Partial purification of the NADPH-dependent aldehyde reductase from bovine cardiac muscl

ARCHIVES

OF

Partial

BIOCHEMISTRY

Purification

JOSEPH Department

AND

BIOPHYSICS

173, 649-657 (1976)

of the NADPH-Dependent Aldehyde from Bovine Cardiac Muscle’ C. KAWALEK

of Biochemistry, Faculty School of Dental Medicine,

AND

JOHN

Reductase

R. GILBERTSON

of Arts and Sciences, and Department of Pharmacology-Physiology, University of Pittsburgh, Pittsburgh, Pennsylvania 15261 Received June 18, 1975

An aldehyde reductase catalyzing the NADPH-dependent reduction of long-chain aldehydes has been purified 690-fold from bovine cardiac muscle. Based on the results obtained during gel filtration, this enzyme has an apparent molecular weight of 34,000. The p1 of the aldehyde reductase was 6.1 and the enzymatic activity had a sharp pH optimum at 6.4. The enzyme catalyzed the reduction of aromatic aldehydes and aliphatic aldehydes having eight or more carbon atoms. Short-chain aldehydes, aldoses, or ketoses or long-chain methyl ketones were not utilized as substrates by this enzyme. However, the methyl ketone, pentadecan-2-one, was a competitive inhibitor of this enzyme with an apparent Ki = 10 pM when tetradecanal was the variable substrate. The reaction was not reversible when ethanol or hexadecanol was employed as substrate, utilizing either NAD+, or NADP+ as a cofactor. The addition of 10 mM pyrazole to the incubation medium had no effect on the enzymatic activity.

Analytical studies have indicated that 0-alkyl lipids account for 1525% of the total phospholipids in cardiac muscle (1) and 45% of the phospholipids of functionally important enzymes, such as mitochondrial succinic dehydrogenase (2). Considering these facts, it is interesting to note that only recently have there been any definitive studies regarding the metabolic origin of this lipid type. Several workers have demonstrated that a fatty alcohol and acyl-dihydroxyacetone phosphate are the final substrates directly involved in Oalkyl glycerol biosynthesis (3, 4), while others have reported that fatty aldehydes are also incorporated into the fatty chains of the 0-alkyl lipids (5, 6). The facile interconversion of fatty acids and alcohols has been demonstrated in several mammalian tissues (7, 8). Work in this laboratory has culminated in the isolation of a cytoplasmic NADH-dependent acyl-CoA reductase from bovine cardiac muscle that catalyzes the formation of 1 This study was supported by grants, No. AM16837 and CA14914, from the National Institutes of Health, U.S. Public Health Service. 649 Copyright 0 1976 by Academic Press, Inc. All rights of reproduction in any form reserved.

long-chain aldehydes (9). Preliminary reports concerning the existence (10) and partial purification (11) of a soluble NADPH-dependent aldehyde reductase from bovine cardiac muscle have also been presented. The information reported here concerns the procedures employed in the partial purification and some of the molecular characteristics of this aldehyde reductase from bovine cardiac muscle. MATERIALS

AND

METHODS

All chemicals were of the highest quality commercially available and all solvents, except methanol, were redistilled before use. Nucleotide cofactors were supplied by Sigma Chemical Company. Longchain aldehydes were synthesized by oxidation of the corresponding alcohols via the tosylate (12) or by reduction of the fatty acids (6). Pentadecan-2-one was purchased from Pfaltz and Bauer Chemical Co. and purified to one component via tic* (13). Whatman ion-exchange celluloses, CM-11 and DE-32, were washed according to the manufacturer’s instructions. * Abbreviations used: tic, thin-layer chromatography; glc, gas-liquid chromatography; CM-cellulose, carboxymethyl cellulose; DEAE-cellulose, diethylaminoethyl cellulose.

