of the Malate-Lactate Transhydrogenase

Transcription

1 Eur. J. Biocheni. 35, (1973) Molecular Weight and Subunit Structure of the Malate-Lactate Transhydrogenase S. H. George ALLEN Fachbereich Biologie der Universitat Konstanz, and Biochemistry Department, Albany Medical College, Albany, New York (Received December 27, 1972/February 26, 1973) The molecular weight of the malate-lactate transhydrogenase as determined by sedimentation-diffusion and high-speed-equilibrium methods is approximately Dissociation of the protein with guanidine-hc1, sodium dodecylsulfate or citraconic acid anhydride yields a homogeneous preparation for which molecular weight values ranging from to have been found. The transhydrogenase thus appears to be a dimer, composed of two subunits and an NAD+/ NADH equivalent binding weight of Addition of a 33-fold excess of citraconic acid anhydride based on the lysine content of the transhydrogenase was necessary for complete dissociation. This was accompanied by a complete loss of enzymatic activity and release of the bound NAD+/NADH, which, in the native enzyme, is very tightly bound. Attempts to restore enzymatic activity as well as to reform the dimer structure after removal of the citraconyl groups, even in the presence of NADf were unsuccessful. Partial citraconylation while destroying most of the enzymatic activity did not cause release of the NAD+/NADH prosthetic groups. The enzymatic activity of these preparations can be partially restored upon removal of the citraconyl groups. Apparently, removal of the prosthetic groups is associated with irreversible denaturation and the transhydrogenase represents a case where native configuration is extremely dependent on the binding of the prosthetic group. The malate-lactate transhydrogenase contains tightly bound NAD+/NADH and catalyzes a coupled oxidation-reduction without the utilization of exogenous NADf or NADH [1,2], as shown in the following equation : L-Lactate + oxalacetate + pyruvate + L-malate. The bound coenzymes are released from the protein only under those conditions which bring about denaturation and dissociation of the protein [3,4]. Because of the close association between the binding of the coenzyme and the maintenance of native configuration, this enzyme may serve as a good model for studies on the relationship between coenzyme binding and protein conformation. Before careful dissociation and conformational studies could be done with the transhydrogenase it was necessary to determine its molecular weight and subunit structure. A weight of was reported by Dolin [l] who used the short-column sedimentation-equilibrium method of Yphantis [5]. These measurements were performed at 3 "C and assumed a partial specific Enzymes. Malate-lactate transhydrogenase or L-lactate : oxalacetate oxidoreductase (EC ) ; malate dehydrogenase (EC ). volume of 0.75 mg/g. Allen [2] reported a molecular weight of & loo/, using the sucrose-gradient centrifugation method of Martin and Ames [6]. Also using this method, Allen and Patil [2] had also reported the presence of a subunit with a molecular weight of following succinylation of the transhydrogenase. Thus, it was suggested that the enzyme was composed of three subunits. In view of the discrepancy in the molecular weight of the native enzyme and the necessity of proving a trimer structure, a more careful study was performed to elucidate the molecular weight and subunit structure of the transhydrogenase. MATERIAL AND METHODS The transhydrogenase was isolated from Veillonella alcalescens (ATCC 27215) and purified as described previously [2,4]. The colorless protein was homogeneous (Fig. 1) as measured in the analytical ultracentrifuge (Beckman Model E) and had a specific activity of approximately 250 pmol x min-1 xmg-l as measured in the assay coupled with malate dehydrogenase [2]. Protein was determined by absorbance at 280 nm (absorption coefficient = 1.27 cm2 - mg-i). The enzyme was stable when

