31P Nuclear Magnetic Relaxation Studies Ofphosphocreatine

Proc. Natl. Acad. Sci. USA Vol. 74, No. 10, pp. 4271-4275, October 1977 Biochemistry

31P nuclear magnetic relaxation studies of phosphocreatine in intact muscle: Determination of intracellular free magnesium (nuclear Overhauser effect/13C nuclear magnetic resonance) SHEILA M. COHEN*t AND C. TYLER BURTt *Department of Chemistry, University of Illinois at Chicago Circle, Chicago, Illinois 60680; and *Department of Biological Chemistry, University of Illinois at the Medical Center, Chicago, Illinois 60612 Communicated by Irving M. Klotz, July 25, 1977

ABSTRACT 31P nuclear magnetic relaxation rates for nipulations were performed in a cold room at 40 and NMR phosphocreatine in intact frog gastrocnemius were compared accumulations in a probe maintained at 40 were begun im- with those observed in model solutions at 40, a temperature at which muscle maintains its physiological state for at least 5 hr. mediately. Both nuclear Overhauser effect and spin-lattice relaxation rate PCr was from Sigma Chemical Co.; Mg(NO3)2 (ultrapure) (1/Tj) experiments indicate that interactions form was from Alfa Products. Metal-free solutions of PCr were the dominant relaxation path fordirle-dipoleP in intact muscle and model prepared by passing constituents through a column of Chelex. solutions, independent of phosphocreatine and Mg concentra- The pH was adjusted to 7.4 (pD = meter reading + 0.4) at room tions. Spin-spin relaxation rates (1/T2) measured by modified temperature with HCO (or DCl) prepared by passing Carr-Purcell-Melboom-Gill spin-echo experiments suggest the HCl (DCl) importance of scalar coupling modulated by chemical exchange gas through H20 (D20). Solutions were 0.1 M in KCI. Solutions with free Mg. From these results, we estimate the free intra- in deuterated solvent were lyophilized three to four times in cellular Mg in intact muscle as 4.4 mM and demonstrate that 99.8% D20. The final concentration of PCr in solution was 31p T2 experiments can be used as a tool for studying free Mg determined by total P analysis (9). Analyses for Mg and Mn in levels with minimum disturbance of the intact cell. stock and final solutions were by Trace Elements Inc. (Park Ridge, IL); these analyses showed Mn in final solutions to be 31P nuclear magnetic resonance (NMR) studies of intact cells <0.1 ,uM. All solutions were degassed either by nitrogen bub- to date have concentrated on high-resolution spectra of me- bling or several freeze-pump-thaw cycles. tabolites in intact muscle (1, 2), erythrocytes (3), yeast (4), and High-resolution NMR spectra were obtained at 36.43 MHz Escherichia coli and Ehrlich ascites tumor cells (5, 6). We report on a hybrid Bruker HFX-90 spectrometer with 2H stabilization here on the feasibility of measuring the relaxation parameters operating in the Fourier transform mode as described (10). of phosphocreatine (PCr) in intact frog muscle at 4°. The Spinning 10-mm samples were used; the field was locked either spin-lattice relaxation time (TI) and the spin-spin relaxation to a capillary of D20 (muscles and PCr/H20 solutions) or D20 time (T2) of PCr in intact muscle are compared with those solvent. Typical conditions included a 900 pulse of ca 20 lisec, observed in model solutions at 4°. Both relaxation rates are 32-128 scans of 4000 data points each, sweep width of 1 kHz, valuable as dynamic probes of the internal environment in exponential multiplication introducing 0.6-Hz line broadening, muscle because T2 can be sensitive to processes that do not affect and 'H irradiation. T, at all. These relaxation studies also enable us to assign a value T, measurements were made with the following pulse se- for the free intracellular Mg concentration in intact gastroc- quence: 900 pulse, spoil field homogeneity, wait delay time r, nemius via an intrinsically nonintrusive method. 