31P saturation transfer spectroscopy predicts differential intracellular macromolecular association of ATP and ADP in skeletal muscle

The kinetics of phosphoryl exchange involving ATP and ADP have been investigated successfully by in vivo ³¹P magnetic resonance spectroscopy using magnetization transfer. However, magnetization transfer effects seen on the signals of ATP also could arise from intramolecular cross-relaxation. This relaxation process carries information on the association state of ATP in the cell. To disentangle contributions of chemical exchange and cross-relaxation to magnetization transfer effects seen in ³¹P magnetic resonance spectroscopy of skeletal muscle, we performed saturation transfer experiments on wild type and double-mutant mice lacking the cytosolic muscle creatine kinase and adenylate kinase isoforms. We find that cross-relaxation, observed as nuclear Overhauser effects (NOEs), is responsible for magnetization transfer between ATP phosphates both in wild type and in mutant mice. Analysis of ³¹P relaxation properties identifies these effects as transferred NOEs, i.e. underlying this process is an exchange between free cellular ATP and ATP bound to slowly rotating macromolecules. This explains the β-ATP signal decrease upon saturation of the γ-ATP resonance. Although this usually is attributed to β-ADP ↔ β-ATP phosphoryl exchange, we did not detect an effect of this exchange on the β-ATP signal as expected for free [ADP], derived from the creatine kinase equilibrium reaction. This indicates that in resting muscle, conditions prevail that prevent saturation of β-ADP spins and puts into question the derivation of free [ADP] from the creatine kinase equilibrium. We present a model, matching the experimental result, for ADP ↔ ATP exchange, in which ADP is only transiently present in the cytosol.     

Analysis of compartmentation of ATP in skeletal and cardiac muscle using 31P nuclear magnetic resonance saturation transfer.

We have developed a model for the analysis of the forward creatine kinase reaction in muscle as measured by the nuclear magnetic resonance (NMR) technique of magnetization transfer. The model, accounting for the double-exponential behavior observed in some NMR magnetization transfer data, allows for the existence of two ATP pools, one that is NMR-visible (NMR-VIS) and another that is NMR-invisible (NMR-INVIS). We have applied the model to experimental data for the forward creatine kinase reaction in skeletal and cardiac muscles to study the dependence of the creatine kinase rate constants and fluxes on workload and to account for the differences between heart and skeletal muscle. The results suggest that an NMR-distinct ATP pool exists in both heart and skeletal muscles, and that phosphate exchange with this pool catalyzed by creatine kinase increases with increased workload. The results also agree with previously published estimates of the rates of mitochondrial translocase and net ATP synthesis obtained by traditional biochemical methods.     

31P NMR saturation transfer measurements of phosphorus exchange reactions in rat heart and kidney in situ

31P NMR spectra of rat kidney and heart, in situ, were obtained at 97.2 MHz by using chronically implanted radio-frequency coils. Previous investigators have used magnetization transfer techniques to study phosphorus exchange in perfused kidney and heart. In the current experiments, saturation transfer techniques were used to measure the steady-state rate of exchange between inorganic phosphate (Pi) and the gamma-phosphate of ATP (gamma ATP) in kidney, and between phosphocreatine (PCr) and gamma ATP, catalyzed by creatine kinase, in heart. The rate constant for the exchange detected between Pi and gamma ATP in kidney, presumably catalyzed by oxidative phosphorylation, was 0.12 +/- 0.03 s-1. This corresponds to an ATP synthesis rate of 12 mumol min-1 (g wet weight)-1. Comparison of previously published O2 consumption and Na+ reabsorption rates for the intact kidney with the NMR-derived rate for ATP synthesis gave flux ratios of JATP/JO2 = 1.6-3.3 and JNa+/JATP = 4-10. The rate constants for the creatine kinase reaction, assuming a simple two-site exchange, were found to be 0.57 +/- 0.12 s-1 for the forward direction (PCr----ATP) and 0.50 +/- 0.16 s-1 for the reverse direction (ATP----PCr). The forward rate (0.78 +/- 0.18 intensity unit/s) was significantly larger (p less than 0.05) than the reverse rate (0.50 +/- 0.16 intensity unit/s). This difference between the forward and reverse rates of creatine kinase has been previously noted in the perfused heart. The difference has been attributed to participation of ATP in other reactions.(ABSTRACT TRUNCATED AT 250 WORDS)     