2 Vol.35, No.2, 1973 S. H. G. ALLEN 339 a 32 4a 64 Time (min 1 Fig. 1. Bedimentation of native transhydrogenase. The enzyme concentration was 10 mg/ml in 0.05 M potassium phosphate buffer ph 7.1 and sedimentation was performed at rev./min at 20 "C. Exposures were taken at the times (in min) indicated, at a bar angle of 60" and an 5 ~ 0 of, ~ 4.57 S was calculated stored at -20 "C as an ammonium sulfate suspension in 0.05M Tris-HCI buffer ph 7.9. Absorbance was measured with a Beckman model DB-G (Beckman Instruments, Miinchen) and fluorescence with a Hitachi Model MPF-3 (Perkin-Elmer & Co. GmbH, Norwalk, Conn.). Circular dichroism measurements were performed at 20 & 0.5 "C using a Cary 60 spectropolarimeter equipped with a Cary 6002 CD accessory. Citraconic acid anhydride was purchased from Schuchardt (Munchen), guanidine-hc1 from Heico Inc. (Delaware Gap, Pa. U.S.A.), malate dehydrogenase from Boehringer Mannheim GmbH (Mannheim, Germany). All other chemicals were of reagent grade. The viscosity (r) of buffer solutions was measured with an Ostwald viscosimeter at 20 "C. The density (e) was measured with a digital densitometer (Model DMA 10, Anton Paar KO, Graz, Austria). RESULTS Molecular Weight of Native Enzyme Prior to all measurements the transhydrogenase preparation was dialyzed at 4 OC against 0.05 M potassium phosphate buffer ph 7.1. Diffusion experiments were carried out in double-sector, capillary-type cells at five different protein concentrations from 1.6 to 11.4 mg/ml. The centrifuge (Spinco Model E, with a Photoelectric Scanning System, Beckman Instruments, Miinchen, Germany). was run at 4000 rev./min and exposures were taken at 2 and 4-min intervals. The diffusion coefficients, which were found to be independent of protein concentration were calculated according to the height area method as described by Sund et al. [7], and an average D ~ o value, ~ of Fick Units (1 Fick unit = cmz/s) was found ri) - 3. I: v) ; m 0._ L L m 0._ c m c c m._ E u I $ Protein concn. (mg/rnl) Fig. 2. Dependence of sedimentation coefficient on protein concentration for native transhydrogenase. The protein was dialyzed thoroughly against 0.05 M potassium phosphate buffer ph 7.1 and sedimentation was performed at 20 "C at rev./min. The SZO,~ extrapolated to zero by the method of least squares was 5.26 S Sedimentation experiments were carried out in single-sector, 12-mm cells, either with normal or wedge windows. The centrifuge was run at 20 C at rev./min and either schlieren optics or photoelectric scanner at 280nm were used. The sedimentation coefficients, s p ~ were, ~ extrapolated to zero protein concentration (Fig.2). An sgosw value of 5.26 S was found. This compares well with the previously reported value of 4.6 S [2] which was not corrected for protein concentration. Calculation of the molecular weight was based on the Svedberg equation [8,9]. The partial specific volume of 0.726mgIg was calculated from the amino acid content of the transhydrogenase [4] as described by Cohn and Edsall [lo]. From these data the molecular weight was calculated to be 71000

3 340 The Molecular Weight and Subunits of Malate-Lactate Transhydrogenase Eur. J. Bioehem L8 Time ( min 1 Fig. 3. Sedhnentation of transhydrogenase in 5 M guanidine- HCI. The transhydrogenase was treated with guanidine-hci and 0.01 M 2-mercaptoethanol as described in Methods. The protein at a concentration of 7.2 mg/ml was sedimented in a synthetic boundary cell at 20 "C and rev./min. 128 Exposures were taken at 16-min intervals at a bar angle of 60' and an SZO,~ value of 1.20 S was obtained, after correction for the density (pzo) of g/cm3 and viscosity (qz0) of m2/s, both measured at 20 "C as described in Methods &3600 [Ill. A frictional ratio was also calculated and found to be 1.19 which corresponds to a prolate ellipsoid with an axial ratio of approximately 4. Since a value of is considerably lower than that reported previously [I, 21, the molecular weight of the transhydrogenase was also determined with the high-speed (meniscus-depletion) sedimentationequilibrium method [ 113. Four protein concentrations (0.21 to 0.57 mg/ml) were placed in the six-channel cell which was run in an AN-G rotor at rev./ min at 20 "C for 2 h, and then at rev./min. Results obtained with the photoelectric scanner indicate that equilibrium was attained and a molecular weight of & 2500 was calculated from a plot of log absorbance at 280nm versus r2 where r = distance from centre of rotation described by Chervenka [ll]. Thus the molecular weight of the native transhydrogenase appears to be rather than Molecular Weight of Subunits Previous results [4] with sucrose gradient centrifugation [6] indicated that the native enzyme could be dissociated into subunits with a molecular weight of approximately A more accurate determination with the sedimentation-diffusion method was carried out. I ml of a 10 M guanidine- HC1 solution containing 0.01 M 2-mercaptoethanol and 0.05M phosphate buffer ph 7.1 was carefully layered under 1 ml of dialyzed transhydrogenase at room temperature. The contents of this tube were rapidly mixed to yield a protein solution containing 5M guanidine-hc1. With care taken to prevent evaporation, the sample was dialyzed for two days against a 5 M guanidine-hc1 solution Area (cd) Fig. 4. Dependence of sedimentation coefficient on protein concentration of the transhydrogenase in 5 M guanidine-h C1. The protein was thoroughly dialyzed against 0.05 M potassium phosphate buffer, ph 7.1, containing 5 M guanidine-hcl and 0.01 M 2-mercaptoethanol and sedimentation in the same solution was performed at 20 "C and rev./min. The SZO,~ was extrapolated to zero protein concentration by the method of least squares and was found to be 1.87 S Sedimentation experiments were performed at rev./min and a single sedimenting species was observed (Fig.3). The ~ 2 0 was, ~ determined at 9 protein concentrations and when extrapolated to zero protein concentration (Fig.4!, a value of 1.87 S was obtained. Diffusion measurements on the enzyme in 5M guanidine-hc1 were made as described with the native enzyme, except that in this case a rotor speed of 10000rev./min was used. The D ~ O was, ~ determined at 4 protein concentrations and found to be Fick units. Because of the difficulty in estimating protein concentration in the presence of 5 M guanidine-hc1, these determinations were related to the area under the initial schlieren patterns

4 Vo1.35, No.2,19?3 S. K. G. ALLEN 341 as measured on a 10-fold enlargement on millimeter Table 1. The effect of citraconylation on the activity and paper with a planimeter. Using both the D Z ~, ~ dissociation of the transhydrogenase Citraconylation was performed as described in Methods. and the s ~,,~ the frictional ratio was calculated to The specific activity of the enzyme was measured by the be 2.28 which corresponds to a prolate elipsoid indirect spectrophotometric method coupled with malate having an axial ratio of approximately 28. Assuming dehydrogenase as described previously [2]. Dissociation was no change in the partial specific volume of the protein measured by comparing the area on the schlieren plates as measured with a planimeter on 10-fold enlargements on in 5 M guanidine-hcl, the molecular weight was millimeter paper, and expressed as percentage total area. calculated to be f Reactivation was with ph 4.5 as described in Methods The molecular weight of the subunits was also Excess specific Dissociation Reactivation measured using dodecysulfate-polyacrylamide-gel citraconic activity into electrophoresis as described by Weber and Osborn anhvdride siihnnits + [12]. The transhydrogenase which had been treated vniol x min-' -fold with guanidine-hc1 was diluted approximately x mg-' Olio total 'I. "0 10-fold, to give a protein concentration of approx- None& imately 0.5 mg per ml, in lo/o sodium dodecyl sulfate containing I Olio 2-mercaptoethanol and 0.01 M sodium phosphate buffer ph 7.0. The mixture was heated at 100 "C for 5 min and then held at 37 "C overnight. The preparation was dialyzed at room temperature against a