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The interaction of rabbit muscle aldolase with NADH
274
Biochimica et Biophysica Acta, 1038 (1990) 274-276 Elsevier
BBA Report
BBAPRO 30265
The interaction of rabbit muscle aldolase with N A D H Yuri V. Chumachenko, Alexander I. Sytnik and Alexander P. Demchenko A. V. Palladin Institute of Biochemistry, Academy of Sciences of the Ukrainian SSR, Kiev (U. S. S.R.) (Received 25 September 1989)
Key words: Fluorescence; NAD binding domain; Aldolase; (Rabbit muscle)
Fluorescence studies on both the emission of aldolase and N A D H bound to the enzyme were carried out. Aldolase was found to bind four molecules of N A D H with K o = 6.0 + 0 . 3 / t M . K D values for N A D P H and N A D + were 41 ± 4 / t M and 140 q - 3 0 / t M , respectively. The affinity to N A D H was comparable with that of some NAD-dependent dehydrogenases, and was not affected by the substrate or the inhibitor.
Rabbit muscle fructose 1,6-bisphosphate aldolase (D-fructose-l,6-bisphosphate D-glyceraldehyde-3-phosphate-lyase, EC 4.1.2.13) is the enzyme of the glycolytic pathway which catalyzes the aldole cleavage of fructose 1,6-bisphosphate (FBP) into two phosphotrioses. The enzyme consists of four identical subunits, with three tryptophans in each of them. Aldolase was long believed to be among the well-explored proteins, but the data of the last decade has shown that still little is known about this or other so-called 'soluble' enzymes and their plausible function in vivo. Some authors have demonstrated the possibility of interactions of aldolase with the ligands of adenine nature [1-5], but their findings were sometimes contradictive. Therefore, the evaluation of the specificity of the binding process still needs clarification. Our previous experiments have shown that a 10-fold molar excess of N A D H can cause the enhancement of aldolase activity by some 70%, and the amount of the phosphorus of phoshpotrioses formed in aldolase reaction increased from 23.5 + 3.5 to 40.0 + 3.0/tg per 1 mg of protein in 1 min [6]. In the present work we concentrated on the specificity and stoichiometry of the aldolase-NADH complex formation. In all experiments a homogeneous, five-times recrystallized aldolase was used. The enzyme was separated as described previously [7]. For the quantification of aldolase an absorbtion coefficient of z~ A0.1~ 280 equal to 0.91
Abbreviation: FBP, fructose 1,6-bisphosphate. Correspondence: Y.V. Chumachenko, 9, Leontovich Street, A.V. Palladin Institute of Biochemistry, Academy of Sciences, Ukrainian SSR, Kiev, U.S.S.R.
was used. Concentration of N A D H , N A D P H and N A D ÷ was determined using molar absorptions (e260) of 14.1.10 3, 15.0.10 3 and 17.6.10 3 M -1 . c m -1, respectively. All of them were from Boehringer and were used without further purification. For the concentration of A T P (Sigma) an e259 of 15.4.103 M - 1 . cm -1 was used. Fluorimetric assays were carried out with a 'Hitachi MPF-4' spectrofluorimeter at 22 ° C. Since the results obtained with or without a correction unit were the same an instrument sensitivity correction of spectra was not necessary. Rectangular cells of 1 × 1 cm were used. The inner filter effect was corrected as mentioned before [8]. When high ligand concentrations were used, the fluorescence was registered from the cell surface. In this case no correction was needed. Intrinsic protein fluorescence was excited at 293 nm and the fluorescence of N A D H and N A D P H was excited at 340 nm. The binding of aldolase to the dinucleotides was followed by intrinsic fluorescence quenching and expressed by Hill [9,10], Scatchard [11] and Stinson and Holbrook [12] plots. The dissociation constant (KD) values and the number of binding sites were determined by the fluorescence of N A D H and N A D P H as well. It is noteworthy to mention that in the presence of aldolase the fluorescence of N A D H was quenched with a concomitant shortwave shift in its maximum. The binding parameters, as determined with extrinsic fluorescence [13,14], were similar to those obtained with the intrinsic fluorescence quenching. For the evaluation of K D, the method of Senkewich et al. [15] was found to be highly efficient. All experiments were carried out in 0.01 M sodium phosphate buffer ( p H 7.4). The dissociation of aldolase into subunits was performed in 1.2 M MgC12 [16]. Since the enhancement of aldolase activity could have a structural origin, the N A D H - i n d u c e d changes in the enzyme fluorescence should be expected. For com-
