ARCHIVES
OF BIOCHEMISTRY
AND
BIOPHYSICS
Interaction
168, 531-535 (1975)
of Muscle
Pyruvate
1,8-Anilinonaphthalene K. C. INGHAM2 Biochemistry
Department,,
Michigan
Kinase with
Sulfonate’ C. H. WELTER3
AND
State University,
East Lansing, Michigan
48824
Received October 21, 1974 The interaction of the fluorescent probe l&anilinonaphthalene sulfonate with rabbit muscle pyruvate kinase is reported. Four moles of 1,8-anilinonaphthalene sulfonate interact per mole of the 4subunit enzyme with a dissociation constant of 1.7 x lo-’ M at 30”. The interaction is not competitive with substrates ADP and phosphoenolpyruvate, with cations K+ and Mg2+, nor with effector phenylalanine.
Rabbit muscle pyruvate kinase is a tetrameric enzyme (M, 237,000) catalyzing a phosphoryl transfer between phosphoenolpyruvate (PEP)’ and adenosine diphosphate (ADP). The reaction requires both a divalent (Mg2+) and a monovalent (K+) cation for optimum activity. The binding of these activators results in a conformational change manifested by a partial quenching and increased polarization of the tryptophan fluorescence (l), a negative difference absorption spectrum in the 280-310 nm region (2, 3), and increased ellipticity in the 260-290 nm region (4). Substrate binding and changes in temperature also affect the conformation (5). ANS (l,%anilinonaphthalene sulfonate) is known to bind to a variety of proteins (6). The binding is manifested by an enhanced fluorescence quantum yield and a blue shift in the maximum of the fluorescence spectrum. Furthermore, the changes in ANS ’ Supported in part by the National Science Foundation, BG 25116, and Michigan State Agricultural Station Journal Article 6995. *Supported in part by NIH Training Grant GM 1091. Present address: National Institute of Arthritis, Metabolism, and Digestive Diseases, NIH, Bethesda, MD 20014. 3 NIH Carrier Development Awardee (L-K3GM-9725-02). ’ Abbreviations used: ANS, l&anilinonaphthalene sulfonate, and PEP, phosphoenolpyruvate.
fluorescence which accompany the addition of substrates, activators, and inhibitors are often quite dramatic, offering a convenient method for monitoring these interactions under conditions which utilize a minimum amount of protein. In hopes of developing a sensitive probe for activator, substrate, and temperature-induced conformational transitions in pyruvate kinase, we have investigated the interaction of this enzyme with ANS. EXPERIMENTAL
Pyruvate kinase was prepared from frozen rabbit muscle by the method of Tietz and Ochoa (7) with a final ammonium sulfate fractionation as previously described’ (3). The final precipitate was dissolved in imidazole hydrochloride. Enzyme was stored as a solution (50-80 mg/ml) in 0.05 M imidazole HCl, pH 7, at 4°C. Immediately prior to use, the enzyme was passed through a Sephadex G-25 column, eluting with 0.1 M tetramethylammonium cacodylate buffer at pH 7.5. The protein fraction was heated to 35-39°C for 20-30 min followed by centrifugation to remove the small amount of precipitate thus formed. This prevented the later appearance of turbidity during the course of some of the fluorescence titrations. Enzyme concentrations were determined spectrophotometritally using cZBO= 1.28 x lo5 M-’ cm-’ (3). The magnesium salt of ANS (Aldrich) was recrystallized three times from H,O and the concentrations determined using caeO= 5000Me1 cm-’ (8). Fluorescence measurements were made on an Aminco-Bowman spectrofluorimeter equipped with a jacketed cell holder for controlling the temperature at
531 Copyright 0 1975by Academic Press, Inc. All rights of reproduction in any form reserved.
PROCEDURES
532
INGHAM AND SUELTER
30°C. The optical density was kept below 0.1 at the excitation wavelength to minimize errors due to absorption artifacts. Corrections were made for any dilution factors as well as for any background fluorescence. In order to compensate for fluctuations in the lamp intensity and to compare results from one experiment to the next, all fluorescence intensities were relative to those of a reference solution of quinine sulfate in 0.1 N sulfuric acid using the same excitation and emission wavelength (410 nm and 470 nm, respectively, unless otherwise stated). Titration of ANS with protein was accomplished by successive removal of small aliquots from a concentrated protein solution containing ANS and replacing by an equal volume of a solution of ANS at the same concentration. In this way, the protein concentration was continuously varied while keeping ANS constant. The usual method of measuring pyruvate kinase catalytic activity by coupling to lactate dehydrogenase could not be employed because of binding of ANS to the dehydrogenase. Therefore, the pH stat (3) and direct spectrophotometric assays (9) were used. Lineweaver-Burk plots were subjected to the statistical analysis suggested by Wilkinson (10).
