©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Creatine Kinase Equilibration Follows Solution Thermodynamics in Skeletal Muscle
P NMR STUDIES USING CREATINE ANALOGS (*)

(Received for publication, October 20, 1994; and in revised form, March 3, 1995)

Robert W. Wiseman (1)(§) Martin J. Kushmerick (1) (2) (3)

From the  (1)Departments of Radiology and (2)Physiology and Biophysics and (3)Center for Bioengineering, University of Washington Medical Center, Seattle, Washington 98195

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
APPENDIX
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The hypothesis tested was whether creatine kinase (CK) equilibrates with its substrates and products in the cytosol as if in solution. We used the creatine analogs cyclocreatine (cCr) or beta-guanidopropionate (betaGPA) to test if mass action ratios () for CK in muscle could be predicted from combined equilibrium constants (K) measured in solutions mimicking the intracellular environment. Mice were fed cCr or betaGPA and their muscles assayed for substrates and products of the CK reaction by P NMR spectroscopy and high performance liquid chromatography. After three weeks of feeding, was indistinguishable from K in cCr-treated muscles demonstrating both PCr/Cr and phospho-analog/analog must have equilibrated with a constant and uniform cellular ATP/ADP ratio. In betaGPA-treated muscles, was smaller than K due to a higher content of muscle betaGPA. Feeding betaGPA for 9-12 weeks resulted in a closer agreement between K and , suggesting ATP/ADP ratios are not uniform within the muscle perhaps due to transient metabolic stress in some cells. From this analysis it follows that calculation of free ADP from the CK equilibrium for a heterogeneous population of cells with respect to total Cr and ATP content is correct only if chemical potentials of these cells are uniform.


INTRODUCTION

One of the key tenets of bioenergetics is the near-equilibrium property of creatine kinase (CK) (^1)coupled with cytosolic substrates which are freely mixing and available to the enzyme(1, 2, 3, 4) . Three criteria have been used to demonstrate that a near-equilibrium condition exists within the cytoplasm: (a) kinetic limitations are considered minimal because concentrations of substrates are in the approximate range of their Kvalues(5, 6) ; (b) CK activity is far in excess of maximal ATPase activity within the cell(1) ; (c) P NMR spin transfer methods have shown that forward and reverse fluxes for CK are equal (7) and are far greater than net ATPase activity(8) . Thermodynamic control of PCr/Cr by ATP/ADP through CK equilibration implies that ATP/ADP is the same in all loci within the cell(1, 9, 10) . Alternatively, the cytoplasmic fraction of ATP and ADP must be so large that other ``compartments'' (which may or may not be at the same chemical potential) do not influence physicochemical properties of bulk cytoplasm. For a simple system which is not diffusion limited, it has been shown that this near-equilibrium formulation can be used for calculation of ADP and ATP chemical potential (DeltaG)(11, 12, 13, 14) . For complex populations of cells, comprising tissues or organs, a crucial assumption for proper calculation of ADP and DeltaG (which are derived values from measured parameters (PCr, Cr, ATP, P(i), and pH) on a population of cells) is that not only is ATP/ADP within each cell uniform, but it is uniform between cells as well.

By a variety of criteria, heterogeneity of cell types in individual muscles have been documented(15, 16, 17, 18, 19) , and chemical contents of these cell types have shown that their metabolite contents differ. Furthermore it has been shown that these differences are reflected in chemical potentials of predominantly fast versus slow muscles (-68 to -61 kJ/mol ATP)(20) . In addition to heterogeneity of metabolite distribution, there is also considerable information on the existence, properties, and intracellular distribution of CK isoforms (3, 21, 22) . Although the major isoform is MM-CK (which is thought to be in solution in the cytoplasm as well as bound to the M-line of myofibrils), there is also a substantial amount of ``mitochondrial'' isoforms existing in dynamic multimers in the intermembrane space of mitochondria(3) . Based on the existence of multiple isoforms of CK taken together with heterogeneity of metabolite contents(23, 24) , it has been argued that the simple concept of CK equilibration, with its substrates and products mixing in the bulk cytoplasm as if in solution, is not completely valid.

In view of the large number of physiological and biochemical studies that have assumed a simple behavior of the cytosol (with respect to CK function), we investigated this issue by capitalizing upon the competitive inhibitor nature of two creatine analogs (beta-guanidopropionic acid (betaGPA) and cyclocreatine (cCr)) to establish a criterion for testing CK function in vivo. Application of creatine analogs to study cellular functions of CK was pioneered by Fitch et al.(25, 26, 27) and Walker(28, 29, 30, 31) . Analogs of creatine deplete total Cr over a period of several weeks when fed to rodents and chickens. Both phosphorylated and free forms of analog accumulate in brain, heart, and skeletal muscle(27, 28, 29, 32, 33, 34, 35, 36, 37) . As competitive inhibitors, these compounds can be exploited to answer questions in cellular biology concerning organization of enzymatic activity in cells, because these analogs are also utilized by cells as substrates for cellular energy metabolism(34, 37, 38, 39, 40, 41) . So it is possible to test whether competitive inhibitors and endogenous substrates obey simple rules of enzyme kinetics and equilibration.

Feeding synthetic analogs of creatine (which as competitive inhibitors are not expected to disturb equilibration) partially displaces PCr and Cr from the cell. By choosing duration of feeding such that there were approximately equal concentrations of total creatine and total analog with minimal adaptive changes, (^2)we optimized the ability to measure mass action ratios for CK and to test whether ratios in tissue equaled those predicted from combined equilibrium constants measured in solutions mimicking the intracellular ionic environment. There are a number of reasons why this equality may not hold true: (a) analogs and their phosphorylated forms may not mix and equilibrate with endogenous substrates and products of CK (as would occur in a solution); (b) mixing occurs, but metabolites may be separated in compartments with significantly different ATP/ADP or PCr/Cr ratios; or (c) ATP/ADP ratios between individual muscle cells may not be uniform. Our results show that in steady state and during long-term exposure to these analogs none of these possibilities is true, except for an instructive case of feeding betaGPA for short (3 week) duration, where heterogeneity of ATP/ADP ratios must be considered.


