COTTON EFFECTS IN
CREATINE PHOSPHOKINASE
References Burton, K. (1951), Biochem. J . 48,458. Chlumecka, V., Mitra, S. K., D'Obrenan, P., and Smith, C. J. (1970), J . Biol. Chem. 245,2241. Cramer, F., Doepner, H., v. d. Haar, F., Schlimme, E., and Seidel, H. (1968), Proc. Nat. Acad. Sci. U. S. 61, 1384. Daniel, V., and Littauer, U. Z . (1963), J . Biol. Chem. 238, 2102. Daniel, V., and Littauer, U. Z . (1969, J . Mol. Biol. 11,692. Furth, J. J., Hurwitz, J., Krug, R., and Alexander, M. (1961), J . Biol. Chem. 236,3317. Hashimoto, S., Kawata, M., and Takemura, S. (1969), Biochem. Biophys. Res. Commun. 37,777. Herbert, E., and Canellakis, E. S. (1963), Methods Enzymol. 6,28. Hirst-Bruns, M. E., and Philipps, G. R. (1970), Biochim. Biophys. Acta 21 7, 189. Holley, R. W . , Madison, J. T., and Zamir, A. (1964), Biochem. Biophys. Res. Commun. 17,389. Levitt, M. (1969), Nature (London)224,759.
Makman, M. H., and Cantoni, G. L. (1966), Biochemistry 5, 2246. Miller, J. P., Hirst-Bruns, M. E., and Philipps, G. R . (1970), Biochim. Biophys. Acta 21 7,76. Miller, J. P., and Philipps, G. R. (1970), Biochern. Biophys. Res. Commun. 38, 1174. Miller, J. P., and Philipps, G . R. (1971a), J . Biol. Chem. (in press). Miller, J. P., and Philipps, G. R. (1971b), J . Biol. Chem. (in press). Nihei, T., and Cantoni, G. L. (1963), J. Bio/. Chem. 238, 3991. Philipps, G. R. (1969), Nature (London) 223,374. Philipps, G. R. (1970), J . Biol. Chem. 245,859. Preiss, J., Berg, P., Ofengand, E. J., Bergmann, F. H., Dieckmann, M. (1959), Proc. Nat. Acad. Sci. U.S. 45,319. Preiss, J., Dieckmann, M., and Berg, P. (1961), J . Biol. Chem. 236,1748. Starr, J. L., and Goldthwait, D. A. (1963), J. Biol. Chem. 238, 682. Yaniv, M., andGros, F. (1969), J. Mol. Biol. 44,1,17,31.
Extrinsic Cotton Effects in Complexes of Creatine Phosphokinase with Adenine Coenzymes* Jeremias H. R. Kagi,? Ting-Kai Li, and Bert L. Valleet.
ABSTRACT : Rotatory dispersion and circular dichroic properties of creatine phosphokinase (CPK) and its complexes with adenine coenzymes were compared between 190 and 600 mp. Binding of ADP, dADP, or ATP, either alone or in combination with bivalent metal ions and substrates, generates a large positive extrinsic Cotton effect at the absorption band of the bound chromophores, but does not alter the far-ultraviolet rotatory dispersion of CPK. The extrinsic Cotton effects contribute to the rotatory dispersion in the near-ultraviolet and visible region accounting for the increased dextrorotation of the enzyme-coenzyme-substrate complex reported previously (Samuels, A. J., Nihei, T., and Nada, L. (1961), Proc. Nut. Acad. Sci. US.47, 1992). The amplitude of the Cotton effect increases in proportion to the extent of complex formed until 2 moles of coenzyme is bound per mole of of the CPK. The maximum total amplitude, CPK .ADP complex is 95,000" compared to the maximum span of only 5000" for the negative Cotton effect of the free coen-
[ws4$
G
eneration of rotatory power by asymmetric binding of chromophoric molecules to macromolecules is well recognized (Ulmer and Vallee, 1965). In proteins and polypeptides, such Cotton effects have been termed extrinsic Cotton effects
* From the Biophysics Research Laboratory, Department of Biological Chemistry, Harvard Medical School, and the Division of Medical Biology, Peter Bent Brigham Hospital, Boston, Massachusetts. Receiced Jdj. 13, 1970. This work was supported by Grant-in-Aid GM15003 from the National Institutes of Health of the Department of Health, Education, and Welfare.
zyme. Binding of MgZ+ and substrates to the complex does not affect the extrinsic Cotton effect, but substitution of Mgz+ by Mn2+ tends to reduce its amplitude slightly. The Cotton effect of the C P K . d A D P complex is similar in amplitude to that induced by ADP, but, in contrast, that of the CPK ATP complex is 30-40x smaller. The differences are considered to reflect different modes of attachment of the coenzymes to CPK. The association of these Cotton effects with a strong absorption band of the coenzyme and their accompaniment by bathochromic and hypochromic changes of this band are suggestive of their generation by a dipole coupling mechanism (Kuhn-Kirkwood), Theoretical considerations show that they could arise from intermolecular coupling of the adenine transition of the coenzyme and strongly allowed transitions in the protein. An interaction with farultraviolet transitions of a juxtaposed tryptophanyl residue is proposed to account for the extrinsic Cotton effects in this system.
to distinguish them from those of the peptide backbone and of amino acid side chains which are designated as intrinsic Cotton effects (Blout, 1964). In enzymes, such extrinsic Cotton effects can arise from noncovalent complexes with chromophoric coenzymes, prosthetic groups, metal ions,
t Investigator of the Howard Hughes Medical Institute. Present address: Biochemisches Institut der Universitat Zurich, Zurich, Switzerland. $. To whom to address correspondence. B I O C H E M I S T R Y , VOL.
