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Query: UNIPROT:Q9UMR3 (NMR)
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The paramagnetic effects of the bound manganese ion and of a covalently attached spin label on proton nuclear spin relaxation rates have been used to calculate distances for a structural model of the MnADP and creatine complexed to creatine kinase from rabbit muscle. The nucleotide and guanidino substrates are so aligned on the enzyme that the transferable phosphoryl group on one substrate is in apposition to the acceptor moiety on the second substrate. The divalent metal ion is most probably liganded to the alpha and beta phosphates of the nucleotide substrate, both in the abortive MnADP-creatine-enzyme complex and in the active MnATP-creatine-enzyme complex. The metal ion-formate distance approximately 5 A in the Mn(II)ADP-formate-creatine-enzyme complex and less than 5 A in the Co(II)ADP-formate-creatine-enzyme complex is consistent with the suggestion that the monovalent anion is binding at the site normally occupied by the transferable phosphoryl group, thus producing a complex which mimics the transition state. Although only an upper limit of the distance from Mn(II) to the guanidino substrate could be determined in the presence of formate, it could be concluded that the disposition of the guanidino substrate changes upon addition of formate, since the relative distances of the methyl and methylene group are inverted. The effect of formate and nitrate on increasing the residence time of creatine in the MnADP-creatine-enzyme complex as determined by NMR provides evidence that the complexes observed by NMR are identical with those involved in the catalytic mechanism, since a parallel effect of formate and nitrate is observed in the kinetics of the enzymatic reaction, where the dissociation constant of creatine from the abortive quaternary complex decreases in the presence of the anions as had been determined from their inhibition of the forward reaction (Milner-White, E.J., and Watts, D.C. (1971) Biochem. J. 122, 727-740). Although the guanidino substrate is not directly liganded to the divalent metal ion, the electron paramagnetic resonance spectrum of manganese in the transition state analog complexes, i.e. nitrate-ADP-guanidino substrate-enzyme, is strongly dependent on catalytic activity of the guanidino substrate. The structural differences observed by EPR among transition state analog complexes with various guanidino substrates were not reflected in distances from Mn(II) to the guanidino substrate, which were 10% and 0.3% as active as creatine. Within the experimental error of 1 A, the distances were the same. The enzyme or the enzyme-substrate complexes may be considered to exist in a number of structurally distinct conformations in equilibrium based on the EPR spectra and on the anomalous temperature-dependence of the relaxation rates of the formate proton of the transition state analog complexes...
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PMID:Magnetic resonance study of the three-dimensional structure of creatine kinase-substrate complexes. Implications for substrate specificity and catalytic mechanism. 17 21

Changes in the creatine kinase system, cellular energetics, regulation of respiration and alterations in parameters of contractility in experimental animals (myopathic hamsters), and in patients with dilated cardiomyopathy were studied. 31P-NMR methods were used to show that cardiomyopathic hearts are characterized by decreased work index, lower tissue ATP, phosphocreatine, and total creatine contents and diminished creatine kinase activity and energy fluxes. In isolated mitochondria, only the creatine kinase activity was decreased. Both in cardiomyopathic hamsters and human hearts a share of mitochondrial creatine kinase in the total tissue enzyme activity was decreased from 33% to 18% and that of BB elevated from 5% in control to 20%, at an unchanged relative level of MM. In saponins-skinned cardiac fibers on cardiomyocytes creatine (Cr, 25 mM) decreased Km for ADP in regulation of respiration from 133 +/- 20 to 20 +/- 4 microM due to activation of coupled mitochondrial creatine kinase-oxidative phosphorylation reactions in control hamster hearts. In the case of cardiomyopathy it decreased Km for ADP only to 81 +/- 13 microM. In endocardial biopsy samples from the hearts of patients with dilated cardiomyopathy taken during angiography, creatine stimulated respiration was decreased by 36% of control value, which correlated well with increase of end-diastolic pressure and fall in ejection fraction. Thus, changes in mitochondrial creatine kinase expression diminished the efficiency of cellular regulation of respiration in cardiomyopathic hearts that may have functional consequences for hemodynamics or may be adaptive alterations in response to decreased contractility.
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PMID:The creatine kinase system and cardiomyopathy. 129 99

