Energy Of Adenosine Triphosphate Hydrolysis Research Articles

Generation of infrared radiation with modulated amplitude at terahertz frequencies

The role of infrared radiation in stimulating cellular metabolism can be dual. Firstly, the energy of light quanta is used by the cell instead of the energy of adenosine triphosphate hydrolysis. Secondly, light absorption cannot replace this hydrolysis. However, it can accelerate its rate and thus the productivity of metabolic reactions associated with it. This process is significantly enhanced by the use of terahertz modulation of infrared radiation, which is manifested, for example, in optically stimulated transport of ions through biological membranes. For therapeutic effects on the above-described processes in biological tissues, a device has been developed that generates infrared radiation with wavelengths in the range of maximum transparency of biological tissues modulated in amplitude in a frequency range of 1011–1012 Hz. The modulation of radiation has been produced by the method of interference.

A bio-pen for direct writing of single molecules on user-functionalized surfaces.

Advancing ultrahigh resolution (below 10 nm) direct writing technologies could lead to impacts in areas as diverse as disease detection, genetic analysis and nanomanufacturing. Current methods based on electron-beams and photo- or dip-pen nanolithography are laborious and lack flexibility when aiming to create single molecule patterns for application specific integration. We hypothesize that a novel strategy could be developed to allow for writing of parallel and yet individually addressable patterns of single molecules on user-controlled surfaces. The strategy is based on using in vitro self-recognition of tubulin protein to assemble rigid protofilaments of microtubules, with one such microtubule to be subsequently used as a “bio-pen” capable of writing “inks” of single kinesin molecules in user-defined environments. Our results show that single kinesin inks could be written under the energy of adenosine triphosphate hydrolysis and observed by both atomic force and optical microscopy. Upon extending ink functionalities, the integration of soft and hard materials for nanostructure assembly and complex single molecule pattern formation is envisioned.

The A-B-C of small-molecule ABC transport protein modulators: From inhibition to activation-a case study of multidrug resistance-associated protein 1 (ABCC1).

Several mechanisms of pharmacokinetic, metabolic, and regulatory nature have been elucidated to take part or act in concert in the phenomenon of multidrug resistance (MDR). MDR is characterized by cross-resistance of cells against chemotherapeutic agents, which are used for treatment of e.g., cancer, bacterial infections, or human immunodeficiency virus (HIV) infections. One group of proteins that combines all three stated aspects-the metabolism and distribution of drugs as well as their own regulation-is adenosine triphosphate-binding cassette (ABC) transporters. These efflux pumps use the energy of adenosine triphosphate hydrolysis for drug translocation from the membrane and the cytosol to the extracellular space, often with cotransport of a cosubstrate. Multidrug resistance-associated protein 1 (MRP1, ABCC1) had been discovered as one major key player in cancer-related MDR. The xenobiotic substrates include anthracyclines, vinca alkaloids, podophyllotoxins, as well as glutathione (GSH)-adducts of certain cytostatics. Contrary to other transport proteins involved in cancer-related MDR the activity of MRP1 is related to the GSH content of cells. A modern strategy to overcome MRP1-associated MDR is besides its inhibition the activation of GSH efflux, enforcing cell death due to cellular stress. In addition, it has recently been found that MRP1 contributes to the β-amyloid protein clearance in Alzheimer's disease (AD). Collectively, transport activation of MRP1 is of therapeutic value, and furthermore helps to elucidate the transport protein function and the mechanisms behind it. This review is meant to summarize the known concepts of MRP1 activation, which might contribute to a further understanding of MRP1 in particular and ABC transporters in general.