10e5 M) with protein and the circles represent the titration of fixed protein (4.0 x 1O-5 M in subunits) with ANS. The double reciprocal plots inserted in Fig. 1 give the same limiting slope and intercept at the ordinate axis. The latter is reciprocally related to the maximum fluorescence (F,,,) which would be obtained under saturating conditions. Both titrations extrapolate to the same maximum fluorescence suggesting that each subunit binds one molecule of ANS. The deviation of the fixed ANS titration data (triangles) at low protein concentration may be due to slight dissociation of the tetramer exposing new sites. The fraction of dye bound at any point can be calculated from the relation X = The data can then be treated FodF,,,. according to Scatchard (11) using the following equation: 6 &(1-X)
RESULTS AND DISCUSSION
Binding of ANS The binding of ANSto pyruvate kinase is accompanied by an enhanced quantum yield of fluorescence and a blue shift of the maximum to -470 nm. This enhancement is illustrated in Fig. 1 where the triangles represent the titration of fixed ANS (1.0 x
n = Kd-
6 EJ
where D, is the total dye concentration, v is the ratio of the concentration of bound dye (XD,) to the total subunit concentration, Kd is the dissociation constant, and n is the number of ANS binding sites (per subunit). The results for the fixed protein titration are illustrated by the circles in 1
I
2 [ANS]
3 w [SUBUNITS].
4
s
X IO4
FIG. 1. Enhancement of ANS fluorescence by pyruvate kinase: (O), titration of fixed protein (4.0 x 10e5 M subunits) with dye; (A), titration of fixed dye (4.0 x 10e5 M) with protein. All measurements were at 30°C in 0.1 M tetramethylammonium cacodylate buffer, 0.1 M KCl, 0.001 M MgCI,, pH 7.5.
l,R-ANS
INTERACTION
WITH
Fig. 2 which give an excellent straight line corresponding to n = 1.0 and Kd = 1.7 x 10m4 M. An analogous treatment of the fixed ANS titration data (triangles, Fig. 2) gives good agreement except at very low protein concentration as previously mentioned. Effect
of Substrates and Activators ANS Fluorescence
on
In order to gain insight into the nature of the ANS binding site, the fluorescence of a solution of pyruvate kinase (3.3 PM) and ANS (1.0 x 1O-4 M) was examined in the presence and absence of saturating levels of various substrates and activators. The results are summarized in Table I where the relative fluorescence intensities are normalized to a value of 100 in the absence of any added cations or substrates. All mea-
MUSCLE
PYRUVATE
surements were made in 0.1 M tetramethylammonium cacodylate buffer, at pH 7.5 and 30°C. Several points are worth noting. First of all, 0.1 M (CH,),N+ (a nonactivating cation) and 0.1 M K+ have essentially the same effect, reducing the ANS fluorescence to about 80% of its value in the absence of these ions. Thus, the effect of the activating cation K+ can be explained entirely in terms of ionic strength. The presence of 0.1 M Mg2+, in addition to 0.1 M K+, causes a further reduction to about 66% of the value in their absence. If saturating levels of ADP or PEP are introduced, the fluorescence drops to less than half the reference level, either.in the presence or absence of the activating cations. If both ADP and PEP are present, the fluorescence is further reduced. However, even at saturating levels of these substrates, substantial ANS fluorescence persists. If ANS were simply competing with substrate for the active site, the fluorescence should have dropped to less than 1% of its level in the absence of substrate. This suggests that the ANS binding site is not identical with the active site although there could be some overlap. Alternatively, ANS may be bound at a remote site whose properties are altered by the binding of substrates and/or activators. The decrease in ANS fluorescence produced by Mg2+ is reversed by the addition of phenylalanine. This is consistent with the observed decrease in Mn2+ binding produced by phenylalanine (12). It is not due to chelation of cation since the association constants for cation binding with enzyme and phenylalanine are not consistent with this interpretation. Effect
FIG. 2. Scatchard analysis of the binding of ANS to pyruvate kinase. Conditions and symbols are identical to those in Fig. 1. The straight line is theoretical obtained by linear regression analysis of the circled data and has a correlation coefficient of 0.992.