650

KAWALEK

AND GILBERTSON

(pH 6.2; 1 mM 2-mercaptoethanol) and dialyzed for Two different techniques were employed to meas- 16 h against three changes (2 liters each) of the same ure aldehyde reductase activity: (A) quantitative glc buffer. of the total lipid extracts from enzymatic reaction Column Chromatography mixtures and (B) spectrophotometric measurement of the rate of NADPH oxidation. A column of CM-cellulose (2.5 x 80 cm) was equilMethod A: quantitative glc. Long-chain alcohols ibrated with 0.01 M potassium phosphate buffer (pH formed by the enzymatic reduction of the corre- 6.2; 1 mM 2-mercaptoethanol). Approximately 125 sponding long-chain aldehydes were quantitated by ml of the dialyzed AR-2 fraction containing 4.4 g of glc of the total lipids extracted from the reaction protein were applied to the column. Fractions conmedium as described previously (10). Unless inditaining 13.5 ml of effluent were collected. The CMcated otherwise, the concentration of the assay comcellulose column was washed with the equilibration ponents in a final volume of 1 ml were: 75 mM buffer until the A,,, approached zero. Method A was potassium phosphate buffer (pH 6.4; 1 mM 2-mercap- employed to determine the enzymatic activity of toethanol), 0.50 mM NADPH and/or NADH, 0.10 each fraction since a nonspecific NADPH-oxidase mM hexadecanal dispersed in Tween 20 (final con- activity negated use of the spectrophotometric ascentration, lpg/ml) and 0.25-4.0 mg of protein. Con- say. trols contained all the components except protein or The aldehyde reductase activity was not absorbed reduced pyridine nucleotide. The incubations were onto the column and was washed out with the equilicarried out in 15-ml glass-stoppered conical centribration buffer. Enzymatically active fractions were fuge tubes for 10 min at 37°C. pooled, ammonium sulfate was added to achieve 90% Method B: spectrophotometric assay. This method saturation (57 g/lOOml), and the protein was allowed measured the rate of NADPH oxidation by continuto precipitate overnight at 4°C. The precipitated ously recording the decrease in absorbance at 340 protein, collected by centrifugation at 10,OOOgfor 20 nm (e = 6.22 x lo6 M-I cm-‘). The assays were min, was dissolved in a minimal volume of buffer performed in a Gilford 2400 spectrophotometer for 10 and dialyzed for 16 h against three changes (2 liters min at 37°C in 3-ml (l-cm path length) glass cu- each) of 0.01 M Tris-HCl (pH 7.5; 1 mM 2-mercaptovettes. Unless indicated otherwise, the concentra- ethanol). tion of the components utilized in this assay method A 2.5 x 40-cm column of washed and degassed were as follows: 75 mM potassium phosphate buffer DEAE-cellulose was equilibrated with the above (pH 6.4; 1 mM 2-mercaptoethanol), 0.125 mM 0.01 M Tris-HCl buffer. About 400 mg of the protein NADPH, 0.10 mM 3-pyridinecarboxyaldehyde or a fraction from the CM-cellulose column, dissolved in long-chain aldehyde dispersed in Tween 20, and 50 ml of the equilibrating buffer, were loaded onto 0.05-1.0 mg of protein in a total volume of 2 ml. The the DEAE-cellulose column. After the protein solutotal reaction mixture minus the substrate served as tion had penetrated the resin bed, any protein adthe control. The 3-pyridinecarboxaldehyde was used hering to the sides of the column was washed onto previously by Bosron and Prairie in studying the the column with 10 ml of the equilibrating buffer. aldehyde reductase activity from procine kidney cor- The column was eluted with 2 liters of a linear salt tex (14). This aromatic aldehyde has the advantage gradient, O-O.25M NaCl in 0.01 M Tris-HCl (pH 7.5; of being soluble in water, thus eliminating the re- 1 mM 2-mercaptoethanol). Ten-milliliter fractions quirement for solubilization with detergents which were collected and assayed for protein, A**,,, and are inherent in studies with long-chain aldehydes. enzymatic activity (Fig. 1). Both assay methods Protein concentrations were measured using the could be employed now, since the aldehyde reducbiuret method (15), with bovine serum albumin as tase eluted from the DEAE-cellulose column did not the standard, or spectrophotometrically for more di- contain any NADPH-oxidase activity. Both Methods lute solutions (16, 17). A and B were employed to determine the elution pattern. Each assay method gave the same quantiEnzyme Purification tative and qualitative results, indicating that either Unless stated otherwise, all preparative proce- technique could be used to assay enzymatic activity. dures were carried out at 4°C. Preparation of the Fractions containing the highest activity were com105,OOOgsupernatant and ammonium sulfate frac- bined and the protein precipitated by the addition of ammonium sulfate as described before. The precipitionations were performed as described previously (10). The ammonium sulfate procedure gave two tated protein was recovered by centrifugation, disprotein fractions: AR-l, precipitating at O-38% satu- solved in a minimal volume of 0.075 M potassium ration and the other, AR-2, precipitating between 38 phosphate buffer (pH 6.4; 1 mM 2-mercaptoethanol) and 90% saturation. The AR-2 fraction was dissolved giving a protein concentration of 4.3 mg/ml and in 50-100 ml of 0.01 M potassium phosphate buffer stored at -20°C. Assay Procedures

