Evidence for mitochondrial localization ofbetaine aldehyde dehydrogenase in rat liver:purification, characterization, and comparisonwith human cytoplasmic E3 isozyme
Ming-Kai Chern and Regina Pietruszko
Abstract: Betaine aldehyde dehydrogenase has been purified to homogeneity from rat liver mitochondria. Theproperties of betaine aldehyde dehydrogenase were similar to those of human cytoplasmic E3 isozyme in substratespecificity and kinetic constants for substrates. The primary structure of four tryptic peptides was also similar; onlytwo substitutions, at most, per peptide were observed. Thus, betaine aldehyde dehydrogenase is not a specific enzyme,as formerly believed; activity with betaine aldehyde is a property of aldehyde dehydrogenase (EC 1.2.1.3), which hasbroad substrate specificity. Up to the present time the enzyme was thought to be cytoplasmic in mammals. This reportestablishes, for the first time, mitochondrial subcellular localization for aldehyde dehydrogenase, which dehydrogenatesbetaine aldehyde, and its colocalization with choline dehydrogenase. Betaine aldehyde dehydrogenation is an importantfunction in the metabolism of choline to betaine, a major osmolyte. Betaine is also important in mammalian organismsas a major methyl group donor and nitrogen source. This is the first purification and characterization of mitochondrialbetaine aldehyde dehydrogenase from any mammalian species.
Key words: betaine, aldehyde, dehydrogenase, mitochondria, rat liver.
Résumé: La déshydrogénase de l’aldéhyde de la bétaïne a été purifiée jusqu’à homogénéité à partir de mitochondriesde foie de rat. Ses propriétés sont semblables à celles de l’isoenzyme E3 cytoplasmique humaine quant à la spécificitéenvers les substrats et les constantes cinétiques. Les structures primaires de quatre peptides produits par la trypsinesont également semblables; seulement, au plus, deux substitutions par peptide sont notées. Ainsi, la déshydrogénase del’aldéhyde de la bétaïne n’est pas une enzyme spécifique comme on le pensait; l’activité catalytique envers l’aldéhydede la bétaïne est attribuable à l’aldéhyde déshydrogénase (EC 1.2.1.3) qui a une spécificité envers plusieurs substrats.Jusqu’à présent, on pensait que l’enzyme se trouvait dans le cytoplasme des cellules de mammifères. Cet article établitpour la première fois que l’aldéhyde déshydrogénase qui catalyse la déshydrogénation de l’aldéhyde de la bétaïne estlocalisée dans les mitochondries, comme la choline déshydrogénase. La déshydrogénation de l’aldéhyde de la bétaïneest importante dans le métabolisme de la choline en bétaïne, un important osmolyte. La bétaïne est égalementimportante dans l’organisme des mammifères en tant que donneur de groupes méthyle et source d’azote. C’est lapremière fois que la déshydrogénase de l’aldéhyde de la bétaïne des mitochondries d’un mammifère est purifiée etcaractérisée.
Mots clés: bétaïne, aldéhyde, déshydrogénase, mitochondries, foie de rat.
[Traduit par la Rédaction] Chern and Pietruszko 187
Introduction
NAD-linked aldehyde dehydrogenase (EC 1.2.1.3) cata-lyzes both the irreversible dehydrogenation of aldehydes and
hydrolysis of esters. The enzyme occurs in several molecularforms, all homotetramers with molecular masses of 213 000– 219 000 Da that exhibit differences in primary structureand substrate specificity. Human aldehyde dehydrogenase,with broad substrate specificity and high affinity for shortchain aliphatic aldehydes, occurs as three known isozymes,E1, E2, and E3. E1 and E3 are cytoplasmic and E2 is mito-chondrial (Pietruszko 1989). The human genes coding forthe isozymes, are known asALDH1, which codes for E1,ALDH2, which codes for E2, andALDH9, which codes forE3 (Human GenBank Database). Human E3 isozyme wasfirst purified and characterized in 1989 (Kurys et al. 1989)as an aldehyde dehydrogenase with broad substrate specific-ity that includedγ-aminobutyraldehyde among its substrates.The substrate list gradually expanded to include otheraminoaldehydes (Ambroziak and Pietruszko 1991) and most
M.-K. Chern and R. Pietruszko.1 Center of Alcohol Studiesand Department of Molecular Biology and Biochemistry,Rutgers, the State University of New Jersey, 607 AllisonRoad, Piscataway, NJ 08854-8001, U.S.A.
