©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Alcohol:NAD Oxidoreductase Is Present in Rat Liver Peroxisomes (*)

(Received for publication, October 10, 1994)

Haruhiko Sakuraba Tomoo Noguchi (§)

From the Department of Biochemistry, Kyushu Dental College, Kokura, Kitakyushu 803, Japan

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

Alcohol:NAD oxidoreductase was found in the peroxisomes of animal liver for the first time as follows. The distribution of alcohol:NAD oxidoreductase activity with nonanol as substrate in the light mitochondrial fraction (peroxisome-enriched fraction) of rat liver was examined by centrifugation in a sucrose density gradient. Most of the enzyme activity was localized in the mitochondria, with some activity in the peroxisomes. The administration of clofibrate, a peroxisome proliferator, to rats resulted in a marked increase of the enzyme activity in the peroxisomes, but not in the mitochondria. The enzyme was found to be located in the matrix of the peroxisomes. The evidence was obtained that the enzyme differed from alcohol dehydrogenases and alcohol oxidizing systems found previously. The enzyme activity was not affected by pyrazole, an inhibitor of alcohol dehydrogenase and sodium azide, an inhibitor of catalase. The enzyme was NAD-dependent and oxidized straight chain aliphatic alcohols with a variety of carbon chains (C(2)-C(18)), showing the maximum on nonanol. K values toward these aliphatic alcohols decreased with increasing chain length. The major reaction product was identified as the carboxylic acid by using high performance liquid chromatography.


INTRODUCTION

Three separate enzymes have been reported to be involved in conversion of ethanol to acetaldehyde in mammalian liver. 1) The most widely known is the NAD-linked cytosolic alcohol dehydrogenase (EC 1.1.1.1), which catalyzes the reversible interconversion of a wide variety of alcohols and their corresponding aldehydes(1, 2) . 2) A mixed function oxidase (EC 1.6.2.4, 1.14.14.1), known as the microsomal ethanol-oxidizing system, catalyzes the NADPH and oxygen-dependent oxidation of short chain alcohols to corresponding aldehydes(3) . 3) Catalase, which is present in the peroxisomes, can also catalyze the oxidation of short chain alcohols to corresponding aldehydes by its peroxidatic activity(4) . The latter two enzymes are NAD-independent and cannot oxidize higher primary alcohols(5) .

In the present study, we report that a fourth enzyme that can oxidize aliphatic alcohols with a variety of carbon chain (C(2)-C(18)) to corresponding aldehydes is present in the peroxisomes of rat liver. The enzyme is NAD-dependent and induced by the administration of clofibrate, a peroxisome proliferator.


EXPERIMENTAL PROCEDURES

Animals

Thirty-day-old male rats (body weight, 100-110 g) of the Wistar strain were maintained ad libitum on either a normal diet (Oriental Yeast Co., Ltd., Tokyo, Japan) or a normal diet supplemented with clofibrate (0.3%, v/w) for 3 weeks.

Subcellular Fractionation of Rat Liver by Differential Centrifugation

All procedures were carried out at 0-4 °C. Rat livers were cut into small pieces with scissors and homogenized by one excursion in nine volumes of 0.25 M sucrose in 20 mM glycylglycine, pH 7.5, in a Potter-Elvehjem homogenizer with a Teflon pestle at 600 rpm. The homogenate was filtered through two layers of cheesecloth and subjected to differential centrifugation to obtain heavy mitochondrial, light mitochondrial, microsomal, and soluble fractions as described by de Duve et al.(6) .

Sucrose Density Gradient Centrifugation of Light Mitochondrial Fraction with a Vertical Rotor

A portion (3 ml) of the light mitochondrial fraction (peroxisome-enriched fraction), corresponding to 2 g of liver, was layered on a 31.5-ml sucrose gradient (24-54%, w/w) with 2.5 ml of 57% sucrose solution as a cushion in the bottom and centrifuged at 132,000 times g for 70 min in a Hitachi (Tokyo, Japan) 55P-72 centrifuge with a vertical rotor (RPV 50T-160). Fractions (2.2 ml) were collected from the bottom of the gradient.

Assay of Enzyme

Alcohol:NAD oxidoreductase activity was, unless specified, assayed spectrophotometrically by measuring the rate of NAD reduction(7) . The reaction system contained 100 mM Tris/HCl, pH 8.0, 0.5 mM nonanol, 2 mM NAD, 0.5 mg of bovine serum albumin, and enzyme in a total volume of 1.0 ml. Catalase (EC 1.11.1.6)(8) , glutamate dehydrogenase (EC 1.4.1.3)(9) , NADH-cytochrome c reductase (EC 1.6.99.3)(10) , and palmitoyl-CoA-dependent NAD reduction (over all beta-oxidation activity of peroxisomes) (11) were assayed by the methods of the cited references. A unit of enzyme activity is defined as the amount of enzyme that catalyzes a formation of product or a decrease in substrate of 1 µmol/min at 37 °C. Protein was determined by the method of Bradford(12) . Bovine serum albumin was used as the standard.

