Microsomal NADH Research Articles

Decavanadate interacts with microsomal NADH oxidation system and enhances cytochrome c reduction

Oxidation of NADH with accompanying oxygen consumption (NADH:O(2) = 1:1) was observed in the combined presence of metavanadate (MV), decavanadate (DV) and microsomes. Oxygen consumption was negligible in the absence of MV, but NADH was oxidized and DV was reduced to a form of vanadyl-V(IV), colored blue like vanadyl sulfate but differed from it in having a 23-fold higher absorbance at 700 nm. DV can interact with the NADH oxidation system of microsomes as an electron acceptor, in addition to the known ferricyanide and cytochrome c. DV enhances rate of cytochrome c reduction significantly at microM concentrations. These studies indicate potential of DV as a redox intermediate.

Roles for NAD(P)H oxidases and reactive oxygen species in vascular oxygen sensing mechanisms

Observations that physiological levels of O 2 control the rates of production of reactive O 2 species by systems including NAD(P)H oxidases and that certain of these species have signalling mechanisms that regulate vascular tone has resulted in consideration of these systems in processes that mediate the sensing of changes in P O 2 . Evidence exists for the participation of hydrogen peroxide-dependent regulation of prostaglandin production and soluble guanylate cyclase activity, resulting from the metabolism of peroxide by cyclooxygenase and catalase, respectively, in P O 2 -elicited signalling mechanisms that regulate vascular force generation. A microsomal NADH oxidase whose activity is controlled by the redox status of cytosolic NAD(H) appears to function as a P O 2 sensor in bovine pulmonary and coronary arteries where changes in O 2 levels control the production of superoxide anion-derived hydrogen peroxide and a cGMP-mediated relaxation response. Interactions with nitric oxide and superoxide anion, and the activity of glutathione peroxidase appear to influence the function of these O 2 sensing systems, and some of these interactions, along with the activation of other oxidases, may contribute to alterations in P O 2 sensing mechanisms under pathophysiological conditions that affect vascular function.

NADH:Fe(III)-chelate reductase of maize roots is an active cytochrome b5 reductase

Microsomal NADH:Fe(III)-chelate reductase (NFR) of maize roots has been purified as a monomeric flavoprotein of 32 kDa with non-covalently bound FAD. In the presence of NADH, NFR efficiently reduced the physiological iron-chelate Fe(III)-citrate ( K cat/ K m(Fe(III)-citrate)=6.0×10 6 M −1 s −1) with a sequential reaction mechanism. Purified NFR was totally inhibited by the sulfhydryl reagent PHMB at 10 −9 M, and it could use cyt b 5 as alternative electron acceptor with a maximal reduction rate as high as with Fe(III)-citrate. We conclude that in maize roots the reduction of Fe(III)-citrate is chiefly performed by a cytochrome b 5 reductase, mostly associated with intracellular membranes and in part with the plasma membrane.

Lactate and PO2 modulate superoxide anion production in bovine cardiac myocytes: potential role of NADH oxidase.

Lactate increases lucigenin chemiluminescence (CL)-detectable superoxide anion (O2.-) generation in bovine vascular smooth muscle and endothelium, and a microsomal flavoprotein-containing NADH oxidase whose activity is regulated by PO2 and cytosolic NAD(H) redox appears to be the detected source of O2.- production. Little is known about the importance of this O2.(-)-producing system in cardiac myocytes. In isolated bovine cardiac myocytes, lactate (10 mmol/L) increased lucigenin-detectable O2.- levels to approximately 1.8 times baseline, whereas pyruvate (10 mmol/L) and mitochondrial probes did not increase the detection of O2.-. A nonmitochondrial NADH oxidase activity, found in microsomes containing a cytochrome b558, was a major source of O2.- production in the homogenate of myocytes, because NADH (0.1 mmol/L) increased basal lucigenin CL >100-fold. NADPH oxidases, mitochondria, and xanthine oxidase were minor sources of detectable O2.- production. However, mitochondria released H2O2 in the presence of 5 mmol/L succinate and 30 micromol/L antimycin, based on its detection as catalase-inhibitable luminol (+horseradish peroxidase)-elicited CL. Diphenyliodonium (DPI), an inhibitor of flavoprotein-containing oxidases, significantly attenuated basal, lactate, and NADH-elicited lucigenin CL. Hypoxia eliminated myocyte lucigenin CL, and posthypoxic reoxygenation caused an 8.6-fold increase in the detection of O2.- that was potentiated by lactate and inhibited by DPI. NADH oxidase activity linked to cytosolic NAD(H) redox appears to be a key source of O2.- production in cardiac myocytes that could contribute to oxidant signaling mechanisms and injury upon exposure to changes in PO2 and metabolites produced under hypoxia, such as lactate. These processes could contribute to the previously observed potentiation of injury caused by lactate in cardiac ischemia/reperfusion.