1Author to whom all correspondence should be addressed(e-mail: [email protected].).
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recently betaine aldehyde (Chern and Pietruszko 1995).Thus, substrates of the E3 isozyme include those previouslythought to belong to specific aldehyde dehydrogenases. In somespecies, the enzymes frequently calledγ-aminobutyraldehydedehydrogenases or betaine aldehyde dehydrogenases are re-ported to be cytoplasmic (Ambroziak et al. 1991; Rothschild andGuzman-Barron; Testore et al. 1995) in others mitochondrial(Dragolovich and Pierce 1994). Their peroxisomal localizationhas been also described (Nakamura et al. 1997). In plants,betaine aldehyde dehydrogenase, known to function inosmoregulation via synthesis of betaine from choline, is local-ized in the chloroplast (Weretilnyk and Hanson 1989).
Primary structure of the E3 isozyme is up to the presenttime incomplete (Kurys et al. 1993; Lin et al. 1996; Chernand Pietruszko 1998). One reason appears to lie in cDNA,which is exceptionally rich in C and G bases, producing un-expected frame shifts (Chern and Pietruszko 1998). Al-though it has been claimed that a complete amino acidsequence was obtained (Lin et al. 1996), the enzyme couldnot be expressed. Moreover, our more recent results (Chernand Pietruszko 1998), indicate that the amino terminal se-quence of the claimed complete clone is incorrect. Our ownattempts to complete the amino acid sequence have beenalso unsuccessful. Employing E3 isozyme cDNA libraryfrom fetal human brain, we were able to isolate a clone thatwas larger than that expected from subunit with a molecularmass of 54 000 Da. This clone coded for 525 amino acidresidues. But even this longer clone did not appear to pos-sess the start methionine codon.
Choline dehydrogenase, an enzyme that oxidizes cholineto betaine aldehyde is localized in the mitochondria (Mannet al. 1938). Mitochondria were employed for synthesis ofbetaine aldehyde from choline when formation of betainewas observed (Zhang et al. 1992), suggesting that betaine al-dehyde dehydrogenase in rat liver was also localized in themitochondria. In this investigation, an attempt was made tocharacterize betaine aldehyde dehydrogenase that was local-ized in the mammalian mitochondria. Human mitochondriaare difficult to obtain as is fresh, postoperative human livertissue. Purification and characterization of rat liver mito-chondrial equivalent of human E3 isozyme is described inthis paper. This is the first purification and characterizationof mitochondrial betaine aldehyde dehydrogenase from anymammalian species.
Materials and methods
Materialsβ-Nicotinamide adenine dinulcleotide (β-NAD), Grade 1, was
from Boehringer-Mannheim. Betaine aldehyde, glycolaldehyde,β-nicotinamide adenine dinucleotide phosphate (β-NADP), D-sorbitol,β-NAD N6 (11 atom spacer) agarose, cimetidine, andp-nitrophenylacetate were from Sigma. Propionaldehyde and acetaldehyde,redistilled before use, were from Aldrich Chemical Co.γ-Aminobutyraldehyde diethyl acetal (Aldrich) was redistilled be-fore storage at 4°C andhydrolysed before use (Ambroziak andPietruszko 1986). Pharmalyte (pH 3–10) carrier ampholyte, agaroseisoelectric focusing, isoelectric focusing calibration kit (pH 3–10), andHMW electrophoresis calibration kit (for native gel), CM Sephadex C-50, and 5′ AMP Sepharose 4B were from Pharmacia Biotech. Gelcodecolor silver stain was from Pierce Chemical Co. Ultrafiltration productswere from Amicon and Schleicher & Schuell. All other chemicals werereagent grade.
Activity assay, kinetics, and assay of substratesThe assay mixture contained 0.1 M sodium pyrophosphate (pH
9.0), 500 mM NAD, 1 mM EDTA, and 1 mM betaine aldehyde.KmandVmax values for aldehyde substrates were determined in 0.1 Msodium phosphate buffer (pH 7.4) or 0.1 M sodium pyrophosphatebuffer (pH 9.0) containing 500µM NAD and 1 mM EDTA at25°C. TheVmax values were adjusted to maximal specific activityof the enzyme. All data were analyzed using nonlinear computerprograms HYPER or COMP of Cleland (1979). The NADH extinc-tion coefficient of 6.22 mM–1·cm–1 was used. At the completion ofthe reaction, the concentration of substrates was determined fromthe amount of NADH formed using the E2 isozyme forpropionaldehyde and acetaldehyde and the E3 isozyme forγ-aminobutyraldehyde and betaine aldehyde. The E2 and E3isozymes were purified from human liver as previously described(Kurys et al. 1989; Hempel et al. 1982).