Identification of Reaction Products by High Performance Liquid Chromatography

For identification of the reaction products, the UV-absorbing derivatives of aldehydes and carboxylic acids were prepared as previously described(13) . The reaction was stopped by addition of 0.5 ml of HCl to the assay mixture, and the products were extracted with 3 ml of diethyl ether by vigorous shaking in a sealed test tube. After centrifugation at 3,000 times g for 5 min, the layer of diethyl ether was transferred into a reaction vial and dried up under nitrogen. To prepare the 4-nitrobenzyl oxime, the UV-absorbing derivative of aldehyde, 5 µmol of O-(4-nitrobenzyl)hydroxylamine hydrochloride (Dojindo, Kumamoto, Japan) in 100 µl of methanol and 1 µl of triethylamine were added to the vial. Then it was sealed and heated for 1 h at 65 °C. On the other hand, to prepare the 4-nitrobenzyl ester, the UV-absorbing derivative of carboxylic acid, 2.4 µmol of O-(4-nitrobenzyl)-N,N`-diisopropylisourea (Dojindo) in 100 µl of methylene chloride was added to the another vial. Then it was sealed and heated for 2 h at 80 °C. After cooling, an aliquot of the each solution of derivatives was subjected to a column (4.6 mm times 25 cm) of TSK gel ODS-120T (Toyo Soda Manufacturing Co., Ltd.). Acetonitrile-water (70:30) was used as the mobile phase at a flow rate of 1.0 ml/min. The effluent from the column was monitored by a UV detector at a wavelength of 262 nm. 0.5 µmol of nonanal or nonanoic acid in 1 ml of 100 mM Tris/HCl, pH 8.0, was used as a standard solution of aldehyde or carboxylic acid, respectively. Instead of the assay mixture, the standard solutions were treated in the same way as described above.


RESULTS AND DISCUSSION

Alcohol:NAD oxidoreductase activities with nonanol as substrate were determined in subcellular fractions from normal and clofibrate-treated rat livers (Table 1).



Clofibrate treatment resulted in the increase of the specific activity of the enzyme only in the light mitochondrial fraction (peroxisome-enriched fraction) but not in other fractions (Table 1).

The result suggests that the enzyme is present in the peroxisomes of rat liver, because clofibrate treatment of rats is known to cause proliferation of the peroxisomes and increase of peroxisomal enzyme activities in liver(14) .

Fig. 1shows the representative sedimentation profiles of the light mitochondrial fractions (peroxisome-enriched fractions) from normal and clofibrate-treated rat liver in a sucrose density gradient. The peroxisomes and mitochondria were separated; the peroxisomes, marked by catalase, was only at a density of about 1.25 g/ml, and the mitochondria, marked by glutamate dehydrogenase, only at a density of about 1.18 g/ml (Fig. 1A). Compared with normal rats, liver from clofibrate-treated rats showed a marked increase (about 10-fold) in the peroxisomal beta-oxidation activity (palmitoyl-CoA-dependent NAD reduction) (Fig. 1B). These results show that liver peroxisomes proliferated in clofibrate-treated rats(15, 16) .


Figure 1: Subcellular distribution of alcohol:NAD oxidoreductase in normal and clofibrate-treated rat livers. The light mitochondrial fractions were prepared from livers of normal and clofibrate-treated rats and were separately subjected to sucrose density gradient centrifugation as described in the text. Fractions of 2.2 ml were collected from the bottom of each tube. A, glutamate dehydrogenase (bullet) and catalase (circle) of normal rat liver; B, palmitoyl-CoA-dependent NAD reduction activities of normal (bullet) and clofibrate-treated (circle) rat liver; C, alcohol:NAD oxidoreductase activity of normal (bullet) and clofibrate-treated (circle) rat liver.



On the other hand, in normal rat, most of alcohol:NAD oxidoreductase activity with nonanol as substrate, was recovered in the mitochondria, with some activity in the peroxisomes (Fig. 1C). However, the treatment with clofibrate resulted in a marked increase (about 5-fold) of the peroxisomal enzyme activity with no effect on the mitochondrial one. These results clearly demonstrate that alcohol:NAD oxidoreductase is located in the peroxisomes of rat liver.

