Eur. J. Biochem. 81, 1x5-I92 (1977)

Purification and Characterization of Two Aldehyde Dehydrogenases from Pseudomonas aeruginosa Luc GUERRTLLOT and Jean-Paul VANDECASTEELE Service de Biochimie, Institut Franqais du Pttrole, Rueil-Malmaison (Received June 27, 1977)

Two soluble aldehyde dehydrogenases isoenzymes have been purified and separated from extracts of a paraffin-assimilating bacterium, Pseudomonas aeruginosa. The first one, obtained at an estimated purity of 20% (spec. act. with butanal 0.33 kat/kg) was NAD-dependent. It was rapidly inactivated at pH 8.6 but was efficiently protected by NAD. It had a molecular weight of 225000 and presented a high affinity for aldehydes of short and middle chain lengths. The second enzyme, obtained in a nearly homogenous state (spec. act. with pentanal 0.62 katlkg) was NADP-dependent. It was activated by ions, in particular potassium ions, and had a good affinity for aldehydes of higher chain lengths. Both enzymes were stabilized by thiols and glycerol and were inactivated by reagents of sulfhydryl groups. These enzymes are 'constitutive' and their physiological function is uncertain. When the bacteria were grown on n-paraffin a new membrane-bound NAD-dependent aldehyde dehydrogenase activity was produced.

In a previous work, purification of the alcohol dehydrogenases of Pseudomoms aeruginosa and studies of their properties in relation to the capacity of this species to assimilate n-paraffins have been completed [l-31. A similar study has been undertaken with aldehyde dehydrogenases of the same strain. As in the case of alcohol dehydrogenases [2], NADdependent and NADP-dependent aldehyde dehydrogenase activities were observed in the soluble fraction of extracts from cells grown on n-paraffin as well as on glucose. The enzymes involved have been purified and characterized and the results obtained are presented here.

containing 7 mM 2-mercaptoethanol and 1 mM dithiothreitol. 100 pg DNase (Sigma DHC) and 0.5 ml of 0.2 M magnesium sulfate were added. The cell suspension, immersed in a ice bath, was disintegrated with a Branson S75 sonifier (six periods of 15 s at the maximal output), then centrifuged at 3 "C for 60 min at 100000 x g. 16 yi glycerol (final volume) was added to the supernatant. The sedimented particles were washed once in the same buffer, centrifuged in the same conditions and then resuspended in the same buffer in a final volume of 10 ml. This constituted the 100000 x g pellet. Enzllme Assays

MATERIALS AND METHODS Preparation of Extracts '

The strain used, Pseudomonas aeruginosa 196 Aa, was grown on glucose as carbon source as previously described [2]. Cell-free extracts were prepared as follows. 5 g (wet weight) of cells were suspended in 10 ml of 10 mM potassium phosphate buffer (pH 7.2) This work has been carried out as a partial fulfilment of a doctoral thesis to he submitted by L. Guerrillot to thc Universiti de Paris-Sud. Enzymes. Aldehyde-NAD oxidoreductase (EC 1.2.1.3); aldchydc-NADP oxidoreductase (EC 1.2.1.4); DNase (EC 3.1.4.5).

The reduction of NAD or NADP was measured at 30 "C with an Eppendorf recording photometer at 334 nm. The aldehydes used were present as true solutions in the buffer used to avoid the artefacts resulting from the use of emulsions, as previously reported in the case of long-chain alcohols [l]. The concentrations of NAD and NADP were 1 mM and 0.5 mM respectively. The buffers used were either 50 mM glycine potassium hydroxide (pH 9.4) or 50 mM potassium pyrophosphate (pH 8.6) as indicated. The reaction was initiated by addition of the enzyme. Enzymic activities are expressed in microkatals (pkat) or nanokatals (nkat) corresponding to

186

Aldehyde Dehydrogenases from Pseudomonas aeruginom

reaction rates of micromoles or nanomoles per second, respectively, in these conditions.