Protein assayProtein was assayed by the microbiuret procedure of Goa (Goa
1953) using bovine serum albumin as a standard. The protein con-centration in the last step of purification was estimated byabsorbance at 205 nm using an extinction coefficient of 31(1 mg·mL–1·cm–1; Goldfarb et al. 1951). The enzyme was dialyzedagainst four changes of 100-mL saline (0.9% NaCl). The dialysateof the last change of saline was used as a blank.
Isoelectric focusingIsoelectric focusing was carried out on agarose plates (114 ×
225 mm) composed of 1% w/v agarose, 12% sorbitol, and 0.063%Pharmalyte (pH 3–10). The enzyme was stained for activity using100 mM Tris–HCl buffer (pH 8.5), containing NAD (20 mg/30 mL),nitro blue tetrazolium chloride (20 mg/30 mL), phenazinemethosulfate (2 mg/30 mL), and 1.5 mM betaine aldehyde orpropionaldehyde. For staining with γ-aminobutyraldehyde(300 µM), sodium phosphate buffer (100 mM, pH 7.4) containing1 mM EDTA was used. Protein bands were stained with CoomassieBrilliant Blue G.
Molecular mass determinationThe discontinuous sodium dodecyl sulfate – polyacrylamide gel
electrophoresis (SDS-PAGE) of Laemmli (1970) was used; sam-ples were concentrated by precipitation in 5% cold trichloroaceticacid. The native molecular mass was estimated using continuous4–20% polyacrylamide Tris–glycine precast gels obtained fromNovex. The Tris–glycine buffer in gel was exchanged with Tris bo-rate – EDTA buffer containing 0.09 M Tris, 0.08 M boric acid, and0.0025 M EDTA (pH 8.4) by a 12 h pre-electrophoresis step. Oth-erwise, the gels were used as suggested by the manufacturer’s in-structions.
Amino acid composition analysis and sequencing ofpeptides
10% Laemmli gels with 0.75 mm thickness were used to sepa-rate proteins. The gel was stained with 0.1% Coomassie Blue in10% acetic acid, 50% methanol, and 40% H2O for 1 h and thendestained by soaking for 3 h in 10% acetic acid, 50% methanol,and 40% H2O with three solvent changes until the background wasnearly clear. The band was excised and placed in an Eppendorftube and frozen. A similar size piece of gel that did not contain anyprotein was excised and put in a separate Eppendorf tube as a con-trol. Both samples were kept frozen on dry ice during shipment.The hydrolysis – amino acid analysis, in situ tryptic digest in gel,elution of the resulting peptides, HPLC separation, and amino acidsequencing were then done by W.M. Keck Facilities (Williams et al.1996) at Yale University, New Haven, Conn.
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Purification procedure
BuffersAll buffers used for enzyme purification were made with
deionized water, degassed, and saturated with nitrogen. Two buff-ers were used. Buffer 1 was 30 mM sodium phosphate at pH 6.0and buffer 2 was 30 mM sodium phosphate at pH 8.0. Both bufferscontained 1 mM EDTA and 0.1% v/v 2-mercaptoethanol. All of thefollowing steps were carried out at 4°C.
Isolation of the mitochondrial matrixLivers from male Sprague–Dawley rats (2–3 months old) were
used. Mitochondria were isolated according to the procedure ofHogeboom (1955). Immediately after the rat was decapitated, theliver was excised and homogenized in 9 mL of cold 0.25 M sucrosefor every gram of liver using a Potter homogenizer. The homogenatewas centrifuged at 700 ×g for 10 min in a Sorvall RC-5B centri-fuge; the sediment was discarded. The supernatant was centrifugedat 5000 × g for 10 min. The resulting mitochondrial pellet waswashed three times by resuspending at a ratio of 9 mL of 0.25 M su-crose to 1 g ofmitochondrial pellet and then centrifuged again at5000 ×g for 10 min. The isolated mitochondria were resuspended in1.5 mL of buffer 1 for every gram of mitochondrial pellet andsonicated at a pulse cycle of 2 s and 40% duty cycle for 40 s with aW-380 sonicator from Heat Systems-Ultrasonics, Inc. The suspen-sion after sonication was centrifuged at 100 000 ×g for 30 min in aBeckman L50 ultracentrifuge at 4°C.
