ARCHIVES
OF
BIOCHEMISTRY
AND
BIOPHYSICS
176, 136-143 (1976)
Pyridine Nucleotide Transhydrogenase from Pseudomonas aeruginosa: Purification by Affinity Chromatography and Physicochemical Properties’ BENDICHT Department
of Chemistry,
WERMUTH2 University
AND
of California,
Received
February
NATHAN
0. KAPLAN
San Diego, La Jolla,
California
92093
9, 1976
Pyridine nucleotide transhydrogenase from Pseudomonas aeruginosa was purified 150-fold by affinity chromatography on immobilized 2’-AMP. The binding of the enzyme is pH dependent. Elution was achieved with 2’-AMP, NADP+, or NADPH but not with Y-AMP, NAD+, or NADH. The enzyme preparations appeared to be homogeneous in gel chromatography and ultracentrifugation, but only if these procedures were carried out in the presence of 2’-AMP or NADP+. With 2’-AMP a sedimentation coefficient of 34 S, a molecular weight of 1.6-1.7 million, and a Stokes’ radius of 11.7 nm were determined. In the presence of NADP+ the sedimentation coefficient was 42 S and the molecular weight was 6.4 million. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate revealed one kind of subunit with a molecular weight of 54,000. This was consistent with results from amino acid analyses and paper chromatography of peptides. Eight molar urea inactivated the enzyme but did not dissociate it into subunits. Full activity was restored after dialysis against urea-free buffer by mercaptoethanol and flavin-adenine dinucleotide.
Pyridine nucleotide transhydrogenase from Pseudomonas aeruginosa catalyzes the reaction NADPH + NAD+ % NADP+ + NADH. 2’-AMP-nucleotides are enzyme activators (1, 21, with the exception of NADP+, which is a potent inhibitor (2). Cohen and Kaplan (1) and Louie et al. (3) have demonstrated that this enzyme is heterogeneous and that 2’-AMP-nucleotides influence an association-dissociation equilibrium. In the absence of nucleotides the enzyme occurs in a polymeric, rodlike structure with a sedimentation coefficient of 120 S. Upon addition of 2’-AMP or NADP+ cylindrical protomers with an S value of 34 are formed. Van den Broek and co-workers (4) have shown that pyridine ’ This work was supported by Grant CA 11683 from the National Institutes of Health and Grant BC-SO-Q from the American Cancer Society. 2 Recipient of a Swiss National Science Foundation Postgraduate Fellowship. Present address: Medizinisch-Chemisches Institut der Universitat Bern, CH-3012 Bern, Switzerland.
nucleotide transhydrogenase from Acetovinelandii also exists in different molecular states. The NADP+ stabilizes small molecular forms and NADPH favors the formation of aggregates of this enzyme. Although the kinetics of the reactions regulated by adenine nucleotides have been studied in detail (2, 3, 51, there is little information concerning the structural properties of the enzyme. This was caused by a laborious and time-consuming purification procedure (l), which hampered the production of large amounts of enzyme. We have shown recently (6) that several dehydrogenases could be purified by affinity chromatography on &substituted immobilized adenine nucleotides. In this report we demonstrate that the transhydrogenase from Pseudomonas aeruginosa can be partially purified by affinity chromatography on immobilized 2’-AMP. The physicochemical properties of the enzyme were investigated and a model of the molecular structure is suggested. A pre-
batter
136 Copyright All rights
0 1976 by Academic Press, Inc. of reproduction in any form reserved.
PHYSICOCHEMICAL
PROPERTIES
liminary account of this work has been presented previously (7). MATERIALS
AND
METHODS
Bovine serum albumin, ovalbumin, beef liver catalase, and horse spleen ferritin were purchased from Sigma. Chicken heart malate dehydrogenase, rabbit muscle pyruvate kinase, and fibrinogen were gifts from Mr. B. Bachman, Dr. L. H. Lazarus, and Dr. R. F. Doolittle, respectively, of the Chemistry Department (University of California, San Diego). Acetylcholinesterase form D was prepared from electric eel tissue (8). DEAE-cellulose3 (DE-52) was obtained from Whatman and Sepharose 4B was obtained from Pharmacia Fine Chemicals. 2’-AMP-Sepharose and 5’-AMP-Sepharose were prepared as described earlier and contained both 10 to 12 pmol ligand/ml of settled Sepharose (6). Acrylamide, N, N’-methylenebis-acrylamide and sodium dodecyl sulfate were obtained from Bio-Rad Laboratories. Pyridine nucleotides and analogs were supplied by P-L Biochemicals; 2’-AMP, 5’-AMP, and FAD by Sigma; 2-mercaptoethanol, dithiothreitol, and urea by Calbiochem; and CNBr by Aldrich. Fluorescamine was a gift from Hoffman-La Roche.
Enzyme Activity Transhydrogenase was assayed by following the reduction of (TN)AD+ at 400 nm. In a total volume of 1 ml the standard assay mixture combined 0.1 M Tris-Cl, pH 7.4, 0.3 mM NADPH, and 0.5 mM (TN)AD+. The reaction was started by the addition of enzyme. NADPH was replaced by 0.3 mM NADH when a nonactivating assay system was desired. One enzyme unit is defined as the reduction of 1 /*mol of (TN)AD+/min. A molar extinction coefficient of 11.3 x lo3 was used for (TN)ADH at 400 nm (9).
Protein Determination Proteins of crude extracts were determined by the biuret method (10) and protein concentrations of purified enzyme were measured with fluorescamine (11) using bovine serum albumin as standard.
Ultracentrifugal
Analyses
A Spinco Model E analytical centrifuge equipped with a photoelectric scanning system was used. Sedimentation coefficients were determined by sedimentation velocity experiments using interference 3 Abbreviations used: P’-AMP-Sepharose, 8-(6aminohexyll-amino-2’-AMP-Sepharose; 5’-AMPSepharose, 8-(6-aminohexyll-amino-5’-AMP-Sepharose; (TN)AD+, thio-nicotinamide-adenine dinucleotide. FAD, flavin-adenine dinucleotide; DEAE-cellulose, 0-(diethylaminoethyl) cellulose.
137
OF TRANSHYDROGENASE
optics, as well as utilizing active enzyme centrifugation according to Kemper and Everse (12). Molecular weight studies were made by the sedimentation equilibrium method detailed by Yphantis (13). All experiments were carried out in 0.1 M Na phosphate, pH 7.0, containing 1 mM EDTA and 1 mM dithiothreitol.
Determination
of Stokes’ Radii
A calibrated Sepharose 4B column as described by Siegel and Monty (14) was used. Markers were blue dextran (void volume), acetylcholinesterase form D (r s = 13.6 nm), fibrinogen (11.6 nm), ferritin (7.9 nm), catalase (5.2 nm), and bovine serum albumin (3.6 nm).
Sodium Dodecyl Sulfate Gel Electrophoresis
Polyacrylamide
The methods of Weber and Osborn (15) as well as Laemmli (16) were followed. The gels were calibrated with bovine serum albumin (M, = 67,000), pyruvate kinase (57,000), ovalbumin (45,000), and malate dehydrogenase (32,500). The system of Laemmli without sodium dodecyl sulfate was used for regular polyacrylamide gel electrophoresis. Gels were stained for proteins with Coomassie brilliant blue and activity bands were made visible by incubating the gels in 0.1 M sodium phosphate buffer containing NADPH (2 mglml) and nitrobluetetrazolium (0.5 mg/ml).
Amino
Acid Analyses
Hydrolysis of transhydrogenase was performed at 110°C in 6 N HCl for 24, 48, and 72 h, respectively. Amino acids were analyzed on a Beckman Model 119 automatic amino acid analyzer using a single column system. Tryptophan was determined spectrophotometrically by the method of Bencze and Schmid (17).