533
KINASE
of ANS on Enzyme
Activity
The effect of ANS on enzyme kinetics was investigated by measuring the substrate dependence of the initial velocity in the presence and absence of the dye. The results (Table II) are consistent with a noncompetitive inhibition. Apparent K, values for ADP and PEP were not significantly changed by the presence of ANS. By contrast, V decreased significantly with increasing ANS concentration. The K, for ANS (assuming simple noncompetitive in-
534
INGHAM
EFFECT
OF SUBSTRATES AND ACTIVATORS Molar KC1
(CK),NCl
0 0 0.1 0.1 0.1 0.1 0.1 0 0 0 0.1 0
“The ANS pH 7.5, 30°C.
concentration The excitation
WELTER
TABLE I ON THE ENHANCEMENT
OF ANS
FLUORESCENCE
concentrations
M&L
0 0.1 0 0 0 0 0 0 0 0 0 0
AND
0 0 0 0.01 0.01 0.01 0.01 0 0 0 0.01 0 was 1.0 x lo-’ and emission
Ionic strength
ADP
PEP
Phe
0 0 0 0 0.01 0 0.01 0.01 0 0.01 0 0
0 0 0 0 0 0.01 0.01 0 0.01 0.01 0 0
0 0 0 0 0 0 0 0 0 0 0.01 0.01
M. Pyruvate wavelengths
BY PYRUVATE KINASEO Fluorescence intensity
0.10 0.20 0.20 0.23 0.24 0.24 0.25 0.11 0.11 0.12 0.23 0.10
kinase was 3.3 PM in 0.1 M (CH,),N were 350 and 470 nm, respectively.
100 78 82 66 45 42 37 47 46 35 78 83 cacodylate
buffer,
binding is weaker than we had hoped, it should be sufficient to make ANS useful as a probe of conformational transitions in ANS Apparent K, (mM) Relative this enzyme. Preliminary results indicate a V (rnM) (ADP) (PEP) substantial change in ANS fluorescence upon binding of activators, substrates, and 0.000 0.40 l 0.01 lOOA 4 the allosteric inhibitor, phenylalanine (12). 0.250 0.29 i 0.04 44+ 4 Further studies are required to determine 0.000 0.22 * 0.04 100 f 12 whether these changes reflect a change in 0.081 0.24 zt 0.03 75i 8 affinity or a change in the quantum yield of 0.139 0.13 * 0.04 35 f 17 bound ANS (or both). 0.000 0.021 l 0.003 lOOk 4 Although 4 mol of ANS bind per mole of 0.058 0.033 +c 0.006 72+ 8 45 f 14 0.116 0.019 l 0.011 protein, which is equivalent to the number of catalytic or phenylalanine binding sites, 0 All solutions contained 0.1 M KCI, 0.01 M MgCl,, the evidence does not support interaction and saturating levels of fixed substrate at pH 7.5 and at either site. First, saturating levels of 30°C. V for all three groups of data was arbitrarily cations or substrates which are known to normalized to a value of 100 in the absence of ANS. bind (2) do not eliminate the fluorescence Data in the first two rows were obtained by the enhancement. Second, the kinetic inhibipH-stat method in unbuffered solutions. The remaintion by ANS was not competitive when ing data were obtained by observing the disappearance of PEP absorption at 240 nm in 0.1 M (CH,),N ADP or PEP were varied (Table II). Neicacodylate buffer. Higher concentrations of ANS ther is ANS bound at the phenylalanine could not be used due to interfering absorption. binding site (13), since addition of phenylalanine increases the fluorescence of ANS. hibition) ranged from 1.0 to 1.9 x lo-’ M in The data are consistent with yet another reasonable agreement with the dissociation site on the protein surface for which no constant determined fluorimetrically in physiological function is known. the absence of substrates, TABLE
EFFECT
OF ANS
II
ON PYRUVATE
KINASE
ACTIVITY~
REFERENCES SUMMARY
AND
CONCLUSIONS
Pyruvate kinase binds 4 mol of ANS per mole of tetrameric enzyme with a dissociation constant of 1.7 x 1O-4 M. Although the
1. SUELTER, C. H. (1967) Biochemistry 6, 418-423. 2. SVELTER, C. H., SINGLETON, R., JR., KAYNE, F. J., ARRINC~TON, S., GLASS, J., AND MILDVAN, A. S. (1966) Biochemists 5, 131-139.
