Localization of Aldehyde in Rat Liver1 A. HORTON University
Received September 12, 1974 Evidence supporting the existence of three aldehyde dehydrogenases, betaine aldehyde dehydrogenase and two aldehyde dehydrogenases ALDH-I and ALDH-II, in rat liver has been confirmed. Subcellular fractionation indicates that betaine aldehyde dehydrogenase is predominantly in the cytosol with about 5% in the mitochondria. Of the aldehyde dehydrogenase activity (ALDH-I plus ALDH-II) 80% is found in the mitochondria and 20% in the microsomal fraction. Of the two enzymes ALDH-I is exclusively mitochondrial and ALDH-II is distributed between mitochondria and microsomes. Submitochondrial fractionation indicates that betaine aldehyde dehydrogenase and ALDH-I are located in the matrix, and that ALDH-II is chiefly located in the outer membrane.
This paper reports on the subcellular distribution of aldehyde dehydrogenases in rat liver. Since 1953 when a rat liver mitochondrial enzyme with broad substrate specificity was described (1) and confirmed later by independent investigators (2), a number of reports have appeared in the literature describing the presence of aldehyde dehydrogenase in a variety of subcellular fractions. Two independent reports (3, 4) described an aldehyde dehydrogenase localized chiefly in the cytoplasm of rat liver whilst other investigators (5) have demonstrated the presence of a similar enzyme in the soluble and microsomal fractions of rat liver. Aldehyde dehydrogenase activity has been demonstrated in a mitochondrial extract (6) and the results obtained by other groups (7, 8) indicate that the mitochondria are the predominant site for the oxidation of acetaldehyde during the metabolism of ethanol. Another report (9) on the subcellular distribution of aldehyde dehydrogenase assigned 80% of the enzymic activity to the mitochondria and the remaining 20% to the supernatant, ‘Supported in part by a grant from the Medical Research Council. Copyright All rights
0 1975 by Academic Press, Inc. of reproduction in any form reserved.
there being no activity in the microsomes. The enzymic oxidation of a wide range of aldehydes by rat liver mitochondria has been described (10) and more detailed investigations revealed the presence of enzymic aldehyde oxidizing activity in both the inner and outer membranes (ll), whilst another report (12) assigns most of the mitochondrial aldehyde dehydrogenase to the matrix. Aldehyde dehydrogenase has been purified from a variety of mammalian sources (13-18) and the possible existence of more than one enzyme was discussed in several reports (12, 14-17). Two NAD+dependent aldehyde dehydrogenases have since been isolated and partially purified from the supernatant fraction of a rat liver homogenate (19), whilst another report describes two aldehyde dehydrogenases, one located in the cytosol and the other in the mitochondria of rat liver (20). During the course of the work described in this paper, evidence has been presented in support of the existence of two NAD+dependent aldehyde dehydrogenases in rat liver, one located exclusively in the mitochondria and the other in both mitochondria and microsomes (21). Another dehydrogenase that reacts with betaine alde-
hyde has been reported to be present in the cytosol and with low activity in mitochondria (22). This paper confirms evidence supporting the existence of three aldehyde dehydrogenases in rat liver and in addition reports on the localization of these enzymes within the liver cell based on extensive and careful subcellular fractionation studies. One enzyme designated ALDH-I has been located in the mitochondrial matrix whilst a secone enzyme designated ALDH-II has been shown to be located in the outer mitochondrial membrane and the endoplasmic reticulum. Betaine aldehyde dehydrogenase activity has been shown to be predominantly in the cytosol (87%) in agreement with Wilken et al. (22) and the remainder in the mitochondrial matrix. MATERIALS
Fractionation of rot liuer. All rats used in these experiments were three-month-old females bred in this laboratory from an original Wistar strain, raised on the Oxoid pasteurized breeding diet and fasted for 12 h before death. Rats were killed by cervical fracture and the liver removed immediately and placed in ice-cold 0.25 M sucrose containing 0.5 mM EDTA (pH 7.8) where it was weighed, cut into small pieces and washed with the same medium to remove blood. The liver (5 g) was then homogenized first by hand in a small Potter-Elvehjem homogenizer and then in a similar but larger homogenizer with the Teflon pestle, rotating at 1,000 rpm for 2 min; six up-and-down movements of the homogenizer tube were employed. The radial clearance between pestle and tube was 0.01 in. Throughout ,this procedure, both homogenizer tubes were kept in an ice-water mixture. The volume of the liver homogenate was then increased to approximately 60 ml by addition of the sucrose-EDTA medium and then fractionated according to the method of De Duve et al. (23) to yield a nuclear fraction (N) and a cytoplasmic fraction (E) with the modification that the homogenate was passed through four layers of cheese-cloth to remove connective tissue to facilitate accumte measurements of enzymic activities, and the nuclear fraction was sedimented at 4,750g min. The cytoplasmic fraction was further fractionated (23) to yield the following fractions: heavy mitochondria (M) (29,000g min), light mitochondria (L) (100,OOOg min), microsomes (P) (4,600,OOOg min), and supematant (S). All particulate fractions were washed three times with isolation medium. The microsomal fraction was further washed with isotonic saline to reduce the likelihood of adsorption
