The Binding of Divalent Cations to Myosin

Vol.250,

THE JOURNAL OF BIOLOGICAL CHEMISTRY No. lG,Issue of August 25, pp. 6282-6287, Printed in U.S.A.

The

Binding

1975

of Divalent

Cations

to Myosin” (Received

MARGERY

C. BEINFELD,$

From the Department

DAVID

A.

of Biochemistry,

BRYCE,

DANIEL

KOCHAVY,

School of Medicine,

SUMMARY

Monovalent and divalent cations strongly influence the binding of ADP (1, 2), inorganic pyrophosphate (3), and ATP (4) to myosin which contributes to their well known effects upon the rate of ATP hydrolysis (5, 6). The K+-moderated ATPase activity of myosin is inhibited by Mg2+, Mnzf, and Ca2f and the free metal ion concentrations producing 50% inhibition were estimated to be about lop8 M for Mg2$ (6), 10e5 to 10m6 M for Mn2+ (5), and 1O-3 M for Cazf (5). Equilibrium binding studies indicate that myosin has two binding sites for Mg2f with a binding constant greater than 2 X lo6 M-l (1) in addition to a large number of low affinity binding sites (7). No comparable data are available on the binding of other divalent metal ions. Prompted by the interesting effects of Mn2+ upon the Pi’*OHJ60 exchange catalyzed by myosin in the presence of ATP or * This work was supported by Research Grants NS 07749 from the National Institutes of Health, United States Public Health Service, GB 33867-X from the National Science Foundation, and a grant-in-aid from the Missouri Heart Association. A preliminary report of portions of this work was presented at the 65th Annual Meeting of the American Society of Biological Chemists held in June, 1974 in Minneapolis, Minnesota. t. Recipient of a postdoctoral fellowship from the Missouri Heart Association. 5 To whom all correspondence may be directed. 6282

ANTHONY

University,

January

24, 1975)

MARTONOSI$

St. Louis, Missouri

63104

ADP (8) and the unique possibilities offered by the paramagnetic properties of Mn *+ for the study of the structure of enzyme-metal-substrate and enzyme-metal-product complexes by NMR and EPR spectroscopy (g-12), we investigated by equilibrium techniques the interaction of Mn2+ with myosin. Centrifuge transport and equilibrium dialysis measurements using the 5’Mn isotope and electron paramagnetic resonance studies according to the method of Reuben and Cohn (13) indicate the existence of two Mn’ r+ binding sites on myosin with intrinsic affinity constants of the order of lo5 to lo6 and 20 to 30 low affinity sites with affinity constants of about lo3 M-l. Mg2+, Ca2+, Sr2+, Zn2+, Co2+, and Ni2+ compete with Mn2+ for the binding sites. N-Ethylmaleimide was without effect upon the binding of Mn2+ to myosin. EXPERIMENTAL

PROCEDURE

Purification of Myosin-Myosin was isolated from predominantly white back and leg muscles of rabbit by the procedure described earlier (1). To the purified myosin solution (pH 6.8 to 7.0) containing 10m3 M EDTA, solid (NHa)$Oa was added to 35yo saturation and the resulting precipitate was discarded. The (NHa)#O, concentration of the supernatant was adjusted to 50yo saturation (14). After stirring for 1 hour at 4”, the precipitated myosin was collected by centrifugation and stored as an ammonium sulfate paste at -10” (5) or in 50yo glycerol at -15” (2). Fresh myosin or myosins stored under these conditions for less than 1 month gave similar results. For the removal of contaminating Mg2+ and other divalent metal ions from myosin the ammonium sulfate precipitate of myosin was dissolved in 0.6 M KC1 and dialvzed for 4 to 5 hours at 4” aeainst a 200- to 300.fold excess of 0.6 M ,Cl containing lo+ M EDNA. This was followed by an overnight dialysis against the same volume of fresh solvent. The dialysis tubing was treated to remove heavy metal ions and other impurities as described by Westhead and McLain (15). All subsequent steps were carried out using polyethylene containers which were cleaned by washing in succession with 0.1 N HCI, deionized water, 10e3 M EDTA, and finally with large volumes of deionized water. KC1 solutions were freed of divalent metal contamination by passing them through a column of Chelex 100 resin (1). After dialysis the myosin was precipitated by dilution with cold deionized H20; the precipitated protein was washed once with 0.04 M KC1 and 5 X low3 M EDTA followed by three washings with 0.04 M KCl. The final precipitate was dissolved in a small volume of Chelex-treated KC1 to yield a final concentration of 0.6 M KCl. After centrifugation the concentration of myosin solutions ranged between 18 and 20 mg of protein/ml as determined by biuret (16). The Mg2+ content measured by atomic absorption spectrophotometry (1) using the Perkin-Elmer model 303 instrument was 0.2 to 0.3 atom of Mg2+/mol of myosin. The same instrument was used for the assay of MnZ+ concentration. ATPase activity was measured by following the liberation of incorganic phosphate according to the Fiske-SubbaRow method (17). Measurement of Mn2f Binding by EPR Spectroscopy-The bind-