1.8-ANS
INTERACTION
WITH
3. KAYNE, F. J., AND SUELTER, C. H. (1965) J. Amer. Chem. Sot. 87, 897-900. 4. WILDES, R. A., EVANS, H. J., AND BECKER, R. R. (1971) Biochim. Biophys. Acta 229, 850-854. 5. KAYNE, F. J., AND SUELTER, C. H. (1968) Biochemistry 7, 1678-1684. 6. BRAND, L., AND GOHLKE, J. R. (1972) Annu. Reu. Biochem. 41, 843-868. 7. TIETZ, A., AND OCHOA, S. (1958) Arch. Biochem. Biophys. 78, 477-493.
MUSCLE
PYRUVATE
KINASE
535
8. STRYER, L. (1965) J. Mol. Biol. 13, 482-495. 9. PON, N. G., AND BONDAR, R. J. L. (1967) Anal. Biochem. 19, 272-279. 10. WILKINSON, G. N. (1961) Biochem. J. 80,324-332. 11. SCATCHARD, G. (1949) Ann. N.Y. Acad. Sci. 51, 660-672. 12. KAYNE, F. J., AND PRICE, N. L. (1972) Biochemistry 11, 4415-4420. 13. KAYNE, F. J., AND PRICE, N. L. (1973) Arch. Biochem. Riophys. 159, 292-296.
OF BIOCHEMISTRY
AND
BIOPHYSICS
Interaction
168, 531-535 (1975)
of Muscle
Pyruvate
1,8-Anilinonaphthalene K. C. INGHAM2 Biochemistry
Department,,
Michigan
Kinase with
Sulfonate’ C. H. WELTER3
AND
State University,
East Lansing, Michigan
48824
Received October 21, 1974 The interaction of the fluorescent probe l&anilinonaphthalene sulfonate with rabbit muscle pyruvate kinase is reported. Four moles of 1,8-anilinonaphthalene sulfonate interact per mole of the 4subunit enzyme with a dissociation constant of 1.7 x lo-’ M at 30”. The interaction is not competitive with substrates ADP and phosphoenolpyruvate, with cations K+ and Mg2+, nor with effector phenylalanine.
Rabbit muscle pyruvate kinase is a tetrameric enzyme (M, 237,000) catalyzing a phosphoryl transfer between phosphoenolpyruvate (PEP)’ and adenosine diphosphate (ADP). The reaction requires both a divalent (Mg2+) and a monovalent (K+) cation for optimum activity. The binding of these activators results in a conformational change manifested by a partial quenching and increased polarization of the tryptophan fluorescence (l), a negative difference absorption spectrum in the 280-310 nm region (2, 3), and increased ellipticity in the 260-290 nm region (4). Substrate binding and changes in temperature also affect the conformation (5). ANS (l,%anilinonaphthalene sulfonate) is known to bind to a variety of proteins (6). The binding is manifested by an enhanced fluorescence quantum yield and a blue shift in the maximum of the fluorescence spectrum. Furthermore, the changes in ANS ’ Supported in part by the National Science Foundation, BG 25116, and Michigan State Agricultural Station Journal Article 6995. *Supported in part by NIH Training Grant GM 1091. Present address: National Institute of Arthritis, Metabolism, and Digestive Diseases, NIH, Bethesda, MD 20014. 3 NIH Carrier Development Awardee (L-K3GM-9725-02). ’ Abbreviations used: ANS, l&anilinonaphthalene sulfonate, and PEP, phosphoenolpyruvate.
