plus GDP. Brefeldin A is a macrolide antibiotic inhibitor of membrane trafficking. A model is presented where the NADH oxidase functions as a thiol-disulfide oxidoreductase activity involved in the formation and breakage of disulfide bonds. The thiol-disulfide interchange is postulated as being associated with physical membrane displacement as encountered in cell enlargement or in vesicle budding. The model, although speculative, does provide a basis for further experimentation to probe a potential function for this enzyme system which, under certain conditions, exhibits a hormone- and growth factor-stimulated oxidation of NADH.     

Physical membrane displacement: Reconstitution in a cell-free system and relationship to cell growth+

Summary Physical membrane displacement is a process common to all forms of vesicle budding as well as cell enlargement and pleomorphic shape changes. Cell-free reconstitution of membrane budding has been achieved with transitional endoplasmic reticulum fractions from both plants and animals where 50 to 70 nm transition vesicles have been observed to bud from the part-rough, part-smooth membrane elements that define transitional endoplasmic reticulum. This budding phenomenon requires ATP, is facilitated by cytosol and guanine nucleotides, and is both time- and temperature-dependent. The transitional endoplasmic reticulum buds that form when concentrated by preparative free-flow electrophoresis will attach specifically to cis Golgi apparatus membranes immobilized on nitrocellulose as an acceptor compartment. Golgi apparatus membranes derived from the trans compartment do not serve as an efficient acceptor compartment. Transfer of the vesicles once formed is rapid, nearly complete and no longer dependent upon added ATP. Transfer shows a strict temperature dependency corresponding to that of the intact cell where at temperatures of 16°C or below, vesicles form but do not attach to cis Golgi whereas at temperatures of greater than 16°C, vesicles both form and fuse. The principle ATPase of transitional endoplasmic reticulum which may be involved in the budding process has been identified, characterized and isolated. A 38 kDa cis Golgi apparatus associated protein also has been identified as a potential candidate as a docking protein. Transfer between trans Golgi apparatus and the plasma membrane also has been studied by cell-free analysis. Here, transfer has been found to be stimulated by NADH or NADH plus ascorbate. The role of NADH is unknown but the ability of plant and Golgi apparatus to oxidize NADH is inhibited by brefeldin A, a compound known to block membrane trafficking even at the level of the trans Golgi network. NADH oxidase activity of plasma membranes also has been described and is inhibited as well by brefeldin. Recent observations suggest that brefeldin A may block both the formation of vesicles at the trans Golgi apparatus as well as auxin hormone-stimulated cell elongation in plants. This once again raises the possibility of whether or not plant cell elongation is obligatorily mediated by membrane input from the Golgi apparatus. The latter seems unlikely based on two additional lines of evidence. The first is that auxin-induced cell elongation in plants shows no sharp temperature transition over the range of 4 to 24°C, whereas production of secretory vesicles from the trans Golgi apparatus appears to be largely prevented at temperatures of 18°C or less. Secondly, the sodium selective ionophore, monensin, which effectively blocks the formation of functional secretory vesicles at the trans Golgi apparatus, is also largely without effect on auxin-induced cell elongation for periods of 4 h or longer. Taken together the findings suggest that the action of brefeldin A on vesicle budding at the Golgi apparatus and cell enlargement, are not directly correlated but may represent a common action of the drug on some constituent essential to membrane displacement mechanisms.Abbreviations BFA brefeldin A - IAA indole-3-acetic acid; 2, 4-D 2, 4-dichlorophenoxyacetic acid - NSF N-ethylmaleimide-sensitive factor Much of the information summarized in this report was presented as a plenary lecture at the XV International Botanical Congress Tokyo, Yokohama, Japan, August 28–September 3, 1993.     

