Frederick L. Crane

Biochemical Functions of Coenzyme Q10

Coenzyme Q is well defined as a crucial component of the oxidative phosphorylation process in mitochondria which converts the energy in carbohydrates and fatty acids into ATP to drive cellular machinery and synthesis. New roles for coenzyme Q in other cellular functions are only becoming recognized. The new aspects have developed from the recognition that coenzyme Q can undergo oxidation/reduction reactions in other cell membranes such as lysosomes, Golgi or plasma membranes. In mitochondria and lysosomes, coenzyme Q undergoes reduction/oxidation cycles during which it transfers protons across the membrane to form a proton gradient. The presence of high concentrations of quinol in all membranes provides a basis for antioxidant action either by direct reaction with radicals or by regeneration of tocopherol and ascorbate. Evidence for a function in redox control of cell signaling and gene expression is developing from studies on coenzyme Q stimulation of cell growth, inhibition of apoptosis, control of thiol groups, formation of hydrogen peroxide and control of membrane channels. Deficiency of coenzyme Q has been described based on failure of biosynthesis caused by gene mutation, inhibition of biosynthesis by HMG coA reductase inhibitors (statins) or for unknown reasons in ageing and cancer. Correction of deficiency requires supplementation with higher levels of coenzyme Q than are available in the diet.

Tightly bound cardiolipin in cytochrome oxidase.

Tightly bound cardiolipin has been found in cytochrome oxidase. The molar ratio of bound cardiolipin to cytochrome oxidase approaches 1:1. The tightly bound cardiolipin cannot be removed by many solvents which extract loosely bound lipids, but can be extracted with alkaline chloroform-methanol. Phospholipase A does not hydrolyze the bound lipid. After partial removal by repeated cholate-(NH4)2SO4 treatment maximum activity can be restored in the oxidase by cardiolipin but not by detergents. The bound cardiolipin has a fatty acid composition similar to the bulk mitochondrial cardiolipin but small amounts of three other phospholipid fractions with a high level of saturated fatty acid are also found to be closely associated with the oxidase.

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.

NADH OXIDATION IN LIVER AND FAT CELL PLASMA MEMBRANES

1. Introduction Evidence that transformed cells have an increased NADH/NAD ratio [I] and decreased adenylate cyclase [2] and evidence that NADH will inhibit the AMP cyclase of plasma membrane [3] has brought up the question of a sensor of NADH in the plasma membrane. The fact that the flavin antagonist atebrin inhibits cyclase suggested that an NADH dehydrogenase might be the site of the NADH inhibition [3] . There have been reports of NADH dehydrogenase activity in plasma membrane preparations. In most

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.

The diversity of coenzyme Q function

Coenzyme Q is uniquely designed as an electron and proton carrier within the lipid phase of membranes. It now appears that this unique chemistry has diverse application to important functions in all cellular membranes. The first function of coenzyme Q was defined in the energy transduction process in mitochondria. New studies show that the presence of coenzyme Q in other cellular membranes has dynamic rather than passive significance. Coenzyme Q functions in the plasma membrane electron transport involved in activation of signalling protein kinases related to gene activation for cellular proliferation. Furthermore, the antioxidant potential of the reduced coenzyme Q is now taken on a new significance in the evidence that the reduced quinone can act to maintain tocopherol in the reduced state in membranes and ascorbate reduced both inside and outside the cell.

[37] Quinones in algae and higher plants

Publisher Summary This chapter discusses the determination of quinones from algae and higher plants. In the case of determination of quinones from higher plants, spinach leaves in 200-g quantities are ground in 1.5 liters of grinding medium for 1 minute at low speed (15,500 rpm without a load). The chlorophyll content of spinach chloroplasts is determined by Arnons method. The green supernatant is used for a spectrophotometric assay for chlorophyll a (663 nm) and b (645 nm) read against an acetone blank. The extract from chloroplasts or whole leaves containing plastoquinones and other quinones is transferred to a separatory funnel. After separation of the initial two phases, the green lipid epiphase is set aside while the aqueous hypophase is rewashed with 500 ml of heptane, which is later added to the previously collected heptane epiphase. Plastoquinones of the A, B, and C types, which occur in all taxonomic divisions of higher plants have also been found in all classes of algae, including representatives of greens, yellow-greens, blue-greens, reds, browns, and the flagellate, Euglena. Quinones from algae can be extracted by the heptane–isopropanol–water (1:1:1) method, or by the alternative quinone extraction procedures with acetone.

