Vanadate-dependent NAD(P)H oxidation by microsomal enzymes

Vanadate-dependent NAD(P)H oxidation, catalyzed by rat liver microsomes and microsomal NADPH-cytochrome P450 reductase (P450 reductase) and NADH-cytochrome b5 reductase (b5 reductase), was investigated. These enzymes and intact microsomes catalyzed NAD(P)H oxidation in the presence of either ortho- or polyvanadate. Antibody to P450 reductase inhibited orthovanadate-dependent NADPH oxidation catalyzed by either purified P450 reductase or rat liver microsomes and had no effect on the rates of NADH oxidation catalyzed by b5 reductase. NADPH-cytochrome P450 reductase catalyzed orthovanadate-dependent NADPH oxidation five times faster than NADH-cytochrome b5 reductase catalyzed NADH oxidation. Orthovanadate-dependent oxidation of either NADPH or NADH, catalyzed by purified reductases or rat liver microsomes, occurred in an anaerobic system, which indicated that superoxide is not an obligate intermediate in this process. Superoxide dismutase (SOD) inhibited orthovanadate, but not polyvanadate-mediated, enzyme-dependent NAD(P)H oxidation. SOD also inhibited when pyridine nucleotide oxidation was conducted anaerobically, suggesting that SOD inhibits vanadate-dependent NAD(P)H oxidation by a mechanism independent of scavenging of O2-.     

Characterization of vanadate-dependent NADH oxidation stimulated by Saccharomyces cerevisiae plasma membranes.

Plasma membrane-stimulated vanadate-dependent NADH oxidation has been characterized in Saccharomyces cerevisiae. This activity is specific for vanadate, because molybdate, a similar metal oxide, did not substitute for vanadate in the reaction. Vanadate-dependent plasma membrane-stimulated NADH oxidation activity was dependent on the concentrations of vanadate, NADH, and NADPH and required functional plasma membranes; no stimulation occurred in the presence of boiled membranes or bovine serum albumin. The dependence of membrane-stimulated vanadate-dependent NADH oxidation was not linearly dependent on added membrane protein. The activity was abolished by the superoxide anion scavenger superoxide dismutase and was stimulated by paraquat and NADPH. These data are consistent with the previously proposed chain reaction for vanadate-dependent NADH oxidation. The role of the plasma membrane appears to be to stimulate superoxide radical formation, which is coupled to NADH oxidation by vanadate. 51V-nuclear magnetic resonance studies are consistent with the hypothesis that a phosphovanadate anhydride is the stimulatory oxyvanadium species in the phosphate buffers used at pHs 5.0 and 7.0. In phosphate buffers, compared with acetate buffers, the single vanadate resonance was shifted upfield at both pH 5.0 and pH 7.0, which is characteristic of the phosphovanadate anhydride. Since the cell contains an excess of phosphate to vanadate, the phosphovanadate anhydride may be involved in membrane-mediated vanadate-dependent NADH oxidation in vivo.     

Superoxide is responsible for the vanadate stimulation of NAD(P)H oxidation by biological membranes

Vandate augments the oxidation of NAD(P)H, but not of NMNH, by rat liver microsomes. Paraquat increases the vanadate effect on NADPH, but not on NADH, oxidation. Substoichiometric levels of NADPH caused the co-oxidation of NADH or NMNH and SOD inhibited in all cases. The ratio of NADH or NMNH co-oxidized per NADPH added allowed estimation of average chain length, which increased as the pH was lowered from 8.0 to 7.1. The initial rate of this co-oxidation of NMNH was a saturating function of the concentration of microsomes, reflecting a decrease in chain length with an increase in number of concomitant reaction chains, and due to increasing radical-radical termination reactions. Mitochondrial outer membranes behaved like the microsomal membranes, but mitochondrial inner membranes catalyzed a rapid oxidation of NADH which could be augmented by vanadate, whose action was enhanced by paraquat and inhibited by antimycin or rotenone. These and related observations support the view that vanadate stimulates NAD(P)H oxidation by biological membranes, not by virtue of interacting with enzymes, but rather by interacting with O-2.     

