Effects of temperature and field strength on the NMR relaxation times of Na in frog striated muscle

Pulsed NMR techniques have been applied to the study of the relaxation parameters characterizing ²³Na within frog striated muscle. Experiments were performed at 3°C, 22–24°C and 39°C at a Larmor frequency of 15.7 MHz; at 22–24°C, measurements were obtained both at 15.7 MHz and at 7.85 MHz.As previously reported, only a single spine-lattice relaxation time (T₁) was observed, but both slow (T₂)_I and fast (T₂)_II components of the spin-spin relaxation time were measured. The effect of temperature (θ) upon (1/T₁) was qualitatively similar to that reported for ²³Na in free solution; (θ) did not significantly affect (1/T₂) over the range of temperatures studied. (1/T₂)_I, and to a lesser degreee, (1/T₁) exhibited a modest inverse dependence of doubtful significance on the Larmor frequency.The data are examined within the framework of a simple specific model; a conservative values in assumed for the quadrupolar coupling constant characterizing immobilized intracellular Na⁺. Within this framework, the results suggest that the fraction of bound ions whose molecular tumbling is severely restricted does not exceed some few percent of the total sodium population.     

Specificity of Na+ binding to phosphatidylserine vesicles from A 23Na NMR relaxation rate study

²³Na NMR relaxation rate measurements show that Na⁺ binds specificially to phosphatidylserine vesicles and is displaced partially from the binding site by K⁺ and Ca²⁺ but to a considerably less extent by tetraethylammonium ion. The data indicate that tetraethylammonium ion affects the binding of Na⁺ only slightly, by affecting the surface potential through its presence in the double layer, without competing for a phosphatidylserine binding site. Values for the intrinsic binding constant for the Na⁺-phosphatidylserine complex that would be consistent with the competition experiments (and the dependence of the relaxation rate on concentration of free Na⁺) fall in the range 0.4–1.2 M^?1 with a better fit towards the higher values. We conclude that in the absence of competing cations in solution an appreciable fraction of the phosphatidylserine sites could be associated with bound Na⁺ at 0.1 M Na⁺ concentration.     

Kinetics of monensin complexation with sodium ions by 23Na NMR spectroscopy

The kinetics of the sodium binding to the ionophore monensin (Mon) in methanol has been studied by ²³Na NMR spectroscopy. Fast quadrupole relaxation of the bound sodium affected the relaxation rate of the free sodium through an exchange process between these two species. The exchange was found to be dominated by the reaction: Na⁺ + Mon^? ? MonNa. The dissociation rate constant at 25°C is 63 s^?1, with an activation enthalpy of 10.3 $\text{kcal}\text{mol}$ and activation entropy of ?15.8 $\text{cal}\text{mol}$ deg. These results indicate that the specificity of the binding of sodium ions to monensin is reflected in the relatively slow dissociation process. The entropy changes indicate that the activated monensin-sodium complex undergoes a conformational change, but the existence of a conformational change in monensin anion prior to complexation is excluded.     

A 59Co NMR relaxation probe of macromolecule anionic binding sites

⁵⁹Co NMR linewidth measurements are reported which show that hexacyanocobaltate-(III) ion may be used as a sensitive probe of protein interactions with anions in aqueous solutions. Applications are demonstrated to bovine serum albumin where the probe ion binding is monitored as a function of pH and is displaced from the protein sites by hexacyanoferrate(III) ion. The general utility of complex metal ions is suggested as a generally useful approach to the analysis of ion-macromolecule interactions.     

Pulsed NMR studies of water in striated muscle non-freezing factor of water II. Spin-lattice relaxation times and the dynamics of the non-freezing fraction of water

Spin-lattice relaxation times for the water protons in frog gastrocnemius muscle are reported over the temperature range 193 to 283 °K at Larmor frequencies of 30 and 60 MHz. Results of measurements under similar conditions of the transverse relaxation times are also reported. The relaxation times of the non-freezing 20% of the muscle water are interpreted in terms of water molecules, absorbed on or interacting with the proteins, and which are undergoing anisotropic motion, probably with a distribution of correlation times. Proton spin-lattice relaxation times are also reported for muscles under tension, the tension being produced by loading of the muscles with varying weights.     