ATP-phosphocreatine metabolism catalyzed by creatine kinase. Comparison of saturation transfer (NMR) and isotope labeling technics

Unidirectional fluxes from ATP to phosphocreatine (PCr) catalyzed by MM-isoenzyme of creatine kinase (CK) were measured by using 31P-NMR saturation transfer technique and by means of radioactively labeled [gamma-32P]ATP. At 30-37 degrees C and pH 7.4 in a wide range of [PCr]/[creatine] ([PCr]/[Cr]) ratios (0.2 to 3.0) both of these methods gave similar results, thus showing that magnetization (saturation) transfer allows to determine fluxes close to real ones under "physiological" conditions. However, at [PCr]/[Cr] ratio higher than 5 ([ADP] less than 30 microM) or at decreased temperatures (7-15 degrees C, [PCr]/[Cr] approximately 1) fluxes determined by saturation transfer substantially exceeded those measured with the radioactive label. These data imply that under "physiological" conditions phosphoryl group transfer is actually rate-determining step of the CK reaction. On the contrary, at high [PCr]/[Cr] values or at low temperature the control step could be shifted from the phosphoryl group transfer or distributed among other steps of the reaction.     

Reaction rates of creatine kinase and ATP synthesis in the isolated rat heart. A 31P NMR magnetization transfer study

The NMR technique of magnetization transfer can be used to define intracellular reaction kinetics. In order to determine the relationship between ATP synthesis and flux through the creatine kinase reaction in the intact heart, we used this technique to measure flux through the creatine kinase reaction in the isolated, isovolumic rat heart at five levels of cardiac performance and oxygen consumption. The unidirectional reaction rate constants (s-1) calculated from a two-site exchange model for both the forward and reverse creatine kinase reactions increased with cardiac performance and oxygen consumption. As the rate-pressure product varied from 0 to 44.7 X 10(3) mm Hg/min and oxygen consumption rose from 5.9 to 45.8 mumol of O2/g dry weight/min, kforward increased from 0.27 to 1.30 and kreverse increased from 0.31 to 1.14. The relationship between creatine kinase flux and oxygen consumption, and thus ATP synthesis, took the form of the Michaelis-Menten equation. Rates of ATP synthesis estimated from magnetization transfer were similar to values calculated from oxygen consumption. The longitudinal relaxation time of creatine phosphate (2.06 s), the gamma-phosphorus atom of ATP (0.75 s), and inorganic phosphate (0.81 s) did not change with cardiac performance. These results show that myocardial energy transfer via the creatine kinase reaction is closely coupled to energy production.     

31P NMR measurements of myocardial pH in vivo

A 31P NMR magnetization transfer method for measuring myocardial pH in vivo is demonstrated in the lamb, dog and cat. The method involves measuring the difference in chemical shift between the resonances of phosphocreatine and inorganic phosphate in magnetization transfer difference spectra in which the gamma-phosphate resonance of ATP has been saturated. The method has been verified by measuring the chemical shift difference between the resonances of 2-deoxyglucose 6-phosphate and phosphocreatine following infusion of the animals with 2-deoxyglucose. The measured pH values are significantly lower than those obtained in previous studies on the heart in vivo.     