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dialysis, and electron Centrifuge transport, equilibrium paramagnetic resonance studies on the binding of Mn2+ to myosin revealed two sets of noninteracting binding sites which are characterized at low ionic strength (0.016 M KCl) by affinity constants of lo6 M-I (Class I) and lo3 M-' (Class II), respectively. At 0.6 M KC1 concentration, the affinity of MnZ+ for both sets of sites is reduced. The maximum number of binding sites is 2 for the high affinity and 20 to 25 for the low affinity set. Other divalent metal ions displace Mn2+ from the high affinity sites in the following order of effectiveness: Ca > Mg = Zn = Co > Sr > Ni. The inhibitory effects of Mg2+ and Ca2+ upon the Mn 2+ binding are competitive with inhibitor constants of 0.75 to 1 mM which is similar to that of the low affinity divalent metal ion binding sites. Exposure of myosin to 37” partially inhibits Mn2+ binding to Class I parallel with inhibition of ATPase activity. The binding of Mn2+ to the high affinity binding sites is not significantly influenced by ADP or PP;, although Mn2+ increases the affinity of ADP binding to myosin at high ionic strength.

St. Louis

AND

for publication,

6283

r/A

= K,

-

RESULTS

The

binding

of Mn?+

was

measured

at, low

(0.02 to 0.05 in KCl), by the centrifuge transport method using 54Mn (Fig. 1) and at high ionic strength (0.6 M KCl) by equilibrium dialysis or by electron spin resonance techniques (Fig.

2). Under

both

conditions

the binding

of Mn2+

FIG. 2. Scatchard plots of the binding of Mn2+ to myosin in 0.6 M KCl. The binding was measured by EPR (0, 0) or by equilibrium dialysis (A, 0) methods. For EPR measurements myosin (14.9 to 15.7 mg of protein/ml) was dissolved in solutions containing 0.6 M KC1 and 2 X 10-j to 6 X 10e3 M MnC&, pH 7.4 and the free Mn*+ concentration was determined at 25”, as described under “Experimental Procedure” (0) In some experiments (0 ) myosin was inactivated by exposure to 37” for 1 hour in 0.6 M KC1 prior to the EPR measurements. After this treatment about 7.37, of the Ca2+-activated and 11.47, of the EDTA-activated ATPase was retained compared with control enzyme stored at 0”. The equilibrium dialysis studies were performed using QMnC12 at 2-5” in the presence of 0.6 M KC1 and 0.5 M Tris-HCl, pH 8.0 (0) or pH 7.4 (a) as described under “Experimental Procedure.”

ionic

strength

is satisfactorily described binding sites with widely

FIG. 1. Scatchard plots of Mn %+binding to myosin at low ionic strength. The binding of 64Mn2+ was measured in a medium of 0.05 M Tris-HCl buffer and 0.016 M KCl, pH 7.4 at 2-5” by the centrifuge transport method as described under “Experimental Procedure.” O-0, no addition; m---m, 1.1 X 10m4 M ADP; a---n, 1.1 X 1O-4 M inorganic pyrophosphate. In this and subsequent figures the free manganese concentration (A) is expressed in micromolars.

K,

where r is the number of Mn2+ ions bound per 500,000 g of myosin, n is the maximum number of Mn 2+ binding sites per 500,000 g of myosin, K is the affinity constant in M-l and A is the free concentration of Mn2+ expressed in micromoles. The effect of other divalent metal ions and enzyme inhibitors upon the Mn2+ binding was evaluated according to Dixon (21) and characterized by inhibitor constants (K,) expressed as M. Materials-[8-i”C]ADP, @Mn, and %a were obtained from New England Nuclear Co. Standard reference solutions for the authentication of Mn2+ concentration were obtained from Fisher Chemical Co. All reagents were of analytical grade and distilled ion-exchanged water was used throughout.

to myosin

1

to myosin

in terms of two sets of noninteracting different affinities for Mn2+.