of aldehyde dehydrogenase to the membranes. Enzyme assays were carried out on these fractions within a few hours of preparation where possible, or the fractions were frozen and assayed the following day. Subfractionation of rat liver mitochondria. Three submitochondrial fractions representing outer membranes, inner membranes and matrix plus intermembrane space were isolated by the procedure of Sottocasa et al. (24) with minor modifications. Mitochondria, isolated by the procedure of Parsons and Williams (25) were shown to be practically free from microsomal contamination (less than 4%) by determining the specific activities (on the protein basis) of the preparation with respect to glucose-6-phosphatase and NADPH-cytochrome c reductase, and by comparing these values with those found for microsomes prepared from the same homogenate. Careful and thorough washing of the mitochondria was found to be essential for removal of microsomal contamination. The mitochondria were resuspended in 10 mM Trisphosphate buffer pH 7.5 containing 0.02% bovine serum albumin and after standing at 0°C for 5 min the suspension was diluted with one third volume of 1.8 M sucrose, 2 mM ATP and 2 mM MgSO,. Following a further 5 min at O’C the suspension was sonicated in aliquots of 5 ml with a MSE 100 W ultrasonic disintegrator for 3 x 12 s at 20 kHz. The sonicated suspension was then layered over 5 ml of 0.76 M sucrose and 10 ml 1.32 M sucrose and centrifuged at 98,000g for 3 h, to yield three subfractions: a tightly packed dark brown pellet at the bottom of the tube (heavy subfraction) a band at the interface between the 0.76 and 1.32 M sucrose layers (light subfraction), and a clear supernatant in the 0.45 M sucrose layer of the gradient (soluble subfraction). The heavy subfraction represents the inner membrane system of the mitochondria with some of the matrix retained, whereas the light subfraction is the outer membrane. The soluble subfraction probably contains part of the matrix content plus material originating from the intermembrane space. Enzyme Assays. Aldehyde dehydrogenase (EC 126.96.36.199) was measured spectrophotometrically at 25°C by the method of Racker (26) in 50 mM sodium pyrophosphate pH 8.8 in the presence of 1 mM sodium amytal. The reaction was started by the addition of substrates. The concentrations of cofactors and substrates used in the assays of different enzymic activities were as follows: total aldehyde dehydrogenase activity, 0.5 mre NAD+ and 5.0 mM acetaldehyde; ALDH-I-NAD+, 0.5 mM NAD+ and 0.025 rnM acetaldehyde; ALDH-II-NADP’, 2.5 mM NADP+ and 5.0 mM acetaldehyde: betaine aldehyde dehydrogenase, 0.5 mM NAD+ and 1.0 mM betaine aldehyde. ALDHII-NAD+, is the difference between the total activity and the activity of ALDH-I; acetaldehyde was selected as the most suitable substrate on account of the
observation that one of the two rat liver aldehyde dehydrogenases separated by Shum and Blair (19) showed a restricted substrate specificity. The activity of aldehyde dehydrogenase was found to be completely stable for several days when stored at -2O’C and only slight losses of activity occurred after several weeks. Hydroxylamine (0.5 mM) or pyrazole (0.1 mM) was added to inhibit alcohol dehydrogenase. Samples of the liver mitochondrial fractions were treated with sodium deoxychoiate (with the pH adjusted to that of the assay) at a concentration of 0.20 mg deoxycholate per mg protein prior to the assay of aldehyde dehydrogenase to release latent activity and to disperse the particulate material to facilitate spectrophotometric measurements. Treatment with deoxycholate has been shown to be without effect on aldehyde dehydrogenase (11, 21) and this observation was confirmed in the course of the present investigation. However, betaine aldehyde dehydrogenase activity is affected and deoxycholate was not used in assays of this enzyme. Latent enzyme activity was released either by repeated freezing and thawing (six cycles) or sonication. Enzyme latency was not found in the microsomal fractions. No substrate controls were used to correct the results of aldehyde dehydrogenase activity in fractions containing cytoplasmic enzymes. Monoamine oxidase (EC 188.8.131.52) was assayed at 30°C by a modification of the method of Tabor et al. (27) as described by Schnaitman et al. (28) with e2:zzm = 1.34 x 10’ for benzaldehyde (29). Cytochrome c oxidase (EC 184.108.40.206) was measured polarographically at 25’C by the procedure of Schnaitman et al. (28). Glutamate dehydrogenase (EC 220.127.116.11) was assayed by the method of Brdiczka et al. (29) in a medium containing sodium amytal (1 mM) to inhibit the oxidation of NADH. NADPH-cytochrome c reductase (EC 18.104.22.168) was measured by the method of Phillips and Langdon (30) with e&o’,“, = 2.11 x 10’ for the reduction of cytochrome c. Acid phosphatase, (EC 22.214.171.124) using (?I-glycerophosphate as substrate, was measured by the method of Furth and Robinson (31) and the inorganic phosphate released assayed by the procedure of Robinson et al. (32). Glucose-6-phosphatase (EC 126.96.36.199) was assayed as described by Hiibscher and West (33) and the inorganic phosphate released was measured by the method of King (34). Adenylate kinase (EC 188.8.131.52) was measured by the method of Schnaitman and Greenawalt (35). Protein was assayed by the method of Lowry et al. (36). Determination dehydrogenase.