0167-4838/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
275 parison two more structurally related ligands were selected: N A D P H and N A D +. Some attempts to describe the interaction of aldolase with the adenine-containing structures have been made before [2-5]. In our experiments, the affinity of aldolase to N A D H , N A D P H and N A D ÷ was investigated by the quenching of the intrinsic fluorescence accompanying the enzyme-ligand interaction. This tactic is quite mastered, well described [10,12] and needs a small, almost physiological, concentration of the solutes. When added to aldolase, N A D H , N A D P H or N A D ÷ caused protein fluorescence quenching, the degree of which greatly depended on the ligand used. The Hill representation [9,10] of a gradual satuaration of aldolase solutions with these ligands has shown that N A D H had the highest affinity to the enzyme (Fig. 1). The K D for the aldolase-NADH complex was found by this method to be 6.0 + 0.3/~M, which is 1-2 orders smaller than that for N A D P H and N A D ÷ (41 + 4/~M and 140 + 30/xM, respectively). The K D value for aldolase-NADH interaction was close to that of some NAD-dependent enzymes [12,17], which are known to have the NAD-binding site in their structure. The Hill coefficients for N A D H and N A D P H were equal to 1, and that for N A D + was equal to 1.1. Such a result leads one to believe that the interaction of the enzyme with these three dinucleotides is noncooperative in its character. Since N A D H has the highest affinity to the enzyme, we applied the Scatchard technique to resolve the NADH-binding pattern. Plotting the data according to the Scatchard relationship resulted in a biphasic saturation curve (Fig. 1, Inset). Such a representation suggests the existence in aldolase of two NADH-binding sites with K D values of 6.0 + 0.3 /~M and 130 + 40 /xM, respectively. Using Stinson and Holbrook representation [12], it was calculated that tetrameric aldolase can bind four molecules of N A D H . Of interest is the NADH-induced blue shift in the aldolase emission maximum from 322 to 314 nm. Neither N A D P H nor N A D + generated such a shift. This effect is the subject of a separate discussion. It was tempting to compare the data obtained for the tetrameric aldolase with those for the monomeric form. For this purpose the fluorescence of N A D H fitted well. The enzyme readily dissociates in 1.2 M MgC12 [16], and since the spectral properties of N A D H at such salt concentration remained unaffected, its fluorescence was used for K D evaluation. The dissociation of the enzyme manifested itself in a significant fall in NADH-binding capacity ( K D = 29 + 4 ptM). This fact, and the lack of cooperativity in the ligand-binding process, led us to believe that the binding site is somehow related to intersubunit interface contact. Binding to aldolase has resulted in both the N A D H emission quenching and in the shift in its maximum from 460 to 440 nm. The results of these experiments were also used for the
I
2 I 0
-1
-10
-14 -is
-2 -3 Fig. 1. Evaluationof the binding parametersof aldolaseby tryptophan fluorescencequenching with NADH (1), NADPH (2) and NAD ÷ (3). o and [] represent quenchingwith NADH in the presence of 100 #M of FBP and ATP, respectively. (Fo- F) ~=
(to- rm~)
where F 0 is the initial fluorescence, F is the fluorescence in the presence of ligand and Fmas is the fluorescence at the end of quenching; [ligand]free = [ligand]tot- E 0 where [ligand]tree is the concentration of free ligand in the solution, [ligand]tot is the total concentration of ligand and E 0 is the initial enzyme normality. Slope equals Hill coefficient; ordinate intercept equals - I n K o . Experimental data for quenching with N A D H are presented for high-affinity site with K D = 6.0+0.3 #M. The excitation wavelength is 293 nm; emission wavelength is 322 nm. Inset: Scatchard plot for the binding of N A D H to aldolase. N A D H (final concentrations from 1.4.10 -6 M to 300.10-6 M) was added to the enzyme solution (7.0-10 -7 M). / i F is the difference between the fluorescence of the solution containing enzyme and N A D H , and that containing the enzyme only. K D is equal to the slope. The excitation wavelength is 293 nm; emission wavelength is 322 nm.