At low ionic strength (Fig. 1) the maximum number of high affinity sites (nr) is about 1.9 in reasonable agreement with the number of binding sites reported for ADP (l), ATP (4), and PPi (l-3). The value of the affinity constant K1 = 1.58 X lo6 M-l. The maximum number of low affinity sites is 20 to 25 with affinity constant Kz N 2 X lo3 M-I, ADP (1OP M), and inorganic pyro-


ing measurements were performed in a Varian model V-4500 EPR spectrometer as described by Cohn and her collaborators (13, 18). The EPR signal of free MnZ+ ion has a hyperfine structure of six lines. When Mn2+ becomes bound to myosin the signal is broadened to such extent that it disappears. The free Mn2+ concentration may be determined from the average peak to peak distance by calibration against solutions of known Mn2+ concentration. Calibration of the instrument was performed daily and the settings were left unchanged for a series of runs. The field was scanned from 2900 to 3900 G in a period of 10 min. The solutions were placed in a quartz flat cell about 5 inches in height and 0.1 mm in thickness. The same cell was used for all measurements. To assure reproducible positioning of the cell, the spectrum of each solution was scanned several times and between scans the cell was removed from the cavity. The reproducibility of the measurement was generally better than 10%. Between samples the EPR cell was washed with concentrated nitric acid followed by deionized water and was dried in a stream of nitrogen. All runs were carried out at 23-25” temperature. A stream of N, gas was allowed to flow through the cavity in order to maintain constant temperature. Measurement of @Mn2+ Binding by Centrifuge Transport-The experiments were performed essentially as described earlier (1) in a medium of 0.016 M KC1 and 0.05 M Tris buffer, pH 8.0 or pH 7.4 at 2-5” with total s4Mn2+ concentrations ranging from 1.1 X 10-E M to lo+ M. The final concentration of myosin was about 1.7 to 2.7 mg of protein/ml. Under these conditions myosin was essentially insoluble. The incubation was started by the addition of myosin to the otherwise complete test system. After 10 min of incubation the precipitated myosin was removed by centrifugation at 1600 X g for 10 min. The concentration of myosin-bound manganese was evaluated from the decrease in the radioactivity of the supernatant compared with a myosin-free control sample of otherwise identical composition. Protein was determined in the supernatant by the Lowry method (19)) and in the sediment by biuret (16). For radioactivity measurements a Packard model 3001 AutoGamma spectrometer or a Packard model 3320 liquid scintillation counter was used. The concentrations of competing metal ions and inhibitors are given in the legends. Measurement of Mn2+ Binding by Equilibrium Dialysis-The measurements were performed at 2-5” essentially as described earlier (1) in a medium of 0.6 M KC1 and 0.05 M Tris buffer, pH 8.0 or 7.4, at total 54Mn concentrations ranging from 5 X 10es M to 10m3M. The concentration of myosin in the dialysis tubing was 5 to 8 mg of protein/ml. After equilibration for 22 to 26 hours the inside and outside solutions were separated and their radioactivity was measured, as described above. Measurement of Ca2+ Bindi?lg-The binding of 4”Ca was measured at low and high ionic strength by the centrifuge transport and equilibrium dialysis techniques outlined above. Analysis of Binding Data-The binding data were usually represented in Scatchard plots (20) based upon the equation

6284

I

2

3

4

5

6

1i 7

FIG. 3. Competition between Mg2+ and Mn2+. The Scatchard plots represent data obtained at low ionic strength with the centrifuge transport method as described under “Experimental Procedure.” O-O, no magnesium; O--O, 1.1 X 1OW M MgC12; A-A, 2.2 X lo-+ M MgCL; O-0, 4.4 X 10e5 M MgCl,. The Dixon plots (inset) contain data from a similar set of experiments at the following total MnC12 concentrations: n -m, 4.4 X 10-T M; O-0, 2.2 X lo-6 M; O-0, 5.5 X 1o-6 M; A-A, 3.3 X 1OW M. Intercept in the upper left quadrant is consistent with competitive inhibition yielding a K; of about 0.75 mM. Although as originally described (21) Dixon plots refer to l/v versus I, analogous considerations apply to the l/r versus I plots presented in this report (20).