To study the relative roles of creatine kinase (CK) and adenylate systems in cardiac energy turnover, the effect of CK inhibitor, iodoacetamide- (IAA, 0.5 mM), and 2-deoxyglucose-(DOG, 2 mM) induced) 65% depletion of adenine nucleotides at slightly decreased CK flux was determined in isolated rat heart. Both substances did not substantially affect contractile parameters of the isovolumic heart. However, an augmentation of cardiac work induced by isoproterenol addition was feeble and transient in IAA-treated hearts while the response of DOG-treated hearts was well preserved. The cardiac failure after IAA treatment was associated with irreversible fall in myocardial ATP content as evidenced by 31P-NMR technique. Furthermore, these hearts were unable to perform cardiac pump function due to insufficient cardiac filling and distensibility. The DOG-treated hearts exhibited 50% reduction in the pump function and were able to increase their work in elevated resistance. The results suggest that CK pathway is extremely important for both full cardiac relaxation and maximal contractile function.
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PMID:[Functional significance of 2 pathways of energy transport in cardiomyocytes]. 140 43

ATP synthesis from PCr through creatine kinase reaction was measured in vivo in rat leg muscle using 31P NMR magnetization transfer and progressive saturation. Both techniques determined a spin-lattice relaxation time for PCr of 3 s at rest and an identical forward rate constant of 0.22-0.26 s-1. In stimulated muscles, magnetization transfer showed that flux was not changed with a steady-state PCr of 54% of initial level. During stimulation inducing a PCr decrease to 38% of initial value, flux was significantly lowered by 30%. These findings could result from an accumulation of ions and water increases or from compartmentation of ATP and PCr in different pools either in the muscle cell or in the different muscle fibers. In addition, these results could reinforce the hypothesis against a crucial role for creatine kinase shuttle in the ATP supply in skeletal muscle.
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PMID:Creatine kinase activity in rat skeletal muscle with intermittent tetanic stimulation. 156 71

In a series of three papers, we demonstrate and validate an approach for concurrent absolute quantification in situ of blood flow and energy metabolism with a modification of the NMR method for absolute concentration determination put forth by Thulborn and Ackerman [J. Magn. Reson. 55, 357 (1983)] and later expanded upon by Tofts and Wray. In this first paper of the series, we briefly review the theoretical basis for the concentration measurement and present, for the first time, a successful paired validation of metabolite quantification via 31P surface-coil NMR through corroborative in vitro enzymatic assays. The paired radiolabeled microsphere validation of blood flow measurement via 2H surface-coil NMR employing D2O as a freely diffusible tracer and the concurrent determination of blood flow and energy metabolism in a septic rat model are presented in the accompanying second and third paper to complete the series. In this article a classical RF tank circuit is employed to describe the effect of conductive sample loading on the NMR receiver by considering its apparent series resistance. It is shown in an easily visualized generalizable manner that the effect of sample loading on the observed NMR signal intensity can be accounted for quantitatively by monitoring changes in 90 degrees pulse width at constant power at a fixed reference point, i.e., Ssample = Sphantom (PW90phantom/PW90sample). In a series of paired experiments the absolute concentrations of high energy phosphates obtained from resting rat leg muscle (n = 4) in situ (NMR) and in vitro (enzymatic) were determined as follows: [PCr]NMR = 17.2 +/- 0.8 SD, [PCr]enzymatic = 17.3 +/- 2 SD, [ATP]NMR = 5.1 +/- 0.8 SD, [ATP]enzymatic = 5.0 +/- 0.2 SD mmol/kg tissue wet wt. Results of these two independent methods of concentration determination were not statistically different (P = 0.94 and P = 0.74 respectively) and serve to rigorously validate the Thulborn approach for absolute quantification of phosphorous metabolites in situ via NMR. Furthermore, these results strongly suggest that ATP and PCr in resting rat leg muscle under normal physiologic conditions are 100% NMR visible. The free cytosolic [ADP]NMR was estimated from the creatine kinase reaction equilibrium expression to be 0.022 +/- 0.003 SD mmol/kg tissue wet wt.
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PMID:Concurrent quantification of tissue metabolism and blood flow via 2H/31P NMR in vivo. I. Assessment of absolute metabolite quantification. 159 56