Creatine kinase-catalyzed ATP-phosphocreatine exchange: comparison of 31P-NMR saturation transfer technique and radioisotope tracer methods

Unidirectional fluxes from ATP to phosphocreatine, catalyzed by the MM isoenzyme of creatine kinase, were measured by both the 31P-NMR saturation transfer technique and radioisotope tracer ([gamma-32P]ATP) method. It was found that at 30-37 degrees C and pH 7.4, over a wide range of [phosphocreatine]/[creatine] (from 0.2 to 5.0) ratios, both methods gave the same results, showing that magnetization transfer allows determination of real fluxes under 'physiological' conditions. However, at [PCr]/[Cr] ratios higher than 5 ([ADP]free less than 30 microM) or at lower temperatures (t less than 15 degrees C, [PCr]/[Cr] approximately 1), the fluxes assessed by saturation transfer were somewhat faster than those detected by the radioisotope tracer method. These data imply that under physiological conditions phosphoryl group transfer is actually the rate-determining step of the creatine kinase reaction. In contrast, at high [PCr]/[Cr] ratios or at lower temperatures, control may be shifted from phosphoryl group transfer or distributed among other steps of the reaction.     

Direct activation of purified protein kinase C by unsaturated fatty acids (oleate and arachidonate) in the absence of phospholipids and Ca2+

31P saturation transfer techniques have been used to measure phosphate kinetics in the yeast Saccharomyces cerevisiae. The phosphate consumption rate observed in acetate grown mid-log cells was combined with measurements of O2 consumption to yield P/O ratios of 2.2 and 2.9, for cells respiring on glucose and ethanol, respectively. However, no phosphate consumption activity was observed in saturation transfer experiments on anaerobic glucose fed cells. The phosphate consumption rates measured by saturation transfer in cells respiring on glucose and ethanol was attributed to the unidirectional rates of mitochondrial ATP synthesis.     

Nucleosidediphosphate kinase of Escherichia coli, a periplasmic enzyme.

The ATP-ADP exchange activity previously described in a membrane farction of Escherichia coli appeared after a cold osmotic shock according to Neu and Heppel ((1965) J. Biol. Chem. 240, 3685--3692) in the shock fluid. Membranes derived from shocked cells had no activity. The enzyme responsible for this activity has been purified 125-fold and catalyzed the transfer of a phosphoryl radical from ribonucleosidetriphosphates (NTPs) to ribonucleosidediphosphates (NDPs); this is, therefore, a non-specific nucleosidediphosphate kinase (ATP:nucleosidediphosphate phosphotransferase, EC 2.7.4.6). The activity required the presence of a divalent cation, Mg2+, Mn2+ or Ca2+ at a unity mol/mol ratio of nucleotide for maximal activation. The enzyme exhibited simple saturation kinetics with respect to the phosphate donor but inhibition by excess substrate was observed upon increasing phosphate acceptor. The kinetics of the reaction indicated an ordered bi-molecular ping-pong reaction mechanism. Differential heat sensitivity of the enzyme whether it is heated alone with ATP, ADP or Mg2+ opens possibilities to study different enzyme-substrate complexes.     

Computer simulation of the P31 NMR equations governing the creatine kinase reaction

A method for solving the Bloch equations that govern magnetization transfer NMR experiments is presented. It requires the numerical evaluation of a matrix exponential and lends itself to computer simulation. It is a simple, versatile method for evaluating P31-NMR magnetization transfer experiments designed to measure biochemical exchange rates. We apply the method to the saturation and inversion transfer experiments and find that the "initial slope" method of determining flux is subject to at least two interpretations. This verifies the accepted concept that biochemical models representing compartmentation and competing reaction hypotheses cannot be reliably distinguished by simply selecting values for NMR and biochemical parameters that give a "best fit" to experimental data. However, our results do indicate that a controlled manipulation of biochemical exchange rate may distinguish between these two models.     