%-

Celi suspension

Ultrasonic treatment, ultracentrifugation

Protein Assay

The modified Folin method previously described [2]has been utilized. Analytical Gel Electrophoresis

Protamine suifate addition, centrifugation, glycerol addition, dialysis

Polyacrylamide gel electrophoresis was carried as previously described [2] except that the p H of the gel was lowered to 8.5 and that the substrates utilized to reveal the activity were either 0.1 M propanal or 4 mM octanal in 0.05 M potassium pyrophosphate buffer (pH 8.6). Optical analysis of the gels was carried out using a gel scanner (Isco, Lincoln, Nebraska, U S.A.) connected to recording photometer (Isco UA-5) at 58Onm (for protein after coloration with Coomassie blue) or 470 nm (for enzyme activity). Chromatographic Techniques

OE A € -cellulose chromatography

A

Fractions 80r.94

The techniques and materials utilized have been previously indicated [2]. Tn some cases, protein concentration in column effluents was monitored at 280 nm with a recording photometer (Isco UA-5). Potassium phosphatc buffer containing 16 glycerol and 7 mM 2-mercaptoethanol was routinely used in handling the enzymes and consequently will be simply referred to as phosphate buffer. Molecular weights were carried out by filtration on a Sephadex G-200 column (750 x 25 mm) according to Andrews [4]. The column was calibrated with enzymes of known molecular weights.

:/:

Rcugents

Aldehydes utilized were from Fluka. Their purity was examined by gas-liquid chromatography in conditions previously described [3]. The predominant impurities detected were the corresponding acids (2 - 3 %). Contamination by lower or higher aldehydes was small (below 1%). RESULTS

PuriJication ofthe NAD-Dependent and N A DP- Dependmt Aldehyde Dehydrogenases The scheme followed for the separation and purification of the two aldehyde dehydrogenases studied is given in Fig. 1 and the results of the various purification steps are shown in Tables 1 (for the NADdependent enzyme) and 2 (for the NADP-dependent enzyme). The standard ultrasonic and ultracentrifugation treatments were applied to 80 g (wet weight)

Hydroxyapatite Chromatography

Hydroxyapatite chromatography

I

1

Sephadex G - 200 chromatography

Sephadex G -200 chromatography

I

Aldehyde dehydrogenase (NADP)

i

ALdehyde dehydrogenase ( N A D )

Fig. 1. Purification scheme of' rlie NAD-dependent and NADPdepmiieni uldehyde dehydrogenases

of cells by portions of 10 g; 7.2 ml of a 2 protamine sulfate solution were added dropwise to the resulting 100000 x g supernatant (138 ml) which was stirred for 30 min, then centrifuged 20 min at 48 000 x g. Protamine sulfate has been preferred to streptomycin sulfate for the precipitation of nucleic acids because use of the latter resulted in a lower recovery of the NAD-dependent activity due to partial precipitation during the subsequent dialysis step. Glycerol (final concentration 167;) was added to the protamine sulfate supernatant. 16 :L glycerol and 7 mM 2-mercaptoethanol were routinely added to all enzyme preparations and chromatography buffers (unless otherwise stated) because their presence was found necessary to impart the enzymes studied with sufficient stability to allow their purification and study. The protamine sulfate supernatant was dialyzed against 20 mM phosphate buffer pH 7.3 (178 i d ) thcn adjusted to pH 7.8 and adsorbed on the top of a DEAE-cellulose column previously equilibrated with 20 mM phosphate buffer pH 7.8 and eluted with a phosphate gradient.

187

L, Guerrillot and J.-P. Vandecasteele Table 1 . Purification of NAD-dependent aIdehj.de dehydrogenuse Activity determinations were performed in 50 mM K3HPZ07(pH 8.6) with 0.2 mM butanal Preparation

100000 x g supernatant Protamme wpernatant Dialyzed supernatant D EAE-ceilulose eluate (fractions 95- 120) Concentrated DEAF-cellulose eluate (fractions 95 - 120) Hydroxyapatite eluate Sephadex G-150 eluate (64-90 ml) Sephadex G-200 eluate (173 -240 ml) Fraction (198 -203 ml) of Sephadex G-200 a

Volume

Proteins

Total activity

Specific activity

Yield

Purification

ml

mg,/ml

pkat

mkat/kg

%

-fold

138 142 178

44 44.8 36

13.8 14.7 11.8

2.3 2.2 1.9

100

1

254

1.9

4.7

9.5

40

5.1

39.5 232

11.7 0.18

4.4 2.3

9.5 57

38 20

5.1 30

24

0.75

2.0

112

17

60

67

0.099

1.7

253

14

135

5

0.11"