Physiological control of the plasma membrane sodium pump, (Na+,K+)-ATPase, is essential for proper function of eukaryotic cells. In the electric organ of the elasmobranch Narcine brasiliensis, the normal demands placed upon the pump during the process of generation of electrical currents call for large and rapid changes in activity of this enzyme, making this a good model for the study of its cellular regulation. 31P NMR spectroscopic techniques were used to study metabolic regulation of membrane pump function in resting and stimulated electric organ and in skeletal muscle of the live, intact N. brasiliensis. Because the ATP synthetic abilities of the electric organ by glycolysis or oxidative phosphorylation are extremely limited, depletion of phosphocreatinine (PCr) could be used to determine the activity of the (Na+,K+)-ATPase after the electric organ was stimulated to discharge, and to measure the net flux from PCr to ATP through the creatine phosphokinase (CPK) reaction in the electric organ. Saturation transfer, an NMR technique which measures exchange rates, was applied to determine the unidirectional flux in the forward direction through the same reaction in the electric organ and in skeletal muscle as a control. The pseudo first-order rate constant kf for the CPK reaction at 24 degrees C in resting electric organ was 0.000 +/- 0.002 s-1 (n = 10) and in skeletal muscle was 0.08 +/- 0.03 s-1 (n = 3). The results demonstrate that in resting electric organ, which is well supplied with CPK, there was no measurable flux through this reaction, although CPK when extracted is highly active. Measured and calculated levels of all substrates for the creatine kinase reaction in the electric organ are similar to those in unstimulated skeletal muscle, where the creatine phosphokinase reaction rates are high in vivo. In contrast to the resting electric organ, during stimulation of the electric organ the measured net rate constant was greater than 0.08 s-1. In addition, as shown by lack of PCr depletion, there was virtually no net turnover of ATP in the resting organ compared to the stimulated organ. The marked difference in the (Na+,K+)-ATPase activity in the resting and activated electric organ confirmed earlier results (Blum, H., Nioka, S., and Johnson, R. G., Jr. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 1247-1251). Together, these results suggest that there is a novel method of coordinate regulation of cellular enzymes of great sensitivity and rapidity.
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PMID:Coupled in vivo activity of creatine phosphokinase and the membrane-bound (Na+,K+)-ATPase in the resting and stimulated electric organ of the electric fish Narcine brasiliensis. 164 45

The aim of this study was to determine whether acute changes in [Mg2+]free occur during increased hydrolysis of cytosolic ATP, and whether these changes were of sufficient magnitude to be involved in the modulation of myocardial metabolism. 31P-NMR was used to estimate free cytosolic Mg2+ levels ([Mg2+]free) during hypoxia, isoproterenol stimulation, and graded low-flow ischemia in crystalloid perfused, isovolumic rat hearts. Cytosolic [Mg2+]free was calculated to be 0.73 +/- 0.12 mM in control hearts (100 mmHg hydrostatic pressure, 95% O2, n = 18). Cytosolic [Mg2+]free increased gradually during 10 min periods of hypoxia (65%, 50%, 35%, 5% O2), and 20 min infusions of isoproterenol (0.4, 3.0, 75 nM), to maximum values greater than 250% of control (P less than 0.05). During 8 min periods of graded low-flow ischemia (12.0, 7.2, 5.3, 3.4, and 1.6 ml/min/g), [Mg2+]free did not change significantly. [Mg2+]free displayed an inverse linear correlation with total cytosolic [ATP] during isoproterenol infusion (r = 0.87), and an exponential correlation during hypoxia (r = 0.82). The data indicate that acute changes in cytosolic [Mg2+]free can occur during conditions of net ATP hydrolysis although changes in ATP alone do not appear to be solely responsible for the changes in [Mg2+]free. Since the magnitude of the changes in [Mg2+]free are sufficient to alter equilibria of enzymes such as creatine kinase and myokinase, it is possible that these changes are involved in the acute modulation of myocardial metabolism.
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PMID:Cytosolic free magnesium in stimulated, hypoxic, and underperfused rat heart. 165 49