Initial rates of enzymic activity were measured as described for the general assay except that pyrazole was omitted with mitochondrial and microsomal fractions because of the absence of alcohol dehydrogenase. Duplicate measurements, with a reproducibility within 2.5% were made in all cases and the results used in a double reciprocal plot. Release of
any latent enzyme activity was by one of the methods already described, depending upon the enzyme concerned. Chemicals. Acetaldehyde, benzylamine, fl-glycerophosphate and N,iV,N’,N’-tetramethyl-p-phenylene diamine dihydrochloride (TMPD)z were obtained from BDH Chemicals Ltd., Poole, Dorset, U.K. The acetaldehyde was redistilled before use and was stable at -20°C in the form of an aqueous solution (500 mM) for several weeks. Amytal (5-ethyl-5-isoamyl barbituric acid), glucose-6-phosphate, ascorbic acid, sodium deoxycholate, NAD+ NADH, NADP+, NADPH, ADP, cytochrome c (Type VI) and yeast alcohol dehydrogenase were supplied by Sigma (London) Chemical Co., London S.W.6. a-Ketoglutarate was purchased from Boehringer Corporation (London) Ltd., London W.5. 2,2-Diethoxyethyltrimethylammonium iodide was obtained from the Aldrich Chemical Co., Inc., Milwaukee, WI. All other reagents were of A. R. grade. Synthesis of betaine aldehyde. This substrate was synthesized by a slight modification of the procedure of Speed and Richardson (37). The pale yellow crystals obtained after removal of the acid were washed twice in anhydrous acetone to yield white crystals and a yellow supernatant which was decanted. Thin layer chromatography on a silica gel plate with methanol/acetone/concn HCl (90:10:4) as solvent revealed only one spot on treatment with iodine vapour. The mp of the washed crystals (123”124°C) was identical with that reported elsewhere (38). RESULTS
This paper describes the results of investigations of three NAD+-dependent aldehyde dehydrogenases in rat liver cells. During the course of this work, Tottmar et al. (21) provided evidence from kinetic experiments for the possible existence of at least two different NAD+-dependent aldehyde dehydrogenases in rat liver. Distribution studies showed that one enzyme designated enzyme I was exclusively located in the mitochondria and that another enzyme, designated enzyme II was located in both the mitochondrial and microsomal fractions. It was suggested that an NADP+-dependent aldehyde dehydrogenase found in both mitochondrial and mi‘Abbreviations used: TMPD, N,N,N’,N’-tetramethyl-p-phenylene diamine dihydrochloride; and HEPES, N-2-hydroxyethyl piperazine-Ai’-2-ethane sulfonic acid.
crosomal fractions was identical with enzyme II. The kinetic: data from experiments with isolated mitochondria yielded a curved double reciprocal plot from which apparent K, values for these two similar enzymes acting on acetaldehyde were calculated by the method of Reiner (39). An apparent K, value for acetaldehyde of well below 10 PM was obtained for enzyme I and a value of 1.7 mM for en:syme II. The latter figure was obtained from the linear double reciprocal plot which resulted when the rate for enzyme I was subtracted from the total rate. Measurements made in our laboratory on isolated mitochondrial fractions yielded two similar double reciprocal plots, and an apparent K, of less than 5 PM for acetaldehyde for enzyme I (designated ALDH-I) and 1.5 mM for enzyme II (designated ALDH-II) when calculated by the same method. Kinetic data obtained in our laboratory with the microsomal fraction yielded a linear double reciprocal plot and an apparent K, for acetaldehyde of 0.8 mM; no activity was detected at concentrations of acetaldehyde below 50 PM. This result is in agreement with the previously reported apparent K, for acetaldehyde of 0.9 mM obtained with the microsomal fraction (21). Optimal pH values of 8.8-9.0 for these reactions with pyrophosphate buffer were also confirmed. Apparent K, values for NAD+ for ALDH-I (mitochondrial) and ALDH-II (microsomal) were 22 PM and 33 pM, respectively, which are in close agreement with previous results (21). This kinetic data wa,s measured at pH 8.8, which is close to the optimum, to ensure that any slight variation in pH would have only a minimal effect on the rates of enzymic activity measured. However, apparent K, values for acetaldehyde and NAD+ for ALDH-I and ALDH-II were also obtained from assays performed at the more physiological pH of ‘7.6 using either 0.1 M sodium pyrophosphate or 0.1 M HEPES (N-2hydroxyethyl piperazine-N’-2-ethane sulfonic acid) as buffer. For both acetaldehyde and NAD+ the apparent K, values for both enzymes were very close to the values obtained at pH 8.8. The value of V