The internal rotenone‐insensitivae NADPH dehydrogenase contributes to malate oxidation by potato tuber and pea leaf mitochondria

Inside-out submitochondrial particles from both potato (Solanum tuberosum L. cv. Bintje) tubers and pea (Pisum sativum L. cv. Oregon) leaves possess three distinct dehydrogenase activities: Complex I catalyzes the rotenone-sensitive oxidation of deamino-NADH, ND_in(NADPH) catalyzes the rotenone-insensitive and Ca²⁺-dependent oxidation of NADPH and ND_in(NADH) catalyzes the rotenone-insensitive and Ca²⁺-independent oxidation of NADH. Diphenylene iodonium (DPI) inhibits complex I, ND_in(NADPH) and ND_in (NADH) activity with a K_i of 3.7, 0.17 and 63 µM, respectively, and the 400-fold difference in K_i between the two ND_in made possible the use of DPI inhibition to estimate ND_in (NADPH) contribution to malate oxidation by intact mitochondria. The oxidation of malate in the presence of rotenone by intact mitochondria from both species was inhibited by 5 µM DPI. The maximum decrease in rate was 10–20 nmol O₂ mg^?1 min^?1. The reduction level of NAD(P) was manipulated by measuring malate oxidation in state 3 at pH 7.2 and 6.8 and in the presence and absence of an oxaloacetate-removing system. The inhibition by DPI was largest under conditions of high NAD(P) reduction. Control experiments showed that 125 µM DPI had no effect on the activities of malate dehydrogenase (with NADH or NADPH) or malic enzyme (with NAD⁺ or NADP⁺) in a matrix extract from either species. Malate dehydrogenase was unable to use NADP⁺ in the forward reaction. DPI at 125 µM did not have any effect on succinate oxidation by intact mitochondria of either species. We conclude that the inhibition caused by DPI in the presence of rotenone in plant mitochondria oxidizing malate is due to inhibition of ND_in(NADPH) oxidizing NADPH. Thus, NADP turnover contributes to malate oxidation by plant mitochondria.     

Hydroperoxide peroxidase activity in liver microsomes

The oxidation of either NADH or NADPH by cumene hydroperoxide in rat liver microsomes is described. The Km′ for the hydroperoxide varied with the pyridine nucleotide utilized (NADPH, Km′ = 0.91 mM; NADH, Km′ = 3.3 mM). Carbon monoxide did not inhibit the peroxidase activity although a variety of other agents which interact with cytochrome P₄₅₀ did produce inhibitory effects. Moreover, aminotriazole, which stimulated NADPH peroxidase activity, had an inhibitory action on NADPH peroxidase. These various experiments suggest that NADH- and NADPH-dependent peroxidase activity may be mediated by separate components of the microsomal electron transport chain, which may be distinct from but closely interacting with cytochrome P₄₅₀.     

The effect of o-diphenols upon the microsomal NADPH and NADH oxidase activities

Catechol and catecholamines have been assayed upon the microsomal NADPH and NADH oxidase activities. Epinephrine shows a catalytic effect on the NADPH oxidation characterized by a small lag. The two to threefold increase in rate can be suppressed by Superoxide dismutase if the enzyme is added before the reaction begins. The catalytic effect is ascribed to a quinone formed by two electron oxidation of epinephrine by the Superoxide ion. The quinone, which is not catalytically active in the NADH chain, appears to mediate electrons between the NADPH-cytochrome c reductase and oxygen. The four electron oxidation product adrenochrome is also active upon the NADPH chain but inactive upon the NADH chain.Epinephrine did not change the menadione-stimulated NADPH oxidase activity. Presumably, during this and the NADH oxidase activities, two electrons are simultaneously transferred to the oxygen molecule.Catechol and catecholamines doubled the rate of autoxidation of NADH in the presence of catalytic amounts of NADH-cytochrome b₅ reductase and cytochrome b₅, a result which suggests Superoxide ion formation in the autoxidation of the cytochrome.Epinephrine does not act upon the desaturation of endogenous substrate or upon endogenous lipid peroxidation.     