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KXGI, or inhibitors and may be considered manifestations of the steric specificity of enzyme structure and function (Ulmer et ai., 1961). Thus, recognition of such Cotton effects offers a n important means for the study of the modes of noncovalent intermolecular interactions in such complexes. Extrinsic Cotton effects of enzymeecoenzyme complexes have been investigated extensively in systems which possess chromophoric groups in the visible and near-ultraviolet region (Ulmer and Vallee, 1965). But, until recently, instrumental limitations have impeded extension of such studies into the farther ultraviolet regions where nucleotide chromophores of coenzymes absorb radiation. The present account reports optical rotatory dispersion and circular dichroic spectra of complexes of creatine phosphokinase with A D P and ATP between 190 and 600 mp and demonstrates the occurrence of large positive, extrinsic Cotton effects at the adenine absorption band of the bound coenzymes. These Cotton effects are superimposed upon the rotatory dispersion of the protein and are responsible for the coenzyme-induced changes in rotation reported previously for this system (Saniuels ef ai., 1961). The extrinsic Cotton effects of the A D P and ATP complexes differ significantly in amplitude suggesting that the two coenzymes are bound differently to creatine phosphokinase. Theoretical considerations indicate that the extrinsic Cotton effects of the magnitude observed could arise from intermolecular dipole--dipole coupling of transitions of the adenine moiety of the coenzymes and an adjacent tryptophan residue of the protein. A preliminary report of some of the data has been made (Kagi and Li, 1965). Experimental Section Creatine phosphokinase from rabbit skeletal muscle was obtained as a salt-free preparation from Boehringer-Mannheim Corp., West Germany. Phosphotransferase activity measured by the rate of creatine phosphate formation (Kuby Pf al., 1954a) was 45 units mg-'. ATPase activity measured by the formation of inorganic phosphate in a reaction mixture containing 10 mg ml-I of enzyme, 0.01 M ATP, 0.02 M MgCI,, pmole and 0.1 M glycine, pH 9, 25", was less than 5 X mg-1 min--l. Concentrated stock solutions of CPK were prepared in metal-free distilled water and stored at 0" for.not more than 2 weeks. Protein concentration was determined spectrophotometrically in 0.1 M sodium phosphate buffer (pH 7.5) using a n absorptivity at 280 mp of 0.88 ml mg-' cm-I (Kuby et d., 1962). Since the enzyme has a molecular weight of 82,600 and two active sites (Yue et ul., 1967) equivalent molar concentrations were calculated on the basis of half the molecular weight, i.e., 41,300. All calculations of molar rotation and ellipticity of CPKI and its complexes are based on this value and are designated equivalent molar rotation or ellipticity. Sodium salts of ADP, dADP, ATP, and AMP were obtained from Sigma Chemical Co. Stock solutions of the coenzymes were prepared in metal-free distilled H 2 0 and stored frozen a t -20" for not longer than 2 weeks. Sodium creatine phosphate and creatine were products of Nutritional Biochemical Corp. and J. T. Baker Chemical Co., respectively. Stock solutions were prepared in metal-free distilled water before L I S ~ . ~
- -.~
~~
- ..
~
~
~
' Abbreviations used are: CPK, creatine phosphokinase; C P K . ADP, C P K . dADP, etc., tleiiotc the binary enzyme-coenzyme complex irrespcctive of thc presence or absence of activating metal ions.
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Magnesium chloride, manganese chloride, Na9EDTA, and glycine were reagent grade chemicals. Glycylglycine was obtained from Sigma Chemical Co. N-Ethylmorpholine (practical grade, Eastman) was redistilled twice and stored at 3". Urea (analytical grade, Merck) was recrystallized from absolute ethanol (Korsgaard Christensen, 1952). Samples for optical measurements were prepared immcdiately prior to analysis by mixing equal volumes of C P K stock solutions and a buffered solution containing appropriate concentrations of coenzymes, substrates, and metals either alone or in combination. Calibrated Lang-Levy pipets were employed throughout. Optical rotation was measured with a Cary Model 60 recording spectropolarimeter. The slit width was programmed to yield constant energy over the entire spectral range. F o r measurements below 210 mp the cell compartment and monochromator were flushed with nitrogen. The cell compartment was thermostated tit 27 ". All measurements were performed in calibrated fused-quartz cells (Opticell Co., Brentwood, Md.). Measurements at temperatures other than 27" were made in water-jacketed cells. At wavelengths longer than 220 nip the instrument was operated with a response time of 3 sec and scan speeds of 2-5 mp;min. At wavelengths shorter than 220-mp response times were 10 and 30 sec with correspondingly lower x x n speeds. All measured rotations were the mean of three successive scans yielding a precision of approximately .=0.001' between 190 and 220 mp and =0.0004" at longer wavelengths. Base-line rotations, recorded in the same cell an solutions containing all components except the enzyme, wcrt subtracted algebraically from those of the solutions containing enzyme. For difference dispersion studies corresponding pairs of solutions were measured in alternating sequence in the same cell. Enzyme concentrations were held identical within -1-0.2%. Specific rotation. molar, or equivalent molar rotation. and reduced mean residue rotation, were evaluated from standard expressions (Fasnian, 1963). The mean residue weight of 112.5 g was evaluated from the amino acid coniposition of rabbit inusclc C P K (Noltmann rt a / . , 1962). Circular dichroic spectra were measured at 25' in a Durrum-Jasco recording spectropolarimeter. 2 Molar and equivalent molar ellipticities, [e],, expressed in units of deg cm2 dmo1c.- !. were evaluated from the relationship: [o]A = (ei --- t,,).3300, where f l - trl is the molar or equivalent molar dichroic difference absorptivity. The rotational strength, uncorrected for the polarizability of the solvent was calculated according to Moscowitz (1960) a s
where I S the maximum ellipticity, ,,A ,, the wavelength position of the circular dichroic band, and AA the wavelength interval in which [(?]A falls to 0.368 of [e],,,,,. The degree to which CPK combined with the coenzymes was calculated in each instance from the dissociation constant 6 X 10-j %f for MgADP-1 or MnADP-l, 1 X lop4 M for M for MgATP-', and 3 X M for ATP-4 ADP-3 1 X (Kuby ef nl., 1962; O'Sullivan and Cohn, 1966a). Unless otherwise specified all concentrations of coenzymes employed were sufficient to saturate at least 95 of the binding sites. . .. ~~-~ __ _ _ ~ ~ The authors are indebted to Dr. E. Blout, Department of Biological Chcmistry, Harvard Medical School, Boston, Mass,, for permission to usc this instrument, ~~
2
COTTON EFFECTS IN CREATINE PHOSPHOKINASE
A
I\
+401
1 I
.