for ALDH-I was reduced by 40% on changing the pH from 8.8 to 7.6 but the V for ALDH-II showed little response to this change of pH with mitochondria (less than 10% reduction) and no change with microsomes. Investigation of the NADP+dependent aldehyde dehydrogenase in mitochondrial and microsomal fractions yielded linear double reciprocal plots in both cases both for acetaldehyde and NADP+ indicating one enzyme. Apparent K, values for acetaldehyde and NADP+ were 0.6 mM and 3.0 mM, respectively, for the mitochondrial enzyme and 0.5 mM and 2.7 mM for the microsomal enzyme which are consistent with other data (21). The NADP+ used was shown to be free of NAD+ by the use of yeast alcohol dehydrogenase which is absolutely NAD+ specific (40). Investigations in this laboratory have extended this study to include betaine aldehyde dehydrogenase, an enzyme shown to be present in the mitochondria and cytosol of rat liver cells (22). Apparent K, values for enzymes of mitochondria and cytosol for betaine aldehyde are 100 PM and 110 pM, respectively, and 65 pM and 75 PM for NAD+; NADP+ did not react. Subcellular Distribution of Aldehyde Dehydrogenases and Marker Enzymes
Each fraction isolated as described from the rat liver homogenate was assayed for the activities of three aldehyde dehydrogenases (assuming ALDH-II to be identical with ALDH-NADP+) and for the activities of five marker enzymes. To facilitate comparison between the activities of different enzymes, the results of this investigation are presented after the manner of de Duve (23) and are expressed as the mean relative specific activity plotted against the percentage protein content of each fraction (Fig. 1). The area of each block is thus proportional to the percentage of activity recovered in the corresponding fraction, and its height to the degree of purification achieved over the homogenate (i.e., the nuclear (N) and cytoplasmic (E) fractions). Recovery of enzymes was excellent and ranged between 93 and 100%. The
ALDEHIOE DfHIDROCfNASE (AlDWI
ALDEHYDf DfHIDROCENASE (TOTAL-ALDH-1)
2 ACID PHOSPH*TASf
FIG. 1. Distribution patterns of aldehyde dehydrogenase and marker enzymes in rat liver. The ordinate represents the mean relative specific activity of fractions (percentage of total activity divided by percentage of total protein) and the abscissa the percentage of total protein. Results are means of three experiments + SD. Subcellular fractions are represented by the following letters; N = nuclear. M = heavy mitochondrial, L = light mitochondrial and lysosomal, P = microsomal and S = final supernatant. Percentage recoveries * SD and total activities of enzymes + SD (i.e., activities in nuclear plus cytoplasmic fractions expressed as nmol/min/mg protein) were as follows; aldehyde dehydrogenase (total) 94.3 * 3.7; 30.0 + 3.3; ALDH-I 93.6 * 4.1; 8.8 + 1.2; ALDH-II 94.1 * 3.8; 21.2 l 1.9; ALDH-NADP+ 95.8 + 2.3, 21.6 l 2.2; betaine aldehyde dehydrogenase 94.8 * 3.5, 17.9 * 2.5; monoamine oxidase 99.3 l 2.5, 2.8 + 0.3; cytochrome c oxidase 99.0 l 3.5, 590.7 + 24.8; glutamate dehydrogenase 98.6 * 2.2, 963.4 l 8.5; NADPH-cytochrome c reductase 98.5 + 3.5, 21.9 + 2.3; acid phosphatase (total activity) 932.0 + 3.8, 1238.3 i 18.8.
percentage distribution of the marker enzymes, protein and the absolute values for different enzyme activities in fractions N and E corresponded well with data in the literature (21, 23) and so did the specific enzyme activities of the marker enzymes in the different fractions. Assays of the five marker enzymes made on each fraction provided a means of as-
sessing the integrity of the mitochondria, and the distribution of different subcellular particles in the fractions. Glutamate dehydrogenase, cytochrome c oxidase, and monoamine oxidase were used as marker enzymes for the mitochondrial matrix, inner membrane, and outer membrane, respectively, NADPH-cytochrome c reductase as marker enzyme for the microsomal
fraction, and acid phosphatase (total activity) as marker for lysosomes. The relative specific activity (i.e., percentage of total activity/percentage of total protein) of glutamate dehydrogenase in the final supernatant was low and similar to that of cytochrome c oxidase indicating that no substantial leakage of enzyme from the matrix or inner membrane of the mitochondria had occurred. A comparison of the relative specific activities of monoamine oxidase, glutamate dehydrogenase and cytochrome c oxidase revealed that the activity of the former enzyme in the microsomal fraction was greater than the activities of the other two enzymes indicating that some rupture of the outer mitochondrial membrane had occurred, but the extent of this damage was slight. The graphs in Fig. 1, which in addition to the relative specific activities of the aldehyde dehydrogenases include the relative specific activities of mitochondrial and microsomal marker enzymes, show that aldehyde dehydrogenase activity, measured with acetaldehyde as substrate, is distributed between mitochondrial and microsomal fractions, with the greater percentage of the total activity residing in the mitochondria. The very low aldehyde dehydrogenase activity found in the final supernatant is probably attributable to release from mitochondria and possibly microsomes as indicated by marker enzymes. From a consideration of the relative specific activities of mitochondrial marker enzymes, the low relative specific activity of aldehyde dehydrogenase in the nuclear