EXPERIMENTAL PROCEDURES

Measurements of Apparent Equilibrium Constants

K for the CK reaction in the presence of its endogenous substrates and a competitive inhibitor with CK in a solution in vitro can be defined as the sum of two separate reactions, each with its own uniquely defined equilibrium constant but coupled by substrates common to both reactions. Our approach was to measure this K in solution and the corresponding mass action ratio in intact muscle as a test of equilibration in vivo. K can be expressed as the ratio of individual equilibrium constants for the combined reaction. This derivation has the advantage of simplifying chemical analyses, since substrates common to both reactions, ADP, ATP (and Mg), as well as pH, factor out of algebraic expressions; its limitations and implications are discussed in detail (see ``Discussion'') and illustrated mathematically (see ``Appendix'').

In our experiments, combined reactions are measured in solution at the temperature of interest with a similar set of measurements then performed on muscles of animals treated with analogs. If the reaction achieved equilibration with its substrates in muscle as in solution, the value for K in solution and the mass action ratio in muscle should agree within experimental error. The following equations specify the approach taken.

For the CK reaction,

consider the reaction for the analog (X) which is phosphorylated at equilibrium (PX),

At pH 7.0, equilibrium expression for and are written as follows.

With adenylates at equilibrium with both analog and creatine species, then we can define a K as follows.

In muscle, when the mixture of substrates and a competitive inhibitor comes to equilibrium with the same adenylate pool, the observed mass action ratio () for the combined reaction will equal K. Unlike solution experiments in which there is no net ATP utilization (ATPase flux) and ATP/ADP does not change, equilibration may not be exact. In muscle, even at rest, there is a small net ATPase activity. In this case, equilibration will only occur if forward and reverse fluxes of CK are far in excess of steady state fluxes of ATP through ATP synthesis and ATPase reactions.

Measurements of Combined Equilibrium Constants in Solution

K for CK in the presence of creatine and either analog was measured using a combination of P NMR spectroscopy and HPLC methods. Solutions were constructed to model cytoplasmic conditions at 23 °C and contained in mM: 100 KCl, 5.3 ATP, 15 PCr, 100 MOPS, 4 EGTA, 1 KH(2)PO(4), 92 KC(2)H(3)O(
2), and 70 Tris at pH 7.0 as is standard for skinned fiber experiments(42) . Aliquots of 3.5 ml each were used for NMR experiments to which were added unphosphorylated forms of either betaGPA or cCr prior to adjusting final volume and pH of each solution. At the start of NMR experiments and before addition of CK, a 50-µl aliquot of this mixture was assayed for ATP content using an optical spectrophotometer at 259 nm. Absorbance due to analogs was negligible at this wavelength. A control NMR spectrum was acquired under fully relaxed conditions, then the phosphorylation reaction was initiated by adding 2 mg/ml of rabbit CK to each tube (260 units of CK activity/ml). Serial spectra were acquired during the time course to equilibration and were halted when change in peak areas for PCr and phospho-analog differed by less than 5% from previous acquisition. At equilibration, samples were removed from the magnet, kept at constant temperature, and CK-denatured by addition of 4% SDS/ml, chilled to 4 °C and centrifuged to remove precipitated SDS and protein(43) . This method was used to avoid acid hydrolysis of PCr which could significantly alter the result. Supernatant samples were frozen at -70 °C for later analysis by HPLC.

Materials

beta-Guanidopropionic acid was synthesized from beta-alanine and cyanamide as described previously(40) . Cyclocreatine was synthesized from chloroacetate and ethylamine as reported by Griffiths and Walker (36) and recrystallized twice from hot water. Product purity and verification of structure were assessed by proton NMR spectroscopy and elemental analysis (Galbraith, Knoxville, TN). HPLC standards for PcCr and betaGPAP were produced from equilibration solutions and calibrated in the following manner. Quantification of PCr was accomplished by integration of P NMR spectra of solutions prior to addition of CK and after equilibration with analog (PCr and PX). As the total integral of this region (PCr + PX) did not change, the amount of PX equals initial less final PCr area. Resonances were scaled to absolute chemical content by using ATP content measured spectrophotometrically at 259 nm. These calibrated solutions provided standards for HPLC quantification of PcCr and betaGPAP. Rabbit creatine kinase, ATP, ADP, PCr, and Cr were obtained from Sigma.

Analog Administration

Male Swiss Webster mice were obtained at age 21 days and allowed to acclimate to the cage for a period of 1 week prior to feeding. Analogs were administered by mixing with standard rodent chow (betaGPA 2% w:w, cCr 1% w:w; BioServ, Frenchtown, NJ). Animals were allowed food and water ad libitum. Feeding experiments were performed for a period of 3 weeks at which time tissues from control, betaGPA, and cCr fed mice were excised from animals under surgical anesthesia (ketamine and xylazine). EDL (fast-twitch) and SOL (slow-twitch) muscles were allowed to recover in a bath of physiologic saline from surgical perturbations for 30 min, after which they were either removed from the bath, blotted, and rapidly frozen at -80 °C with brass Wollenberger tongs or placed in a custom-built P NMR probe for analysis after which these samples were also frozen. In both cases, HPLC analysis of neutralized perchloric acid extracts were performed.

Nuclear Magnetic Resonance Spectroscopy

Phosphorus NMR spectroscopy was performed on a 7T GN 300 (General Electric) using either a 10-mm probe (for model solutions) or a custom-built phosphorus probe for isolated mouse muscles(44) . For solutions, data were acquired with a /2 pulse width (18 µs at 90 watts), 15-s delay, and 4096 data points. Transformed data were the sum of 64 acquisitions which were apodized with a 3-Hz exponential filter prior to the Fourier transform. For muscle samples, data were acquired with a /2 pulse width (6.6 µs at 50 watts), 15 s delay, and 2048 data points. Summed data were the result of either 300-400 data transients apodized with a 15-Hz exponential filter prior to the Fourier transform. The coil for this probe was a six-turn solenoid made from 30-gauge Formvar-coated copper wire, driven by a balance matched tank circuit, serially tuned to the phosphorus frequency. Muscle samples were held in place within a 1.5-mm inner diameter capillary. Magnetic field homogeneity was shimmed on the available proton signal prior to the start of the experiment to a line width of less than 0.07 ppm (for solutions) and 0.1 ppm (for muscle).

Muscles were superfused with phosphate-free saline equilibrated with 95% O(2), 5% CO(2) and contained (mM) 116 NaCl, 4.6 KCl, 26.2 MOPS (titrated to pH 7.4 with NaOH), 2.5 CaCl(2), 1.2 MgSO(4), and gentamycin (10 mg/l) at pH 7.4. Since these preparations gave reproducible P spectra for up to 4 h, we concluded that they were in a metabolic steady state during the 2-h duration of our experiment. Muscles were freeze-clamped after completion of spectral acquisition. Contralateral muscles were prepared similarly and frozen when its mate was put into the probe. Muscles frozen about 30-45 min after dissection rather than after 3 h in the NMR probe showed no differences in metabolite contents.