ALDEHYDE

REDUCTASE

FROM

HEART

80

160

200

120 Fraction

651

number

FIG. 1. Chromatography of the aldehyde reductase from the CM-cellulose column on DEAE-cellulose. The column was eluted with a linear salt gradient from 0 to 0.25 M NaCl in 0.01 M Tris-HCI buffer. Assay Method D with hexadecanal as the substrate was used to determine the elution profile. Units are defined as nanomoles of hexadecanol formed per 10 min. The protein elution profile is designated by (O-O), units of activity by (A-A) and the salt gradient by (O=O). -

Isoelectric

Focusing

An LKB-8101 electrofocusing column, water jacketed at 4”C, was used in these procedures. The instrument was adjusted so as to have the cathode as the lower electrode and the anode as the upper electrode. The ampholine solutions contained 1 mM 2mercaptoethanol. The aldehyde reductase recovered from the DEAE-cellulose column was dialyzed overnight against 0.01 M Tris-HCl buffer (pH 7.5; 1 mM I-mercaptoethanol) to reduce the salt concentration. The dialyzed protein solution (about 5-6 ml, 24 mg of protein) was substituted for the less dense ampholine solution in some of the middle fractions of the gradient. After the gradient was constructed, a constant potential of 300 V was applied to the column for 30 h. At the end of this interval, 2.5-ml fractions were collected and their pH and A,,, measured. Enzymatic activity was determined by Method B with 0.2-ml aliquots of the column fractions using 3-pyridinecarboxyaldehyde as the substrate. Fractions containing the highest specific activity were pooled and an aliquot from the combined solutions was assayed. Gel Filtration

Chromatography

A Sephadex G-200 column (2.6 x 84 cm), preequilibrated with 0.1 M NaCl in 0.04 M potassium phosphate buffer (pH 6.4; 1 mM 2-mercaptoethanol), was prepared for upward flow. The void volume of the column was determined by measuring the elution volume of blue dextran. The column was calibrated by determining the elution volumes of aldolase (X8,000), ovalbumin (45,000), chymotrypsinogen A (25,000) and ribonuclease (13,700). A plot of log molecular weight versus K,, was constructed, where K,, = (V, - V,MV, - V,); V, is the elution volume of the protein; V, is the void volume of the column, and V, is the bed volume of the column. Aliquots of the aldehyde reductase from the elec-

trofocusing column (7 mg of protein in 2 ml of buffer) were applied to this Sephadex G-200 column and the protein eluted with the equilibrating buffer. Fractions containing 3.2 ml of eluate were collected. The protein elution profile was followed by measuring fractions were assayed for the &15-225. Individual enzymatic activity using Method B with 3-pyridinecarboxyaldehyde as the substrate. Fractions having the highest specific activity were pooled, concentrated with an Amicon ultrafiltration apparatus containing a UM-2 membrane, and assayed again with hexadecanal as the substrate. Polyacrylamide