240
i
JOO
360
wAVEWGT,H, m y
200
230
245
260
215
280
320
WAVELENGTH, mp FIGURE 1 : Optical rotatory dispersion of CPK and of the CPK 'ADP complex. Specific rotation of CPK (---) and CPK.ADP complex (--) is plotted against wavelength. The complex is formed by addition of 10 mM ADP and 20 mM MgCL to CPK in 0.1 M glycine, pH 9, 27". Right: 270-360 mp. CPK.ADP complex is less levorotatory than CPK and shows anomalous dispersion below 300 mp. CPK: 2-5 mg m1-I; cell path length: 0.1-2 cm. Center: 240-280 mp, Crossover of the rotatory dispersion of the CPK .ADP complex and of CPK indicating a positive Cotton effect centered about the adenine absorption band of the bound coenzyme (A, 262 mp). Maximum dextrorotatory and levorotatory deflections from the plain dispersion of CPK are located near 278 and 243 mp. Below 243 mp the dispersion of the complex approaches that of CPK. CPK: 60 mg ml-l; cell path length: 0.01 cm. Left: 19@245 mp. CPK and CPK .ADP exhibit identical intrinsic Cotton effects with extrema at 232 mp (-5600 + 100") and 198 mp (+26,000 i 1ooO"). CPK: 0.85 mg ml-I; cell path length: 0.01-0.20 cm.
Results The optical rotatory dispersion of native creatine phosphokinase is plain throughout the visible and near-ultraviolet spectral regions; in the far-ultraviolet region the enzyme displays characteristic intrinsic Cotton effects with a trough a t 232 mp, a n inflection point a t 225 mp, a shoulder at 216 mp, and a peak at 198 mp (Figure 1). Complex formation with the coenzyme, ADP, induces distinct alterations in the rotatory dispersion. In the visible and near-ultraviolet region the dispersion curve of the enzyme-coenzyme complex remains plain but becomes more dextrorotatory than that of the enzyme alone. At 600 mp their specific rotations differ by 4 and this value increases progressively toward shorter wavelengths (Figure 1 , right). At 300 m p the increment is +45", equivalent to a reduction of 10% in total levorotation. These changes are virtually identical with those reported earlier for ternary complexes of CPK formed in the presence of a n equilibrium mixture of substrates and coenzymes (Samuels et a/,, 1961). Below 300 mp, a spectral region not previously examined, the dispersion curve of the complex is anomalous. A positive Cotton effect can be recognized against the background of the descending dispersion curve of the enzyme characterized by a maximum dextrorotatory deflection at 278 mp, a crossover at 262 mp, and a maximum levorotatory deflection at 243 mp (Figure 1 , center . This spectral location of the Cotton effect coincides with that of the absorption band of the adenine portion of the bound coenzyme providing evidence for its origin in this chromophoric group of the complex (extrinsic Cotton effect). Analogous Cotton effects are also observed in binary complexes of C P K with dADP and ATP. Ternary complexes with ADP and creatine or with the full equilibrium mixture O
2: Rotatory dispersion of ADP and difference rotatory dispersion of the CPK.ADP complex us. CPK. Molar rotation of ADP and the difference in rotation of the CPK.ADP complex and CPK (data of Figure 1) expressed as equivalent molar rotation, [MI:', are plotted against wavelength. The difkrence rotatory dispersion (-) shows a large positiae Cotton effect formed on binding of ADP to CPK. Its midpoint is located at 262 mp and its extrema at 278 mp ([M]i:8 +49,000") and 243 mp ([M];:, -46,000'). The crosses (x) represent the contribution of the positive Cotton effect to the difference dispersion in the visible and near-ultraviolet region of the spectrum as calculated from its amplitude and width parameters by the expressions given by Schellman (1968). Free ADP, 10 mM in 0.1 M glycine, pH 9, (---) exhibits a much smaller negurioe Cotton effect at 259 mp with extrema at 273 mp ([MI;:, -3200") and 247 mp ([Ml;:, +1800°).3
FIGURE
of coenzymes and substrates exhibit rotatory dispersion curves indistinguishable from that of the CPK . ADP complex (for conditions, see Table I). Substrates alone d o not affect the dispersion curve of the enzyme. The intrinsic Cotton effects of CPK are not affected by formation of complexes with coenzymes or with combinations of coenzyme and substrate. Below 240 mp the rotatory dispersion curve of the C P K . A D P complex coincides with that of CPK (Figure 1, left). Estimates of helicity derived from the amplitude of the trough at 232 mp of CPK and of complexes formed under various conditions are identical within the error of measurement. The helical features of both are largely abolished, however, in 6 M urea (Table I). The rotatory contribution arising from ADP binding can be isolated from the total rotation by subtraction of the dispersion curve of the enzyme from that of the enzyme-ADP complex, to yield the difference rotatory dispersion (Figure 2). The single extrinsic Cotton effect is opposite in sign and nearly 20 times larger than that of free ADP. It has a total amplitude of approximately 95,000' and is centered at 262 mp, the absorption maximum of the bound coenzyme. The extrinsic Cotton effect appears to account fullyfor the rotatory difference between the complex and the free enzyme since there is excellent agreement between the experimental curve and the difference rotatory dispersion calculated from the height 3 Magnesium ions have no effect on the Cotton effect of ADP except for a small positive displacement of the entire dispersion curve accounting for previously reported differences in dispersion parameters (McCormick and Levedahl, 1959), and presumably originating from changes in the far-ultraviolet transitions of the ribose-diphosphate component. 4 The formation of the enzyme-coenzyme complex shifts the absorption maximum of the coenzyme from 259 to 262 m p as judged by the CPK.ADP cs. CPK difference spectrum. There is also substantial hypochromism as the molar absorbancy at the maximum decreases by 1 5 % from 15.4 X IO3 to 13.2 X IO1 I. mole-' cm-I. Similar absorptive changes occur i n complexes of CPK \h ith dADP, ATP, and AMP.
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Reduced Mean Residue Rotation a t 232 mp and Apparent Helix Content of C P K and Its Complexes with Adenine Coenzymes and Substrates:
TABLE I :
__ -
--
__ ________
Coenzyme
Metal
_________
_
- -
.