Two methods of quantification were used for spectral analysis. In solutions where signal-to-noise and spectral resolution were high and base lines strictly flat, spectral peaks were integrated by summing digitized data symmetrically around each peak. Each integral value was expressed as fraction of the total phosphorus integral within the spectrum and reported in absolute chemical content based upon ATP concentration measured by optical spectroscopy as described above. In isolated hindlimb muscles, time domain fitting of free induction decay was used (45) in a commercially available package (FITMASTERS, Philips Medical Systems). As with solution data, integrals were expressed as fractional areas and normalized to chemical content by HPLC-determined ATP content in µmolesbulletgww. ATP content by P NMR spectroscopy was the average of , alpha, and beta ATP resonances.

HPLC Analysis

Chromatographic analysis was performed on stable neutralized perchloric acid extracts as described previously (46) . Tendons were removed from frozen muscle before weighing. Total creatine content was the sum of Cr and PCr; total analog content for each compound was calculated similarly.


RESULTS

In Vitro Combined Equilibrium Constants

P NMR spectroscopy was used to measure ATP, PCr, and PX in each solution during approach to equilibrium which reached completion within the first spectrum for cCr (less than 10 min), yet took approximately 8 h for betaGPA (data not shown). Phosphorylation rates based on time to equilibration after addition of CK were substantially faster for cCr than betaGPA as expected from kinetic constants for these compounds(47) . Fig. 1shows spectra of representative solutions at equilibrium. Note that phosphorylated analog species and PCr are resolved, and ADP was below limits of detection. Summed areas of PCr and phospho-analog resonances in final equilibrium solutions were the same as total PCr peak areas in initial spectra prior to addition of CK, and there was no change in ATP resonance intensities. This stoichiometry demonstrates that the increase in phospho-analog equaled the decrease in PCr. Besides verifying that equilibrium was reached, P NMR experiments provided HPLC standards for phospho-analogs, which were not otherwise available. At the conclusion of each NMR experiment reaction mixtures were analyzed by HPLC for their components of free analog necessary for proper calculation of K; HPLC analysis of phosphorylated species provided a cross-calibration of methods. Both HPLC and P NMR measurements permit separate calculation of a PCr/ATP ratio. Correlation between PCr/ATP ratio for NMR versus PCr/ATP by HPLC were linear with unity slope and intercepts that were not significantly different from zero at a level of p < 0.05. Regression equations were for solutions containing cCr (n = 7) and betaGPA (n = 8), respectively.


Figure 1: P NMR solution spectra for determination of combined equilibrium constants. A depicts a typical spectrum for betaGPA experiments. Chemical shift values were 2.5, -2.54, -2.98, -5.0, -10.0, and -18.5 for P(i), PCr, betaGPAP, and , alpha, and beta resonances of ATP, respectively . B depicts solutions used for cCr. Note that chemical shifts are identical with the exception of PcCr at -2.38 and absence of betaGPAP. Solutions contained (in mM): 100 KCl, 5.3 ATP, 15 PCr, 100 MOPS, 4 EGTA, 1 KH(2)PO(4), 92 KC(2)H(3)O(
2), 70 Tris (pH 7.0), and 1 mg/ml CK. Note chemical shift differences of PCr to betaGPAP (0.44) and PcCr (0.16) and that there is no ADP detected in either solution, indicating that the source of phosphate for each analog is from PCr hydrolysis.



Two such experiments (with least variance) were used to construct HPLC standards for phospho-analog in HPLC assays. These standards are essential for measuring PX concentrations in freeze-clamped muscles and for cCr treated animals where P NMR chemical shift differences are small.

The values in all experiments for K with CK for both analogs are given in Table 1. K values for both analogs with CK agree with previous reports(34, 40, 48) .



Analysis of Mouse Muscles

P NMR Spectroscopy

Representative P NMR spectra of resting EDL muscles from both control and analog-fed animals are shown in Fig. 2. There is an accumulation of phosphorylated synthetic analog within 21 days of feeding as indicated by the chemical shift change in the region of PCr (-2.54 ppm). Chemical shift differences (Delta = 0.44 ppm) between PCr and betaGPAP (-2.98 ppm relative to phosphoric acid) were readily identifiable using 15 Hz apodization with near base-line resolution achieved using only 2 Hz filtering (see inset of panel B). In contrast, chemical shift differences (Delta = 0.16 ppm) between PcCr (-2.38 ppm relative to phosphoric acid) and PCr were much smaller with betaGPA and not resolved as well in vivo due to inherently broader line widths compared with solutions. The inset of Fig. 2C contains the same region reprocessed with 2-Hz exponential filtering and shows PCr as a shoulder upfield from the PcCr resonance.


Figure 2: P NMR spectra of control (A) and betaGPA (B)- and cCr (C)-treated extensor digitorum longus muscles from mouse hindlimb. Data acquisition parameters were as follows: /2 pulse width (6.6 µs), 15-s predelay, 5-KHz sweep width, and 400 acquisitions. Summed data were filtered with a 15-Hz exponential prior to the Fourier transform. Inset of each panel contains an expanded region surrounding the PCr resonance which was processed using 2-Hz line broadening to show chemical shift differences of PCr to betaGPAP (0.44, B) and PCr and PcCr (0.16, C). Peak assignments are as follows: P = inorganic phosphate, PCr = phosphocreatine, betaGPAP = beta-guanidophosphonic acid phosphate, PcCr = phosphocyclocreatine, and , alpha, and beta reson ances of ATP.



Fractional peak areas of EDL and SOL muscles of control and analog-fed mice are presented in Table 2. Control values for PCr, P(i), and ATP are consistent with previous reports using Swiss-Webster (20) and C-57 strains of mice (49, 50) from this laboratory.