Co,

Mx103

FIG. 4. Competition between Ca2+ and Mn2+. The experiments were performed at low ionic strength by the centrifuge transport method. Scatchard plots: O---O, No Ca2+; m---w, 1.1 X 10m5 M Cazf; O-0, 4.4 X 10m5 M CaZf; A-A, 2.2 X lop4 M Ca*+; O---O, 1.1 X lop3 M Ca2+. Dixon plots: m--m, 4.4 X lo-’ M MnCl*; O-0, 2.2 X 10M6 M MnC12; O---O, 5.5 X 10e6 M MnC12; A--A, 3.3 X 10m5 M MnC12. CaClz (Fig. 4) indicating negative cooperativity. develop an upward convex curvature at Mg2+ (Fig. (Fig. 4) concentrations higher than 3 to 4 mM (not significance of these nonlinearities is not clear but likely to influence the principal conclusions drawn and 4. In 0.6 hf KC1 on the basis of equilibrium dialysis electron spin resonance data (Fig. 6) Ca2+ may more effective competitor for hW+ binding than value of Ki from Fig. 5 for Mg2+ is 0.9 mM and for

Dixon plots 3) and Ca2f shown). The they are not from Figs. 3 (Fig. 5) and be a slightly Mg2+. The Ca2+ 0.3 mM,


phosphate (1O-4 M) had little effect on the MnQ+ binding to either group of sites over a limited range of Mn2+ concentration, where precipitation of insoluble manganese salts did not occur. In 0.6 M KC1 (Fig. 2) the range of values for the maximum number of high affinity Mn2f sites (nJ is 1.5 to 2.1 with affinity constants (K1) ranging from 0.86 X lo5 to 3.6 X lo5 M-I. The corresponding values for the low affinity sites are n2 = 20 to 25 and K2 = 5.8 X lo2 calculated from EPR data. Initial estimates of these values were obtained by least squares fitting of the linear portions of the Scatchard plots corresponding to each set. The final values were derived by iteration calculation based on equations developed by Klotz and Hunston (20). These data indicate that 0.6 M KC1 moderately decreases the affinity of Mnzf for myosin at both sets of sites, in comparison with experiments at low ionic strength. Data obtained by equilibrium dialysis using 54AMn or by electron paramagnetic resonance measurements showed reasonable agreement. The estimated affinity constant of about 10” M-l for the binding of Mn2+ to the high affinity sites is consistent with the observed dependence of the inhibition of potassium-moderated ATPase activity of myosin upon Mn2+ concentration (5). Replacement of 0.6 M KC1 m-ith 0.6 M NaCl had little or no effect on the binding of Mn 2+ to myosin, although the ATPase activity of myosin is inhibited by sodium ions (22). The ATPase activity of myosin is inhibited after a l-hour exposure to 31” and essentially destroyed at 37”. In heat-inactivated myosin, the number of the “high affinity” sites is substantially reduced compared with the native enzyme (Fig. 2) indicating a possible relationship between ATPase activity and Mn2+ binding. Heat treatment has no effect on the number and affinity of low affinity Mn2+ binding sites. The low affinity Mn2+ binding sites are similar to those found in actin (23) and other proteins and probably represent nonspecific binding of Mn2+ to myosin. The large number of binding sites and the low value of K2 are consistent with this interpretation. The relationship of the two high affinity NIn2+ binding sites to the previously observed high affinity sites for Mg2+ (l), ADP (I), and ATP (4) was further investigated by studying the competition of Ma*+ or Ca2+ with the Mn2+ binding. Competition by Mg2+ and Ca2+ for Mn2+ Binding Sites--Mg2+ binds tightly to two sites on myosin with an estimated affinity constant greater than 2 X lo6 and probably close to 10’ hl-’ (1). There is a clear correlation between Mg2f binding to these sites and the inhibition of potassium-activated myosin-ATPase (1, 3, 6) by Mg 2f. A similar Mg2+ binding site is involved in the interaction of inorganic pyrophosphate (3) and ADP (1) with myosin. Ca2+ binds to myosin with much lower affinity. If the two high affinity Mn*+ binding sites (Figs. 1 and 2) are identical with those involved in the binding of the two functionally important YIg2+ eons to myosin, u-c would expect effective displacement of Mn2+ from the binding sites at 10-j to 1OV M Mg2+ concentrations. This follows from the fact that the reported affinity constant of ML 12+ for the high affinity sites on myosin (1, 6) is greater than the affinity of Mn2+. The relationship between the binding sites of Mn2f and Mg2+ was investigated by competition studies at low ionic strength using varying concentrations of both metal ions (Fig. 3). In Scatchard as well as in Dison plots the inhibition of Mn2+ binding by Mg2+ is competitive with an inhibitor constant of 0.75 m&l which is close to the affinity of YIg2+ for the low affinity divalent ion binding sites. Essentially similar data were obtained with Ca2+ (Fig. 4). The Scatchard plots arc nonlinear at low 11n2+ concentrations in the presence of 0.05 to 0.2 m&r MgCIZ (Fig. 3) or 0.01 to 0.2 Inhl

