aldehyde dehydrogenase - jbc. · pdf file2692 aldehyde dehydrogenase. i vol. 243, no. 10...

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 243, No. 10, Issue of May 25, PP. 2691-2702,1968

Printed in U.S.A.

Aldehyde Dehydrogenase

I. PURIFICATION AND PROPERTIES OF THE ENZYME FROM PSEUDOMONAX AERUGINOSA*

(Received for publication, August 21,1967)

RICHARD G. VON TIGERSTROM$ AND W. E. RAZZELL

From the Department of Microbiology, University of Alberta, Edmonton, Canada

SUMMARY

1. An aldehyde dehydrogenase was found in cell extracts of Pseudomonas aeruginosa ATCC 9027 grown on several carbon sources. It was present in highest concentration in cell extracts after growth of the organism on ethylene glycol or ethanol.

2. The enzyme from ethanol-grown cells was purified approximately ZO-fold. A 23% recovery of enzyme was obtained and the purified preparation appeared to be at least 95% homogeneous as evidenced by gel filtration, ultracentrifugation, and electrophoresis.

3. Instability of the enzyme was overcome by using sodium bisulfite in the buffer systems, and the stability of the enzyme under various conditions of temperature, pH, and salt concentration was studied.

4. The aldehyde dehydrogenase requires potassium or ammonium ions for activity. Some activity is also obtained in the presence of rubidium ions. Nicotinamide adenine dinucleotide is a better hydrogen acceptor than nicotinamide adenine dinucleotide phosphate and a wide variety of aldehydes are oxidized. The Michaelis constants for glycolaldehyde, glyceraldehyde, nicotinamide adenine dinucleotide, nicotinamide adenine dinucleotide phosphate, potassium, and ammonium were determined.

5. A mercaptan is required for maximum activity. This requirement can be partly replaced by chelating agents.

6. The enzyme is protected effectively from iodoacetate inhibition, heat inactivation, and trypsin digestion when potassium and nicotinamide adenine dinucleotide are present.

7. A potassium-activated aldehyde dehydrogenase was also found in cell extracts of other strains of the genus Pseudomonas.

* This research was supported by Grant A-2975 from the Na- tional Research Council of Canada. Presented in part at the 51st Meeting of the Federation of American Societies for Experi- mental Biology, Chicago, 1967 (Fed. Proc., 26, 2620). The work reported here is taken from a thesis submitted by Mr. R. G. von Tigerstrom in partial fulfillment of the requirements for the degree of Doctor of Philosophy, University of British Columbia, Vancouver, Canada.

$ Present address, Department of Biochemistry, University of British Columbia, Vancouver 8, Canada.

A great number of aldehyde dehydrogenase activities from diverse tissues and microorganisms have been reported and the subject has been reviewed by Jakoby (1). One of the notable properties of the aldehyde dehydrogenases so far described is their instability. This has prevented extensive purification and study of these proteins on the molecular level, although several catalytic properties have been investigated with the use of partially purified enzyme preparations.

Of special interest in the context of the present report are the potassium-activated yeast enzyme (2, 3) and the phosphate- or arsenate-requiring enzyme from Pseudomonas jtuorescens (4). Beports on the phosphate-requiring enzyme and the yeast enzyme (5) also implicated a special role of the thiol groups on the proteins in reactions catalyzed by them. Protection by monovalent cations and coenzymes from the inhibition with thiol reagents of the yeast aldehyde dehydrogenase was reported by Stoppani and Milstein (6) ; while Sorger and Evans (7) studied the reactivation by monovalent cations of this enzyme after dialysis and suggested that these cations may have an effect on the physical arrangement of the protein. The purification of the yeast enzyme, originally reported by Black (2), was modified by Stoppani and Milstein (6), but a stable preparation was not obtained. A more recent report (8) which implicates the involvement of Zn++ in the active protein also reports some progress in the further purification of the yeast enzyme.’

The genus Pseudomonas is the source of several different aldehyde dehydrogenases. In addition to the phosphate-requiring enzyme, which is active on a wide variety of aldehydes, several enzymes with a high specificity for semialdehydes have been reported (1). An enzyme, specific for aliphatic aldehydes and requiring Fe++ or Ca*, and flavin for activity, was reported from Pseudomonas aeruginosa (10).

The present publication deals with a potassium-activated aldehyde dehydrogenase from P. aerugkosa which has not been described previously. The enzyme has been shown to be paraconstitutive in this organism, obtainable in quantity, and was brought to a high degree of purity in the stable state. Studies of the requirements for maximum catalytic activity,

1 During revision of our manuscript, the publication of the purification and crystallization of aldehyde dehydrogenase from yeast was reported by Steinman and Jakoby (9).

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2692 Aldehyde Dehydrogenase. I Vol. 243, No. 10

stability, and inhibition of this enzyme as well as some physical properties are given, while the molecular properties of the protein will be reported subsequently.

EXPERIMENTAL PROCEDURE

~Vaterials-The following materials were obtained from commercial sources: NXDPt from P-L Biochemicals; NAD+, NADH, dithiothreitol, cysteine hydrochloride, iodoacetate, iodoacetamide, Cellex-T, and Bio-Gel P-60 from Calbiochem; glycolaldehyde, 2,3-dimercaptopropanol, and glyceric acid from Sigma; 2-mercaptoethanol, acetaldehyde, butyraldehyde, isobutyraldehyde, and propionaldehyde from Eastman-Kodak; sodium metabisulfite, benzaldehyde, glycolic acid, and o- phenanthroline from Fisher; protamine sulfate from Lilly; nn-glyceraldehyde from Mann; Sephadex G-200 from Phar- macia; trypsin from Worthington; starch (hydrolyzed) for electrophoresis from Connaught Laboratories, Toronto, Canada.

Organisms-P. aeruginosa ATCC 9027 and P. Jluorescens A 312 were obtained from the Department of Microbiology, University of British Columbia. P. fluorescens 36’, 22’, and 266 and Pseudomonas ovalis were obtained from the Department of hlicrobiology, University of Alberta.

Growth of Organism for Enzyme Production--P. aeruginosa was grown in Roux flasks containing 100 ml of medium con- sisting of NHdHzPOd, 0.3%; K2HP04, 0.4%; FeS04.5H20, 5 ppm; yeast extract, 0.1%; and tryptone, 0.1%; all adjusted to pH 7.4 before sterilization. Separate sterile solutions of MgS04. 7Hz0 and 95% ethanol were prepared and added at the time of inoculation to yield concentrations of 0.05 and 0.3 ‘%, respectively. A 1% inoculum was added and the culture was incubated for 24 hours at 30”, with a further addition of ethanol equal to 0.4% of the medium after 10 hours of growth. Cell yields of 13 to 14 g, wet weight were obtained per liter of medium.