solution containing 0.1 O/, sodium dodecylsulfate. Polyacrylamide gels were run at room temperature at 8mA per gel for approximately 5 h. In one set of experiments, the proteins were visualized by staining with Coomassie brilliant blue. In a second set, the proteins were first dansylated [13,14] and the bands visualized with ultraviolet light. Results with a series of peptide chains of known molecular weight were the same regardless of the method. Mean RE values of 0.22, 0.32, 0.40, 0.55 and 0.82 were obtained with Coomassie blue staining for bovine serum albumin, ovalbumin, lactate dehydrogenase, trypsin and cytochrome c, respectively. Mean RE values of 0.21, 0.36, 0.41, 0.55, and 0.77 were obtained for the same series of proteins which had been dansylated. The malate-lactate transhydrogenase yielded a single band with an RE of 0.33 which corresponded to a molecular weight of & 5O/,. Thus, all values obtained are in the range and suggest that the transhydrogenase is a dimer. Dissociation with Citraconic Anhydride Previous experiments had shown that dissociation of native transhydrogenase could be effected by succinylation [4]. Since succinyl groups are not easily hydrolyzed from the protein, all molecular weight determinations must take into account the presence of a variable number of succinyl groups on each protein molecule. Furthermore, experiments on reaggregation and reactivation of the enzyme are not possible. Dixon and Perham [15] and Gibbons and Perham [I61 describe the use of citraconic anhydride (2-methylmaleic anhydride) as a more desirable blocking agent because it can be hydrolyzed from the modified protein under rather mildly acidic coiiditions. The transhydrogenase was treated for 20 min at 2 "C with various concentrations of a A volume of cold acetone equal to the quantity of citraconic anhydride diluted in acetone was added. Under the conditions of the experiment this had no effect on the specific activity. citraconic anhydride diluted in cold acetone. The molar amount of citraconic anhydride used was based on the molar content of lysine in the transhydrogenase (47 mol per g) and expressed as fold excess. Table 1 shows the effect of citraconylation on the enzymatic activity as well as on the dissociation of the protein into subunits. The activity of the transhydrogenase appears to be extremely sensitive to the modification, for even a 1.6-fold excess of reagent caused a 910/, drop in activity which affected only a slight (8O/,) dissociation into subunits. A 33-fold excess of reagent virtually destroys the enzymatic activity and brings about complete dissociation of the protein. Fig.5A shows the schlieren patterns of the native enzyme (upper) and the enzyme treated with a 33-fold excess of citraconic anhydride which has an s, value of 1.3 S (lower). Care must be taken to control the ph during the reaction with citraconic anhydride. This is best done by monitoring the ph with constant stirring with a ph meter and by adding 5-pl amounts of 5 N NaOH to maintain the ph at 8.0. If the ph fluctuates sharply during the reaction, then the citraconylation at 33-fold excess does not proceed far enough to cause complete dissociation of the protein. In Fig. 5 B are the schlieren patterns of the native (upper) and the enzyme treated with a 3.3-fold excess of reagent (lower). This latter preparation is composed of two components, an undissociated protein which represents about 75O/,, and a smaller component with an sz0 of about 2 S which represents about 25O/, of the total protein. Fluorescence spectra of this partially dissociated protein revealed that this protein had retained all