CNBr Cleavage Peptides
and Chromatography
of
Transhydrogenase was dialyzed in 0.1 N HCl at a concentration of 1 mg/ml. CNBr was added to the enzyme solution at a concentration of 5 mg/ml and incubated for 24 h at room temperature. The peptides were then lyophilized. Separation of the peptides was performed by descending chromatography in n-butanol-acetic acid-water-pyridine (15:3:12:10) on Whatman 3MM and stained with a Cd/ninhydrin reagent.
Purification of Pyridine hydrogenase
Nucleotide
Trans-
Cell growth. Pseudomonas aeruginosa cells were grown from a stock culture in a medium described
138
WERMUTH
AND
by Cohen and Kaplan (1). The cells were frozen immediately after harvesting and stored at -20°C. Disruption of cells. Frozen cells (800 g) were thawed and suspended in 2 liters of 0.1 M sodium phosphate buffer, pH 7.0, containing 1 mM EDTA. Cells were broken by passing through a Sorvall Ribi cell fractionator (Model RF-11 at 700 atm. Two liters of phosphate buffer were added and the cell debris was removed by centrifugation at 18,000g for 30 min. The precipitate was resuspended in 2 liters of buffer and centrifuged as before. The supernatants were combined and made up to contain 0.05 M mercaptoethanol. 2’-AMP-Se&rose. To the crude extract were added 40 ml of 2’-AMP-Sepharose and it was kept suspended for 18 h by light stirring. The Sepharose was then collected by filtration through a Buchner funnel (Corning, 36060, C) and washed with 3 liters of 0.1 M phosphate buffer, pH 7.0, containing 1M NaCl, 0.05 M mercaptoethanol, and 1 m&r EDTA: followed by 2 liters of 0.1 M sodium borate buffer, pH 8.0, containing 0.05 M mercaptoethanol and 1 mM EDTA. Transhydrogenase was eluted by 0.1 M sodium borate, pH 8.5, containing 10 mM 2’-AMP. The eluate was adjusted to pH 7 by 0.5 M phosphoric acid and dialyzed against three changes, 6 liters each of 0.02 M sodium phosphate buffer, pH 7.0 containing 0.05 M mercaptoethanol and 1 mM EDTA. DEAE-cellulose. The dialyzed enzyme solution was applied to a column 2 cm in diameter, containing 150 ml of packed DE-52, equilibrated in dialysis buffer. The column was washed with 5 column volumes of buffer. Bound proteins were then eluted by a 0 to 0.5 M NaCl gradient made up in column buffer. Fractions were collected and the tubes containing transhydrogenase were pooled. The enzyme was concentrated by precipitation with ammonium sulfate to 80% of saturation. After 1 h the suspension was centrifuged at 18,OOOg for 30 min. The precipitate was dissolved in 0.1 M sodium phosphate buffer containing 1 mM dithiothreitol and 1 mM EDTA and dialyzed once against 2 liters of the same buffer. Sepharose 4B. The dialyzed enzyme preparation was subjected to gel chromatography on Sepharose 4B by applying 2-ml portions on a column, 1.1 cm in diameter, containing 55 ml of Sepharose, equilibrated in 0.1 M sodium phosphate buffer, pH 7.0, TABLE PURIFICATION
OF PYRIDINE
Volume Extract 2’-AMP-Sepharose DEAE-Cellulose Sepharose 4B a Elution
volume
5,800 365 230 10” corresponding
NUCLEOTIDE
(ml)
KAPLAN
containing 1 mM dithiothreitol and 1 mM EDTA. Fractions were collected and analyzed for transhydrogenase activity. The enzyme was checked for homogeneity by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Fractions containing homogeneous enzyme were pooled and stored at 4°C. RESULTS
Purification
A summary of the purification procedures for pyridine nucleotide transhydrogenase from Pseudomonas aeruginosa is given in Table I. In a first step the enzyme was bound to 2’-AMP-Sepharose and then eluted with free 2’-AMP. In order to get optimum binding and elution conditions various parameters were tested for their influence on the binding of the enzyme to the immobilized 2’-AMP. Binding studies at different pH values showed that the capacity of 2’-AMP-Sepharose for transhydrogenase depended upon the pH. Between pH 6 and 7.5, approximately 450 enzyme units were bound per milliliter of settled 2’-AMP-Sepharose. An increase in pH above pH 8 resulted in a decrease of the binding capacity. At pH 9 only 200 units of transhydrogenase were adsorbed by the gel-bound 2’-AMP. The same pH dependence was observed for the elution of gel-bound transhydrogenase, Any pH lower than 7.5 made the 10 mM 2’AMP or 1 M NaCl ineffective. Elution, hqwever, was achieved by these substances at pH 8. In addition to the influence of the pH, the effects of various adenine nucleotides were investigated. 2’-AMP, 5’-AMP, NAD+, NADP+, NADH, and NADPH were tested for the ability to elute the enzyme from the 2’-AMP-Sepharose. The results are summarized in Fig. 1. Only the 2’-AMP nucleotides, 2’-AMP, NADP+, and I
TRANSHYDROGENASE
Total units (IU) 14,760 10,452 6,480 3,305
to the high molecular
of Transhydrogenase
FROM Pseudomonas
Total protein (ma)
Specific activity W/md
40,600 219 78 15
0.36 48 83 220
enzyme forms.
aeruginosa Yield 100 71 44 22
(%)
PHYSICOCHEMICAL Control S.-AMP
-
2.-AMP NAD+ NADP+ NADH NADPH Na Po40.5M. NaCI
1M 25
50
139
PROPERTIES OF TRANSHYDROGENASE
75
ACTIVITY(%) FIG. 1. The influence of adenine nucleotides and salts on the elution of pyridine nucleotide transhydrogenase from 2’-AMP-Sepharose. Transhydrogenase (40 IU) was incubated with 2 ml of 2’-AMPSepharose in 0.1 M Tris-citric acid buffer, pH 8, containing 0.05 M mercaptoethanol and 0.1 mu FAD. After 30 min the Sepharose was freed from buffer on a Buchner funnel and 100-mg aliquots were suspended in 0.9 ml of the Tris-citric acid buffer containing the desired amounts of salts or adenine nucleotides. The samples were agitated for 30 min and centrifuged and the supernatants were assayed for enzyme activity. Controls with underived Sepharose 4B were carried out in parallel to correct for activating or inhibiting effects of the adenine nucleotides. The control with underived Sepharose in the absence of any effecters was taken as 100% activity. The phosphate used was adjusted to pH 8 by NaOH.
NADPH, eluted the enzyme. Y-AMP, NAD+, and NADH eluted less than 25% of the bound enzyme activity. Some elution was also observed with 1 M NaCl but not with 0.5 M sodium phosphate. A 130- to GO-fold purification was obtained by the 2’-AMP-Sepharose step. However, this enzyme preparation was not yet homogeneous as shown by polyacrylamide gel electrophoresis, which revealed four protein bands, but only one activity band. Further purification was achieved by ion exchange chromatography on DEAE-cellulose and gel filtration on Sepharose 4B. Figure 2 shows the elution profile from a Sepharose 4B gel filtration experiment. About 60% of the enzyme activity was eluted in the void volume of the column. The rest emerged as a broad shoulder, and rechromatography of these
fractions yielded a symmetrical peak. From a calibrated column a Stokes’ radius of 11.7 nm was estimated for this enzyme form (inset, Fig. 2). The homogeneity of the two obtained enzyme forms was tested by electrophoresis on polyacrylamide gels. The enzyme which was eluted in the void volume did not penetrate a 4% polyacrylamide gel. However, after addition of 5 mM 2’-AMP to the enzyme samples, a single protein band was observed which coincided with a band stained for activity. A specific activity of 220 IU/mg of protein was obtained for this form independent of the presence or absence of 2’-ATP. The low molecular form showed two protein bands on polyacrylamide gels, but only one staining for activity. The band which was also stained for activity had the same migration rate as the 2’-AMP-treated high molecular form. A specific activity of 104 IU/ mg of protein was determined. When the activity of the fractions from the Sepharose 4B gel filtration was measured with NADH it was for all fractions less than
Eluate
volume
Iml)
FIG. 2. Gel filtration of pyridine nucleotide transhydrogenase on Sepharose 4B. A sample (2 ml) of pyridine nucleotide transhydrogenase after purification on DEAE-cellulose was chromatographed on a Sepharose 4B column (1 x 60 cm) equilibrated with 0.1 M Na phosphate buffer, pH 7.0, containing 1 mM EDTA and 1 mM dithiothreitol (0). The eluate between 30 and 37 ml was concentrated and rechromatographed on the same column (Al. Activity for this profile is one-tenth of the given scale. The inset shows a plot of normalized elution volume (K,,) versus the Stokes’ radius of various proteins. B, bovine serum albumin; C, catalase; Fe, ferritin; Fi, fibrinogen; A, acetylcholinesterase. The position of transhydrogenase (T) is marked by a vertical bar.
