Aldehyde Dehydrogenase

(Received for publication, May 24, 1968)

ltrc11.41~ G. vos TICXHSTROJI~ ASD W. I5 RKXELI,

From the Lkpartmenl vf I7ficrobiology, I’niwsity of Alberta, Edmonfon, C’amxla

SUMMARY

1. A molecular weight of 187,000 was determined for aldehyde dehydrogenase by calculation from the determined sedimentation and diffusion coefficients, by the method of Archibald, and from gel filtration and sucrose gradient centri- fugation with reference enzymes.

2. Aldehyde dehydrogenase contained relatively large amounts of all common amino acids. The lowest proportion was obtained for cysteic acid-22 residues per molecule of active enzyme.

3. Aldehyde dehydrogenase from Pseudomonas aeruginosa did not contain zinc.

4. The point of isoelectric precipitation for aldehyde dehy- drogenase was pH 4.8. The enzyme was soluble below pH 4.0 but its sedimentation, diffusion, and viscosity behaviors indicated that it was denatured at low pH, to a subunit of molecular weight 95,000.

5. At low ionic strength the enzyme underwent a partial dissociation, probably into two subunits, with the loss of enzymatic activity. This dissociation could be reversed by increasing the salt concentration. Dissociation and associa- tion could be followed in the ultracentrifuge and on starch gel electrophoresis. The dissociated form of the enzyme was isolated after starch gel electrophoresis, and was found to be inactive, but reassociation occurred in 0.1 M salt, and reacti- vation required a reducing agent.

h prrvious publication (1) dealt with the pot.aasium- or

* This study was supported by Grant A-2075 from the Nat.ional Research Council of Canada. Results were presented in part at the 5lst meeting of the Federat.ion of American Societies for Experimental Biology at Chicago, April 1967 (VOX TIGERWRCXI, R. C;., ;\ND R\ZZEI,I., W. F:., Fed. Proc., 26, 726 (3967)). The work reported here is taken from a thesis submitted by R. G. von Tigerstrom in partial fulfillment of the requirements for the degree of Doctor of Philosophy, University of British Columbia, Vancouver. The preceding paper in this series is Reference 1.

$ Present address, L)epartmcnt of Biochemistry, University of British Columbia, Vancouver 8, Canada.

c\mmoniuln-acti~lltcd nldehydc dchydrogcnnse from Pseu- domonas aerrcginosa. It described the purification of this enzyme from ethanol-grown cells, which resulted in a homogene- ous enzyme preparstion as judged from electrophoresis, ultra- ccntrifugat.ion, and other criteria. Uisulfite buffer stabilized the enzyme, especially in t.hc early stages of purification. This enzyme oxidized a wide variety of aldehydes, and NAD+ was the preferred hydrogen acceptor. In addition to the activating ion a reducing agent was required for maximum enzymatic activity. Although chelating agents replaced the reducing agent to some degree, ortho-phen:lnt.hroline, in the presence of a reducing agent, inhibited the enzyme. This indicated the possible involvement of zinc in the enzymatic activity. The presence of activat.ing ion aud NAD+ protected the enzyme from inhibition by iodoacetate, as well as from trypsin dig&on and heat inact.ivat.ion. These observations led to the conclusion that aldehyde dehydrogenase underwent a conformational change in the presence of the activating ion which was enhanced by the binding of NAD+.

The great. stability of the purified enzyme preparation made it, possible to investigate more characteristics of this aldehyde dehydrogenase. Such studies have not been possible with most other aldehyde dehydrogenases, owing to their inatnbilit.y (1, 2). This paper describes some physical and molecular properties of t.he cnzymr. The molecular wright was estimated by several methods. The amino acid composition of the enzyme was determined, and the enzyme was analyzed for zinc. The reversible dissociation of the active molecule and its physical properties below the isoelectric point are reported.

EXPEHIMEKTAL PROCEDURE

Jlaterials

,411 chemicals were obtained from commercial sources, as previously noted (1).

Yeast alcohol dehydrogcnase and trypsin were products of Worthington. Catalasr was prepared from horse blood.’

-~Ildehyde Deh2/drogenase-hldeh~~e dehydrogenase at. the

* Cat&se was the gift of I>r. C. R. Schonbaum, Department of 13iochemistry, University of Alberta.

6495

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6496 Aldehyde Dehydrogcnase. II Vol. 243, Ko. 24

limit of purificat.ion from P. aeruginosa, A’I’CC 9027, stored at. -20” (l), was used throughout this study.

Methods

Bnzyme ~~lasays .Udehydc dehydrogenasc was diluted and assayed as reported previously (I) except where indicated otherwise. C’atalasc activity was followed at 230 mp (3). Alcohol drhydrogcnase activity was determined by the rnethod of Vallee and Hoch (4).

Starch Gel Bleclrophoresis-Starc~h gel elrctrophorel;is was performed essentially as dcscribed previously (l), except for a temperature of 4” and a running tirne of 3f to 4 hours.

Sucrose Gradient Centrifugntion-Sucrose gradient centrifuga- tion, at 35,000 rprn, was pcrforrncd in a 5 to 20yc sucrose solution, dissolved in the desired buffer, for 13 hours at 5” with the use of the Bcckrnan model L-2 ultracentrifuge with the SW 391, swinging bucket rotor. Fractions of approximately 0.2 ml were collected, starting from the bottom of the tube.

Studies with Analytical Ultracentrifuge--A Beckman model E ultracentrifuge was used for all esperinients.2 Sedimentation cocflicient.s of aldehyde dchydrogcnase were determined in several buffer solutions. Dialysis against these solutions for 16 to 24 hours preceded each ultraccntrifugation.

The diffusion constant for alclchyde dchydrogcnase was obtained according to the method of Mbllcr (5). Diffusion of the boundary was observed at 16,200 rpm.