Properties of a superoxide anion-generating microsomal NADH oxidoreductase, a potential pulmonary artery PO2 sensor.

In this study, we describe properties of a microsomal NADH oxidoreductase that is a potential PO2-dependent source of vasoactive reactive O2 species in the calf pulmonary artery. Microsomes show an NADH-dependent production of superoxide anion (O2-.), as detected by lucigenin-elicited chemiluminescence, a superoxide dismutase inhibited reduction of nitro blue tetrazolium (NBT) and 2,6-dichlorophenol-indophenol, and O2 consumption. The microsomal production of O2-. was modulated by physiologically relevant levels of NADH and PO2, and O2-. production was reduced by inhibitors of NADH-dependent microsomal electron transport. Microsomes catalyzed an NADH-mediated reduction of several electron acceptor dyes, cytochrome c (rotenone insensitive) and methemoglobin. On reduction with dithionite, a cytochrome with an absorbance at approximately 558 nm was observed. Arterial O2-. levels (chemiluminescence) were also reduced by NBT and microsomal electron transport inhibitors. In pulmonary arteries, NBT selectively inhibited PO2 and lactate elicited changes in force generation, presumably by trapping O2-. and preventing H2O2 formation. Thus these studies are consistent with an involvement of O2-.-derived H2O2 generation via a microsomal NADH-cytochrome b558 electron transport system in calf pulmonary artery smooth muscle PO2 and lactate-elicited tone responses.

Application of electron-donor properties of glucose oxidase and xanthine oxidase for reduction of microsomal NAD(P)H-dependent electron-transport chains.

The reduction of cytochromes b5 and P-450 in mammalian hepatic microsomes by glucose oxidase and xanthine oxidase has been investigated. Under anaerobic conditions cytochrome b5 is reduced by glucose oxidase to the "dithionite" level, while cytochrome P-450 remains oxidized. Under the same conditions xanthine oxidase completely reduces both hemoproteins. Besides, neither glucose oxidase nor xanthine oxidase reduces isolated cytochromes. They can be reduced only after addition of microsomes to incubation media. Only in this case are the cytochromes, both isolated and included in microsomal membranes, reduced. The participation of microsomal flavoproteins in the reduction reaction is discussed. The method suggested makes it possible to substantially decrease the rates of reduction of microsomal hemoproteins, thus permitting the investigation of interactions between microsomal NADH- and NADPH-dependent electron-transport chains and electron carriers.

The Role of Reactive Oxygen Species in the Antitumor Activity of Bleomycin

Calf thymus DNA was incubated with bleomycin and FeCl3 in the presence of isolated rat liver microsomal NADH-cytochrome b5 reductase, cytochrome b5 and NADH which catalyze redox cycling of the bleomycin-Fe-complex. Furthermore, isolated rat liver nuclei were incubated with bleomycin, FeCl3 and NADH, a system in which redox cycling of bleomycin-Fe leads to DNA damage. In both systems free bases from DNA were released. Furthermore, 8-hydroxy-guanine was also found in the supernatant. On the other hand, 8-hydroxy-deoxyguanosine was detected in DNA of cell nuclei indicating that hydroxylation of the guanine molecule occurred in intact DNA. The release of bases correlated with the release of malondialydehyde as well as with NADH and oxygen consumption. These results indicate that NADH-cytochrome b5 reductase catalyzes redox cycling of the bleomycin-Fe-complex which results in the formation of reactive oxygen species which oxidize deoxyribose as well as bases of DNA. Both mechanisms may contribute to the cytotoxic and cytostatic effects of bleomycin observed in intact cells.