Metabolite Quantitation by HPLC

Quantitative measurements of all metabolites of CK reaction were performed using HPLC methods because: (a) creatine and free analog, necessary for calculation of K, are not detected by P NMR, nor are their resonances resolved by in vivo^1H NMR spectroscopy^2; (b) ATP content is needed to scale chemical content in vivo by NMR; and (c) chemical shift differences in PCr and PcCr are small, so HPLC estimates provide important cross comparisons to quantify PcCr concentrations in muscle. Fig. 3presents levels of PCr, Cr, and ATP in neutralized perchloric acid extracts determined by HPLC in control (open bars) and betaGPA (closed bars)- and cCr (left-hatched bars)-treated muscles. Total Cr values for control SOL and EDL were 19.4 ± 1.04 µmolbulletgww and 28.7 ± 1.71 µmolbulletgww and decreased in both treatment groups after 21 days of analog feeding to 28 and 22% of control values for betaGPA SOL and EDL and to 37 and 42% of control for cCr SOL and EDL, respectively. betaGPA-treated mice contained only 29 and 31% of PCr in SOL and EDL, respectively. In cCr fed mice, PCr values were only 32 and 38% of control levels of PCr in SOL and EDL, respectively. The contents of free Cr decreased in both EDL and SOL with both analogs to approximately 50% of control levels. There was also a concurrent decrease in ATP concentrations for each analog treatment, however decreases were larger in betaGPA group (54 and 73% of control for SOL and EDL) than in cCr group (75 and 95% of control for SOL and EDL).


Figure 3: Endogenous substrates for CK (except ADP) as determined by anion (PCr and ATP) and cation (free creatine) exchange HPLC for control (open bars) and betaGPA (closed bars)- and cCr (hatched)-treated EDL and soleus. Data are presented as mean + S.E. with units of µmolesbulletgww.



The contents of phosphorylated and free analog from betaGPA (closed bars) and cCr (left-hatched bars) fed animals are presented in Fig. 4. Accumulation of each phospho-analog reached similar levels in both EDL and SOL during 21 days of feeding while levels of free analog were 11-12-fold lower in cCr-treated muscles than those from mice-administered betaGPA.


Figure 4: Synthetic substrates for CK in betaGPA (closed bars)- and cCr (hatched)-treated EDL and soleus. PcCr and betaGPAP were determined by anion exchange HPLC (left panel), whereas free cCr and betaGPA were determined using a cation exchange column (right panel). In addition to the measured value for free analog, B shows predicted values (cross-hatched) for each analog calculated from solution equilibrium constants and therefore contain no error bars. Data are presented as mean + S.E. with units of µmolesbulletgww.



Cross-calibrations

Integration of areas from P NMR spectra yields in vivo values for metabolite ratios which can be compared with HPLC data obtained from extracts of the same or contralateral muscle from the same animal under stable resting conditions. Therefore, we have independent validation by NMR and HPLC of relative metabolite content with respect to phosphorus containing metabolites. As with solution experiments, the test of quantitative agreement between the two techniques was performed by measuring PCr/ATP in addition to PCr/PX in NMR spectra and HPLC. Results of these comparisons are presented in Fig. 5. Note that compounds had no difference in mean values for either method used. That NMR ratios (closed bars) are indistinguishable from those determined by HPLC (open bars) demonstrates that phosphorus-containing substrates for CK are 100% NMR visible, both under normal resting states and under conditions of partial creatine depletion. Quantitative agreement in stoichiometry with PCr, PX, and ATP by both methods in control and treated muscles (where P(i) is lowered by creatine depletion and incorporated into PX) suggests that, although P(i) was only measured by the NMR method, all P(i) is NMR-visible in these muscles.


Figure 5: Comparison of HPLC (closed bars) and NMR (open bars) ratios of PCr/ATP, PCr/betaGPAP, and PCr/PcCr (presented as PCr/PX). ATP values for NMR were calculated as mean of + alpha + beta ATP resonances. Value s are presented as mean + S.E.



In Vivo Tests of Equilibration

Fig. 6contains results of calculations of K for CK using HPLC analysis of neutralized perchloric acid extracts. The upper panel contains data from cCr-treated mice at 3 weeks of feeding and compares mass action ratios for EDL (cross-hatched), SOL (right-hatched) with K values determined from solution experiments (left-hatched) which were 33.9 ± 2.15, 33.6 ± 1.67, and 34.3 ± 3.26, respectively. The lower panel contains data for betaGPA-treated mice presented in the same order. Values of for EDL and SOL and K in solution are 2.05 ± 0.16, 1.58 ± 0.11, and 3.06 ± 0.31, respectively. Agreement of for EDL and SOL in neutralized perchloric acid extracts with solution K in the instance of cCr-treated muscles shows that these substrates equilibrate in the cytoplasm. This does not rule out existence of smaller local ``pools'' of metabolites but contributions of these pools to total metabolite content would have to be large to influence this result.


Figure 6: Combined equilibrium constants for cCr/Cr and betaGPA/Cr via CK reaction compared for in vitro (solution) and in vivo. Values are presented as mean + S.E. (n = 6-8 for each group). The upper panel represents data from cCr experiments, whereas the lower panel contains data from betaGPA experiments: left- hatched bars = solutions, right-hatched bars = soleus, and cross-hatched bars = EDL. The lower panel contains data from experiments performed at 3 weeks as well as on a longer time course (9-12 week) betaGPA feeding (solid bars) to test for transient changes in tissue . Asterisks were placed over 3-week data to indicate that these values deviate significantly from solution K.



The result in betaGPA-treated muscles suggests there is a lack of equilibration between PCr/Cr and betaGPAP/betaGPA, since for both tissues is considerably lower than solution K values. Because P NMR spectra and HPLC results agree in both analog treatments, the apparent lack of equilibration can only be explained by either low free Cr or high betaGPA values (see ). Fig. 4shows depletion of free Cr occurs to approximately the same extent in EDL and SOL of both analog-treated groups; therefore lack of equilibration may result from the presence of too much betaGPA. This finding leads to the hypothesis that there may be some nonspecific binding of betaGPA in the intact cell which is released during perchloric acid extraction and contributes to HPLC measurement.

To test whether there is any significant binding of betaGPA to subcellular fractions we performed an equilibrium binding experiment using differential centrifugation with a whole muscle homogenate. Rabbit tibialis anterior muscle was homogenized (1:10 w/v) in 125 mM Tris buffer (pH 7.0). After an initial incubation period of 30 min, the homogenate was divided into 4 equal volumes. Either betaGPA or cCr (approximately 40-50 mM) was added to a pair of samples by diluting 1:1 with stock solutions of each analog (also in 125 mM Tris), then incubated for an additional period of 30 min at 25 °C. Differential centrifugations were performed at 600, 5,000, and 13,000 times g, and supernatants and pellets of each fraction were subjected to perchloric acid extraction. The test for binding of analog as compared with Cr was to measure the ratio of synthetic analog to endogenous Cr in each fraction. Neutralized extracts were assayed by cation exchange HPLC for free betaGPA, cCr, and Cr. If some betaGPA binding were sufficient to account for the observed lack of equilibration, then there should have been excess betaGPA in at least one of the fractions, and a different ratio of betaGPA/Cr should have been detected. Cyclocreatine was used as a negative control for this experiment. The results presented in Table 3were decisive: the ratio of free Cr to analog was constant in all fractions tested. On this basis we exclude an unsuspected selective binding of betaGPA as an explanation for apparent disequilibration of betaGPA experiments in animals fed the diet for 3 weeks.