fraction is clearly attributable to mitochondrial contamination. The possible existence of two aldehyde dehydrogenases was indicated by the results of kinetic experiments using different concentrations of acetaldehyde or NADP+ instead of NAD+. The enzymic activity observed at low concentrations of acetaldehyde (25 PM) had a subcellular distribution similar to that of the mitochondrial markers which indicates that this enzyme (ALDH-I) is located entirely in the mitochondrial fraction. By subtracting the activity of ALDH-I from the total activity obtained in the presence of a saturating concentration of acetaldehyde, the remaining enzymic activity (ALDH-II) may be seen to be distributed between the mitochondrial and microsomal fractions although predominantly in the latter; a similar distribution was obtained for the NADP+-dependent enzyme indicating that these two enzymes may be identical. The distribution of enzymes represented in Fig. 1 are in general agreement with results of experiments reported during the course of this work (21). The specific activities of these aldehyde dehydrogenases which appear in Table I are consistent with other reported data (21). In addition, Fig. 1 shows a graph of the relative specific activity of betaine aldehyde dehydrogenase in the subcellular fractions. The major part of the total activity of this enzyme is clearly in the final supernatant and is probably located in the cytosol, but in spite of numerous washings, a small amount of the activity (5%) is found in the mitochondrial
TABLE SPECIFIC ACTIVITIES
AND NADP+ MICROSOMAL,
DEPENDENT ALDEHYDE DEHYDROGENASES AND SUPERNATANT FRACTIONS’
Aldehyde dehydrogenase (total) ALDH-I ALDH-II (total - I) ALDH-NADP+ Betaine aldehyde dehydrogenase D Results protein.
of five experiments
50.6 25.5 25.1 24.2 4.3 + SD. Specific
zt * zt i *
6.1 2.0 2.7 2.1 0.7
86.7 1.5 85.2 83.7 0.2 is nmol
zt 9.8 l 0.4 zt 9.8 zt 10.3 f 0.02
3.0 0.4 2.6 1.3 42.8
* 0.5 * 0.05 ziz 0.5 f 0.2 zt 6.3
fraction. No activity was detected in the microsomal fraction. These findings are in general agreement with data reported by Wilken et al. (22). The distribution of this enzyme within the cell and its substrate specificity clearly distinguish it from the other aldehyde dehydrogenases described. The above data, therefore, support the conclusions that at least three different aldehyde dehydrogenases are present in the rat liver cell. Distribution
of Aldehyde Dehydrogenase in
Mitochondria, previously washed three times in 0.25 M sucrose, and allowed to swell in 10 IIIM K+ phosphate buffer pH 7.5 for 20 min were then removed from the suspension by centrifugation to leave a supernatant which possessed very little glutamate dehydrogenase or any aldehyde dehydrogenase but about 80% of the total adenylate kinase. These results indicate that neither ALDH-I, ALDH-II nor betaine aldehyde dehydrogenase is located in the mitochondrial intermembrane space. More information concerning the distribution of aldehyde dehydrogenases in mitochondria was obtained by the fractionation of mitochondria as described into three fractions; the soluble fraction representing the contents of the intermembrane space and matrix, the light fraction the outer membrane, and the heavy fraction the inner membrane plus a part of the matrix. ALDH-I, ALDH-II, ALDHNADP+, betaine aldehyde dehydrogenase and certain appropriate marker enzymes were assayed in all three fractions. Enzymes used as markers were glutamate dehydrogenase for the matrix, cytochrome c oxidase for the inner membrane, adenylate kinase for the intermembrane space and monoamine oxidase for the outer membrane. The results of these experiments, as in the case of the subcellular fractionation, are expressed as the mean relative specific activity plotted against the percentage protein content of each fraction (Fig. 2). The distribution of ALDH-I and betaine aldehyde dehydrogenase is similar to the distribution of
glutamate dehydrogenase and quite different to that of adenylate kinase indicating that these enzymes are located in the matrix. ALDH-II and ALDH-NADP+ have submitochondrial distributions that are similar and to a considerable extent resemble the distribution of monoamine oxidase, although they have a lower percentage activity in the light fraction and much more in the soluble fraction indicating a location in the outer membrane and possibly to some extent in the matrix. However, it is possible that the enzymes are only loosely bound to the outer membrane resulting in some appearing in the soluble fraction. From this data, it appears that one enzyme (ALDH-II) is chiefly membrane bound and the other two (ALDH-I and betaine aldehyde dehydrogenase) are located in the matrix. DISCUSSION