None None MgClz (20 mM) MnC12 (9 5 mM) MgC12 (20 m ~ ) MnCls (9 5 m ~ ) None MgCII (20 mM) MgCh (20 mM)
ADP (10 mM) ADP (10 mv) None ADP (5 mM)'
MgC12 (20 mM) MnCI? (9 5 mM) None MgCl? (20 mM)
- --
Bufferb
None None None None None None None None Creatine (21 2 mM) creatine phosphate (2 4 mM)g Creatine (50 mv) Creatine (50 mM) None None
J-
--
-
___
-
c
C A
4 A
-
-4710 - 4730 - 4770 - 4770 - 4770 - 4640 - 4760 - 4740 - 4740
21s 21 21 21 21 20 21 21 21
- 4680
21 21 4 3
-4710 - 2550h -2360
6 M urea
+ 6 M urea
-
Helix' -
A. B, C A A, B, C C
C C A A
__-
[~~']232~
-
-
_______.__
None A D P (10 mM)' ADP (2 6-10 mM) ADP (10 mM) dADP (10 mM) dADP (10 mhr) ATP (1 3 mM) ATP (1 3 mbi) ADP (2 6 mM) ATP (1 3 mM)
+
-
Substrate
All samples contained between 58 and 70 mg ml-l CPK. Measurements were made in cells of 0.01-cm path length. 1) A, 0.1 M glycine-NaOH, p H 9, 27"; B, 0.1 M glycylglycine-NaOH, pH 7.4, 27"; C, 0.05 M N-ethylmorpholine-HCI, p H 8, 10". e Precision .t85".dPer cent helix content is computed from [n2'IYa.using as standards the reduced mean residue rotations at 233 mp of completely helical (- 1 5,000O) and random coil (-2000") poly-a-L-glutamic acid (Yang, 1967). The helix content estimated from [ I I I ' ] , ~ , ~= +21,800 is approximately 3 0 z based on poly-a-L-glutamic acid standard of = +80,000 (Blout et ul., 1962). e Helix content calculated from hu = - 178 and X, = 252 mp (Yang, 1961) is 28 and 2 9 z , respectively. Dispersion parameters are evaluated from rotations between 600 and 320 mp. taking Xo = 212 nip (Fasman, 1963) and assuming bo = -630 for a completely helical protein. Owing to superimposition of the coenzyme-induced Cotton effect upon the plain dispersion curve of CPK, these parameters cannot yield a meaningful measure of helicity in CPK-coenzyme complexes. Both Moffit and Drude plots of the complexes exhibit curvature when evaluated from rotations between 600 and 320 mp. 20 Inhi EDTA. Equilibrium mixture at pH 9 (Samtiels et id., 1961). In 6 M urea the features of the peptide Cotton effects of native CPK are abolished. The residual optical rotatory dispersion curve is similar to that generally observed with proteins and polypeptides in random coil conformation but the high absorbancy of urea precludes measurement below 210 nip. Urea increases levorotation of CPK in visible and near-ultraviolet region. Helix content calculated from bois 6 %. j The extrinsic Cotton effect of the CPK. ADP complex is abolished in 6 51 urea, presumably since ADP no longer binds. 1%
*
and width parameters of the Cotton effect (Figure 2). Even as far removed from the crossover point as the Na D line (586 mp), there is fair coincidence of the measured (+1500 ;i; 500") and the calculated ( t 1 2 6 0 " ) molar difference rotation. The Cotton effect generated by coenzyme binding increases in proportion to the extent of complex formed, offering a means to measure the stoichiometry of the interaction (Figure 3, left). A plot of the amplitude of the peak of the Cotton efrect of the C P K ' C D P complexes cs. the molar proportion of the reactants indicates binding of 2 moles of ADP/mole of C P K (Figure 3, right), in agreement with previous data obtained by equilibrium dialysis, ultracentrifugation (Kuby cf c i / . , 1962), and difference absorption spectroscopy (Rotistan e / ( I / . ! 1968). The circular dichroic spectrum displays the optical asymmetry of the CPK ' A D P complex equally prominently (Figure 4, top). The spectrum of C P K shows a negative dichroic band in the region of the aromatic side-chain chromophores = 16,000 deg cm? dniole-') and the beginning of the large negative dichroic band of the nearest peptide bond transition. Binding of ADP generates a positive band in the region of the adenine chromophore. The difference circular dichroic spectrum (Figure 4,bottom) shows a single positivi. syiiimetrical band centered a t 262 nip with a n~nxiiiiuiii equivalent molar ellipticity [e],,..of 1-6S.000 deg cm2 dmole
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corresponding to a rotational strength RZ6?= f5.0 X cgs unit. Free ADP and complexes of ADP with Mg2+ exhibit a negative circular dichroic band at 259 mp with a maximum molar ellipticity of [&, = - 3400 deg cm2dmole--'. The complex of C P K with dADP, a coenzyme for the transphosphorylase reaction (Morrison er id., 1961) also reveals a large extrinsic Cotton effect (Figure 5). Its total amplitude (91,000O) is virtually identical with that induced by ADP, a significant observation considering that the optical asymmetry of ADP and dADP differs substantially (Yang and Samejinia, 1963). By contrast, a significantly smaller extrinsic Cotton effect is seen in the complex with ATP.> While similar in shape. its total amplitude (62:OOO") and its dextrorotatory contribution to the enzyme in the visible region of the spectrum is about 60% of that observed with the diphosphate analogs. No extrinsic Cotton effect is discerned in mixtures of C P K with AMP (Table 11). The extrinsic Cotton effects generated by binding of ADP or its analogs are unchanged under a variety of conditions of pH, trmperattire, and ionic environment (Table 11). Simi-
