Inhibition of the NADH oxidase activity of plasma membranes isolated from xenografts and cell lines by the antitumor sulfonylurea,N-(4-methylphenylsulfonyl)-N′-(4-chlorophenyl)urea (LY 181984) correlates with drug susceptibility of growth

Summary Inhibition of NADH oxidase activity of plasma membranes isolated from a series of human xenografts and cell lines by the antitumor sulfonylurea, N-(4-methylphenylsulfonyl)-N-(4-chlorophenyl) urea (LY 181984), correlated with the ability of the sulfonylurea to inhibit cell growth. Growth of rat kidney cells either untransformed or transformed with Kirsten-ras (K-ras) were unaffected by the sulfonylurea. Similarly, the NADH oxidase activity of isolated plasma membranes from K-ras transformed cells was unaffected by LY 181984. In contrast, when transformed with Harvey-ras (H-ras), both growth and NADH oxidase activity were inhibited. With the inactive but structurally related LY 181985 (N-4-methylphenyl-sulfonyl)-N-(phenyl)urea), neither growth nor plasma membrane NADH oxidase activity of either sulfonylurea-susceptible or -resistant tissues or cell lines was inhibited. Both sulfonylureas were inactive with rat liver plasma membranes but NADH oxidase activity of plasma membranes and growth with HeLa cells was inhibited by the active (LY 181984) but not by the inactive (LY 181985) sulfonylurea. The findings suggest a possible correlation between inhibition of plasma membrane NADH oxidase activity by the antitumor sulfonylureas and their oncolytic action.     

The Sulfonylurea-Inhibited NADH Oxidase Activity of HeLa Cell Plasma Membranes has Properties of a Protein Disulfide–Thiol Oxidoreductase with Protein Disulfide–Thiol Interchange Activity

Plasma membrane vesicles of HeLa cells are characterized by a drug-responsive oxidation of NADH. The NADH oxidation takes place in an argon or nitrogen atmosphere and in samples purged of oxygen. Direct assay of protein thiols by reaction with 5,5-dithiobis-(2-nitrobenzoic acid) (DTNB; Ellman's reagent), suggests that protein disulfides may be the natural electron acceptors for NADH oxidation by the plasma membrane vesicles. In the presence of NADH, protein disulfides of the membranes were reduced with a concomitant stoichiometric increase in protein thiols. The increase in protein thiols was inhibited in parallel to the inhibition of NADH oxidation by the antitumor sulfonylurea LY181984 with an EC₅₀ of ca. 30 nM. LY181984, with an EC₅₀ of 30 nM, also inhibited a protein disulfide–thiol interchange activity based on the restoration of activity to inactive (scrambled) RNase and thiol oxidation. The findings suggest that thiol oxidation, NADH-dependent disulfide reduction (NADH oxidation), and protein disulfide–thiol interchange in the absence of NADH all may be manifestations of the same sulfonylurea binding protein of the HeLa plasma membrane. A surface location of the thiols involved was demonstrated using detergents and the impermeant thiol reagent p-chloromercuriphenylsulfonic acid (PCMPS). The surface location precludes a physiological role of the protein in NADH oxidation. Rather, it may carry out some other role more closely related to a function in growth, such as protein disulfide–thiol interchange coupled to cell enlargement.     

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.     

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.     

Thiol groups of chicken liver LDH (author's transl)]

Crystallized chicken liver H4 lactatedehydrogenase with PCBM and DTNB, proved to have sic thiol groups per enzyme molecule. Sulphydryl groups seemed necessary for activity since the enzyme became inactive when the groups were blocked by PCMB, DTNB or by Zn (II), Cu (II) or Hg (II). LDH inhibited by Hg (II) recovered its activity after treatment with beta-mercaptoentanol. LDH reversible inactivation, caused by PCMB, was partially impeded by NAD, NADH hand L-lactate but inactivation caused by DTNB was impeded in any way by coenzymes or substrates. PCMB is a competitive inhibitor with the coenzymes but is non-competitive with the substrates whereas DTNB is a competitive inhibitor with NADH or L-lactate. Kinetic studies of the DTNB inactivation suggest the possible formation of a DTNB-LDH-NADH complex. The formation of LDH-NADH and LDH-NAD pyruvate inactive complexes have been detected by U.V. absorbancy measurements. Such inactive complexes have equally been observed experimenting with the PCMB of Hg (II) previously treated enzyme. The results showed that these essential sulphydryl groups are not involved in th attaching of coenzymes or substrates to the chicken liver LDH molecule, but they seem to suggest the participation of --SH groups during the reversible hydrogen transfer between NADH and pyruvate.     