For calculat.ion of the molecular weight, the method of Archi- bald, as described by Schnchman (6), was used. Calculations were made from the pattern obtained at the meniscus.

Determination of Point of Zsoelectric Precipitatiolz-‘Len microliters of a solution of 4.8 mg of aldchyde dchydrngcnnsc per ml of 10 rnM potassium phosphate, pH 7.0, were added to each of a series of tubes cont,aining 1 ml of cold 10 mM potassium phosphate adjusted to the desired hydrogen inn concentrations with IICl. Tubes covering the pH mnge from pH 3.0 to pH 7.0 naro prepared and incubat.cd for 9 hours at 5”. They ncre then centrifuged at 27,000 X g for 15 min. Protein determinations according to the method of Lowry et al. (7) were performed on the precipitated protein. The point. of isoelectric precipitation was dct.crmined from a graph showing the protein precipitated at the different hydrogen ion concentrations.

Viscos4y Determinations-Viscosity measurements were carried out in a capillary viscomcter tube immersed in a constant temperature water bath at 5”. X volume of 1.75 ml was used for these determinations.

Atid IZydrolysisSamples of aldchyde dchydrogcnase, pre- pared for acid hydrolysis according to the method of Moore and Stein (S), were treated at 110’ for 12, 24, 48, or 72 hours. Du- plicate samples were prepared for each hydrolysis time.

Performic Acid Oridation-Duplicate samples, each .cnn- taining 0.1 mg of protein, were lyophiliaed and treated according to the method of Moore (9).

Amino Acid Analy.G--The hydrolyzed samples of aldchyde dehydrogcnase and performic acid-oxidized aldehydc dehy- drogenase were taken to dryness by flash evaporation and dis- solved in 1 ml of sodium citrate buffer, pH 2.2. .4n aliquot, usually 200 ~1, of these solutions was subjected to amino acid

* Studies with the ultracentrifugc were carried out with the kind cooperation of Dr. C. M. Kay of the Department of Bio- chemistry, IJniversit,y of Alberta, and the technical assistance of Mr. M. R. Aarbo

analysis with the use of the Beckman aut.ornat.ic amino acid analyzer, model 120B.3 The results were expressed as grams of amino acid per 100 g of protein, and as number of residue equivalents per 187,000 g of protein.

Protein-Protein was determined by the method of Lowry et al. (7). Crystalline bovine serum albumin was used as the standard. The protein concentration of purified aldehyde dehydrogcnasc also was determined by absorbance at 280 nip after the relation of absorbance at 280 nip to dry weight had been established (1).

.Yitrogen-Nitrogen was determined by the method of .\linari and Zilversmit (10). Protein samples (approximately 100 pg) were digested by refluxing for 90 min in 16-mm test tubes in the prcscnce of SeOC12 in HzSOl and subjected to nesslerizntion. Xmmonium sulfate dried to constant weight was used as the standard.

Zinc-.Udehyde dehydrogcnasc was dialyzed for 40 hours RI. 0” against 10 mhr Kc‘1 adjusted to pH 7.2 with Tris. This enzyme solut.ion (4.8 mg per ml) was analyzed for Zr? with the use of a F’erkin-Elmer 303 atomic absorption spectrnl~hntometer.’ Standard solutions, containing 0.5 to 4.0 pg of zinc per nil, were prepared with ZIGSO,, with the use of the same buffer solution as for the dialysis of the enzyme. -1 preparation of yeast alcohol dehytlrogenase (5.0 mg per ml) was dialyzed in parallel with the aldrhydc dchgdrngenasr and analyzed for Zn++ content.

.liolecukr Properties of .4 ldehyde Dehydrogenase

.lmi~o rlcid Compositiolz-;\mino acid analyses were performed with the Beckman automatic amino acid snalyzcr; t.he results arc shown in Table I. The vnlurs shown are the mean values calculated from the results ohtttined after hydrolysis times of 12, 24, 48, and 72 hours, with the following cxcrptions: the values for ralinr and isnlcucine are mean values obtained after 72 hours of hydrolysis, and the values for serino and threonine were obtained by extrapolation to zero hours of hydrolysis. Cyst& acid and methinnine sulfonc were determined separately after performic acid oxidation (9) of a sample of aldrhydc dehytlrogennse followed by 24 hours of hydrolysis.

Total nitrogen content of aldehyde dchydrogenase, det.crmincd according to the rnct.liod of Minari and Zilversmit (lo), was 16.0?;.

Tryptophun was estimated by t.he spectrophotometric method of Beaven and Holiday (II). From the absorbance of aldehyde dehydrogenase in 0.1 M sodium hydrosidc at 280 rnp and 294.4 rnp, it was calculated that there were 31 moles of tryptophnn and 110 moles of tgrosine per mole of aldchgde dchydrogenasc. The value obtained for tyrosine is not in agreement with the value obtained by the automatic amino acid analysis and, therefore, the value for tryptophan can bc accepted only as an approximation.

Table I also shows the number of amino acid residues per molecule of enzyme, assuming the molecular weight of the

3 We are indebted to J)r. L. B. Smillie of the 1)epartmont of Biochemistry, University of Alberta, for the use of the amino acid analyzer and to Mr. 5:. Pnradowski for performing the analyses.

4 The use of this equipment was made possible through the kind permission of 1)r. S. Pawluk, Department of Soil Science, University of Alberta.

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Issue of December 25, 196s R. G. von Tige&Tom and W. E. Raxxell 6497

TABLE I Amino acid composition of aldehyde dehydrogenase

Amino acid I

content

Lysine ........................ Histidine ..................... Arginine ...................... Aspartic acid + asparagine. .. Threonine, .................... Serine ........................ Glutamic acid + glutamine. .. Proline ....................... Glycine ....................... Alanine ....................... Valine ........................ Isoleucine. .................... Leucine. ...................... Tyrosine. ..................... Phenylalanine. ................ Cystcic acid. ................. Methionine. .................. Tryptophan ...................