140
WERMUTH
AND KAPLAN
10% compared to the activity obtained with NADPH. An approximately lo-fold increase of the activity was achieved when 1 mu 2’-AMP or 1 mM CaCl, was added to the NADH/(TN)AD+ assay system. Oligomeric
Fkms
1.6 -
and Subunits
It has been shown by Cohen and Kaplan (1) that pyridine nucleotide transhydrogenase from Pseudomonas aeruginosa is dissociated into a low molecular form by 2’-AMP and NADP+. In the presence of either of these two nucleotides they obtained a sedimentation coefficient of 33.8 S. As anticipated an S value of 34 was obtained for our enzyme preparation in the presence of at least 1 mM 2’-AMP. At 2’AMP concentrations below 1 mM or in the absence of the effector, the high molecular form gave a sedimentation pattern indicating nonhomogeneous enzyme forms. Most of the low molecular enzyme sedimented as the 34 S form even in the absence of 2’-ATP, but a small percentage of the material always sedimented at a much faster rate. In the presence of 1 mM NADP+ a welldefined boundary was observed again; however, a sedimentation coefficient of 42 S was calculated from its sedimentation rate. Molecular weight determinations were performed for the 34 and 42 S forms. In both cases a plot of the logarithm of fringe displacement versus the square of rotational distance yielded straight lines (Fig. 3). The calculated weight average molecular weight for the 34 S form was 1.6 x 106. A value of 1.7 x lo6 was obtained from the Stokes’ radius and the sedimentation coefficient. For the 42 S form a weight average molecular weight of 6.4 x lo6 was calculated from the sedimentation equilibrium experiments. Table II gives a summary of physicochemical parameters of transhydrogenase obtained by gel flltration and ultracentrifugation. In order to get some information on the subunit composition of transhydrogenase, the high molecular form was submitted to gel electrophoresis in the presence of sodium dodecyl sulfate. A single protein band was obtained in either a phosphate buffer or a Tris-glycine buffer system. The
-1
,
I 49.2 SQUARE
OF
I 49.4
I 49.6
ROTATIONAL
DISTANCE
FIG. 3. Molecular weight determination of pyridine nucleotide transhydrogenase from sedimentation equilibrium experiments. The solvent was 0.1 M Na phosphate, pH 7.O, containing 1 mM dithiothreito1 and 1 mM EDTA. The initial protein concentration was 0.4 mg/ml. Sedimentation was for 20 h at (0) 6994 rpm in the presence of 10 mM P’-AMP and at 2994 rpm in the presence of 1 mM NADP+ (0). TABLE II SUMMARY OF PHYSICOCHEMICAL PARAMETERS OF PYRIDINE NUCLEOTIDE TRANSHYDROGENASE FROM Pseudomonas aeruginosa
Enzyme in presence of 2’-AMP Stokes’ radius (nm) Sedimentation coefficient” (s~~,~) Molecular weight;a ultracentrifugation gel filtration
11.7 34 1.6 x lo6 1.7 x 106
NADP+ 42 6.4 x 10”
a A value of 0.73 was used for the partial specific volume as calculated from the amino acid analysis (18).
band migrated between the marker proteins pyruvate kinase and ovalbumin. No difference in the relative migration rate was observed in 10% as well as in 12.5% gels (Fig. 4). A molecular weight for the subunit of 54,000 M, was estimated from the calibration curves. When transhydrogenase was incubated in 8 M urea and then submitted to gel electrophoresis in the presence of 8 M urea, it did not-in contrast to the marker protein bovine serum albumin - penetrate into 10% acrylamide gel.
PHYSICOCHEMICAL
PROPERTIES OF TRANSHYDROGENASE
141
ever, was much more pronounced when inactivated transhydrogenase was reactivated. In the absence of FAD and mercaptoethanol no reactivation was observed. Three percent reactivation was observed with 0.1 mM FAD, 36% with 1% mercaptoethanol, and 95% of the original activity
si B+Gsee+ MOBILITV FIG. 4. Determination of the subunit molecular weight of pyridine nucleotide transhydrogenase by sodium dodecyl sulfate polyacrylamide gel electrophoresis. A Tris-glycine buffer system, pH 8.3, as described by Laemmli (16) was used. B, bovine serum albumin; P, pyruvate kinase; 0, ovalbumin; M. malate dehydrogenase. The position of transhydrogenase (T) is marked by vertical bars. (0) 10% acrylamide, (A) 12.5% acrylamide.
The fact that only one protein band was seen on sodium dodecyl sulfate polyacrylamide gels, however, does not rule out the possible existence of more than one type of subunit with the same molecular weight. In order to rule out this possibility, amino acid analyses and chromatography of CNBr-peptides were carried out. The result of the amino acid analyses is given in Table III. A partial specific volume of 0.73 cm3/g was calculated from this amino acid composition (18). Paper chromatography of cyanogen bromide-treated transhydrogenase revealed a total of six ninhydrin spots. This is almost in agreement with the methionine content of the enzyme and suggests that the protein is composed of only one kind of subunit.
TABLE III
AMINO ACID COMPOSITION IN RESIDUES PER SUBUNIT (54,000 MOLECULAR WEIGHT) OF P~RIDINE NUCLEOTIDE TRANSHYDROGENASE FROM Pseudomonas aeruginosa”
Aspartic acid Threonineb Serine* Glutamic acid Proline Glycine Alanine Valine Methionine
60 23 38 44 19 48 37 38 6
Isoleucine’ Leucine’ Tyrosine Phenylalanine Histidine Lysine Arginine Tryptophan ‘/&ystine
36 39 18 21 9 17 28 7 nd’l
a Average of determinations after 24,48, and 72 h of hydrolysis. * Values extrapolated from 24, 48, and 72 h to zero hour. ’ Values after 72 h of hydrolysis. rl Not determined.
Urea Inactivation
Gel electrophoresis in the presence of 8 M urea has shown that transhydrogenase is resistant to dissociation by this denaturing agent. The influence of different urea concentrations on the enzyme activity was therefore tested. Since it was known that FAD and mercaptoethanol protect the enzyme against activity losses (1) their effect on the urea inactivation was also investigated. Figure 5 shows the dependence of enzyme activity on the urea concentration in presence or absence of FAD and/or mercaptoethanol. Indeed, some protection by FAD and mercaptoethanol was observed. The influence of these two reagents, how-
DREA]
(M)
FIG. 5. Inactivation of pyridine nucleotide transhydrogenase by urea. Test tubes containing 0.95 ml of 0.1 M sodium phosphate, pH 7.0 and various concentrations of urea, FAD, and/or mercaptoethanol were prepared. Fifty microliters of transhydrogenase (200 IU/ml) were added and incubated at 4°C for 1 h before the activity was measured. Samples of fully inactivated transhydrogenase were dialyzed once against 100 vol of 0.1 M sodium phosphate buffer, pH 7.0, containing different amounts of FAD and/or mercaptoethanol. The points on the right indicate the effects aRer dialysis and removal of the urea. (0) Urea only; (A) urea in presence of 1% mercaptoethanol, (0) 0.1 mM FAD, (0) 1% mercaptoethanol plus 0.1 mM FAD. See text for further explanations.
