Pulsed nuclear magnetic resonance study of 39K in frog striated muscle.

Samples of 1 M KCl solution and 10 samples of intact frog striated muscle were studied at 4-7 degrees C and/or at 21-22 degrees C. Field inhomogeneity was minimized by using small sample volumes and by using a superconducting magnet designed specifically to provide highly homogeneous fields. In the present experiments, magnetic field inhomogeneity was measured to contribute less than 15% to the free induction decay observed for intracellular 39K. The signal-to-noise ratio of the measurements was enhanced by means of extensive time-averaging. The rates of nuclear relaxation for 39K in aqueous solution were 22 +/- 3 (mean +/- 95% confidence limits) s-1 at 4-7 degrees C and 15 +/- 2 s-1 at 21-22 degrees C. For intracellular 39K, (1/T2) was measured to be 327 +/- 22 s-1 and 229 +/- 10 s-1 at the lower and higher temperatures, respectively. The corresponding values for (1/T1) in the same muscle samples were 198 +/- 31 s-1 and 79 +/- 15 s-1 at 4-7 degrees C and at 21-22 degrees C, respectively. These results for 39K are similar to those previously obtained for intracellular 23Na. Since less than 1% of the intracellular 23Na has been estimated to be immobilized, fractional immobilization of intracellular 39K is also likely to be insubstantial.     

Pulsed nuclear magnetic resonance study of 17O from H217O in rat lymphocytes.

Lymphocytes obtained from thymus glands of normal rats and culture lines of malignant rat thymocytes were enriched with H217O. The longitudinal and transverse relaxations of the 17O were determined separately in samples of packed cells and supernatant solutions. The longitudinal relaxation of intracellular 17O of fresh viable lymphocytes was nonexponential, becoming simply exponential with eventual necrosis. The rate of spin-lattice relaxation (1/T1) was fitted by a sum of two exponentials. The average mole fraction of the molecules subject to the slower relaxation rate (1/T1s) was two-thirds of the total water. Lowering the Larmor frequency (omega) from 7.72 to 4.36 MHZ increased the faster component (1/T1f) by 12% without altering (1/T1s). The value of the single exponential decay of the nonviable cells was not appreciably different from the initial rate of relaxation of the fresh cells. Similar results were obtained in studies of the transverse relaxation rates. The simplest interpretation is that two-thirds of the cell water is located within the nucelus and is characterized by a slower rate of relaxation than the one-third of the cell water in the cytoplasm because of the different macromolecular compositions of the two-subcellular compartments. The malignant lymphocytes were characterized by prolonged values for the slow and fast components of both the longitudinal and transverse relaxations of 17O.     

Phosphorus nuclear magnetic resonance studies of phosphorus metabolites in frog muscle.

31P-nuclear magnetic resonance was applied to living muscles of bullfrogs, and the time courses of metabolic changes of ATP, creatine phosphate, inorganic phosphate, and sugar phosphates were studied under anaerobic and aerobic conditions. A decrease in creatine phosphate was observed in the resting muscle under anaerobic conditions with a concomitant decrease in the intracellular pH, while the ATP level remained constant. With the use of 2,4-dinitro-1-fluorobenzene and iodoacetic acid, ATP disappeared quickly. When the resting muscle was perfused with oxygen-saturated glucose-Ringer's solution, the amount of creatine phosphate increased gradually. These findings indicate that anaerobic glycolysis is insufficient for even the resting energy consumption whereas oxidative phosphorylation is sufficient. The effects of tetanic stimulation on living muscles were also studied. When glycolysis and oxidative phosphorylation were suppressed, the intracellular energy store was depleted by the tetanic contraction. Anaerobic glycolysis produced rapid recovery of the energy store level, although it was insufficient to reach the initial level. Aerobic oxidative phosphorylation produced sufficient energy to reach the initial level, and this level was never exceeded. This finding suggests the existence of a regulatory mechanism for the energy store level.     