Enzyme Assay-The enzyme reaction was followed by measur- ing the rate of appearance of NADH at 340 rnp in a l&cm light path at 35” with a Gilford automatic spectrophotometer model 2000. Unless otherwise specified, the incubation mixture contained the following constituents in a final volume of 1 ml: potassium phosphate buffer, pH 7.2, 100 mM; 2-mercaptoethanol, 10 InM; NAD+, 2 InM; an appropriate amount of enzyme; and glycolaldehyde, 1 mM. The reaction was started by addition of the aldehyde after prior incubation for 5 min at 35” of all other components. Dilutions of the enzyme were made with 100 mM potassium phosphate buffer-lo.0 mM dithiothreitol-1.0 mM EDTA, pH 7.0. A measurable reaction is obtained with approximately 1.0 pg of purified aldehyde dehydrogenase. One unit of enzyme activity is defined as the amount catalyzing the formation of 1 pmole of NADH per min and specific activity as units per mg of protein.

dIethods-Protein concentrations were determined by the method of Lowry et al. (11). In purified enzyme preparations, protein was also measured by absorption at 280 mp. The dry weight of protein in an enzyme solution was determined after extensive dialysis against 1.0 mM pota,ssium phosphate buffer, pH 7.0, by drying l.O-ml samples to constant weight at 80” and 90” and correcting for the weight of buffer.

Starch gel electrophoresis (12, 13) was performed at 20-22’ on gel slices 3 cm wide, 20 cm long, and 0.3 cm thick. A current of 2.0 ma per slice was applied for 2 hours. The starch blocks were prepared with 12.5% starch in 30 mM Tris-borate buffer, pH 8.5, 8.0, and 7.6, or in 10 mM potassium phosphate buffer,

pH 7.0. The electrode compartments contained the same buff- ers, respectively, at 10 times the above concentration. Protein was detected with a 0.2% Amido black solution in methanol- water-acetic acid (5 : 5: 1). Polyacrylamide disc electrophoresis and staining of the gels was performed according to the method of Davis (14). Electrophoresis was performed at 2(r22” with a current of 2 ma per gel tube for 110 min. A modification of the method of Fine and Costello (15) was used to detect aldehyde dehydrogenase activity in starch or acrylamide gels. The gels were incubated for 3 min at 2&22” in a reaction mixture containing potassium phosphate, pH 7.2, 100 mM; NAD+, 1.0 InM;

and 2-mercaptoethanol, 5 mM. To initiate the reaction, 0.1 ml of 20 mM glycolaldehyde, 0.1 ml of p-nitroblue tetrazolium (10 mg per ml), and 0.02 ml of phenazine methosulfate (5 mg per ml) were added per 10 ml of reaction mixture. Formation of a dark band indicat.ed a region of aldehyde dehydrogenase activity.

For isolation of the products of enzyme reaction, glycolaldehyde and glyceraldehyde (100 pmoles) were permitted to react in lo-ml assay mixtures modified to contain excess purified enzyme and 50 mM potassium phosphate. For the reaction with glycolaldehyde, four subsequent additions of NAD+ and six subsequent additions of aldehyde were made. For the reaction with glyceraldehyde, three subsequent additions of NAD+ were made when the rate of oxidation had decreased. The reaction mixtures were reduced in volume by lyophilization and desalted. The products were identified by comparison with known compounds with the use of descending chromatography on Whatman No. 40 paper. The solvent systems butanol- acetic acid-water (12 : 3 : 5) and ethyl acetate-pyridine-water (12:5:4) were employed. The compounds were detected by spraying with bromcresol green reagent or dipping in silver nitrate reagent (16).

RESULTS

Induction of Aldehyde Dehydrogenase

The specific activity of aldehyde dehydrogenase in cell extracts prepared from P. aeruginosa grown on five different substrates is shown in Table I. When ethanol was used as the carbon source the specific activity of the enzyme was 65 times greater than when glucose was used. Ethanol was therefore selected as the substrate for growth of this organism in mass culture to obtain a high yield of aldehyde dehydrogenase for purification.

Purijkation of Aldehyde Dehydrogenase

All operations were performed at O-3” unless otherwise stated. Step 1: Preparation of Cell E&act-Cells from 6 liters of culture

medium were harvested by centrifugation at 13,200 x g, washed once with 50 mM potassium phosphate-10 mM mercaptoethanol, pH 7.0, and suspended in 336 ml of 100 mM sodium bisulfite-10 InM mercaptoethanol, pH 7.0. The cells were disrupted by sonic treatment of 50-ml quantities of the suspension for 7 min, with the use of a Bronwill Biosonik at maximum output. The temperature of the suspension during this operation was kept below 10”. The sonic extract was diluted to 560 ml and the final buffer concentration was adjusted to 50 mM potassium phosphate-100 mM sodium bisulfite-10 mM mercaptoethanol, pH 7.0. After centrifugation at 31,700 x g for 60 min, 527 ml of cell extract were obtained.

Step 2: Protamine Sulfate Treatment-Protamine sulfate solu-


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Issue of May 25, 1968 R. G. von Tigerstrom am-l W. E. Raze11 2693

TABLE I Aldehyde dehydrogenase activity in cell extracts of P. aeruginosa

The organism was grown in the medium described under “Ex- perimental Procedure” for 48 hours at 25”. The carbon sources, with the exception of acetate, were added at 0.2% concentration at the time of inoculation and again after 24 hours. Potassium acetate, pH 4.0, was added at 0.1% concentration at the time of inoculation and a further O.l5oj, was added at 15 and 30 hours. Inoculum from a culture grown on the respective carbon source was used. The cells were harvested, washed once in 50 mM potassium phosphate containing 10 mM 2-mercaptoethanol, pH 7.0, and suspended in the same buffer. Cell extracts were prepared by sonic treatment and centrifugation at 31,700 X g for 45 min. Aldehyde dehydrogenase activity was determined as described under “Experimental Procedure.”

Specific activity

&m&x/min/ng protein

Glucose................................... 0.012 Acetate................................... 0.051 Glycerol.................................. 0.071 Ethylene glycola. . 0.364 Ethanol”. 0.780

0 Less than 10% of the activity was obtained in the absence of K+ as found by comparing activity in a Tris-chloride-buffered system with and without KCI.

tion (20 mg per ml, pH 5.0) to a concentration of 20% by volume

was added with stirring to 527 ml of cell extract. Stirring was continued for 20 min and the precipitate was sedimented by centrifugation at 31,700 X g for 30 min. The volume of the supernatant solution was 608 ml.

Step S: Ammonium Sulfate Fractionation-Solid ammonium sulfate, 127 g, was added, with stirring, to 6008 ml of the solution obtained in Step 2. Stirring was continued for an additional 30 min and the precipitate was removed by centrifugation at 37,700 X g for 30 min. The volume of thesupernatant solution was 655 ml. To this a further 61.5 g of ammonium sulfate were added as above. After centrifugation the precipitate, which contained the enzyme activity, was dissolved in 5 mM potassium phosphate-10 mM sodium bisultite-10 mM mercaptoethanol-0.5% ethylene glycol, pH 7.0, to give a total volume of 100 ml.