90° pulse, sample free-induction decay, and then spoil field It is now clear that metal ions are of central importance in homogeneity. This sequence (11), which was repeated n times, many biological reactions and knowledge of free Mg levels has afforded a considerable saving of time over the inversion-re- particular significance. For example, Stephenson and Podolsky covery method in the T, regimen of 31P in PCr, thus making (7) showed that in skinned muscle fibers, Ca transport by intact it possible to carry out T, measurements on muscle during the sarcoplasmic reticulum is strongly influenced by Mg concen- interval in which there was no more than a 10% change in tration. The present nonperturbing technique for free Mg is of PCr. special interest; of the few alternate methods, the one of choice The Carr-Purcell-Meiboom-Gill (CPMG) (12) T2 mea- is probably a differential absorption procedure involving the surements were performed on a Bruker B-KR pulsed spec- microinjection of a Mg-binding dye to measure ionized Mg in trometer operating in conjunction with the same Nicolet data the volume surrounding a dialysis capillary (8). system and a 21.14 kG magnet used for high-resolution spectra. Two home-built modifications to the probe of this pulsed MATERIALS AND METHODS spectrometer were made so that satisfactory results could be Adult frogs, 5-12.5 cm in length, from Lake Champlain Frog obtained on biological samples in which phosphate metabolites Farm (Alberg, VT) were stored at 4°. Frogs appearing to be in are in the mM range-namely: (i) because field-frequency drift good condition were decerebrated and the rear legs were iso- is known to have a deleterious effect on the results of the CPMG lated. The gastrocnemius was dissected out and placed in a method, we installed an external deuterium lock system in the 10-mm NMR tube with standard frog Ringer's solution (113 pulse probe, which was capable of holding the 31p field-fre- mM NaCI/2.5 mM KCI/1.8 mM CaCl, pH 7.2). These ma- quency stability within 1-2 Hz over several hours, and (ii) in Abbreviations: NMR, nuclear magnetic resonance; PCr, phospho- The costs of publication of this article were defrayed in part by the creatine; T1, spin-lattice relaxation time; T2, spin-spin relaxation time; payment of page charges. This article must therefore be hereby marked CPMG, Carr-Purcell-Meiboom-Gill; NOE, nuclear Overhauser effect; "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate DD, dipole-dipole. this fact. t Present address: Bell Laboratories, Murray Hill, NJ 07974. 4271 Downloaded by guest on September 26, 2021 4272 Biochemistry: Cohen and Burt Proc. Natl. Acad. Sci. USA 74 (1977)

_ 100 0 0 ~0 80 .0 0 0~ "F 60- 40 (a) (b) %4- 0 T= 277 K 20 A B A B 0 _ I w0 50 100I- 150 200 250 300 Minutes FIG. 1. Change in concentration (expressed as % of the original value) of whole-muscle phosphate metabolite as a function of time. There was less than a 20% change in PCr concentration over 5 hr at 40. This demonstrates the feasibility of long-term relaxation experiments at this temperature. The insets show two representative 31p spectra ofthe time course for change in PCr concentration (peak B) in whole muscle relative to a sealed reference.capillary of trimethyl phosphate (peak A). Experimental conditions were maintained constant throughout. (Inset a) The 0 min spectrum; the ratio of the integrated peak areas (B/A) is 0.73. (Inset b) A spectrum taken 290 min later in which this ratio is 0.64. order to use the significant gain in sensitivity offered by the of muscle were maintained within normal limits longer than large nuclear Overhauser effect (NOE) of 31P of PCr in muscle at room temperature (15). Typical 31P NMR spectra of the time (see below), we added proton irradiation capability to the 31P course for change in PCr concentration in whole muscle at 40 pulse probe. Whereas heteronuclear spin decoupling has a are shown in Fig. 1 inset. Long-term experiments requiring 