COTTON EFFECTS IN CREATINE PHOSPHOKINASE
I
,q\
+ 401
I
1'
i
-40 I1
, .\
x
i n j
260 280 3Do WAVELENGTH, rnp
0 2 4 6 8 MOLES ADP/MOLE CPK
3: Rotatory dispersion titration of CPK with ADP. Left: difference rotatory dispersion of CPK 'ADP complex us. CPK as a function of ADP concentration. Differences in rotation expressed as equivalent molar rotation, [MI?, are plotted against wavelength. CPK: 116 mg m1-I; ADP: (a) none, (b) 1.3 mM, (c) 2.6 mM, (d) 5.2 and 10.3 mM; in 0.1 M glycine, pH 9, 27", containing 20 mM MgC12, cell path length 0.01 cm. Right: the positive amplitude of the Cotton effect, [MI&, is plotted against the molar ratio of ADP and CPK. The extrapolated linear portions of the titration curve intersect at 2.1 moles of coenzyme/mole of enzyme. The stippled line is the theoretical titration curve calculated from the data of Kuby et ai. (1962). FIGURE
larly, a t the high concentration of coenzyme employed in these studies the presence of bivalent metal ions is not essential for their formation. Although Mg2+ is a component in most experiments described here, Cotton effects of the same magnitude are obtained in the absence of added Mgz+ or in the presence of EDTA included to remove metal ions present adventitiously. In marked contrast, however, substitution of Mg2+ by Mn*+ reduces the amplitude of the extrinsic Cotton effects of both C P K . A D P and C P K . d A D P by about 2 0 Z . Addition of substrates has also no effect on the extrinsic Cotton effect. Difference rotatory dispersion of ternary complexes of CPK with ADP, creatine, and MgZf, or of complexes formed in presence of the complete equilibrium mixture of coenzymes and substrates and Mg2+ are super-
%C
~
.-
-
260 290 WAVELENGTH. mp
~
320
5 : Difference rotatory dispersion of the CPK.ATP and CPK .dADP complex os. CPK. Differences in rotation expressed as equivalent molar rotation [ M ] r are plotted against wavelength. All measurements are made in 0.1 M glycine, pH 9, 27'; cell path exhibits a positive length: 0.01 cm. The CPK.ATP complex (-) Cotton effect with midpoint near 260 mp and extrema at 278 mp -30,000"). CPK, 66 mg ([MI::, +32,000") and 244 mp m1-l; ATP, 13 T I M ; MgC12,20 mM. The Cotton effect of the CPK. dADP complex (---)has a midpoint near 262 mp and extrema at 278 +48,000") and 244 mp ([MI$*,-43,000"). CPK, 68 mg m1-I; dADP, 9 mM; MgC12,20 mM.
FIGURE
Extrinsic Cotton Effects of CPK Complexes with Adenine Coenzymes : Effect of Analogs and Metal Ions.
TABLE 11:
Coenzymea
Metal
Buffer*
ADP 5 mM 10 mM 5 mM 10 mM 10 mM 10 mM
None None MgCl?, 20 mM Same Same MnC12, 9 . 5 mMd
dADP 9 mM 10 m M 10 mM
MgC12, 20 mM Same MnC12, 9 . 5 m M d
ATP 13 mM 13 mM
None MgCIg, 20 mM
A A A B C
C A
C C A
AMPe 5 mM
Total Amplitude.
95,000 93,000 95,000 95,000 98,000 76,000 91,000 93,000 77,000
A
67,000 62,000
A
0
All samples contained between 58 and 70 mg ml-1 C P K ; measurements were made in cells of 0.01-cm path length. * A, 0.1 M glycine-NaOH, p H 9, 27"; B, 0.1 M glycylglycineNaOH, p H 7.4, 27"; C, 0.05 M N-ethylmorpholine-HC1, p H 8, IO". c Difference of peak and trough amplitudes (in equivalent molar rotation) evaluated from the difference rotatory dispersion of CPK-coenzyme complexes us. CPK in same buffer. The precision of each measurement is i 7 0 0 0 " . d To avoid precipitation of manganese hydroxide from the solution coenzymes were kept in slight excess over the metal ion. Based on the apparent dissociation constant, K'SI,,AD~-I = 4 X measured in the same buffer system, 9 5 Z of the metal is complexed by the coenzyme (O'Sullivan and Cohn, 19662). "Formation of a C P K . A M P complex was verified by its ultraviolet difference absorption spectrum. 0
240
- -
-
-
260
280
300-
WAVELENGTH, mp FIGURE 4: Top: circular dichroic spectrum of CPK 'ADP complexes and of CPK. Equivalent molar ellipticity, [e],, is plotted against generated in presence wavelength. The CPK .ADP complex (--) of 10 mM ADP and 20 mM MgC1, shows coenzyme induced positive circular dichroic band superimposed upon the spectrum of CPK (---I, CPK: 60 mg ml-l, in 0.1 M glycine, pH 9, 27"; cell path length: 0.01 cm. Base lines obtained on solutions containing all components except CPK are subtracted. Bottom: circular dichroic difference spectrum of the CPK.ADP complex cs. CPK. The difference in equivalent molar ellipticity, A[O]x, obtained by algebraic subtraction, is plotted against wavelength. The maximum difference equivalent molar ellipticity, A[L~],,,is +68,000 deg cm2dmole-1.