0.18

333

178

Spectrophotometric determination of protein

Table 2 Ptirificarion of AlADY-dependent aldehyde &hydrogenax Activity determinations were performed in 50 mM K?HPzO7(pH 8 6) with 4 mM pentanal ~

Preparation

Volumc

Protein

Total activity

Specific activity

Yield

Purification

~

100000 x g supernatant Protamine supernatant Dialyzed supernatant DEAE-cellulose eluate (fractions 80 - 94) Concentrated DEAE-cellulose eluale (fractions 80 - 94) Hydroxyapatite eluate Sephadex G-200 eluate (175-235 ml) Fraction (200 - 205 ml) of Sephadex G-200 a

ml

mg/ml

pkat

mkat/kg

:4

-fold

138 142 178

44 44.8 36

14.2 33.8 10.8

2.3 2.5 1.7

100

1

148

1.5

5.2

22

48

12.8

35 170

6 0.065

5.0 3.0

24 283

46 28

13.9 166

60

0.08

2.6

533

23.5

314

5

0.09"

0.28

61 7

363

Spectrophotometric determination of' protein

As shown in the elution diagram (Fig.2) separation of the two aldehyde dehydrogenases took place on this column. In spite of various attempts however, complete separation could not be achieved in one step and the results obtained in the conditions adopted are given in Fig.2. From this step on, purification of the two enzymes was carried out independently. For purification of the NAD-dependent enzyme, fractions 95- 120 of this step were dialyzed against 20 mM phosphate buffer (pH 6.8), then concentrated to a volume of 39.5 ml and subjected to chromatography on a hydroxyapatite column (25 x 340mm) previously equilibrated with the same buffer. Elution was performed with a concentration gradient of the phosphate buffer (pH 6.8). The elution concentration was 50 mM

for the NAD-dependent enzyme and 40 mM for the contaminating NADP-dependent enzyme. Further purification of the NAD-dependent enzyme was carried out on a Sephadex G-200 column. 100 mM potassium phosphate (pH 7.1) was used as equilibration and elution buffer. (In the experiment reported in Table 1, however, a 25 x 370-mm Sephadex G-150 column was first used in the same conditions.) On the purest preparation from the Sephadex G-200 column (fractions 198-203 ml) with a specific activity of 0.33 katikg, electrophoresis on polyacrylamide gel was performed and the results are given in Fig.3. The recordings show a single peak of enzyme activity but several protein peaks, enzyme activity corresponding to the second major protein peak. This allows a

188

Aldehyde Dehydrogenases from Pseiidomonas aeruginnsa

loo

I

+

rI 1 I

I I

0

20

40

80

60

100

120

140

Fraction number

Fig. 2. Separation of' the NAD-dependent and NADP-dependent aldehyde clehydrogenases by DEAE-cellulose cliromatography. A 3X mm x 370-mm column was used. The elution rate was 95 mlih. Fractions of 10 ml were collected. (A--A) NADP-dependent aldehyde dehydrogenase aclivity ; (0-0) NAD-dependent aldehyde dehydrogenase activity; ( 0 4 ) protein (tng/ml) ; (----) concentration of elution phosphate buffer (pH 7.8). Protein concentration in fraction 140 was 22 m g h l

true activity of the NAD-dependent enzyme with NADP. The NADP-dependent enzyme (fractions 80 - 94 of the initial chromatography on DEAE-cellulose) was further purified by chromatography on hydroxyapatite and on Sephadex G-200 in the same conditions as the NAD-dependent enzyme. The purest fraction of the Sephadex G-200 eluate (200 - 205 ml) has been subjected to gel electrophoresis and recordings given in Fig.4 indicate a single enzyme activity band corresponding to a single major protein peak. Purity of the enzyme estimated from this result is in the range 90-952). Preparations of the NADP-dependent enzyme obtained after Sephadex G-200 chromatography contain no NAD-dependent activity detected by spectrophotometry ( < O . l ? < of the activity with NADP).