4-5 complex effect on echo formation, the use of the NOE for signal hr were practical at this temperature; the data in Fig. 1 dem- enhancement does not contain this complication (13). Repro- onstrate that our muscle preparation maintained at least 80% ducible T2 values were obtained with the pulse spacings, 2r, of of this high-energy phosphate at the end of our longest exper- 2-20 msec used here. So that echo amplitudes could be mea- iments. sured relative to the proper steady state, the radio frequency Observed T1 values for several intact muscle samples and pulses were not turned off until the spin-echo decays had model solutions are listed in Table 1. Differences observed proceeded to about 6T2. Time averaging with a recycle time between stretched and unstretched muscle cannot be consid- of 6T, was used (14). Prior to averaging, signals were passed ered significant. However, it is significant that the average T, through a B-KR pulse-gated integrator whose output gave the of 4.8 sec for muscle and the average T1 of 4.9 sec for PCr in envelope of the echoes directly. CPMG measurements were simple aqueous solutions were the same within the stated error made on nonspinning samples in 10-mm tubes with the sample limits. This suggests that the system of 31P nuclear spins in PCr constrained within the radio frequency coil for greater H1 is relaxing by the same mechanisms in both cases. Comparison homogeneity. With muscle samples, this was accomplished by of T1 for PCr in H20 solution with T1 in D20 indicated that the choosing frogs of appropriate size so that a single gastrocnemius 'H-31P dipole-dipole (DD) interaction was the dominant re- almost filled the radio frequency coil space. laxation process here, just as was found for aqueous H3PO4 (16), and was attributable to the formation of hydrogen bonds be- RESULTS tween solvent and the phosphate moiety. If we call (1/T1)H and These studies were conducted at 40 because with cooling there was a reduced rate of loss of contractility and the ionic contents

Table 1. Spin-lattice relaxation times for 31p of PCr in intact Decoupler off muscle and model solutions at 277 K Sample Ti, sec Northern frog gastrocnemius muscle samples* a (stretched) 5.1 I 0.5 b (stretched) 5.2 c (unstretched) 4.7 d (unstretched) 4.8 Decoupler: on e (unstretched) 4.8 f (unstretched) 4.2 II -10 0 5. 10 15 Muscle average 4.8 + 0.5 -5 ppm H,, PCr (H20 solution)t 4.9 + 0.2 FIG. 2. Effect of proton irradiation on the 31p NMR of intact PCr (D20 solution)s 13.7 + 0.2 gastrocnemius muscle from northern frog as 277 K. The intensity differences between these spectra illustrate the NOE. All conditions * Each muscle sample was from a different frog. Samples so desig- were maintained constant for both spectra (64 scans each), except that nated were stretched ca 10% beyond rest length in a modified NMR broad band 1H irradiation was turned on for the lower trace; gated tube. decoupling gave the same results as turning the decoupler off. Because t Average of three measurements on three different solutions. Solu- the proton relaxation times were shorter than the 31P TI, a waiting tions ofPCr in the range of 38-400 mM had T, values that were the period of 5P, (31p) between 90° pulses was sufficiently long so as to same within the stated error limits. avoid underestimation of NOE values (17). Downloaded by guest on September 26, 2021 Biochemistry: Cohen and Burt Proc. Natl. Acad. Sci. USA 74 (1977) 4273