Varied magnetic field, multiple-pulse, and magic-angle spinning proton nuclear magnetic resonance study of muscle water.

The nuclear magnetic resonance linewidth of 1H in water of frog muscle was studied as a function of magnetic field strength and angle of orientation. The results suggest that the observed spectra are dominated by demagnetization field anisotropy and dispersion, but a small static dipolar interaction of the order of a few hertz man be present. Data from line-narrowing, multiple-pulse experiments also indicate the presence of a small dipolar broadening.     

Pulsed nuclear magnetic resonance study of39K within Halobacteria

Summary The³⁹K contents of isolated pellets and supernatant solutions from suspensions ofHalobacterium halobium were studied at 21–22°C by pulsed NMR spectroscopy. The rates of transverse relaxation were measured directly from the free induction decay (FID). The rate of longitudinal relaxation was measured by studying the FID after pairs of pulses of approximately 90°. Care was exercised to minimize the effect of magnetic field inhomogeneity; its contribution to the FID was approximately 25–30 sec^–1. The transverse relaxation process was found to consist of at least two components, whose rates were 321–449 sec^–1 and 1,122–2,067 sec^–1. In one preparation where the longitudinal relaxation process was studied, the data could be well fit to a single exponential relaxing at 253±33 (mean ±95% confidence limits) sec^–1. Comparison of the relative intensities of the NMR signals with the results of atomic absorption photometric analyses indicated that the great bulk of the intracellular³⁹K was detected by the NMR techniques used. The data obtained from the current NMR ofH. halobium are consistent with: (1) fractional binding of <3% of the total intracellular K⁺, (2) a small ordering factor characterizing all of the intracellular K⁺, or (3) some combination of the two.     

Pulsed NMR studies on water in striated muscle III. The effects of water content

Pulsed NMR is used to study the kinetics of dehydration of frog gastrocnemius muscle. In addition, measurements are reported of the variation of the spin-lattice (T₁) and transverse (T₂) nuclear magnetic relaxation times of the water protons as a function of water content. The proton transverse relaxation and freezing properties of the water in muscles which had been dehydrated and then rehydrated are also investigated. Correlation of the double-exponential dehydration kinetics with the transverse relaxation at various water contents provides strong evidence for the evidence of a fraction of muscle water (10–20%) which is sufficiently strongly held to the solid substance of the muscle to make it relatively slowly removed under conditions of zero relative humidity but which is still dynamically very mobile on average. This is supported by the dependence of T₁ on water content. The relaxation times are interpreted qualitatively in terms of a number of possible effects which are at present not distinguishable. The properties of the dehydrated-rehydrated muscles indicated changes in the muscle proteins which affect the transverse relaxation of the water protons and the freezing properties of the muscle water.     

The activity of creatine kinase in frog skeletal muscle studied by saturation-transfer nuclear magnetic resonance.

1. The activity of creatine kinase in intact anaerobic frog muscle at 4 degrees C at rest and during contraction was investigated by using saturation-transfer 31P n.m.r. 2. At rest, the measured forward (phosphocreatine to ATP) reaction flux was 1.7 X 10(-3) M . s-1 and the backward flux was 1.2 X 10(-3) M . s-1. The large magnitude of both fluxes shows that creatine kinase is active in resting muscle, so the observed constancy of [phosphocreatine] demonstrates that the enzyme and its substrates are at equilibrium. 3. The apparent discrepancy between the fluxes must arise largely from an underestimation of the backward flux resulting from interaction of ATP with other systems, e.g. via adenylate kinase. For purposes of further calculation we have therefore adopted 1.6 X 10(-3) M . s-1 as an estimate of both fluxes. 4. During contraction, when the creatine kinase reaction is no longer at equilibrium, the net rate of phosphocreatine breakdown, estimated directly from the change in area of the inorganic phosphate peak, was 0.75 X 10(-3) M . s-1. Saturation transfer indicates that the forward reaction flux remains at approx. 1.6 X 10(-3) M . s-1 and the backward flux decreases to about 0.85 X 10(-3) M . s-1. 5. The activity of creatine kinase during contraction is large enough to account for the well-established observation that, during contraction, the concentration of ATP falls by less than 2-3%. The reaction catalysed by creatine kinase is driven forward during contraction by the large relative increase in the concentration of free ADP, which is more than doubled. 6. The observation that the forward flux does not increase during contraction and that the backward flux decreases can most simply be explained on the basis of competition of reactants for a limited amount of enzyme.     