CM Sephadex C-50 chromatography and 5′ AMPSepharose 4B chromatography
CM Sephadex C-50 chromatography and 5′ AMP Sepharose 4Bchromatography were done as previously described (Kurys et al.1989).
NAD agarose chromatographyCimetidine was dissolved in the pooled active fractions from 5′
AMP Sepharose to reach a final concentration of 1.5 mg cimetidineper 1 mL of pooled sample. The sample was then concentrated byultrafiltration in an Amicon stirred cell to 5.7 mL (averaged vol-ume from two experiments) and applied to an NAD agarose col-umn (12.4 × 1.6 cm) pre-equilibrated with buffer 1 containing1.5 mM cimetidine (Brandt et al. 1987). The column was washedthoroughly with 200 mL buffer 1 containing 1.5 mM cimetidine toremove extraneous protein and then washed with 5 mL buffer 2 toremove cimetidine. The enzyme was eluted by NAD gradient em-ploying 80 mL of buffer 2 in the mixing chamber and 80 mL ofbuffer 2 containing 1 mg NAD/mL in the reservoir. The individualactive fraction was adjusted to pH 6.0 with saturated monobasicsodium phosphate and mixed with glycerol to make a final concen-tration of 16–18% v/v. The enzyme was stored at –12°C under ar-gon.
Results
Preliminary evidence for mitochondrial localization ofrat liver betaine aldehyde dehydrogenase
Preliminary experiments using rat mitochondrial cholinedehydrogenase for synthesis of labeled betaine aldehydeconfirmed previously reported results (Zhang et al. 1992).Labeled betaine was obtained each time, suggesting thatbetaine aldehyde dehydrogenase was present in the mito-chondria. In further experiments, betaine aldehyde was usedfor determination of activity in separated cytoplasm and mi-tochondria. The mitochondrial matrix contained about 5% oftotal rat liver betaine aldehyde dehydrogenase activity; 95%was in the cytoplasm. Activities of alcohol dehydrogenase,
lactate dehydrogenase, and glutamate dehydrogenase weredetermined on the mitochondrial matrix and the cytoplasmicfraction. Alcohol dehydrogenase activity (1.5% of total) and2.6% of total lactate dehydrogenase activity were also foundin the mitochondrial matrix. Glutamate dehydrogenase activ-ity was exclusively confined to the mitochondrial matrix, in-dicating that the mitochondria were intact. Rat mitochondriallactate dehydrogenase was first reported by Brandt et al.(1987); functional importance of this enzyme in the intra-cellular shuttle of lactate has been recently described(Brooks et al. 1999). Thus it appears more than likely thatmitochondrial alcohol dehydrogenase is also present inmammalian mitochondria. The mitochondrial matrix, fromwashed mitochondria of male Sprague–Dawley rats, whensubjected to isoelectric focusing, showed two betaine alde-hyde dehydrogenase active bands: a major band of pI 5.4and a minor band of pI 5.1 (Fig. 1, lane 1). Mitochondrialbetaine aldehyde dehydrogenase migrated more cathodallythan the rat cytoplasmic enzyme and could be readily distin-guished on isoelectric focusing gels. Before attempting en-zyme purification the mitochondria were washed severaltimes before sonication and were sonicated only when nobetaine aldehyde dehydrogenase activity was detectable inthe wash (Fig. 2).
Enzyme purificationThe purification steps, involving use of three chromato-
graphic columns, are shown in Table 1, which represents
Fig. 1. Isoelectric focusing of rat liver mitochondrial matrixbetaine aldehyde dehydrogenase before and after purification.Lane 1, mitochondrial matrix developed with betaine aldehyde;lane 2, pI standards developed with Coomassie Brilliant Blue G;lane 3, betaine aldehyde dehydrogenase after purificationdeveloped with Coomassie Brilliant Blue G; lane 4, betainealdehyde dehydrogenase after purification developed with betainealdehyde. The agarose gel was run at 250 V and 15 W for 12 h.