Rats fed betaGPA over longer time periods did not exhibit this apparent lack of equilibration(40) , suggesting that the present results showing excess free betaGPA might be transient, and if measured later in the time course, would not be present. To test this hypothesis, we fed mice over equivalent time periods as in rat studies (9-12 weeks) and performed the same HPLC measurements as in the present study. The results presented in Fig. 6show a marked deviation of mass action ratios from K in muscles from betaGPA animals with several weeks of feeding but with typical feeding regimens used, 9-12 weeks, mass action ratios are statistically indistinguishable from K, indicating that betaGPA effects on are transient.


DISCUSSION

One of the key tenets of bioenergetics of excitable tissues is the near-equilibrium property of CK coupled with the assumption that cytosolic metabolites involved in the reaction are freely mixing and hence available to the enzyme. The existence of multiple isozymes of CK and demonstration of their localization into subcellular compartments, perhaps with their associated substrate pools(23, 24) , has suggested that the concept of freely diffusing metabolites mixing cytoplasm may be too simplistic. In light of the importance of this concept to interpretation of biochemical data, in particular by P NMR spectroscopy, we employed two competitive inhibitors of CK, cCr, and betaGPA to investigate these issues. Feeding synthetic analogs of creatine (which as competitive inhibitors are not expected to disturb equilibration) partially displaced the content of PCr and Cr from the cell. By choosing the duration of feeding such that there were approximately equal concentrations of total Cr and total analog, it was possible to have an accurate measure of mass action ratios for CK in resting muscle and test the hypothesis that these ratios equal those predicted from K measured in solution as would be the case if the reaction is equilibrating and the cytosol is freely mixing with respect to substrates.

In cCr experiments, in muscle was indistinguishable from those predicted by K in solution. Therefore results with this analog could not disprove the central hypothesis of this work. In contrast, in betaGPA experiments (3 weeks of analog feeding), in muscle was clearly different from those predicted by K in solution. However, with longer periods of feeding in muscle were indistinguishable from that predicted by K in solution, as has been shown in other preparations(40) . Thus our results show that muscles in the presence of betaGPA undergo a significant bioenergetic transformation from the onset of analog feeding to the time they demonstrate large phenotype changes in contractile and metabolic proteins(51, 52) . The primary observation of near-equilibration of CK with its natural substrates and exogenous competitive inhibitors is consistent with the idea that cytoplasmic phosphorylation potential displays thermodynamic characteristics of substrates and enzymes freely mixing. Thus, these data establish a solid biochemical basis for the widely used concept of simple solution dynamics for interpretation of P NMR data.

The remaining discussion considers three further issues that show cell to cell differences in ATP/ADP are the only physiologically important factors determining whether observed mass action ratio from tissue equals K measured in solution. This conclusion is derived from the principle that PCr/Cr (and also PX/X) ratios are set by cell ATP/ADP ratios (which are determined by cytosolic chemical potential) acting through CK thereby rendering calculation of independent of total Cr or analog content(1, 9, 10) . By extension, this conclusion can be related to calculation of free ADP in whole tissue where metabolite heterogeneity is present either as a characteristic of phenotype (20) or induced via differential cellular responses to metabolic stress (e.g. fiber recruitment during exercise) or abnormal physiologic states (e.g. localized ischemia).

Quantitative Analysis

Analysis of solution NMR experiments test validity of using both HPLC and NMR methods to assay mass action ratios in muscle. Absolute concentrations for PCr and PX were obtained from NMR by ratio to the mean of ATP resonances, which was measured by optical spectroscopy, yielding mM concentrations of PCr and PX in each solution. For HPLC, both ATP and PCr were determined from enzymatically calibrated standards. PCr/ATP, obtainable from each method, was used as a method of cross-calibration. In solution these ratios of chemical contents are very well correlated for both analogs, since a plot of PCr/ATP has a slope of unity and a zero intercept. Our conclusion from these experiments is that both assays are fully internally consistent and proper concentrations can be derived from both methods for phosphorus metabolites in solution.

To test whether quantitative agreement occurs in muscle between NMR and HPLC of their respective perchloric acid extracts, a similar analysis was performed as in solutions for in vivo data from control and treated muscles. From Fig. 5, PCr/ATP for all muscles are in excellent agreement using P NMR and anion exchange HPLC. Quantitative agreement with NMR and chemical methods illustrates that artifacts of freeze clamping and extraction are absent using our technique irrespective of total Cr content. Calculation of might also be influenced by NMR visibility of PCr and PX. NMR visibility of ATP and P(i) have been examined in other tissues such as heart and liver, particularly under different metabolic states (53, 54, 55, 56) . Our analysis using metabolite ratios allowed unambiguous investigation of this issue in muscle. To maintain agreement with stoichiometry of the chemical results, any NMR ``invisible'' portion of these metabolites would require an equivalent fraction of the other metabolites to be ``invisible'' as well. We illustrate this important point as follows: consider PCr/ATP in control EDL (from Fig. 3) with approximate metabolite contents of 20 µmolebulletgww PCr and 5 µmolbulletgww ATP. If a ``pool'' of ATP or PCr corresponding to 20% of total cellular content was not NMR visible by some mechanism (e.g. rapid T(2) relaxation), this possibility is equivalent to 4 µmol of PCr being invisible but only 1 µmol of ATP. This ``NMR-invisible'' pool would have to be nonstoichiometric (4 PCr/1 ATP) to be consistent with observed ratios and these ``invisible fractions'' would have to be extracted and quantified to the same extent for HPLC and NMR ratios to agree. Additionally, in muscles from treated animals, PCr/PX ratios for both methods also agree. Therefore, any NMR-invisible fraction which exists must influence invisible metabolites in this pool via an identical nonstoichiometric mechanism. We consider this scenario which so affects equal fractions of PCr, PX, and ATP untenable and favor the simpler explanation that phosphorus metabolites involved in the CK reaction, namely PCr, ATP (and in analog-treated muscles PX) must be fully NMR-visible.