Action of ebselen on rat hepatic microsomal enzyme-catalyzed fatty acid chain elongation, desaturation, and drug biotransformation

In the previous study, the organoselenium-containing anti-inflammatory agent, Eb-selen, was found to disrupt both hepatic microsomal NADH- and NADPH-dependent electron transport chains. In the current investigation, we focus on the action of Ebselen on three separate metabolic reactions, namely, fatty acid chain elongation, desaturation, and drug biotransformation, which utilize reducing equivalents via these microsomal electron transport pathways. Both NADH-dependent and NADPH-dependent chain elongation reactions showed (i) that the condensation step was inhibited by Ebselen; all three substrates, palmitoyl CoA (16:0), palmitoleoyl CoA (16:1), and γ-linolenyl CoA (18: 3), were differentially affected by Ebselen; for example, the apparent K i 's of Ebselen for the condensation of 16:0, 16:1, and 18:3 in the absence of bovine serum albumin (BSA) preincubation were 7, 14, and 34 μ m, and those in the presence of BSA preincubation were 35, 62, and 150 μ m, respectively, supporting earlier data for multiple condensing enzymes; (ii) that the β-ketoacyl CoA reductase-catalyzed reaction step which appears to receive electrons, at least in part, from the cytochrome b 5 system, was also markedly inhibited by varying Ebselen concentrations; and (iii) that similar results were obtained with the dehydrase and the enoyl CoA reductase. Hence, each of the four component steps was significantly inhibited by Ebselen. Another important fatty acid biotransformation reaction, Δ9 desaturation of stearoyl CoA to oleoyl CoA, was significantly inhibited (90%) by 30 μ m Ebselen. This effect appeared to be directly related to the NADH-dependent electron transport chain rather than to a direct action on the desaturase enzyme. Last, Ebselen also inhibited both aminopyrine and benzphetamine N-demethylations, two cytochrome P450-catalyzed reactions, in untreated rats, in rats on a high carbohydrate diet, and in phenobarbital-treated rats.

Hydrocarbon content and microsomal BPH and reductase activity in mussel, Mytilus sp., from the Venice area, north-east Italy

During 1988, wild mussels were collected bimonthly from two stations having different pollution load, the first located close to the city of Venice and the second offshore in the Gulf of Venice, north-east Italy. Composite samples of digestive glands were analysed for their aliphatic and aromatic hydrocarbon content and for microsomal BPH, NADH-ferricyanide reductase (NADH-FERRIRED) and NADH- and NADPH-cytochrome c reductase (NAD(P)H-CYTCRED) activities. Results show that significant differences exist between the two stations in digestive gland hydrocarbon content while the enzymatic activity appears to be at the same level; as a consequence no significant correlations have been found between biochemical parameters and hydrocarbon concentrations in digestive glands.

Effects of pregnenolone-16 α - carbonitrile on drug metabolizing enzymes in hypophysectomized female rats

Treatment of intact and hypophysectomized female rats with pregnenolone-16α-carbonitrile (PCN) resulted in a significant increase in hepatic aryl hydrocarbon hydroxylase (AHH) activity. However, the total cytochrome P-450 concentration, as measured by CO difference spectra, was increased to a greater extent in hypophysectomized rats than in intact rats. Total cytochrome P-450 was found to be 0.82 ± 0.16 vs 2.43 ± 0.31 nmoles/mg protein for control and PCN-treated hypophysectomized rats, respectively, and 0.68 ± 0.23 vs 1.28 ± 0.05 nmoles/mg protein for control and PCN-treated intact rats respectively. The concentration of metyrapone complex in microsomes from intact control and PCN-treated rats was found to be 0.4 ± 0.11 vs 1.88 ± 0.23 M respectively. Treatment of hypophysectomized rats with PCN resulted in an approximate 10-fold increase in the concentration of the metyrapone complex (0.42 ± 0.15 M for control and 4.46 ± 0.44 M for PCN-treated). Microsomal NADPH and NADH cytochrome c reductase activities were also altered by PCN-treatment. Aminopyrine demethylase activity was stimulated approximately three-fold by PCN treatment in both intact and hypophysectomized rats. Benzphetamine demethylase activity was not significantly affected by PCN treatment. The results of these studies suggest that the absence of the pituitary gland can markedly influence PCN induction of cytochrome P-450 in the liver in female rats. PCN also differentially affects microsomal mixed-function oxidase activities associated with drug and xenobiotic metabolism.

Microsomal detoxication enzyme responses of the marine snail, Thais haemastoma, to laboratory oil exposure.