Measuring inorganic phosphate and intracellular pH in the healthy and hypertrophic cardiomyopathy hearts by in vivo 7T 31P-cardiovascular magnetic resonance spectroscopy

BackgroundCardiovascular phosphorus MR spectroscopy (31P-CMRS) is a powerful tool for probing energetics in the human heart, through quantification of phosphocreatine (PCr) to adenosine triphosphate (ATP) ratio. In principle, 31P-CMRS can also measure cardiac intracellular pH (pHi) and the free energy of ATP hydrolysis (ΔGATP). However, these require determination of the inorganic phosphate (Pi) signal frequency and amplitude that are currently not robustly accessible because blood signals often obscure the Pi resonance.Typical cardiac 31P-CMRS protocols use low (e.g. 30°) flip-angles and short repetition time (TR) to maximise signal-to-noise ratio (SNR) within hardware limits. Unfortunately, this causes saturation of Pi with negligible saturation of the flowing blood pool. We aimed to show that an adiabatic 90° excitation, long-TR, 7T 31P-CMRS protocol will reverse this balance, allowing robust cardiac pHi measurements in healthy subjects and patients with hypertrophic cardiomyopathy (HCM).MethodsThe cardiac Pi T1 was first measured by the dual TR technique in seven healthy subjects. Next, ten healthy subjects and three HCM patients were scanned with 7T 31P-MRS using long (6 s) TR protocol and adiabatic excitation. Spectra were fitted for cardiac metabolites including Pi.ResultsThe measured Pi T1 was 5.0 ± 0.3 s in myocardium and 6.4 ± 0.6 s in skeletal muscle. Myocardial pH was 7.12 ± 0.04 and Pi/PCr ratio was 0.11 ± 0.02. The coefficients of repeatability were 0.052 for pH and 0.027 for Pi/PCr quantification. The pH in HCM patients did not differ (p = 0.508) from volunteers. However, Pi/PCr was higher (0.24 ± 0.09 vs. 0.11 ± 0.02; p = 0.001); Pi/ATP was higher (0.44 ± 0.14 vs. 0.24 ± 0.05; p = 0.002); and PCr/ATP was lower (1.78 ± 0.07 vs. 2.10 ± 0.20; p = 0.020), in HCM patients, which is in agreement with previous reports.ConclusionA 7T 31P-CMRS protocol with adiabatic 90° excitation and long (6 s) TR gives sufficient SNR for Pi and low enough blood signal to permit robust quantification of cardiac Pi and hence pHi. Pi was detectable in every subject scanned for this study, both in healthy subjects and HCM patients. Cardiac pHi was unchanged in HCM patients, but both Pi/PCr and Pi/ATP increased that indicate an energetic impairment in HCM. This work provides a robust technique to quantify cardiac Pi and pHi.

Identifying Cell Cycle Modulators That Selectively Target ARID1A Deficiency Using High-Throughput Image-Based Screening

ARID1A, a component of the chromatin remodeling complex SWI/SNF, is an evolutionarily conserved complex that uses the energy of adenosine triphosphate hydrolysis to remodel chromatin structure and functions as a master regulator of gene transcription. Recent genomic studies have revealed that ARID1A is one of the most frequently mutated genes in human cancers. However, therapeutic approaches that selectively target ARID1A-mutant tumors are not yet clinically available. Our previous study showed that ARID1A facilitates chromatin response and cell cycle checkpoint activation after DNA damage. Therefore, an ARID1A deficiency may result in therapeutic vulnerabilities in cell cycle modulators. The goals of our study were to develop a novel screening approach, based on fluorescent ubiquitination-based cell cycle indicators (FUCCI), and to identify chemical agents that can selectively modulate the cell cycle transition in ARID1A-deficient cancer cells. Using this high-throughput assay, we screened 2643 compounds and identified six potential chemical modulators that can selectively modulate the cell cycle in ARID1A-deficient cells; these agents may be useful for developing new therapeutics for ARID1A-mutant tumors. In summary, our study demonstrates that FUCCI cell-based high-content screening is a powerful and effective approach for identifying cell cycle modulators and can be applied to multigenotypic screening for targeted cancer therapeutics.