142
WERMUTH
AND
was recovered in the presence of 0.1 mM FAD and 1% mercaptoethanol. Five molar guanidine appears to inactivate the enzyme irreversibly. DISCUSSION
Cohen and Kaplan (1) have described a purification method which starts with two ammonium sulfate precipitations, a timeconsuming and laborious procedure. A fivefold purification in a yield of 44% was obtained in these two purification steps. Affinity chromatography on 2’-AMP-Sepharose, on the other hand, gave a 150-fold purification and a yield of 70% in one step. No centrifugations of large volumes are necessary and the small amounts of 2’AMP-Sepharose which are used to achieve complete binding of transhydrogenase are easy to handle. Barry and O’Carra (19) have suggested some criteria to discriminate between biospecific and nonspecific binding of dehydrogenases to affinity adsorbants. Elution of the protein must be accomplished by low concentrations of biospecific ligands, such as substrates, inhibitors, or activators. Nonspecific agents such as salts or organic solvents should not elute the enzymes or change the elution pattern. According to these criteria the binding of transhydrogenase to 2’-AMP-Sepharose is nearly all biospecific. Neither NaCl up to 0.5 M nor the adenine nucleotides 5’-AMP, NAD+, or NADH eluted the enzyme from the column and elution was only obtained by 2’-phosphate adenine nucleotides. The strong pH dependence observed is typical for many dehydrogenases. From their studies, Dean and co-workers (20) concluded that each enzyme is eluted at a characteristic pH value corresponding to the pK of a specific amino acid at the binding site. Based on this hypothesis an amino acid with an apparent pK of about 9 interacts with the 2’AMP nucleotides. This finding could be related to the activation of the enzyme. From FAD titration studies, Cohen and Kaplan determined a subunit molecular weight for transhydrogenase of 40,000 to 45,000. This value, however, was based on a protein determination using bovine serum albumin as a standard. The direct
KAPLAN
approach of sodium dodecyl sulfate polyacrylamide gel electrophoresis described in this paper yielded a subunit molecular weight of 54,000. Assuming a molecular weight of 1.6 to 1.7 million for the protomer we find approximately 30 subunits per active enzyme unit, which is in agreement with the results of Louie et al. (3). Their electron micrographs show seven membered rings and regular rectangular images which are assumed to be on the side view of the rings. Four of these rings form one enzyme unit, which amounts to 28 subunits per protomer. Since 8 M urea did not dissociate the enzyme, the subunits must be held together by strong forces. Covalent bonds, however, can be excluded since complete dissociation into 54,000 units was obtained by sodium dodecyl sulfate even in the absence of mercaptoethanol. Cohen and Kaplan (1) have shown that in the presence of NADP+, the transhydrogenase exists in a dissociated form with a sedimentation coefficient of 34 S. Reexamination of these results indicates that in the presence of NADP+, transhydrogenase occurs as a small aggregate of four 34 S units.4 The occurrence of small aggregates, on the other hand, is supported by electron micrographs of transhydrogenase which in the from Acetobacter vinelandii, presence of NADP+ show small particles and short rods composed of three to five of these particles (4). It is, however, difficult to compare our results with the earlier study of Cohen and Kaplan, since they had obtained their values at a higher pH. Sabo et al. (211, for example, have shown that the decameric lysine decarboxylase from Escherichia coli aggregates at lower pH values forms long rods and dissociates into dimers at elevated pH. It was suggested by Louie et al. (3) that the dissociation-association equilibrium between high and low molecular transhydrogenase forms could regulate the enzyme activity. The present results, however, show that all active forms of the enzyme require nucleotides * As yet, it has not been clarified whether these small particles induced by NADP+ play a role in the regulation of the transhydrogenase activity (Widmer and Kaplan, submitted for publication).
PHYSICOCHEMICAL
PROPERTIES OF TRANSHYDROGENASE
containing a 2’-AMP moiety for full activity and that the state of aggregation has no direct influence on the catalytic activity (see also Widmer and Kaplan).4 The biological significance of the association-dissociation equilibrium, which is greatly influenced by 2’-AMP-nucleotides therefore rests unclear. Further investigations concerning this problem are in progress in this laboratory. Note added in proof. We would like to thank Dr. Peter Brodelius for calling our attention to a paper now in press which is in agreement with the data reported in our manuscript (B. Hbjeberg, P. Brodelius, J. Rydsttim, and K. Mosbach, Affinity chromatography and binding studies on immobilized 5’AMP and 2’,5’-ADP of nicotinamide nucleotide transhydrogenase from Pseudomonas aeruginosa, Eur. J. B&hem., in press).
REFERENCES 1. COHEN, P. T., AND KAPLAN, N. 0. (1970). J. Biol. Chem. 245, 2825-2836. 2. COHEN, P. T., AND KAPLAN, N. 0. (1970)5. Biol. Chem. 245, 4666-4672. 3. LOUIE, D. D., KAPLAN, N. O., AND MCLEAN, J. D. (1972) J. Mol. Biol. 70, 651-664. 4. VAN DEN BROEK, H. W. J., SANTEMA, J. S., AND VEEGER, C. (1971)Eur. J. B&hem. 24,46-54. 5. RYDSTR~M, J., HOEK, J. B., AND HOJEBERG, B. (1973) Biochem. Biophys. Res. Commun. 52,
421-429. 6. LEE, C.-Y., LAPPI, D. A., WERMUTH, B., EvERSE, J., AND KAPLAN, N. 0. (1974) Arch. B&hem. Biophys. 163, 561-569.
143
7. WERMUTH, B. (1975) Fed. Proc. 34, 682. 8. WERMIJTH, B. (1973) Dissertation, Universitat
Bern. 9. PABST LABORATORIES (1961) “Ultraviolet
Absorption Spectra of Pyridine Nucleotide Coenzymes and Coenzyme Analogues,” Pabst Laboratories Circular OR-18. 10. HENRY, R. J., SOBEL, C., AND BERKMAN, S. (1957) Anal. Chem. 29, 1491-1495. 11. %HLEN, P., STEIN, S., DAIRMAN, W., AND UDENFRIEND, S. (1973) Arch. B&hem. Bit+ phys. 155, 213-220.
12. KEMPER, D. L., AND EVERSE, J. (1973) in Methods in Enzymology (Hirs, C. W. H., and Timasheff, S. N., eds.), Vol. XVII, pp. 67-82, Academic Press, New York. 13. YPHANTIS, D. (1964) Biochemistry 3, 297-317. 14. SIEGEL, L. M., AND MONTY, K. J. (1966) Biochim. Biophys. Actu 112, 346-362. 15. WEBER, K., AND OSBORN, M. (1969). J. Biol. Chem. 244, 4401-4412. 16. LAEMMLI, U. K. (1970) Nature (London) 227, 680-685. 17. BENCZE, W. L., AND SCHMID, K. (1957) Anal. Chem. 29, 1193-1196. 18. COHN, E. J., AND EDSALL, J. T. (1943) in Pro-
teins, Aminoacids and Peptides (Cohn, E. J. and Edsall, J. T., eds.), pp. 370-381, Hafner, New York. 19. BARRY, S., AND O’CARRA, P. (1973) Biochem. J. 13’5, 595-607. 20. LOWE, C. R., HARVEY, M. J., AND DEAN, P. D. G. (1974) Eur. J. Biochem. 41, 347-351. 21. SABO, D. L., BOEKER, E. A., BEYERS, B., WARON, H., AND FISCHER, E. H. (1974) Biochemistry
13, 662-670.