The cytochrome P-450 monooxygenase or mixed function oxidase (MFO) system is a widely distributed enzyme system involved in the detoxication of foreign organic compounds (xenobiotics) taken up by organisms. Increases in the activities of the MFO system, occur with exposure of the organism to organic xenobiotics and such responses in the field have been proposed as a means of identifying biological impact by organic pollution. The carnivorous marine gastropod Thais haemastoma, or southern oyster drill, rapidly accumulated polynuclear aromatic and other hydrocarbons from the environment, through both the food source and the water-column. In laboratory experiments T. haemastoma were exposed to the water soluble fraction (WSF) of South Louisiana crude oil and the responses of the MFO system examined. Preliminary characterization of the snail MFO system was carried out using methodology developed from studies on the common mussel Mytilus edulis. Microsomal benz(a)pyrene hydroxylase (BPH), NADH- and NADPH- dependent cytochrome c reductase (NAD(P)H-CYTCRED) and NADH-dependent ferricyanide reductase (NADH-FERRIRED) activities were measured but it was not possible to determine cytochrome P-450 or b/sub 5/.

Biosynthesis of gamma-linolenic acid in cotyledons and microsomal preparations of the developing seeds of common borage (Borago officinalis).

The developing seeds of Borago officinalis (common borage) accumulate a triacylglycerol oil that is relatively rich in the uncommon fatty acid gamma-linolenate (octadec-6,9,12-trienoic acid). Incubation of developing, whole, cotyledons with [14C]oleate and [14C]linoleate showed that the gamma-linolenate was synthesized by the sequential desaturation of oleate----linoleate----gamma-linolenate. Microsomal membrane preparations from the developing cotyledons contained an active delta 6-desaturase enzyme that catalysed the conversion of linoleate into gamma-linolenate. Experiments were designed to manipulate the [14C]linoleate content of the microsomal phosphatidylcholine. The [14C]linoleoyl phosphatidylcholine labelled in situ was converted into gamma-linolenoyl phosphatidylcholine in the presence of NADH. The substrate for the delta 6-desaturase in borage was, therefore, the linoleate in the complex microsomal lipid phosphatidylcholine, rather than, as in animals, the acyl-CoA. This was further confirmed in experiments that compared the specific radioactivity of the gamma-linolenate, in acyl-CoA and phosphatidylcholine, that was synthesized when [14C]linoleoyl-CoA was incubated with microsomal membranes, NADH and non-radioactive gamma-linolenoyl-CoA. The delta 6-desaturase was positionally specific and only utilized the linoleate in position 2 of sn-phosphatidylcholine. Analysis of the positional distribution of fatty acids in the endogenous microsomal sn-phosphatidylcholine showed that, whereas position 1 contained substantial linoleate, only small amounts of gamma-linolenate were present. The results shed further light on the synthesis of C18 polyunsaturated fatty acids in plants and in particular its relationship to the regulation of the acyl quality of the triacylglycerols in oilseeds.

Methyl ethyl ketone peroxide damage to cytochrome P-450 peroxidase activities

Some new aspects of damage to cytochrome P-450 peroxidase activities by a toxic peroxide were investigated. Methyl ethyl ketone peroxide (MEKP) damage to cytochrome P-450 and inhibition of cytochrome P-450-mediated peroxidase activities were studied. In vivo, hepatic microsomal NADH- and NADPH-peroxidase activities were induced in rats by phenobarbital and 3-methylcholanthrene. Phenobarbital treatment induced more NADH- and NADPH-peroxidase activity than did 3-methylcholanthrene treatment. In vitro, microsomal cytochrome P-450 from rat liver was more sensitive to damage by MEKP than was this protein from rat kidney. Destruction of cytochrome P-450 hemoprotein and inhibition of its associated peroxidase activities by MEKP increased as a function of time of exposure to the peroxide. The addition of MEKP to microsomes resulted in a progressive increase in spectral absorbance at 433 nm. MEKP was an irreversible inhibitor of NADH- and NADPH-peroxidase, and NADH-peroxidase was more sensitive to damage than was NADPH-peroxidase. At 0 time and low concentration, MEKP was immediately bound to microsomal cytochrome P-450 and it exhibited competitive inhibition of NADH- and NADPH-peroxidase. At high concentration, MEKP was a mixed inhibitor of NADH- and NADPH-peroxidase. This study also showed that MEKP is a substrate for microsomal P-450 peroxidase activities and suggests that these peroxidase activities may play an important role in metabolism of toxic lipophilic organic peroxides that enter the endoplasmic reticulum.

The inhibitory effect of halides and carboxylates on hepatic NADH: cytochrome b5 oxidoreductase.