1H- and 31P-NMR studies on smooth muscle of bullfrog stomach

³¹P-NMR spectra of bullfrog stomach smooth muscle showed peaks for creatine phosphate (4.8 μmol·g⁻¹ wet wt.), ATP (3.6), inorganic phosphate (P_i, 2.4), phosphomonoesters (3.0) and phosphodiesters (3.3). The intracellular pH was 7.3, and calculated from the chemical shift of P_i. ¹H-NMR spectra of smooth muscle yielded peaks of 2.9 for lactate, 6.6 for total creatine (creatine phosphate + creatine) and methyl protons of choline tentatively assigned to glycerolphosphorylcholine or to membrane phospholipids. Creatine phosphate and ATP decreased under anaerobic conditions, and intracellular acidification was observed with the concomitant increase in lactate. ³¹P saturation transfer studies showed that saturation of the γ-ATP resonance reduced the intensity of creatine phosphate to 60% of its control value, and the measured T₁ value of creatine phosphate was 2.4 s with saturation. The calculated forward flux of the creatine kinase reaction (decomposition direction of creatine phosphate) was 0.77 μmol·g⁻¹ wet wt.·s⁻¹. The creatine kinase flux was approx. 100-times larger than the ATP turnover rate, calculated from the oxygen consumption rate with the assumption, P/O = 3. In conclusion, the creatine kinase reaction is at equilibrium in resting smooth muscle of bullfrog stomach.     

Carbamyl phosphate-dependent ATP synthesis catalyzed by formyltetrahydrofolate synthetase.

Formyltetrahydrofolate synthetase (formate:tetrahydrofolate ligase (ADP-forming), EC 6.3.4.3) from Clostridium cylindrosporum catalyzes phosphate transfer from carbamyl phosphate to ADP. This activity is lost when monovalent cations are removed and is recovered when K+ is added back. Carbamyl phosphate is an inhibitor of the formyltetrahydrolfolate synthetase forward reaction, and formate as well as phosphate inhibit the ATP synthesis reaction. Acetyl phosphate and phosphonoacetate are inhibitors of both reactions. The results of kinetic studies support the concept that carbamyl phosphate is an analog of the putative intermediate of the formyltetrahydrofolate synthetase reaction, formyl phosphate.     

Measurement of an individual rate constant in the presence of multiple exchanges: application to myocardial creatine kinase reaction

Forward [creatine phosphate (CP)----adenosine 5'-triphosphate (ATP)] and reverse (ATP----CP) fluxes of myocardial creatine kinase (CK) measured by using 31P nuclear magnetic resonance (NMR) and conventional saturation transfer (CST) methods are unequal; this is a paradoxical result because during steady state fluxes into and out of the CP pool must be the same. These measurements, however, treat the CK reaction as a two-site exchange problem and ignore the presence of the ATP gamma in equilibrium Pi exchange involving the ATPases. We have applied a method [U?urbil, K. (1985) J. Magn. Reson. 64, 207] based on the saturation of multiple resonances, by which a single unidirectional rate constant can be measured unequivocally in the presence of multiple exchanges, to the measurement of CK fluxes in isovolumic rat hearts perfused under three different conditions; two of the three perfusion conditions showed a large discrepancy in the CK fluxes determined by CST, and one did not. In contrast, when the effect of the ATP gamma in equilibrium Pi exchange on the CK rate measurements was eliminated, multiple saturation transfer (MST) measurements on the same hearts yielded equal forward and reverse fluxes in all cases. The rate constant for the ATP gamma----CP conversion measured by MST was larger than the value obtained by the conventional methodology whereas both methods gave the same rate constant in the CP----ATP direction. These results demonstrate that the cause of the paradoxical data obtained by CST measurements of CK kinetics is the ATP gamma in equilibrium Pi exchange and that CK rates when determined rigorously are consistent with the CK reaction being in equilibrium.(ABSTRACT TRUNCATED AT 250 WORDS)     

ATP-Induced phosphorylation of the sarcoplasmic reticulum Ca2+ ATPase: molecular interpretation of infrared difference spectra.