evaluation of the number of binding sites. All the findings were parallel to those obtained for the intrinsic fluorescence quenching and provide additional evidence of the complex formation. Quenching of N A D H fluorescence with aldolase was for us somewhat unexpected, because in the presence of NAD-dependent dehydrogenases the emission of this ligand usually increased [13,18,19]. Similar observations, i.e., quenching and blue shift in N A D H fluorescence maximum, have been reported for the other glycolytic enzyme, glyceraldehyde3-phosphate dehydrogenase [18]. Some authors have attempted to locate the aldolase sequence responsible for the binding of adenine-containing ligands [1,2,5,20], but the subject still remains a
276 matter of guesswork. In itself, the fact of augmentation of aldolase activity suggests the existence of an N A D binding site outside the active centre of the enzyme and noncompetitive relations between its substrate F B P and N A D H , i.e., ternary complex formation. The fluorimetric investigation of a l d o l a s e - N A D H interaction in the presence of F B P showed that the substrate does not interfere with this process (Fig. 1, line 1). It is well documented that aldolase can interact with ATP, and in each subunit of this enzyme there are two sites with K D values of 0.024 and 0.45 m M , respectively [3]. These authors observed that saturation of the first site with A T P induced an inhibition of the aldolase reaction, and this fact was interpreted as evidence for the concurrence between A T P and FBP. We observed that when the same site was saturated with ATP, the affinity of the enzyme to N A D H remained unaltered (Fig. 1, line 1). A subsequent rise in A T P concentration and saturation of the second site (data not shown), resulted in a significant fall in a l d o l a s e - N A D H interaction. Retaining of the N A D H - b i n d i n g capacity by aldolase in the presence of substrate or inhibitor suggests that its active site and the high-affinity N A D H - b i n d i n g site are either completely separated or that their overlapping does not h a m p e r the binding process. It was previously documented [21,22] that aldolase possesses one of the lowest, if not the lowest, catalytic capacity of all enzymes in the glycolytic pathway. If this is so, the augmentation of the enzyme activity might be attributed to the necessity to bring its function into conformity with the mean velocity level of glycolysis. However, this logical pattern arises from the experiments with the separate purified enzyme. The situation in vivo m a y appear to be more complicated. The structural and functional properties of any enzyme can be greatly dependent on m a n y factors, which are destroyed or eliminated during protein separation and purification. N e w experiments will be needed to shed light on
this problem. W e n o w have experimental evidence that the interaction with N A D H is accompanied by significant c o n f o r m a t i o n a l changes in aldolase, and the work with m o r e complex systems involving other glycolytic enzymes and m e m b r a n e s is in progress.