5 342 The Molecular Weight and Subunits of Malate-Lactate Transhydrogenase Eur. J. Biochem LO Time (mid 72 a 16 2L 32 Time (min) Fig. 5. Comparison of the sedimentation characteristics of native The sz0 values were 4.97 S for the native and 1.33 S for the and citraconylated transhydrogenase. (A) Upper schlieren citraconylated protein. (B) Upper schlieren pattern is the pattern in wedge-window cell is the native transhydrogenase native enzyme which is the same as in (A). The lower schlieren at a concentration of approximately 3 mg/ml. In the lower pattern is the transhydrogenase (approximately 5 mg/ml) schlieren pattern is the transhydrogenase (approx. 3 mg/ml) treated with a 3.3-fold excess of citraconic anhydride. Both treated with a 33-fold excess of citraconic anhydride. Both samples were dialyzed against 0.05 M Tris-HC1 buffer ph 7.9, samples were dialyzed against 0.05 M Tris-HC1 buffer ph 7.9 and sedimentation was performed at 20 "C at rev./min. and sedimentation was performed at 20 "C at rev./min. The szo values were 4.84 S for the native and 4.6 S for the larger and approximately 2.0 S for the smaller component of its bound NADH (Fig.6A, curve I). The protein concentration for the native, untreated enzyme (Fig.6B, curve 1) was mg/ml while that of the modified protein was mg/ml. This represents 8701, as much protein in the control as in the treated and corresponds with the fact that the native protein exhibits 8901, as much fluorescence as the treated. Thus both contain an equivalent concentration of NADH, and the NADH appears to remain bound to the protein even after quite extensive citraconylation, and loss of 99O/, of the enzymatic activity. Lack of enzymatic activity cannot therefore be attributed to the removal of NADH from the enzyme. That the loss in activity preceeds loss of NADH was also noted when the transhydrogenase was exposed to 7M urea, as previously described [4], and probably reflects the unfolding of the protein which is necessary before the NADH can be released. When 0.4pmol sodium pyruvate are added to the 3.6 pmol of citraconylated enzyme (Fig.6A, curve 11) very little (about 4O/,) quenching of enzyme fluorescence can be seen. In the case of the native enzyme this concen- tration of pyruvate causes an almost complete (92O/,) quenching of the fluorescence (Fig. 6B, curve 11). Citraconylation while not removing the NADH has apparently affected the pyruvate binding site and it can be seen (Fig.6A, curve 111) that the addition of 14 kmol of pyruvate to this preparation is necessary to bring about significant (64O/,) quenching of fluorescence. Fig.6A, curve IV also shows that enzyme which has been treated with a 33-fold excess of citraconic anhydride is almost completely devoid of NADH as measured in the fluorimeter. Even with the addition of an excess of L-malate, which was added to reduce any NAD+ on the enzyme, there was no increase in the fluorescence, indicating that the protein, when fully dissociated is completely devoid of prosthetic group, i.e. both NADH and NAD+. Reactivation of the Citraconylated Transhydrogenuse Gibbons and Perham [I61 found that dialysis of the citraconylated aldolase against 0.01 N HC1 ph 2 for 6 h regenerated 95O/, of the amino groups.

6 Vol. 35, No. 2, 1973 S. H. G. ALLEN Y) -4 ; -8 m U a I m Wavelength (nrn) Fig. 6. Pluorescence emission spectra of native and citraconylated transhydrogenase. (A) Citraconylated transhydrogenase, mg/ml, in a volume of 2.0 ml in 0.05 M Tris- HCI buffer ph 7.9. (I) No addition, (11) contains in addition, 0.4 pmol sodium pyruvate, (111) contains a total of 14 pmol of sodium pyruvate, and (IV) is the transhydrogenase treated with 33-fold excess of citraconic anhydride and the same plot was obtained even with the addition of 4pmol L-malate. (B) Native transhydrogenase, mg/ml in a volume of 2.0 ml in 0.05 M Tris-HC1 buffer ph 7.9: (I) no additions, and (11) contains 0.4 pmol sodium pyruvate. All fluorescence measurements were carried out at 20 C in a thermostatically controlled cuvette holder. Excitation wavelength was 340 nm They then attempted to anneal the regenerated protein by dialysis against 20 mm ammonium acetate buffer ph 5.5, containing 20 mm 2-mercaptoethanol as described by Schachman[17]. They found that with aldolase which was 50 /, citraconylated and which was only slightly dissociated into subunits, a recovery of 75O/, of the enzymatic activity