Standard Gibbs energy of metabolic reactions: II. Glucose-6-phosphatase reaction and ATP hydrolysis

ATP (adenosine triphosphate) is a key reaction for metabolism. Tools from systems biology require standard reaction data in order to predict metabolic pathways accurately. However, literature values for standard Gibbs energy of ATP hydrolysis are highly uncertain and differ strongly from each other. Further, such data usually neglect the activity coefficients of reacting agents, and published data like this is apparent (condition-dependent) data instead of activity-based standard data. In this work a consistent value for the standard Gibbs energy of ATP hydrolysis was determined. The activity coefficients of reacting agents were modeled with electrolyte Perturbed-Chain Statistical Associating Fluid Theory (ePC-SAFT).The Gibbs energy of ATP hydrolysis was calculated by combining the standard Gibbs energies of hexokinase reaction and of glucose-6-phosphate hydrolysis. While the standard Gibbs energy of hexokinase reaction was taken from previous work, standard Gibbs energy of glucose-6-phosphate hydrolysis reaction was determined in this work. For this purpose, reaction equilibrium molalities of reacting agents were measured at pH7 and pH8 at 298.15K at varying initial reacting agent molalities. The corresponding activity coefficients at experimental equilibrium molalities were predicted with ePC-SAFT yielding the Gibbs energy of glucose-6-phosphate hydrolysis of −13.72±0.75kJ·mol−1. Combined with the value for hexokinase, the standard Gibbs energy of ATP hydrolysis was finally found to be −31.55±1.27kJ·mol−1. For both, ATP hydrolysis and glucose-6-phosphate hydrolysis, a good agreement with own and literature values were obtained when influences of pH, temperature, and activity coefficients were explicitly taken into account in order to calculate standard Gibbs energy at pH7, 298.15K and standard state.

Conformational Motions and Functionally Key Residues for Vitamin B12 Transporter BtuCD-BtuF Revealed by Elastic Network Model with a Function-Related Internal Coordinate.

BtuCD–BtuF from Escherichia coli is a binding protein-dependent adenosine triphosphate (ATP)-binding cassette (ABC) transporter system that uses the energy of ATP hydrolysis to transmit vitamin B12 across cellular membranes. Experimental studies have showed that during the transport cycle, the transporter undergoes conformational transitions between the “inward-facing” and “outward-facing” states, which results in the open–closed motions of the cytoplasmic gate of the transport channel. The opening–closing of the channel gate play critical roles for the function of the transporter, which enables the substrate vitamin B12 to be translocated into the cell. In the present work, the extent of opening of the cytoplasmic gate was chosen as a function-related internal coordinate. Then the mean-square fluctuation of the internal coordinate, as well as the cross-correlation between the displacement of the internal coordinate and the movement of each residue in the protein, were calculated based on the normal mode analysis of the elastic network model to analyze the function-related motions encoded in the structure of the system. In addition, the key residues important for the functional motions of the transporter were predicted by using a perturbation method. In order to facilitate the calculations, the internal coordinate was introduced as one of the axes of the coordinate space and the conventional Cartesian coordinate space was transformed into the internal/Cartesian space with linear approximation. All the calculations were carried out in this internal/Cartesian space. Our method can successfully identify the functional motions and key residues for the transporter BtuCD–BtuF, which are well consistent with the experimental observations.