Disc-Gel

Electrophoresis

Twenty micrograms of the aldehyde reductase isolated after chromatography on the Sephadex G200 column were subjected to electrophoresis in (5 mm x 7 cm) 7% polyacrylamide gels according to the method described by Davis (18). The current level was adjusted to 2.0 Ma/gel. Electrophoresis was carried out at room temperature in a Canalco Model 6 apparatus at pH 9.5. Protein was stained on the gels with 0.25% Coomassie blue in 10% trichloroacetic acid for 1 h (19) and then destained overnight and stored in 10% trichloroacetic acid. The gels were scanned at 575 nm on a Gilford linear transport, Model 2410. RESULTS

Enzyme tion

Purification

and

Characteriza-

Cell-free homogenates were prepared from bovine cardiac muscle and separated into 100,OOOgparticulate and supernatant fractions as described before (10). Enzymatic activity in each fraction was assayed by Method A. In these studies, it was repeatedly noted that the total enzymatic

652

KAWALEK

AND

GILBERTSON

activity of the 100,OOOgsupernatant frac- electric focusing, the aldehyde reductase tion was approximately six times greater migrated as a symmetrical peak with an than that of the 700g supernatant fraction. apparent p1 of 6.1. This isoelectric focusing The reason for this difference was not eval- step was included in the final purification uated. Comparison of the total enzymatic scheme, despite the fact that no significant activity of the 100,OOOgsupernatant and purification was achieved and only 65% of particulate fractions indicated that the en- the enzymatic activity applied to the isoezymatic activity of the supernatant frac- lectric focusing column was recovered in tion was about 11 times greater than that toto. The reason for this was that omission of the particulate fraction. Thus the of this step in the purification procedure 100,OOOgsupernatant fraction was rou- resulted in the presence of significant amounts of enzymaticially inactive protein tinely utilized in further purification which migrated in the same molecular steps. As reported previously, the aldehyde re- weight range as the aldehyde reductase ductase activity present in the 105,OOOg during Sephadex G-200 chromatography. supernatant fraction precipitated between Therefore, inclusion of the electrofocusing 38 and 90% saturation with ammonium step removed the majority of these consulfate (10). NADPH was the preferred co- taminants which otherwise would have defactor, since the activity noted with creased the effectiveness of the gel filtraNADH represented about lo-20% of that tion procedure. The molecular weight of the aldehyde obtained with NADPH. When both cofactors were present in equimolar amounts, reductase was determined by gel filtration the activity was not greater than that ob- on a previously calibrated Sephadex G-200 served with NADPH alone. This nonaddi- column (Fig. 2). The molecular weight was tivity rules out the possibility of more than calculated as a function of the K,, for the one enzyme, each having a different cofac- observed enzymatic activity. Several determinations gave an average K,, = 0.550, tor requirement. The protein precipitated at 38-90% satu- corresponding to an apparent molecular ration with ammonium sulfate was consid- weight of 34,000. Although the activity ered the source of enzymatic activity in and protein profiles were not coincident, calculating purification. The results of the fractions 89-97 had the highest overall speprocedures employed to purify the alde- cific activity and, when pooled, contained hyde reductase (Table I) indicate that the 35% of the enzymatic activity applied to enzyme was purified 690-fold with a recov- the column. Purity of the enzyme eluted from the Sephadex G-200 column was evalery of 11%. When the aldehyde reductase obtained uated by polyacrylamide disc-gel electroafter CM-cellulose chromatography was phoresis (Fig. 3). The results of this proceassayed as a function of pH, a sharp pH dure indicate that a single component is optimum was found at 6.4. During the iso- not present. However, that a considerable TABLE

I

SUMMARY OF THE PARTIAL PURIFICATION OF THE ALDEHYDE REDUCTASE FROM BOVINE CARDIAC MUSCLE Protein bxd

Specific activitp

Yield (8)

4400 400 60 21.9 0.7

0.7 7.1 40.0 43.6 480.0

100.0 92.0 77.5 31.0 10.8

formed per 10 min per milligram

of protein.