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mean values from two purifications. While CM-Sephadexremoved over 60% of proteins, 5′AMP Sepharose removedmitochondrial ALDH2. Chromatography on NAD agaroseachieved a >19-fold increase in specific activity over theprevious step. By using cimetidine, a potent aldehyde sub-strate competitive inhibitor for the human E3 isozyme(Kikonyogo and Pietruszko 1997), the binding of the en-zyme to NAD agarose could be improved from 60 to 90%.The final specific activity (3.24 ± 0.44µmol·min–1·mg–1,mean ± SD) was found to be constant in the active fractions.
Isoelectric focusing, gradient gel electrophoresis, andSDS-PAGE
Enzyme eluted from NAD agarose was analyzed by anisoelectric focusing gel and isoelectric points were deter-mined using the pI standards. Activity staining with betainealdehyde showed one major band and one minor band withisoelectric points of 5.4 and 5.1, respectively (Fig. 1, lane 4).The same two bands were visualized during Coomassie Bril-liant Blue G staining (Fig. 1, lane 3), showing that the en-zyme was homogeneous. One major (58.8 kDa) and oneminor (54 kDa) band were seen on SDS-PAGE (Fig. 3). Gra-dient gel electrophoresis of the native enzyme (4–20%), fol-lowed by silver staining, gave one band with an estimatedmolecular mass of 255 kDa.
Kinetic propertiesThe Km and Vmax values of the purified enzyme for
betaine aldehyde,γ-aminobutyraldehyde, glycolaldehyde,NAD, and acetaldehyde are shown in Table 2. The enzymeutilized NADP (500µM) at about 6% of the rate that it uti-lized NAD (500µM). The velocity with acetaldehyde as thesubstrate was low (0.058µmol·min–1·mg–1 in 0.1 M sodiumphosphate (pH 7.4) containing 0.5 mM NAD, 1 mM EDTA,
and 126µM acetaldehyde), and it was difficult to directlydetermine itsKm value. TheKi value was determined insteadbecause theKi of an alternate substrate equalsKm (Segel1975). Acetaldehyde inhibited the enzyme in a competitivemanner versus betaine aldehyde (Fig. 4). Since the rate withacetaldehyde was less than 2% of that with betaine alde-hyde, the contribution of acetaldehyde to NADH formationwas ignored. TheKi value of 14µM was one order ofmagnitude smaller than theKm value for betaine aldehyde(Table 2). Cimetidine inhibited the enzyme in a competi-tive manner versus varied betaine aldehyde (Fig. 5). TheKi was 33.2 ± 3.6µM. Enzyme activity was inhibited by5 mM and 55 mM chloral by 9 and 75%, respectively.Disulfiram (40µM) inhibited enzyme activity by 27% in thestandard assay system for betaine aldehyde. No effect wasobserved when the enzyme was assayed in the presence ofMgCl2 (100µM), CaCl2 (110µM), and MnCl2 (110µM) in30 mM PIPES buffer (pH 7.0) containing 500µM NAD and1 mM betaine aldehyde.
Comparison of amino acid composition, peptide maps,and peptide sequences
Amino acid composition (not shown) of rat mitochondrialenzyme was similar to that of human E3 isozyme. HPLCchromatograms of tryptic digests of the rat mitochondrialand human cytoplasmic betaine aldehyde dehydrogenasewere similar but not identical. Three peaks, Nos. 27, 66, and
Fig. 2. Distribution of betaine aldehyde dehydrogenase activityin the cytoplasm and mitochondria. 1, postmitochondrialsupernatant; 2–4, consecutive washes of the mitochondrial pelletwith 9 mL of 0.25 M sucrose for each gram of pellet; 5,mitochondrial matrix after sonication.
Fig. 3. SDS-PAGE of purified rat mitochondrial betaine aldehydedehydrogenase. 16, 18, 19, and 20 represent fraction numberscontaining betaine aldehyde dehydrogenase following NADagarose chromatography. St, molecular mass standards withbands from the top of the gel corresponding toβ-galactosidase(116 kDa), phosphorylase (97.4 kDa), bovine serum albumin (66kDa), egg albumin (45 kDa), glyceraldehyde-3-phosphatedehydrogenase (36 kDa), and carbonic anhydrase (29 kDa). TheSDS-polyacrylamide (10%) gel was stained with CoomassieBrilliant Blue R.