fluorescence which accompany the addition of substrates, activators, and inhibitors are often quite dramatic, offering a convenient method for monitoring these interactions under conditions which utilize a minimum amount of protein. In hopes of developing a sensitive probe for activator, substrate, and temperature-induced conformational transitions in pyruvate kinase, we have investigated the interaction of this enzyme with ANS. EXPERIMENTAL
Pyruvate kinase was prepared from frozen rabbit muscle by the method of Tietz and Ochoa (7) with a final ammonium sulfate fractionation as previously described’ (3). The final precipitate was dissolved in imidazole hydrochloride. Enzyme was stored as a solution (50-80 mg/ml) in 0.05 M imidazole HCl, pH 7, at 4°C. Immediately prior to use, the enzyme was passed through a Sephadex G-25 column, eluting with 0.1 M tetramethylammonium cacodylate buffer at pH 7.5. The protein fraction was heated to 35-39°C for 20-30 min followed by centrifugation to remove the small amount of precipitate thus formed. This prevented the later appearance of turbidity during the course of some of the fluorescence titrations. Enzyme concentrations were determined spectrophotometritally using cZBO= 1.28 x lo5 M-’ cm-’ (3). The magnesium salt of ANS (Aldrich) was recrystallized three times from H,O and the concentrations determined using caeO= 5000Me1 cm-’ (8). Fluorescence measurements were made on an Aminco-Bowman spectrofluorimeter equipped with a jacketed cell holder for controlling the temperature at
531 Copyright 0 1975by Academic Press, Inc. All rights of reproduction in any form reserved.
PROCEDURES
532
INGHAM AND SUELTER
30°C. The optical density was kept below 0.1 at the excitation wavelength to minimize errors due to absorption artifacts. Corrections were made for any dilution factors as well as for any background fluorescence. In order to compensate for fluctuations in the lamp intensity and to compare results from one experiment to the next, all fluorescence intensities were relative to those of a reference solution of quinine sulfate in 0.1 N sulfuric acid using the same excitation and emission wavelength (410 nm and 470 nm, respectively, unless otherwise stated). Titration of ANS with protein was accomplished by successive removal of small aliquots from a concentrated protein solution containing ANS and replacing by an equal volume of a solution of ANS at the same concentration. In this way, the protein concentration was continuously varied while keeping ANS constant. The usual method of measuring pyruvate kinase catalytic activity by coupling to lactate dehydrogenase could not be employed because of binding of ANS to the dehydrogenase. Therefore, the pH stat (3) and direct spectrophotometric assays (9) were used. Lineweaver-Burk plots were subjected to the statistical analysis suggested by Wilkinson (10).
10e5 M) with protein and the circles represent the titration of fixed protein (4.0 x 1O-5 M in subunits) with ANS. The double reciprocal plots inserted in Fig. 1 give the same limiting slope and intercept at the ordinate axis. The latter is reciprocally related to the maximum fluorescence (F,,,) which would be obtained under saturating conditions. Both titrations extrapolate to the same maximum fluorescence suggesting that each subunit binds one molecule of ANS. The deviation of the fixed ANS titration data (triangles) at low protein concentration may be due to slight dissociation of the tetramer exposing new sites. The fraction of dye bound at any point can be calculated from the relation X = The data can then be treated FodF,,,. according to Scatchard (11) using the following equation: 6 &(1-X)
RESULTS AND DISCUSSION
Binding of ANS The binding of ANSto pyruvate kinase is accompanied by an enhanced quantum yield of fluorescence and a blue shift of the maximum to -470 nm. This enhancement is illustrated in Fig. 1 where the triangles represent the titration of fixed ANS (1.0 x
n = Kd-
6 EJ
where D, is the total dye concentration, v is the ratio of the concentration of bound dye (XD,) to the total subunit concentration, Kd is the dissociation constant, and n is the number of ANS binding sites (per subunit). The results for the fixed protein titration are illustrated by the circles in 1
I
2 [ANS]
3 w [SUBUNITS].