Step 4: Acetone Fractionation-This operation was performed in a salt-ice bath at - 15”. During the addition of acetone the temperature of the preparation was kept below 3”. Then 60 ml of acetone, chilled to - 15”, were added with stirring to 100 ml of the ammonium sulfate fraction. The precipitate was removed by centrifugation at 23,300 X g for 10 min at -5” and set aside (I). This procedure was repeated on the supernatant fraction with the addition of 25 ml (II) and then 40 ml of cold acetone (III). Precipitates I, II, and III, thus obtained, were each suspended in 100 mM potassium phosphate-100 mM sodium bisulfite-10 InM mercaptoethanol-1.0% ethylene glycol, pH 7.0, to a final volume of 100 ml. Fraction III alone usually contained 73% of the enzyme activity. To this fraction 43 g of solid ammonium sulfate were added and, after being stirred for 2 hours, the mixture was centrifuged at 27,000 x g for 20 min. The precipitate was recovered, suspended in 50 ml of 100 mM potassium phosphate-10 mM mercaptoethanold mM EDTA, pH 7.0, and the suspension was centrifuged as before to remove any de- natured protein. The volume was then adjusted to 78 ml with the same buffer.

Step 6: Isoelectric Fractionation-To 78 ml of the acetone fraction 12.9 g of ammonium sulfate were added. The slight amount of precipitate which formed after 30 min of stirring was removed by centrifugation at 27,000 X g for 10 min. Acetic acid (1 M,

containing 16.5 g of ammonium sulfate per 100 ml) was used to lower the pH of the preparation: thus, 5.1 ml were added with stirring to bring the pH from 6.55 to 5.4; and after centrifugation at 27,000 X g for 10 min, 5.2 ml of the acid mixture were added to adjust the pH to 4.8. The fraction precipitating between pH 5.4 and pH 4.8, a.fter a further 10 min of stirring, was collected by centrifugation as above. It was dissolved with 100 mM potassium phosphate-10 mM mercaptoethanol-5 mM EDTA-1.0% ethylene glycol, pH 7.0, to yield 15 ml of solution.

Step 6: Ion Exchange Chromatography-This step was performed at room temperature (20-22”). Cellex-T in the chloride form was packed to a height of 49 cm in a column 2.1 cm in diameter, converted to the phosphate form with 0.1 M potassium phosphate, pH 7.0, and equilibrated with the starting buffer (5 m&i potassium phosphate-10 mM mercaptoethanol-I mM EDTA- 1.0% ethylene glycol, pH 7.0). The enzyme preparation obtained in Step 5 was dialyzed against 500 ml of the starting buffer for 4 hours, with one change of buffer at 2 hours, and applied to the column. Then 20 ml of starting buffer were added to wash the enzyme into the column. A linear gradient was used to elute the enzyme. This consisted of 450 ml of starting buffer and 450 ml of 300 mM potassium phosphate-10 mM mercaptoethanol-l mM EDTA-1.0% ethylene glycol, pH 7.0. Fractions of 8.9 ml were collected at a flow rate of 3 ml per min. The protein concentration and aldehyde dehydrogenase activity of the fractions were determined, yielding the elution curve shown in Fig. 1. Fractions 50 to 63 were pooled (volume, 124 ml) and to this were added, with stirring, 6.2 ml of M potassium phosphate, pH 7.0, and 56 g of ammonium sulfate. After the suspension had been kept at 4” for 15 hours the precipitated protein was collected by centrifugation, dissolved, and made to a volume of 4.0 ml with 50 mM potassium phosphate-10 mM mercaptoethanol-1 mM EDTA, pH 7.0.

Step 7: Gel Filtration-This step was performed at room tem-

FIG. 1. Chromatography of aldehyde dehydrogenase on Cellex- T. Protein (315 mg) was applied to a column (2.1 X 49 cm) and eluted with a linear gradient of phosphate as described in the text. The enzyme activity was determined by the standard assay procedure described under “Experimental Procedure” except that the assay was carried out at 20-22”. The results are expressed as units per 1.0 ml of the fraction.


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2694 Aldehyde Dehydrogenme. I Vol. 243, No. 10

3 50

40

I 2

30

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TUBE NUMBER

FIG. 2. Gel filtration of aldehyde dehydrogenase on Sephadex G-200. Protein (116 mg) in 4.0-ml volume was applied to a column of Sephadex G-200 (2.5 X 38 cm) and eluted in 50 mM potassium phosphate-10 mM 2-mercaptoethanol-1 mM EDTA, pH 7.0. The enzyme activity was determined as described under “Experi- mental Procedure” with 10 ~1 of a 1: 10 dilution of each fraction. The results are expressed as units per 1.0 ml of the fraction.

TABLE II Summary of purification of aldehyde dehydrogenase from

P. aeruginosa ATCC 902Y

ml %

Cell extract ................. 527 8450 Protamine sulfate. .......... 608 6400 Ammonium sulfate. ......... 100 2300 Acetone precipitation. ...... 78 675 Isoelectric precipitation ..... 15 315 Cellex-T. ................... 4 116 Sephadex G-200. ............ 4 86

5500 0.65 100 5380 0.84 98 4430 1.9 81 3520 5.2 64 2280 7.3 42 1690 14.5 30 1250 14.7 23

perature. A column, 2.5 cm in diameter, was packed with Sephadex G-200 in 50 mad potassium phosphate-10 mM mercaptoethanol-1 mu EDTA, pH 7.0, to a height of 38 cm and washed with 2 volumes of the same buffer. The enzyme preparation obtained in Step 6 was applied to the column and eluted with the above buffer at a flow rate of 0.6 ml per min. Fractions of 2.4 ml were collected after 23 ml of the eluent had passed through the column. Protein concentration and aldehyde dehydrogenase activity were determined, yielding the results shown in Fig. 2. Fractions 38 to 56 (44 ml) were pooled and to this were added, with stirring, 4.4 ml of M potassium phosphate, pH 7, and 21 g of ammonium sulfate. After being kept at 4” for 15 hours the suspension was centrifuged and the precipitate was dissolved, made to 4.0 ml with 100 mu potassium phosphate-l0 mM dithiothreitol-1 mu EDTA, pH 7.0, and stored at -22’.

The cell extract of P. aeru~~osa has a strong reddish brown color. This is gradually removed during the purification. The last traces of color, still observed after the ion exchange chromatography, are removed in the final step. A solution of 20 mg per ml of protein of the final product is colorless. A summary of the purification is shown in Table II. Thii procedure has been carried out many times with similar results and almost identical final specific activities and yields. Additional steps, performed

on a purified preparation, such as elution from calcium phosphate gel, ammonium sulfate precipitation, ion exchange chromatography at pH 6.0 or pH 8.0, and precipitation of the enzyme by dialysis at low ionic strength did not result in any increase of specific activity.

Purity and Physical Properties

Electrophoretic Mobility of Aldehyde Dehydrogenase-In starch gel electrophoresis, the purified protein migrates toward the anode as one major band at pH 6.8, 7.4, and 8.5. The electrophoresis of two preparations of different purity is shown in Fig. 3. Enzymatic activity (see “Experimental Procedure”) is associated with the major band under these conditions. The faint band with faster electrophoretic mobility, which can be observed in the gel electrophoresis of the purified preparation, is believed to be the dissociated and inactive form of the enzyme. Exposure of the protein to low salt concentration (1.0 mM) for prolonged periodsof time prior to electrophoresis results in an increase in the protein concentration of the faint, faster moving band. This can be reversed by the addition of appropriate salts and indicates a dissociation-association of the protein, which will be dealt with in detail in a separate communication.