Purification and properties of the membrane-bound NADH oxidase of a facultatively anaerobic alkaliphile

A membrane-bound NADH oxidase of an anaerobic alkaliphile, M-12 (a strain of Amphibacillus sp.), was solubilized with decanoyl N-methylglucamide and purified by chromatography on DEAE-Sepharose and hydroxyapatite. The purified enzyme appears to consist of a single polypeptide component with an apparent molecular mass of 56 kDa. The enzyme catalyzed the oxidation of NADH with the formation of H₂O₂ and exhibited a specific activity of 46 μmol NADH min^–1 (mg protein)^–1. NADPH did not serve as a substrate for the enzyme. The K _m for NADH was estimated to be 0.05 mM. The enzyme exhibited a pH dependence for activity, with a pH optimum at approximately 9.5. The enzyme required a high concentration of salt and exhibited maximum activity in the presence of 600 mM NaCl. Received: 3 August 1998 / Accepted: 23 December 1998     

The stimulation of NADH oxidase activity of rat liver plasma membranes by guanine nucleotides may involve both guanine nucleotide-binding proteins of the plasma membrane and responses not mediated by classic heterotrimeric G proteins

Summary The stimulation of NADH oxidase activity of plasma membranes of rat liver observed with guanine nucleotides may involve both guanine nucleotide-binding proteins of the plasma membrane and responses not mediated by classic heterotrimeric G proteins. These conclusions are based on findings that detergent treatment and peptide antisera to a consensus guanine nucleotidebinding domain (GAGES) of G subunits of heterotrimeric G proteins reduced but did not eliminate the stimulation of NADH oxidase activity by guanine nucleotides. The proteins immunoprecipitated by the antisera, when added back to plasma membranes, stimulated the NADH oxidase activity. This stimulated rate was further stimulated by the addition of GTP but was not dependent upon guanine nucleotide presence. Additions of cytosol, either fractionated or unfractionated did not appear to stimulate the NADH oxidase activity of rat liver plasma membranes. The activities of the plasma membranes and the activities introduced by the cytosol fractions were nearly, but not entirely, additive. The results are suggestive of a subunit composition of the NADH oxidase but one distinct from that involving solely heterotrimeric G proteins. Also a strong dependence on cytosolic components, as found with the NADPH oxidase complex of neutrophils, is not obvious. In addition, the possibility that the NADH oxidase may exhibit an intrinsic re-sponse to guanine nucleotides, not dependent on accessory proteins, cannot be ruled out. Among the several bands immunoprecipitated with the antisera and reactive with the antisera on Western blots, were peptide bands in the molecular weight range ascribed to the NADH oxidase.     

Inhibition of plasma membrane NADH oxidase activity and growth of HeLa cells by natural and synthetic retinoids

Several retinoids, both natural and synthetic, were evaluated for their ability to modulate NADH oxidase activity of plasma membranes of cultured HeLa cells and the growth of HeLa cells in culture. Both NADH oxidase activity and the growth of cells were inhibited by the naturally-occurring retinoids all trans-retinoic acid (tretinoin) and retinol as well as by the synthetic retinoids, trans-acitretin, 13-cis-acitretin, etretinate and arotonoid ethylester (Ro 13-6298). For all retinoids tested, inhibition of NADH oxidase activity and inhibition of growth were correlated closely. With tretinoin, etretinate and arotonoid ethylester, NADH oxidase activity and cell growth were inhibited in parallel in proportion to the logarithm of retinoid concentration over the range of concentrations 10-8 to 10-5 M. Approximately 70% inhibition of both NADH oxidase activity and growth was reached at 10 µM. With retinol, trans-acitretin and 13-cis-acitretin, inhibition of NADH oxidase activity and growth also were correlated but maximum inhibition of both was about 40% at 10 µM. The possibility is suggested that inhibition of the plasma membrane NADH oxidase activity by retinoids may be related to their mechanism of inhibition of growth of HeLa cells in culture. (Mol Cell Biochem 166: 101-109, 1997)     

Influence of salts and pH on the growth as well as NADH oxidase of the halotolerant bacterium A505