was possible. However, with higher degrees of modification needed for full dissociation (800/, and higher) very little recovery of activity could be noted. The transhydrogenase is not as stable to acid ph as is the rabbit muscle aldolase and a less drastic treatment of the enzyme was necessary to check for regeneration of activity. Sia and Horecker [18], using maleic anhydride, report extensive hydrolysis of maleyl groups from maleyl-aldolase after exposure to ph 4.5 in the presence of 1 mm dithiothreitol for 45min. Under these conditions they report obtaining about 45,Ilo reappearance of catalytic activity and most of the protein being found in the tetrsmeric form. Since citraconyl groups are reported to be more easily hydrolyzed than maleyl groups, this procedure was used in an attempt to regenerate the catalytic activity of the transhydrogenase (Table 1). Results indicate that some reactivation is indeed possible in spite of the fact that the native enzyme is also extensively damaged having only Wavelength (nm) Fig. 7. Circular dichroism spectra of native and citraconylated transhydrogenase. (I) The native enzyme, 0.2 mg/ml in a 0.05 M Tris-HC1 ph 7.9; (11) the native enzyme treated with a 33-fold excess of citraconic anhydride, protein concentration is 0.2 mg/ml also in 0.5 M Tris-HC1 ph 7.9; (111) the same sample as in (11) which has been treated with 0.01 N HCI and the circular dichroism spectrum is also made in 0.01 N HC1. Measurements were made at FR 0.04", velocity 140pm/s and a light path of 1 mm. The temperature was 20 & 0.5 "C about 40 /, of the original activity after identical treatment with acid. Thus, when protein which was treated with a I.6-fold excess of citraconic anhydride is treated at ph 4.5 there is a return of activity to approximately 84O/, of the control. This protein was, however, only sightly dissociated. With the protein which had been more extensively citraconylated and which showed a significant degree of dissociation, the reactivation was less extensive, and the fully dissociated protein showed practically no significant return of catalytic activity. These results were not due to a lack of coenzyme alone for when 14mM NAD+ was incorporated into the reactivation mixture of protein treated with 3.3-fold excess of citraconic anhydride only a 30 /, higher activity was noted over the control without NAD. Thus results with the transhydrogenase indicate that after extensive dissociation of the protein, reactivation is not possible. Ultracentrifuge studies appear to confirm these results since in no instance could 5.3-S protein be found in fully dissociated preparations from which the citraconyl groups had been hydrolyzed. Fig. 7 shows the circular dichroism spectra of the native (I) as well as the dissociated transhydrogenase (11). The native enzyme shows a pattern typical of many proteins, while the citraconylated enzyme has the spectrum of a completely denatured protein, a random coil configuration. The return of some secondary structure can be seen in the spec-

7 344 The Molecular Weight and Subunits of Malate-Lactate Transhydrogenase Eur. J. Biocheni. trum of the enzyme from which the citraconyl groups have been hydrolyzed (Fig. 7, curve 111). This spectrum lacks the peak at 210 nm typical of the native enzyme and may suggest a structure containing a pleated sheet conformation. Molecular- Weight Determination of Citraconylated Protein Sedimentation-equilibrium at 20 "C was carried out at 5 different protein concentrations as previously described [I 11. The transhydrogenase was modified as described above using a 33-fold excess of citraconic anhydride. The citraconyl groups were then hydrolyzed from the protein with 0.01 N HC1 as described by Gibbons and Perham [16]. The resulting protein was soluble in 0.01 N HCl and with acetate and citrate buffer up to ph 3.5. Above this ph it was insoluble even in the presence of 0.1 N LiCl or 0.1 M sodium citrate. For the molecular weight determinations, the protein was dialyzed against N HC1 ph 