Procedure

Ammonium sulfate precipitate CM-celluloseb DEAE-celluloseb~ c Isoelectric Focusing Sephadex G-20w a Nanomoles of hexadecan-l-01 b Assayed using Method A. c Assayed using Method B.

(38-90%)6

Purification (nfold) 10 57 62 686

ALDEHYDE

REDUCTASE

Fraction

FROM

HEART

653

number

FIG. 2. Elution

profile of aldehyde reductase activity from a Sephadex G-200 column. concentration in each 3.2-ml fraction was determined spectrophotometrically Enzyme activity was determined by using assay Method B (A-A).

Protein (0-O).

g

16-

2 y

12-

Marker dye 1

8 6 - 0.8 $ :: =I 0.4~ Origin

n

40

2.0 Length

of gel,

60 cm

FIG. 3. Scan of polyacrylamide disc-electrophoresis gels. The gels were scanned at 575 nm with the Gilford linear transport, Model 2410. Full scale was 2.OA. Twenty micrograms of the aldehyde reductase from the Sephadex G-200 column were subjected to electrophoresis as described in the methods.

degree of purification has been achieved is indicated by the presence of one major protein band and several minor components. Catalytic

Activity

Employing the enzyme preparation for the CM-cellulose column, the rate of the reaction was found to be linear with respect to protein concentration over a range of 0.3 to 4 mg of protein/ml. Using 2.3 mg of the enzyme preparation for the CM-cellulose column the rate of the reaction, employing Method A to quantitate the product, was linear for over 15 min. A linear increase in the rate of hexadecanol formation was observed as the concentration of NADPH in the incubation medium was increased up to a maximum of 90 PM. The

apparent K, for NADPH, calculated from a Lineweaver-Burk plot, was 50 PM. This value is similar to that obtained with the aldehyde reductase from porcine kidney (14) and greater than that reported for a similar enzyme from brain (20-22). With 2.3 mg of protein from the CM-cellulose column, the rate of the reaction was directly proportional to the concentration of hexadecanol up to 30 PM. Addition of larger amounts of substrate resulted in little increase in enzymatic activity. The aparent K, for hexadecanal was 18 PM. Substrate

Specificity

Following chromatography of the enzyme on DEAE-cellulose, the substrate specificity was assessed by Method B, utilizing a number of aldehydes other than hexadecanal. The apparent Km’s and V’s calculated for each substrate from Lineweaver-Burk plots are in Table II. From these data, it is apparent that no correlation exists between the chain length or structural conformation of the aldehydes and their corresponding Km’s. The Km’s ranged from 690 PM for phenylacetaldehyde to 18 PM for hexadecanal. These values are of similar magnitude to those reported previously for the brain aldehyde reductase when phenylacetaldehyde or other aromatic aldehydes were utilized as substrates (20, 22), but the K, for 3-pyridinecarboxaldehyde is approximately 20fold less than that reported for the aldehyde reductase of porcine kidney (14). No detectable activity was observed with pentadecan-2-one, n-ribose, n-glucuronic acid,

654

KAWALEK

AND GILBERTSON

TABLE II SUBSTRATE SPECIFICITY OF THE ALDEHYDE REDUCTASE?

Substrate

3-Pyridinecarboxaldehyde Phenylacetaldehyde O&anal Dodecanal Tridecanal Tetradecanal Pentadecanal Hexadecanal Heptadecanal Octadecanal Oleyl-aldehyde Linoleyl-aldehyde Linolenyl-aldehyde

K, (PM)

V (nmol min-’ mg-9 nmolesl min/mg

114.0 670.0 134.0 28.6 25.0 89.0 50.0 18.0 33.0 114.0 114.0 200.0 160.0

60.0 78.0 93.0 116.5 104.8 58.0 34.9 19.5 14.5 11.6 14.0 89.5 145.0

a Substrate specificity of the aldehyde reductase was assessed by Method B, with the enzyme eluted from the DEAE-cellulose column.