~
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KXGI, imposable on that of the Mg2+ containing binary CPK ADP complex. Discussion Creatine phosphokinase can be added t o the list of enzymes in which the stereospecific interaction with the coenzyme manifests as a n extrinsic Cotton effect (Ulmer and Vallee, 1965). Such effects are superimposed upon the rotatory features of the protein and can generally be distinguished from the Cotton effects of the polypeptide backbone and of amino acid side chains by their spectral location which coincides with a prominent absorption band of the bound coenzyme. The Cotton effects of complexes of CPK with adenine coenzymes occur near 260 m p , the region of maximum absorption of these coenzymes, and, to our knowledge, are the first example of a n extrinsic Cotton effect associated with the adenine chromophore in a n enzyme. They are coniparable to analogous Cotton effects observed previously with DPNH, FAD, and pyridoxal phosphate dependent enzymes (Ulmer and Vallee, 1965), but because of their superimposition upon a more steeply descending portion of the rotatory dispersion of the protein moiety, they are manifested less conspicuously in the dispersion curve of these complexes (Figure 1). Hence, recognition of these extrinsic Cotton efyects by spectropolarimetry proved more difficult and their delineation against the large background rotation made more strenuous demands on the experimental precision of such measurements. In some cases reduction of the error of measurement to less than 1 % of the total measured rotation has been required. These demands are less severe for circular dichroic measurements (Figure 4), but it is apparent that with both techniques the increasing contribution of the protein sets an ultimate limit to the detection of such Cotton effects in the low-ultraviolet region. The presence of the extrinsic adenine Cotton effect alters the rotatory dispersion of the complex appreciably, both in the near ultraviolet and the visible regions, causing an apparent reduction in the levorotation of the enzyme by as much as 10% (Figure 1). The difference in rotation coincides accurately with that computed from the amplitude and width parameters of the Cotton effect (Figure 2). This confirms earlier suggestions that changes in specific rotation of a protein on binding of a chromophoric ligand need not imply any alteration in protein conformation but may arise from the superimposition on the background rotation of the protein of the rotational contribution of a newly generated, optically active absorption band (Ulmer and Vallee, 1965). As a corollary, it was stressed that in such complexes variations in rotational parameters, derived from measurements in regions of plain dispersion, should not be ascribed to conformational changes in the protein unless the possible presence of new Cotton effects in other spectral regions is eliminated. This is reemphasized by the present data which show that the generation of the extrinsic C P K . A D P Cotton effect accounts for the increased dextrorotation of CPK above 300 mp in the presence of a mixture of coenzymes, substrates, and Mg2+ previously reported (Samuels et nl., 1961). The intrinsic Cotton effects of C P K are typical of a n 01helical protein (Blout et d., 1962) and indicate a n a-helix content of about 21 (Table 1). Importantly, these features remain completely unaffected on binding of coenzymes. substrates, and bivalent metal ions, added either separately or jointly. They lend no support to the suggestion of gross conformational differences between a “working” and
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“resting” mode of the enzyme (Samuels et id., 1961). These findings also imply that effects of coenzymes and substrates on enzyme conformation inferred from kinetic studies (Hammes and Hurst, 1969) or from measurements of hydrogen-deuterium exchange (Lui and Cunningham, 1966), susceptibility to chemical modification (Watts, 1963 ; O’Sullivan ei ul., 1966; O’Sullivan and Cohn, 1966b), and resistance to proteolytic digestion (Lui and Cunningham, 1966; Jacobs and Cunningham, 1968) arc either restricted to nonhelical regions or reflect only minor localized structural perturbations of the molecule. The exirinsic Cotton effects of the enzyme-coenzyme complexes, as displayed by difference rotatory dispersion curves, are nearly symmetrical and consistent with the occurrence of a single positive band in the circular dichroic difference spectrum (Figure 4). The total amplitude of nearly 100,000” in equivalent molar rotation for the Cotton effect of the C P K . A D P complex (Figure 2) is of the order seen with other enzyme-coenzyme complexes and is typically much larger than that of the negative Cotton effect of the free coenzyme (Ulmer and Vallee, 1965). Its amplitude is also much larger than that reported for Cotton efects of side-chain chromophores of proteins (Beychok and Fasman, 1964: Beychok, 1965; Simpson and Vallee, 1966). Its rotational strength of + 5 X cgs unit (Figure 4) is coniparable, in fact, to that reported for transitions of a-helically oriented peptide groups such as the T - - K * bands at 198 mp cgs unit) and at 191 mp (-8.1 X l(l.3’Jcgs (-2.9 X unit) (Holzwarth and Doty, 1965), the most potent sources of rotatory power known in proteins. Compared to free ADP the extrinsic Cotton effect of the enzyme-coenzyme complex is also shifted toward longer wavelengths by 3 mp (Figures 2 and 3). The same bathochromic shift and a concomitant decrease in absorbance is apparent in its absorption spectrum4 and is consistent with the appearance of marked difference spectra upon formation of complexes of CPK with adenine coenzymes (Noda, 1Y63; Roustan et ul., 1968). Studies currently in progress indicate that the extents of the spectral change vary with dill’erent analogs of ADP and appear to be correlated to a certain degree with the rnagnittide of the Cotton effect, suggesting il related origin of these different optical phenomena. Extrinsic Cotton effects are considered to be manifestations of structural asymmetry existing at the bound chromophore or in its vicinity (Ulmer and Vallee, 1965). Hence, variations in the magnitude of Cotton effects of saturated complexes of CPK with different analogs of A D P (Figure 5 , Table 11) can be attributed to some steric differences in the mode of coenzyme binding. Thus, the Cotton effect of the complex of CPK with ATP, smaller as compared to that with ADP, provides evidence for a distinit difference in attachment of these two coenzymes to the enzyme supporting a similar inference drawn earlier from the absorption properties of the two complexes (Noda, 1963; Roustan er d.,1968). It could indicate that the interconversion of the two coenzymes during the enzymatic phosphate transfer is accompanied either by a local conformational transition in the enzyme or by a translocation of the bound coenzyme. In similar fashion, the failure to observe an extrinsic Cotton effect in complexes of C P K with AMP, a n inhibitor of the phosphotransferase reaction (Kuby er til., 1954b) implies that this analog is bound in yet another way (Table 11). On the other hand, the similarity of the Cotton effects of the CPK ADP and CPK dADP complexes suggests nearly identical geometry about the common adenine chromophore