Selective Inhibition of Auxin-Stimulated NADH Oxidase Activity and Elongation Growth of Soybean Hypocotyls by Thiol Reagents

The NADH oxidase activity of isolated vesicles of soybean (Glycine max cv Williams 82) plasma membranes and elongation growth of 1-cm-long hypocotyl segments were stimulated by auxins (indole-3-acetic acid or 2,4-dichlorophenoxyacetic acid [2,4-D]). The auxin-induced stimulations of both NADH oxidase and growth were prevented by the thiol reagents N-ethylmaleimide, p-chloromercuribenzoate, 5,5[prime]-dithiobis(2-nitrophenylbenzoic acid), dithiothreitol, and reduced glutathione. These same reagents largely were without effect on or stimulated slightly the basal levels of NADH oxidase and growth when assayed in the absence of auxins. In the presence of dithiothreitol or reduced glutathione, both 2,4-D and indole-3-acetic acid either failed to stimulate or inhibited the NADH oxidase activity. The rapidity of the response at a given concentration of thiol reagent and the degree of inhibition of the 2,4-D-induced NADH oxidase activity were dependent on order of reagent addition. If the thiol reagents were added first, auxin stimulations were prevented. If auxins were added first, the inhibitions by the thiol reagents were delayed or higher concentrations of thiol reagents were required to achieve inhibition. The results demonstrate a fundamental difference between the auxin-stimulated and the constitutive NADH oxidase activities of soybean plasma membranes that suggest an involvement of active-site thiols in the auxin-stimulated but not in the constitutive activity.     

A growth factor- and hormone-stimulated NADH oxidase from rat liver plasma membrane.

NADH oxidase activity (electron transfer from NADH to molecular oxygen) of plasma membranes purified from rat liver was characterized by a cyanide-insensitive rate of 1 to 5 nmol/min per mg protein. The activity was stimulated by growth factors (diferric transferrin and epidermal growth factor) and hormones (insulin and pituitary extract) 2- to 3-fold. In contrast, NADH oxidase was inhibited up to 80% by several agents known to inhibit growth or induce differentiation (retinoic acid, calcitriol, and the monosialoganglioside, GM3). The growth factor-responsive NADH oxidase of isolated plasma membranes was not inhibited by common inhibitors of oxidoreductases of endoplasmic reticulum or mitochondria. As well, NADH oxidase of the plasma membrane was stimulated by concentrations of detergents which strongly inhibited mitochondrial NADH oxidases and by lysolipids or fatty acids. Growth factor-responsive NADH oxidase, however, was inhibited greater than 90% by chloroquine and quinone analogues. Addition of coenzyme Q10 stimulated the activity and partially reversed the analogue inhibition. The pH optimum for NADH oxidase was 7.0 both in the absence and presence of growth factors. The Km for NADH was 5 microM and was increased in the presence of growth factors. The stoichiometry of the electron transfer reaction from NADH to oxygen was 2 to 1, indicating a 2 electron transfer. NADH oxidase was separated from NADH-ferricyanide reductase, also present at the plasma membrane, by ion exchange chromatography. Taken together, the evidence suggests that NADH oxidase of the plasma membrane is a unique oxidoreductase and may be important to the regulation of cell growth.     

Is the Drug-Responsive NADH Oxidase of the Cancer Cell Plasma Membrane a Molecular Target for Adriamycin?