References 1 Stellwagen, E. (1976) J. Mol. Biol. 106, 903-911. 2 Kasprzak, A.A. and Kochman, M. (1980) Biochim. Biophys. Acta 612, 455-459. 3 Kasprzak, A.A. and Kochman, M. (1980) Eur. J. Biochem. 104,
443-450. 4 Kasprzak, A.A. and Kochman, M. (1981) J. Biol. Chem. 256, 6127-6177. 5 Matteuzzi, M., Bellini, T., Bergamini, C.M. and Dallocio, E. (1985) Biochem. Int. 10, 53-62. 6 Chumachenko, Y.V., Lototskaya, L.S. and Kudlai, T.N. (1985) Ukr. Biokhim. Zh. 57, 33-38. 7 Guly, M.F., Kolomiichenko, M.A., Dvornikova, P.D. and Popadiuk, E.J. (1954) Ukr. Biokhim. Zh. 26, 130-138. 8 London, E. (1988) Anal. Biochem. 154, 57-63. 9 Brown, W.E.L. and Hill, A.V. (1922-1923) Proc. R. Soc. Ser. B. 94, 297-334. 10 Luisi, P.L. and Favilla, R. (1970) Eur. J. Biochem. 17, 91-94. 11 Scatchard, G. (1949) Ann. N.Y. Acad. Sci. 511,660-668. 12 Stinson, R.A. and Holbrook, J.J. (1973) Biochem. J. 131, 719-728. 13 Winer, A.D., Schwert, G.W. and Millar, D.B.S. (1959) J. Biol. Chem. 234, 1149-1154. 14 Daniel, E. and Weber, G. (1966) Biochemistry 5, 1893-1900. 15 Senkewich, S.B., Strumilo, S.A., Zavodnik, I.B. and Vinogradov, V.V. (1986) Biochimia 51, 1534-1539. 16 Hsu, L.-S. and Neet, K.E. (1973) Biochemistry 12, 586-595. 17 Luisi, P.L., Olomucki, A., Baici, A. and Karlovic, D. (1973) Biochemistry 12, 4100-4106. 18 Velick, S.F. (1958) J. Biol. Chem. 233, 1455-1467. 19 Ross, J.B.A., Subramanian, S. and Brand, L. (1982) in The Pyridine Nucleotide Coenzymes (Everse, J., Anderson, B. and KwanSaYou, eds.), pp. 19-49, Academic Press, New York. 20 Grazi, E., Dilasio, A., Trombetta, G. and Magri, E. (1978) Arch. Biochem. Biophys. 190, 405-408. 21 Scrutton, M.C. and Utter, M.F. (1968) Annu. Rev. Biochem. 37, 249-302. 22 Walsh, T.R., Masters, C.J., Morton, D.J. and Clarke, F.M. (1981) Biochim. Biophys. Acta 675, 29-39.
Biochimica et Biophysica Acta, 1038 (1990) 274-276 Elsevier
BBA Report
BBAPRO 30265
The interaction of rabbit muscle aldolase with N A D H Yuri V. Chumachenko, Alexander I. Sytnik and Alexander P. Demchenko A. V. Palladin Institute of Biochemistry, Academy of Sciences of the Ukrainian SSR, Kiev (U. S. S.R.) (Received 25 September 1989)
Key words: Fluorescence; NAD binding domain; Aldolase; (Rabbit muscle)
Fluorescence studies on both the emission of aldolase and N A D H bound to the enzyme were carried out. Aldolase was found to bind four molecules of N A D H with K o = 6.0 + 0 . 3 / t M . K D values for N A D P H and N A D + were 41 ± 4 / t M and 140 q - 3 0 / t M , respectively. The affinity to N A D H was comparable with that of some NAD-dependent dehydrogenases, and was not affected by the substrate or the inhibitor.
Rabbit muscle fructose 1,6-bisphosphate aldolase (D-fructose-l,6-bisphosphate D-glyceraldehyde-3-phosphate-lyase, EC 4.1.2.13) is the enzyme of the glycolytic pathway which catalyzes the aldole cleavage of fructose 1,6-bisphosphate (FBP) into two phosphotrioses. The enzyme consists of four identical subunits, with three tryptophans in each of them. Aldolase was long believed to be among the well-explored proteins, but the data of the last decade has shown that still little is known about this or other so-called 'soluble' enzymes and their plausible function in vivo. Some authors have demonstrated the possibility of interactions of aldolase with the ligands of adenine nature [1-5], but their findings were sometimes contradictive. Therefore, the evaluation of the specificity of the binding process still needs clarification. Our previous experiments have shown that a 10-fold molar excess of N A D H can cause the enhancement of aldolase activity by some 70%, and the amount of the phosphorus of phoshpotrioses formed in aldolase reaction increased from 23.5 + 3.5 to 40.0 + 3.0/tg per 1 mg of protein in 1 min [6]. In the present work we concentrated on the specificity and stoichiometry of the aldolase-NADH complex formation. In all experiments a homogeneous, five-times recrystallized aldolase was used. The enzyme was separated as described previously [7]. For the quantification of aldolase an absorbtion coefficient of z~ A0.1~ 280 equal to 0.91
Abbreviation: FBP, fructose 1,6-bisphosphate. Correspondence: Y.V. Chumachenko, 9, Leontovich Street, A.V. Palladin Institute of Biochemistry, Academy of Sciences, Ukrainian SSR, Kiev, U.S.S.R.