COTTON EFFECTS IN CREATINE PHOSPHOKINASE
irrespective of their differences in the sugar moiety (Figure 5 and Table 11). Similarly, the insensitivity of the CPKsADP Cotton effect to the presence or absence of Mgz+ (Table 11) and to ternary complex formation with substrates and products implies that the steric environment of the adenine chromophore remains undisturbed on binding of these agents to the binary complex. This suggests that neither Mg2+ nor the substrate induce a n appreciable structural rearrangement at the binding site. By contrast, the lower magnitude of the extrinsic Cotton effect of the CPK . A D P and CPK 9dADP complexes in the presence of Mn2+ might imply a somewhat different mode of binding of the magnesium and manganese complexes of ADP to CPK. Conceivably such differences could be related to the known dissimilarity of the coordinative properties of the two metals (Cohn and Hughes, 1962). Recent studies on spin-labeled CPK have revealed a perhaps analogous structural difference between CPK . ADP complexes formed in the presence of Mg2+and of Zn2+, a metal similar to Mnz+in its binding affinities for ADP (Taylor et a[., 1969). The physical mechanism by which extrinsic Cotton effects arise has not been identified as yet. The large magnitude of the extrinsic adenine Cotton effects and their accompaniment by hypochromism and spectral shifts is very reminiscent, however, of the changes which occur when adenine nucleotides are incorporated into polynucleotides and nucleic acid structures, Thus, single-stranded and double-stranded polyadenylic acid exhibit large double Cotton effects near 260 mp, encompassing total molar amplitudes of up to 100,000 and 170,000”, respectively, i.e., egects 20 to 40 times larger than that of the single negative Cotton effect of the monomer (Lamborg et d.,1965; Holcomb and Tinoco, 1965; Sarkar and Yang, 1965). Similar nonadditive rotatory dispersion curves displaying either double or single Cotton effects are observed in adenine-containing dinucleotides (Warshaw et al., 1965; Van Holde et al., 1965; Bush and Tinoco, 1967), and have been thought to arise from coupling of the transition moment dipoles of pairs of neighboring, stacked chromophores when held in fixed, mutually asymmetric positions. By analogy, the extrinsic Cotton effects reported here could be considered to arise from similar asymmetric stacking of the adenine moiety of the coenzyme on one of the aromatic amino acid side chains of the protein. Generation of optical activity by such an arrangement would require coupling of the adenine transition dipole with a sterically fixed transition dipole in the plane of the aromatic side chain. Because of the * of the indole ring, linear polarization of the ~ - f transitions tryptophan, in particular, would meet this requirement.6 This residue has been suggested previously to play a role in coenzyme binding to CPK based on ultraviolet difference spectra (Noda, 1963; Roustan et a/., 1968).7 A simple calcula6 I n contrast to tryptophan the benzenoid transitions of phenylalanine should have no preferred direction of polarization in the plane of the ring and, hence, should not be capable of producing rotatory power in the assumed stacked-card arrangement. Tyrosine, on the other hand, is known to exhibit some linear polarization in the plane of the ring (Weber, 1960) and might thus be expected to generate optical activity by this mechanism. Of course, a nonvanishing contribution to the rotational strength could result from all aromatic side chains if they were tilted with respect to the plane of the adenine ring. In this case the nondegenerate tryptophan transitions would also be the largest contributors. Interaction of adenine coenzymes with a tryptophan residue is supported also by the observation that tryptophan fluorescence of CPK is quenched approximately 25 and 1 5 % on complex formation with ADP and ATP, respectively (J. Kagi, in preparation).
tion detailed in the appendix shows that the magnitude of the rotational strength to be expected from optimal interaction of the 259-mp transition of the adenine group with the ultraviolet transitions of a contiguous tryptophanyl residue (9 X 10-39 cgs unit) would be indeed sufficient to account for the value observed experimentally (R262= +5 X lO+9 cgs unit). Assessment of the contributions arising from each of the four resolvable tryptophan transitions indicates furthermore that the rotational strength of the adenine transition generated by such a mechanism would derive mainly from the interaction with the strong far-ultraviolet transitions. The occurrence of but a single Cotton effect at the adenine band would be consistent with such coupling. The same mechanism could also produce the bathochromic and hypochromic changes noted in the absorption spectrum of the bound coenzymes (DeVoe and Tinoco, 1962). The capacity of this model to account for the large rotations of the complex suggests that this or an analogous intermolecular coupling mechanism may, indeed, play the dominant role in the generation of this extrinsic Cotton effect. This is true, particularly, since other known sources of rotatory power such as hindrance of free rotation about the N-glycosidic bond in the bound coenzyme (Emerson et a/., 1966) or restrictions imposed on the ribose or phosphate conformation on binding seem unable to account for more than a fraction of the magnitude of the extrinsic Cotton effect (Warshaw and Tinoco, 1966). Acknowledgments We are deeply indebted to Dr. S. A. Latt and Dr. D. D. Ulmer for advice and criticism in the preparation of the manuscript. Appendix Estimates of the maximum rotational strength attainable from dipole-dipole interaction of the 259-mp transition of ADP with linearly polarized T-T* transitions of a juxtaposed stacked tryptophan residue can be made from expressions derived from coupled oscillator theory of optical rotation (Tinoco, 1962; Schellman and Schellman, 1964; Urry, 1965; Schellman, 1968). In general, the total rotational strength, Ri, of the i transition of ADP arising from coupling with different j transitions in a vicinal chromophore is given by the sum of the contributions from each interacting pair
The rotational strength Rii resulting from interaction of a single pair of electrically allowed transitions is
where V i j is the coulombic interaction potential, ut and u, the frequencies, g t and gj the electric dipole moments of the transitions, rtj the vector distance between their origins, and c and h the speed of light and Planck’s constant, respectively. To a first approximation, the interaction potential is
B I O C H E M I S T R Y , VOL.