2.8 for 24 h. Sedimentation in the analytical ultracentrifuge revealed a single boundary with an uncorrected szo of 1.3 S. The sedimentation equilibrium was carried out at rev./min after a 2.5 h overspeeding at rev./ min. Calculations revealed the molecular weight to be f This molecular weight is somewhat lower than that obtained in guanidine-hcl but is identical with that previously reported for the succinylated-transhydrogenase [4]. Since the molecular weight of the native enzyme is 71000, the molecular weight of each subunit should be Thus the true value appears to lie between that obtained from the guanidine-hcl experiment (i.e ) and dodecylsulfate-polyacrylamide-gel electrophoresis and the citraconylated protein (i.e ), and best estimates indicate that the native enzyme is composed of two subunits. DISCUSSION Although previous results indicated values of [2] and [I] for the molecular weight of the native transhydrogenase these values appear to be too high. The molecular weight as determined by sedimentation-equilibrium and highspeed sedimentation equilibrium appears to be & Furthermore, the s& of 5.26 S is more consistent with a molecular weight of approximately 70000, than with a value of [19]. The molecular weight of the subunit previously reported on the succinylated transh ydrogenase was approximately The results reported here suggest that the weight of this subunit is between and i.e. approximately Furthermore, assuming the same absorption coefficient for the bound as for the free NADH, then there appears to be 1 mol coenzyme bound per g protein. Many pyridine-nucleotide-dependent dehydrogenases have this coenzyme equivalent weight as was pointed out by Jaenicke and Pfleiderer [20] but many others, of course, do not, as can be seen in the complilation of protein subunits presented by Darnall and Klotz [21]. The transhydrogenase appears to be similar in size and subunit structure to the pig-heart and horse-heart mitochondria1 malate dehydrogenase [22]. The transhydrogenase differs from the malate dehydrogenase and other pyridinenucleotide-dependent dehydrogenases in that the NAD+/NADH is much more firmly bound, and that renaturation as measured by association of subunits to form active dimers has thus far not been possible. All the denaturing agents tested, 7 M urea [4], 5 M guanidine-hc1, modification of the protein with succinic [4] or citraconic anhydrides, bring about a release of the NAD+/NADH and a concomitant dissociation into subunits. The transhydrogenase is not dissociated into subunits by 5 M lithium chloride, or by freezing and thawing as is lactate dehydrogenase [23]. Furthermore, after the release of the bound NAD+/NADH, the subunits which do appear are quite insoluble at ph values from 4 to 9. Addition of 2-mercaptoethanol and coenzyme does not prevent this precipitation, and in no case has significant return of either enzymatic activity or dimer formation been noted in the completely dissociated protein. As expected the fully succinylated and citraconylated apoenzymes which are in the dissociated state, are soluble at all ph values and as noted in Fig. 7 the citraconylated protein shows very little secondary structure in the circular dichroism spectrum, being mainly random coil. After removal of the citraconyl groups there is a return to some structure in the apoenzyme (Fig. 7, curve 111) but this is quite different from the native structure, perhaps containing some pleated sheet conformation. It appears that any procedure for the renaturation of this protein must involve an unfolding of this structure and a refolding into the structure of the native enzyme. The role of coenzyme binding in the renaturation is as yet unknown, but in view of the tightness of this binding it is tempting to postulate that this is a very important factor. As shown in experiments on the reversible denaturation of lactate dehydrogenase by Levi and Kaplan [24], the three-dimensional structure of a protein is not entirely determined by the amino acid sequence of the polypeptide chains. These workers present evidence which indicates that the inclusion of the coenzyme in the reassociation media of lactate dehydrogenase causes the protein to refold into a structure whose properties more closely resemble those of the native enzyme. In the case of the transhydrogenase, the simple inclu-