Enhanced growth inhibition and antitumor responses to adriamycin have been observed repeatedly from several laboratories using impermeant forms of adriamycin where entry into the cell was greatly reduced or prevented. Our laboratory has described an NADH oxidase activity at the external surface of plasma membrane vesicles from tumor cells where inhibition by an antitumor sulfonylurea, N-(4-methylphenylsulfonyl)-N-(4-chlorophenyl)urea (LY181984), and by the vanilloid, capsaicin (8-methyl-N-vanillyl-6-noneamide) correlated with inhibition of growth. Here we report that the oxidation of NADH by isolated plasma membrane vesicles was inhibited, as well, by adriamycin. An external site of inhibition was indicated from studies where impermeant adriamycin conjugates were used. The EC₅₀ for inhibition of the oxidase of rat hepatoma plasma membranes by adriamycin was several orders of magnitude less than that for rat liver. Adriamycin cross-linked to diferric transferrin and other impermeant supports also was effective in inhibition of NADH oxidation by isolated plasma membrane vesicles and in inhibition of growth of cultured cells. The findings suggest the NADH oxidase of the plasma membrane as a growth-related adriamycin target at the surface of cancer cells responsive to adriamycin. Whereas DNA intercalation remains clearly one of the principal bases for the cytotoxic action of free adriamycin, this second site, possibly related to a more specific antitumor action, may be helpful in understanding the enhanced efficacy reported previously for immobilized adriamycin forms compared to free adriamycin.     

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)     

Effect of cadmium,mercury and copper on partially purified hepatic flavokinase of rat

The effect of cadmium (Cd2+), mercury (Hg2+) and copper (Cu2+) was studied with partially purified flavokinase (ATP:riboflavin 5-phosphotransferase EC 2.7.1.26) from rat liver. All the divalent heavy metal cations inhibited flavokinase activity in a concentration-dependent manner. The inhibitory effect of cadmium on the enzyme was completely reversed by increasing concentration, of Zinc (Zn2+) indicating a competition between Zn2+ and Cd2+ for binding with the enzyme. A competition between riboflavin and Cd2+ is also evident from the present investigation. These observations hint at the possibility that Zn2+ and Cd2+ probably compete for the same site on the enzyme where riboflavin binds. However, inhibition of flavokinase by Hg2+ could not be reversed by Zn2+. Our studies further reveal that hepatic flavokinase appears to contain an essential, accessible and functional thiol group(s) which is evident from a concentration dependent inhibition of activity by sulfhydryl reagent s like parachloromercuribenzoate (PCMB), 5,5-dithiobis (2-nitrobenzoic acid)(DTNB), and N-ethylmaleimide (NEM). Inhibition of flavokinase by sulfhydryl reagents were protected, except in case of NEM inhibition, when the enzyme was incubated with thiol protectors like glutathione (GSH) and dithiothreitol (DTT). Furthermore, the enzyme could also be protected from the inhibitory effect of Cd2+ and Hg2+ by GSH and DTT suggesting that Cd2+ probably interacts with a reactive thiol group at or near the active site of enzyme in bringing about its inhibitory effect. (Mol Cell Biochem 167: 73-80, 1997)     

NADH oxidase of liver plasma membrane stimulated by diferric transferrin and neoplastic transformation induced by the carcinogen 2-acetylaminofluorene

NADH oxidase of purified plasma membranes (electron transfer from NADH to oxygen) was stimulated by the growth factor diferric transferrin. This stimulation was of an activity not inhibited by cyanide and was not seen in plasma membranes prepared from hyperplastic nodules from liver of animals fed the hepatocarcinogen, 2-acetylaminofluorene, nor was it due to reduction of iron associated with diferric transferrin. With plasma membranes from nodules, the activity was already elevated and the added transferrin was without effect. The stimulation by diferric transferrin did not correlate with the absence of transferrin receptors which were increased at the nodule plasma membranes. With liver plasma membranes, the stimulation by diferric transferrin raised the plasma membrane NADH oxidase specific activity to approximately that of the nodule plasma membranes. In contrast to NADH oxidase, which was markedly stimulated by the diferric transferrin, NADH ferricyanide oxidoreductase or reduction of ferric ammonium citrate by liver plasma membranes was approximately equal to or slightly greater than that of the nodule plasma membrane and unaffected by diferric transferrin. The results suggest the possibility of coupling of NADH oxidase activity to a growth factor response in mammalian cells as observed previously for this enzyme in another system.     