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75, from rat mitochondrial enzyme digest were selected forsequencing after examination with laser desorption–ioniza-tion mass spectrometry. The peptide from peak No. 66 ex-actly matched the human E3 isozyme sequence reported byKurys et al. (1993), as well as the corrected ALDH9 se-quence (Lin et al. 1996; Chern and Pietruszko 1998) fromposition 227 to 238 (shown in Fig. 6). The repurified prod-uct of peak No. 75 revealed a mixture of two peptides with aratio of 9:1. The primary peptide from peak No. 75 was ho-mologous with human E3 (Kurys et al. 1993) and ALDH9from position 326 to 337 with an arginine 332-to-alaninesubstitution. The secondary peptide from peak No. 75 washomologous with E3 (Kurys et al. 1993) and ALDH9 fromposition 89 to 97 with two position substitutions: threonine92 to valine and cysteine 95 to threonine. Five amino acidresidues from the 11 amino acid residue peptide from peakNo. 27 matched exactly ALDH9 residues 25–29; it turnedout that the reported ALDH9 (see Fig. 6) sequence was inthe wrong frame (Chern and Pietruszko 1998). When the se-
quence was read in correct frame, it was quite similar to thatof the peak No. 27 peptide. Recently sequenced betaine al-dehyde dehydrogenase from cod liver is also shown in Fig. 6for comparison. This comparison suggests that alanine 18may be also incorrect. The cod enzyme, which shows 70%positional identity with human ALDH9, has an arginine resi-due in that position, as would be expected from tryptic pep-tide hydrolysis. Please note that the cod enzyme (sequencedfrom protein) is longer than ALDH9 by 10 amino acid resi-dues and has a subunit molecular mass of 54 367 Da.
Comparison of other properties of rat mitochondrialand human cytoplasmic enzymes
Rat cytoplasmic betaine aldehyde dehydrogenase was pre-viously purified; the Km for betaine aldehyde (110µM,Rothschild and Guzman Barron 1965; 260µM, Goldbergand McCaman 1968) was similar to that determined duringthis investigation for the mitochondrial enzyme. Because theenzyme was only partially purified, physicochemical proper-
Step Total activity (µmol·min–1) Total protein (mg) Specific activity (µmol·min–1·mg–1) Yield (%)
Note: Activity was determined with 1 mM betaine aldehyde in 100 mM pyrophosphate (pH 9.0) containing 1 mM EDTA and 500µM NAD.
Table 1. Purification of betaine aldehyde dehydrogenase from rat liver mitochondria.
Varied substrate Constant substrate (mM) Km (µM)a Vmax (µmol·min–1·mg–1)a Vmax/Km
Betaine aldehyde NAD (0.5) 117.8±9.5 3.36±0.13 0.028Betaine aldehydeb NAD (0.5) 156.2±27.2 3.90±0.35 0.025γ-Aminobutyraldehyde NAD (0.5) 3.94±0.35 0.243±0.007 0.062Glycolaldehyde NAD (0.5) 152.5±22.9 0.50±0.00 0.003NAD Betaine aldehyde (1.0) 34.0±0.9 3.27±0.03 0.096Acetaldehydec NAD (0.5); betaine aldehyde (1.0) 13.9±0.8 0.058d 0.004
Notes: Measurements were carried out at pH 7.4 and 25°C in 0.1 M sodium phosphate containing 1 mM EDTA unless otherwise indicated.aValues are means ± SE.bDetermined at pH 9.0 and 25°C in 0.1 M sodium pyrophosphate containing 1 mM EDTA.cThe Ki value for acetaldehyde competitive inhibition on betaine aldehyde activity was taken to represent itsKm value.d89% of Vmax at 126µM acetaldehyde.
Table 2. Kinetic properties of betaine aldehyde dehydrogenase from rat liver mitochondrial matrix.
Property Rat mitochondrial Human cytoplasmic
Specific activity of D (µmol·min–1·mg–1) 3.3 8.4Specific activity of E (µmol·min–1·mg–1) 0.11 0.33Native molecular mass (kDa) 255 230Subunit molecular mass (kDa) 53, 59 54Isoelectric point 5.4, 5.1 5.4, 5.3Km for BAL (µM) 118 260Km for NAD (µM) 34 35Km for GABAL (µM) 4 5Km for acetaldehyde (µM) 14 50Km for glycolaldehyde (µM) 153 240Ki for cimetidine (µM) 33 1
Notes: D, dehydrogenase activity determined with 1 mM betaine aldehyde in 0.1 M sodium pyrophosphate (pH 9.0) containing 1 mM EDTA; E,esterase activity determined at 100µM p-nitrophenyl acetate in 50µM sodium phosphate (pH 7.0) containing 1 mM EDTA; BAL, betaine aldehyde;GABAL, γ-aminobutyraldehyde.Km and Ki values were determined in 0.1 M sodium phosphate (pH 7.4) (see Materials and methods).