0.6 0

rn m c

04 0

c n m L

5: 0

n

2

4

0 Direction o f gel scanning

Fig. 3. Gel electrophoresis of the purifird NAD-dependent aldehyde clehydrogenuse. (A) Scanning of gel for enzyme activity (developed by incubation with tetrazolium red). (B) Scanning of gel for protein (stained with Coomassie brillant blue). (1) Top of gel; (2) bottom of gel

rough estimation of enzyme purity of about 20%. A low residual NADP-dependent activity was still detectable by spectrophotometry in the purified preparations of the NAD-dependent enzyme (1.1 kat of NADP-dependent activity in 100 kat of NAD-dependent activity). It is not known whether this activity results from a residual contamination or represents

PROPbRTTES OF THE NAD-DEPENDENT AND NADP-DkPENDENT ALDEHYDE DEHYDROGENAStS

Molecular Weigllt The determination of molecular weights using gel filtration on Sephadex G-200 gave, as shown in Fig. 5, a molecular weight of 225000 -t 15000 for the NADdependent enzyme and 215000 & 15000 for the NADP-dependent enzyme.

hfluence of p H and Buffer on Kinetic Constants

From the results given in Table 3. it can be observed that for the NAD-dependent enzyme small

189

L. Guerrillot and J.-P. Vandecasteele

Table 3. Influence o f p H and buffkr on kinetic constants of N A D dependent and NADP-dependent aldehyde dehydrogenuses Purified enzyme preparations obtained after hydroxyapatite chromatography were used. The aldehyde used was hexanal. The concentrations were 1 mM for NAD and 0.5 mM for NADP Conditions of assay

NAD-dependent enzyme

kat/kg KHZP04 pH7.4 K ~ H P ~ O T pH 8.2 pH 8.6 K3IIP207 pH 9.4 K3HP207 GlycineiKOH pH 8.6 Glycine/KOH pH 9.4 Tris-HC1 pH 8.6

0.6

8 c

0.125 0.069 0.142 0.139

pM

9.5

NA DPdependent enzyme

katikg

pM

0.050 0.194 0.292 0.514

150 165 250 690

0.319

450

I 12 I00

04 c

$

2 6

0.2

0 Direction of gel scanning

Fig. 4. Gel electrophoresis of the purified NADP-dependent aldehyde dehydrogenase. Details as in Fig. 3

potassium hydroxide respectively. However, as discussed later, enzyme inactivation takes place at these higher pH values. A particular case is that of TrisHCI buffer in which lower activities were recorded because of the high K, value found in this buffer. This higher value is probably related to the formation of imine bonds between Tris and aldehyde as observed in the case of other enzymes utilizing aldehydes [ 5 ] . For the NADP-dependent enzyme, results given in Table 3 indicate an increase of Vvalues at higher pH values. No results are given with TrisHCl buffer in which very low activities are observed. Inactivation of the enzyme at higher pH values was not observed in this case. Influence of Various Ions on Enzyme Activity

0

'

105

'

~

'

!

I 1

1 , ~

1

2.10~ Molecular weight

I

I , / , ~

~

I

~

3 105

Fig. 5. Moleculur weighf determination 01 NAD-dependent and NADP-dependent aldehyde dehydrogenases by Sephadex G-200 chromutography. A 25 x 750-mm column was used. Elution was performed with 100 mM phosphate buffer (pII 7.1) at a flow rate of 5.6 ml/h. The marker proteins used were: (0)ox liver lactate dehydrogenase ( M , 140000); (A) hog liver fumarase ( M , 204000); (0)ox liver catalase ( M , 247000); (v) cow milk xanthine oxidase ( M , 275000). Kav constants, where K,, = ( V e - Vo)/(Vr-V0) [4], of the purified enzymes: (0)NADP-dependent aldehyde dehydrogenase ; (m) NAD-dependent aldehyde dehydrogenase

variations of K,,, values with pH and buffer composition are observed. V values are more affected and increase with pH. Optimal values of 9 and 9.4 were obtained in potassium pyrophosphate and in glycine!