Fig. 4 shows the results of disc electrophoresis of three prep-

FIG. 3. Starch gel electrophoresis of aldehyde dehydrogenase at two stages of the purification. The gel in the center shows a sample after acetone precipitation (Step 4) and the gels on left and right show the enzyme solution after gel filtration (Step 7). Approximately 100 pg and 50 pg of protein were applied to the gel slices, respectively, and electrophoresis was performed with Tris- borate buffer, pH 8.5, as described under “Experimental Pro- cedure.” The anode is at the bottom of the picture. The difference in mobility of the enzymes results merely from variations in the gel bed and not from any change in the protein.


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arations at different stages of the purification procedure. As in the starch gel electrophoresis, the purified enzyme shows only one major protein band. Enzymatic activity could be shown in this band only if the enzyme was applied to the prepared gel imme- diately prior to the electrophoresis, which was carried out for approximately 2 hours. Incorporation of the protein into a sample during the additional 90 min required for gel preparation resulted in loss of enzymatic activity. However, the electropho- retie pattern of the protein was not changed significantly by this alternate method of application. To estimate the possible amount of contaminating protein in the purified enzyme, 2,4,8, 20, 40, and 80 pg of enzyme protein were subjected to electrophoresis. In the two gels containing 40 pg and 80 /lg of protein, diffuse bands with a faster electrophoretic mobility than the major enzyme band were observed. These were estimated to represent less than 2.5% of the total protein applied. Because of the dissociation phenomenon of the enzyme described above, the faint fast band may not necessarily be a protein contaminant but could represent, at least in part, dissociated aldehyde dehydrogenase.

In any case, the results of electrophoresis by these two methods indicate a degree of homogeneity of the purified aldehyde dehydrogenase in excess of 95%.

Sedimentation Behavior in Ultracentrifuge-As mentioned above, aldehyde dehydrogenase has a tendency to undergo re- versible dissociation. Fig. 5 shows the schlieren pattern of re- activated aldehyde dehydrogenase in the ultracentrifuge. Two peaks were identified: a major, fast component which represents the enzyme in the fully associated state, and a minor, slow component which is thought to be aldehyde dehydrogenase which had failed to reassociate under these conditions. (The minor component increases upon exposure of the enzyme to low salt.) Fig. 6 shows the relationship of the logarithm of the distance sedimented to time (17) for a 72-mm interval for each peak. The linear relationships indicate that the sedimentation econstants did not change during the course of the experiment. s20,W values of 9.0 and 5.5 S were calculated for the major and theminor component, respectively.

Abswption Spectrum-Aldehyde dehydrogenase in 50 mM potassium phosphate, pH 7.0, has an absorbance maximum at 277 rnp and shoulders at 282 rnp and 289 mM. From dry weight measurements and determinations of the absorbance at 280 rnp, it was calculated that a purified enzyme solution at 1.0 mg per ml concentration in 1.0 MM potassium phosphate, pH 7.0, has an absorbance of 1.04 at 280 mp. A2so: A260 was 1.83.

Requirements for Enzyme Activity

Assay of Maximum Activit~Aldehyde dehydrogenase was assayed as outlined under “Experimental Procedure.” The aldehyde substrate was added last to the otherwise complete reaction mixture after 5 mm of incubation at 35”. This resulted in maximum activity. Fig. 7 shows the results obtained when other components of the reaction mixture were added to initiate the reaction. The reaction rate was markedly reduced in all cases where the enzyme was not previously incubated, at least briefly, with K+, NAD+, and reducing agent before the aldehyde was introduced. The effect was greatest when K+ and NAD+ were added last.

2 Ultracentrifuge data were obtained through the kind coopera- tion of Dr. C. M. Kay, Department of Biochemistry.

FIG. 4. Polyacrylamide disc electrophoresis of aldehyde dehydrogenase at three different stages of the purification. The gel at the left shows cell extract (Step 1,240 rg of protein), the gel in the center the acetone precipitate (Step 4,70 pg of protein), and the gel at the right the enzyme solution after gel filtration (Step 7, 40 pg of protein). Electrophoresis was carried out as described under “Experimental Procedure.” The anode is at the bottom of the picture.

Ionic Requirements---Fig. 7 also shows the dependence of the reaction on the activating ion, K+. In a Tris-chloride-buffered assay system K+ was effectively replaced by NHat and, to a lesser extent by Rb+. These results and the effect of other monovalent and divalent cations are shown in Table III. It should be noted, in particular, that phosphate did not activate the enzyme. The possible inhibitory effect of some monovalent cations on the enzyme reaction was tested in a potassium phosphate-buffered assay system. Of the salts tested, only LiCl and NaAsOh were inhibitory. The effect of the latter was ascribed to inhibition by the anion. The results are shown in Table IV.

In view of the report of an aldehyde dehydrogenase from P. fluorescens which is dependent on phosphate or arsenate for activity (4), a study was made of the requirements of aldehyde dehydrogenase from several other members of the genus Pseu- domas. Six different organisms were grown with ethanol as the carbon source in the medium described under “Experimental Procedure” and the cell extracts were assayed for their aldehyde- oxidizing capacity in a potassium phosphate- and a sodium phosphate-buffered assay system. The results are shown in Table V. None of the activities in the sodium phosphate-buffered assay system were greater than 8% of those measured in the potassium phosphate assay system. The same requirement for potassium was observed in comparing the activity of cell extracts in a Tris-


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2696 Aldehyde Dehydrogmase. I Vol. 243, No. 10

SObI 8.29 053-

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., ,,s’obI. 507

078. l / HoA B 18 24 32 .o 48 e4 a4 ,e TlME (MINUTES)

FIG. 6. Determination of sedimentation coefficient of aIdehyde dehydrogensse. The log of distance sedimented (2) is plotted against time. The conditions of centrifugation are stated in the legend of Fig. 5. l , major component; 0, minor component.

ENZYME + ALDEHYOE LAST

6.4 ALDEHYDE LAST

DI ENZYME LAST P

x

E SH + ALDEHYDE LAST

: K+ + NAD+ LAS.1

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TIME (MINUTESI

FIG. 7. Effect of sequence of addition of assay components to the reaction mixture on measurement of aldehyde dehydrogenase activity. The assay was performed as outlined under “Experi- mental Procedure” except that 0.1 M potassium buffer was replaced by 0.1 M T&chloride buffer, pH 7.4, and 0.1 M KCl. The components listed beside each line were added last to an otherwise complete reaction mixture which had been incubated for 5 min at 35”. Wherever two components were added last, the one noted first was added 20 set before the one noted second.

chloride-buffered assay system with or without the addition of K+ as KCl. Therefore, the aldehyde dehydrogenase activity of alI the Pseudomonas species tested was dependent on K+ for ac-

tivity. Enzyme Specijkity-Aldehyde dehydrogenase oxidizes a va-

riety of aldehydes. NAD+ or, to a lesser extent, NADP+ can serve as the hydrogen acceptor. The specific activities of crude cell extracts and the purified enzyme preparation were determined with several substrates. The constant ratio of the specific activity of the purified enzyme preparation to the specific activity of the crude cell extract (Table VI) indicates that the activity with the different substrates is due to one enzyme.