Table 3. Comparison of properties of rat mitochondrial with human cytoplasmic E3 isozyme.
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ties are not available for comparison. For this reason proper-ties of rat mitochondrial and human cytoplasmic enzymesare compared in Table 3. It can be seen that there is a greatsimilarity between the enzymes, the differences are inKi forcimetidine, specific activity, subunit molecular mass, andperhapsKm for acetaldehyde.
Discussion
Choline performs several biological functions. It is a pre-cursor of phosphatidylcholine, sphingomyelin, and the neu-rotransmitter acetylcholine. Recent work also points to itscritical role in brain development (Musztajn 1998). Cholineis also enzymatically oxidized via betaine aldehyde tobetaine, which is a methyl group donor and nitrogen sourceas well as an important osmoregulator in some organisms(Musztajn 1998). In mammalian organisms choline is oxi-dized by choline dehydrogenase, localized in the mitochon-dria (Mann et al. 1938), to betaine aldehyde.
Because the majority of betaine aldehyde dehydrogenaseactivity of rat liver was known to be cytoplasmic, before at-tempting purification from rat liver mitochondria, precau-tions were taken to avoid contamination by the cytoplasm bywashing mitochondria with sucrose before sonication. Ex-tensive washing did not remove betaine aldehyde de-hydrogenase from the mitochondria (see Fig. 2). Theprocedure for purification was similar to that used for hu-
man E3 isozyme (Kurys et al. 1989) but considerably sim-plified. Instead of six chromatographic columns, only threecolumns were used in succession: CM-Sephadex, 5′AMP-Sepharose 4B, and NAD Agarose (Table 1). It was foundpreviously in our laboratory that cimetidine (an aldehyde-competitive inhibitor for the E3 isozyme; Kikonyogo andPietruszko 1997) improved binding of the E3 isozyme to theNAD Agarose column. Cimetidine improved binding of ratmitochondrial betaine aldehyde dehydrogenase to NADAgarose and stabilized the enzyme, resulting in better activityrecovery (about 90%). The specific activity of the homoge-neous enzyme with betaine aldehyde was 3.3µmol·min–1·mg–1
protein; comparable to that of betaine aldehyde dehydrogenasefrom chloroplast (3.3µmol·min–1·mg–1 protein; Weretilnyk andHanson 1989).
The above experiments demonstrate that betaine aldehydedehydrogenase is localized in the rat liver mitochondrial ma-trix. Thus, in the mitochondria, it is colocalized with cholinedehydrogenase. This localization may be connected with itsrole on the metabolic pathway from choline to betaine—aphysiologically important metabolite. Mitochondrial local-ization of betaine aldehyde dehydrogenase also explains for-mation of radioactive betaine when radioactive choline wasincubated with rat liver mitochondria in our preliminary ex-periments and that of Zhang et al. (1992).
Even though the specific activity is lower than that of thehuman E3 isozyme (Table 3), the similarities to E3
Fig. 4. Inhibition of betaine aldehyde dehydrogenase from rat liver mitochondria by acetaldehyde. Acetaldehyde concentrations were0 µM (+), 2.4 µM (s), 4.7µM (j), 9.5µM (n), 10.7µM (u), and 16.6µM (r). The reaction velocity (V) is expressed as micromolesof NADH per minute per millilitre of enzyme solution. The inset shows a plot of the slopes represented in the main figure.
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isozyme are numerous. In almost all of the propertieslisted in Table 3, rat mitochondrial betaine aldehydedehydrogenase resembles human E3 isozyme. Amino acidsequence, although notidentical, also resembles human cy-toplasmic E3 isozyme (see comparison of four sequencedpeptides with ALDH9 in Fig. 6). Thus, these results alsoshow that betaine aldehyde dehydrogenase is not a specificenzyme, as formerly believed, but is a rat mitochondrialequivalent of human cytoplasmic E3 isozyme. Based onthese results, we suggest that human E3 isozyme may bealso localized in the mitochondria.