For some time, reports of investigations on aldehyde dehydrogenase have indicated the existence of more than one enzyme. In 1966, Deitrich (4) reported that on the basis of gel filtration measurements the mitochondrial and supernatant aldehyde dehydrogenases of rat liver were different proteins. More recently, (41) he has demonstrated the existence of two aldehyde dehydrogenases in rat liver supernatant, one inducible by phenobarbital and the other not. An aldehyde dehydrogenase in the mitochondrial fraction was not inducible by phenobarbital. Two fractions from rat liver have been reported (20) to have enzymes which have different apparent K, values for acetaldehyde; the value for the mitochondrial enzyme was less than 10 PM and that for the other enzyme described as cytosolic was 30 pM. Shum and Blair (19) purified two NAD+-dependent aldehyde dehydrogenases from rat liver cytosol both of which had apparent K, values for acetaldehyde in the mM range; their enzyme I had an apparent K, value of 1 mM for acetaldehyde and 34 PM for NAD+ which are similar to the values reported for ALDH-II from this laboratory and for enzyme II by Tottmar et al. (21). Neither of the purified enzymes isolated by Shum and
( TOTAL - ALDH
DEHYDROCENASE 3 2
GLUTAMATE DEHY DROCENASE
0Ii--a H 0
FIG. 2. Distribution patterns of aldehyde dehydrogenase and marker enzymes in rat liver mitochondria. Ordinate and abscissa as for Fig. 1. Results are means of three experiments. Submitochondrial fractions are represented by the following letters; H = heavy, L = light, S = soluble. Percentage recoveries l SD and specific activities * SD (expressed as nmol/mitimg protein) of enzymes were as follows. ALDH-I 97.0 + 7.3, 25.3 * 2.2; ALDH-II (total minus ALDH-I) 101.3 + 8.5, 25.0 f 2.5; ALDH-NADP+ 98.6 f 6.8; 24.1 + 2.6; hetaine aldehyde dehydrogenase 97.2 + 6.6, 4.2 * 0.7; cytochrome c oxidase 99.1 + 8.3, 1440.0 + 220; glutamate dehydrogenase 103.7 * 5.6, 2380.0 * 310; monoamine oxidase 100.6 + 8.1, 7.2 * 1.2; adenylate kinase 97.5 + 5.3. 410.0 * 51.
Blair, in common with purified horse liver aldehyde dehydrogenase (17) was reactive with NADP+. Apparent K, values for aldehyde for purified horse liver enzyme lie in the range 0.1 -1.0 PM and for NAD+ 57
(17) which are similar to values obtained for ALDH-I of rat liver, but for purified human liver aldehyde dehydrogenase the reported apparent K, value for acetaldehyde was 0.5 PM and for NAD+ 0.6
mM (13). Although only one enzyme was hyde dehydrogenase and in view of this fact and evidence from several laboratories for purified from horse and human livers, aldehyde dehydrogenase from rabbit liver the existence of two enzymes of widely has been separated into two isoenzymes by differing apparent K, values coupled with classical techniques of enzyme purification the results of distribution studies reported here and elsewhere (11, 21) support for the (42). From investigations on the NAD+alternative explanations is lacking. The reported presence of betaine aldedependent aldehyde dehydrogenase of rat liver mitochondria, Marjanen (9) reported hyde dehydrogenase in the cytosol and an apparent K, of 10 pM for acetaldehyde mitochondrial fraction of rat liver (22) has and Lundquist et al. (43) from experiments been confirmed and apparent K, values with a rat liver homogenate reported an ap- for the aldehyde are intermediate between parent K, value of less than 1 pM for the the values recorded for acetaldehyde for same substrate. A possible reason for the ALDH-I and ALDH-II. A previously resingle low apparent K, values reported is ported stimulatory effect of cysteine (45) that both investigators used low concen- on the enzymic activity could not be contrations of substrate (0.1-0.6 mM). By con- firmed and NADP+ did not serve as cofactrast, Deitrich (4) using mM concentrations tor. of substrate in experiments on rat liver hoSubcellular fractionation of the rat liver mogenates has reported apparent K, val- cell with the aid of appropriate marker ues for aldehyde in the range l-4-4.5 mM. enzymes, which afforded a measure of the Using a partially purified 100,000 g super- purity of each fraction, provided a reliable natant from mitochondria, Grunnet (12) subcellular distribution pattern for aldereported two apparent K, values for both hyde dehydrogenase activity. Of the total acetaldehyde (0.1 I.~M and 1.0 mM) and aldehyde dehydrogenase activity recorded NAD+ (2 /IM and 50 PM) for aldehyde 80% was located in the mitochondria and dehydrogenase at pH 7.4. Two apparent 20% in the microsomes for which the endoK, values for acetaldehyde for mitochonplasmic reticulum enzyme NADPH-cytodrial aldehyde dehydrogenase from bovine chrome c reductase was marker. Marjanen brain (15) and from mouse liver (16) have (9) reported that no aldehyde dehydrogenalso been reported. Tottmar et al. (21) ase activity could be detected in his microfound two apparent K, values for acetaldesomal fraction but despite three washings hyde (one in the @M range and one in the of our microsomal fraction with a medium mM range) and two apparent K, values for identical with that used by him, the enNAD+ (24 PM and 35 PM), for NAD+zymic activity was not removed. The disdependent aldehyde dehydrogenase in rat tribution study indicates that ALDH-I is liver mitochondrial