6285

Mg

or Co,

Mx103

5. The effect of Ca2f and Mg2+ upon the MnZ+ binding in 0.6 M KCl. Equilibrium dialysis was carried out as described under “Experimental Procedure,” at free Mn2+ concentrations of 3.3 X (A, LJ.) and 1OW M (0, A) or 1.6 X 1OW M (0, a). Magnesium calcium (0, 0) concentrations are indicated on the abscissa. FIG.

FIG. 7. The binding of Ca2+ to myosin. The binding of 45Ca was measured by the centrifuge technique in a medium of 0.033 M KCl, 50 mM Tris, pH 7.4, and myosin (1.66 mg/ml) at 4’, as described under “Experimental Procedure.”

r

0.3

1

lull

I 10-z

Metal

mhlbltor,

M

FIG. 8. The effect of various divalent metal ions upon the binding of Mn2+ to myosin. The binding of MnZ+ was measured at low

ionic strength

1

r

2

FIG. 6. The effects of Mg2+ and Ca2+ upon the binding of Mn2+ to myosin. EPR measurements were performed at 25” in the presence of 0.6 M KC1 at pH 7.4 under the conditions described under “Experimental Procedure” and in legend to Fig. 2. The broken line is the regression line of data presented in Fig. 2 without Mgz+ or Ca2+. O-O, 5 X 10M3 M MgC12; B--W, 5 X 10-d M CaC12;

Total

Mna+ concentrations

ranged between 2

X

using the centrifugation

method.

Medium

contained

0.05 M Tris, pH 7.4, 0.05 M KCl, 2.2 X 10-G M 5’MnC12, 2.12 mg of myosin/ml, and the various divalent metal ions in concentrations indicated on the abscissa. 0-0, CaC12; B--B, CoCl,; O-O, MgC12; A-A, ZnCL; n--A, SrC12; O-O, NiC12.

10e5 to 6

X

10V3 M.

which are close to those given in Figs. 3 and 4 at low ionic strength. The affinity constant of Mg2+ binding calculated from the slope of the Scatchard plot shown in Fig. 6 is about 0.94 x lo3 M-‘, i.e. essentially that of the low affinity divalent cation binding sites. Similar measurements in the presence of 5 x 1O-4 M Ca2+ (Fig. 6) give a Kca = 3.2 x IO3 M-‘. The inhibitory effect of Mg2+ and Ca2+ on the binding of Mn2+ to myosin was not altered significantly in the presence of 1O-4 M ADP. Binding of CaQf to Myosin-The binding of Ca2+ to myosin was measured directly using Wa at low ionic strength. Scatchard plots of the data (Fig. 7) indicate the existence of two sets of binding sites with affinity constants of 1.4 x lo5 M-l (Kl) and about lo3 M-l (K2) respectively. The maximum number of binding sites in the high affinity set is n1 = 1.4, i.e. similar to the number of high affinity Mg*+ (1) and Mn*+ binding sites. The inhibitor constant of Ca2+ derived from competition with Mn2+ is similar to the apparent affinity constant of the weak binding sites.