Reduced Pyridine Nucleotide Oxidases of Euglena gracilis var. bacillaris*

SYNOPSIS. Cell-free extracts of a streptomycin-bleached strain of Euglena gracilis var. bacillaris have been examined for enzyme systems primarily responsible for the oxidation of reduced pyridine nucelotides. NADH lipoyl dehydrogenase, NADH and NADPH oxidase, NADH and NADPH diaphorase, and NADH and NADPH cytochrome c reductase have been demonstrated. The NADPH-linked enzymes had lower activity rates and were less sensitive to N-ethyl maleimide and p-hydroxymercuribenzoate than their NADH-linked counterparts. NADH cytochrome c reductase was the most sensitive to antimycin A. Michaelis-Menten constants (K_m) determined were as follows: NADH diaphorase, 350 μM; NADPH diaphorase, 200 μM; NADH cytochrome c reductase, 13 μM; NADPH cytochrome c reductase, 9 μM; NADH oxidase, 100 μM; NADPH oxidase 150 μM; NADH lipoyl dehydrogenase, 0.35 μM. Enzyme activities after storage at –5 C indicate that the diaphorases are less labile than the other tested enzymes, and the differential activities of the NADH and NADPH linked enzymes suggest that functionally they may have different roles.     

Kinetic and functional characterization of a membrane-bound NAD(P)H dehydrogenase located in the chloroplasts of Pleurochloris meiringensis (Xanthophyceae)

Using isolated chloroplasts or purified thylakoids from photoautotrophically grown cells of the chromophytic alga Pleurochloris meiringensis (Xanthophyceae) we were able to demonstrate a membrane bound NAD(P)H dehydrogenase activity. NAD(P)H oxidation was detectable with menadione, coenzyme Q₀, decylplastoquinone and decylubiquinone as acceptors in an in vitro assay. K _m-values for both pyridine nucleotides were in the molar range (K _m[NADH]=9.8 M, K _m[NADPH]=3.2 M calculated according to Lineweaver-Burk). NADH oxidation was optimal at pH 9 while pH dependence of NADPH oxidation showed a main peak at 9.8 and a smaller optimum at pH 7.5–8. NADH oxidation could be completely inhibited with rotenone, an inhibitor of mitochondrial complex I dehydrogenase, while NADPH oxidation revealed the typical inhibition pattern upon addition of oxidized pyridine nucleotides reported for ferredoxin: NADP⁺ reductase. Partly-denaturing gel electrophoresis followed by NAD(P)H dehydrogenase activity staining showed that NADPH and NADH oxidizing proteins had different electrophoretic mobilities. As revealed by denaturing electrophoresis, the NADH oxidizing enzyme had one main subunit of 22 kDa and two further polypeptides of 29 and 44 kDa, whereas separation of the NADPH depending protein yielded five bands of different molecular weight. Measurement of oxygen consumption due to PS I mediated methylviologen reduction upon complete inhibition of PS II showed that the NAD(P)H dehydrogenase is able to catalyze an input of electrons from NADH to the photosynthetic electron transport chain in case of an oxidized plastoquinone-pool. We suggest ferredoxin: NADP⁺ reductase to be the main NADPH oxidizing activity while a thylakoidal NAD(P)H: plastoquinone oxidoreductase involved in the chlororespiratory pathway in the dark acts mainly as an NADH oxidizing enzyme.Abbreviations Coenzyme Q₀-2,3-dimethoxy-5-methyl-1,4-benzoquinone - FNR ferredoxin: NADP⁺ reductase - MD menadione - MV methylviologen - NDH NAD(P)H dehydrogenase - PQ plastoquinone - PQ₁₀ decylplastoquinone - SDH succinate dehydrogenase - UQ₁₀ decylubiquinone (2,3-dimethoxy-5-methyl-6-decyl-1,4-benzoquinone)     

Decreased prereceptorial glucocorticoid activating capacity in starvation due to an oxidative shift of pyridine nucleotides in the endoplasmic reticulum

Redox state of pyridine nucleotides of the endoplasmic reticulum (ER) lumen was determined in different nutritional conditions. NADPH-dependent cortisone reduction and NADP⁺-dependent cortisol oxidation were measured in rat liver microsomes, by utilizing the luminal 11β-hydroxysteroid dehydrogenase type 1 activity. Cortisone reduction decreased, while cortisol oxidation increased during onward starvation, showing that the luminal NADPH/NADP⁺ ratio was substantially decreased. Cortisone or metyrapone addition caused a smaller decrease in NADPH fluorescence in microsomes from starved rats. The results demonstrate that nutrient supply is mirrored by the redox state of ER luminal pyridine nucleotides.     