Growth yield of the halotolerant bacterium A505 was increased by the supplement of Na⁺, K⁺, or Rb⁺ into the culture media with pH 7.5, and inhibited by Li⁺ or Cs⁺. In the presence of less than 0.1 M NaCl or KCl alkaline growth media, pH 9.2 to 9.7, afforded optimal growth of this strain. Intracellular ion content of this microbe changed reflecting on the Na⁺ or K⁺ concentration in the media, although it tended to accumulate K⁺ and extrude Na⁺ in the media without NaCl supplemented. A 1.2 to 1.4-fold stimulation of in vitro NADH oxidase activity was obtained by supplement of salts, except for LiCl. The rate of NADH oxidation in the absence of salts correlated with the pH and showed clear maxima at pH about 8, irrespective of growth conditions. In the presence of 0.5 M NaCl or KCl, on the other hand, pH dependence was less significant and showed only a flat maximum at pH around 7. Effects of anions on NADH oxidase were realized following the lyotropic series: SO ₄ ^2- >F^->CH₃COO^->Cl^->I^->SCN^-, aside from NO ₃ ^- , which exhibited the largest stimulation on enzyme activity in all the anions examined.Abbreviations HEPES 4-(2-hydroxyethyl)-1-piperazine-ethanesulfonic acid - HQNO 2-heptyl-4-hydroxyquinoline-N-oxide - MES 4-morpholineethanesulfonic acid - Tris tris(hydroxy-methyl)methylamine     

Fractionation of suspension cultures of wild carrot and kinetics of membrane labeling

Summary Wild carrot (Daucus carota L.) cells, grown in suspension culture, were labeled with radioactive precursors and fractionated into constituent membranes to be analyzed for specific radioactivity. Results show rapid incorporation of [³H] leucine into endoplasmic reticulum (ER)-, Golgi apparatus-, and plasma membrane/tonoplast-enriched fractions. The time lag between incorporation into ER and its appearance in Golgi apparatus or plasma membrane/tonoplast were less than 5 minutes. With an average time of 3–4 minutes for cisternal formation estimated from studies with monensin, and an average of 5 cisternae per dictyosome (total transit time of 15–20 minutes), it was not possible to account for early incorporation of radioactivity into plasma membranes by passage of proteins from ER to plasma membrane via the Golgi apparatus. To account for the findings, it would appear that at least some proteins were delivered to the plasma membrane via the first membranes that exited (i.e., mature face vesicles) from the Golgi apparatus post-pulse and that some of these proteins had been translated and inserted into membranes at or near the mature face of the Golgi apparatus.     

NADH oxidase of plasma membranes

NADH oxidase is a cyanide-resistant and hormone-responsive oxidase intrinsic to the plasma membrane of both plant and animal cells. The activity has many unique characteristics that distinguish it from other oxidases and oxidoreductases of both organelles and internal membranes and from other oxidoreductases of the plasma membrane. Among these are resistance to inhibition by cyanide, catalase, superoxide dismutase, and phenylchloromer-curibenzoate. Activity is stimulated by hormones and growth factors and inhibited by quinone analogs such as piericidin, the flavin antagonist atebrin, and growth inhibiting gangliosides such as G_M3. In marked contact to the NADH-ferricyanide oxidoreductase of the plasma membrane, the NADH oxidase is activated by lysophospholipids and fatty acids, products of phospholipase A₂ action, in a time-dependent manner suggestive of stabilization of an activated form of the enzyme. The hormone-responsive NADH oxidase of the plasma membrane is not a peroxidase and may function as a terminal oxidase to link transfer of electrons from NADH to oxygen at the plasma membrane. The functional significance of the NADH oxidase of the plasma membrane is unknown but some relationship to growth or growth control is indicated. In both animal and plant plasma membranes, the oxidase is activated by growth factors and hormones to which the cells or tissues of origin have functional hormone or growth factor receptors. In addition, substances that inhibit the oxidase, the associated transmembrane reductase or both, inhibit growth. In transformed cells and tissues, the hormone and growth factor responsiveness of the NADH oxidase is reduced or absent. With human keratinocytes which exhibit an increased sensitivity to the anti-proliferative action of both retinoic acid and calcitriol, the NADH oxidase of the plasma membrane is strongly inhibited by these agents and shows the same increased sensitivity. If transfer of electrons from NADH to oxygen across or within the eukaryotic plasma membrane is an important aspect of growth or growth control, then the hormone- and growth factor-responsive NADH oxidase associated with the plasma membrane could be of fundamental importance. Because of its low basal activity, stimulation by growth factors and hormones, and the inhibition of growth in direct proportion to inhibition of the oxidase, the activity is a candidate as a rate-limiting step in the growth process. Completely unknown is the mechanism whereby NADH oxidization and growth or growth control may be coupled. This, together with further characterization of the activity and the mechanism of loss of control with neoplastic transformation, represent important challenges for future investigations.     