was used. Concentration of N A D H , N A D P H and N A D ÷ was determined using molar absorptions (e260) of 14.1.10 3, 15.0.10 3 and 17.6.10 3 M -1 . c m -1, respectively. All of them were from Boehringer and were used without further purification. For the concentration of A T P (Sigma) an e259 of 15.4.103 M - 1 . cm -1 was used. Fluorimetric assays were carried out with a 'Hitachi MPF-4' spectrofluorimeter at 22 ° C. Since the results obtained with or without a correction unit were the same an instrument sensitivity correction of spectra was not necessary. Rectangular cells of 1 × 1 cm were used. The inner filter effect was corrected as mentioned before [8]. When high ligand concentrations were used, the fluorescence was registered from the cell surface. In this case no correction was needed. Intrinsic protein fluorescence was excited at 293 nm and the fluorescence of N A D H and N A D P H was excited at 340 nm. The binding of aldolase to the dinucleotides was followed by intrinsic fluorescence quenching and expressed by Hill [9,10], Scatchard [11] and Stinson and Holbrook [12] plots. The dissociation constant (KD) values and the number of binding sites were determined by the fluorescence of N A D H and N A D P H as well. It is noteworthy to mention that in the presence of aldolase the fluorescence of N A D H was quenched with a concomitant shortwave shift in its maximum. The binding parameters, as determined with extrinsic fluorescence [13,14], were similar to those obtained with the intrinsic fluorescence quenching. For the evaluation of K D, the method of Senkewich et al. [15] was found to be highly efficient. All experiments were carried out in 0.01 M sodium phosphate buffer ( p H 7.4). The dissociation of aldolase into subunits was performed in 1.2 M MgC12 [16]. Since the enhancement of aldolase activity could have a structural origin, the N A D H - i n d u c e d changes in the enzyme fluorescence should be expected. For com-
0167-4838/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
275 parison two more structurally related ligands were selected: N A D P H and N A D +. Some attempts to describe the interaction of aldolase with the adenine-containing structures have been made before [2-5]. In our experiments, the affinity of aldolase to N A D H , N A D P H and N A D ÷ was investigated by the quenching of the intrinsic fluorescence accompanying the enzyme-ligand interaction. This tactic is quite mastered, well described [10,12] and needs a small, almost physiological, concentration of the solutes. When added to aldolase, N A D H , N A D P H or N A D ÷ caused protein fluorescence quenching, the degree of which greatly depended on the ligand used. The Hill representation [9,10] of a gradual satuaration of aldolase solutions with these ligands has shown that N A D H had the highest affinity to the enzyme (Fig. 1). The K D for the aldolase-NADH complex was found by this method to be 6.0 + 0.3/~M, which is 1-2 orders smaller than that for N A D P H and N A D ÷ (41 + 4/~M and 140 + 30/xM, respectively). The K D value for aldolase-NADH interaction was close to that of some NAD-dependent enzymes [12,17], which are known to have the NAD-binding site in their structure. The Hill coefficients for N A D H and N A D P H were equal to 1, and that for N A D + was equal to 1.1. Such a result leads one to believe that the interaction of the enzyme with these three dinucleotides is noncooperative in its character. Since N A D H has the highest affinity to the enzyme, we applied the Scatchard technique to resolve the NADH-binding pattern. Plotting the data according to the Scatchard relationship resulted in a biphasic saturation curve (Fig. 1, Inset). Such a representation suggests the existence in aldolase of two NADH-binding sites with K D values of 6.0 + 0.3 /~M and 130 + 40 /xM, respectively. Using Stinson and Holbrook representation [12], it was calculated that tetrameric aldolase can bind four molecules of N A D H . Of interest is the NADH-induced blue shift in the aldolase emission maximum from 322 to 314 nm. Neither N A D P H nor N A D + generated such a shift. This effect is the subject of a separate discussion. It was tempting to compare the data obtained for the tetrameric aldolase with those for the monomeric form. For this purpose the fluorescence of N A D H fitted well. The enzyme readily dissociates in 1.2 M MgC12 [16], and since the spectral properties of N A D H at such salt concentration remained unaffected, its fluorescence was used for K D evaluation. The dissociation of the enzyme manifested itself in a significant fall in NADH-binding capacity ( K D = 29 + 4 ptM). This fact, and the lack of cooperativity in the ligand-binding process, led us to believe that the binding site is somehow related to intersubunit interface contact. Binding to aldolase has resulted in both the N A D H emission quenching and in the shift in its maximum from 460 to 440 nm. The results of these experiments were also used for the