?tLole/lOO g

0.0397 0.0181 0.0343 0.0727 0.044 0.037 0.0949 0.0382 0.0818 0.1029 0.055 0.0535 0.0710 0.0300 0.0335 0.0118 0.0151

(0.0155)”

roles/naol

74 34 64

136 82 G9

177 71

153 192 103 100 132 56 62 22

gllO0 g

5.80 2.81 5.97 9.68 5.24 3.88

13.94 4.39 6.14 9.17 6.44 7.02 9.33 5.43 5.54 1.43 2.25

(3.16)

Total. ........................ 107.61

= Parentheses indicate spectrophotometric data.

enzyme is 187,000 (see below). Each amino acid is present in relatively large quantities, and the composition does not lend itself to speculation or calculation of the molecular weight or number of subunits. If, a’s suggested below, the enzyme is composed of two subunits of equal composition, the odd num- bered values must be &l.

Analysis of Enzyme for Zinc-Yea,st aldehyde dehydrogenase ha,s been reported to contain zinc (12). The inhibition of the P. aeruginosa aldehyde dehydrogenase by o-phenanthroline suggested that zinc might be present in this enzyme as well, although addition of zinc to the inhibited enzyme after separa- tion of the inhibitor and enzyme by gel filtration did not restore a,ctivity and other chelating agents, such as EDTA, did not inhibit the enzyme (1). Purified aldehyde dehydrogena,se wa,s, therefore, analyzed for zinc with the use of an atomic absorption spectrophotometer. A solution of the enzyme (4.8 mg per ml) which retained at least 65% of the original specific activity after extensive dialysis contained no detectable zinc. From the results obtained with standard solutions it was estimated that 0.1 pg of zinc per ml could have been detected. A solution of yeast a,lcohol dehydrogenase of similar concentration was found to contain more than 5 pg of zinc per ml, in agreement with the results of Vallee and Hoch (4). It was concluded that the aldehyde dehydrogenase of P. aeruginosa does not contain this metal ion.

Molecular Weight-The results of analytical ultracentrifuga- tion had previously led to the report of a sedimentation coeffi-

cient, SZO,~, of 9.0 S for active aldehyde dehydrogenase (1). Subsequent experiments under similar conditions gave values of 9.2 S and 9.4 S. Fig. 1 shows the sedimentation of purified aldehyde dehydrogenase at 59,780 rpm, and in this case a sedimentation coefficient of 9.4 was obtained. The diffusion coefficient was determined by the method of Miiller (5), in which the diffusion of the boundary of a protein in a centrifugal field

over a period of time is measured. The values of

J- tiq1 - SW?) 4Y2

were calculated from these measurements, and Fig. 2 shows their relation to time. A value for the diffusion coefficient, D 20,w, of 4.4 x lop7 cm2 set was obtained from the slope of this line after appropriate corrections for temperature and buffer concentration. In all calculations the partial specific volume (0) was assumed to be 0.73 cm3 per g (13), since calculations made from the amino acid analyses according to the procedure of Cohn and Edsall (14) indicate B = 0.736, without reference to the amino group contributions of glutamine and asparagine or to the uncertainties in the tyrosine and phenylalanine values. From the SZ~,~ (9.4 S) and D20,w (4.4 X 1O-7 cm2 set) a molecular weight of 191,000 was calculated with the use of the Svedberg equation. The use of an s20.W value of 9.0 S yields a molecular weight of 183,000, and the use 9.2 S yields a molecular weight of

FIG. 1. Sedimentation of aldehyde dehydrogenase at pH 7.0. Aldehyde dehydrogenase (3.7 mg per ml) in 100 mM potassium phosphate-10 mM dithiothreitol-1 mM EDTA, pH 7.0, was cen- trifuged at 59,780 rpm at 20”. Sedimentation was from right to left. Pictures were taken at 8, 24, and 40 min after full speed was attained.

I 2 3

SECONDS x IO-’

FIG. 2. Determination of the diffusion coefficient for aldehyde dehydrogenase. Aldehyde dehydrogenase (1.2 mg per ml) in 80 mM potassium phosphate-8 mat dithiothreitol-0.8 mM EDTA, pH 7.0, was centrifuged at 59,780 rpm for 40 min and then at 16,200 rpm for 512 min.

& t?(l - SC.&)

was calculated from the slope of the protein boundary and plotted against time. The diffusion coefficient was obtained from the slope of this line.

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6498 Aldehyde Ilehydrogenuw. II Vol. 233, Iio. 24

4 B 12 I6 20

TUBE NUMBER

FIG. 8. St~crose densit,y gradient centrifugation of aldchydc dehydrogenasc and rcfercnce enzymes. A solution of uldehyde dchydrogenase, catalase (mol wt, 225,000 to 250,000), and yeast alcohol dehydrogcnase (mol wt 151,000), in a volume of 0.1 ml, was applied t,o a 5 to 20’;; sucrose gradient in 100 mM pokassinm phosphate-1 m\t 2-mcrcaptoethanol, pTI i.0. Fractions were collected after centrifttgation at 35,000 rpm for 13 hours at. 5”. Enzymat.io act.ivity was dotermined as dcscrihed under “Expcri- mental Procedure.” O- - -0, uldehydc dehydrogenase; O----O, catalase; APA, yeast alcohol dehgdrogenase.

187,000. Thus, the average molecular weight from scdimmta- tion studies is 18i,OOO + 4,000.

MAecular weight determinations carried out by the method of Archibald (6) indicated values of 168,000 and 207,000; thus, the average molecular weight was 187,000 f 20,000.

The sedimcmation constant of aldehyde dehydrogenase was also estimated from the results of comparative sucrose densky gradient centrifugatiorr studies (Fig. 3). Aldehyde dehydrogen- ase was locat.ed in t.he gradient between Catalan (s = 11.3) and yeast alcohol dehydrogenase (s = 7.6). Calculat,ions according to the method of Martin and .4mes (15) yielded values of s of 9.4 and 8.4 for aldehyde dehydrogenase, which are in agreement with the value, s = 9.0 to 9.4, obtainctd by means of analytical ultracentrifugation.