The specific activity of a purified enzyme preparation determined with several aldehyde substrates at different concentrations is shown in Table VII. The lower specsc activities for <

FIG. 5. Sedimentation pattern of purified aldehyde dehydrogenase at 20”. The pictures were taken 16 min (top), 40 min (mid- dle), and 64 min (bottom) after the centrifuge had reached 59,780

The protein concentration was 5.8 mg per ml in 80 mM zykiurn phosphat,e8 rnM dithiothreitol-0.8 mu EDTA, pH 7.0. Sedimentation in each frame proceeds from right to left.


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Issue of May 25, 1968 R. G. von Tigerstrom and W. E. Razzell 2697

acetaldehyde, propionaldehyde, butyraldehyde, and isobutyraldehyde at the higher concentrations indicate substrate inhibition. This effect is not observed with glycolaldehyde, benzaldehyde, and glyceraldehyde at the concentrations used.

Products from glycolaldehyde and glyceraldehyde oxidation were isolated with the use of descending paper chromatography. They were identified as glycolic acid and glyceric acid, respectively, by comparison with similar amounts of known standards.

Michaelis Constants-The apparent Michaelis constants of the enzyme for several substrates and activating ions were determined. All results are calculated from the double reciprocal plot proposed by Lineweaver and Burk (18). When all other react- ants were maintained at the levels employed in the standard assay system, variations in the concentration of NAD+ yielded a K, of 3.7 x 1W4 M, whereas for NADP+ the value was 3.0 x 1OF M. With glyceraldehyde, the K, was 1.4 x lo+ M and glycolaldehyde, which showed substrate inhibition above 4 x

1w4 M, yielded a K, of 4 X low4 M from data obtained below inhibitory substrate concentrations. Such inhibition has been observed with several other aldehydes at high concentrations

TABLE III

Ionic requirements for aldehyde dehydrogenase activity

The assays were performed as outlined under “Experimental Procedure” except that 0.1 M potassium phosphate was replaced by 0.1 M Tris-chloride, pH 7.4, and the enzyme was dissolved in 0.1 M Tris-chloride-O.01 M dithiothreitol, pH 7.4. All salts listed in the table were added to give a final concentration of 0.1 M.

Additions Relative rate

None <6 KC1 1ooa Potassium phosphate 100 NH&l 110 RbCl 65 LiCl <6 NaCl <6 Sodium phosphate <6 CSCI <6 MgC12 <6 MnClz <6

Q The reaction in the presence of KC1 is arbitrarily assigned a value of 100.

TABLE IV

Effect of monovalent cations on potassium-activated aldehyde oxidation

The enzyme was assayed as outlined under “Experimental Procedure,” with potassium phosphate as the buffer. The salts listed were added to give a final concentration of 0.1 M.

Additions Relative rate

None lOOa KC1 91 NH&l 100 RbCl 91 NaCl 91 LiCl 86 CsCl 91 Na arsenate 51

5 The reaction in the presence of potassium phosphate with no additions is arbitrarily assigned a value of 100.

TABLE V

Aldehyde dehydrogenase activity in cell extracts of six different strains of Pseudomonas

To prepare the inoculum all organisms were transferred twice in the medium described under “Experimental Procedure” with ethanol as the carbon source. The organisms were then grown in the same medium for 48 hours at 25”. Ethanol, 0.4%, was added at the time of inoculation and a further 0.3% was added after 24 hours. The cells were harvested and cell extracts prepared as described in the legend of Table I. Aldehyde dehydrogenase activity was determined after dialysis and dilution of the cell extracts with 50 mM Tris-chloride buffer containing 10 mM 2-mercaptoethanol, pH 7.0. Enzyme activity was determined as stated under “Experimental Procedure” except that 0.1 M potassium phosphate buffer, pH 7.2, or 0.1 M sodium phosphate buffer, pH 7.2, was used in the reaction mixture.

Source of cell extract Specific activity

With KC 1 With Na+

P. jluorescens 36’. .......................... P. jZuorescens 22’. ......................... P. Jluorescens 266. ......................... P. jtuorescens A 312. ............... ....... P. ovalis B 8. .............................. P. aeruginosa ATCC 9027 ..................

wide/min/mg protein

0.80 0.054 0.65 0.035 0.32 0.022 0.23 0.018 0.40 0.023 0.55 0.021

TABLE VI

Specijic activity of crude and purified enzyme preparations with di$erent substrates and hydrogen acceptors

The specific activity of aldehyde dehydrogenase in the cell extract and purified enzyme preparation was determined by the assay described under “Experimental Procedure.” The following aldehyde and hydrogen acceptor concentrations were present in the reaction mixture: glyceraldehyde, 5 mM; glycolaldehyde, 1 mM; acetaldehyde, 0.05 mM; NAD+, 2 mM; and NADP+, 2 mad. The enzyme concentrations were adjusted to obtain a measurable reaction rate.

Substrate

Ratio of Specific activity specific

activities of purified to

Crude ) Purified c~$~%%

*mdes/min/mg pro1ein

Glyceraldehyde + NAD+. 0.16 2.8 17.5 Glycolaldehyde + NAD+. 0.73 12.7 17.4 Glycolaldehyde + NADP+. 0.03 0.48 16.0 Acetaldehyde + NAD+. 0.88 16.0 18.2

(Table VII). Activating cations K+ and NH4+ permitted the attainment of almost identical Tr,,, values, but the K, for Kf was 6 x 10U3 M, whereas the K, for NH,+ was 12 X lo+ M.

Reducing Agents-The enzyme was assayed routinely in the presence of 10 mM 2-mercaptoethanol. This reagent could be replaced, in part, by EDTA, or replaced effectively by lower concentrations of cysteine, dithiothreitol, or 2,3-dimercaptopropanol. The activities obtained with EDTA and 2-mercaptoethanol were not additive. These results are shown in Table VIII. At high concentrations of cysteine (5 to 10 mM) the initial fast reaction rate was arrested after approximately 4 mm.

This loss of activity could be reversed by the addition of glycolaldehyde. This may indicate the formation of a thiohemiacetal as


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TABLE VII

Substrate specijkity

The specific activity of purified aldehyde dehydrogenase was determined as described under “Experimental Procedure.” The concentration of aldehydes used is shown in the table.

Substrate

Glycolaldehyde. . Acetaldehyde. . . Propionaldehyde Butyraldehyde. . Isobutyraldehyde. Benealdehyde. Glyceraldehyde..

Specific activity with substrate at a concentration of

3.2 5.3 11.4 9.25

7.6 5.6 3.9 2.9 7.7 7.3 0.3 0.5

10.2 4.85 4.2 1.7 4.9 1.9 0.4 1.8

TABLE VIII

Activity of aldehyde dehydrogenase in presence of EDTA and diferent reducing agents

Purified aldehyde dehydrogenase (55 pg per ml) dissolved in 0.1 M potassium phosphate, pH 7.0, was assayed as described under “Experimental Procedure” except that 2-mercaptoethanol was replaced by the additions listed in the table.

Addition Concentration

None EDTA

2-Mercaptoethanol

2-Mercaptoethanol + EDTA Cysteine

Dithiothreitol

2,3-Dimercaptopropanol (British anti Lewisite)

_-

?nM

0.1 1.0

10.0 20.0

0.1 1.0

10.0 20.0

10.0 + 10.0 0.1 1.0

10.0 0.01 0.1 1.0

10.0 0.01 0.1 1.0

10.0

-

I 1

Specitic activity

moles/ e&/m&! protein

1.7 1.9 3.8 4.8 5.2 3.1 6.4

12.5 12.8 11.0

3.5 11.1 14.2 6.3

11.3 13.2 14.2

8.1 11.3 12.8 14.9

a result of the reaction of the aldehyde substrate and this reducing agent. This phenomenon was not observed when the other reducing agents or glutathione were employed at similar high concentrations.