Table 2. NOE enhancement for 31p ofPCr in muscle and model solutions at 277 K Determina- NOE (1 + 7obs) tions, no. Muscle 2.04 + 0.06 3 PCr in H20* 2.00 I 0.03 5 PCr in D2O* 1.15 I 0.05 5 * Solutions were 0.3 M in PCr and 0.1 M in KCl and had T1 values within the limits given in Table 1. e (1/TL)D the observed 31P relaxation rates in H20 and D20 solvents, respectively, and let (1/T1)X be a combined term for a spin-rotation contribution (12) and an intramolecular DD contribution-other possible contributions to T1 such as chemical-shift anisotropy (4) or scalar relaxation (16) having a minimal effect for small phosphate metabolites-then the following relation (16) obtains: [(l/T,)H - (/Tl)x]/[(I/T,)D - (/Tl)x] = (3/4)92H/2'yD2 = 15.91. Thus, by using the data in Table 1, we found that (T1)x = 15.7 sec and that ca 70% of the observed relaxation rate in muscle and H20 solvent was attributable to a DD interaction between 31P and the water protons, with T1DD (intermolecular) estimated to be 6.6 sec. Complementary information was contained in NOE measurements. Typical NOE experiments on whole muscle are shown in Fig. 2 and NOE results for muscle and model solutions are summarized in Table 2. The NOE values listed here are to be compared with the theoretical NOE, 1 + nt, of 2.24 for a 31p nucleus relaxing predominantly by DD interactions with protons, with no distinction being made between intramolecular and intermolecular interactions (18). For 31P DD interactions with deuterium, the theoretical NOE, 1 + qt, is 1.19, again in the "extreme narrowing limit." An estimate of T1DD (inter- L I O 1 2 3 4 5 TIME (sec) Table 3. Spin-spin relaxation times for 31p of PCr in intact muscle and model solutions at 277 K FIG. 3. The CPMG method for the determination of the spin- spin relaxation time T2 of 31p in PCr at 36.433 MHz and 277 K. The Sample T2, msec l/T2, sec' pulse sequence used was: 900,r, 180, 2Tr, 1800, 2r, . . ., with the phase of the radio frequency of the 900 pulse shifted by 900 relative to the Northern frog gastrocnemius phase of the 1800 pulse. The pulse spacing 2r was 2 msec. The time- muscle samples* averaged results of five representation CPMG experiments are shown: a 834 1.20 (a) 0.7 M PCr, (b) 37.5 mM PCr, (c) 37.5 mM PCr with 8.5 mM Mg, b 901 1.11 (d) 37.5 mM PCr with 5.3 mM Mg, and (e) intact northern frog gas- c 1000 1.00 trocnemius muscle (average of about 500 decays requiring 4 hr). Muscle average (3) 912 1.10 k 0.1 molecular + intramolecular) could be obtained from T1DD (intermolecular + intramolecular) = (ft/lobs) T1 (obs). The PCr (D20 solutions) 2460 0.407 equivalence of the muscle NOE and that of a simple aqueous PCr (H20 solutions) 1503 0.665 solution of PCr again suggests that the same relaxation mech- PCr/3.47 mM Mg 1290 0.775 anisms were operative. Using the average NOE of 2.02, we PCr/5.30 mM Mg 1061 0.943 + = 5.7 PCr/6.90 mM Mg 931 1.07 estimated that TIDD (intermolecular intramolecular) PCr/7.63 mM Mg 904 1.11 sec which indicated that ca 82% of the observed relaxation rate PCr/8.32 mM Mg 834 1.20 was due to all DD interactions. The remainder, PCr/9.70 mM Mg 784 1.28 1/Ti (other) = (1/T1)H PCr/9.70 mM Mg 757 1.32 - + PCr/10.67 mM Mg 731 1.37 l/T1DD (intermolecular intramolecular), PCr/11.1 mM Mg 708 1.41 contained the spin-rotation interaction contribution. In both PCr/13.9 mM Mg 626 1.60 muscle and H20 model solution, the following contributions The estimated accuracy was 5%. The reproducibility for a given PCr could be isolated: T1DD (intramolecular) = 39.1, T1DD (inter- solution over a 4-month period was within 5%. Solutions contained molecular) = 6.6, and T1 (other) = 26.3 sec. In D20 model so- 37.5 mM PCr and 0.1 M KCl. lutions, these contributions were estimated to be 39.1, 106, and * Each muscle sample was from a different frog. 26.3 sec, respectively. Downloaded by guest on September 26, 2021 4274 Biochemistry: Cohen and Burt Proc. Natt. Acad. Sci. USA 74 (1977)

I I I -- I I I I I I I I I I , 1.6

1.4 1.2F 1.0F I- 0.8

0.6 11

0.4 - I I I-- I I I 11 I I i I I F 0.0 0.02 0.04 0.06 0.08 0.10 0.12 0.14 Fraction PCr bound (for Kapp = 20 M-) FIG. 4. Dependence ofthe 31p relaxtion rate lfJ2 on the fraction ofP ~rbound by Mg at 277 K. The fraction bound (FB = [MgPCrJ/[PCrjtOi) was determined from the formal concentrations of Mg and PCr with the apparent stability constant measured by Smith and Alberty (23).