and microsomal frac- clearly located in the mitochondrial fractions; data from this laboratory confirm tion and ALDH-II appears to be distribthese results. uted between the mitochondrial and miAlthough the abrupt transition in the crosomal fractions. The results of kinetic double reciprocal plot for aldehyde dehy- experiments indicate that the microsomal drogenase activity has been interpreted as enzyme and ALDH-II of mitochondria are indicative of the existence of two enzymes, very similar if not identical. These results other possible explanations do exist. Glutaare in general agreement with a similar mate dehydrogenase appears to exhibit study published during the course of this this phenomenon even though it is believed work (21). The marked discrepancies beto be a single enzyme (44). It is also tween reports of many investigators conpossible that the apparent presence of an cerning the subcellular distribution of alenzyme of low K, may be due to nonendehyde dehydrogenase in rat liver may zymic activity. However, nonenzymic ac- possibly be attributed to the use of either tivity has not been observed in experi- partial fractionation procedures or differments with mitochondrial rat liver alde- ent assay methods and conditions, for ex-
ample, because of the concentration of substrate used, only one of the two aldehyde dehydrogenase activities was measured. Reports (4, 9) of the presence of aldehyde dehydrogenase activity in the final supernatant from cell fractionation may most probably be attributed to the release of enzyme from the mitochondria or microsomes due to the severe conditions used for homogenization during their isolation. The similar distribution patterns of ALDH-NADP+ and ALDH-II agree with the suggestion that these two activities probably represent a single enzyme. Other NAD+-dependent dehydrogenases which have low affinities for NADP+ compared with NAD’ but have similar maximum rates with both cofactors have been described (40). Grunnet (12) suggested that aldehyde dehydrogenase activity with NADP+ as cofactor is due to the formation of NAD+ from NADP.+ by the action of alkaline phosphatase in the mitochondria but our data do not support this view. The conversion of NADP+ to NAD+ was investigated under assay conditions in the presence of ethanol and yeast alcohol dehydrogenase with is absolutely NAD+ specific (40) but no enzymic activity was detected until NAD+ was added. It was also found that on ageing mitochondria, the activities of ALDH-II (NAD+) and ALDH-NADP+ decreased at similar rates. In addition, at low concentrations of acetaldehyde (less than 50 pM) no activity was detected with NADP+ as only ALDH-I is active at such low substrate levels and this enzyme requires NAD+ as cofactor. The reported (12) activation of aldehyde oxidation by extramitochondrial NAD+ in intact mitochondria was confirmed using the same assay conditions. NADP+ was also effective and this fact together with the finding that acetaldehyde at low concentrations (less than 50 PM) was not oxidised indicates that ALDH-II is probably the enzyme involved. The activities of the aldehyde dehydrogenases., especially ALDH-I were considerably enhanced when assayed in 50 mM sodium pyrophosphate buffer pH 8.8 in preference to the 0.1 M potassium phos-
phate buffer pH 7.4 used by Grunnet. The results of the submitochondrial fractionation, which again used appropriate marker enzymes to afford a measure of the purity of the three fractions, indicated that ALDH-I and probably betaine aldehyde dehydrogenase are located in the matrix. This localization of the latter enzyme agrees with data published elsewhere (22). Mitochondrial swelling experiments in which there was a release of adenylate kinase, the marker enzyme for the intermembrane space, but little or no aldehyde dehydrogenase activity eliminated the possibility that these two aldehyde dehydrogenases are located between the membranes. ALDH-II appears to be predominantly located in the outer membrane, but unless it is assumed that the enzyme is only loosely bound to the membrane, there exists a possibility that some may be located in the matrix. Grunnet (12) concluded that the main aldehyde dehydrogenase activity was in the matrix although there was low activity in a fraction representing the outer membrane and the intermembrane space. Smith and Packer (11) have presented results indicating an enrichment of aldehyde dehydrogenase activity in outer membranes compared with whole mitochondria and inner membrane. This paper confirms the existence of three aldehyde dehydrogenases in the rat liver cell and has reported on the subcellular and submitochondrial distribution of these enzymes. ACKNOWLEDGMENT We are grateful to Mrs. J. Flint for technical assistance. REFERENCES S. S.,
Biol. 2. GLENN,
200, 515-523. VANKO, M. (1959) Arch. 82, 145-152. (1965) Biochem. Z. 341, 300-314. A. (1966) Biochem. Pharmacol. AND
3. BUTTNER, 4. DEITRICH,
1911-1922. 5. TIETZ,
(1964) J. Biol. Chem. 239, 4081-4090. 6. MCGUIRE, J. S. (1965) Arch. Biochem. 110,