pared to reactions carried out in the absence of the detergent, an average decrease in enzymatic activity of 13.5% was observed. These results suggest that the detergent may have caused at least a proportionate decrease in the relative reaction rates for those lipid substrates solubilized with Tween 20. Correlation of the Spectrophotometric Gas Chromatographic Assay

and

Several substrates were used to correlate the spectrophotometric assay, Method B, with the quantitative glc assay, Method A. The oxidation of NADPH was measured spectrophotometrically for 10 min, then the cuvettes were immediately extracted with chloroform:methanol (1:l) and the products quantitated using Method A Figure 4 shows the correlation between the nanomoles of NADPH oxidized and the nanomoles of fatty alcohol synthesized with palmityl-, oleyl-, linoleyl-, and linolenyl-aldehydes as substrates. From these data, it is apparent that the reductase studied here does not catalyze the reduction of a double bond and, that for every mole of fatty alcohol produced, one mole of NADPH was oxidized.

nglucuronolactone, acetaldehyde, or butyraldehyde as substrate. Biosynthesis of long-chain aldehydes could not be demonstrated when an aliquot of the enzyme purified through the CM-cellulose step was incubated with albumin-bound palmitate or palmityl-CoA Inhibitors of Enzymatic Activity under conditions previously shown to be These studies were performed with the optimal for the acyel-CoA reductase (9). aldehyde reductase obtained after DEAEOxidation of a long-chain alcohol by an cellulose chromatography. Aqueous solualiquot of the AR-2 enzyme could not be demonstrated in an assay system containing 200 PM hexadecanol dispersed in Tween 20,500 PM NADP+ and/or NAD+ in a final volume of 1 ml of 0.04 M pyrophosphage buffer, pH 8.5. The mixture was incubated at 37°C for 10 min and the extent of the reaction evluated by glc. Using assay Method B, the enzyme eluted from 4.0 the DEAE-cellulose column could not catai lyze the oxidation of hexadecanol or E 2.0 ethanol when either NAD+ or NADP+ was utilized as a cofactor under the conditions noted above.

10.0 6.0 & 5 ii 6.0 E s

Effects of Detergent

The effect of Tween 20 (1 pg/ml) on the aldehyde reductase activity was evaluated by Method B with the water-soluble subComstrate, 3-pyridinecarboxaldehyde.

2.0

4.0

nmd.,

FATTY

6.0

6.0

10.0

ALCOHOL FORMED

FIG. 4. Correlation of the spectrophotometric assay, Method B, with the glc assay, Method A. Pal(O-O), oleyl(M-M), linoleylmityl(O-O), and linolenyl-aldehydes (A-A) were employed as substrates.

ALDEHYDE

REDUCTASE

tions of the inhibitor were incubated for 2 min at 25°C with 40 pg of protein, specific activity 60 nmol of NADPH oxidized per minute per milligram of protein. Method B was utilized to assay enzymatic activity with tetradecanal as the substrate. Reactions were initiated by the addition of NADPH. Additions of mercuric chloride, 0.05 and 2.5 PM caused a 36 and 76% inhibition of enzymatic activity, respectively. Preincubation with either hexadecanol or NADPH alleviated the observed inhibition by about 50%. Sodium barbital, pentabarbital, phenobarbital, and propranolol caused only a 15% inhibition of enzymatic acitivity as compared to the untreated enzyme. At the above concentration, chlorpromazine and chlordiazepoxide caused a 30% decrease in enzymatic activity. Pyrazole at a concentration of 10 mM had no effect on the rate of NADPH oxidation. A further increase in the concentration utilized for the above drugs resulted in no further increase in inhibitory activity. The possibility that the aldehyde reductase studied here might also reduce a methyl ketone was evaluated with pentadecan-2-one as the substrate. The methyl ketone was not a substrate for the enzyme; however, because of the similarity in structure between tetradecanal and the methyl ketone, the ability of the ketone to act as an inhibitor of this reductase was evaluated. Studies were performed at two different concentrations of pentadecan-2one using tetradecanal as the substrate. From the results shown in Fig. 5, it is apparent that the ketone is a competitive inhibitor of the enzyme. As shown in the inset, replotting the slopes as a function of the ketone concentration resulted in a straight line, which intersected the abscissa at a point corresponding to a Ki = 10 PM.