betaGPA Equilibration

The results with cCr-treated muscles and quantitative agreement of HPLC and NMR lead to the conclusion that there is thermodynamic control of PCr/Cr and PcCr/cCr through CK in the cytoplasm. These results argue that the apparent lack of equilibration in betaGPA-treated muscles at the 3-week time point cannot be explained by artifacts of the methodology. Furthermore, since PCr and PX values from HPLC are the same as those measured in vivo, lack of equilibration observed in 3-week betaGPA feeding must be explained in terms of either free Cr or free betaGPA content. One possible explanation for apparent lack of equilibration of betaGPA with CK might be that a fraction of free betaGPA, which is present in muscle but bound to an unknown location thus unavailable for reaction, is subsequently released during perchloric acid extraction. This notion is similar to binding of ADP to actin. The key tenet of this hypothesis is that CK actually equilibrates with both betaGPA and betaGPAP and any bound and unbound pools have equilibrated as well, but extraction of the muscles with perchloric acid extraction releases bound metabolite which is then assayed by HPLC. Predicted values for unphosphorylated analog are presented in Fig. 4for both analogs (cross-hatched bars) as well as HPLC measured values for betaGPA (closed bars) and cCr (right-hatched bars). The difference between predicted value and that measured depicts the amount that must be bound (by hypothesis) and subsequently released during extraction to account for disequilibration in betaGPA treated muscles. This putative bound fraction, calculated from the combined equilibrium constants for each analog (assuming the dependent variable is the unphosphorylated analog) is 50-100% less than what was actually measured in perchloric acid extracts of EDL and SOL by HPLC. Thus, the prediction from equilibrium binding experiments is that if betaGPA had some binding affinity for a subcellular component, a different ratio of Cr/X should have been detected in at least one of the fractions tested. Results of equilibrium binding experiments (see Table 3) demonstrate that Cr/X is constant regardless of the fraction sampled. The 600 times g pellet of intact cells and tissue fragments contained only 10% of the Cr and analog. The remainder was found in the supernatant of each differential centrifugation, demonstrating that these compounds behave similarly and are primarily found in solution. Finally, if nonspecific binding were occurring, animals subjected to longer feeding time courses (possessing higher total analog concentrations) would also exhibit an apparent disequilibration with betaGPA, perhaps to a greater extent. But betaGPA fed animals on longer feeding regimes have tissue mass action ratios which are not distinguishable from solution K values at 3 weeks. One would expect the opposite result if binding were a plausible explanation. We therefore exclude nonselective binding of betaGPA as the explanation for the apparent disequilibration after 3 weeks of feeding.

The second possibility to explain lack of equilibration is based upon enzyme kinetics and suggests that CK fluxes under conditions of our muscle experiments may not be sufficient to maintain equilibrium in resting muscle in vivo in the presence of competitive inhibitors during 3 weeks of feeding or following metabolic perturbations during tissue handling in the experiment. Arguments against this hypothesis are derived from in vivo muscle experiments from other investigators. P NMR spectroscopy performed on intact hindlimb of betaGPA-treated rats have shown that betaGPAP levels decrease 20% (approximately 5 mM) during acute exercise in rat hindlimb muscles and return to initial levels within 13 min(40) . This translates into a phosphorylation rate of 0.37 mM/min for rat fast-twitch muscles. During the course of our NMR experiments (3 h), approximately 60 mM of betaGPAP could have been produced. This flux is far in excess of what is required to meet equilibrium conditions assuming rat and mouse fast-twitch muscles have similar CK activities. Thus a kinetic limitation under resting conditions cannot explain the observations for betaGPA-treated muscles.

Heterogeneity of Metabolite Distribution

In solution experiments, equilibration of CK with a uniform ATP and ADP concentration is certain even in the presence of competitive inhibitors (see and ). In intact tissues this may not be completely valid because of heterogeneity. There are two possibilities where PCr/Cr and PX/X might equilibrate with different ATP/ADP ratios in intact muscle: (a) either ATP/ADP in some subcellular location of some or all cells is different than bulk cytoplasm or (b) a subpopulation of cells within the tissue might be at a different cytosolic ATP/ADP ratio than the rest. These alternatives reflect intracellularversusintercellular compartments. Creatine kinase localization within the cell, including mitochondrial isoforms, have been reported (3) , but their existence does not de facto demonstrate that associated substrates are segregated from the cytosol as well. Heterogeneity of chemical contents within muscle fiber types has been reported previously(20) , raising the possibility that apparent lack of equilibration with betaGPA is due to intercellular heterogeneity of ATP/ADP ratios, perhaps induced by exposure to analog itself.

What aspects of metabolite distribution could explain the observed disequilibration with betaGPA at 3 weeks? From our analysis based upon an expansion of from the ``Experimental Procedures'' (see in the ``Appendix''), lack of agreement for solution K values with from muscles of betaGPA-treated animals cannot be explained by varying distributions of Cr or its analogs. In fact, observed disequilibration can only be explained if ATP/ADP ratios are no longer constrained to be equal (e.g.fPCr fPCr, Appendix, and ) between volume fractions. Although adenylates do not directly enter into any of these calculations, under equilibrium conditions, fractional PCr content is determined by ATP/ADP through CK (9, 10) and therefore ultimately sets PX/X ratio as well. Thus, if ATP/ADP, or equivalently fPCr, in 1 volume fraction is significantly different from the other then (s) no longer equals K. This is possible while physicochemical constraints for thermodynamic equilibrium are still met in each volume fraction comprising the sample. This analysis demonstrates that apparent lack of equilibration observed is a physiologic phenomenon resulting from different fractional PCr contents (hence different ATP/ADP ratios) in each volume fraction. Thus measurements from whole muscle may appear to violate CK equilibration, i.e. (s) does not equal K, despite the fact that each volume fraction is in equilibrium as defined by and in the ``Appendix.'' We favor the hypothesis that betaGPA is selectively effecting a set of muscle fibers in a cell-specific manner, and the nature of this perturbation is related to metabolism of Cr ultimately altering cellular energetics. This fiber-specific effect may in part explain adaptive changes in Cr analog administration in murine muscle(51, 52, 57) .