To examine intraperoxisomal localization of alcohol:NAD oxidoreductase activity with nonanol as substrate, the light mitochondrial fraction was prepared from livers of clofibrate-treated rats. The light mitochondrial fraction, containing 60 µg of protein, was suspended in 20 mM glycylglycine, pH 7.5, containing 0.25 M sucrose and various concentrations (0-0.3 M) of KCl. After incubation at 4 °C for 15 min, each suspension was centrifuged at 12,500 times g for 30 min and each resulting supernatant was assayed for unsedimentable activity. KCl (up to 0.3 M) did not solubilize any the catalase as the peroxisomal matrix marker or alcohol:NAD oxidoreductase activity, showing that alcohol:NAD oxidoreductase is not the peripheral membrane protein that is simply associated with peroxisomes by ionogenic interaction (data not shown).

Next, the peroxisomal fractions prepared from livers of clofibrate-treated rats (Fig. 1C, fractions2-4) were combined and diluted with the same volume of 0.01 M pyrophosphate buffer, pH 10.5, which is known to break rat liver peroxisomes(17) . After being stored overnight at 4 °C, about 6 ml of the suspension was subjected to sucrose density gradient centrifugation as described previously (10) (Fig. 2). Catalase as the peroxisomal matrix marker was completely solubilized and recovered in the soluble top fraction, and NADH-cytochrome c reductase in the membrane is distributed over a broad density range with a peak of about 1.17 g times ml(10, 18) . Nearly all of the alcohol:NAD oxidoreductase activity was recovered in the soluble top fraction. These results show that alcohol:NAD oxidoreductase is located in the peroxisomal soluble matrix.


Figure 2: Intraperoxisomal localization of alcohol:NAD oxidoreductase. Peroxisomal fractions (fractions 2-4 in Fig. 1C), isolated on a sucrose density gradient, were diluted with the same volume of 0.01 M pyrophosphate butter, pH 10.5, which is known to break rat liver peroxisomes. After being stored overnight at 4 °C, the suspension (about 6 ml) was layered on 28 ml of a sucrose gradient (30-56%, w/w) solution and centrifuged at 132,000 times g for 70 min. Fractions of 2.2 ml were collected from the bottom of the tube. A, NADH-cytochrome c reductase (circle) and catalase (bullet); B, alcohol:NAD oxidoreductase activity (bullet).



Fig. 3represents high performance liquid chromatograms of the UV-derivatives of the reaction products. The solubilized fractions of the peroxisomes in Fig. 2(fractions 14-16) were combined and used as the peroxisomal alcohol:NAD oxidoreductase preparation. The reaction system contained 100 µl (0.1 milliunit) of the enzyme preparation in the standard assay mixture. After incubation at 37 °C for 17 h, the products were extracted and converted to the appropriate derivatives as described under ``Experimental Procedures''. The 4-nitrobenzyl derivatives of nonanal and nonanoic acid as the standards were separated on an ODS column; the 4-nitrobenzyl oxime, corresponding 30 nmol of nonanal, was at a retention time of about 12.7 min and the 4-nitrobenzyl ester, corresponding 10 nmol of nonanoic acid, was at a retention time of about 9.9 min (Fig. 3A). Most of the 4-nitrobenzyl derivative of the reaction product was associated with authentic 4-nitrobenzyl ester of nonanoic acid (Fig. 3B), whereas the only minor peak that was associated with authentic 4-nitrobenzyl oxime of nonanal was detected (Fig. 3C). When these samples were coinjected with authentic derivatives, an enhancement of each peaks was observed. Amounts of produced nonanoic acid and nonanal were estimated at 88.9 and 6.1 nmol, respectively. These results suggest that the nonanol as substrate was oxidized to nonanoic acid with nonanal as a possible intermediate by this reaction system.


Figure 3: Identification of the reaction products of alcohol:NADoxidoreductase by high performance liquid chromatography. The UV-absorbing derivatives of nonanoic acid, nonanal, and the reaction products were prepared as described in the text. A, the 4-nitrobenzyl derivatives of nonanoic acid (10 nmol) and nonanal (30 nmol). B, 4-nitrobenzyl ester of the reaction products. 10 µl of the sample (100 µl) was injected. C, 4-nitrobenzyl oxime of the reaction products. 50 µl of the sample (100 µl) was injected.



Table 2shows kinetic constants of the peroxisomal alcohol:NAD oxidoreductase for straight chain aliphatic alcohols. The same preparation of the enzyme in Fig. 2was used. The K(m) values of the enzyme toward straight chain aliphatic alcohols decreased with increasing chain length. The enzyme has K(m) values in the millimolar range only for ethanol and in the micromolar range for longer chain length aliphatic alcohols (C(4)-C(18)) (Table 2). On the other hand, the enzyme showed high activities toward medium chain length aliphatic alcohols (C(8)-C), showing the maximum activity on nonanol (C(9)). The enzyme showed low activities toward longer chain length (C(14)-C(18)) or shorter chain length (C(2)-C(7)) alcohols (Table 2). Methanol did not serve as the substrate even at the high concentration of 2 M. NADP did not serve as a cofactor in all experiments.