Influence of various ions has been studied in more detail with preparations twice dialyzed against a 10 mM triethanolamine-HC1 buffer (pH 7.3) containing 16 % glycerol and 7 mM 2-mercaptoethanol. Results given in Table 4 indicate a slight activation by potassium and ammonium ions of the NADdependent enzyme. These activations do not seem due to the increase of ionic strength. Results obtained for the NADP-dependent enzyme indicate a clear activation by addition of salts. Some effect of ionic strength may be involved but the highest activation was obtained by addition of potassium ions. Enzyme Stability

As already stated, glycerol improves the stability of both enzymes. Regarding the NAD-dependent enzyme, a crude extract at 4 "C lost 60% activity in 44 h in the absence of glycerol but only 10% in the presence of 16 % glycerol. Stabilization by reducing

190

Aldehyde Dehydrogenases from Pseudornonas aeruginosa

Table 4.Injluence of various ions on the activity of NAD-dependent and NADP-dependent dehydrogenases The NAD-dependent dehydrogenase was assayed with 1 mM NAD and 0.2 mM butanal, the NADP-dependent dehydrogenase with 0.5 mM NADP and 4 mM pentanal. Triethanolamine buffer contained 0.05 M triethanolamine-HC1 buffer pH 8.6 Buffer

Relative velocities of

K ~ H P z O0.05 ~ , M, pH 8.6 Triethanolamine Triethanolamine 0.1 M KCl Triethanolamine + 0.1 M NaCl Triethanolamine + 0.1 M NH4CI Triethanokdmine + 0.05 M (NH4)zSOa Triethanolaminc + 0.1 M Tris-HCI pH 8.6 Triethanolamine + 0.1 M Na2HP04 pH 8.6 Triethanolamine 0.1 M KzHPOLpH 8.6

+

+

NADdependent enzyme

NADPdependent enzyme

100 65 86 67 83 84 21 75 100

100 7 85 52 49 52 10 72 100

The NAD-dependent aldehyde dehydrogenase was found unstable at alkaline pH values. As shown in Fig.6, rapid inactivation took place at 30 "C at pH 8.6. At pH 7.4 the enzyme is stable in the absence or in the presence of butanal. NAD efficiently protected the enzyme against inactivation thus allowing activity determinations at this pH value. Butanal had no protective effect. The protective effect of glycerol as well as stabilization by thiols were also observed for the NADPdependent enzyme : a fraction from hydroxyapatite chromatography lost 93 % activity in 7 days at 4 "C after removal of 2-mercaptoethanol by dialysis and regained 75 % or 65 % activity upon addition of 1 mM dithiothreitol or 7 mM 2-mercaptoethanol respectively. A purified preparation kept at - 8 'T in presence of 16% glycerol and 3 mM dithiothreitol lost 12% activity in 6 months. Action of Inhibitors of Suljhydryl Groups

Both enzymes were completely inhibited by a 3-min incubation with 0.1 mM p-hydroxy-mercuribenzoate or 1 mM o-iodosobenzoate. A similar incubation with 1 m M sodium iodoacetate inhibited the NADPdependent enzyme by 92"/, but did not inhibit the NAD-dependent enzyme. Determination of Apparent Kinetic Constants

0

Ll

I

0

I

I

I

,

I

I

I 10

5

I

, I

6 25

Time (min)

Fig. 6. Inactivation of NAD-dqendcni uldehyde dehydrogenuse at high p H values. The buffers used were 50 mM potassium pyrophosphate (pH 8.6) or S 0 m M potassium phosphate (pH 7.4). The pH 8.6; (v - a ) incubation temperature was 30 "C. ((r-4) pH 8.6 -t 0.2 mM butanal; (M pH )8.6 1 m M NAD; (A-A) pH 7.4 + 0.2 mM butanal

+

agents was also observed: an NAD-dependent enzyme preparation from hydroxyapatite chromatography lost 87 % activity in 7 days at 4 "C after dialysis in the absence of 2-mercaptoethanol but addition of 1 mM dithiothreitol or 7 mM 2-mercaptoethanol restored 80 % or 70 % activity, respectively. Purified preparations kept at - 8 "C in the presence of 16 glycerol and 1 mM dithiothreitol lost 25 %activity in 6 months.