Response of NADH oxidation and growth in K-562 cells to the antitumor sulfonylurea,N-(4-methylphenylsulfonyl)-N′-(4-chlorophenyl)urea (LY181984)

Summary Growth of K-562 cells in culture is inhibited by the antitumor sulfonylureaLY181984 (N-(4-methylphenylsulfonyl)-N-(4-chlorophenyl)urea) with an ED₅₀ of about 30 M. LY181984 was shown previously to inhibit NADH oxidation by plasma membranes from HeLa cells and other sources and to influence mitochondrial oxidative phosphorylation. With K-562 cells, NADH oxidation by plasma membranes was transiently stimulated and then inhibited by LY181984. NADH oxidation by whole cells was transiently stimulated and then inhibited by 0.1 to 100 M LY181984 as well. Both the stimulations and inhibitions of activity were time-dependent. NADH oxidation by lower phase membranes depleted of plasma membranes by aqueous two-phase partition also was inhibited by micromolar and submicromolar concentrations of LY181984. Inhibition did not correlate with mitochondrial presence but rather with membranes that appeared to be fragments of the Golgi apparatus. The oxidation of NADH by whole cells and of plasma membranes that was inhibited by LY181984 was distinguished from mitochondrial NADH oxidation by resistance to inhibition by cyanide and by proceeding under oxygen-depleted conditions or an argon atmosphere. In contrast to the active antitumor agent LY181984, the inactive but chemically-related analog, LY181985 (N-(4-methylphenyl-sulfonyl)-N-(4-phenylurea), inhibited neither growth nor NADH oxidation with K-562 cells or cell fractions.     

Inhibition of oxidative stress produced by plasma membrane NADH oxidase delays low-potassium-induced apoptosis of cerebellar granule cells

From 1 to 3 h after the onset of cerebellar granule cells (CGC) apoptosis in a low-K+(5 mm KCl) medium there was a large decay of NADH and a 2.5-fold increase of the rate of reactive oxygen species (ROS) production (measured using CGC loaded with dichlorodihydrofluorescein). During the same time period, the ascorbate-dependent NADH oxidase activity, which accounted for more than 90% of both total NADH oxidase activity and NADH-dependent *O2- production of CGC lysates, increased 2.5- to threefold. The stimulation of the ascorbate-dependent NADH oxidase activity by oxidized cytochrome c, 2.5-fold at saturation with a K(0.5) of 4-5 microm cytochrome c, can at least partially explain this activation. The plasma membrane ascorbate-dependent NADH oxidase activity accounted for more than 70% of the total activity (both in terms of NADH oxidase and *O2- release) of CGC lysates. 4-Hydroxyquinazoline (4-HQ), which was found to block this apoptotic process, prevented the increase of ROS production. 4-HQ protection against cell viability loss and DNA fragmentation correlated with the inhibition by 4-HQ of the ascorbate-dependent NADH oxidase activity of CGC lysates, showing the same K(0.5)-value (4-5 mm 4-HQ). The efficient blockade of CGC apoptosis by addition of superoxide dismutase to the medium further supports the neurotoxic role of *O2- overproduction by the plasma membrane ascorbate-dependent NADH oxidase.     