The oxidation of malate and exogenous reduced nicotinamide adenine dinucleotide by isolated plant mitochondria

Exogenous NADH oxidation by cauliflower (Brassica oleracea L.) bud mitochondria was sensitive to antimycin A and gave ADP/O ratios of 1.4 to 1.9. In intact mitochondria, NADH-cytochrome c reductase activity was only slightly inhibited by antimycin A. The antimycin-insensitive activity was associated with the outer membrane. Malate oxidation was sensitive to both rotenone and antimycin A and gave ADP/O values of 2.4 to 2.9. However in the presence of added NAD⁺, malate oxidation displayed similar properties to exogenous NADH oxidation. In both the presence and absence of added NAD⁺, malate oxidation was dependent on inorganic phosphate and inhibited by 2-n-butyl malonate.     

The Effect of Respiratory Inhibitors on NADH, Succinate and Malate Oxidation in Corn Mitochondria

The effect of a series of respiratory inhibitors on the oxidation of NADH in state 4 and state 3 conditions was studied with corn shoot mitochondria. Comparisons were made using malate and succinate as substrates. The inhibitors, rotenone, amytal, antimycin A and cyanide, inhibited oxidation of NADH in state 3 but rotenone and amytal did not inhibit oxidation in state 4. The inhibition by antimycin A was partially overcome by the presence of cytochrome c. The results indicate the presence of alternative pathways available for NADH oxidation depending on the metabolic condition of the mitochondria. Under state 4 conditions, NADH oxidation bypasses the amytal and rotenone sensitive sites but under state 3 conditions a component of the NADH respiration appears to be oxidized by an internal pathway which is sensitive to these inhibitors. Still a third pathway for NADH oxidation is dependent on the addition of cytochrome c and is insensitive to antimycin A. Succinate oxidation was sensitive to cyanide and antimycin A under both state 4 and state 3 conditions as well as amytal and rotenone under state 3 conditions but was not inhibited by amytal and rotenone under state 4 conditions. Malate oxidation was inhibited by cyanide, rotenone and amytal under both state 4 and state 3 conditions. Antimycin A inhibited state 3 but did not appreciably alter state 4 rates of malate oxidation. With all substrates tested inhibition by antimycin A was greatly facilitated by preswelling the mitochondria for 10 min. This was interpreted to indicate that swelling increases the accessibility of antimycin A to the site of inhibition.     

Vanadate stimulates oxidation of NADH to generate hydrogen peroxide

Vanadate in the polymeric form of decavanadate, but not other forms, stimulated oxidation of NADH to NAD⁺ NADPH was also oxidized with comparable rates. This oxidation of NADH was accompanied by uptake of oxygen and generated hydrogen peroxide with the following stoichiometry: NADH + H⁺ + O₂ → NAD⁺ + H₂O₂. The reaction followed second-order kinetics. The rate was dependent on the concentration of both NADH and vanadate and increased with decreasing pH. The reaction had an obligatory requirement for phosphate ions. Esr studies in the presence of the spin trap dimethyl pyrroline N oxide indicated the involvement of Superoxide anion as an intermediate. The reaction was sensitive to Superoxide dismutase and other scavengers of superoxide anions.     

A primary respiratory Na+ pump of an anaerobic bacterium: the Na+-dependent NADH:quinone oxidoreductase of Klebsiella pneumoniae

Membranes of Klebsiella pneumoniae, grown anaerobically on citrate, contain a NADH oxidase activity that is activated specifically by Na⁺ or Li⁺ ions and effectively inhibited by 2-heptyl-4-hydroxyquinoline-N-oxide (HQNO). Cytochromes b and d were present in the membranes, and the steady state reduction level of cytochrome b increased on NaCl addition. Inverted bacterial membrane vesicles accumulated Na⁺ ions upon NADH oxidation. Na⁺ uptake was completely inhibited by monensin and by HQNO and slightly stimulated by carbonylcyanide-p-trifluoromethoxy phenylhydrazone (FCCP), thus indicating the operation of a primary Na⁺ pump. A Triton extract of the bacterial membranes did not catalyze NADH oxidation by O₂, but by ferricyanide or menadione in a Na⁺-independent manner. The Na⁺-dependent NADH oxidation by O₂ was restored by adding ubiquinone-1 in micromolar concentrations. After inhibition of the terminal oxidase with KCN, ubiquinol was formed from ubiquinone-1 and NADH. The reaction was stimulated about 6-fold by 10 mM NaCl and was severely inhibited by low amounts of HQNO. Superoxide radicals were formed during electron transfer from NADH to ubiquinone-1. These radicals disappeared by adding NaCl, but not with NaCl and HQNO. It is suggested that the superoxide radicals arise from semiquinone radicals which are formed by one electron reduction of quinone in a Na⁺-independent reaction sequence and then dismutate in a Na⁺ and HQNO sensitive reaction to quinone and quinol. The mechanism of the respiratory Na⁺ pump of K. pneumoniae appears to be quite similar to that of Vibrio alginolyticus.     