I
2 I 0
-1
-10
-14 -is
-2 -3 Fig. 1. Evaluationof the binding parametersof aldolaseby tryptophan fluorescencequenching with NADH (1), NADPH (2) and NAD ÷ (3). o and [] represent quenchingwith NADH in the presence of 100 #M of FBP and ATP, respectively. (Fo- F) ~=
(to- rm~)
where F 0 is the initial fluorescence, F is the fluorescence in the presence of ligand and Fmas is the fluorescence at the end of quenching; [ligand]free = [ligand]tot- E 0 where [ligand]tree is the concentration of free ligand in the solution, [ligand]tot is the total concentration of ligand and E 0 is the initial enzyme normality. Slope equals Hill coefficient; ordinate intercept equals - I n K o . Experimental data for quenching with N A D H are presented for high-affinity site with K D = 6.0+0.3 #M. The excitation wavelength is 293 nm; emission wavelength is 322 nm. Inset: Scatchard plot for the binding of N A D H to aldolase. N A D H (final concentrations from 1.4.10 -6 M to 300.10-6 M) was added to the enzyme solution (7.0-10 -7 M). / i F is the difference between the fluorescence of the solution containing enzyme and N A D H , and that containing the enzyme only. K D is equal to the slope. The excitation wavelength is 293 nm; emission wavelength is 322 nm.
evaluation of the number of binding sites. All the findings were parallel to those obtained for the intrinsic fluorescence quenching and provide additional evidence of the complex formation. Quenching of N A D H fluorescence with aldolase was for us somewhat unexpected, because in the presence of NAD-dependent dehydrogenases the emission of this ligand usually increased [13,18,19]. Similar observations, i.e., quenching and blue shift in N A D H fluorescence maximum, have been reported for the other glycolytic enzyme, glyceraldehyde3-phosphate dehydrogenase [18]. Some authors have attempted to locate the aldolase sequence responsible for the binding of adenine-containing ligands [1,2,5,20], but the subject still remains a
276 matter of guesswork. In itself, the fact of augmentation of aldolase activity suggests the existence of an N A D binding site outside the active centre of the enzyme and noncompetitive relations between its substrate F B P and N A D H , i.e., ternary complex formation. The fluorimetric investigation of a l d o l a s e - N A D H interaction in the presence of F B P showed that the substrate does not interfere with this process (Fig. 1, line 1). It is well documented that aldolase can interact with ATP, and in each subunit of this enzyme there are two sites with K D values of 0.024 and 0.45 m M , respectively [3]. These authors observed that saturation of the first site with A T P induced an inhibition of the aldolase reaction, and this fact was interpreted as evidence for the concurrence between A T P and FBP. We observed that when the same site was saturated with ATP, the affinity of the enzyme to N A D H remained unaltered (Fig. 1, line 1). A subsequent rise in A T P concentration and saturation of the second site (data not shown), resulted in a significant fall in a l d o l a s e - N A D H interaction. Retaining of the N A D H - b i n d i n g capacity by aldolase in the presence of substrate or inhibitor suggests that its active site and the high-affinity N A D H - b i n d i n g site are either completely separated or that their overlapping does not h a m p e r the binding process. It was previously documented [21,22] that aldolase possesses one of the lowest, if not the lowest, catalytic capacity of all enzymes in the glycolytic pathway. If this is so, the augmentation of the enzyme activity might be attributed to the necessity to bring its function into conformity with the mean velocity level of glycolysis. However, this logical pattern arises from the experiments with the separate purified enzyme. The situation in vivo m a y appear to be more complicated. The structural and functional properties of any enzyme can be greatly dependent on m a n y factors, which are destroyed or eliminated during protein separation and purification. N e w experiments will be needed to shed light on
this problem. W e n o w have experimental evidence that the interaction with N A D H is accompanied by significant c o n f o r m a t i o n a l changes in aldolase, and the work with m o r e complex systems involving other glycolytic enzymes and m e m b r a n e s is in progress.