Several CPMG spin-echo experiments are shown in Fig. 3 describe the observed dependence of l/T2 on total Mg con- and observed spin-spin relaxation times for intact muscle and centration. Further experiments are required before a quan- model solutions are given in Table 3. It is important to note that titative estimate of the relative importance of these two the muscle average T2 was significantly shorter than T2 ob- mechanisms to the observed T2 can be made. served in the simple aqueous PCr solution. Whereas T2 was In Fig. 4, l/T2 is plotted against FB, the fraction of PCr greatly affected by Mg concentration, we note that not only was bound by Mg. This fraction was calculated with the apparent T1 independent of PCr concentration as indicated in Table 1 stability constant Kapp measured by Smith and Alberty (23) at but also T1 and the 31P chemical shift were.independent of Mg 25° and 0.2 ionic strength as 20 + 2 M-1. Standard Debye- concentration-in this range. The PCr chemical shift remained Huckel theory considerations indicated that at 40 and ca 0.1 constant at 19.52 h 0.02 ppm from a reference capillary con- ionic strength Kap was unlikely to fall outside these limits. taining 37 mM methylene diphosphonic acid in D20, when the Relying upon the observed similarity of the relaxation behavior PCr concentration was varied from 2-30 mM at 40 in solutions of PCr in intact muscle and our model solutions as well as results containing 13 mM Mg and 0.1 M KCL. Similarly, Mg titrations (24) showing that diffusion through internal field gradients in of 20mM PCr solutions showed that even in the presence of 70 striated muscle had a negligible effect on water proton l/T2, mM Mg, the PCr chemical shift varied from the above value we used the average observed 1/T2 for muscle from Table 3 and by only 0.2 ppm. Experiments using glycerol-3-phosphoryl- the observed relation between l/T2 and FB of Fig. 4 to estimate choline as an internal standard gave similar results. It is repre- FB in muscle as 0.08. Because the free Mg concentration in mM sentative of the independence of T1 of the presence of Mg that is simply 103 FB/[Kapp (1 - FB)], independent of total PCr, the solution containing 6.9 mM Mg and 37 mM PCr described these results indicate that the intracellular free Mg in frog in Table 3 had a T1 of 4.75 sec and the one with 37 mM PCr and gastrocnemius was 4.4 mM. The total Mg content of frog muscle no Mg had a T1 of 4.66 sec. (This also indicates that our Mg was ca 11.3 mM (25), of which 3.1 mM was tied up as MgATP solutions were free of paramagnetic impurities because in that (1, 25). Based on this data combined with the muscle FB, Kapp case Tj would have been greatly affected.) These data are for PCr, and the accepted total PCr content of frog muscle of highly suggestive of relaxation by scalar coupling (19). Thus, 27 mM (26), the following distribution of intracellular con- the total spin-spin relaxation rate is given by 1/T2 total = (1/ stituents was indicated: free Mg, 4.4; MgPCr, 2.2; free PCr, T1)H + 1/T2SC because the same processes contributing to T, 24.8; MgATP, 3.1; and Mg-bound myosin, 1.6 mM. These re- also contribute to T2,-e.g., T1DD = T2DD. sults are in good agreement with calculations based on apparent The scalar-coupling contribution to l/T2 can be substantial binding constants for Mg+2, Ca+2, and