OF
BIOCHEMISTRY
AND
BIOPHYSICS
176, 136-143 (1976)
Pyridine Nucleotide Transhydrogenase from Pseudomonas aeruginosa: Purification by Affinity Chromatography and Physicochemical Properties’ BENDICHT Department
of Chemistry,
WERMUTH2 University
AND
of California,
Received
February
NATHAN
0. KAPLAN
San Diego, La Jolla,
California
92093
9, 1976
Pyridine nucleotide transhydrogenase from Pseudomonas aeruginosa was purified 150-fold by affinity chromatography on immobilized 2’-AMP. The binding of the enzyme is pH dependent. Elution was achieved with 2’-AMP, NADP+, or NADPH but not with Y-AMP, NAD+, or NADH. The enzyme preparations appeared to be homogeneous in gel chromatography and ultracentrifugation, but only if these procedures were carried out in the presence of 2’-AMP or NADP+. With 2’-AMP a sedimentation coefficient of 34 S, a molecular weight of 1.6-1.7 million, and a Stokes’ radius of 11.7 nm were determined. In the presence of NADP+ the sedimentation coefficient was 42 S and the molecular weight was 6.4 million. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate revealed one kind of subunit with a molecular weight of 54,000. This was consistent with results from amino acid analyses and paper chromatography of peptides. Eight molar urea inactivated the enzyme but did not dissociate it into subunits. Full activity was restored after dialysis against urea-free buffer by mercaptoethanol and flavin-adenine dinucleotide.
Pyridine nucleotide transhydrogenase from Pseudomonas aeruginosa catalyzes the reaction NADPH + NAD+ % NADP+ + NADH. 2’-AMP-nucleotides are enzyme activators (1, 21, with the exception of NADP+, which is a potent inhibitor (2). Cohen and Kaplan (1) and Louie et al. (3) have demonstrated that this enzyme is heterogeneous and that 2’-AMP-nucleotides influence an association-dissociation equilibrium. In the absence of nucleotides the enzyme occurs in a polymeric, rodlike structure with a sedimentation coefficient of 120 S. Upon addition of 2’-AMP or NADP+ cylindrical protomers with an S value of 34 are formed. Van den Broek and co-workers (4) have shown that pyridine ’ This work was supported by Grant CA 11683 from the National Institutes of Health and Grant BC-SO-Q from the American Cancer Society. 2 Recipient of a Swiss National Science Foundation Postgraduate Fellowship. Present address: Medizinisch-Chemisches Institut der Universitat Bern, CH-3012 Bern, Switzerland.
nucleotide transhydrogenase from Acetovinelandii also exists in different molecular states. The NADP+ stabilizes small molecular forms and NADPH favors the formation of aggregates of this enzyme. Although the kinetics of the reactions regulated by adenine nucleotides have been studied in detail (2, 3, 51, there is little information concerning the structural properties of the enzyme. This was caused by a laborious and time-consuming purification procedure (l), which hampered the production of large amounts of enzyme. We have shown recently (6) that several dehydrogenases could be purified by affinity chromatography on &substituted immobilized adenine nucleotides. In this report we demonstrate that the transhydrogenase from Pseudomonas aeruginosa can be partially purified by affinity chromatography on immobilized 2’-AMP. The physicochemical properties of the enzyme were investigated and a model of the molecular structure is suggested. A pre-
batter
136 Copyright All rights
0 1976 by Academic Press, Inc. of reproduction in any form reserved.
PHYSICOCHEMICAL
PROPERTIES
liminary account of this work has been presented previously (7). MATERIALS
AND
METHODS
Bovine serum albumin, ovalbumin, beef liver catalase, and horse spleen ferritin were purchased from Sigma. Chicken heart malate dehydrogenase, rabbit muscle pyruvate kinase, and fibrinogen were gifts from Mr. B. Bachman, Dr. L. H. Lazarus, and Dr. R. F. Doolittle, respectively, of the Chemistry Department (University of California, San Diego). Acetylcholinesterase form D was prepared from electric eel tissue (8). DEAE-cellulose3 (DE-52) was obtained from Whatman and Sepharose 4B was obtained from Pharmacia Fine Chemicals. 2’-AMP-Sepharose and 5’-AMP-Sepharose were prepared as described earlier and contained both 10 to 12 pmol ligand/ml of settled Sepharose (6). Acrylamide, N, N’-methylenebis-acrylamide and sodium dodecyl sulfate were obtained from Bio-Rad Laboratories. Pyridine nucleotides and analogs were supplied by P-L Biochemicals; 2’-AMP, 5’-AMP, and FAD by Sigma; 2-mercaptoethanol, dithiothreitol, and urea by Calbiochem; and CNBr by Aldrich. Fluorescamine was a gift from Hoffman-La Roche.
Enzyme Activity Transhydrogenase was assayed by following the reduction of (TN)AD+ at 400 nm. In a total volume of 1 ml the standard assay mixture combined 0.1 M Tris-Cl, pH 7.4, 0.3 mM NADPH, and 0.5 mM (TN)AD+. The reaction was started by the addition of enzyme. NADPH was replaced by 0.3 mM NADH when a nonactivating assay system was desired. One enzyme unit is defined as the reduction of 1 /*mol of (TN)AD+/min. A molar extinction coefficient of 11.3 x lo3 was used for (TN)ADH at 400 nm (9).
Protein Determination Proteins of crude extracts were determined by the biuret method (10) and protein concentrations of purified enzyme were measured with fluorescamine (11) using bovine serum albumin as standard.
Ultracentrifugal
Analyses
A Spinco Model E analytical centrifuge equipped with a photoelectric scanning system was used. Sedimentation coefficients were determined by sedimentation velocity experiments using interference 3 Abbreviations used: P’-AMP-Sepharose, 8-(6aminohexyll-amino-2’-AMP-Sepharose; 5’-AMPSepharose, 8-(6-aminohexyll-amino-5’-AMP-Sepharose; (TN)AD+, thio-nicotinamide-adenine dinucleotide. FAD, flavin-adenine dinucleotide; DEAE-cellulose, 0-(diethylaminoethyl) cellulose.
137
OF TRANSHYDROGENASE
optics, as well as utilizing active enzyme centrifugation according to Kemper and Everse (12). Molecular weight studies were made by the sedimentation equilibrium method detailed by Yphantis (13). All experiments were carried out in 0.1 M Na phosphate, pH 7.0, containing 1 mM EDTA and 1 mM dithiothreitol.
Determination
of Stokes’ Radii
A calibrated Sepharose 4B column as described by Siegel and Monty (14) was used. Markers were blue dextran (void volume), acetylcholinesterase form D (r s = 13.6 nm), fibrinogen (11.6 nm), ferritin (7.9 nm), catalase (5.2 nm), and bovine serum albumin (3.6 nm).
Sodium Dodecyl Sulfate Gel Electrophoresis
Polyacrylamide
The methods of Weber and Osborn (15) as well as Laemmli (16) were followed. The gels were calibrated with bovine serum albumin (M, = 67,000), pyruvate kinase (57,000), ovalbumin (45,000), and malate dehydrogenase (32,500). The system of Laemmli without sodium dodecyl sulfate was used for regular polyacrylamide gel electrophoresis. Gels were stained for proteins with Coomassie brilliant blue and activity bands were made visible by incubating the gels in 0.1 M sodium phosphate buffer containing NADPH (2 mglml) and nitrobluetetrazolium (0.5 mg/ml).
Amino
Acid Analyses
Hydrolysis of transhydrogenase was performed at 110°C in 6 N HCl for 24, 48, and 72 h, respectively. Amino acids were analyzed on a Beckman Model 119 automatic amino acid analyzer using a single column system. Tryptophan was determined spectrophotometrically by the method of Bencze and Schmid (17).