Effect of pH-The assay for aldehyde dehydrogenase activity was performed in a Tris-chloride-potassium phosphate buffer system at pH values between pH 5.7 and pH 9.3. Prior incubation times of 5 and 10 min were used at 35’ before initiation of the reaction by addition of the aldehyde. The results are shown in Fig. 8. The pH optimum for the initial reaction rate is be-

tween pH 8.0 and 8.6. The values obtained after different prior incubation times are in close agreement. This indicates that the previous incubation itself, even at hydrogen ion concentrations which result in instability of the enzyme, does not affect significantly the measurement of initial reaction rate. Since a linear rate was obtained for a longer period of time at pH 7.2 than at hydrogen ion concentrations close to the pH optimum, the standard assay was carried out at pH 7.2.

Stability

Initially, the instability of aldehyde dehydrogenase had been a serious problem. When the cell extract was prepared in phosphate buffer containing 2-mercaptoethanol, losses of 30 to 50% of the activity occurred upon storage at 4” for 16 hours or during the course of ammonium sulfate fractionation. Activity losses were reduced by storage at -22” or after gel filtration on Bio- Gel P-60. Bisulfite, used as NazSz06, adjusted to pH 7.0 with KOH, was found to be very effective in reducing activity losses. Table IX shows the effect of 2-mercaptoethanol, dithiothreitol, and bisuliite on the stability of aldehyde dehyrogenase in crude cell extracts. When the cell extract was prepared in 100 mM

bisulfite-10 mM mercaptoethanol, pH 7.0, insignificant losses of activity were observed during storage at -22” for several weeks or at 4” or 25” for several days. Bisulfite could not be replaced by hydrazine, hydroxylamine, or ascorbate.

During the early stages of puriCcation the enzyme preparation can be stored in phosphate buffer containing 2-mercaptoethanol at -22” without significant loss of activity. Preparations carried past Step 6 in the purification were found to denature upon freezing and thawing with the loss of 90 to 100% of the activity. This was prevented by the addition of 50 mM bisulfite or 1.0% ethylene glycol to the buffer system or by substitution of dithiothreitol for 2-mercaptoethanol. The enzyme preparation after the final step of purification, dissolved to a concentration of 15 mg per ml in 100 mM potassium phosphate-10 mM di-

FIG. 8. The effect of pH on glycolaldehyde oxidation. The assays were performed as outlined under “Experimental Pro- cedure” except that the reaction mixture also contained 0.1 M

Tris-chloride. The pH of reaction mixture was determined im- mediately after the assay was completed. O-O, aldehyde added after 5 min of prior incubation; O----O, aldehyde added after 10 min of prior incubation at 35”.


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thiothreitol-1 mM EDTA, pH 7.0, was found to be stable at -22” for several months. Dilute enzyme preparations (50

pg per ml) at the final stage of purification deteriorated more readily upon freezing and thawing but could be stored at -22’ without loss of activity in the presence of 30% ethylene glycol. Dilute purified enzyme preparations stored at 4” in 100 mu potassium phosphate-10 mM dithiothreitol-1 mM EDTA, pH 7.0, were stable for several days and 10 mM dithiothreitol could be replaced by 100 mM 2-mercaptoethanol. At this temperature bisulfite showed a favorable effect on the stability of the purified enzyme preparation. These results are shown in Table X.

The enzyme stability at different hydrogen ion concentrations was determined with a dilute, purified enzyme preparation (40 pg per ml). The pH region from 6.8 to 7.2 was found to be the most favorable for storage at 4”.

Inactivation by Heat-The stability of aldehyde dehydrogenase at temperatures from 40-60” was determined in two buffer systems; 150 mu potassium phosphate-10 mu dithiothreitol- 1.0 mM EDTA, pH 7.0, and 100 mu potassium phosphate-50 mM bisulfite-10 mM dithiothreitol-1.0 mM: EDTA, pH 7.0. The results are shown in Fig. 9. They indicate a. relatively low resistance of the enzyme to heating and also the stabilizing effect of bisullite on the enzyme which was especially noticeable in the temperature range 45-53’.

A dilute, purified enzyme preparation in a potassium phosphate-buffered system was effectively protected from heat inactivation at 46” by the presence of 0.4 mM NAD+. In the presence of NAD+, 82% of the initial activity remained after heating for 20 min, whereas 44% remained without NAD+. In a parallel experiment with sodium phosphate buffer, 33% and 13% of the initial activity remained in the presence and absence of NADf, respectively. Therefore, NAD+ in the presence of the potassium ion is much more effective in protecting the enzyme from heat inactivation than in the presence of the sodium ion.

Inhibition and Protection

Aldehyde dehydrogenase is inhibited by reagents reacting with sulfhydryl groups, such as iodoacetate, iodoacetamide, CL?, and p-chloromercuribenzoate. Purified aldehyde dehydrogenase (70 pg per ml) dissolved in 100 mM Tris-chloride- 1 mM dithiothreitol, pH 7.2, and incubated at 20-22” for 1 hour was inhibited 59% in the presence of 1.0 mM iodoacetate and 63 ‘$$ in the presence of 0.1 mM iodoacetamide. The inhibition was found to be greater (67 ‘% with iodoacetate and 81% with iodoacetamide) when K+ was included in the incubation mixture. This enhanced inhibition was not observed in the presence of Na+. The enzyme was protected from iodoacetate inhibition by 0.4 MM NAD+ when K+ or NH*+ were also present. The specificity of this protection with K+ and NAD+ or NADH is shown in Table XI.

A similar specificity was observed when the loss of activity due to trypsin digestion was followed. K+ plus NAD+, or K+ plus NADH, protected the enzyme whereas K+ plus NADP+ did not. These results are shown in Table XII. In contrast to the results for iodoacetate and iodoacetamide inhibition, K+ and to a lesser extent Na+, provided some protection from trypsin in the absence of NAD+.

Arsenite, a common inhibitor of aldehyde dehydrogenases (4) also inhibits the enzyme isolated from P. aeruginosa. Ar- senite at 0.5 m&q 1.0 mM, and 5.0 mM in the reaction mixture resulted in 38, 60, and 92y0 inhibition, respectively. The

Conditions

2-Mercaptoethanol

Dithiothreitol Bisulfitea

Phosphate0

TABLE IX

E$ect of reducing agents, bisulj%e, and phosphate on stability of aldehyde dehydrogenase in cell extract

Cell extracts were prepared by sonic disruption in either 5 mM potassium phosphate containing 10 mM 2-mercaptoethanol, pH 7.0, or 5 mM potassium phosphate containing 10 mM dithiothreitol, pH 7.0. 2-Mercaptoethanol, bisulfite (pH 7.0), and potassium phosphate (pH 7.0) were added to the cell extracts to give the final concentrations shown in the table. Activity of aldehyde dehydrogenase was determined in each sample before and after storage at 4” for 6 days.