An In Silico Classification Model for Putative ABCC2 Substrates

ABCC2 (MRP2; also known as cMOAT or cMRP) belongs to the adenosine triphosphate (ATP)-binding cassette (ABC) transporter superfamily, which represents a large family of transmembrane proteins that use the energy of ATP hydrolysis to transport a wide variety of physiological substrates across biological membranes.1 Phylogenetically, ABC transporters are classified into seven subfamilies of 49 transporter genes (ABCA to ABCG).2 The major physiological role of these transporters is to protect cells and tissues against xenobiotics. Consequently, they also play a critical role in the disposition of drugs and their metabolites, altering their pharmacokinetics and pharmacological profile.1

Identification of a novel post-hydrolytic state in CFTR gating

Adenosine triphosphate (ATP)-binding cassette (ABC) transporters, ubiquitous proteins found in all kingdoms of life, catalyze substrates translocation across biological membranes using the free energy of ATP hydrolysis. Cystic fibrosis transmembrane conductance regulator (CFTR) is a unique member of this superfamily in that it functions as an ATP-gated chloride channel. Despite difference in function, recent studies suggest that the CFTR chloride channel and the exporter members of the ABC protein family may share an evolutionary origin. Although ABC exporters harness the free energy of ATP hydrolysis to fuel a transport cycle, for CFTR, ATP-induced dimerization of its nucleotide-binding domains (NBDs) and subsequent hydrolysis-triggered dimer separation are proposed to be coupled, respectively, to the opening and closing of the gate in its transmembrane domains. In this study, by using nonhydrolyzable ATP analogues, such as pyrophosphate or adenylyl-imidodiphosphate as baits, we captured a short-lived state (state X), which distinguishes itself from the previously identified long-lived C2 closed state by its fast response to these nonhydrolyzable ligands. As state X is caught during the decay phase of channel closing upon washout of the ligand ATP but before the channel sojourns to the C2 closed state, it likely emerges after the bound ATP in the catalysis-competent site has been hydrolyzed and the hydrolytic products have been released. Thus, this newly identified post-hydrolytic state may share a similar conformation of NBDs as the C2 closed state (i.e., a partially separated NBD and a vacated ATP-binding pocket). The significance of this novel state in understanding the structural basis of CFTR gating is discussed.

Allopurinol Acutely Increases Adenosine Triphospate Energy Delivery in Failing Human Hearts

This study tested the hypothesis that acute administration of the xanthine oxidase (XO) inhibitor allopurinol improves cardiac high-energy phosphate concentrations in human heart failure (HF) and increases the rate of adenosine triphosphate (ATP) synthesis through creatine kinase (CK), the primary myocardial energy reserve. Studies of patients and animal models implicate impaired myocardial high-energy phosphate availability in HF. The XO reaction is a critical terminal step in ATP and purine degradation and an important source of reactive oxygen species. Thus, XO inhibition is a potentially attractive means to improve energy metabolism in the failing human heart. We randomized 16 patients with nonischemic cardiomyopathy in a double-blind fashion to allopurinol (300 mg intravenously) or placebo infusion, 4-to-1, the latter for purposes of blinding only. The myocardial concentrations of ATP and creatine phosphate (PCr) and the rate of ATP synthesis through CK (CK flux) were determined by (31)P magnetic resonance spectroscopy. Allopurinol infusion increased mean cardiac PCr/ATP and PCr concentration by ∼11% (p < 0.02), and mean CK flux by 39% (2.07 ± 1.27 μmol/g/s to 2.87 ± 1.82 μmol/g/s, p < 0.007). Calculated cytosolic adenosine diphosphate concentration decreased, whereas the free energy of ATP hydrolysis (ΔG(∼ATP)) increased with allopurinol. The increased CK flux was disproportionate to substrate changes, indicating increased CK enzyme activity. Intravenous administration of the XO inhibitor allopurinol acutely improves the relative and absolute concentrations of myocardial high-energy phosphates and ATP flux through CK in the failing human heart, offering direct evidence that myofibrillar CK energy delivery can be pharmaceutically augmented in the failing human heart. (Intravenous Allopurinol in Heart Failure; NCT00181155).