Results with cCr and previously established equilibration of CK with betaGPA in other preparations exposed to long duration feeding (confirmed here in mouse hindlimb muscles) are highly significant for describing and understanding functional organization of CK in muscle cells. That we observed muscle to be indistinguishable from solution K indicates that these major bioenergetic reactions function in cells of resting muscles as they do in homogeneous solutions in vitro, despite the presence of mitochondrial and cytoplasmic isoforms, their partial binding to macromolecular structures, and compartmentalization of the cell volume (sarcoplasmic reticulum, mitochondria, etc.). Corollaries of this conclusion, supported by other studies of CK function, are several: (a) calculations of metabolically active ADP concentration from CK equilibration are valid; (b) concentrations of PCr, phospho-analog, and ATP measured by P NMR are equal to those measured in perchloric acid extracts, thus there is no evidence for NMR-invisible pools of these metabolites; (c) the net quantitative contribution of metabolites in diffusion-limited compartments to total measured quantities is small; (d) effects of such compartmentalization, however important for certain functions, are negligible with respect to total cellular bioenergetics and metabolism. Thus our experiments unambiguously demonstrate that thermodynamic characteristics of the cytosol can be predicted as if these metabolites were freely mixing in solution. Finally, if there is an apparent lack of equilibration due to two compartments in the muscle with differing ATP/ADP ratios, bioenergetics can still be solved by knowledge of either ATP/ADP (or fPCr) in one of the two components.


APPENDIX

The following equations illustrate the effects of analog, Cr, and adenylate distribution within the sample on calculation of . From expansion of in ``Experimental Procedures,'' consider 2 volumes which comprise the entire sample. This expression can be expanded to include mass action ratios for more than 2 fractional volumes. Contribution of each volume fraction to the total mass action ratio can be written as follows,

where (s) is the mass action ratio for the total sample volume (V(s)), alpha is the first fractional volume, and beta is the second fractional volume and these are constrained to be alpha + beta = V(s). Mass action ratios for each fractional volume ( and ) can be written as follows.

Case 1: Distribution of Total Analog

From the total analog content for each fractional volume (TX), content of PX and X can be expressed in terms of PCr and Cr content as follows.

If we constrain each fractional volume to its thermodynamic equilibrium by setting the solution combined equilibrium constant K = = , we see that for any analog concentration (TX), the PX/X ratio is now entirely defined by the only independent variables in and , namely PCr and Cr. Under these circumstances the PX/X ratio reflects the concentrations of PCr and Cr in each volume fraction independent of total analog content. More importantly, one can also conclude that as long as concentrations of PCr and Cr are equal in both fractional volumes then K = (s). These conclusions are independent of total analog present or its distribution between volume fractions.

Case 2: Distribution of Total Creatine

Independent variables from and can be expressed as fractional PCr (fPCr) content with respect to total Cr present as follows.

As in Case 1, it follows from these equations that as long as fPCr for both volume fractions is identical then = , inde pendent of total Cr content of either volume fraction or distributions. Thus K remains equal to (s).

Case 3: Distribution of Adenylates

If adenylates are in equilibrium PCr and Cr within each volume fraction, then we write,

where K and K are equilibrium constants for CK for each fractional volume . Thus only if ATP/ADP ratios for both frac-tional volumes are equal will PCr/Cr and PX/X ratios agree and thus K = .


FOOTNOTES

*
This work was supported by National Institutes of Health Grants F32 AR08105 and R29 AR41793 (to R. W. W.) and AR36281 (to M. J. K.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dept. of Radiology, SB-05, University of Washington Medical Center, Seattle, WA 98195. Tel.: 206-685-1262; Fax: 206-543-3495; rwiseman{at}u.washington.edu(emb).

(^1)
The abbreviations used are: CK, creatine kinase (EC 2.7.3.2); Cr, creatine; PCr, phosphocreatine; cCr, cyclocreatine; betaGPA, beta-guanidopropionate; X, creatine analog; PX, phosphorylated analog; HPLC, high performance liquid chromatography; MOPS, 4-morpholinepropanesulfonic acid; EDL, extensor digitorum longus; SOL, soleus.

(^2)
R. W. Wiseman, unpublished observations.


ACKNOWLEDGEMENTS

We gratefully acknowledge the technical assistance of Rudolph Stuppard for HPLC analysis of tissue extracts and Dr. Thomas W. Beck for assistance in solution NMR experiments and HPLC. Many thanks to Drs. P. Bryant Chase, W. Ross Ellington, and Jeroen Jeneson for critical reading of the manuscript and providing thoughtful comments.