Inhibition of plasma membrane NADH dehydrogenase by adriamycin and related anthracycline antibiotics

Doxorubicin (adriamycin) is cytotoxic to cells, but the biochemical basis for this effect is unknown, although intercalation with DNA has been proposed. This study suggests that the cytotoxicity of this drug may be due to inhibition of the plasma membrane redox system, which is involved in the control of cellular growth. Concentrations between 10^–6–10^–7 M adriamycin inhibit plasma membrane redox reactions >50%. AD32, a form of adriamycin which does not intercalate with DNA, but is cytotoxic, also inhibits the plasma membrane redox system. Thus, the cytotoxic effects of adriamycin, which limit its use as a drug, may be based on the inhibition of a transplasma membrane dehydrogenase involved in a plasma membrane redox system.     

Vanadate-stimulated NADH oxidation in plasma membrane

The rate of NADH oxidation with oxygen as the acceptor is very low in mouse liver plasma membrane and erythrocyte membrane. When vanadate is added, this rate is stimulated 10- to 20-fold. The absorption spectrum of vanadate does not change with the disappearance of NADH. The reaction is inhibited by superoxide dismutase, and there is no activity under an argon atmosphere. This indicates that oxygen is the electron acceptor and the reaction is mediated by superoxide. The vanadate stimulation is not limited to plasma membrane. Golgi apparatus and endoplasmic reticulum show similar increase in NADH oxidase activity when vanadate is added. The endomembranes have significant vanadate-stimulated activity with both NADH and NADPH. The vanadate-stimulated NADH oxidase in plasma membrane is inhibited by compounds, which inhibit NADH dehydrogenase activity: catechols, anthracycline drugs and manganese. This activity is stimulated by high phosphate and sulfate anion concentrations.     

Aqueous two-phase partition and detergent precipitation of a drug-responsive NADH oxidase from the HeLa cell surface

The partitioning behaviour of a drug (capsaicin)-responsive NADH oxidase (tNOX) activity released from HeLa ceIls by low pH treatment followed by heat and proteinase K was determined. When partitioned in a standard 6.4% PEG 3350/6.4% dextran T-500 two-phase system, the bulk of the tNOX activity was in the dextran-rich lower phase. The activity was inhibited by and bound to the triazine dye, Cibacron blue. Affinity partition, where the Cibacron blue was coupled to amino PEG 5000 and added to the first two-phase separation step, resulted in the partitioning of activity to the upper PEG phase. A second partition with PEG-salts resulted in the release of the tNOX from the Cibacron blue–amino PEG enriched phase into the salt-enriched lower phase. The phase-purified protein exhibited anomalous behavior and tended to multimerize in sodium dodecyl sulphate (SDS) prior to SDS-polyacrylamide gel electrophoresis (PAGE). Multimerization appeared to be enhanced by PEG. The multimerization was enhanced with the reduced protein in the presence of detergent prior to SDS–PAGE. In addition, the activity was precipitated by PEG 8000 at concentrations between 6 and 30% by weight. In the presence of or after exposure to PEG 3350 or PEG 8000, the protein could not be detected by Western blot analysis after SDS–PAGE suggesting that the protein failed to enter the gel even though other HeLa cell surface proteins were unaffected. The anomalous multimerization behavior has thus far precluded the use of phase partition as a practical purification step for the oxidase.     