Insights into the mechanisms of myosin and kinesin molecular motors from the single-molecule unbinding force measurements

In cells, ATP (adenosine triphosphate)-driven motor proteins, both cytoskeletal and nucleic acid-based, operate on their corresponding 'tracks', that is, actin, microtubules or nucleic acids, by converting the chemical energy of ATP hydrolysis into mechanical work. During each mechanochemical cycle, a motor proceeds via several nucleotide states, characterized by different affinities for the 'track' filament and different nucleotide (ATP or ADP) binding kinetics, which is crucial for a motor to efficiently perform its cellular functions. The measurements of the rupture force between the motor and the track by applying external loads to the individual motor-substrate bonds in various nucleotide states have proved to be an important tool to obtain valuable insights into the mechanism of the motors' performance. We review the application of this technique to various linear molecular motors, both processive and non-processive, giving special attention to the importance of the experimental geometry.

Kinesin-13s form rings around microtubules

Kinesin is a superfamily of motor proteins that uses the energy of adenosine triphosphate hydrolysis to move and generate force along microtubules. A notable exception to this general description is found in the kinesin-13 family that actively depolymerizes microtubules rather than actively moving along them. This depolymerization activity is important in mitosis during chromosome segregation. It is still not fully clear by which mechanism kinesin-13s depolymerize microtubules. To address this issue, we used electron microscopy to investigate the interaction of kinesin-13s with microtubules. Surprisingly, we found that proteins of the kinesin-13 family form rings and spirals around microtubules. This is the first report of this type of oligomeric structure for any kinesin protein. These rings may allow kinesin-13s to stay at the ends of microtubules during depolymerization.

Directed Binding: A Novel Physical Mechanism That Describes the Directional Motion of Two-Headed Kinesin Motor Proteins

We propose a novel physical mechanism to describe the mode of processive propagation of two-headed kinesin motor proteins along microtubule (MT) filaments. Binding and unbinding of the kinesin heads to and from the MT filament play a crucial role in producing movement. The chemical energy of adenosine triphosphate hydrolysis is used in large part for the unbinding process of kinesin from the MT filament. Importantly, in our model, the binding of each head is to be directionally oriented to the MT filament. Therefore, we treat the two motor domains (heads) as extended objects that are connected with each other by a neck region that contains the kinesin dimerization domain. The head domains recognize tubulin binding sites by feeling the two-dimensional periodic potential from the MT surface and are also subjected to thermal noise. Using experimentally determined results regarding physical parameters of the walk, we develop a simple mathematical and mechanical model in which directed binding of the heads to tubulin results in a directed twist of the molecule, probably in the neck linker region, away from its relaxed state. Unbinding of the head from the filament relaxes the twist and defines the propagation direction. We showed that there must be at least two torsional springs (one for every head) involved that can store elastic energy. Consequently, in our model, it is the internal structure both of the relaxed and tensed-up state and the transition mode between them that define the walking direction of kinesin. We present calculations based on the model that are in good quantitative agreement with experimental observations for kinesin.

Severe energy deprivation of human hibernating myocardium as possible common pathomechanism of contractile dysfunction, structural degeneration and cell death