Apparent kinetic constants have been determined for aldehydes of various chain lengths and are given in Table 5. For the NAD-dependent enzyme, lower K, values were observed for aldehydes of higher chain lengths. Inhibition by excess substrate has been observed with all the aldehydes studied. Variations of V values were small. Apparent K , values for NAD of 44 pM in the presence of 200 pM butanal in 50 mM potassium pyrophosphate buffer (pH 8.6) and 20 pM in the presence of 330 pM butanal in 50 mM potassium phosphate (pH 7.4) have been found. With the NADP-dependent enzyme, the apparent K, values obtained for various aldehydes were higher than with the NAD-dependent enzyme especially for lower aldehydes. They decreased as the chain length increased (by factor of 3500 from ethanal to dodecanal). The V values presented two maxima, for pentanal and for decanal. The presence of more than one enzyme appears an unlikely explanation for this fact as this result was obtained with the homogenous preparation obtained after Sephadex G-200 chromatography and also because the mixed substrate method [I31 applied to the purified enzyme using pentanal and decanal as the two substrates also indicated the presence of a single enzyme. Kinetic constants were impossible to determine for this enzyme in crude extracts probably because of

191

L. Gucrrillot and J.-P. Vandecasteele Table 5. Apparent kinetic constants of NAD-dependent and NADPdependent aldehyde dehydragenases The determinations were done with purified preparation from hydroxyapatite chromatography in 0.05 M potassium pyrophosphate buffer (pH 8 . 6 ) in the presence of 1 mM NAD+ or 0.5 mM NADP+ Substrate chain length

NAD-dependent enzyme

NADP-dependent enzyme

Kill

V

K,

V

PM

mkat/kg

phf

mkat/kg

165 64 45

107 87 132

35000 11 000 2 200 435 250 108 62 25 23 16 10 23 15

22 30 92 95 70 47

9.5

115

% 5.0

92

-1.3

70

so 80

92 63 33 12 8

the presence of the NADP-dependent alcohol dehydrogenases [2] which may also be responsible for the impossibility of detecting the enzyme activity in these extracts at neutral pH. The removal of alcohol dehydrogenases which took place on the DEAE-cellulose column gave preparations of NADP-dependent aldehyde dehydrogenase in which the determination of K, values was possible and gave results similar to those obtained with the most purified preparations. No inhibition by excess substrate was observed with aldehydes of various chain lengths for this enzyme. A K,,, value for NADP of 65 yM was obtained on a preparation from Sephadex G-200 chromatography with 4 mM pentanal in 50 mM potassium pyrophosphate bufYer (pH 8.6). Influence of the Carbon Source Usedfor Growth on the Synthesis of the Enzymes

From the results shown in Table 6, it is apparent that the high-speed supernatants of the extracts which contained the enzymes under study had very similar activities when the carbon source used for growth was glucose, succinate, malonate, n-heptane or n-hexadecane. However, the particulate fraction which sedimented at high speed (100000xg pellet) of extracts from cells grown on n-heptane or n-hexadecane exhibited a high NAD-dependent aldehyde dehydrogenase activity which was absent in extracts from cells grown on other carbon sources. NADP-dependent aldchyde dehydrogenase activity was consistently

Table 6. Effect of carbon source on the aldehyde dehydrogenase activities of Pseudomonas aeruginosa Specific activity was measured in 50 mM pyrophosphate buffer (pH 8.6) with 0.96 mM octanal and 1 mM NAD or 0.5 mM NADP. Carbon source

Specific activity of 1oooooxg pellet

100000 x g supernatant with NAD

with NADP

with NAD

mkatikg 50 mM sodium succinate 50 mM sodium malonate 55 mM glucose 0.4 7; n-heptane 0.6 '4n-hexadecane 0.6% ethanol

1.5 1.2 1.75 I .8 2.1 4.3"

1.25 I .o 1.4 0.92 0.93 17.5

0.2 0.33 0.33 3.5 11.2 0.32

a This value was not significantly increased by addition of 0.1 M KC1.

low in the particulate fractions from these various preparations. These results clearly indicate the 'constitutive' nature of the two soluble aldehyde dehydrogenases studied and the existence of a new NADdependent particulate aldehyde dehydrogenase activity that is induced by growth on n-paraffins. In addition, the results obtained with ethanol as a carbon source suggest the presence in this case of a new soluble NADP-dependent activity.