CNBr Cleavage Peptides
and Chromatography
of
Transhydrogenase was dialyzed in 0.1 N HCl at a concentration of 1 mg/ml. CNBr was added to the enzyme solution at a concentration of 5 mg/ml and incubated for 24 h at room temperature. The peptides were then lyophilized. Separation of the peptides was performed by descending chromatography in n-butanol-acetic acid-water-pyridine (15:3:12:10) on Whatman 3MM and stained with a Cd/ninhydrin reagent.
Purification of Pyridine hydrogenase
Nucleotide
Trans-
Cell growth. Pseudomonas aeruginosa cells were grown from a stock culture in a medium described
138
WERMUTH
AND
by Cohen and Kaplan (1). The cells were frozen immediately after harvesting and stored at -20°C. Disruption of cells. Frozen cells (800 g) were thawed and suspended in 2 liters of 0.1 M sodium phosphate buffer, pH 7.0, containing 1 mM EDTA. Cells were broken by passing through a Sorvall Ribi cell fractionator (Model RF-11 at 700 atm. Two liters of phosphate buffer were added and the cell debris was removed by centrifugation at 18,000g for 30 min. The precipitate was resuspended in 2 liters of buffer and centrifuged as before. The supernatants were combined and made up to contain 0.05 M mercaptoethanol. 2’-AMP-Se&rose. To the crude extract were added 40 ml of 2’-AMP-Sepharose and it was kept suspended for 18 h by light stirring. The Sepharose was then collected by filtration through a Buchner funnel (Corning, 36060, C) and washed with 3 liters of 0.1 M phosphate buffer, pH 7.0, containing 1M NaCl, 0.05 M mercaptoethanol, and 1 m&r EDTA: followed by 2 liters of 0.1 M sodium borate buffer, pH 8.0, containing 0.05 M mercaptoethanol and 1 mM EDTA. Transhydrogenase was eluted by 0.1 M sodium borate, pH 8.5, containing 10 mM 2’-AMP. The eluate was adjusted to pH 7 by 0.5 M phosphoric acid and dialyzed against three changes, 6 liters each of 0.02 M sodium phosphate buffer, pH 7.0 containing 0.05 M mercaptoethanol and 1 mM EDTA. DEAE-cellulose. The dialyzed enzyme solution was applied to a column 2 cm in diameter, containing 150 ml of packed DE-52, equilibrated in dialysis buffer. The column was washed with 5 column volumes of buffer. Bound proteins were then eluted by a 0 to 0.5 M NaCl gradient made up in column buffer. Fractions were collected and the tubes containing transhydrogenase were pooled. The enzyme was concentrated by precipitation with ammonium sulfate to 80% of saturation. After 1 h the suspension was centrifuged at 18,OOOg for 30 min. The precipitate was dissolved in 0.1 M sodium phosphate buffer containing 1 mM dithiothreitol and 1 mM EDTA and dialyzed once against 2 liters of the same buffer. Sepharose 4B. The dialyzed enzyme preparation was subjected to gel chromatography on Sepharose 4B by applying 2-ml portions on a column, 1.1 cm in diameter, containing 55 ml of Sepharose, equilibrated in 0.1 M sodium phosphate buffer, pH 7.0, TABLE PURIFICATION
OF PYRIDINE
Volume Extract 2’-AMP-Sepharose DEAE-Cellulose Sepharose 4B a Elution
volume
5,800 365 230 10” corresponding
NUCLEOTIDE
(ml)
KAPLAN
containing 1 mM dithiothreitol and 1 mM EDTA. Fractions were collected and analyzed for transhydrogenase activity. The enzyme was checked for homogeneity by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Fractions containing homogeneous enzyme were pooled and stored at 4°C. RESULTS
Purification
A summary of the purification procedures for pyridine nucleotide transhydrogenase from Pseudomonas aeruginosa is given in Table I. In a first step the enzyme was bound to 2’-AMP-Sepharose and then eluted with free 2’-AMP. In order to get optimum binding and elution conditions various parameters were tested for their influence on the binding of the enzyme to the immobilized 2’-AMP. Binding studies at different pH values showed that the capacity of 2’-AMP-Sepharose for transhydrogenase depended upon the pH. Between pH 6 and 7.5, approximately 450 enzyme units were bound per milliliter of settled 2’-AMP-Sepharose. An increase in pH above pH 8 resulted in a decrease of the binding capacity. At pH 9 only 200 units of transhydrogenase were adsorbed by the gel-bound 2’-AMP. The same pH dependence was observed for the elution of gel-bound transhydrogenase, Any pH lower than 7.5 made the 10 mM 2’AMP or 1 M NaCl ineffective. Elution, hqwever, was achieved by these substances at pH 8. In addition to the influence of the pH, the effects of various adenine nucleotides were investigated. 2’-AMP, 5’-AMP, NAD+, NADP+, NADH, and NADPH were tested for the ability to elute the enzyme from the 2’-AMP-Sepharose. The results are summarized in Fig. 1. Only the 2’-AMP nucleotides, 2’-AMP, NADP+, and I
TRANSHYDROGENASE
Total units (IU) 14,760 10,452 6,480 3,305
to the high molecular
of Transhydrogenase
FROM Pseudomonas
Total protein (ma)
Specific activity W/md
40,600 219 78 15
0.36 48 83 220
enzyme forms.
aeruginosa Yield 100 71 44 22
(%)
PHYSICOCHEMICAL Control S.-AMP
-
2.-AMP NAD+ NADP+ NADH NADPH Na Po40.5M. NaCI
1M 25
50
139
PROPERTIES OF TRANSHYDROGENASE
75
ACTIVITY(%) FIG. 1. The influence of adenine nucleotides and salts on the elution of pyridine nucleotide transhydrogenase from 2’-AMP-Sepharose. Transhydrogenase (40 IU) was incubated with 2 ml of 2’-AMPSepharose in 0.1 M Tris-citric acid buffer, pH 8, containing 0.05 M mercaptoethanol and 0.1 mu FAD. After 30 min the Sepharose was freed from buffer on a Buchner funnel and 100-mg aliquots were suspended in 0.9 ml of the Tris-citric acid buffer containing the desired amounts of salts or adenine nucleotides. The samples were agitated for 30 min and centrifuged and the supernatants were assayed for enzyme activity. Controls with underived Sepharose 4B were carried out in parallel to correct for activating or inhibiting effects of the adenine nucleotides. The control with underived Sepharose in the absence of any effecters was taken as 100% activity. The phosphate used was adjusted to pH 8 by NaOH.
NADPH, eluted the enzyme. Y-AMP, NAD+, and NADH eluted less than 25% of the bound enzyme activity. Some elution was also observed with 1 M NaCl but not with 0.5 M sodium phosphate. A 130- to GO-fold purification was obtained by the 2’-AMP-Sepharose step. However, this enzyme preparation was not yet homogeneous as shown by polyacrylamide gel electrophoresis, which revealed four protein bands, but only one activity band. Further purification was achieved by ion exchange chromatography on DEAE-cellulose and gel filtration on Sepharose 4B. Figure 2 shows the elution profile from a Sepharose 4B gel filtration experiment. About 60% of the enzyme activity was eluted in the void volume of the column. The rest emerged as a broad shoulder, and rechromatography of these
fractions yielded a symmetrical peak. From a calibrated column a Stokes’ radius of 11.7 nm was estimated for this enzyme form (inset, Fig. 2). The homogeneity of the two obtained enzyme forms was tested by electrophoresis on polyacrylamide gels. The enzyme which was eluted in the void volume did not penetrate a 4% polyacrylamide gel. However, after addition of 5 mM 2’-AMP to the enzyme samples, a single protein band was observed which coincided with a band stained for activity. A specific activity of 220 IU/mg of protein was obtained for this form independent of the presence or absence of 2’-ATP. The low molecular form showed two protein bands on polyacrylamide gels, but only one staining for activity. The band which was also stained for activity had the same migration rate as the 2’-AMP-treated high molecular form. A specific activity of 104 IU/ mg of protein was determined. When the activity of the fractions from the Sepharose 4B gel filtration was measured with NADH it was for all fractions less than
Eluate
volume
Iml)
FIG. 2. Gel filtration of pyridine nucleotide transhydrogenase on Sepharose 4B. A sample (2 ml) of pyridine nucleotide transhydrogenase after purification on DEAE-cellulose was chromatographed on a Sepharose 4B column (1 x 60 cm) equilibrated with 0.1 M Na phosphate buffer, pH 7.0, containing 1 mM EDTA and 1 mM dithiothreitol (0). The eluate between 30 and 37 ml was concentrated and rechromatographed on the same column (Al. Activity for this profile is one-tenth of the given scale. The inset shows a plot of normalized elution volume (K,,) versus the Stokes’ radius of various proteins. B, bovine serum albumin; C, catalase; Fe, ferritin; Fi, fibrinogen; A, acetylcholinesterase. The position of transhydrogenase (T) is marked by a vertical bar.