K+ interacting with (20) through the term proportional to (27rJ)2S(S + 1)r0 in which ATP, PCr, and myosin in frog muscle, which estimate the free j is the spin-spin coupling between spins S (25Mg) and 31P and Mg level as 3.4 mM (25). (See Note Added in Proof.) As = Tex, the chemical exchange time for Mg binding. The scalar Both proton-coupled and proton-decoupled 13C spectra of coupling is transmitted through a chemical bond that is tran- PCr solutions with and without Mg were recorded at 22.63 sient in this case and Tex is directly proportional (21) to the MHz and 4°. The carbon chemical shifts, independent of the concentration of the Mg-bound PCr in agreement with the presence of Mg, were: 175.2 (C={O), 156.4 (C=N), 54.0 (CH2), observed relation between l/T2 and total Mgconcentration for and 37.3 (CH3) ppm from tetramethylsilane. The C-P coupling a fixed level of PCr given in Table 3. There is also a scalar constant, however, was sensitive to Mg; 12JPNCI was 3.66 Hz coupling of the first kind modulated by chemical exchange with with Mg present and 2.44 Hz without Mg. This change is highly the water solvent; this can be considered to provide a constant suggestive of a conformational change in PCr upon Mg binding contribution when temperature and PCr concentration are held because it is known that 2JPOC can vary with changes in bond constant. In addition, there is the scalar relaxation of the second angle (27). kind (19) in which contribution to 1/T2 is due to the direct coupling between 31P and 14N, for which -r is the nitrogen quadrupolar relaxation time Tq. Because the chemical exchange DISCUSSION of Mg is also in effect an exchange of N between two sites, Tq The present experiments show the usefulness of 31P NMR re- could be sensitive to Mg exchange in a way (22) that would also laxation studies in probing the intracellular environment in Downloaded by guest on September 26, 2021 Biochemistry: Cohen and Burt Proc. Natl. Acad. Sci. USA 74 (1977) 4275

intact muscle. We demonstrate that our muscle preparation 2. Hoult, D. I., Busby, S. J. W., Gadian, D. G., Radda, G. K., Rich- maintains its physiological state throughout long-term experi- ards, R. E. & Seeley, P. J. (1974) Nature 252, 285-287. ments at 4°. Early studies using NMR-e.g., to examine water 3. Henderson, T. O., Costello, A. J. R. & Omachi, A. (1974) Proc. proton relaxation in muscle-often did not measure the met- Nati. Acad. Sci. USA 71, 2487-2490. abolic state of the muscle. That is, neither high-energy phos- 4. Salhany, J. M., Yamane, T., Shulman, R. G. & Ogawa, S. (1975) phates nor mechanical properties were measured. Rapoport and Proc. Nati. Acad. Sci. USA 72,4966-4970. 5. Navon, G., Ogawa, S., Shulman, R. G. & Yamane, T. (1977) Proc.. Bidinger (15) reported that in long-term (>6 hr) aerobic ex- Natl. Acad. Sd. USA 74,87-91. periments, the ionic contents and contractility of frog muscle 6. Navon, G., Ogawa, S., Shulman, R. G. & Yamane, T. (1977) Proc. changed with time. It is almost axiomatic then that during an- Nati. Acad. Sci. USA 74,888-891. aerobic experiments the metabolic state of muscle must be 7. Stephenson, E. W. & Podolsky, R. J. (1977) J. Gen. Physiol. 69, carefully monitored because rigor is progressively setting in 1-16. (28). 