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KXGI, Substitution of V i jin eq 2 yields (4) where D i = p i 2 , and D, = p i 2 is the dipole strength of the two transitions and Gij is the geometric term determined by the mutual position of the transition moment dipoles (Tinoco, 1962). It is given by the vector expression
where the e’s are unit vectors in the direction of the transition moments. When the interacting transition moment dipole vectors lie superimposed in parallel planes, forming right angles with the distance vector and an angle of 45” to each other G,, attains a maximum value and eq 5 reduces to
with r l j being equal to the vertical distance between the stacked chromophores. Numerical values of the dipole strength are obtainable from the absorption spectra of the interacting chromophores (Moscowitz, 1960). For the 259-mp transition of ADP, a value of D259= 15.2 X 10-36cgs unit was determined from the absorption spectrum of adenosine (Pabst, Circular, 1956). For the far-ultraviolet transitions of the indole side chain of tryptophan values of DIg5= 21 X and DZl9= 24 X cgs unit were obtained from the spectra of McDiarmid (1965). Approximate values for the near-ultraviolet transitions of D L ~=S 3.5 X and D259= 3.3 X cgs unit were calculated from the total integrated absorption intensity of the unresolved bands (Donovan et a/., 1961) assuming equal intensity of the two electronic transitions as suggested by Konev (1967). On the assumption that the vertical distance, rij, between the optimally positioned adenine and indole rings be the same as between bases in DNA, Le., 3.4 X 1 0 P cm (Langridge et ui., 1960) the magnitudes (Ri,, calculated for the maximum rotational strength arising from coupling of the 259 mp/196 mp and the 259 mp/219 mp pairs are of the order of 8 X and 17 X cgs unit, respectively. If the 219-mp and the 195-mp bands of the indole ring are attributed to in-plane transitions analogous to the perpendicularly polarized lB,, ‘A and lB:, ‘A transitions of the naphthalene spectrum as suggested by Platts vary-conjugate sequence (Platt, 1951) their rotatory contribution would be of opposite sign. Hence the likely maximum total rotational strength contributed to the adenine transition from these two strong bands is equal to their difference, i.e., Y X cgs unit. The rotatory contribution to be expected from coupling of the adenine transition with the near-ultraviolet transitions of tryptophan is much lower. The calculated maximum rotational strength arising from interactions with the 272and 289-mp transitions (Konev, 1967) is of the order of 2 X cgs unit for each if an estimated 60 and 30% overlap of the coupled bands is taken into account. Since these tryptophan transitions are thought to exhibit nearly perpendicular polarization along the same axes as the far ultraviolet transitions (Konev, 1967) their contribution to the ellipticity of the adenine band again would be of opposite +
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sign and thus largely canceling. Hence, in the model assumed, the rotational strength of the ADP transition would seem to derive mainly from the interaction with the far-ultraviolet bands of tryptophan. References Beychok, S. (1965), Proc. Nut. Acad. Sci. U. S. 53,999. Beychok, S., and Fasman, G. D. (1964), Biochemistry 3, 1675. Blout, E. R. (1964), Biopolymers Symp. I , 397. Blout, E. R., Schmier, I., and Simmons, N. S. (1962), J. Amer. Chem. Soc. 84,3193. Bush, C. A., and Tinoco, I., Jr. (1967), J. Mol. Biol. 23,601. Cohn, M., and Hughes, T. R. (1962), J. Biol. Chem. 237, 176. DeVoe, H., and Tinoco, I., Jr. (1962), J . Mol. Biol. 4,518. Donovan, J. W., Laskowski, M., Jr., and Scheraga, H. A. (1961), J . Amer. Chem. SOC.83,2686. Emerson, T. R., Swan, R. J., and Ulbricht, T. L. V. (1966), Biochern. Biophys. Res. Commun. 22,505. Fasman, G. D. (1963), Methods Enzymol. 6,928. Hammes, G. K., and Hurst, J. K . (1969), Biochemistry 8, 1083. Holcomb, D. N., and Tinoco, I., Jr. (1965), Biopolymers 3,121. Holzwarth, G., and Doty, P. (1965), J. Amer. Chem. SOC. 87,218. Jacobs, G., and Cunningham, L. W. (1968), Biochemistry 7,143. KLgi, J., and Li, T.-K. (1965), Fed. Proc., Fed. Amer. Soc. Exp. Biol. 24,285. Konev, S . V. (1967), Fluorescence and Phosphorescence of Proteins and Nucleic Acids, New York, N. Y., Plenum, p 52. Korsgaard Christensen, L. (1952), C. R. Trav. Lab. Curisberg 28, 37. Kuby, S. A:, Mahowald, Th. A., and Noltmann, E. A. (1962), Biochemistry I , 748. Kuby, S. A., Noda, L., and Lardy, H. A. (1954a), J. Biol. Chem. 209,191. Kuby, S. A , , Noda, L., and Lardy, H. A. (1954b), J. Bioi. Chem. 210,65. Lamborg, M. R., Zamecnik, P. C., Li, T.-K., Kagi, J., and Vallee, B. L. (1965), Biochemisfry 4, 63. Langridge, R., Wilson, H. R., Hooper, C. W., Wilkins, M. H. F.,andHamilton,L. D. (1960), J. Mol. Biol. 2,19. Lui, N. S. T., and Cunningham, L. (1966), Biochemistry 5, 144. McCormick, W. G., and Levedahl, B. H. (1959), Biochim. Bioph~,s. Acta 34, 303. McDiarmid, R. S. (1965), Ph.D. Thesis, Harvard University, Cambridge, Mass. Morrison, J. F., O’Sullivan, W. J., and Ogston, A. G. (1961), Biochim. Biophys. Acta 52, 82. Moscowitz, A. (1960), in Optical Rotatory Dispersion, Djerassi, C., Ed., New York, N. Y., McGraw-Hill, p 150. Noda, L. (1Y63), I43rd National Meeting of the American Chemical Society, Cincinnati, Ohio, Jan 1963, p 1YA. Noltmann, E. A,, Mahowald, T. A., and Kuby, S. A. (1962), J. Biol. Chem. 237, 1146. O’Sul!ivan, W. J., and Cohn, M. (1966a), J. Biol. Chetn. 241, 3104. O’Sullivan, W . J.: and Cohn, M. (1966b), J . Bid. Chetn. 241, 31 16. O’Sullivan, W. J.. Diefenbach, M., and Cohn, M. (1966), Hiochetiiistry 5 , 2666. Pabst Laboratories, Circular OR-10 (1Y56), Milwaukee, Wis.
GLUTAMIC DEHYDROGENASE
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Urry, D. W. (1965), Proc. Nut. Acad. Sci. U. S . 54,640. Van Holde, K. E., Brahms, J., and Michelson, A. M. (1969, J. Mol. Biol. 12,726. Warshaw, M. M., Bush, C. A., and Tinoco, I., Jr. (1965), Biochem. Biophys. Res. Commun. 18,633. Warshaw, M. M., and Tinoco, I., Jr. (1966), J . Mol. Biol. 20,29. Watts, D . C . (1963), Biochem. J . 89,220. Weber, G . (1960), Bi0chem.J. 75,335. Yang, J. T. (1961), Tetrahedron 13,143. Yang, J. T. (1967), in Poly-a-Amino Acids, Fasman, G. D., Ed., New York, N. Y . ,Marcel Dekker, p 267. Yang, J. T., and Samejima, T. (1963), J. Amer. Chem. SOC. 85,4039. Yue, R . S., Palmieri, R. J., Olson, 0. E., and Kuby, S. A. (1967), Biochemistry 6,3204.
Sedimentation Equilibrium Studies on Glutamic Dehydrogenase" M. Cassmant and H. K. Schachmanj
Sedimentation equilibrium experiments were performed on commercial preparations of beef liver glutamic dehydrogenase (GDH). Initial observations of a deviation from a simple associating system, as well as the presence of low (