The phosphate metabolites, PCr, ATP, ADP and inorganic phosphate (Pi), were quantitated in the brain of the newborn, neonatal, juvenile and adult dog to investigate the potential control mechanisms responsible for increased ATP demands during development. The concentrations of PCr and Pi were measured in vivo by MRS using the enzymatic-measured ATP as the internal standard. Phosphocreatine values increased during development from 2.08 mmol/kg wet weight in the 0-2 day newborn to 5.11 mmol/kg wet weight in the adult brain and paralleled the increases in the total creatine pool (PCr + Cr) from 4.12 to 10.05 mmol/kg wet weight. Brain ATP concentrations increased approximately 40% during postnatal development; however, when expressed as intracellular concentration, no increase in ATP was apparent due to the age-dependent decrease in extracellular space. The Pi concentration, estimated by MRS, increased significantly during postnatal development with a range of 1.78 to 2.52 mmol/kg wet wt, then decreased to 1.97 mmol/kg wet weight at adulthood. In those developmental stages where total Pi was measured enzymatically on freeze-clamped tissue, the NMR visible Pi comprised about 48 to 93% of the total, with the highest percentage being visible in the newborn brain. The intracellular pH decreased from 7.21 in the newborn to 7.10 in the adult. With development, the free ADP concentration, calculated from the components of the creatine kinase equilibrium, ranged from 27 to 34 microM. These values are close to the apparent in vitro Km of ADP for oxidative phosphorylation.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Quantitation of high energy phosphate compounds and metabolic significance in the developing dog brain. 167 45

To measure free creatine in the isolated perfused rat heart, the concentration of phosphocreatine, and phosphocreatine plus creatine (sigma Cr) were measured by 31P- and 1H-NMR, respectively. Quantification was performed in the presence and absence of an intraventricular balloon filled with a known amount of PCr, which acted as an external standard. Total (free plus bound) phosphocreatine and creatine were measured by HPLC analysis of extracts from the same hearts, freeze-clamped at the end of the perfusions. A greater concentration of creatine (mumol/g dry wt.) in the perfused rat heart was measured by HPLC analysis (40.3 +/- 2.38 (11)) as compared to NMR (34.6 +/- 1.95 (11)), whilst no significant difference was observed in the measurement of phosphocreatine between the two assay methods. Consequently, a greater sigma Cr was measured by HPLC. This work suggests that the majority of Cr in the heart is NMR visible and unbound, so available to interact with creatine kinase. The lower free ADP concentration calculated from NMR measurements (53.3 +/- 3.80 microM (9)) was not significantly different from that determined by HPLC analysis (56.9 +/- 5.90 microM (9)). This suggests that the concentration of free ADP in the heart is higher than values where it can regulate oxidative phosphorylation most effectively.
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PMID:Determination of free creatine and phosphocreatine concentrations in the isolated perfused rat heart by 1H- and 31P-NMR. 173 53

NMR in vivo spectroscopy is one of the few methods available for non-invasive investigations of cerebral metabolism in animals and humans. 31P and 1H spectroscopy are particularly suitable for monitoring the cerebral energy metabolism by determining the cerebral levels of ATP, ADP, phosphocreatine (PCr), inorganic phosphate (Pi), lactate and intracellular pH (pHi). These techniques also seem to be suitable for studying the effects of anesthetics by directly comparing the anesthetized and unanesthetized states in the same subject. The effects of halothane and isoflurane on the changes elicited in the cerebral energy metabolism by experimental hypercapnia were investigated by in vivo NMR spectroscopy. Halothane was found to aggravate the decrease in PCr attributed to the shift in creatine kinase equilibrium induced by the cerebral acidosis associated to hypercapnia, while the level of cerebral ADP was decreased to a lesser extent than in unanesthetized animals. In contrast isoflurane did not modify the changes in cerebral energy metabolism elicited by hypercapnia except that the decrease in PCr was significantly slowed, suggesting a lower creatine kinase activity. These data indicate that isoflurane and halothane act by two different mechanisms to produce a decrease in oxygen consumption. Halothane could interfere with oxidative metabolism by disturbing ATP metabolism, while isoflurane could decrease oxygen consumption by a general sedative action, slowing both cerebral functional activity and cerebral energy homeostasis.
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PMID:[Value of in vivo NMR spectroscopy in the study of cerebral metabolism under inhalation anesthesia]. 184 37

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