Table 3shows the effect of inhibitors (1 and 10 mM) on alcohol:NAD oxidoreductase activity of the same preparation in Table 2. The enzyme activity was strongly inhibited by cyanide and N -Ethylmaleimide. However the activity was not affected by addition of iodoacetate, pyrazole (inhibitor of alcohol dehydrogenase)(19) , or sodium azide (inhibitor of catalase) (20) to the incubation mixture (Table 3).



In the present study, alcohol:NAD oxidoreductase was found to be present in the peroxisomes of rat liver. The enzyme was induced by the administration of a peroxisomal proliferator. The enzyme was NAD-dependent and oxidized straight chain aliphatic alcohols of a variety of carbon chain (C(2)-C(18)) and not inhibited by sodium azide, an inhibitor of catalase (Table 3), showing that the enzyme differs from the peroxisomal catalase. Catalase, in addition to the decomposition of H(2)O(2), can also catalyze the oxidation of short chain alcohols to corresponding aldehydes by the NAD-independent peroxidatic activity, but cannot oxidize higher primary alcohol(5) . Furthermore, peroxisomal alcohol:NAD oxidoreductase is also distinguishable from cytosolic alcohol dehydrogenase and microsomal alcohol-oxidizing systems as follows. 1) The peroxisomal enzyme is insensitive to pyrazole (Table 3), a potent inhibitor for cytosolic alcohol dehydrogenase(19) . 2) The microsomal ethanol-oxidizing system catalyzes the NADP-dependent oxidation(3) . However the peroxisomal enzyme cannot utilize NADP as cofactor. 3) Recently, it has been reported that the alcohol oxidoreductase that can metabolize long chain aliphatic alcohols (C-C(18)) to the corresponding fatty acids with NAD as cofactor is present in rat liver(21) . The enzyme is located in the microsomal fraction and named fatty alcohol:NAD oxidoreductase. However, the enzyme cannot oxidize ethanol(21) .

We do not know whether the peroxisomal alcohol:NAD oxidoreductase is involved in ethanol metabolism in vivo or not. Two forms of alcohol dehydrogenase have been reported to be present in the cytosol of rat liver, designed class I and class III isozymes(22, 23) . Only class I isozyme is active with ethanol at the blood concentrations of this compound(22, 23) . Class I isozyme exhibits K(m) value of 1,400 µM for ethanol (22, 23) somewhat higher than that of the peroxisomal enzyme. This minor difference suggests a potential role for the peroxisomal activity in ethanol metabolism.

On the other hand, the specific activity of the peroxisomal enzyme for octadecanol is nearly identical with that of rat liver microsomal fatty alcohol:NAD oxidoreductase (1.3 milliunits/mg microsomal protein)(21) . Recently, it has been reported that alkyl dihydroxyacetone phosphate synthase is mainly localized in peroxisomes (24) . The enzyme catalyzes the biosynthesis of ether lipids from acyl-dihydroxyacetone phosphate and fatty alcohol. These reports and the present data suggest a potential role for the peroxisomal activity in regulating the cellular levels of ether-linked lipids. Quantitative data on the physiological role of the peroxisomal alcohol:NAD oxidoreductase are required.

Rizzo et al.(25) have reported that a fatty alcohol cycle is present in human skin fibroblasts. In this cycle, fatty acyl-CoA is converted to the corresponding alcohols by acyl-CoA reductase and generated fatty alcohol is converted to the corresponding fatty acid by fatty alcohol:NAD oxidoreductase. Sjögren-Larsson syndrome, a metabolic disease characterized by abnormal accumulation of fatty alcohols in skin fibroblasts, congenital ichthyosis, and mental retardation, is caused by a defect of fatty alcohol:NAD oxidoreductase in this cycle(26, 27) . Fatty alcohol:NAD oxidoreductase activity has been reported to be associated with the particulate fraction of skin fibroblasts homogenate(27) . It is of interest whether this disease is caused by disorder of the peroxisomes or not.


FOOTNOTES

*
This work was supported in part by a grant-in-aid for scientific research from the Ministry of Education, Science and Culture of Japan and by the Cancer Research Fund from Fukuoka Cancer Research Association, Fukuoka, Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dept. of Biochemistry, Kyushu Dental College, Kokura, Kitakyushu 803, Japan. Tel.: 93-582-1131; Fax: 93-591-7086.


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©1995 by The American Society for Biochemistry and Molecular Biology, Inc.