ObjectivesWe tested the hypothesis that severe alterations in myocardial energy metabolism play an important role in the pathophysiology of human hibernating myocardium (HHM). BackgroundIt is well established that a disturbed myocardial energy metabolism results in impairments of contractile performance, structure and viability. All of these are important characteristics of HHM. MethodsIn 16 patients with documented coronary artery disease and impaired left ventricular function, HHM was preoperatively detected by thallium-201 scintigraphy, radionuclide ventriculography and low-dose dobutamine echocardiography. These regions were validated as HHM by their recovery of contractile function three months following revascularization. During open-heart surgery, transmural biopsies were removed from the hibernating areas and analyzed both biochemically and morphologically. These findings were compared to normal human myocardium. All metabolite contents given were normalized for the degree of fibrosis (control: 9.8 ± 0.5%; HHM: 28.1 ± 3.0%; p < 0.05), providing myocellular contents. ResultsIn HHM, decreased contents (μmol/g wet weight) in adenosine triphosphate (ATP) (control: 4.17 ± 0.26; HHM: 1.72 ± 0.25; p < 0.001), creatine phosphate (5.67 ± 0.70 vs. 0.84 ± 0.13; p < 0.001) and creatine (27.6 ± 3.19 vs. 11.2 ± 1.56; p < 0.0001) were found, but contents in lactate (2.22 ± 0.26 vs. 25.38 ± 3.53; p < 0.001), purine bases (0.58 ± 0.09 vs. 1.26 ± 0.13; p < 0.001) and protons (pH units: 7.199 ± 0.01 vs. 6.59 ± 0.07; p < 0.001) were increased. Levels in adenosine diphosphate, adenosine monophosphate and inorganic phosphate remained unchanged. Energy depletion in HHM was reflected by decreases in the free energy of ATP hydrolysis and in energy charge. ConclusionsThese data confirm our hypothesis that HHM is energy-depleted myocardium, exhibiting signs of chronic reduction in resting blood flow and a downregulation of energy turnover. The alterations in energy metabolism observed may become operative in triggering and maintaining contractile dysfunction, continuous tissue degeneration and cardiomyocyte loss.

Factors affecting the rate of phosphocreatine resynthesis following intense exercise.

Within the skeletal muscle cell at the onset of muscular contraction, phosphocreatine (PCr) represents the most immediate reserve for the rephosphorylation of adenosine triphosphate (ATP). As a result, its concentration can be reduced to less than 30% of resting levels during intense exercise. As a fall in the level of PCr appears to adversely affect muscle contraction, and therefore power output in a subsequent bout, maximising the rate of PCr resynthesis during a brief recovery period will be of benefit to an athlete involved in activities which demand intermittent exercise. Although this resynthesis process simply involves the rephosphorylation of creatine by aerobically produced ATP (with the release of protons), it has both a fast and slow component, each proceeding at a rate that is controlled by different components of the creatine kinase equilibrium. The initial fast phase appears to proceed at a rate independent of muscle pH. Instead, its rate appears to be controlled by adenosine diphosphate (ADP) levels; either directly through its free cytosolic concentration, or indirectly, through its effect on the free energy of ATP hydrolysis. Once this fast phase of recovery is complete, there is a secondary slower phase that appears almost certainly rate-dependent on the return of the muscle cell to homeostatic intracellular pH. Given the importance of oxidative phosphorylation in this resynthesis process, those individuals with an elevated aerobic power should be able to resynthesise PCr at a more rapid rate than their sedentary counterparts. However, results from studies that have used phosphorus nuclear magnetic resonance ((31)P-NMR) spectroscopy, have been somewhat inconsistent with respect to the relationship between aerobic power and PCr recovery following intense exercise. Because of the methodological constraints that appear to have limited a number of these studies, further research in this area is warranted.

Simplification of DNA topology below equilibrium values by type II topoisomerases.

Type II DNA topoisomerases catalyze the interconversion of DNA topoisomers by transporting one DNA segment through another. The steady-state fraction of knotted or catenated DNA molecules produced by prokaryotic and eukaryotic type II topoisomerases was found to be as much as 80 times lower than at thermodynamic equilibrium. These enzymes also yielded a tighter distribution of linking number topoisomers than at equilibrium. Thus, topoisomerases do not merely catalyze passage of randomly juxtaposed DNA segments but control a global property of DNA, its topology. The results imply that type II topoisomerases use the energy of adenosine triphosphate hydrolysis to preferentially remove the topological links that provide barriers to DNA segregation.