Our previous work (Izaguirre et al. 1997), based on as-sumed cytosolic localization of the human E3 isozyme, usedmRNA, betaine aldehyde dehydrogenase activity, and West-ern blot analysis to study tissue distribution. The results withmRNA indicated main subcellular localization in the skeletaland heart muscle, which had very little E3 protein or betainealdehyde dehydrogenase activity. One possibility, not con-sidered at that time, was that the enzyme might have beenlocalized in muscle mitochondria, instead of the cytoplasmas previously assumed. As a result, no steps were taken toassure breakage of the mitochondria. It is possible thatsubcellular distribution could be different in different tis-sues. While there is very little mitochondrial enzyme inliver, all of the muscle or of heart muscle enzyme could bemitochondrial, if the subcelular distribution is determined bythe physiological function. In the horseshoe crab cardiac tis-
sue the majority of betaine aldehyde dehydrogenase is mito-chondrial (Dragolovich and Pierce 1994). Differences in dis-tribution of mitochondrial lactate dehydrogenase (Brandt etal. 1987) in different tissues are well established. Recently, acomplete amino acid sequence of betaine aldehydedehydrogenase from Baltic cod (Gadus callarias) liver, ob-tained by sequencing protein, has been deposited in theGenBank database (accession No. P56533). The completesubunit consists of 503 amino acid residues with a molecularmass of 54 367 Da. Alignment of this sequence withALDH9 (Fig. 6) shows regions of positional identity heavilyconcentrated towards the center of the molecule and thecarboxy terminal end, where the active site is located. Evenafter correction, there are two stretches of sequence at theamino terminal end where no similarities are seen. Thus, thefact that human ALDH9 sequence is shorter than that of thecod enzyme may be real or it may be incomplete.
The following differences between rat mitochondrialbetaine aldehyde dehydrogenase and human E3 isozymewere noted: specific activity (about 40% of human E3isozyme);Ki value for cimetidine (about 30 times higher forthe rat enzyme); subunit heterogeneity with one subunit(58.8 kDa) being considetably larger than that of the humanenzyme (54 kDa); and larger molecular mass. There werealso some differences found in the four peptides that we se-quenced (Fig. 6). At the present time, it is impossible to de-cide whether the mitochondrial and cytoplasmic enzymes
Fig. 5. Dixon plot of cimetidine inhibition of betaine aldehyde dehydrogenase from rat liver mitochondria. Betaine aldehydeconcentrations were 250µM (+), 125 µM (n), and 62.5µM (s). The reaction velocity (V) is expressed asµmoles of NADH perminute per milligram of enzyme protein. The inset shows a plot of the slopes represented in the main figure to show that inhibition iscompetitive.
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are products of the same or different genes. Differences ob-served above could represent species differences betweenhumans and rats. Sequences of cytoplasmic and mitochon-drial isozymes from the same species are needed, otherwisevariations owing to species difference could be confusedwith gene difference. There are examples of cytoplasmic andmitochondrial enzymes coded for by the different genes orby the same nuclear gene. Mammalian ALDH1 and ALDH2are examples of enzymes coded for by distinct genes. On theother hand, mammalian fumarase (Moss 1982) andglutathione reductase (Tamura et al. 1996) are examples ofenzymes where a single gene locus codes for the cytoplas-mic and mitochondrial forms. In rat liver, mitochondrial andcytoplasmic fumarases are translationally regulated by twoATG initiation codons present in one species of mRNA
(Suzuki et al. 1992). The yeast fumarase, however, isprocessed in the mitochondria and then released into the cy-toplasm (Stein et al. 1994). Presence of a much larger sub-unit (58.8 vs. 54 kDa) (Fig. 3) in rat mitochondrial betainealdehyde dehydrogenase suggests a different gene. It couldalso mean that there is a single gene but that cDNA is muchlarger than currently believed and has a mitochondrial leadersequence. If this is the case, the enzyme may be processed inthe mitochondria and then released into cytoplasm.
Acknowledgements
Financial support of United States Public Health ServiceGrant 1R01 AA00186 from the National Institute on Alco-hol Abuse and Alcoholism is acknowledged.
Fig. 6. Sequence alignment of four peptide sequences from rat liver mitochondrial betaine aldehyde dehydrogenase and of cod liverbetaine aldehyde dehydrogenase with human ALDH9. The human sequence (ALDH9) is deduced from cDNA (Lin et al. 1996). Thecod sequence (CODBADH) (GenBank accession No. P56533, 01 Feb. 1998) has been obtained by direct sequencing of cod protein.Identical residues are connected with vertical lines. The residues that are underlined were read by Lin et al. (1996) in a wrong frame;sequence shown is that corrected by Chern and Pietruszko (1998).
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