Pulsed nuclear magnetic resonance studies of human bone marrow.

Pulsed nuclear magnetic resonance (NMR) studies of human bone marrow reveal that the proton spin-lattice relaxation times (T1) of acute myeloid leukemia (AML) and chronic myeloid leukemia (CML) are much greater than the T1 of normal bone marrow.     

C nuclear magnetic resonance study of 17α-substituted estradiols

We report the ¹³C NMR data for 20 compounds bearing a substituent (alkyl, alkenyl, alkynyl, alkylamide, spiro-γ-lactone, phenyl, benzyl, naphthyl, etc.) at the 17α-position of estradiol. The carbon assignments were done using 1D and 2D NMR experiments (distortionless enhancement by polarization transfer, homonuclear correlated spectroscopy, heteronuclear shift correlation, and heteronuclear shift correlation via long-range couplings). Only the chemical shifts of carbons 12–18, which surround the substitution site, were affected by the addition of a substituent. The magnitude of the effects (shielding or deshielding) was influenced by the 17α-substituent. The individual effects at these carbons were sufficiently distinctive to identify specific centers and should be valuable for signal assignment of a variety of 17α-derivatives of estradiol. In addition to carbon-skeleton assignment, we also report the carbon-substituent assignments.     

1H nuclear magnetic resonance double resonance study of oxytocin in aqueous solution.

Peptide NH resonances in the 250 MHZ 1H nuclear magnetic resonance (NMR) spectrum of oxytocin in H2O were assigned to specific amino acid residues by the "underwater decoupling" technique (i.e., decoupling from corresponding CalphaH resonances, which are buried beneath the intense water peak). These experiments confirm previous assignments of A. I. Brewster an V. J. Hruby ((1973), Proc. Natl. Acad. Sci. U.S.A. 70, 3806) and A. F. Bradbury et al. ((1974), FEBS Lett. 42, 179). Three methods of assigning NH resonances of peptides--solvent titration, underwater decoupling, and isotopic labeling--are compared. As the solvet composition is gradually changed from dimethyl sulfoxide to H2O, oxytocin undergoes a conformational change at 70-90 mol % of H2O. Exposure to solvent of specific hydrogens of oxytocin in H2O was studied by monitoring intensity changes of solute resonances when the solvent peak was saturated. Positive nuclear Overhauser effects (NOE's) of 14 +/- 5 were observed for the Tyr ortho CH and meta CH resonances, respectively. Comparative studies with deamino-oxytocin indicate that these effects result predominantly from intermolecular dipoledipole interaction between aromatic side chain CH protons and protons of the solvent. The NOE's therefore indicate intimate contact between water and the aromatic CH hydrogens of the Tyr side chain. The extent of saturation transferred by proton exchange between water and NH group varies with Ph in a manner which appears to reflect the acid-base catalysis of the protolysis reaction. There is no indication that any NH protons are substantially shiedled from the solvent.     

Study of anisotropy in nuclear magnetic resonance relaxation times of water protons in skeletal muscle.

The anisotropy of the spin-lattice relaxation time (T1) and the spin-spin relaxation times (T2) of water protons in skeletal muscle tissue have been studied by the spin-echo technique. Both T1 and T2 have been measured for the water protons of the tibialis anterior muscle of mature male rats for theta = 0, 55, and 90 degrees, where theta is the orientation of the muscle fiber with respect to the static field. The anisotropy in T1 and T2 has been measured at temperatures of 28, -5 and -10 degrees C. No significant anisotropy was observed in the T1 of the tissue water, while an average anisotropy of approximately 5% was observed in T2 at room temperature. The average anisotropy of T2 at -5 and -10 degrees C was found to be approximately 2 and 1.3%, respectively.     