Oxidation of Reduced Nicotinamide Adenine Dinucleotide Phosphate by Potato Mitochondria: INHIBITION BY SULFHYDRYL REAGENTS

Potato tuber mitochondria oxidized exogenous NADH and exogenous NADPH at similar rates; the electron transfer inhibitor rotenone did not inhibit the oxidation of either substrate. Submitochondrial particles, prepared from potato tuber mitochondria, exhibited a greater capacity to oxidize NADH than NADPH; rotenone inhibited the oxidation of NADH by 29% and the oxidation of NADPH by 16%. The oxidation of both NADH and NADPH by potato mitochondria exhibited pH optima of 6.8, and although substantial NADH oxidase activity was observed at pH 8.0, little NADPH oxidase activity was detected at that pH. The oxidation of NADPH by the mitochondria was more sensitive to inhibition by EDTA than was the oxidation of NADH.     

The oxidation and reduction of pyridine nucleotides by Rhodopseudomonas spheroides and Chlorobium thiosulfatophilum

Summary The light-induced formation of NADH by whole cells of Rhodopseudomonas spheroides has been followed fluorimetrically and found to lag slightly behind cytochrome c oxidation. The uncoupler, FCCP¹, abolished NADH formation which was also inhibited by HOQNO¹. Electron flow from NADH to oxygen or cytochrome c was inhibited in chromatophores of R. spheroides by HOQNO, antimycin A and rotenone. From the known properties of the inhibitors used it is deduced that NADH formation in the light is dependent upon reversed electron flow. No light-induced formation of NAD(P)H by whole cells or chromatophores of Chlorobium thiosulfatophilum was detected either fluorimetrically or by extraction followed by enzymic assay although cytochrome c oxidation was extensive in whole cells. Extracts of C. thiosulfatophilum catalysed the rapid reduction of endogenous or mammalian cytochrome c; unlike R. spheroides this activity was found almost entirely in the soluble fraction and was insensitive to HOQNO, antimycin A and rotenone. No cytochrome b was detected in C. thiosulfatophilum by difference spectroscopy of pyridine haemochromes of acetone powders. The K _m for NADH of NADH-cytochrome c reductase in both organisms was about 3 mol; the reductase was inhibited by NAD. The rates of NADPH-cytochrome c reductase in R. spheroides particles were too low for K _m determination; for C. thiosulfatophilum particles the K _m for NADPH was about 300 mol. The addition of NADH to soluble extracts of either organism caused the reduction of endogenous flavin that was reoxidised by ferricyanide. The NADH-cytochrome c reductase of C. thiosulfatophilum was not separated from ferredoxin on a DEAE column. It is concluded that in C. thiosulfatophilum the formation of NADH in an energy-linked reaction is unlikely; the possibility of a cyclic electron flow involving chlorophyll, ferredoxin, flavoprotein and cytochrome c is discussed.     

An electron-transport system associated with the outer membrane of liver mitochondria. A biochemical and morphological study

Preparations of rat-liver mitochondria catalyze the oxidation of exogenous NADH by added cytochrome c or ferricyanide by a reaction that is insensitive to the respiratory chain inhibitors, antimycin A, amytal, and rotenone, and is not coupled to phosphorylation. Experiments with tritiated NADH are described which demonstrate that this "external" pathway of NADH oxidation resembles stereochemically the NADH-cytochrome c reductase system of liver microsomes, and differs from the respiratory chain-linked NADH dehydrogenase. Enzyme distributation data are presented which substantiate the conclusion that microsomal contamination cannot account for the rotenone-insensitive NADH-cytochrome c reductase activity observed with the mitochondria. A procedure is developed, based on swelling and shrinking of the mitochondria followed by sonication and density gradient centrifugation, which permits the separation of two particulate subfractions, one containing the bulk of the respiratory chain components, and the other the bulk of the rotenone-insensitive NADH-cytochrome c reductase system. Morphological evidence supports the conclusion that the former subfraction consists of mitochondria devoid of outer membrane, and that the latter represents derivatives of the outer membrane. The data indicate that the electron-transport system associated with the mitochondrial outer membrane involves catalytic components similar to, or identical with, the microsomal NADH-cytochrome b₅ reductase and cytochrome b₅.     