Molecular weight comparisons were: also made by means of gel filtration on Sephades G-200 according to the procedure of Andrews (16), with catnlase (mol wt 238,000) and yeast alcohol dehydrogenase (mol wt 151,900) again used for reference. The results, in Fig. 4, permit a calculation of relative elution ratios which yield a molecular weight estimate for aldehgde dehydrogenase of 250,000. This great a disparity from the previous values of 183,000 to 207,060 for the molecular weight of aldehyde dehgdrogennse suggests that catalasc, at least., was retarded in its elution from the column--or else that the effects of 100 nm salt on aldchyde dehydrogenase diminish its non- specific affinity for the destran of the column, more so than in the case of catalnse. Indeed, Xndrews (16) has pointed out that catalase behaves as if it had a molecular weight of 195,000,

no more than 207,000 determined for aldchyde dehydrogenase by this method.

isoelectric Point-The evidence provided by the precipitation of enzyme at low hydrogen ion concentrations and movement toward the anode in electrophorcsis (1) indicate t.hat aldehyde dehydrogenase is negatively charged at pH 7.0 or above.

The point of isoelectric precipitation of the enzyme was determined by measuring, quantitatively, the amount of protein precipitated in 0.1 hr potassium phosphate adjusted to the desired pH values with hydrochloric acid. The results, shown in Fig. 5, indicate that this point was pH 4.8. Although aldehyde dehydrogenase is soluble below the poiut of isoelectric precipita- tion, at.trmpts to neutralize an acidified sample led to repre- cipitation of the protein, with loss of all enzgmnt.ic activity. It appears that the enzyme is denatured in the course of ncidifica- tion, and the denatured form is insoluhlr at neutral p11.

Turnover Number /or ;I kkhyde Lkhydrogennse-Because of the present uncertainty regarding the number of active sites on the enzyme, estimation of the turnovrr number is not. practical. llowever, the “molecular activity” can bc calculated from the specific activity of the enzyme and its molecular wclight. The result is 3140 moles per min per mole of enzyme.

Dksokattin and Deactivation of Aldehyde Dehydrogenase

Enzyme preparations dissolved in 0.1 Y potassium phosphate buffer, pH 7.0, in the presence of redming agent. wcrr found to have maximum specific activity compared to prcparstions in buffers without reducing agent. It was further observed t.hat

J

TUBE NUMBER

FIG. 4. Gel filtration of aldehyde dehydrogctntsc nttd reference cnzvmcs. A solrttion of aldehyde dchgdrogemlse, catalase (mol wt 225.000 to 250,000)) and yeast alcohol dehytlrogcmrsc (mol wt 151 ,OOO), in a volume of I ml, was applied to a colnmn of Sephadcx (i-200, 3.0 X 37.5 cm, eqnilibrnted with 100 m\r potassium phos- phate-l mM 2-mercaptocthariol, pT1 7.0. Fr:n:tioil.-; of approxi- mately 1 .!I ml were collected at a flow rate of 0.3 ml per min. Enzyme assays were carried orrt as outlined Itndcr “I~sperimental Procednre.” O- - -0, aldchyde dehytlrotq~nasc; l -- 0, cata-

which would correlate well enough with a molecular weight of Iase; A A, ycaet alcohol dehydrogcnase

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Issue of December 25, 1968 R. G. von Tigerstrom and W. E. Raze11 6499

FIG. 5. Isoelectric precipitation of aldehyde dehydrogenase. See “Experimental Procedllre.” The figure shows the protein precipitated at the different hydrogen ion concentrations.

dialysis against solutions of low salt concentration (1 mti1 EDTA, pH 7.2) resulted in a rapid loss of enzyme activity (deactiva- tion). Such deactivation was accompanied by a change in the sedimentation psttern observed ii1 the ultracentrifuge and in the electrophoretic mobility of the protein upon starch gel electrophoresis (dissociation). Although addition of pot.assium phosphate reversed the dissociation observed when dialysis was performed with either dilute EDTA or Tris, reactivation was obtained only in the former case; reactivation of enzyme reassociated with phosphate after dialysis against Tris required a reducing agent. Thus, reassociated enzyme was not fully reactivated in the absence of reducing agent, whereas reactivated enzyme was always found to be reassociated. The experimental

FIG. 6. Sedimentation of dissoci- ated and reassociated aldehyde de- hydrogcnase in the ultracentrifuge. The enzyme preparations (5.7 mg per ml) were centrifuged at 59,780 rpm at 20”. Sedimentation was from righl to Zefl. Pictures were taken at 16, 40, and 64 min after the rotor attained full speed. The enzyme was dis- sociated by dialysis against 1 mM EDTA, pH 7.2 (rl), and an aliquot of this preparation was reassociated by the addition of potassium phos- phate, pH 7.0, and d’thiothreitol to final concentrations of 80 rnhx and 8 m&f, respectively (n). Both prepara- tions were incubated at 30” for GO min prior to centrifugation.

results of a detailed study of this phenomenon are presented below.

Pattern of Dissociated and Reassociated -Aldehyde Dehydrogenase in UZtracentr$uge-Native aldehyde dehydrogenase with a specific activity of 13.5 shows only one component with an .sz~,~ value of 9.4 (Fig. 1). In contrast, Fig. 6A shows the pattern of a sample of partially dissociated enzyme. Two major components, with sZO.~ values of 7.1 and 5.0, and a minor components with an ~20,~ value of 2.8 were observed. After reassociation (Fig. 6B), one major component with an .sz~.~ value of 9.0 and a minor component, ~20,~ of 5.5, were present. The specific activities of the preparations were 4 for the dis- sociated enzyme and 10.2 for the reassociated enzyme. As noted in the legend to Fig. 6, the salt concentration in A was much lower than that in B during the centrifugation itself.