FROM

655

HEART

-1 0050100 0150

l/Tetradecanal,

0200

PM-’

FIG. 5. Aldehyde reductase inhibition by pentadecan-Z-one as a function of the aldehyde concentration. The concentration of pentadecan-2-one (micromolar) applicable to a line is inscribed beside that line. The NADPH concentration was 125 PM, u =nanomoles of NADPH oxidized per minute. Inset: plot of the slopes as a function of the ketone concentration.

tiated from alcohol dehydrogenase, since NADH was not utilized as a cofactor, and neither acetaldehyde nor butyraldehyde was a substrate for the enzyme. This enzyme is considered to function only as a aldehyde reductase, since neither ethanol nor hexadecanol was oxidized under conditions optimal for liver alcohol dehydrogenase (23). In addition, it was shown that the enzyme discussed here is not inhibited by pyrazole, an effective inhibitor of alcohol dehydrogenase (23). The functional role of this enzyme as reductase is further reinforced by the high NADPH:NADP+ ratio observed in most mammalian tissues, thus providing an environment conducive to the reduction of aldehydogenic substances (24). This observation, coupled with the fact that the aldehyde reductase described here has a specific activity at least twice that of the acylCoA reductase from this tissue, could account for the low concentration of longchain aldehydes detected in cardiac muscle (9, 25). The inability of the aldehyde reductase studied here to utilize acetaldehyde and DISCUSSION butyraldehyde as substrates or NADH as a The studies reported here demonstrate cofactor, coupled with its ability to reduce that the aldehyde reductase in bovine car- saturated aliphatic aldehydes, 16 carbon diac muscle catalyzes the reduction of aro- atoms or greater in chain length, differenmatic and aliphatic aldehydes to the corre- tiates this enzyme from the retinal reducsponding alcohols. This enzyme is differen- tase of rat intestinal mucosa (26). The solu-

656

KAWALEK

AND

ble, NADPH-linked aldehyde reductase, purified from porcine kidney cortex (14) differs significantly from the enzyme described here with respect to its substrate specificity and reversibility of the reaction even though these enzymes are quite similar with respect to their molecular weight and p1. That the enzyme characterized here may be similar to the aldehyde reductase (EC 1.1.1.2) studied previously in bovine brain is suggested by its insensitivity to pyrazole and its utilization of NADPH as a cofactor (20, 22). The major difference between the enzyme studied here and the aldehyde reductase from brain is that we could not demonstrate reversibility of the reaction at pH 8-9 and that the bovine cardiac muscle enzyme is not nearly as susceptible to inhibition by barbiturates or other drugs (14, 20, 21). Previous studies of cardiac muscle have demonstrated that the 0-alkyl glycerols are a major component of the phosphoglycerides but that free fatty aldehydes and fatty alcohols are present in only trace amounts (25). These studies have also indicated that in this tissue the fatty chains of the free fatty alcohols and 0-alkyl glycerols are qualitatively similar (25). As noted earlier, other workers have demonstrated that long-chain alcohols are direct precursors of the fatty chains of the Oalkyl glycerols (3,4). Based on these observations, it is suggested that the aldehyde reductase reported here functions in tandem with the acyl-CoA and/or fatty acid reductase of this tissue to provide the fatty alcohols required in the biosynthesis of the 0-alkyl glycerols (9, 27). Considering the lack of substrate specificity of this reductase and the report that a wide spectrum of exogenous fatty alcohols are readily incorporated into the fatty chains of the 0-alkyl glycerides (28), the basis for the limited chain-length distribution observed physiologically in the O-alkyl lipids may be explained by the substrate specificity of the long-chain acylCoA and fatty acid reductases previously isolated from the cytosol of bovine cardiac muscle (9, 27). The in vitro observation reported here