The essential functions of coenzyme Q

SummaryThe essential role of coenzyme Q in biological energy transduction is well established. Coenzyme Q is a unique carrier for two-electron transfer within the lipid phase of the mitochondrial membrane. The function is essential for proton-based energy coupling. The sites of entry and exit of electrons into the quinone are at specific quinone-binding sites which are constructed to allow only two-electron transfer and thus prevent damaging free radical formation by direct reaction of oxygen with the semiquinone. Failure of proper function with diminished energy supply can be related to insufficient quinone, modification of lipid fluidity, or lipid protein interaction and damage or poisoning in binding sites. Supplementation with coenzyme Q can act by reversal of deficiency or decreased mobility, or by overcoming binding site modification. Coenzyme Q has also been shown to increase antioxidant protection in membranes. New sites for coenzyme Q function in Golgi and plasma membrane show evidence for a role in growth control and secretion-related membrane flow.

Quinone interaction with the respiratory chain-linked NADH dehydrogenase of beef heart mitochondria. II. Duroquinone reductase activity.

Abstract 1. Substituted benzoquinones and napthoquinones can function as electron acceptors for the NADH dehydrogenase segment of the mitochondrial electron transport system. Anthraquinones and several hydroxylated quinones are not reduced by the enzyme complex. 2. Piericidin A treatment at concentrations known to inhibit electron transport causes varying degrees of inhibition of quinone reductase activities. The pattern of piericidin A inhibition suggests that certain quinones are reduced at sites either before or after the piericidin A inhibition site. Reduction of quinones such as 5-hydroxy-1,4-napthoquinone (juglone) and 1,2-napthoquinone is inhibited only slightly. Reduction of ubiquinone 1 and 2 and 2,3,5,6-tetramethyl-1,4-benzoquinone (duroquinone) is almost completely inhibited following piericidin A treatment. 3. Comparison of juglone reductase activity with ferricyanide reductase activity suggests that these acceptors are reduced at nonequivalent sites in the NADH dehydrogenase. Juglone reductase activity is stimulated following mercurial treatment while ferricyanide reductase activity is slightly inhibited. Preincubation of NADH dehydrogenase with NADH followed by mercurial treatment causes almost complete inhibition of ferricyanide reductase activity. Activation of juglone reductase activity still occurs under these conditions. The activation is specific for preparations of NADH dehydrogenase, for mercurials, and for napthoquinone reductase activity.

Electron and proton transport across the plasma membrane

Transplasma membrane electron transport in both plant and animal cells activates proton release. The nature and components of the electron transport system and the mechanism by which proton release is activated remains to be discovered. Reduced pyridine nucleotides are substrates for the plasma membrane dehydrogenases. Both plant and animal membranes have unusual cyanide-insensitive oxidases so oxygen can be the natural electron acceptor. Natural ferric chelates or ferric transferrin can also act as electron acceptors. Artificial, impermeable oxidants such as ferricyanide are used to probe the activity. Since plasma membranes containb cytochromes, flavin, iron, and quinones, components for electron transport are present but their participation, except for quinone, has not been demonstrated. Stimulation of electron transport with impermeable oxidants and hormones activates proton release from cells. In plants the electron transport and proton release is stimulated by red or blue light. Inhibitors of electron transport, such as certain antitumor drugs, inhibit proton release. With animal cells the high ratio of protons released to electrons transferred, stimulation of proton release by sodium ions, and inhibition by amilorides indicates that electron transport activates the Na+/H+ antiport. In plants part of the proton release can be achieved by activation of the H+ ATPase. A contribution to proton transfer by protonated electron carriers in the membrane has not been eliminated. In some cells transmembrane electron transport has been shown to cause cytoplasmic pH changes or to stimulate protein kinases which may be the basis for activation of proton channels in the membrane. The redox-induced proton release causes internal and external pH changes which can be related to stimulation of animal and plant cell growth by external, impermeable oxidants or by oxygen.