4
s
X IO4
FIG. 1. Enhancement of ANS fluorescence by pyruvate kinase: (O), titration of fixed protein (4.0 x 10e5 M subunits) with dye; (A), titration of fixed dye (4.0 x 10e5 M) with protein. All measurements were at 30°C in 0.1 M tetramethylammonium cacodylate buffer, 0.1 M KCl, 0.001 M MgCI,, pH 7.5.
l,R-ANS
INTERACTION
WITH
Fig. 2 which give an excellent straight line corresponding to n = 1.0 and Kd = 1.7 x 10m4 M. An analogous treatment of the fixed ANS titration data (triangles, Fig. 2) gives good agreement except at very low protein concentration as previously mentioned. Effect
of Substrates and Activators ANS Fluorescence
on
In order to gain insight into the nature of the ANS binding site, the fluorescence of a solution of pyruvate kinase (3.3 PM) and ANS (1.0 x 1O-4 M) was examined in the presence and absence of saturating levels of various substrates and activators. The results are summarized in Table I where the relative fluorescence intensities are normalized to a value of 100 in the absence of any added cations or substrates. All mea-
MUSCLE
PYRUVATE
surements were made in 0.1 M tetramethylammonium cacodylate buffer, at pH 7.5 and 30°C. Several points are worth noting. First of all, 0.1 M (CH,),N+ (a nonactivating cation) and 0.1 M K+ have essentially the same effect, reducing the ANS fluorescence to about 80% of its value in the absence of these ions. Thus, the effect of the activating cation K+ can be explained entirely in terms of ionic strength. The presence of 0.1 M Mg2+, in addition to 0.1 M K+, causes a further reduction to about 66% of the value in their absence. If saturating levels of ADP or PEP are introduced, the fluorescence drops to less than half the reference level, either.in the presence or absence of the activating cations. If both ADP and PEP are present, the fluorescence is further reduced. However, even at saturating levels of these substrates, substantial ANS fluorescence persists. If ANS were simply competing with substrate for the active site, the fluorescence should have dropped to less than 1% of its level in the absence of substrate. This suggests that the ANS binding site is not identical with the active site although there could be some overlap. Alternatively, ANS may be bound at a remote site whose properties are altered by the binding of substrates and/or activators. The decrease in ANS fluorescence produced by Mg2+ is reversed by the addition of phenylalanine. This is consistent with the observed decrease in Mn2+ binding produced by phenylalanine (12). It is not due to chelation of cation since the association constants for cation binding with enzyme and phenylalanine are not consistent with this interpretation. Effect
FIG. 2. Scatchard analysis of the binding of ANS to pyruvate kinase. Conditions and symbols are identical to those in Fig. 1. The straight line is theoretical obtained by linear regression analysis of the circled data and has a correlation coefficient of 0.992.
533
KINASE
of ANS on Enzyme
Activity
The effect of ANS on enzyme kinetics was investigated by measuring the substrate dependence of the initial velocity in the presence and absence of the dye. The results (Table II) are consistent with a noncompetitive inhibition. Apparent K, values for ADP and PEP were not significantly changed by the presence of ANS. By contrast, V decreased significantly with increasing ANS concentration. The K, for ANS (assuming simple noncompetitive in-
534
INGHAM
EFFECT
OF SUBSTRATES AND ACTIVATORS Molar KC1
(CK),NCl
0 0 0.1 0.1 0.1 0.1 0.1 0 0 0 0.1 0
“The ANS pH 7.5, 30°C.
concentration The excitation
WELTER
TABLE I ON THE ENHANCEMENT
OF ANS
FLUORESCENCE
concentrations
M&L
0 0.1 0 0 0 0 0 0 0 0 0 0
AND
0 0 0 0.01 0.01 0.01 0.01 0 0 0 0.01 0 was 1.0 x lo-’ and emission
Ionic strength
ADP
PEP
Phe
0 0 0 0 0.01 0 0.01 0.01 0 0.01 0 0
0 0 0 0 0 0.01 0.01 0 0.01 0.01 0 0
0 0 0 0 0 0 0 0 0 0 0.01 0.01
M. Pyruvate wavelengths
BY PYRUVATE KINASEO Fluorescence intensity
0.10 0.20 0.20 0.23 0.24 0.24 0.25 0.11 0.11 0.12 0.23 0.10
kinase was 3.3 PM in 0.1 M (CH,),N were 350 and 470 nm, respectively.