Nuclear magnetic resonance transverse relaxation in muscle water.

The origin of the nonexponentiality of proton spin echoes of skeletal muscle has been carefully examined. It is shown that the slowly decaying part of the proton spin echoes is not due to extracellular water. First, for muscle from mice with in vivo deuteration, the deuteron spin echoes were also nonexponential, but the slowly decaying part had a larger weighing factor. Second, for glycerinated muscle in which cell membranes were disrupted, the proton spin echoes were similar to those in intact muscle. Third, the nonexponentiality of the proton spin echoes in intact muscle increased when postmortem rigor set in. Finally, when the lifetimes of extracellular water and intracellular water were taken into account in the exchange, it was found that the two types of water would not give two resolvable exponentials with the observed decay constants. It is suggested that the unusually short T2's and the nonexponential character of the spin echoes of proton and deuteron in muscle water are mainly due to hydrogen exchange between water and functional groups in the protein filaments. These groups have large dipolar or quadrupolar splittings, and undergo hydrogen exchange with water at intermediate rates. The exchange processes and their effects on the spin echoes are pH-dependent. The dependence of transverse relaxation of pH was observed in glycerinated rabbit psoas muscle fibers.     

The relationship between the transverse and longitudinal nuclear magnetic resonance relaxation rates of muscle water.

Whole frog sartorius and gastrocnemius muscles were incubated in Ringer's solutions, either unenriched or enriched with H2 17Oor 2D2O. Subsequently, the rates of transverse (1/T2) and of longitudinal (1/T1) nuclear magnetic relaxation were measured for 17O, 2D, and 1H at room temperature and at 8.1 MHz. The ratio (T1/T2) for 17O was measured to be approximately 1.5-2.0, close to the value roughly estimated from the Larmor frequency dependence of 1/T1 alone over the range 4.3-8.1 MHz. On the other hand (T1/T2) for 2D and 1H were both measured to lie in the range 9-11. Insofar as the entire 17O signal was detected, the data indicate the presence of an exchange mechanism between the major fraction of intracellular water and a minor fraction characterized by enhanced rates of relaxation. Possible molecular mechanisms are presented.     

Proton nuclear magnetic resonance study of cobrotoxin.

Cobrotoxin (Mr 6949), which binds tightly to the acetylcholine receptors, contains no phenylalanines and only two histidines, two tyrosines, and one tryptophan that result in well-resolved aromatic proton resonances in D2O at 360 MHz. His-32, Tyr-25, and the Trp are essential for toxicity and may interact with the acetylcholine receptor. We assign two titratable resonances (pKa = 5.1) at delta = 9.0 and 7.5 ppm at pH 2.5 and at 7.7 and 7.1 ppm at pH 9.5 to the C-2 and C-4 ring protons, respectively, of His-4. Two other titratable resonances (pKa = 5.7) at delta = 8.8 and 6.9 ppm at pH 2.5 and at 7.8 and 6.7 ppm at pH 9.5 are assigned to the C-2 and C-4 ring protons of His-32, respectively. The differences in delta values of the two histidines reflect chemically different microenvironments while their low pKa values could arise from nearby positive charges. A methyl resonance gradually shifts upfield to delta approximately 0.4 ppm as His-4 is deprotonated and is tentatively assigned to the methyl group of Thr-14 or Thr-15 which, from published X-ray studies of neurotoxins, are located in the vicinity of His-4. Further, we have identified the aromatic resonances of the invariant tryptophan and individual tyrosines and the methyl resonance of one of the two isoleucines in the molecule. Several broad nontitrating resonances of labile protons which disappear at pH greater than 9 may arise from amide groups of the beta sheet in cobrotoxin.