Auxin-activated nadh oxidase activity of soybean plasma membranes is distinct from the constitutive plasma membrane NADH oxidase and exhibits prion-like properties

Summary The hormone-stimulated and growth-related cell surface hydroquinone (NADH) oxidase activity of etiolated hypocotyls of soybeans oscillates with a period of about 24 min or 60 times per 24-h day. Plasma membranes of soybean hypocotyls contain two such NADH oxidase activities that have been resolved by purification on concanavalin A columns. One in the apparent molecular weight range of 14–17 kDa is stimulated by the auxin herbicide 2,4-dichlorophenoxyacetic acid (2,4-D). The other is larger and unaffected by 2,4-D. The 2,4-D-stimulated activity absolutely requires 2,4-D for activity and exhibits a period length of about 24 min. Also exhibiting 24-min oscillations is the rate of cell enlargement induced by the addition of 2,4-D or the natural auxin indole-3-acetic acid (IAA). Immediately following 2,4-D or IAA addition, a very complex pattern of oscillations is frequently observed. However, after several hours a dominant 24-min period emerges at the expense of the constitutive activity. A recruitment process analogous to that exhibited by prions is postulated to explain this behavior.     

The Arabidopsis thaliana RER1 gene family: its potential role in the endoplasmic reticulum localization of membrane proteins

Many endoplasmic reticulum (ER) proteins are known to be localized to the ER by a mechanism called retrieval, which returns the molecules that are exported from the ER to the Golgi apparatus back to the ER. Signals are required to be recognized by this retrieval system. In the work on yeast Saccharomyces cerevisiae, we have demonstrated that transmembrane domains of a subset of ER membrane proteins including Sec12p, Sec71p and Sec63p contain novel ER retrieval signals. For the retrieval of these proteins, a Golgi membrane protein, Rer1p, is essential (Sato et al., Mol. Biol. Cell 6 (1995) 1459–1477; Proc. Natl. Acad. Sci. USA 94 (1997) 9693–9698). To address the role of Rer1p in higher eukaryotes, we searched for homologues of yeast RER1 from Arabidopsis thaliana. We identified three cDNAs encoding Arabidopsis counterparts of Rer1p with an amino acid sequence identity of 39–46% to yeast Rer1p and named AtRER1A, AtRER1B, and AtRER1C1. AtRer1Ap and AtRer1Bp are homologous to each other (85% identity), whereas AtRer1C1p is less similar to AtRer1Ap and AtRer1Bp (about 50%). Genomic DNA gel blot analysis indicates that there are several other AtRER1-related genes, implying that Arabidopsis RER1 constitutes a large gene family. The expression of these three AtRER1 genes is ubiquitous in various tissues but is significantly higher in roots, floral buds and a suspension culture in which secretory activity is probably high. All the three AtRER1 cDNAs complement the yeast rer1 mutant and remedy the defect of Sec12p mislocalization. However, the degree of complementation differs among the three with that of AtRER1C1 being the lowest, again suggesting a divergent role of AtRer1C1p.     

Inhibition of erythrocyte plasma membrane NADH dehydrogenase by nucleotides and uncouplers

Erythrocyte ghost NADH dehydrogenase is inhibited in a competitive fashion by ATP and ADP whereas other nucleoside di- and triphosphates, cyclic nucleosides, as well as non-phosphorylating ATP analogs are relatively ineffective. In addition, this enzyme, measured with ferricyanide as electron acceptor, is inhibited by uncouplers of oxidative phosphorylation (proton-conducting reagents), the inhibition being competitive in character (i.e., the uncouplers were without influence upon maximum velocity). The effectiveness of the uncouplers was in the order of their hydrophobic character with the presence of the alkyl side chain rendering nonyl-dinitrophenol much more active than 2,6-dinitrophenol itself. Hydrophobic compounds that are not protonophores (e.g., eosin, proflavin or valinomycin) were not inhibitory. Whereas adenine nucleotides probably inhibit NADH oxidation competitively through structural similarity with the substrate, it appears unlikely that uncouplers compete at the NADH site directly. Rather, the apparently-competitive inhibition in the latter case may reflect competition for proton transfer to an acceptor residing in a hydrophobic region of the enzyme complex.     