A Circulating Form of NADH Oxidase Activity Responsive to the Antitumor Sulfonylurea N-4-(methylphenylsulfonyl)-N′-(4-chlorophenyl)urea (LY181984) Specific to Sera from Cancer Patients

Our laboratory has described a drug-responsive NADH oxidase activity of the external surface of the plasma membrane of HeLa and other cancer cells, but not from normal cells, that was shed into media conditioned by the growth of cancer cells such as HeLa. The shed form of the activity exhibited the same drug responsiveness as the plasma membrane-associated form. In this study, sera from tumor-bearing and control rats, cancer patients, normal volunteers, and patients with diseases other than cancer were collected and assayed for a cancer-specific form of NADH oxidase responsive to the antitumor sulfonylurea N-(4-methylphenylsulfonyl)-N-(4-chlorophenyl)urea (LY181984). With sera from tumor-bearing rats and cancer patients, LY181984 added at a final concentration of 1 M either inhibited or stimulated the activity. With sera from control rats, normal volunteers, or patients with disorders other than cancer, the drug was without effect on the NADH oxidase activity of the sera. The activity altered by the antitumor sulfonylurea was present both in freshly collected sera and in sera stored frozen. Inhibition was half maximal at about 30 nM LY181984. The sulfonylurea-altered activity was found in sera of nearly 200 cancer patients including patients with solid cancers (e.g., breast, prostate, lung, ovarian) and with leukemias and lymphomas. We postulate that the serum presence of the antitumor sulfonylurea-responsive NADH oxidase represents an origin due to shedding from the patient's cancer. If so, the antitumor-responsive NADH oxidase would represent the first reported cell surface change universally associated with all forms of human cancer.     

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.     

The action of paraquat and diquat on the respiration of liver cell fractions

1. Paraquat and diquat produce only a slight increase in the oxygen uptake of rat liver mitochondria, and it is likely that they do not penetrate the mitochondrial membrane. 2. In mitochondrial fragments inhibited by antimycin A or by Amytal, both substances stimulate oxygen uptake with NADH or beta-hydroxybutyrate as substrate but not with succinate. The NADH dehydrogenase of the respiratory chain appears to be involved, at a site only partially inhibited by Amytal. 3. An NADPH oxidase activity is stimulated in rat liver microsomes by diquat, and to a smaller extent by paraquat; diquat also causes an NADH oxidase activity to develop. The effect is not inhibited by carbon monoxide or p-chloromercuribenzoate, and it is probable that a flavoprotein is involved by a mechanism not requiring thiol groups. 4. One molecule of oxygen can oxidize two molecules of NADPH in the stimulated microsomal system, the hydrogen peroxide produced being broken down by a catalase activity in the microsomes. 5. Diquat can stimulate NADH oxidase and NADPH oxidase activity in the postmicrosomal soluble fraction; the enzyme involved may be DT-diaphorase. 6. The mechanism of these reactions and their significance in relation to the toxicity of the dipyridilium compounds are discussed.     

Evidence for inherent differences in the system A carrier from normal and transformed liver tissue. Differential inactivation and substrate protection in membrane vesicles and reconstituted proteoliposomes

Plasma membrane vesicles isolated from intact rat liver (normal hepatocyte) or cultured rat H4 hepatoma cells retain Na+-dependent uptake of 2-aminoisobutyric acid mediated by System A. The carrier was inactivated in normal liver membrane vesicles by either N-ethylmaleimide (NEM) or p-chloromercuribenzene sulfonate (PCMBS). The concentrations required to produce half-maximal inhibition were approximately 370 and 110 microM for NEM and PCMBS, respectively. In contrast, transport of System A in H4 hepatoma membrane vesicles was sensitive to PCMBS (K 1/2 = 180 microM), yet totally unaffected by NEM at concentrations up to 5 mM. Substrate-dependent protection from PCMBS activation was observed for the System A activity in H4 hepatoma membranes, but not in vesicles from normal hepatocytes. Subsequent inactivation of the substrate-protected carrier by sulfhydryl-specific reagents, added following the removal of the protective amino acid, suggests that one or more cysteine residues become less reactive in the presence of System A substrates. Treatment of solubilized membrane proteins with NEM prior to reconstitution into artificial proteoliposomes showed that the selective inactivation by NEM of the carrier in normal liver membranes is not dependent on the lipid environment or on the integrity of the plasma membrane. The results support the hypothesis that there are inherent differences in the System A carriers that are present in normal and transformed liver tissue.     