Time-resolved infrared difference spectra of the ATP-induced phosphorylation of the sarcoplasmic reticulum Ca2+-ATPase have been recorded in H2O and 2H2O at pH 7.0 and 1 degrees C. The reaction was induced by ATP release from P3-1-(2-nitro)phenylethyladenosine 5'-triphosphate (caged ATP) and from [gamma-18O3]caged ATP. A band at 1546 cm-1, not observed with the deuterated enzyme, can be assigned to the amide II mode of the protein backbone and indicates that a conformational change associated with ATPase phosphorylation takes place after ATP binding. This is also indicated between 1700 and 1610 cm-1, where bandshifts of up to 10 cm-1 observed upon protein deuteration suggest that amide I modes of the protein backbone dominate the difference spectrum. From the band positions it is deduced that alpha-helical, beta-sheet, and probably beta-turn structures are affected in the phosphorylation reaction. Model spectra of acetyl phosphate, acetate, ATP, and ADP suggest the tentative assignment of some of the bands of the phosphorylation spectrum to the molecular groups of ATP and Asp351, which participate directly in the phosphate transfer reaction: a positive band at 1719 cm-1 to the C==O group of aspartyl phosphate, a negative band at 1239 cm-1 to the nuas(PO2-) modes of the bound ATP molecule, and a positive band at 1131 cm-1 to the nuas(PO32-) mode of the phosphoenzyme phosphate group, the latter assignment being supported by the band's sensitivity toward isotopic substitution in the gamma-phosphate of ATP. Band positions and shapes of these bands indicate that the alpha- and/or beta-phosphate(s) of the bound ATP molecule become partly dehydrated when ATP binds to the ATPase, that the phosphoenzyme phosphate group is unprotonated at pH 7.0, and that the C==O group of aspartyl phosphate does not interact with bulk water. The Ca2+ binding sites seem to be largely undisturbed by the phosphorylation reaction, and a functional role of the side chains of Asn, Gln, and Arg residues was not detected.     

Mechanism of carbamoyl-phosphate synthetase. Binding of ATP by the rat-liver mitochondrial enzyme.

This paper demonstrates, by pulse-chase techniques, the binding to rat liver mitochondrial carbamoyl phosphate synthetase of the ATP molecule (ATPB) which transfers its gamma-phosphoryl group to carbamoyl phosphate. This bound APTB can react with NH3, HCO-3 and ATP (see below) to produce carbamoyl phosphate before it exchanges with free ATP. Mg2+ and N-acetylglutamate, but not NH3 or HCO-3, are required for this binding; the amount bound depends on the concentration of ATP (Kapp = 10--30 microns ATP) and the amount of enzyme. At saturation at least one ATPB molecule binds per enzyme dimer. Binding of ATPB follows a slow exponential time course (t1/2 8--16 s, 22 degrees C), independent of ATP concentration and little affected by NH3, NCO-3 or by incubation of the enzyme with unlabelled ATP prior to the pulse of [gamma-32P]ATP. Formation of carbamoyl phosphate from traces of NH3 and HCO-3 when the enzyme is incubated with ATP follows the kinetics expected if it were generated from the bound ATPB, indicating that the latter is a precursor of carbamoyl phosphate ('Cbm-P precursor') in the normal enzyme reaction. This indicates that the site for ATPB is usually inaccessible to ATP in solution but becomes accessible when the enzyme undergoes a periodical conformational change. Bound ATP becomes Cbm-P precursor when the enzyme reverts to the inaccessible conformation. Pulse-chase experiments in the absence of NH3 and HCO-3 (less than 0.2 mM) also demonstrate binding of ATPA (the molecule which yields Pi in the normal enzyme reaction), as shown by a 'burst' in 32Pi production. Therefore, (in accordance with our previous findings) both ATPA and ATPB can bind simultaneously to the enzyme and react with NH3 and HCO-3 in the chase solution before they can exchange with free ATP. However, at low ATP concentration (18 micron) in the pulse incubation, only ATPB binds since ATP is required in the chase (see above). Despite the presence of two ATP binding sites, the bifunctional inhibitor adenosine(5')pentaphospho(5')adenosine(Ap5A) fails to inhibit the enzyme significantly. A more detailed modification of the scheme previously published [Rubio, V. & Grisolia, S. (1977) Biochemistry, 16, 321--329] is proposed; it is suggested that ATPB gains access to the active centre when the products leave the enzyme and the active centre is in an accessible configuration. The transformation from accessible to inaccessible configuration appears to be part of the normal enzyme reaction and may represent to conformational change postulated by others from steady-state kinetics. The properties of the intermediates also indicate that hydrolysis of ATPA must be largely responsible for the HCO-3-dependent ATPase activity of the enzyme. The lack of inhibition of the enzyme by Ap5A indicates substantial differences between the Escherichia coli and the rat liver synthetase.     