Insulin control of a transplasma membrane NADH dehydrogenase in erythrocyte membranes

A study of NADH ferricyanide reductase activity in oriented vesicles or open ghosts of human and porcine erythrocytes shows that the dehydrogenase activity can have three types of orientation in the membrane. There is activity which responds only to acceptors and NADH exclusively on the inside face, or exclusively on the outer surface. There is also activity which requires exposure of both sides of the membrane and thus is transmembranous. The transmembrane activity is inhibited by insulin, whereas the internal and external enzymes do not respond to insulin. The transmembrane dehydrogenase can be a basis for proton transport in the plasma membrane.     

Generation of hydrogen peroxide on oxidation of NADH by hepatic plasma membranes

The oxidation of NADH by mouse liver plasma membranes was shown to be accompanied by the formation of H₂O₂. The rate of H₂O₂ formation was less than one-tenth the rate of oxygen uptake and much slower than the rate of reduction of artificial electron acceptors. The optimum pH for this reaction was 7.0 and theK _m value for NADH was found to be 3×10^–6 M. The H₂O₂-generating system of plasma membranes was inhibited by quinacrine and azide, thus distinguishing it from similar activities in endoplasmic reticulum and mitochondria. Both NADH and NADPH served as substrates for plasma membrane H₂O₂ generation. Superoxide dismutase and adriamycin inhibited the reaction. Vanadate, known to stimulate the oxidation of NADH by plasma membranes, did not increase the formation of H₂O₂. In view of the growing evidence that H₂O₂ can be involved in metabolic control, the formation of H₂O₂ by a plasma membrane NAD(P)H oxidase system may be pertinent to control sites at the plasma membrane.     

Effects of Q metabolites and related compounds on mitochondrial succinate and NADH oxidase systems

The effects of Q metabolites (Q acid-I, Q acid-II) and related compounds (dihydro Q acid-I, dehydro Q acid-II, QS-n, and their esters) on mitochondrial succinate and NADH oxidase systems were investigated. The activity restoring succinate oxidation in acetone-treated beef heart mitochondria was found to decrease with descending order of carbon number (n) of the side chain of the Q metabolites; activity was restored with Q acid-I (n = 7) to one-third as much as that with Q-7 and Q-10, but Q acid-II (n = 5) did not restore any activity. Of the related compounds with a carboxyalkyl group (QS-n), QS-16-QS-18 (n = 16–18) were found to be most active, and their activities were also correlated with n. The relationship between the restoration of activity and the partition coefficient was considered. NADH oxidation in pentane-treated beef heart submitochondrial particles could be restored with esters of low molecular weight quinones to the same extent as with Q-10, but not with the metabolites.     

Redox modulation of the response of NADH oxidase activity of rat liver plasma membranes to cyclic AMP plus ATP

NADH oxidase activity of rat liver plasma membranes was inhibited by lowconcentrations (1-100 nM) of ATP. The inhibition was amplified by additionof nanomolar concentrations (0.1-10) of cyclic AMP. The inhibition wascomplex and related to a marked increase in the Km for NADH at high NADHconcentrations together with a concomitant decrease in the Vmax. In theabsence of added or residual ATP, cyclic AMP was without effect. Theresponse of cyclic AMP + ATP was inhibited by low concentrations of theselective inhibitor of cyclic AMP-dependent protein kinase, H-89 but not bystaurosporin. The Vmax but not the Km was modified by treating the plasmamembranes with a mild oxidizing agent, N-chlorosuccinamide, or with thereducing agent, dithiothreitol. In the presence of dithiothreitol, the Vmaxwas reduced by cyclic AMP + ATP. In contrast, in the presence ofN-chlorosuccinamide, the Vmax was increased by cyclic AMP + ATP relative tocyclic AMP + ATP alone. Thus, the effect of cyclic AMP + ATP on the Vmaxcould be either an increase or a decrease depending on whether the membraneswere oxidized or reduced. The results demonstrate regulation of NADH oxidaseactivity of rat liver plasma membranes through cyclic AMP-mediatedphosphorylation by membrane-located protein kinase activities where thefinal response is dependent on the oxidation-reduction status of the plasmamembranes.