100 78 82 66 45 42 37 47 46 35 78 83 cacodylate
buffer,
binding is weaker than we had hoped, it should be sufficient to make ANS useful as a probe of conformational transitions in ANS Apparent K, (mM) Relative this enzyme. Preliminary results indicate a V (rnM) (ADP) (PEP) substantial change in ANS fluorescence upon binding of activators, substrates, and 0.000 0.40 l 0.01 lOOA 4 the allosteric inhibitor, phenylalanine (12). 0.250 0.29 i 0.04 44+ 4 Further studies are required to determine 0.000 0.22 * 0.04 100 f 12 whether these changes reflect a change in 0.081 0.24 zt 0.03 75i 8 affinity or a change in the quantum yield of 0.139 0.13 * 0.04 35 f 17 bound ANS (or both). 0.000 0.021 l 0.003 lOOk 4 Although 4 mol of ANS bind per mole of 0.058 0.033 +c 0.006 72+ 8 45 f 14 0.116 0.019 l 0.011 protein, which is equivalent to the number of catalytic or phenylalanine binding sites, 0 All solutions contained 0.1 M KCI, 0.01 M MgCl,, the evidence does not support interaction and saturating levels of fixed substrate at pH 7.5 and at either site. First, saturating levels of 30°C. V for all three groups of data was arbitrarily cations or substrates which are known to normalized to a value of 100 in the absence of ANS. bind (2) do not eliminate the fluorescence Data in the first two rows were obtained by the enhancement. Second, the kinetic inhibipH-stat method in unbuffered solutions. The remaintion by ANS was not competitive when ing data were obtained by observing the disappearance of PEP absorption at 240 nm in 0.1 M (CH,),N ADP or PEP were varied (Table II). Neicacodylate buffer. Higher concentrations of ANS ther is ANS bound at the phenylalanine could not be used due to interfering absorption. binding site (13), since addition of phenylalanine increases the fluorescence of ANS. hibition) ranged from 1.0 to 1.9 x lo-’ M in The data are consistent with yet another reasonable agreement with the dissociation site on the protein surface for which no constant determined fluorimetrically in physiological function is known. the absence of substrates, TABLE
EFFECT
OF ANS
II
ON PYRUVATE
KINASE
ACTIVITY~
REFERENCES SUMMARY
AND
CONCLUSIONS
Pyruvate kinase binds 4 mol of ANS per mole of tetrameric enzyme with a dissociation constant of 1.7 x 1O-4 M. Although the
1. SUELTER, C. H. (1967) Biochemistry 6, 418-423. 2. SVELTER, C. H., SINGLETON, R., JR., KAYNE, F. J., ARRINC~TON, S., GLASS, J., AND MILDVAN, A. S. (1966) Biochemists 5, 131-139.
1.8-ANS
INTERACTION
WITH
3. KAYNE, F. J., AND SUELTER, C. H. (1965) J. Amer. Chem. Sot. 87, 897-900. 4. WILDES, R. A., EVANS, H. J., AND BECKER, R. R. (1971) Biochim. Biophys. Acta 229, 850-854. 5. KAYNE, F. J., AND SUELTER, C. H. (1968) Biochemistry 7, 1678-1684. 6. BRAND, L., AND GOHLKE, J. R. (1972) Annu. Reu. Biochem. 41, 843-868. 7. TIETZ, A., AND OCHOA, S. (1958) Arch. Biochem. Biophys. 78, 477-493.
MUSCLE
PYRUVATE
KINASE
535
8. STRYER, L. (1965) J. Mol. Biol. 13, 482-495. 9. PON, N. G., AND BONDAR, R. J. L. (1967) Anal. Biochem. 19, 272-279. 10. WILKINSON, G. N. (1961) Biochem. J. 80,324-332. 11. SCATCHARD, G. (1949) Ann. N.Y. Acad. Sci. 51, 660-672. 12. KAYNE, F. J., AND PRICE, N. L. (1972) Biochemistry 11, 4415-4420. 13. KAYNE, F. J., AND PRICE, N. L. (1973) Arch. Biochem. Riophys. 159, 292-296.