The inhibitory effect of several halides and carboxylates on bovine liver microsomal NADH: cytochrome b5 oxidoreductase (EC 1.6.2.2) was examined. The magnitude of inhibition was altered by various anion species but not by cation species. The order of effectiveness was F- less than acetate less than Cl- less than Br- less than I- less than succinate congruent to citrate. The kinetics of these inhibitions was competitive with cytochrome b5, and non-competitive with NADH, indicating that these anions inhibit the interaction of the enzyme with cytochrome b5.

Differential sensitivity of canine cardiac sarcolemmal and microsomal enzymes to inhibition by free radical-induced lipid peroxidation.

Sarcolemmal and microsomal membranes prepared from adult canine cardiac myocytes (sarcolemmal Na+, K+-ATPase = 71.8 mumol/mg per hr and microsomal rotenone-insensitive NADH cytochrome c reductase = 114 mumol/mg per hr) were each preincubated at 37 degrees C in the presence of a free radical generating system consisting of dihydroxyfumarate and Fe -ADP; loss of the Na+, K+-ATPase and reductase activities, as well as the associated increases in lipid peroxidation, measured by malondialdehyde formation, were temporally correlated in both systems. The ATPase was inhibited 70% when the malondialdehyde was 71 nmol/mg protein at 20 minutes and 90% when malondialdehyde was 138 nmol/mg protein at 90 minutes. Inhibition of reductase activity occurred more gradually, displaying a 27% loss of activity when malondialdehyde reached 34 nmol/mg protein at 20 minutes and 60% with a malondialdehyde value of 67 nmol/mg protein at 90 minutes. The greater susceptibility of the sarcolemma to free radical-induced membrane damage may be due to the higher content of unsaturated fatty acids in this membrane, compared to microsomes.

Evidence of an age-related decline in mitochondrial glycerophosphate dehydrogenase activity of isolated rat islets

The activities of three enzymes—two mitochondrial and one microsomal—were measured in isolated islets of Langerhans from 2-month-old and 12-month-old rats. Mitochondrial glycerophosphate dehydrogenase activity (expressed as nanomoles of iodonitrotetrazolium reduced per minute per milligram of protein), decreased ( P < 0.01) from a mean (± SEM) of 73.2 ± 11.2 (2-month-old) to 34.7 ± 5.9 (12-month-old). In contrast, activities of neither mitochondrial monoamine oxidase nor microsomal NADH cytochrome-c reductase changed with age. These results demonstrate that the activity of the glycerophosphate shuttle decreases as rats grow older, and it raises the possibility that the consequent difficulty in regenerating cytosolic NAD + may play a role in the insulin secretory defect associated with aging.

Microsomal electron-transport reductase activities and fatty acid elongation in rat brain. Developmental changes, regional distribution and comparison with liver activity.

Gestational and postnatal changes of microsomal NADH:cytochrome b5 reductase and NADPH:cytochrome c reductase activities were examined in rat brain. The specific activity of NADH:cytochrome b5 reductase was high at 18-19 days of gestational age, decreased to a minimum at 4 to 6 days after birth and increased thereafter. An essentially similar developmental pattern was observed for the specific activity of NADPH:cytochrome c reductase. In contrast, the specific activities of these reductases in liver microsomes were low, did not display a peak during gestation and increased steadily to a maximum at 40-50 days after birth. The rate of incorporation of [2-14C]malonyl-CoA into palmitoyl-CoA in brain microsomes was found to be high in the foetus, sharply decreased to a minimum at the time of birth and increased thereafter. The activity of fatty acid elongation in liver microsomes was much less than that in brain during gestation and increased rapidly after birth to values at 50-60 days 20-fold greater than the foetal activity. NADH and NADPH were equally effective for brain microsomal fatty acid elongation. Regional distribution of cytochrome reductase activities and the activity of fatty acid elongation showed the lowest specific activity in cerebellum. These results suggest that brain microsomal electron transport may be correlated with the developmental alteration in fatty acid elongation.