The effect of calcium and inhibitors on corn mitochondrial respiration

The effects of KCN, antimycin A, malonate, rotenone, and amytal on the oxidation of malate, succinate, and extramitochondrial reduced nicotinamide adenosine dinucleotide (NADH) by corn mitochondria were studied. Potassium cyanide and antimycin A inhibited the oxidation of all three substrates. Rotenone and amytal inhibited only the oxidation of malate, and malonate inhibited only the oxidation of succinate. Rotenone, amytal, and malonate did not inhibit the oxidation of extramitochondrial NADH. The calcium stimulation of the oxidation of extramitochondrial NADH was prevented by KCN and antimycin A but not by amytal, rotenone, or malonate. It is suggested that corn mitochondria possess a flavoprotein specific for extramitochondrial NADH and that this flavoprotein is sensitive to divalent cations.     

Reduced pyridine nucleotide oxidases of Eugena gracilis var. bacillaris.

Cell-free extracts of a streptomycin-bleached strain of Euglena gracilis var. bacillaris have been examined for enzyme systems primarily responsible for the oxidation of reduced pyridine nucelotides. NADH lipoyl dehydrogenase, NADH and NADPH oxidase, NADH and NADPH diaphorase, and NADH and NADPH cytochrome c reductase have been demonstrated. The NADPH-linked enzymes had lower activity rates and were less sensitive to N-ethyl maleimide and p-hydroxymercuribenzoate than their NADH-linked counterparts. NADH cytochrome c reductase was the most sensitive to antimycin A. Michaelis-Menten constants (Km) determined were as follows: NADH diaphorase, 350 muM; NADPH oxidase 150 muM ; NADH lipoyl dehydrogenase, 0.35 muM. Enzyme activities after storage at -5 C indicate that the diaphorases are less labile than the other tested enzymes, and the differential activities of the NADH and NADPH linked enzymes suggest that functionally they may have different roles.     

External Pathway of NADH Oxidation in the Liver of the River Lamprey Lampetra fluviatilis

A possibility of exogenous NADH oxidation via the external pathway has been shown on homogenates and isolated liver cells of the lamprey Lampetra fluviatilis in the presence of rotenone and antimycin A. The homogenates were incubated in isotonic and hypotonic sucrose media, while cells, in isotonic salt medium. At incubating the tissue preparations in isotonic media, digitonin was used to enhance membrane permeability to NADH and cytochrome c. In homogenates, the maximal rate of NADH oxidation via the external pathway in the presence of cytochrome c and digitonin was 5.3 nmol O₂/min/10 mg wet weight. This value in the cells amounted to 12.6, while without addition of exogenous NADH and cytochrome c, to 11.0 nmol O₂/min/10 million cells. Cyanide inhibited completely the NADH oxidation via the external pathway both in homogenates and in cells. The intact lamprey hepatocytes, unlike homogenates, are suggested to contain sufficient concentrations of cytochrome c and extramitochondrial NADH to provide maximal NADH oxidation rate in mitochondria through external pathway. This allows thinking that potential possibilities of NADH oxidation via the external pathway in Cyclostomata and mammals are qualitatively and quantitatively close.     

ω-Hydroxylation of acyclic monoterpene alcohols by rat lung microsomes

Rat lung microsomes were shown to ω-hydroxylate acyclic monoterpene alcohols in the presence of NADPH and O₂. NADH could neither support hydroxylation efficiently nor did it show synergistic effect. The hydroxylase activity was greater in microsomes prepared from β-naphthoflavone (BNF)-treated rats than from phenobarbital (PB)-treated or control microsomal preparations. Hydroxylation was specific to the C-8 position in geraniol and has a pH optimum of 7.8. The inhibition of the hydroxylase activity by SKF-525A, CO, N-ethylmaleimide, ellipticine, α-naphthoflavone, cyt. $\text{c}$ and p-CMB indicated the involvement of the cyt. P-450 system. However, NaN₃ stimulated the hydroxylase activity to a significant level. Rat kidney microsomes were also capable of ω-hydroxylating geraniol although the activity was lower than that observed with lungs.