REFERENCES

  1. Meyer, R. A., Sweeney, H. L., and Kushmerick, M. J. (1984) Am. J. Physiol. 246, C365-C377
  2. Kushmerick, M. J. (1985) J. Exp. Biol. 115, 165-177 [Abstract]
  3. Wallimann, T., Wyss, M., Brdiczka, D., Nicolay, K., and Eppenberger, H. M. (1992) Biochem. J. 281, 21-40 [Medline] [Order article via Infotrieve]
  4. Watts, D. C. (1973) in The Enzymes (Boyer, P. D., ed) 3rd. Ed., pp. 383-453, Academic Press, New York
  5. Kuby, S. A., Noda, L., and Lardy, H. A. (1954) J. Biol. Chem. 210, 65-82 [Free Full Text]
  6. Noda, L., Kuby, S. A., and Lardy, H. A. (1954) J. Biol. Chem. 210, 83-95 [Free Full Text]
  7. Ugurbil, K. (1985) J. Magn. Reson. 64, 207-219
  8. McFarland, E. W., Kushmerick, M. J., and Moerland, T. S. (1994) Biophys. J. 67, 1-13
  9. Connett, R. J. (1988) Am. J. Physiol. 254, R949-R959
  10. Meyer, R. A. (1988) Am. J. Physiol. 254, C548-C553
  11. Veech, R. L., Lawson, J. W. R., Cornell, N. W., and Krebs, H. A. (1979) J. Biol. Chem. 254, 6538-6547 [Abstract]
  12. Lawson, J. W. R., and Veech, R. L. (1979) J. Biol. Chem. 254, 6528-6537 [Abstract]
  13. Alberty, R. A. (1969) J. Biol. Chem. 244, 3290-3302 [Abstract/Free Full Text]
  14. George, P., and Rutman, R. J. (1960) Prog. Biophys. 10, 1-53
  15. Burke, R. E., Levine, D. N., and Zajac, F. E. I. (1971) Science 174, 709-712 [Medline] [Order article via Infotrieve]
  16. Meyer, R. A., Brown, T. R., and Kushmerick, M. J. (1984) Am. J. Physiol. 248, C279-C287
  17. Hintz, C. S., Chi, M. M. Y., Fell, R. D., Ivy, J. L., Kaiser, K. K., Lowry, C. V., and Lowry, O. H. (1982) Am. J. Physiol. 242, C218-C228
  18. Achten, E., VanCauteren, M., Willem, R., Luypaert, R., Malaisse, W. J., VanBosch, G., Delanghe, G., DeMeirleir, K., and Osteaux, M. (1990) J. Appl. Physiol. 68, 644-649 [Abstract/Free Full Text]
  19. Nemeth, P. A., Cope, T. C., Kushner, S., and Nemeth, P. M. (1993) Am. J. Physiol. 264 (33), C411-C418
  20. Kushmerick, M. J., Moerland, T. S., and Wiseman, R. W. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 7521-7525 [Abstract]
  21. Yamashita, K., and Yoshioka, T. (1991) J. Muscle Res. Cell Motil. 12, 37-44 [Medline] [Order article via Infotrieve]
  22. Friedman, D. L., and Perryman, M. B. (1991) J. Biol. Chem. 266, 22404-22410 [Abstract/Free Full Text]
  23. Gellerich, F. N. (1992) FEBS Lett. 297, 55-8 [CrossRef][Medline] [Order article via Infotrieve]
  24. Saks, V. A., Kuznetsov, A. V., Kupriyanov, V. V., Miceli, M. V., and Jacobus, W. E. (1985) J. Biol. Chem. 260, 7757-7764 [Abstract/Free Full Text]
  25. Fitch, C. D., Shields, R. P., Payne, W. F., and Dacus, J. M. (1968) J. Biol. Chem. 243, 2024-2027 [Abstract/Free Full Text]
  26. Fitch, C. D., Jellinek, M., and Mueller, E. J. (1974) J. Biol. Chem. 249, 1060-1063 [Abstract/Free Full Text]
  27. Fitch, C. D., Jellinek, M., Fitts, R. H., Baldwin, K. M., and Holloszy, J. O. (1975) Am. J. Physiol. 228, 1123-1125 [Medline] [Order article via Infotrieve]
  28. Annesley, T. M., and Walker, J. B. (1980) J. Biol. Chem. 255, 3924-3930 [Free Full Text]
  29. Roberts, J. J., and Walker, J. B. (1982) Am. J. Physiol. 243, H911-H916
  30. Turner, D. M., and Walker, J. B. (1985) Arch. Biochim. Biophys. 238, 642-651 [Medline] [Order article via Infotrieve]
  31. Turner, D. M., and Walker, J. B. (1987) J. Biol. Chem. 262, 6605-6609 [Abstract/Free Full Text]
  32. Chevli, R., and Fitch, C. D. (1979) Biochem. Med. 21, 162-167 [Medline] [Order article via Infotrieve]
  33. Mainwood, G. W., Alward, M., and Eiselt, B. (1982) Can. J. Physiol. Pharmacol. 60, 120-127 [Medline] [Order article via Infotrieve]
  34. Annesley, T. M., and Walker, J. B. (1977) Biochem. Biophys. Res. Commun. 74, 185-190 [Medline] [Order article via Infotrieve]
  35. Annesley, T. M., and Walker, J. B. (1978) J. Biol. Chem. 253, 8120-8125 [Medline] [Order article via Infotrieve]
  36. Griffiths, G. R., and Walker, J. B. (1976) J. Biol. Chem. 251, 2049-2054 [Abstract]
  37. Woznicki, D. T., and Walker, J. B. (1980) J. Neurochem. 34, 1247-1253 [Medline] [Order article via Infotrieve]
  38. Shoubridge, E. A., and Radda, G. K. (1987) Am. J. Physiol. 252, C532-C542
  39. Shoubridge, E. A., and Radda, G. K. (1984) Biochim. Biophys. Acta 805, 79-88 [CrossRef][Medline] [Order article via Infotrieve]
  40. Meyer, R. A., Brown, T. R., Krilowicz, B. L., and Kushmerick, M. J. (1986) Am. J. Physiol. 250, C264-C274
  41. Meyer, R. A. (1989) Am. J. Physiol. 257, C1149-C1157
  42. Chase, P. B., and Kushmerick, M. J. (1988) Biophys. J. 53, 935-946 [Abstract]
  43. Chase, P. B., and Kushmerick, M. J. (1995) Am. J. Physiol. 268, C480-C489
  44. Wiseman, R. W., Moerland, T. S., and Kushmerick, M. J. (1993) NMR Biomed. 6, 153-156 [Medline] [Order article via Infotrieve]
  45. de Beer, R., and van Ormondt, D. (1992) NMR Basic Principles and Progress 26, 202-248
  46. Wiseman, R. W., Moerland, T. S., Chase, P. B., Stuppard, R., and Kushmerick, M. J. (1992) Anal. Biochem. 204, 383-389 [Medline] [Order article via Infotrieve]
  47. Rowley, G. L., Greenleaf, A. L., and Kenyon, G. L. (1971) J. Am. Chem. Soc. 93, 5542-5551 [Medline] [Order article via Infotrieve]
  48. LoPresti, P., and Cohn, M. (1989) Biochim. Biophys. Acta 998, 317-320 [Medline] [Order article via Infotrieve]
  49. Phillips, S. K., Wiseman, R. W., Kushmerick, M. J., and Woledge, R. C. (1993) J. Physiol. (Lond.) 462, 135-146 [Abstract]
  50. Phillips, S. K., Wiseman, R. W., Woledge, R. C., and Kushmerick, M. J. (1993) J. Physiol. (Lond.) 463, 157-167 [Abstract]
  51. Shoubridge, E. A., Challiss, R. A. J., Hayes, D. J., and Radda, G. K. (1985) Biochem. J. 232, 125-131 [Medline] [Order article via Infotrieve]
  52. Moerland, T. S., Wolf, N. G., and Kushmerick, M. J. (1989) Am. J. Physiol. 257, C810-C816
  53. Hutson, S. M., Berkich, D., Williams, G. D., LaNoue, K. F., and Briggs, R. W. (1989) Biochemistry 28, 4325-4332 [Medline] [Order article via Infotrieve]
  54. Murphy, E., Gabel, S. A., Funk, A., and London, R. E. (1988) Biochemistry 27, 526-528 [Medline] [Order article via Infotrieve]
  55. Humphrey, S. M., and Garlick, P. B. (1991) Am. J. Physiol. 260, H6-H12
  56. Garlick, P. B., and Townsend, R. M. (1992) Am. J. Physiol. 263, H497-H502
  57. Wiseman, R., and Kushmerick, M. (1994) FASEB J. 8, A10

©1995 by The American Society for Biochemistry and Molecular Biology, Inc.