Electrophoretic Mobility of Dissociated and Reassociated Aldehyde Dehydrogenase-‘l’he purified aldehyde dehydrogenase in the active state shows one major protein zone in starch gel electropho- resis. After dissociation by dialysis against 1 m&f EDT& pH 7.2, a second zone with faster electrophoret’ic mobility was observed (Fig. 7). Reassociation, by addition of potassium phosphate and incubation at elevated temperatures, reversed this change, and only one major zone with the mobility of the unt.reated enzyme was observed, whereas reducing agents had no effect on the electrophoretic mobility of either the fast or the slow zone (Fig. 8).

Deactivated State of Dissociated Enzyme in Protein Zones

Isolated by Starch Gel Electrophoresis-Staining of the active aldehyde dehydrogenase for enzymatic activity after st,arch gel electrophoresis (1) revealed one zone, whereas two zones were observed when the enzyme was applied in the dissocia,ted state. However, since the assay conditions would be espected to permit reassociation and reactivation, other methods were required to

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6500 Aldehyde Dehydrogenase. II Vol. 243, No. 24

FIG. 7 (upper). Starch gel electrophoresis of associated and dissociated aldehyde dehydrogenase. A, aldehyde dehydrogen- ase, dissolved in 100 mM potassium phosphate-10 mM dithio- threitol-1 mlVr EDTA, pH 7.0, was applied to the starch gel. B, aldehyde dehydrogenase was dissociated by dialysis for 48 hours at 0” against 1 mM EDTA, pH 7.2. The enzyme solution was made to 80 mM potassium phosphate-8 rnM dithiothreitol-0.8 rnM EDTA, pH 7.0, immediately before electrophoresis. Electro- phoresis at room temperature and pH 8.5 was carried out as out- lined under “Experimental Procedure” with 50 fig of protein per gel slice. The anode is at the bottom of the figure.

FIG. 8 (lower). Starch gel electrophoresis of dissociated and reassociated aldehyde dehydrogenase. Electrophoresis was carried out at pH 8.5 for 33 hours at 4”. Aldehyde dehydrogenase (5.0 mg per ml) was dissociated by dialysis for 48 hours at 0” against 1 mM EDTA, pH 7.2. Prior to electrophoresis the follow- ing additions were made to the enzyme preparation and the preparations were incubated for 30 min at the temperatures indicated: 1, S, and 6, no additions, incubated at 30”; 2, plus potassium phosphate, pH 7.0, to 90 mM, incubated at 0”; 4, plus potassium phosphate, pH 7.0, to 90 mM, incubated at 30”; 6, plus dithiothreitol to 9 mM, incubated at 30”. In each case, about 40 Mg of protein were applied to each gel slice. The anode is at the bottom of the figure.

show whether both zones had enzymatic activity under cbndi- t,ions which do not readily reassociate the enzyme. A starch gel

prepared by electrophoresis of dissociated aldehyde dehy- drogenase, as in Fig. 7B, was cut into segments perpendicular to the direction of movement of the protein. The segments were eluted with dilute EDTA in the cold and assayed for enzymatic activity, before and after reactivation. The results, shown in Fig. 9, indicate that the slow zone (associated enzyme) is always maximally active whereas the fast zone (dissociated enzyme) is completely inactive unless reactivated.

Conditions for Dissociation and Deactivation-A loss of enzy- matic activity and the appearance of the fast zone on starch gel electrophoresis (deactivation and dissociation) occurred when the enzyme was dialyzed against 1 InM EDTA at pH 7.2, 8.0, and 8.5, or against 1 mM Tris-chloride, pH 7.2. Dialysis against 10 mM EDTA, pH 7.2, however, produced only in- significant changes: that is, there was no dissociation and little deactivation. Also, treatment of the enzyme in 10 mM EDTA with o-phenantbroline at a concentration which completely inhibited aldehyde dehydrogenase (1.5 InM) did not give rise to a faster zone on electrophoresis. These results indicated that the dissociation and deactivation was not due to the chelating action of EDTA, buf rather to the exposure of the enzyme to low salt concentrations. Comparison of the de- activation of 10 mg per ml and 0.1 mg per ml solutions of aldehyde dehydrogenase showed that this process is relatively independent of protein concentration.

Conditions for Reassociation-When aldehyde dehydrogenase was dissociated by dialysis against 1 mM Tris-chloride, pH 7.2, addition of potassium phosphate buffer alone caused a reassocia- tion of the fast zone observed upon electrophoresis to yield the slower one (as in Fig. 8). Similarly, when enzyme was dissoci-

l-

l-

I- A .- - - 07 2 4 6 6

SLICE NUMBER

FIG. 9. Enzymatic activity of isolated dissociated and associ- ated aldehyde dehydrogenase. Aldehyde dehydrogenase (5.8 mg per ml) was dissociated by dialysis against 1 mM EDTA, pH 8.0, and 100 rg were subjected to starch gel electrophoresis at pH 8.5 for 2 hours at 20-22”. The starch gel was then cut into segments and these were eluted with 1 mM EDTA in the cold. Enzymatic activity was determined before and after incubation of the eluted fractions at 30” for 40 min in 80 mM potassium phosphate, pH 8.0, and 8 mM dithiothreitol. ---, enzyme activity eluted with EDTA at 0”; -, enzyme activity after incubation at 30” with 80 mM potassium phosphate-8 mM dithiothreitol.

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Issue of December 25, 1968 R. G. von Tigerstrom and W. E. Raze11 6501

ated by dialysis against 1 mM EDTA, addition of phosphate to 0.1 M caused the enzyme to reassociate. The reassociation did not proceed at 0”, but occurred over a period of time at 30”. In additional experiments of the same kind, reassociation was brought about by incubation after addition of sodium phosphate, sodium chloride, potassium chloride, and lithium chloride at neutral pH and 0.1 AI concentration. Therefore, reassociation is brought about by incubation at increased salt concentrations and is not related to the presence of the activating ion.