Imaging of human brain creatine kinase activity in vivo

Creatine kinase activity and high-energy phosphate concentration have been investigated using localized 31P spectroscopy in the human brain in vivo. The phase-modulated rotating frame imaging technique, incorporating magnetization transfer and inversion recovery, has been used to produce a 1-dimensional rate profile map of steady-state enzyme activity. Large differences in the flux from phosphocreatine (PCr) to ATP have been discovered between volumes of human brain consisting of predominantly gray (2.0 cm) and white (4.5 cm) matter. The concentration of PCr changes slightly (2.0 cm = 5.20 +/- 0.45 mmol.l-1, 4.5 cm = 4.63 +/- 0.31 mmol.l-1), while the ATP concentration remains within limits (3.30 +/- 0.4 mmol.l-1). No change in pHi was detected between the two regions in normal volunteers (n = 6). The forward rate constant of the PCr----ATP reaction in regions of predominantly gray matter (0.30 +/- 0.04 s-1) was twice that of white matter (0.16 +/- 0.02 s-1) in vivo.     

Studies of energy transport in heart cells. Mitochondrial isoenzyme of creatine phosphokinase: kinetic properties and regulatory action of Mg2+ ions.

1. The kinetic properties of mitochondrial creatine phosphokinase (Km for all substrates and maximal rates of the forward and reverse reaction) have been studied. Since (a) Km value for MgADP- (0.05 mM) and creatine phosphate (0.5 mM) are significantly lower than Km for MgATP2- (0.7 mM) and creatine (5.0 mM) and (b) maximal rate of the reverse reaction (creatine phosphate + ADP leads to ATP + creatine) equal to 3.5 mumol times min-1 times mg-1 is essentially higher than maximal rate of the forward reaction (0.8 mumol times min-1 times mg-1), ATP synthesis from ADP and creatine phosphate is kinetically preferable over the forward reaction. 2. A possible regulatory role of Mg2+ ions in the creatine phosphokinase reaction has been tested. It has been shown that in the presence of all substrates and products of the reaction the ratio of the rates of forward and reverse reactions can be effectively regulated by the concentration of Mg2+ ions. At limited Mg2+ concentrations creatine phosphate is preferably synthesized while at high Mg2+ concentrations (more ATP in the reaction medium) ATP synthesis takes place. 3. The kinetic (mathematical) model of the mitochondrial creatine phosphokinase reaction has been developed. This model accounts for the existence of a variety of molecular forms of adenine nucleotides in solution and the formation of their complexes with magnesium. It is based on the assumption that the mitochondrial creatine phosphokinase reactions mechanism is analogous to that for soluble isoenzymes. 4. The dependence of the overall rate of the creatine phosphokinase reaction on the concentration of total Mg2+ ions calculated from the kinetic model quantitatively correlates with the experimentally determined dependence through a wide range of substrates (ATP, ADP, creatine and creatine phosphate) concentration. The analysis of the kinetic model demonstrates that the observed regulatory effect of Mg2+ on the overall reaction rate can be expained by (a) the sigmoidal variation in the concentration of the MgADP- complex resulting from the competition between ATP AND ADP for Mg2+ and (b) the high affinity of the enzyme to MgADP-. 5. The results predicted by the model for the behavior of mitochondrial creatine phosphokinase under conditions of oxidative phosphorylation point to an intimate functional interaction of mitochondrial creatine phosphokinase and ATP-ADP translocase.     

Acetate kinase from Veillonella alcalescens. Regulation of enzyme activity by succinate and substrates.