DISCUSSION The present results establish the existence of two distinct aldehyde dehydrogenases active with aliphatic aldehydes in glucose-grown cells of P . aeruginosa: an NAD-dependent enzyme obtained at a purity of about 20% (spec. act. 0.33 kat/kg) and a NADPdependent enzyme obtained at a purity of about 95 % (spec. act. 0.62 kat/kg). The specific activity obtained for the NADP-dependent enzyme is the highest so far reported for an aldehyde dehydrogenase although recent works report activities of a similar magnitude ~6~71. Both the NADP-dependent and the NAD-dependent enzymes differ from the aldehyde dehydrogenase of P. aeruginosa described by Tiggerstrom and Razzell [S, 91 which is NAD-dependent, induced by growth on ethanol, and dependent for its activity on potassium, ammonium or rubidium ions. In our case, extracts from ethanol-grown cells had a high soluble NADP-dependent aldehyde activity which has not been further characterized. Differences are also apparent with the enzyme of P . fluorescens described by Jakoby [lo] which is activated by phosphate or

192

L. Guerrillot and J.-P. Vandecasteele: Aldehyde Ilehydrogenases from Pseudomonas aeruginosa

arsenate ions. Similitudes however can be found with these two enzymes with respect to involvement of thiol groups and molecular weight. Stabilization by glycerol has also been reported for yeast aldehyde dehydrogenase [ll]. The physiological function of the two enzymes studied here is unknown. Clearly. insufficient evidence is available to conclude on the involvement of any of them in hydrocarbon degradation although their kinetic parameters for middlechain or long-chain aldehydes are compatible with this function. Particular caution in this matter is advisable as a survey of literature indicates the widespread occurrence of non-specific aldehyde dehydrogenases of ill-defined function. A similar situation holds for alcohol dehydrogenases. This point does not seem to have been always given due consideration in research on hydrocarbon metabolism. A further point, bearing on the physiological role of the present enzymes is their ‘constitutive’ nature and the existence of a membrane-bound NADdependent aldehyde dehydrogenase activity induced by growth on n-paraffins, indicating the involvement of the latter enzyme in paraffin metabolism. Bertrand et al. [12] found aldehyde dehydrogenase activity in both the soluble fraction and the particulate fraction of the extracts from paraffin-grown cells of another P. aeruginosa strain, the former activity functioning with both NAD or NADP, and the latter one utilizing only NAD. Although the authors’ interpretation differ, their results suggest that a situation similar to that

which we observed here may be found in other strains of the same species. In view of its involvement in paraffin metabolism, the study of the membrane-bound aldehyde dehydrogenase activity of Pseudonzonas aeruginosa is of great interest. Its purification and characterization are in progress.

REFERENCES I. Tassin, J. P. & Vandecasteele. J. P. (1971) C.R. Hebd. S6ance.v Acad. Sci. Ser. D. Sci. N u t /Pari.yJ 272, 1024- 1027. 2. Tassin, J. P. & Vandecasteele, J. P. (1972) Biochim. Biophys. ActU, 276, 31 -42. 3. Tassin, J. P., Celier, C. & Vandecasteele, J. P. (1973) Biochim. Biophys. Actu, 315, 220- 232. 4. Andrews, P. (1965) Biochem. .I. 96, 595-606. 5. Ogilvie, J. W. & Whitaker, S. C. (1976) Biochim. Biophq’s. Acru, 445, 525 - 536. 6 . Adams, E. & Rosso, G. (1967) J. Bid. Chem. 242, 1802-1814. 7. Callewaert, D. M., Rosemblatt, M. S., Suzuki, K. Sr Tchen; T. T. (1973) J . Biol. Chem. 248. 6009-6013. 8. Van Tigerstrom, R. G. & Razzell, W. E. (1968) J . B i d . Chenz. 243, 269 1 - 2702. 9. Von Tigerstrom, R. G. & Razzell, W. E. (1968) J . Biol. (’hem. 243, 6495 - 6503. 10. Jakoby, W. B. (1958) J . Biol. Chem. 232. 75-87. 2 1 . Bradhury, S. L. & Jakoby, W. B. (1972) Proc. Nut1 Acad. Sci. U . S . A .69,2373 - 2376. 12. Bertrand, J. C., Gallo, M. & Azoulay, E. (1973) Biochimir (Paris) 55,343 350. 13. Dixon, M. & Webb, E. C. (1964) Enzymes 2nd edn, p. 86, Longmans, London.

L. Guerrillot and J.-P. Vandecastcele, Service de Biochimie, Inslitut Frdnqais du Petrole, Boite postale 31 1, F-92506 Rued-Malmaison-Cedex, France

~