140
WERMUTH
AND KAPLAN
10% compared to the activity obtained with NADPH. An approximately lo-fold increase of the activity was achieved when 1 mu 2’-AMP or 1 mM CaCl, was added to the NADH/(TN)AD+ assay system. Oligomeric
Fkms
1.6 -
and Subunits
It has been shown by Cohen and Kaplan (1) that pyridine nucleotide transhydrogenase from Pseudomonas aeruginosa is dissociated into a low molecular form by 2’-AMP and NADP+. In the presence of either of these two nucleotides they obtained a sedimentation coefficient of 33.8 S. As anticipated an S value of 34 was obtained for our enzyme preparation in the presence of at least 1 mM 2’-AMP. At 2’AMP concentrations below 1 mM or in the absence of the effector, the high molecular form gave a sedimentation pattern indicating nonhomogeneous enzyme forms. Most of the low molecular enzyme sedimented as the 34 S form even in the absence of 2’-ATP, but a small percentage of the material always sedimented at a much faster rate. In the presence of 1 mM NADP+ a welldefined boundary was observed again; however, a sedimentation coefficient of 42 S was calculated from its sedimentation rate. Molecular weight determinations were performed for the 34 and 42 S forms. In both cases a plot of the logarithm of fringe displacement versus the square of rotational distance yielded straight lines (Fig. 3). The calculated weight average molecular weight for the 34 S form was 1.6 x 106. A value of 1.7 x lo6 was obtained from the Stokes’ radius and the sedimentation coefficient. For the 42 S form a weight average molecular weight of 6.4 x lo6 was calculated from the sedimentation equilibrium experiments. Table II gives a summary of physicochemical parameters of transhydrogenase obtained by gel flltration and ultracentrifugation. In order to get some information on the subunit composition of transhydrogenase, the high molecular form was submitted to gel electrophoresis in the presence of sodium dodecyl sulfate. A single protein band was obtained in either a phosphate buffer or a Tris-glycine buffer system. The
-1
,
I 49.2 SQUARE
OF
I 49.4
I 49.6
ROTATIONAL
DISTANCE
FIG. 3. Molecular weight determination of pyridine nucleotide transhydrogenase from sedimentation equilibrium experiments. The solvent was 0.1 M Na phosphate, pH 7.O, containing 1 mM dithiothreito1 and 1 mM EDTA. The initial protein concentration was 0.4 mg/ml. Sedimentation was for 20 h at (0) 6994 rpm in the presence of 10 mM P’-AMP and at 2994 rpm in the presence of 1 mM NADP+ (0). TABLE II SUMMARY OF PHYSICOCHEMICAL PARAMETERS OF PYRIDINE NUCLEOTIDE TRANSHYDROGENASE FROM Pseudomonas aeruginosa
Enzyme in presence of 2’-AMP Stokes’ radius (nm) Sedimentation coefficient” (s~~,~) Molecular weight;a ultracentrifugation gel filtration
11.7 34 1.6 x lo6 1.7 x 106
NADP+ 42 6.4 x 10”
a A value of 0.73 was used for the partial specific volume as calculated from the amino acid analysis (18).
band migrated between the marker proteins pyruvate kinase and ovalbumin. No difference in the relative migration rate was observed in 10% as well as in 12.5% gels (Fig. 4). A molecular weight for the subunit of 54,000 M, was estimated from the calibration curves. When transhydrogenase was incubated in 8 M urea and then submitted to gel electrophoresis in the presence of 8 M urea, it did not-in contrast to the marker protein bovine serum albumin - penetrate into 10% acrylamide gel.
PHYSICOCHEMICAL
PROPERTIES OF TRANSHYDROGENASE
141
ever, was much more pronounced when inactivated transhydrogenase was reactivated. In the absence of FAD and mercaptoethanol no reactivation was observed. Three percent reactivation was observed with 0.1 mM FAD, 36% with 1% mercaptoethanol, and 95% of the original activity
si B+Gsee+ MOBILITV FIG. 4. Determination of the subunit molecular weight of pyridine nucleotide transhydrogenase by sodium dodecyl sulfate polyacrylamide gel electrophoresis. A Tris-glycine buffer system, pH 8.3, as described by Laemmli (16) was used. B, bovine serum albumin; P, pyruvate kinase; 0, ovalbumin; M. malate dehydrogenase. The position of transhydrogenase (T) is marked by vertical bars. (0) 10% acrylamide, (A) 12.5% acrylamide.
The fact that only one protein band was seen on sodium dodecyl sulfate polyacrylamide gels, however, does not rule out the possible existence of more than one type of subunit with the same molecular weight. In order to rule out this possibility, amino acid analyses and chromatography of CNBr-peptides were carried out. The result of the amino acid analyses is given in Table III. A partial specific volume of 0.73 cm3/g was calculated from this amino acid composition (18). Paper chromatography of cyanogen bromide-treated transhydrogenase revealed a total of six ninhydrin spots. This is almost in agreement with the methionine content of the enzyme and suggests that the protein is composed of only one kind of subunit.
TABLE III
AMINO ACID COMPOSITION IN RESIDUES PER SUBUNIT (54,000 MOLECULAR WEIGHT) OF P~RIDINE NUCLEOTIDE TRANSHYDROGENASE FROM Pseudomonas aeruginosa”
Aspartic acid Threonineb Serine* Glutamic acid Proline Glycine Alanine Valine Methionine
60 23 38 44 19 48 37 38 6
Isoleucine’ Leucine’ Tyrosine Phenylalanine Histidine Lysine Arginine Tryptophan ‘/&ystine
36 39 18 21 9 17 28 7 nd’l
a Average of determinations after 24,48, and 72 h of hydrolysis. * Values extrapolated from 24, 48, and 72 h to zero hour. ’ Values after 72 h of hydrolysis. rl Not determined.
Urea Inactivation
Gel electrophoresis in the presence of 8 M urea has shown that transhydrogenase is resistant to dissociation by this denaturing agent. The influence of different urea concentrations on the enzyme activity was therefore tested. Since it was known that FAD and mercaptoethanol protect the enzyme against activity losses (1) their effect on the urea inactivation was also investigated. Figure 5 shows the dependence of enzyme activity on the urea concentration in presence or absence of FAD and/or mercaptoethanol. Indeed, some protection by FAD and mercaptoethanol was observed. The influence of these two reagents, how-
DREA]
(M)
FIG. 5. Inactivation of pyridine nucleotide transhydrogenase by urea. Test tubes containing 0.95 ml of 0.1 M sodium phosphate, pH 7.0 and various concentrations of urea, FAD, and/or mercaptoethanol were prepared. Fifty microliters of transhydrogenase (200 IU/ml) were added and incubated at 4°C for 1 h before the activity was measured. Samples of fully inactivated transhydrogenase were dialyzed once against 100 vol of 0.1 M sodium phosphate buffer, pH 7.0, containing different amounts of FAD and/or mercaptoethanol. The points on the right indicate the effects aRer dialysis and removal of the urea. (0) Urea only; (A) urea in presence of 1% mercaptoethanol, (0) 0.1 mM FAD, (0) 1% mercaptoethanol plus 0.1 mM FAD. See text for further explanations.