Eflect of Other Divolent Metal Ions on Binding of Mn2+ to Myosin-The binding of Mn 2+ to the high affinity sites is also inhibited by X2+, Co2+, Srz+, and Zn2+ (Fig. 8). The order of effectiveness of divalent metal ions in displacing MI?‘+ from the binding sites is Ca > Co = Mg = Zn > Sr > Xi. E$ect of Mn2+ Concentration upon Binding of ADP to MyosinThe binding of ADP to myosin in media of high ionic strength requires

Mgzf

(1) while

at a KC1 concentration

of 0.05 M signifi-

cant ADP binding occurs even in the presence of 5 mM EDTA (1). The data of Fig. 9 show a similar relationship between ADP binding and the concentration of Mn2+. The sharp increase in ADP

binding

in the presence

of 0.6 M KC1 at a total

Mn2+

con-

centration of about 1OV M suggests that binding of Mn2+ to the high affinity sites of myosin increases the stability of the myosinADP complex. At 0.05 M KC1 concentration the binding of ADP was not significantly altered by varying the concentration of Mn2+. The increase in the stability of myosin-ADP complex at Mn2+ concentrations higher than 10e5 M is accompanied by inhibition of ATPase activity (5). -SH Groups oj Myosin and Binding of Mn2+-The high affinity sites of Mn2+ binding are not influenced by blocking of groups with large excess of N-ethylmaleimide myosin -SH which causes nearly complete inhibition of both EDTAand


04 !

.15

6286 Although it is plausible to assume that the high affinity binding sites for Mn2+, Ca2+, and MgZi are identical, the situation appears to be somewhat more complex. While Ca2+ and Mg2+ compete for the high affinity 1’1Lln *+ binding sites, the inhibitor

1.0

.

..

. -

l

.

0

constant rADP 05

presence Total

Mn,

M

;: 0: r

‘0c1

1.0

Moles

of N-ethylmale,m,de/Mole

r

of myos~n

10. The effect of Mn*+ to myosin. Myosin pH 7.4 in 0.08 M KC1 for 1 sin mole ratios indicated

N-ethylmaleimide upon the binding of was treated with Wethylmaleimide at hour at 2”, at N-ethylmaleimide to myoof Mn2+ on the abscissa. The binding in a medium containing 0.05 M KCl, 0.05 M Tris,

was measured pH 7.4, and 1.1 X 10m5 M 54MnC12 (0). EDTA-activated ATPase was measured in 0.6 M KCl, 0.10 M Tris, pH 8.0, 5 mM ATP, and 5 mM EDTA at 25” (O ). Calcium-activated ATPase was assayed in a medium of 0.05 M KCl, 0.10 M Tris, pH 7.4, 5 mM ATP, and 5 mM calcium at 25” (w). Ca*+-moderated (24, 25).

from

this

competition

is only

about

1OF

M

ATPase

activity

(Fig.

10) and ADP

binding

DISCUSSIOX

Measurements of Mn*+ binding to myosin by several techniques provided evidence for the existence of two high affinity sites (K1 N- lo5 to lo6 M-‘) and a large number of low affinity binding sites (& N 103) per mol of myosin. Mn*+ binding to the high affinity sites enhances the affinity of ADP binding to myosin and causes inhibition of K+- and Ca2+activated ATPase activity (5). These observations tend to support the assumption that Mn 2+ bound at the high affinity sites is involved in the hydrolysis of ATP by myosin. Myosin contains two high affinity sites for Mg2f (1) and several lines of evidence indicate that Mg*+ bound at these sites has an important physiological role in the energy transduction process during muscle contraction.