Conditions for Reaclio&m--After dialysis against 1 mM EDTA, pH 7.2, the enzyme could be reactivated to a considerable degree with phosphate alone, as shown in Table II. Addition of dithiothreitol in the presence of phosphate further increased the degree of reactivation. However, dialysis against Tris under the same conditions yielded dissociated enzyme which could not be reactivated in the presence of phosphate alone, the addition of dithiothreitol being essential to the reactivation process (Table II). However, no increase in activity was observed after incubation with dithiothreitol or EDTA alone. The reactivation process was relatively independent of the protein concentration, and was essentially complete after 20 min, with a further slight increase up to 60 min. As expected, the process of reactivation, like reassociation, did not proceed rapidly at 0”.

Thus, reactivated enzyme was always found to be reassociated, but reassociated enzyme was more or less dependent on reducing agent for reactivation. It appears that EDTA plays a special role during the dissociation-reassociation process, perhaps protecting the catalytically active sites of the enzyme from an inhibition which is reversible by dithiothreitol. Since EDTA was ineffective in reversing the deactivation of the enzyne resulting from dialysis against Tris, dithiothreitol is specifically required to restore the activity lost in the absence of EDTA. A similar phenomenon was observed, to a lesser extent, under conditions where dissociation does not take place: dialysis of the enzyme against 1 mu Tris chloride-100 mu potassium phosphate, pH 7.0, resulted in a decrease of specific activity to 11% of the original and the activity was increased to 7Oc/, of the original by addition of dithiothreitol to 10 rnM concentration. Dialysis against 1 mM EDTA-100 mM potassium phosphate, pH 7.0, decreased the activity only to 38% of the original, and addition of dithiothrcitol restored 84% of the original activity. Since dissociation had not occurred, the loss of activity on dialysis was entirely due to lack of reducing agent, and EDTA could partially replace the reducing agent.

Size of Dissociated Enzyme-The observed dissociation of the enzyme may result from the liberation of two subunits. The 9 S component of the active enzyme and the 7.1 S component of the dissociated preparation (Fig. 6) are thought to be identical, since the low salt concentration at which the dissociated prcpara- tion was centrifuged may cause partial unfolding of the molecule which would result in a lower sedimentation coefficient. This component corresponds to the slow zone in starch gel elec- trophoresis. Similarly, the 5.5 S component in the reactivated and the 5.0 S component in the dissociated preparation are thought to be identical and to correspond to the fast zone in electrophoresis. When attempts were made to separate the two components by gel filtration and sucrose gradient centrifuga- tion, good separation was not obtained. On Sephadex G-200, when the dissociated preparation was applied, the front of the protein profile was more active than the tail end before reactiva-

TABLE II

Reactivation oj aldehyde dehydrogenase

Aldehyde dehydrogenaae (5.0 mg per ml; specific activity, 13.5) was dialyzed for 48 hours at 0” against 1 mM EDTA, pH 7.2 (Ex- periment A) or 1 mM Tris-chloride, pH 7.2 (Experiment B). For reactivation studies 10 ~1 of dialyzed enzyme were diluted into 60 volumes of 1 mM EDTA, pH 7.2, containing the additions listed. The final potassium phosphate concentration was 80 mM, and that of dithiothreitol, 8 mM. The preparations were assayed at the time of dilution and after 60 min at 30”. Assays were carried out as usual, except that the enzyme was added to the reaction mix- ture approximately 5 set before the aldehyde. Rates obtained after 1 min were recorded.

Additions

Specific activity

At 0 min After 60 min at 300

-_- j

pmolcs/min/mR protrin

Experiment A. Reactivation after dis- sociation by dialysis against 1 mM EDTA

None.... ..__..... .._... .._. 0.6 0.4 Potassium phosphate.. 0.6 4.8 Dithiothreitol. . 0.8 0.7 Potassium phosphate + dithiothreitol 1.2 i 9.7

Experiment B. Reactivation after dis- sociation by dialvsis against 1 mM Tris

None.... .__._..._...... 0.5 0.3 Potassium phosphate. . 0.4 0.5 Dithiothreitol.. 0.6 0.5 Potassium phosphate + dithiothreitol., 0.f 3.7

tion, and the protein in the tail end could be reactivated to a greater extent than the protein in the front. This result is consistent with the conclusion that the active molecule is larger than the disvociated molecule. Activities before and after re- activation of dissociated aldehyde dehydrogenase, recovered from sucrose gradient centrifugation, are shown in Fig. 10. These results indicate, as expected, that the dissociated molecule has a smaller sedimentation constant than the active molecule.

Influence ojO.!her Reagents-Attempts were made to deactivate or dissociate aldehyde dehydrogenase by the addition of urea, sodium lauryl sulfate, and para-hydroxymercuribenzoate, re- agents which have been found to cause dissociation of other en- zymes. The enzyme was precipitated and denatured at approxi- mately 2 M urea. Neither enzymatic activity nor electrophoretic mobility was changed at lower concentrations of urea. Sodium lauryl sulfate and para-hydroxymercuribenzoate also denatured the enzyme at 2 mu and 0.2 mu concentrations, respectively. Little or no change of the electrophoretic mobility was observed at lower concentrations.