142
WERMUTH
AND
was recovered in the presence of 0.1 mM FAD and 1% mercaptoethanol. Five molar guanidine appears to inactivate the enzyme irreversibly. DISCUSSION
Cohen and Kaplan (1) have described a purification method which starts with two ammonium sulfate precipitations, a timeconsuming and laborious procedure. A fivefold purification in a yield of 44% was obtained in these two purification steps. Affinity chromatography on 2’-AMP-Sepharose, on the other hand, gave a 150-fold purification and a yield of 70% in one step. No centrifugations of large volumes are necessary and the small amounts of 2’AMP-Sepharose which are used to achieve complete binding of transhydrogenase are easy to handle. Barry and O’Carra (19) have suggested some criteria to discriminate between biospecific and nonspecific binding of dehydrogenases to affinity adsorbants. Elution of the protein must be accomplished by low concentrations of biospecific ligands, such as substrates, inhibitors, or activators. Nonspecific agents such as salts or organic solvents should not elute the enzymes or change the elution pattern. According to these criteria the binding of transhydrogenase to 2’-AMP-Sepharose is nearly all biospecific. Neither NaCl up to 0.5 M nor the adenine nucleotides 5’-AMP, NAD+, or NADH eluted the enzyme from the column and elution was only obtained by 2’-phosphate adenine nucleotides. The strong pH dependence observed is typical for many dehydrogenases. From their studies, Dean and co-workers (20) concluded that each enzyme is eluted at a characteristic pH value corresponding to the pK of a specific amino acid at the binding site. Based on this hypothesis an amino acid with an apparent pK of about 9 interacts with the 2’AMP nucleotides. This finding could be related to the activation of the enzyme. From FAD titration studies, Cohen and Kaplan determined a subunit molecular weight for transhydrogenase of 40,000 to 45,000. This value, however, was based on a protein determination using bovine serum albumin as a standard. The direct
KAPLAN
approach of sodium dodecyl sulfate polyacrylamide gel electrophoresis described in this paper yielded a subunit molecular weight of 54,000. Assuming a molecular weight of 1.6 to 1.7 million for the protomer we find approximately 30 subunits per active enzyme unit, which is in agreement with the results of Louie et al. (3). Their electron micrographs show seven membered rings and regular rectangular images which are assumed to be on the side view of the rings. Four of these rings form one enzyme unit, which amounts to 28 subunits per protomer. Since 8 M urea did not dissociate the enzyme, the subunits must be held together by strong forces. Covalent bonds, however, can be excluded since complete dissociation into 54,000 units was obtained by sodium dodecyl sulfate even in the absence of mercaptoethanol. Cohen and Kaplan (1) have shown that in the presence of NADP+, the transhydrogenase exists in a dissociated form with a sedimentation coefficient of 34 S. Reexamination of these results indicates that in the presence of NADP+, transhydrogenase occurs as a small aggregate of four 34 S units.4 The occurrence of small aggregates, on the other hand, is supported by electron micrographs of transhydrogenase which in the from Acetobacter vinelandii, presence of NADP+ show small particles and short rods composed of three to five of these particles (4). It is, however, difficult to compare our results with the earlier study of Cohen and Kaplan, since they had obtained their values at a higher pH. Sabo et al. (211, for example, have shown that the decameric lysine decarboxylase from Escherichia coli aggregates at lower pH values forms long rods and dissociates into dimers at elevated pH. It was suggested by Louie et al. (3) that the dissociation-association equilibrium between high and low molecular transhydrogenase forms could regulate the enzyme activity. The present results, however, show that all active forms of the enzyme require nucleotides * As yet, it has not been clarified whether these small particles induced by NADP+ play a role in the regulation of the transhydrogenase activity (Widmer and Kaplan, submitted for publication).
PHYSICOCHEMICAL
PROPERTIES OF TRANSHYDROGENASE
containing a 2’-AMP moiety for full activity and that the state of aggregation has no direct influence on the catalytic activity (see also Widmer and Kaplan).4 The biological significance of the association-dissociation equilibrium, which is greatly influenced by 2’-AMP-nucleotides therefore rests unclear. Further investigations concerning this problem are in progress in this laboratory. Note added in proof. We would like to thank Dr. Peter Brodelius for calling our attention to a paper now in press which is in agreement with the data reported in our manuscript (B. Hbjeberg, P. Brodelius, J. Rydsttim, and K. Mosbach, Affinity chromatography and binding studies on immobilized 5’AMP and 2’,5’-ADP of nicotinamide nucleotide transhydrogenase from Pseudomonas aeruginosa, Eur. J. B&hem., in press).
REFERENCES 1. COHEN, P. T., AND KAPLAN, N. 0. (1970). J. Biol. Chem. 245, 2825-2836. 2. COHEN, P. T., AND KAPLAN, N. 0. (1970)5. Biol. Chem. 245, 4666-4672. 3. LOUIE, D. D., KAPLAN, N. O., AND MCLEAN, J. D. (1972) J. Mol. Biol. 70, 651-664. 4. VAN DEN BROEK, H. W. J., SANTEMA, J. S., AND VEEGER, C. (1971)Eur. J. B&hem. 24,46-54. 5. RYDSTR~M, J., HOEK, J. B., AND HOJEBERG, B. (1973) Biochem. Biophys. Res. Commun. 52,
421-429. 6. LEE, C.-Y., LAPPI, D. A., WERMUTH, B., EvERSE, J., AND KAPLAN, N. 0. (1974) Arch. B&hem. Biophys. 163, 561-569.
143
7. WERMUTH, B. (1975) Fed. Proc. 34, 682. 8. WERMIJTH, B. (1973) Dissertation, Universitat
Bern. 9. PABST LABORATORIES (1961) “Ultraviolet
Absorption Spectra of Pyridine Nucleotide Coenzymes and Coenzyme Analogues,” Pabst Laboratories Circular OR-18. 10. HENRY, R. J., SOBEL, C., AND BERKMAN, S. (1957) Anal. Chem. 29, 1491-1495. 11. %HLEN, P., STEIN, S., DAIRMAN, W., AND UDENFRIEND, S. (1973) Arch. B&hem. Bit+ phys. 155, 213-220.
12. KEMPER, D. L., AND EVERSE, J. (1973) in Methods in Enzymology (Hirs, C. W. H., and Timasheff, S. N., eds.), Vol. XVII, pp. 67-82, Academic Press, New York. 13. YPHANTIS, D. (1964) Biochemistry 3, 297-317. 14. SIEGEL, L. M., AND MONTY, K. J. (1966) Biochim. Biophys. Actu 112, 346-362. 15. WEBER, K., AND OSBORN, M. (1969). J. Biol. Chem. 244, 4401-4412. 16. LAEMMLI, U. K. (1970) Nature (London) 227, 680-685. 17. BENCZE, W. L., AND SCHMID, K. (1957) Anal. Chem. 29, 1193-1196. 18. COHN, E. J., AND EDSALL, J. T. (1943) in Pro-
teins, Aminoacids and Peptides (Cohn, E. J. and Edsall, J. T., eds.), pp. 370-381, Hafner, New York. 19. BARRY, S., AND O’CARRA, P. (1973) Biochem. J. 13’5, 595-607. 20. LOWE, C. R., HARVEY, M. J., AND DEAN, P. D. G. (1974) Eur. J. Biochem. 41, 347-351. 21. SABO, D. L., BOEKER, E. A., BEYERS, B., WARON, H., AND FISCHER, E. H. (1974) Biochemistry
13, 662-670.