A Functional Recombinant Myosin II Lacking a Regulatory Light Chain-Binding Site

Myosin II, which converts the energy of adenosine triphosphate hydrolysis into the movement of actin filaments, is a hexamer of two heavy chains, two essential light chains, and two regulatory light chains (RLCs). Dictyostelium myosin II is known to be regulated in vitro by phosphorylation of the RLC. Cells in which the wild-type myosin II heavy chain was replaced with a recombinant form that lacks the binding site for RLC carried out cytokinesis and almost normal development, processes known to be dependent on functional myosin II. Characterization of the purified recombinant protein suggests that a complex of RLC and the RLC binding site of the heavy chain plays an inhibitory role for adenosine triphosphatase activity and a structural role for the movement of myosin along actin.

The role of magnesium in postischemic cardiac dysfunction : Sommers KE, Ohkado A, Simplaceanu E, et al: Surgery 112:159–165, 1992

The biochemical basis for postischemic myocardial stunning is not fully elucidated. Magnesium is an important regulator of cellular energetic processes and excitation-contraction coupling. We hypothesized that the decrease in function in the postischemic period may be the result of an alteration in magnesium regulation.In a Langendorf perfused rabbit heart model, we used 31P nuclear magnetic resonance spectroscopy to noninvasively determine intracellular Mg2+ and high-energy phosphate levels in the preischemic period and after a 30-minute period of normothermic ischemia. We measured adenosine triphosphate (ATP), phosphocreatine, and the phosphocreatine/inorganic phosphate ratio and calculated the free energy of ATP hydrolysis (delta GATP). On reperfusion, hearts were divided into three groups (n = 7 per group)--those receiving unmodified Krebs-Henseleit (control), 192 ng/ml dobutamine, or 5 mmol/L pyruvate.Function (expressed as the rate-pressure product) was approximately 77% of preischemic values in the control group, whereas in both dobutamine and pyruvate groups it returned to preischemic levels. ATP was decreased similarly in all groups in the postischemic period. Phosphocreatine/inorganic phosphate ratio and delta GATP were higher in the pyruvate group compared with the other groups. Intracellular Mg2+ was elevated significantly in the unmodified control postischemic group compared with preischemic, postischemic dobutamine, and pyruvate groups (1.0 +/- 0.12 vs 0.80 +/- 0.08, 0.64 +/- 0.08, and 0.70 +/- 0.05 mmol/L, respectively; p less than 0.05).We conclude that (1) postischemic "stunned" hearts have elevated Mg2+ levels in association with impaired contractile function, (2) inotropic agents improve contractile function in association with a decline in Mg2+ to preischemic levels despite differing effects on intracellular energetics, and (3) Mg2+ may play an important regulatory role in the heart after ischemia.

Effect of Temperature and Coronary Flow on the Metabolic and Mechanical Function of the Isolated Rat Heart

A number of cardiac metabolic intermediates, namely, adenosine triphosphate (ATP), H+, phosphocreatine (PCr), inorganic phosphate (Pi), adenosine diphosphate (ADP), and related functions of these intermediates, Gibbs' free energy of ATP hydrolysis (delta G) and phosphorylation ratio [ATP/(ADP.Pi)], are thought to adjust mitochondrial oxidative phosphorylation rates to conform to mechanical demand. The effects of hypothermia and altered perfusion pressure on these parameters were evaluated in 12 hearts from Sprague-Dawley rats perfused in the Langendorff mode. 31P-nuclear magnetic resonance (NMR) spectra were obtained at cardiac temperatures between 20 and 37 degrees C, and coronary perfusion pressures between 20 and 145 cm H2O. Coronary flow varied between 0.5 and 15 ml/min throughout this range of intervention. Heart rate (HR), left ventricular systolic pressure (LVSP), and specific volumetric coronary flow (SCF) were determined for each temperature and perfusion pressure. The product HR x LVSP directly correlated with perfusion pressure at all temperatures. The temperature dependence could be represented by an overall activation energy of 72.7 kJ/M. In the constant temperature experiment, SCF and HR x LVSP fell linearly with decreasing perfusion pressure. Quantitative evaluation of the relationship between cardiac function and the metabolic intermediates described above defined these intermediates as nonregulatory with the possible exception of H+.