Concentration Initial activity

w&f % 10 12 20 32 50 55 10 12 30 80 60 83

120 97 30 27

a Also contained 10 mM 2-mercaptoethanol.

TABLE X

Effect of bisul$te on stability of purified aldehyde dehydrogenase

Purified aldehyde dehydrogenase (56 pg per ml) in 106 mM potassium phosphate-10 mM dithiothreitol-1 mm EDTA, pH 7.0, was stored with or without the addition of bisulfite or an equivalent concentration of phosphate. The activity in each preparation was determined as described under “Experimental Procedure” before and after storage at 4” for 16 days.

I I

Addition Concentration Initial activity

rnM % None............................ 46 Bisulfite......................... 40 110 Potassium phosphate. 40 45

40 SO 60 TEMPERATURE (DEGREE 1

FIG. 9. Heat inactivation of aldehyde dehydrogenase. A purified enzyme preparation (50 rg per ml of protein) in 0.5 ml of the buffer system outlined in the text was subjected to 10 min of heat treatment at the temperatures indicated. The activity remaining after the treatment is expressed as percentage of the initial activity. O-O, in potassium phosphate buffer; O--O, in potassium phosphate buffer + bisulfite.


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TABLE XI

Inhibition of aldehyde dehydrogenase by iodoacetate and protection by activating ion and coenzyme

To a purified enzyme preparation (74 pg per ml) in 100 mM Tris- chloride-l mM dithiothreitol, pH 7.2, the additions listed in the table were made to give a final concentration of 190 mM KC1 or NaCl, and 0.4 mru NAD+, NADP+, or NADH. After incubation for 30 min at 25”, iodoacetate was added to give a final concentration of 2 mM. Control samples without iodoacetate were also included in the experiment. The table shows the percentage of the initial activity which remained after a 2-hour incubation at 25”.

Additions

None KC1 NaCl NAD+ KC1 + NAD+ NaCl + NAD+ KC1 + NADP+ KC1 + NADH

Initial activity

%

11 8

17 10 77

8 19 81

TABLE XII

Digestion of aldehyde dehydrogenase by trypsin and protection by activating ion and coenzyme

The details for this experiment are the same as those in the legend to Table XI except that iodoacetate was replaced by trypsin at a final concentration of 0.1 pg per ml. The percentage of the initial activity remaining after 60 min of digestion at 25” is shown in the table.

Additions

None KC1 NaCl NAD+ KC1 + NAD+ NaCl + NAD+ KC1 + NADP+ KC1 + NADH

Initial activity

% 16 51 28 12 91 25 48 89

inhibition with 5.0 IIIM arsenite was reversed by 80% upon the addition of 5.0 mM dithiothreitol. Arsenite inhibition also is reversed completely by dilution.

o-Phenanthroline inhibits aldehyde dehydrogenase by 44‘% at 0.5 mM and 67% at 1.0 InM concentration when added to the reaction mixture during the enzyme assay, but showed a stimu- latory effect on the reaction in the absence of reducing agent. An enzyme preparation (2.8 mg per ml) inhibited to greater than 90% by 2.0 InM o-phenanthroline at 2&22” during a 3- hour period, was not activated after gel filtration on Bio-Gel P-60 to separate the inhibitor from the protein, or by subsequent incubation of the separated protein with 0.01 mM Z&f, Co++, Fe++, or Mg++.

DISCUSSION

The potassium-activated aldehyde dehydrogenase of P. ueruginosa appears to have a major role in the metabolism of

ethanol as indicated by the induction of the enzyme when the organism was grown with ethanol as the carbon source. This enzyme was also present at a high level in extracts of other Pseudomonas species grown on ethanol. Acetaldehyde is assumed to be the natural substrate under these conditions. The enzyme was also induced by growth on ethylene glycol, although this carbon source is quite toxic to these organisms at 0.2yo or higher concentrations.

The conditions chosen for growth of P. aeruginosa for enzyme production permit the preparation of cell extracts with an unusu- ally high concentration of the enzyme. It can be calculated that the enzyme represents as much as 4% of the soluble proteins in the cell extract. 20-fold purification of the enzyme was obtained with high yield in relatively few simple steps and the final enzyme preparation appeared to be homogeneous in the ultracentrifuge and by electrophoresis in starch or polyacrylamide gels. The elution patterns of the ion exchange column and gel filtration column support this assumption and all attempts to improve the specific activity beyond that obtained by the given procedure have failed.

In general, aldehyde dehydrogenases have been reported heretofore to be very unstable proteins. Instability of the aldehyde dehydrogenase from P. aeruginosa was also a problem in initial studies of this enzyme but was overcome by the use of bisulfite as a stabilizing agent and buffer. The exact role of bisulfite in stabilizing the enzyme has not yet been determined. Aldehydes are toxic to the enzyme as evidenced by substrate inhibition and bisulfite is known to form addition products with aldehydes. However, this property of bistite does not appear to account for the stabilizing effect since hydroxylamine and hydrazine, which also form addition products with aldehydes, did not show a stabilizing effect on the enzyme and the addition of aldehydes to cell extracts did not increase instability. The enzyme is stabilized to some degree by gel filtration which may support the idea that bisulfite is combining with, or protecting the enzyme from, some factor in the cell extract. Bisulfite is a very strong reducing agent and this may be the reason for its effect. This is supported by the fact that the presence of high concentrations of 2-mercaptoethanol also resulted in greater stability of the enzyme in cell extracts. The presence of bisulfite in purified enzyme preparations also has a favorable effect as seen from studies on the influence of storage conditions and the inactivation by heat, but further experimentation is required to elucidate the effect of this agent on the physical properties of the enzyme.

The aldehyde dehydrogenase reported here has a wide substrate specificity, but substrate inhibition was observed with all aldehydes tested except benzaldehyde and glyceraldehyde. This inhibition was especially noticeable with acetaldehyde and propionaldehyde. The substrate specificity and substrate inhibition seem to be similar to that reported for the aldehyde dehydrogenases from yeast (2) and the phosphate-requiring enzyme from P. jkmrescens (4). The specificity is different from that shown by the aldehyde dehydrogenase induced in P. aeruginosa by growth on paraffin hydrocarbons (lo), and the aldehyde dehydrogenase of Acetobacter suboxidans (19).

A reducing agent such as 2-mercaptoethanol, cysteine, dithiothreitol, or 2,3-dimercaptopropanol (British anti-Lewisite) is required for enzymatic activity. Although chelating agents can replace mercaptans to a considerable degree (and with EDTA almost 56% of the maximum activity can be obtained),


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Issue of May 25, 1968 R. G. van Tigerstrom and W. E. Raxzell 2701

the activities with reducing agent and EDTA are not additive. A similar observation was made in studies with the yeast aldehyde dehydrogenase (8). These results indicate that aldehyde dehydrogenase is most active when in the fully reduced state and protected from heavy metal ion inhibition. Both conditions can be brought about by mercaptans whereas a chelating agent will only protect from metal ion inhibition. In studies of the effect of mono- and dimercaptans on the aldehyde dehydrogenase from P. jluorescens, the dimercaptans were found to be much better reducing agents (4) ; these results and further studies on the active site of this enzyme and other aldehyde dehydrogenases led Jakoby to conclude that two closely positioned sulfhydryl groups are involved in the active site (5). Similar observations are reported here although cysteine, a monomercaptan, was a very effective reducing agent, at relatively low concentrations.