Physical Properties of Aldehyde Dehydrogenme below Isoelectric Point-Whereas the active molecule at pH 7.0 has an s value of 9.0 to 9.4, aldehyde dehydrogenase in 5.0 mu KCl, pH 3.0, shows a homogeneous peak in the ultracentrifuge with an SZ~,~ value of 2.0 S (Fig. 11). However, gel filtration through Sephadex G-200 in 5.0 mu KCl, pH 3.0, did not indicate that the enzyme has a smaller molecular size than the active enzyme at pH 7.0, since all the protein applied to the column emerged in the effluent at the point where the active enzyme would be expected. Experi- ments in addition to those shown in Fig. 11 permitted further

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Aldehyde Dehydrogenase. II Vol. 243, So. 24

I I I I

5 IO 15 20

TUBE NUMBER

FIG. 10. Sucrose gradient centrifugation of dissociated nldehyde dehydrogenase, Aldehyde dehydrogenase was dissoci- ated by dialysis against 1 my EDTA, pI1 7.2, and 0.8 mg (0.3 ml) was applied to a 5% to 207, sucrose gradient prepared in 1 ml% EDTA, pII 7.2. Fractions were collected after centrifugation at 35,000 rpm for 13 honrs at 5” and assayed before and after reactivation by the addition of potassium phosphate, pI1 7.0, to 80 ml1 and dithiothreitol to 8 mar and incubation at 30” for GO min. O--O, enzyme activity before reactivation; O- - -0, enzyme activity after reactivation.

FIG. 11. Sedimentation of aldehyde dehydrogenase at pI1 3.0. Aldehyde dehydrogenase (5.0 mg per ml) in 5 mM KCI, pH 3.0, was centrifllged at 59,780 rpm at 6”. Sedimentation was from right to left. Pictures were taken at 16, 48, and 80 min after full speed was obtained.

calculation of the molecular weight of the acidified enzyme from the sedimentation coefficient and the diffusion coefficient (DZO,W = 2.72 x 10W7 cm2 set), which was estimated by the method of Mijller (5) ; these indicated a molecular weight approximately half that obtained for aldehyde dehydrogenase at pH 7.0 (95,000). These results could be accounted for if the shape of the enzyme subunit molecule at pH 3.0 differed from the assumed globular shape at pR 7.0, and the much lower diffusion constant in the former condition supports this concept.

To examine this situation further, the viscosity of the enzyme was determined in a capillary viscometer tube at 5”. The en- zyme was dissolved in three different solvent systems at a concen- tration of approximately 5 mg of protein per ml. Each measure- ment was done in duplicate. Aldehyde dehydrogenase in 0.1 M

potassium phosphate-l.0 rnnf dithiothreitol-1.0 mM EDTA, pH 7.0, had a specific viscosity (osr,) of 0.0204; in 1 rnAI EDTA at the

same pH of 7.0, qs,, was 0.0268; whereas in 5.0 mar KC1 at pH 3.0, vsr, was 0.0878. This large increase in viscosity when the enzyme was dissolved at pH 3.0 indicates an unfolding as well as a dissociation of the molecule, which is consistent with the low sediment,ation coefficient and the lower diffusion coefficient.

Solubili@-Aldchyde dehydrogenase is soluble at moderately high salt concentrations above its isoelectric point. At a con- centration of 2 mg per ml, the enzyme was found to be insoluble above 35 70 saturation with ammonium sulfate, a,s well as in very dilute salt solutions (0.05 rnhf EDTA). Almost complete re- covery of enzyme activity was possible after precipit,ation at high salt concent’ration; however, only 25% of the activity was recovered after precipitation by dialysis against a solul;ion of low salt concentration.

DISCUSSION

The general physical characteristics of aldehyde dehydro- genase from P. aeruginosa are those of a typical globular protein, it being insoluble at low and h&h salt concentrations. It is negatively charged at neutral pH, soluble in moderately high salt concentrations, and soluble below the isoelectric point.

Although specific tests for carbohydrate in the purified alde- hyde dehydrogenase have not been carried out, the relation of absorbance to dry weight reported previously (l), the results of the amino acid analysis and t’he nitrogen content of the prepara- tion indicate that the enzyme is not associated with nonprotein material.

Studies on the potassium-activated yeast aldehyde dehydro- genase and the liver aldehyde dehydrogenase with o-phenanthro- line and other chelating agents suggested the involvement of zinc in both enzymes; analysis of the yeast enzyme preparation showed significant amounts of zinc, although qusntitative data mere not presented (12). The inhibition of enzymatic activity by o-phenanthroline is often indicative of the presence of zinc. However, although o-phenanthroline causes inhibition and dis- sociation of glutamic dehydrogenase, nonchelating analogues of o-phenanthroline also inhibit the enzyme and, therefore, inhibi- tion by o-phenanthroline does not necessarily mean that zinc is a constituent of the enzyme (17). In contrast to the potassium- activated aldehyde dehydrogenase of yeast’, analysis of the po- t’assium-activated aldehyde dehydrogenase of P. aeruginosn showed that the enzyme does not cont.ain zinc.

In relatively high salt concentrations (0.1 RI) our aldehyde de- hydrogenase shows one component in the ultracentrifuge and one major zone upon starch gel elect.rophoresis. At low salt concentrations, however, a second component with lower s value is observed in the ultracentrifuge and a second zone, with faster electrophoretic mobilit,y, appears upon electrophoresis. A change of the s value alone could have resulted either from dis- sociation of t’he enzyme or from an unfolding of t.he molecule. The appearance of a zone in electrophoresis with faster mobility was an indication that a smaller molecule was obtained. Starch gel is known to act as a molecular sieve (B), and a smaller mole- cule with the same charge as a larger one is expected to have a faster mobility. An unfolded protein, on the other hand, would be retarded unless the mifolding were outweighed by an increase in charge. The results we obtained therefore indicate a partial dissociation of the active aldehyde dehydrogenase, probably into two subunits of similar or equal size. The dissocia,tion is re- versed by addition of salt to the protein solut’ion, followed by in- cubation at elevated temperature.