Acetate kinase of Veillonella alcalescens has been shown to be highly regulated enzyme exhibiting two levels of control: the requirement for succinate as a heterotropic allosteric effector, and cooperative binding at the substrate level. Succinate addition was necessary for enzymatic activity in both the direction of acyl phosphate synthesis and that of ATP synthesis. Control at the substrate level was apparent in the cooperative binding (Hill coefficients of 2) of acetyl phosphate, ATP, and ADP. Typical Michaelis kinetic data were observed for succinate (Ka = 20 mM for acetyl phosphate synthesis, 0.4 mM for ATP synthesis), acetate, and propionate. The primary effect of succinate was to increase the apparent Vmax of the enzymatic reaction for the variable substrates, ATP, ADP, and acetyl phosphate. The results are interpreted as evidence that, as a heterotropic effector of the acetate kinase reaction, succinate may regulate levels of propionyl-CoA (produced from propionyl phosphate by action of phosphotransacetylase), a compound required for the conversion of succinate to propionate. Acetase kinase has been shown to be a probable dimeric protein composed of two subunits of molecular weight 44,000 each.     

Rotational resonance determination of the structure of an enzyme-inhibitor complex: phosphorylation of an (aminoalkyl)phosphinate inhibitor of D-alanyl-D-alanine ligase by ATP

We have used a newly developed solid-state NMR method, rotational resonance, to establish the structure of an inhibited complex formed upon reaction of D-alanyl-D-alanine ligase, ATP, and the aminoalkyl dipeptide analogue [1(S)-aminoethyl][2-carboxy-2(R)-methyl-1- ethyl]phosphinic acid (Ib). Analogue Ib was determined to be an ATP-dependent, slow-binding inhibitor of the D-Ala-D-Ala ligase from Salmonella typhimurium, with an enzyme-inhibitor half-life of 17 days at 37 degrees C. The inhibited complex shows a 31P NMR spectrum which is very different from that which would arise from a mixture of the free inhibitor and ATP. Four well-resolved lines were observed: two (at -8 and -14 ppm) are assignable as the phosphates of ADP, the third is assignable to an inhibitor resonance (at 53 ppm) that shifts by approximately 19 ppm on binding, and the fourth is assignable to a resonance (at -3 ppm) due to a polyphosphate or phosphate ester moiety. At rotational resonance the spectrum shows evidence for strong dipolar couplings between the phosphinate phosphorus and a phosphate ester species. The dipolar coupling between the phosphorus signals at 53 and -3 ppm was measured at rotational resonance by use of numerical simulations of both the line shape of the signal and the profile of magnetization transfer between the two sites. The measured coupling, 1.0 +/- 0.2 kHz, indicates that the two species are bridged in a P-O-P linkage, with a P-P through-space distance of 2.7 +/- 0.2 A. This proves that the mechanism of inactivation involves phosphorylation of the enzyme-bound inhibitor by ATP to form a phosphoryl-phosphinate adduct.     

Velocity of the creatine kinase reaction in the neonatal rabbit heart: role of mitochondrial creatine kinase

To examine the role of changes in the distribution of the creatine kinase (CK) isoenzymes [BB, MB, MM, and mitochondrial CK (mito-CK)] on the creatine kinase reaction velocity in the intact heart, we measured the creatine kinase reaction velocity and substrate concentrations in hearts from neonatal rabbits at different stages of development. Between 3 and 18 days postpartum, total creatine kinase activity did not change, but the isoenzyme distribution and total creatine content changed. Hearts containing 0, 4, or 9% mito-CK activity were studied at three levels of cardiac performance: KCl arrest and Langendorff and isovolumic beating. The creatine kinase reaction velocity in the direction of MgATP production was measured with 31P magnetization transfer under steady-state conditions. Substrate concentrations were measured with 31P NMR (ATP and creatine phosphate) and conventional biochemical analysis (creatine) or estimated (ADP) by assuming creatine kinase equilibrium. The rate of ATP synthesis by oxidative phosphorylation was estimated with oxygen consumption measurements. These results define three relationships. First, the creatine kinase reaction velocity increased as mito-CK activity increased, suggesting that isoenzyme localization can alter reaction velocity. Second, the reaction velocity increased as the rate of ATP synthesis increased. Third, as predicted by the rate equation, reaction velocity increased with the 3-fold increase in creatine and creatine phosphate contents that occurred during development.