Cardiac microsomal rotenone-insensitive NADH cytochrome C reductase (RINCR): Inhibition by various species of long-chain acyl CoA: W.B. Weglicki, F.F. Kennett, T.E. Knauer and K. Owens, Cardiovascular Research Laboratory, Oklahoma Medical Research Foundation, Oklahoma City, OK 73104

Cardiac microsomal rotenone-insensitive NADH cytochrome C reductase (RINCR): Inhibition by various species of long-chain acyl CoA: W.B. Weglicki, F.F. Kennett, T.E. Knauer and K. Owens, Cardiovascular Research Laboratory, Oklahoma Medical Research Foundation, Oklahoma City, OK 73104

Aryl hydrocarbon hydroxylase of rat brain mitochondria: Properties of, and effect of inhibitors and inducers on, enzyme activity

Aryl hydrocarbon hydroxylase (AHH), a typical example of mixed-function oxidase system, was studied in rat brain mitochondria. The enzyme was found to require oxygen and NADH for optimal expression of the activity. Coaddition of NADPH in the incubation system containing NADH resulted in an additive effect on the enzyme activity. NADH- and NADPH-dependent mitochondrial AHH activity was linear with respect to protein concentration and incubation time. The enzyme exhibited a sharp optima at pH 7.6. Specific activity of NADH-dependent mitochondrial AHH in rat brain was 3–4 and 8–11 times higher than that of NADPH-dependent mitochondrial and microsomal enzyme activity, respectively. Of the species investigated, NADH-dependent mitochondrial AHH followed the order: mice ⩾ guinea pig > rat, while NADPH-supported mitochondrial AHH was in the order: rat > guinea pig ⩾ mice. Specific activity of NADH-dependent mitochondrial AHH in various rat brain regions was similar with the exception of olfactory lobes which exhibited 60% higher activity than other region. When total region activities were added approximately whole brain activity was recovered. The apparent K m value of NADH-dependent mitochondrial AHH was 1.18 μ m with benzo( a)pyrene as a substrate. This K m value was five to six times lower than that of NADPH-dependent microsomal AHH in rat brain (6.66 μ m). NADH-dependent mitochondrial AHH was inhibited by KCN in a concentration-dependent manner while NADPH-supported mitochondrial AHH did not reveal any sensitivity to cyanide. Brain microsomal NADH as well as NADPH-supported AHH was also inhibited by KCN in a concentration-dependent manner. Carbon monoxide inhibited NADH-dependent mitochondrial AHH activity (48%) and had no effect on NADPH-dependent mitochondrial enzyme. Mitochondrial NADH and NADPH-dependent AHH activities were induced by 3-methylcholanthrene (64–73%) and benzo( a)pyrene (91–92%) pretreatments while no induction occurred with phenobarbital administration. 1-Benzylimidazole, SKF 525 A, metyrapone, and α-naphthoflavone inhibited both basal and 3-methylcholanthreneinduced NADH-dependent mitochondrial AHH activity. α-Naphthoflavone was more effective in inhibiting 3-methylcholanthrene-stimulated rat brain NADH-dependent mitochondrial AHH. Mitochondrial NADH-dependent AHH activity increased gradually with the onset of development and attained a steady state after 49–56 days of age. An increase of eight- to ninefold in the specific enzyme activity was observed between 7- and 56-day-old rats. No significant increase in brain mitochondrial AHH activity was observed between 56- and 91-day-old rats.

Vitamin K-Dependent Reactions in Rat Liver: Role of Flavoproteins

The role of flavins in vitamin K function was assessed by examining blood coagulation and in vitro activities of hepatic vitamin K-dependent enzymes from control and riboflavin-deficient rats. One-stage prothrombin times and Factor VII activities were lower in flavin-deficient rats than in ad libitum or pair-fed controls. Fibrinogen, prothrombin, and Factor X activities were normal. Hepatic vitamin K-dependent carboxylase activity was severely depressed in flavin-deficient rats when assayed with [vitamin K + NADH] and somewhat depressed with reduced vitamin K (vitamin KH2) as substrate. One-hour flavin repletion appreciably restored [vitamin K + NADH]-dependent activity, but vitamin KH2-dependent activity was not restored even after 16 hours repletion. These results suggest that the carboxylating enzyme itself is not a flavoprotein, but that the microsomal NADH dehydrogenase required for [vitamin K + NADH]-dependent carboxylation is a flavoprotein. This dehydrogenase may differ from the cytosolic Warfarin-inhibitable ‘DT-diaphorase’ in that the activity of the latter, which is reduced 50% in flavin-deficient rats, is not at all restored by one-hour flavin repletion. Flavin status-dependent differences in NADH-dependent or vitamin KH2-dependent epoxidation of vitamin K paralleled differences in the carboxylase. Flavin deficiency had no effect on vitamin K 2,3-epoxide reductase activity nor on its inhibition by Warfarin.dehydrogenases flavoproteins riboflavin vitamin K