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R. G. uon Tigemtrwn and W. E. Raze11 GO3

Dissociation of proteins is favored by inorganic salts if most newly exposed groups are hydrophilic:, but would be prevented by inorganic salts if most nwly exposrd groups ncre hydrophobic (19). &wrdiugly, upou dissociat.iou in low salt concentrations, aldehyde dehydrogcntie would be espected to expose hydro- phobic groups responsible for the original binding of the .-ubuuits. The binding of subunits of aldchydc dchydrogrnwe by hydro- phobic interaction would bc in agreemeut with the teml)erature dq)endcnce of the reassociation (20).

Although t.here was some difficulty in assessing the specific ac- tivily of a dissociated cuzynie prcpuratioii (since staudnrd assay conditions permit. reassociation), it could be shown that enzyme activity is lost upon dissociat.ion and that. it is regained after re- association. Furthermore, it could be shown that the subunit, isolated after starch gel t,lt~ctrol)lloresis, has uo enzymatic ac- tivity unlrss reassociated. The enzymatic activity of aldehyde

dchydrogcnnse is therefore dependent ou the union of at least two smaller molcculrs to form the active enzyme.

The 10~ sedimentation coefficieut which was observed when aldehyde dehydrogenase was dissolved at pH 3.0 was accom- panied by a wry high viscosity and a relatively low diffusion coefficient as compared to the values obtained for the enzyme at pII 7.0. This indicated an extensive unfolding of the molecule. A molecular weight approximately one-half that of the active molecule was estimated for this material, and therefore it may represent the unfolded subuuitti which were observed upon dis- sociation in low ionic strength buffer.

Apart from the dissociated subunit form of the native euzyme, WC have observed only one active species of aldehyde dehydro- genase from P. aeruginosa. Given the high yields at each step of t.ha purification, there is little reason to believe that multiple forms of the native enzyme occur, in contrast to the situation resulting from the purification of t.he enzyme from yeast (21). In the latter, three molecular species of enzyme apparently arise as a result. of nut&is, the original enzyme having a molecular w-eight of 200,000. Ko studies have been reported on the possi- ble dissociation of the primary form of t.hc yeast enzyme, or in- deed of any other aldehyde dehgdrogenase.

According to the amino acid analysis, there are 22 potential sulfhydryl groups per enzyme molecule. In experiments further

to t.hose reported here treatment of the enzyme with “C-iodo- a&amide, even in a 4-fold excess of dithiothreitol, led to 877; inhibition of enzyme activity when 2.7 moles of inhibitor were taken up per mole of enzyme. Thus, only a fen of the cysteine

residues appear to be involved in the act.ivc site. Such inhibit.inn

studies suggest that there are relatively few sulfhydryl groups re- acting under the conditions tested and, therefore, that most cys- teine moieties arc involved in disulfide bond formation. The great, stability of the enzyme in bisulfitc buffer even at elevated temperatures (I), however, strougly suggests that there are no interchain and possibly fen- or no intrachain disulfide bonds (22) in the active molecule. The requirement of reducing agents for act,ivity and the loss of enzymat.ic activity on dialysis without reducing agents, which would lead to disulfide bond formation, are in accord with this.

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5019 (1967). 22. CECIL, R., ~SD WOKE, R. G., Biochem. J., 82, 401 (1962).

vos ‘I’IGEI~S~IXOLI, Ii. G., AND RA%zELL, W. El., J. Ziiol. Chem., 243, 2691 (1968).

J.monr, W. B., in P. I). BOYER, Il. L.\RDY, .IND K. MYI{- GCK (Editors), The enzynies, I’&. 7, Academic Press, New York, 1983, p. 203.

CHGCE, B., .\NI) M.\EHI.Y, A. C., ir~ S. 1’. COLOWICK AND S. 0. K.\PL.IN (b;ditors), Methods in enzymology, Vol. ZZ, Aca- demic Press, New York, 1955, p. iG4.

VALLEE, B. L., ASD Ilocri, F. I,., Proc. ,Ynl. Acad. Sci. Cr. S. A., 41, 327 (1955).

M~~I.I.E;K, W. .J., Proc. :Ynl. :lcad. Sci. li. S. A., 61, 501 (1964). SCIIACIIYAN, II. J., iu S. I’. COLOWICK I\~~) S. 0. KMLAN

(I’:tlitors); Melhods i,n enzymology, Vol. IV, Academic Press, Sew York, 1957, p. 32.

LOWRY, 0. II., ROSEDRUUGII, S. J., F.\IM, A. I,., .\SD RAND.\LL, R. J., J. Hiol. Chena., 193, 2G5 (1951).

RIOORE, S., .\ND STEIx, w. H., iII S. P. COLO\vIcK :wD N. 0. K.IIX.\N (Kditors), Methods in enzymology, Vol. VI, Acu- demic Press, New York, 1903, p. 819.

~IOORE, S., .I. Biol. Chem., 238, 235 (1963). &IIs,~~I, O., .ISI) ZII.VEIZSYI~~, I>. B., .4nal. Biochem., 6, 320

(1963). BE.\VES, G. IT., .\sD HOLIDAY, E. K., Advance. Prolein Chem.,

7, 319 (1952). STOPP~SI, A. 0. hi., SCII\V~\RCZ, %I. S., M.-I) FREDA. C. E..

ilrclr. Hiochem. Rioph,ys., 113, &X (1966). SMITH. 11. 11.. Biochem. J.. 89. 4511 (19631. COHN,‘E. J., IND EDMLL,’ J. ‘T.,‘P;otei&, amino acids and

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M.\RTIS, Ii. G., .\sI) AMES, B. ?I’., J. Viol. Chem., 236, 1372 (1961).

ASDREW, P., Hiochem. J., 96, 595 (1965). YIELDISG, K. L., AND TOMKISS, G. M., Biochem. Riophys.

Acta, 62, 327 (1962).

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Richard G. von Tigerstrom and W. E. RazzellOF THE ENZYME FROM PSEUDOMONAS AERUGINOSA

Aldehyde Dehydrogenase: II. PHYSICAL AND MOLECULAR PROPERTIES

1968, 243:6495-6503.J. Biol. Chem. 

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