of contaminating

magnesium

in the assay

systems

used

for the measurement of Mn2+ binding since the myosin used in these studies was treated with Chelex 100 resin and with EDTA to remove contaminating Mgzf and all precautions were taken to minimize subsequent Mg2f contamination by the reagents. The Mg2+ content of myosin so treated was shown to be only 0.2 to 0.3 mol of Mg*+ per mol of myosin by atomic absorption spectroscopy. Kinetic effects are not likely to contribute since essentially similar results were obtained by equilibrium dialysis (22 to 26 hours) and by the centrifugation technique (20 to 30 min). It may be argued t,hat most estimates of the affinity of Mgz+ for the enzymatic site were derived from measurements performed in the presence of ATP (B), ADP (l), or inorganic pyrophosphate (3) and therefore may reflect the influence of substrate and substrate analogues upon the Mg*+ binding. However, the binding of Mn*+ to myosin and the inhibitory effect of Ca*+ and Mg2+ upon the Mn *+ binding arc not sufficiently influenced by ATP, ADP, or PPi in order to make this explanation a plausible one. Another puzzling observation is that Ca2+ is a more effective competitor for the Mn*+ binding sites than Mg2+ although from its influence upon the ATPase activity of myosin the affinity of Ca*+ for the active site is relatively weak. Clearly further work is required to establish conclusively the relationship between enzymatic activity and the binding of Mg2+ and Mn2+ to myosin. It is possible that under the conditions of the experiments, in spite of the rather low [Mn*+] concentrations, most of the Mn2+ binding occurs at the low affinity sites. Alternatively, if Mg*+ and Mn*+ are bound at the same site the configuration of bound Mn2f is such as to make competition by Mg*+ and Ca2+ less effective. The lack of influence of ATP, ADP, and PPi on the Mn2+ binding is in general agreement with the proposed ordered sequence of interactions of metal ions and substrates with myosin (3) in which the formation of metal-myosinatc precedes the binding of substrate to the enzyme. It is noteworthy in this regard that in spite of the exceptionally high stability of myosin-ADP complex in the presence of Mn*+ (2) the binding of ADP has only marginal effect? upon the enhancement of proton relaxation rates observed in the presence of myosin-Mn2+. A further study of this problem by nuclear magnetic resonance technique may help to interpret the thermodynamic and kinetic effects of metal ions upon the hydrolysis of ATP (2) by providing a more accurate description of the mode of interaction of ATP and ADP with myosin. N-Ethylmaleimide at relatively high concentration inhibits the binding of ADP and PPi to myosin indicating a requirement for free -SH groups (25). In contrast the binding of Mn2+ to the high affinity sites is entirely unaffected by treatment with up to 1 Unpublished

observations

by Dr. Daniel

Kochavy.

During

the

preparation of this manuscript we learned that Dr. R. G. Yount of the Department of Chemistry, Washington State University, Pullman, Washington made independently similar observations.


9. The effect of MnZ+ upon the binding of ADP to myosin. FIG. The binding of ADP was measured at low ionic strength by the centrifugatihn method in a medium of 0.05 M KCl, 0.009 MeTris, pH 7.0, and 4.4 X 10-e M ADP at MnClz concentrations indicated on the abscissa (0). In control experiments manganese was replaced by 2.2 X 1OW M MgClz (m). At high ionic strength the ADP binding was measured by equilibrium dialysis in a medium of 0.6 M KCI, 0.03 M Tris, pH 8.0, 3.3 X 10-e M ADP at MnClz concentrations indicated on the abscissa (0). In some experiments (0) MnC12 was replaced by 1 mM MgC12.

FIG.

derived

which is similar to that of the nonspecific cation binding site. For comparison the dissociation constants of Mg2+ calculated from the inhibition of ATPase activity (6) or from the Mg*+ dependence of ADP (1) and PPi (3) interaction with myosin are in the range lo-’ to lop6 M (l-3). The relatively high value of the inhibitor constant of Mg*+ cannot be due to the

6287 3000 mol of N-ethylmaleimide per mol of enzyme which excludes the participation of reactive -SH groups from the process. The possibility may be considered that the interaction of bound divalent metal ion with the polyphosphate chain of ADP makes a relatively minor contribution to the over-all affinity of ADP binding to myosin. The 1Mn2+ binding to the high affinity sites is influenced in a similar manner by 0.6 M KC1 or 0.6 M NaCl suggesting that the large difference in the rate of ATP hydrolysis with Naf or K+ as activators is not related to their effect. upon the binding of divalent metal ions to myosin. Acknowledgments--We wish to thank Dr. F. Hertelendy of the Department of Medicine and Dr. R. Layloff of the Department of Chemistry, St. Louis University, for permission to use their equipment and to Mr. E. C. Ernst for collaboration in the early phase of the work. REFERENCES

J. R.,

AND

YOUNT,

R. G. (1966) Biochem.

A. S. (1970) in The Enzymes (BOYER, 2, pp. 445-536, Academic Press, New York

10. MILDVAN, 11. MILDVAN, 246 12. MILDVAN, Areas 13. REUBEN,

A. S., AND COHN, A. S., AND COHN, A. S., AND COHN, Mol. Biol. 33, l-70 J., AND COHN, M.

2. 346,

P. D., ed) Vol.

M. (1963) Biochemistry 2, 910-919 M. (1965) J. Biol. Chem. 240, 238M.

(1970) Adv. Enzymol.

(1970) J. Biol.

Relat.

Chem. 246, 6539-

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