E. P. Biophys.
7. HEDLUND, S. -G. AND KIESSLING, K. -H. (1969) Acta Pharmacol. Toxicol. 27, 381-396. 8. HASSIHEN, I. E., YLMAHRI, R. H., AND KOHONEN, M. T. (1970) Ann. Med. Exp. Biol. Penn. 18, 176-183. 9. MARJANEN, L. (1972) &o&em. J. 127,633-639. 10. HORTON, A. A., AND PACKER, L. (1970) Biochem. J. 116, 19-20p. 11. SMITH, L., AND PACKER, L. (1972) Arch. Biochem. Biophys. 148, 270-276. 12. GRUNNET, N. (1973) Eur. J. Biochem. 35,236-243. 13. KRAEMER, R. J., AND DE~RICH, R. A. (1968) J. Biol. Chem. 243, 6402-6408. 14. BLAIR, A. H., AND BODLEY, F. H. (1969) Can. J. Biochem, 47, 265-272. 15. ERWIN, V. G., AND DEITRICH, R. A. (1966) J. Biol. Chem. 241, 3533-3539. 16. SHEPPARD, J. R., ALBERSHEIM, P., AND MCCLEARN, G. E. (1970) J. Biol. Chem. 245, 2876-2882. 17. FELDMAN, R. J., AND WEINER, H. (1972) J. Biol. Chem. 247, 260-266. 18. DUNCAN, R. J. S., AND TI~ON, K. F. (1971) Eur. J. Biochem. 22, 257-262. 19. SHUM, G. T., AND BLAIR, A. H. (1972) Can. J. Biochem. 50, 741-748. 20. MAFLJANEN, L. A. (1973) Biochim. Biophys. Acta 32’7, 238-246. 21. TO’ITMAR, S. 0. C., PETTERSSON, H., AND KIESSLING, K. H. (1973) Biochem. J. 135,577-586. 22. WILKEN, D. R., MCMACKEN, M. L., AND RODRIQUEZ, A. (1970) Biochim. Biophys. Acta 216, 305-317. 23. DE DWE, C., PRESSMAN, B. C., GIANE~O, R., WATTIAUX, R., AND APPELMANS, F. (1955) Biothem. J. 60, 604-617. 24. SOITOCASA, G. L., KUYLENSTIERNA, B., ERNSTER, L., AND BERGSTRAND. B. (1967) in Methods in Enzymology (Colowick, S. P., and Kaplan, N. O., eds.), Vol. 10, pp. 448-463, Academic Press, New York. 25. PARSONS, D. F., AND WILLIAMS G. R. (1967) in Methods in Enzymology (Colowick, S. P., and Kaplan, N. O., eds.), Vol. 10, pp. 443-448,
BARRETT Academic Press, New York. 26. RACKER, E. (1955) in Methods in Enzymology, (Colowick, S. P., and Kaplan, N. O., eds.), Vol. 1, pp. 514-517, Academic Press, New York. 27. TABOR, C. W., TABOR, H., AND ROSENTHAL, S. M. (1954) J. Biol. Chem. 209, 645-661. 28. SCHNAITMAN, C., ERWIN, V. G., AND GREENAWALT, J. W. (1967) J. Cell Biol. 32, 719-735. 29. BRDICZKA, D., POE, D., BRUNNER, G., AND MILLER, F. (1968) Eur. J. Biochem. 5,294-304. 30. PHILLIPS, A. H., AND LANGDON, R. G. (1962) J. Biol. Chem. 237, 2652-2660. 31. FURTH, A. J., AND ROBINSON, D. (1965) Biochem. J. 97, 59-66. 32. ROBINSON, R., ROUGHAM, M. E., AND WAGSTAFF, D. F. (1971) Ann. Clin. Biochem. 8, 168-170. 33. HUBSCHER, G., AND WEST, G. R. (1965) Nature (London) 205, 799-800. 34. KING, E. J. (1932) Biochem. J. 26, 292-297. 35. SCHNAITMAN, C., AND GREENAWALT, J. W. (1968) J. Cell Biol. 38, 158-175. 36. LOWRY, D. H., ROSEBROUGH, N. J., FARR, A. L., AND RANDALL, R. J. (1951) J. Biol. Chem. 193, 265-275. 37. SPEED, D., AND RICHARDSON, M. (1968) J. Chromatogr. 35, 497-505. 38. BERGEL, F., COHEN, A., AND HINDLEY, N. C. (1950) J. Chem. Sot. 1439-1443. 39. REINER, J. M. (1969) Behavior of Enzyme Systems, 2nd ed., pp. 127-132, Van NostrandReinhold Co., New York. 40. DALZIEL, K., AND DICKINSON, F. M. (1965) Biothem. J. 95, 311-320. 41. DEITRICH, R. A., COLLINS, A. C., AND ERWIN, V. G. (1972) J. Biol. Chem. 247, 7232-7236. 42. RAISON, J. K., HENSON, G., AND RIENITS, K. G. (1966) Biochim. Biophys. Acta 118, 285-298. 43. LUNDQUIST, F., FUGMANN, U., RASMUSSEN, H., AND SVENDSEN, I. (1959) Biochem. J. 72, 409-419. 44. ENGEL, P. C., AND FERDINAND, W. (1973) Biochem. J. 131, 97-105. 45. ROTHSCHILD, H. A., AND BARRON, E. S. G. (1954). J. Biol. Chem. 209, 511-523.