GILBERTSON

that pentadecan-2-one is a competitive inhibitor of the aldehyde reductase of cardiac muscle, Ki = 10 PM, suggests that in future studies, methyl ketones might be employed in cell culture systems to selectively inhibit 0-alkyl phosphoglyceride biosynthesis. Such an approach might be of value in assessing the physiological function of these phospholipids in cell membranes. REFERENCES 1. RAPPORT, M. M., AND NORTON, W. T. (1962) Annu. Rev. Biochem. 31, 103-148. 2. SNYDER, F. (1969) in Progress in the Chemistry of Fats and Other Lipids. (Holman, R. T., ed.), Vol. 10, pp. 289-309, Pergamon, New York. 3. HAJRA, A. K. (1969) Biochem. Biophys. Res. Commun. 37, 486-492. 4. SNYDER, F., MALONE, B., AND BLANK, M. L. (1970). Biol. Chem. 245, 1790-1799. 5. BELL, 0. E., JR., AND WHITE, H. B., JR. (1968) Biochim. Biophys. Acta 164, 441-444. 6. BAUMANN, N. A., HAGEN, P. O., AND GOLDFINE, H. (1965) J. Biol. Chem. 240, 1559-1567. 7. SNYDER, F., AND MALONE, B. (1970) Biochem. Biophys. Res. Commun. 41, 1382-1387. a. FERRELL, W. J., AND KEBBLER, R. J. (1971) Physiol. Chem. Phys. 3, 549-558. 9. JOHNSON, R. C., AND GILBERTSON, J. R. (1972). J. Biol. Chem. 247, 6991-6998. 10. KAWALEK, J. C., AND GILBERTSON, J. R. (1973) Biochem. Biophys. Res. Commun. 51, 10271033. 11. KAWALEE, J. C., AND GILBERTSON. J. R. (1974) Fed. hoc. 33, 1378. 12. MAHADEVAN, V. J. (1964) J. Amer. Oil Chem. Sot. 41, 520. 13. NACCARATO, W. F., AND GILBERTSON, J. R. (1974) Lipids 9, 322-327. 14. BOSRON, W. F., AND PRAIRIE, R. L. (1972) J. Biol. Chem. 247,4480-4485. 15. WARBURG, O., AND CHRISTIAN, W. (1941) Biothem. Z. 310, 384-421. 16. G~RNALL, A. G., BARDAWILL, C. J., AND DAVID, M. M. (1949) J. Biol. Chem. 177, 751-766. 17. MURPHY, J. B., AND KIES, M. W. (1960) B&him. Biophys. Acta 45, 382-384. 18. DAVIS, B. J. (1964) Ann. N.Y. Acad. Sci. 121, 404-427. 19. CHRAMBACK, A., REISFELD, R. A., WYCHOFF, M., AND ZACCARI, J. (1967) Anal. Biochem. 20, 150-154. 20. TABAKOFF, B., AND ERWIN, V. G. (1970) J. Biol. Chem. 245, 3263-3268. 21. TURNER, A. J., AND TIPTON, K. F,. (1972) Eur. J. Biochem. 30, 361-368.

ALDEHYDE

REDUCTASE

22. TABAKOFF, B., ANDERBON, R., AND ALIVISATOS, S. G. A. (1973) Mol. Pharmacol. 9, 428-437. 23. THEORELL, H., AND IONETANI, T. (1963) Biochem. 2. 338, 537-553. 24. GLOCK, G. F., AND MCLEAN, P. (1955) Biochem. J. 61, 388-390. 25. GILBERTSON, J. R., JOHNSON, R. C., GELMAN, P. A., AND BUFFENMYER, C. (1972) J. Lipid Res.

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13, 491-499. 26. FIDGE, N. E., AND GOODMAN, D. S. (1968) J. Biol. Chem. 243, 4372-4379. 27. FERRELL, W. F. (1967) Ph.D. Dissertation, University of Pittsburgh, Pittsburgh, Pa.; Dissertat. Absts. 30, 1969. 28. MARAMATSU, T., AND SCHMID, H. H. 0. (1971) J. Lipid Res. 12, 740-746.