Phosphorylation status of liver by 31P-n.m.r. spectroscopy, and its implications for metabolic control. A comparison of 31P-n.m.r. spectroscopy (in vivo and in vitro) with chemical and enzymic determinations of ATP, ADP and Pi.
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ABSTRACT:
SUBMITTER: Iles RA
PROVIDER: S-EPMC1145160 | biostudies-other | 1985 Jul
REPOSITORIES: biostudies-other
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Project description:The kinetics of phosphoryl exchange involving ATP and ADP have been investigated successfully by in vivo (31)P 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 (31)P 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 (31)P 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.
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Project description:Phase-modulated rotating-frame imaging (p.m.r.f.i.), a localization technique for 31P-n.m.r. spectroscopy, has been applied to obtain information on the heterogeneity of phosphorus-containing metabolites and pH in the skeletal muscle of control and streptozotocin-diabetic rats. Using this method, the metabolic changes in four spatially resolved longitudinal slices (where slice I is superficial and slice IV is deep muscle) through the ankle flexor muscles have been investigated at rest and during steady-state isometric twitch-contraction at 2 Hz. At rest, intracellular pH was lower, and phosphocreatine (PCr)/ATP was higher, throughout the muscle mass in diabetic compared with control animals. The change in PCr/ATP in diabetic muscle correlated with a decrease in the chemically determined ATP concentration. During the muscle stimulation period, the decrease in pH observed in diabetic muscle at rest was maintained, but not exacerbated, by the contractile stimulus. Stimulation of muscle contraction caused more marked changes in PCr/(PCr + Pi), PCr/ATP and Pi/ATP in the diabetic group. These changes were most evident in slice III, which contains the greatest proportion of fast glycolytic-oxidative (type IIa) fibres, in which statistically significant differences were observed for all metabolite ratios. The results presented suggest that some degree of heterogeneity occurs in diabetic skeletal muscle in vivo with respect to the extent of metabolic dysfunction caused by the diabetic insult and that regions of the muscle containing high proportions of type IIa fibres appear to be most severely affected.
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Project description:Transport of creatine in the mouse liver has been investigated in vivo and in the perfused organ. Experiments were carried out with transgenic mice expressing creatine kinase in the liver (brain isoenzyme CKBB; EC 7.2.3.2.) [Koretsky, Brosnan, Chen, Chen and Van Dyke (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 3112-3116] and in the corresponding control mice. The animals were fed a regular chow with or without the addition of 10% creatine (w/w) for 5 days. The kinetics of creatine uptake was measured in the perfused liver by 31P-n.m.r. spectroscopy and biochemical analysis following infusion of creatine at concentrations ranging over 0-15 mM and at an extracellular pH of either 7.40 or 6.40. The results suggest that creatine is actively transported by a pH-dependent mechanism obeying a saturable Michaelis-Menten type of kinetics (Km = 0.80 +/- 0.18 and 5.12 +/- 2.40 mM; Vmax. = 0.57 +/- 0.04 and 1.72 +/- 0.32 mumol.g of liver-1.min-1 at pH 7.40 and 6.40 respectively). Creatine export was evaluated in the perfused liver preloaded with creatine and the results show that less than 2.5 and 5% of the total creatine pool is exported to the perfusate during 80 min of perfusion at pH 7.40 and 6.40 respectively. Taken together, these results seem to explain the observation that creatine accumulates in the mouse liver only when blood creatine is raised by creatine feeding.
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Project description:PurposePhosphorus magnetic resonance spectroscopy ((31)P-MRS) affords unique insight into cardiac energetics but has a low intrinsic signal-to-noise ratio (SNR) in humans. Theory predicts an increased (31)P-MRS SNR at 7T, offering exciting possibilities to better investigate cardiac metabolism. We therefore compare the performance of human cardiac (31)P-MRS at 7T to 3T, and measure T1s for (31)P metabolites at 7T.MethodsMatched (31)P-MRS data were acquired at 3T and 7T, on nine normal volunteers. A novel Look-Locker CSI acquisition and fitting approach was used to measure T1s on six normal volunteers.ResultsT1s in the heart at 7T were: phosphocreatine (PCr) 3.05 ± 0.41s, γ-ATP 1.82 ± 0.09s, α-ATP 1.39 ± 0.09s, β-ATP 1.02 ± 0.17s and 2,3-DPG (2,3-diphosphoglycerate) 3.05 ± 0.41s (N = 6). In the field comparison (N = 9), PCr SNR increased 2.8× at 7T relative to 3T, the Cramer-Ráo uncertainty (CRLB) in PCr concentration decreased 2.4×, the mean CRLB in PCr/ATP decreased 2.7× and the PCr/ATP SD decreased 2×.ConclusionCardiac (31)P-MRS at 7T has higher SNR and the spectra can be quantified more precisely than at 3T. Cardiac (31)P T1s are shorter at 7T than at 3T. We predict that 7T will become the field strength of choice for cardiac (31)P-MRS.