8 Vo1.35, No.2, 1973 S. H. G. ALLEN 345 sion of coenzyme is not sufficient and the changes in conformation appear to be large and virtually irreversible under simple renaturation techniques. In the case of the partially citraconylated enzyme, some return of activity was found after hydrolysis of the citraconyl groups. However, in these cases the NADH+/NADH remained bound to the enzyme and very little dissociation of the protein into subunits took place. It appears then that once the coenzyme is bound, renaturation can take place. In all cases reported here once the coenzyme was removed and the subunits obtained, the protein assumed an unusual structure which resisted renaturation. It is difficult to imagine how such a protein can attain the native configuration in vivo assuming the thermodynamic hypothesis as postulated by Givol et al and Anfinsen [26]. It would appear that, as yet undescribed stabilizing factors which involve the insertion of the coenzyme, must be intimately involved in the biosynthesis of this and perhaps other proteins. Experiments are planned to study the conditions necessary for the reversible denaturation of the malate-lactate transhydrogenase. The author wishes to thank Dr Horst Sund and his entire staff for the excellent facilities and stimulating guidance and encouragement during the course of this work. I am indebted to Frau U. Markau for making the many runs with the analytical ultracentrifuge and to Frau M. Nickel for the hydrodynamic measurements. The author acknowledges with appreciation the Universitat Konstanz for the appointment as guest Professor for one year, the Fulbright Commission for a travel grant and the Sabbatical Support of the Albany Medical College. REFERENCES 1. Dolin, M. I., Phares, E. F. & Long, M. V. (1965) Biochem. Biophys. Res. Cmmun. 21, Allen, S. H. G. (1966) J. Biol. Chem. 241, Doh, M. I. (1969) J. Biol. Chem. 244, Allen, S. H. G. & Patil, J. R. (1972) J. Bwl. Chem. 247, Yphantis, D. A. (1969) Biochemistry, 3, Martin, R. G. & Ames, B. N. (1961) J. BioE. Chem. 236, Sund, H., Weber, K. & Molbert, E. (1967) Eur. J. Biochem. 1, Svedberg, T. & Pedersen, K. 0. (1940) The Ultracentrifuge, Clarendon Press Oxford. 9. Trantman, R. & Crampton, C. F. (1959) J. Am. Chem. Soc. 81, Cohn, E. J. & Edsall, J. T. (1943) Proteins, Amino Acids and Peptides, p. 374, Reinhold Publishing Co. 11. Chervenka, C. H. (1969) A Manual of Methods for the Analytical Ultracentrifuge, Spinco Division, Beckman Inrtruments Inc., Pa10 Alto, California. 12. Weber, K. & Osborn, M. (1969) J. Bkol. Chem. 244, Talbot, D. N. & Yphantis, D. A. (1971) Anal. Biochem. 44, Horton. H. R. & Koshland. D. E.. Jr. (1967)., Methods Enzymol., 11, Dixon, H. B. F. & Perham, R.N. (1968).. Biochem. J. 109, Gibbons, I. & Perham, R. N. (1970) Biochem. J. 116, Schachman, H. K. (1963) Cold dpring Harbor dymp. Quant. Biol. 28, Sia. C. L. & Horecker. B. L ) Bwchem. Biowhus., I 1 " Res. Commun. 31, Svedberg, T. & Pedersen, K. 0. (1940) The Ultracentrifuge Table 48, pp , Clarendon Press, Oxford. 20. Jaenicke. R. & Pfleiderer. - G. (1962) *, Biochim. Biowhus. I " Acta, 60, Darnall, D. W. & Klotz, I. M. (1972) Arch. Biochem. Biophys. 149, Thorne, C. J. R. & Kaplan, -. N. 0. (1963)., J. Biol. Chem. 238, ism. 23. Chilson, D. P., Kitto, 0. B., Pudles, J. & Kaplan, N. 0. (1966) J. Biol. Chem. 241, Levi, A. S. & Kaplan, N. 0. (1971) J. Biol. Chem. 246, Givol, D., LeLorenzo, F., Goldberger, R. F. & Anfinsen, C. B. (1965) Proc. Natl. A d. Sci. U. S. A. 53, Anfinsen, C. B. (1963) Brookhaven Symp. Biol. 15, S. H. George Allen's present address: Department of Biochemistry The Albany Medical College of Union University Albany, New York, U.S.A Eur. J. Biochem., Vo1.35