References 1 Stellwagen, E. (1976) J. Mol. Biol. 106, 903-911. 2 Kasprzak, A.A. and Kochman, M. (1980) Biochim. Biophys. Acta 612, 455-459. 3 Kasprzak, A.A. and Kochman, M. (1980) Eur. J. Biochem. 104,
443-450. 4 Kasprzak, A.A. and Kochman, M. (1981) J. Biol. Chem. 256, 6127-6177. 5 Matteuzzi, M., Bellini, T., Bergamini, C.M. and Dallocio, E. (1985) Biochem. Int. 10, 53-62. 6 Chumachenko, Y.V., Lototskaya, L.S. and Kudlai, T.N. (1985) Ukr. Biokhim. Zh. 57, 33-38. 7 Guly, M.F., Kolomiichenko, M.A., Dvornikova, P.D. and Popadiuk, E.J. (1954) Ukr. Biokhim. Zh. 26, 130-138. 8 London, E. (1988) Anal. Biochem. 154, 57-63. 9 Brown, W.E.L. and Hill, A.V. (1922-1923) Proc. R. Soc. Ser. B. 94, 297-334. 10 Luisi, P.L. and Favilla, R. (1970) Eur. J. Biochem. 17, 91-94. 11 Scatchard, G. (1949) Ann. N.Y. Acad. Sci. 511,660-668. 12 Stinson, R.A. and Holbrook, J.J. (1973) Biochem. J. 131, 719-728. 13 Winer, A.D., Schwert, G.W. and Millar, D.B.S. (1959) J. Biol. Chem. 234, 1149-1154. 14 Daniel, E. and Weber, G. (1966) Biochemistry 5, 1893-1900. 15 Senkewich, S.B., Strumilo, S.A., Zavodnik, I.B. and Vinogradov, V.V. (1986) Biochimia 51, 1534-1539. 16 Hsu, L.-S. and Neet, K.E. (1973) Biochemistry 12, 586-595. 17 Luisi, P.L., Olomucki, A., Baici, A. and Karlovic, D. (1973) Biochemistry 12, 4100-4106. 18 Velick, S.F. (1958) J. Biol. Chem. 233, 1455-1467. 19 Ross, J.B.A., Subramanian, S. and Brand, L. (1982) in The Pyridine Nucleotide Coenzymes (Everse, J., Anderson, B. and KwanSaYou, eds.), pp. 19-49, Academic Press, New York. 20 Grazi, E., Dilasio, A., Trombetta, G. and Magri, E. (1978) Arch. Biochem. Biophys. 190, 405-408. 21 Scrutton, M.C. and Utter, M.F. (1968) Annu. Rev. Biochem. 37, 249-302. 22 Walsh, T.R., Masters, C.J., Morton, D.J. and Clarke, F.M. (1981) Biochim. Biophys. Acta 675, 29-39.