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Project description:Understanding muscle contraction mechanisms is a standing challenge, and one of the approaches has been to create models of the sarcomere-the basic contractile unit of striated muscle. While these models have been successful in elucidating many aspects of muscle contraction, they fall short in explaining the energetics of functional phenomena, such as rigor, and in particular, their dependence on the concentrations of the biomolecules involved in the cross-bridge cycle. Our hypothesis posits that the stochastic time delay between ATP adsorption and ADP/Pi release in the cross-bridge cycle necessitates a modeling approach where the rates of these two reaction steps are controlled by two independent parts of the total free energy change of the hydrolysis reaction. To test this hypothesis, we built a two-filament, stochastic-mechanical half-sarcomere model that separates the energetic roles of ATP and ADP/Pi in the cross-bridge cycle's free energy landscape. Our results clearly demonstrate that there is a nontrivial dependence of the cross-bridge cycle's kinetics on the independent concentrations of ATP, ADP, and Pi. The simplicity of the proposed model allows for analytical solutions of the more basic systems, which provide novel insight into the dominant mechanisms driving some of the experimentally observed contractile phenomena.
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Project description:This minireview is dedicated to the memory of Henryk Eisenberg and honors his major contributions to many areas of biophysics and to the analysis of macromolecular states and interactions in particular. This work reviews the ATP and ADP states of a ubiquitous protein, actins, and considers the present evidence for and against unique, nucleotide-dependent conformations of this protein. The effects of ATP and ADP on specific structural elements of actins, its loops and clefts, as revealed by mutational, crosslinking, spectroscopic, and EPR methods are discussed. It is concluded that the existing evidence points to dynamic equilibria of these structural elements among various conformational states in both ATP- and ADP-actins, with the nucleotides impacting the equilibria distributions.
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Project description:Mitochondrial dysfunction is pivotal in drug-induced acute kidney injury (AKI), but the underlying mechanisms remain largely unknown. Transport proteins embedded in the mitochondrial inner membrane form a significant class of potential drug off-targets. So far, most transporter-drug interactions have been reported for the mitochondrial ADP/ATP carrier (AAC). Since it remains unknown to what extent AAC contributes to drug-induced mitochondrial dysfunction in AKI, we here aimed to better understand the functional role of AAC in the energy metabolism of human renal proximal tubular cells. To this end, CRISPR/Cas9 technology was applied to generate AAC3-/- human conditionally immortalized renal proximal tubule epithelial cells. This AAC3-/- cell model was characterized with respect to mitochondrial function and morphology. To explore whether this model could provide first insights into (mitochondrial) adverse drug effects with suspicion towards AAC-mediated mechanisms, wild-type and knockout cells were exposed to established AAC inhibitors, after which cellular metabolic activity and mitochondrial respiratory capacity were measured. Two AAC3-/- clones showed a significant reduction in ADP import and ATP export rates and mitochondrial mass, without influencing overall morphology. AAC3-/- clones exhibited reduced ATP production, oxygen consumption rates and metabolic spare capacity was particularly affected, mainly in conditions with galactose as carbon source. Chemical AAC inhibition was stronger compared to genetic inhibition in AAC3-/-, suggesting functional compensation by remaining AAC isoforms in our knockout model. In conclusion, our results indicate that ciPTEC-OAT1 cells have a predominantly oxidative phenotype that was not additionally activated by switching energy source. Genetic inhibition of AAC3 particularly impacted mitochondrial spare capacity, without affecting mitochondrial morphology, suggesting an important role for AAC in maintaining the metabolic spare respiration.
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