8. Brinley, F. J., Jr., Scarpa, A. & Tiffert, T. (1977) J. Physiol. The fact that the spin-lattice relaxation behavior of PCr in (London) 266,545-565. muscle is not unlike that in simple model solutions, both being 9. Bartlett, G. R. (1959) J. Biol. Chem. 234, 466-468. dominated by DD interactions and hence directly related to 10. Jameson, C. J., Jameson, A. K. & Cohen, S. M. (1975) J. Chem. in the same way, is in fair agreement with measure- Phys. 62,4224-4226. viscosity 11. McDonald, G. G. & Leigh, J. S., Jr. (1973) J. Magn. Reson. 9, ments by Kushmerick and Podolsky (29) of ionic mobility in 358-362. skinned muscle fibers, which place an upper limit on the vis- 12. Farrar, T. C. & Becker, E. D. (1971) Pulse and Fourier Trans- cosity of cytoplasm at twice that of pure aqueous solutions. Our form NMR (Academic Press, New York). T2 measurements suggest scalar coupling modulated by 13. Freeman, R. & Hill, H. D. W. (1975) in Dynamic Nuclear chemical exchange with water solvent and Mg. This view is Magnetic Resonance Spectroscopy, eds. Jackman, L. M. & supported by observations of the temperature dependence of Cotton, F. A. (Academic Press, New York), pp. 131-162. T1 and T2 which will be reported elsewhere. Brinley et al. (8) 14. Vold, R. L., Vold, R. R. & Simon, H. E. (1973) J. Magn. Reson. have suggested that because it is now known that free Ca in 11,283-298. muscle is in the range, a recalculation would increase the 15. Rapoport, S. I. & Bidinger, J. M. (1974) Am. J. Physiol. 226, AtM 452-457. Nanninga (25) estimate of 3.4 mM free Mg by ca 0.9 mM, 16. Morgan, W. E. & Van Wazer, J. R. (1975) J. Am. Chem. Soc. 97, bringing it very close to our 4.4 mM value derived from T2 6347-6352. measurements. 17. Canet, D. (1976) J. Magn. Reson. 23,361-364. We are now able to use 31P T2 measurements as a tool for 18. Kuhlmann, K. F., Grant, D. M. & Harris, R. K. (1970) J. Chem. determinations of intracellular free Mg levels with minimum Phys. 52,3439-3448. perturbation of the intact cell. The method is nondestructive 19. Abragam, A. (1961) The Principles of Nuclear Magnetism and should provide a monitor of free Mg during physiological (Oxford Univ. Press, London), chap. VIII. investigations. 20. Sharp, R. R. (1972) J. Chem. Phys. 57,5321-5330. 21. Grunwald, E. & Ralph, K. (1975) in Dynamic Nuclear Magnetic Resonance Spectroscopy, eds. Jackman, L. M. & Cotton, F. A. Note Added in Proof. Recently, Roger. C. Woledge (private com- (Academic Press, New York), pp. 621-647. munication), using titration calorimetry, measured Kapp for Mg binding 22. Marshall, A. G. (1970) J. Chem. Phys. 52, 2527-2534. by PCr as 40 ± 2 M-'. Using this improved Kapp value, we estimated 23. Smith, R. M. & Alberty, R. A. (1956) J. Am. Chem. Soc. 78, FB as 0.109 and the intracellular free Mg as 3.0 mM in frog gas- 2376-2380. trocnemius. 24. Packer, K. J. (1973) J. Magn. Reson. 9, 438-443. 25. Nanninga, L. B. (1961) Biochim. Biophys. Acta 54,338-344. This work was supported in-part by the Muscular Dystrophy Asso- 26. Dawson, J., Gadian, D. G. & Wilkie, D. R. (1977) J. Physiol. ciation and Chicago Heart Association. (London) 267, 703-735. 27. Alderfer, J. L. & Ts'o, P.O.P. (1977) Biophys. J. 17,113a. 28. Bendall, J. R. (1951) J. Physiol. (London) 114,71-88. 1. Burt, C. T., Glonek, T. & Barany, M. (1976) J. Biol. Chem. 251, 29. Kushmerick, M. J. & Podolsky, R. J. (1969) Science 166, 1297- 2584-2591. 1298. Downloaded by guest on September 26, 2021