P. jluorescens grown on ethylene glycol produces a phosphate- requiring aldehyde dehydrogenase (4). At the outset of our studies we assumed that the enzyme we were studying was similar to or identical with that of P. fluorescens since potassium phosphate was essential for activity. However, further studies on the ionic requirements for activity showed that this response was due to an absolute requirement for the cation, potassium, which could be replaced by ammonium or, less effectively, by rubidium. These ionic requirements distinguish the enzyme from other, previously reported, pyridine nucleotide-linked aldehyde dehydrogenases of bacterial origin (10, 19). The requirements of the enzyme from P. mruginosa ATCC 9027 do not seem to be unique for the enzyme from this strain, however, as the aldehyde dehydrogenase activity of five other organisms of the genus Pseudomonas was found to be activated by potassium and not by phosphate. Although the potassium salt of phosphate was used in the experiments on the phosphate-requiring enzyme from P. &n-escens, it appears that the anion was im- portant. Therefore, there appear to be two types of pseudo- monads: at least one which possesses an enzyme dependent on phosphate or arsenate, and the several which we have shown to produce an enzyme dependent on potassium or ammonium.

The enzyme investigated here seems to be similar to the potassium-activated yeast enzyme (2) in ionic requirements except that ammonium is a better activating ion than rubidium. The contrary was reported for the yeast enzyme. This could re- flect differences in the molecular structure around the active or activating site. Stoppani and Milstein (6) reported that any one of potassium, NAD+, or acet,aldehyde protected the yeast aldehyde dehydrogenase from inhibition by thiol reagents. An essential role of the thiol groups in the enzyme was implicated. Sorger and Evans (7) showed that monovalent cations, especially the activating ions, had a favorable effect on the stability of the yeast enzyme and suggested that the ions caused a physical rearrangement of the protein.

The inhibition and protection studies with iodoacetate reported in the present paper show that potassium alone does not protect the enzyme but accelerates inhibition. NAD+ alone had no effect, whereas the combination of potassium and NAD+ or potassium and NADH gave good protection from iodoacetate

3 Dr. Jakoby kindly examined his original experimental data on the ph0sphat.e and arsenate effects, which reveal that sodium arsenate was effective in stimulating the enzyme. Apart from possible difficulties inherent in specifying st,rains of pseudomo- nads, the basis of the difference between our observations cannot be explained at present.

2. BLACK, S., Arch. Biochem. Biophys., 34, 86 (1951). Q BLACK, S., in S. P. COLOWICK AND X. 0. KAPLAN (Editors), “ .

Methods in enzymology, Vol. I, Academic Press, New York, 1955, p. 508.

4. 5. 6.

JAICOBY, W. B., J. Biol. Chem., 232, 75 (1958). JAKOBY, W. B., J. Biol. Chem., 232, 89 (1958). STOPPANI, A. 0. M., AND MILSTEIN, C., Biochem. J., 67, 406

(1957). 7. SORGER, G. J., AND EVANS, H. J., Biochim. Biophys. Acta,

118, 1 (1966). 8.

9.

10.

11.

12. 13. 14. 15.

STOPPANI, A. 0. M., SCHWARCZ, M. N., AND FREDA, C. E., Arch. Biochem. Biophys., 113, 464 (1966).

STEINMAN, C. R., AND JAKOBY, W. B., J. Biol Chem., 242, 5019 (1967).

H;YD&AN, M. T., AND AZOULAY, E., Biochim. Biophys. Acta, 77, 545 (1963).

LOWRY, 0: H., ROSEBROUGH, N. J., FARH, A. L., AND RANDALL, R. J., J. Biol. Chem., 193, 265 (1951).

SMITHIES, O., Biochem. J., 61, 629 (1955). SMITHIES, O., Biochem. J., 71, 585 (1959). DAVIS, B. J., Ann. A7. Y. Acad. Sci., 121, 404 (1964). FINE, I. H., AND COSTELLO, L. A., in S. P. COLOWICK AND N.

0. KAPLAN (Editors), Methods in enzymology, Vol. 6, Aca- demic Press, New York, 1963, p. 958.

inhibition. This effect is as specific as the requirement for catalysis of aldehyde oxidation. It is of interest in this respect that maximum activity in the assay is obtained when the enzyme is incubated with the activating ion, reducing agent, and NAD+ before the aldehyde is added to the reaction mixture. A parallel observation was made by Jakoby for the assay of the enzyme from P. Jluarescens. It appears that potassium activates the enzyme, which leads to faster iodoacetate inhibition, and also facilitates the binding of the coenzyme to the active site which, directly or indirectly, blocks the reaction with the thiol reagent. That a physical rearrangement of the enzyme takes place under these conditions is borne out by the fact that the presence of potassium and NAD+ also protects the enzyme from trypsin digestion and heat inactivation. We conclude that this is due to a conformational change of the enzyme caused by the activating ion which is even greater if NAD+ is bound. A non- activating cation like sodium may bring about some conformational change as evidenced by slightly greater resistance to trypsin digestion while activation and NAD+ binding are not facilitated. In contrast, it is interesting to note that in the case of succinic semialdehyde dehydrogenase (20) the presence of the coenzyme leads to a more rapid digestion of the enzyme with trypsin and the coenzyme does not appear to be attached to a thiol group.

Inhibition by arsenite is a common feature of other aldehyde dehydrogenases and two closely positioned thiol groups in the active site of these enzymes are implicated (5). The enzyme reported here is also inhibited by arsenite although detailed studies have not been undertaken, except to note reversal by dithiothreitol.

It is clear that the purity and stability of the aldehyde dehydrogenase preparation which has been achieved will allow investi- gation of some of the molecular properties and give insight into the function of the enzyme and of aldehyde dehydrogenases in general.

REFERENCES

1. JAKOBY, W. B., in P. D. BOYER, H. LARDY, AND K. MYR- s&x (Editors) The enzymes, Vol. 8, Academic Press, New York, 1963, p. 203.


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16. NORDMANN, J., AND NORDMANN, R., in I. SMITH (Editor), 18. LINEWEAVER, H., AND BURIC, D. J., J. Amer. Chem. Sot., 66, Chromatographic and electrophoretic techniques, Vol. I, Inter- 658 (1934). science Publishers, Inc., New York, 1960, p. 272. 19. KING, T. E., AND CHELDELIN, V. H., J. Biol. Chem., 220, 177

17. SCHACHMAN, H. K., in S. P. COLOWICK AND N. 0. KAPLAN (1956). (Editors), Methods in enzymology, Vol. 4, Academic Press, 20. NIRENBERU, M. W., AND JAKOBY, W. B., Proc. Nat. Acad. New York, 1957, p. 32. Sci. U. 8. A., 46, 206 (1960).


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Richard G. Von Tigerstrom and W. E. RazzellENZYME FROM PSEUDOMONAS AERUGINOSA

Aldehyde Dehydrogenase: I. PURIFICATION AND PROPERTIES OF THE

1968, 243:2691-2702.J. Biol. Chem.

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