Adriamycin affects plasma membrane redox functions

Adriamycin (Doxorubicin) stimulates NADH oxidase activity in liver plasma membrane, but does not cause NADH oxidase activity to appear where it is not initially present, as in erythrocyte membrane. NADH dehydrogenase from rat liver and erythrocyte plasma membranes shows similar adriamycin effects with other electron acceptors. Both NADH ferricyanide reductase and vanadate-stimulated NADH oxidation are inhibited by adriamycin, as is a cyanide insensitive ascorbate oxidase activity, whereas NADH cytochrome c reductase is not affected. The effects may contribute to the growth inhibitory (control) and/or deleterious effects of adriamycin. It is clear that adriamycin effects on the plasma membrane dehydrogenase involve more than a simple catalysis of superoxide formation.     

A molecular basis for retinol stimulation of vesicle budding in vivo and in vitro

Retinol stimulates the formation of transition vesicles in situ and in all free systems based on rat liver. The stimulation is on vesicle formation from transitional endoplasmic reticulum and not on vesicle fusion with donor membranes. Vesicle budding in the cell free system requires a nucleoside triphosphate and is sensitive to inhibition by thiol reagents. In this report we develop and test a model whereby a retinol-modulated NADH:protein disulfide reductase (NADH oxidase) with protein disulfide-thiol interchange activity is implicated in the vesicle budding mechanism. The protein has the ability to restore activity to scrambled, inactive RNase A and is stimulated or inhibited by retinol depending on the redox environment. Under reducing conditions and in the presence of a chemical reductant such as GSH, the partial reaction stimulated by retinol appears to be the oxidation of membrane disulfides. This is the first report of an enzymatic mechanism to explain specific retinol effects both in vivo and in vitro on membrane trafficking not given by retinoic acid.     

Different reactivity of two Brain sialyltransferases towards sulfhydryl reagents. Evidence for a thiol group involved in the nucleotide-sugar binding site of the NeuAcα2-3Galβ1-3GalNAc α(2–6)sialyltransferase

We have studied the amino-acid residues involved in the catalytic activity of two distinct brain sialyltransferases acting on fetuin and asialofetuin. These two enzymes were strongly inhibited byN-bromosuccinimide, a specific blocking reagent for tryptophan residues. This result suggests the involvement of such residues in the catalytic process of the two sialytransferases. Furthermore, chemical modifications by various sulfhydryl reagents led to a strong inhibition of the fetuin sialyltransferase while the asialofetuin sialyltransferase was only slightly inhibited. For a more thorough understanding of the thiol inactivation mechanism of the fetuin sialyltransferase, we studied in more detail the reactivity of this enzyme with NEM (N-ethylmaleimide), an irreversible reagent. The time-dependent inactivation followed first-order kinetics and these kinetic data afforded presumptive evidence for the binding of 1 mol NEM per mol of enzyme. Only CMP-NeuAc protected the enzyme against NEM inactivation effectively. MnCl₂ did not enhance the protective effect of CMP-NeuAc. The modifications of the fetuin sialyltransferase kinetic parameters by NEM showed a competitive mechanism between NEM and CMP-NeuAc. The results suggest the involvement of a sulfhydryl residue in or near the nucleotide-sugar binding may induce a change in conformation of the protein, leading to a decreased accessibility of this thiol group located near the nucleotide-sugar binding site). This SH group, is essential to the enzyme activity, which is not the case for the asialofetuin sialyltransferase.Abbreviations p-CMB p-chloromercuribenzoic acid - CPDS 6,6-dithiodinicotinic acid carboxypyridine disulfide - DTNB 5,5-dithiobis-(2-nitrobenzoic acid) - NEM N-ethylmaleimide - DTT dithiothreitol - Mes 2-(N-morpholino)ethane sulfonic acid - NeuAc N-acetylneuraminic acid     

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.