The plasma membrane NADH oxidase of HeLa cells has hydroquinone oxidase activity

Abstract

The plasma membrane NADH oxidase activity partially purified from the surface of HeLa cells exhibited hydroquinone oxidase activity. The preparations completely lacked NADH:ubiquinone reductase activity. However, in the absence of NADH, reduced coenzyme Q10 (Q10H2=ubiquinol) was oxidized at a rate of 15+/-6 nmol min-1 mg protein-1 depending on degree of purification. The apparent Km for Q10H2 oxidation was 33 microM. Activities were inhibited competitively by the cancer cell-specific NADH oxidase inhibitors, capsaicin and the antitumor sulfonylurea N-(4-methylphenylsulfonyl)-N'-(4-chlorophenyl)urea (LY181984). With coenzyme Q0, where the preparations were unable to carry out either NADH:quinone reduction or reduced quinone oxidation, quinol oxidation was observed with an equal mixture of the Q0 and Q0H2 forms. With the mixture, a rate of Q0H2 oxidation of 8-17 nmol min-1 mg protein-1 was observed with an apparent Km of 0.22 mM. The rate of Q10H2 oxidation was not stimulated by addition of equal amounts of Q10 and Q10H2. However, addition of Q0 to the Q10H2 did stimulate. The oxidation of Q10H2 proceeded with what appeared to be a two-electron transfer. The oxidation of Q0H2 may involve Q0, but the mechanism was not clear. The findings suggest the potential participation of the plasma membrane NADH oxidase as a terminal oxidase of plasma membrane electron transport from cytosolic NAD(P)H via naturally occurring hydroquinones to acceptors at the cell surface.

Ultradian oscillators of the circadian clock in <i>Saccharomyces</i> <i>cerevisiae</i>

“…Although externally-supplied NADH was used to assay ENOX activity, hydroquinones resident in the plasma membrane are most likely the natural substrate [10]. When reduced coenzyme Q 10 was assayed, two sets of oxidative maxima were seen (Figure 4) similar to those observed with NADH (Figure 1).…”

“…10 7 cells in 50 µl) were combined with 2.5 ml 50 mM Tris-Mes buffer (pH 7.0), 2 mM KCN and 150 µM NADH. To refold and renature the ENOX proteins of the heat-inactivated yeast, reduced glutathione (GSH) was added to a final concentration of 100 µM, and incubated for 10 min to reduce disulfides to allow for refolding.…”

“…Although enzymatic activity has been most often measured by monitoring NAD(P)H oxidation at 340 nm, the physiological substrates appear to be quinols (i.e., ubiquinols or phytoquinols) [10,11]. The NADH binding site is on the outside of the cell where there is usually little or no NAD(P)H. Quinones, such as Coenzyme Q, act as trans-plasma membrane shuttles to transfer reducing equivalents from cytosolic NAD(P)H to external acceptors, including molecular oxygen and protein disulfide.…”

CNS neurons express two distinct plasma membrane electron transport systems implicated in neuronal viability

“…Macho et al (1999) showed that capsaicin-induced apoptosis was correlated with the level of PMOR activity in different cell types. Capsaicin acts as a competitive inhibitor of ubiquinone (Kishi et al 1999) and perturbs electron flow to oxygen via the plasma membrane NADH oxidase, which normally produces water . Similarly, we found that capsaicin appears to inhibit CoQ 1 -dependent FeCN reduction by chick neurons, although non-specific interference with the assay makes it difficult to collect reliable data.…”

“…An isoform of NADH-cytochrome b 5 reductase (EC. 1.6.2.2) located on the cytoplasmic surface of the plasma membrane reduces ubiquinone and distinct NADH oxidase and FeCN reductase activities use electrons from hydroquinone to reduce oxygen, forming water (Kishi et al 1999), or to reduce FeCN (Kim et al 2002). This ubiquinone-mediated system has yet to be identified in neurons.…”