Sodium-23 NMR relaxation times in nucleated red blood cells and suspensions of nuclei.

The relaxation behavior of intracellular 23Na in suspensions of chicken erythrocytes and of their nuclei was investigated. The transverse magnetization was found to decay biexponentially. The average relaxation rates for the nucleated chicken erythrocytes are considerably shorter than the average relaxation rates obtained for dog and human nonnucleated red blood cells. Of particular significance is the twofold decrease in the short component of T2. Calculations based on the measured 23Na NMR relaxation rates in suspensions of nuclei indicate that most of the difference between the relaxation rates in the mammalian as compared to the chicken erythrocytes, can be accounted for by the contribution of the nuclei in the latter.     

Effect of external sodium and calcium on calcium efflux in frog striated muscle

Summary The effect of media with different ionic composition on calcium efflux from the dorsal head of semitendinosus muscles ofRana pipiens was studied. The reduction in the fractional loss of⁴⁵Ca, when going from normal Ringer's solution to an ONa–OCa medium, was 60%. Withdrawal of only Na or Ca from the external medium also caused a significant drop in the fractional loss (33% and 34%, respectively). The effect of different concentrations of Ca (studied in the absence of the external Na) was also studied. It was found that a linear function could describe the relationship between the calcium-dependent calcium efflux and the external calcium concentration. These results indicate that calcium efflux from frog muscle fibers consists of three major components: one that is dependent on the presence of calcium in the external medium, one that is dependent on the presence of sodium in the external medium, and one that persists in the absence of these two cations.     

67Zn and 1H NMR studies of Zn2+-imidazole and carboxylate complexes

⁶⁷Zn NMR studies of naturally abundant Zn²⁺-imidazole and carboxylate ligands Complexes are shown. Thus, quadrupolar relaxation changes in ⁶⁷Zn NMR caused by adding imidazole ligands are more remarkable than those by carboxylate ligands. The changes caused by adding less bulky imidazole ligands are more prominent than those caused by a bulky imidazole ligan. Changes in Zn²⁺ quadrupolar relaxation rate caused by adding a cyclic hexapeptide consisting of L-histidine, L-cystein(Acm) and D-leucine are larger than those by a corresponding linear hexapeptide. Those changes in the quadrupolar relaxation rate of ⁶⁷Zn NMR among Zn²⁺ complexes can be reasonably interpreted in terms of the differences of equilibrium constants of those complexes to a first approximation.     

Molecular mobilities in chromaffin granules magnetic field dependence of proton T1 relaxation times

The magnetic field dependence of the NMR spin-lattice relaxation time of water protons in intact bovine chromaffin vesicles has been studied over the range 1.00–23.49 kG. The T₁ relaxation time shows a dispersion a t field values near 20 kG. The observed proton resonance arises mainly from solvent protons (¹H₂O), but the relaxation rate, which is a weighted average over all sites with which the solvent protons rapidly exchange (i.e., NH and OH protons), is dominated by exchangeable protons in the most slowly moving soluble component. The field dependence of the T₁ dispersion demonstrates the existence of a site of exchangeable protons for which τ_r = 1.9±0.5 ns at 3°C. This site is assigned to ATP and cationic groups to which its phosphate esters are complexed, since previously measured correlation times of epinephrine and the chromogranin backbone are nearly an order of magnitude too short to explain the T₁ dispersion. Quantitative estimates of the relative numbers of exchangeable protons on the different soluble components support this interpretation. The temperature dependence of T₁ of the peak due to exchangeable protons has also been measured over a temperature range ?3 to 25°C. T₁ lengthens by about 30% over this range and exhibits no discontinuous behavior, as would be expected if a gel transition or structural alterations in the storage complex occurred. T₁ lengthens by less than 10% in chromaffin granule pastes that have been maintained at 25°C for 24 h, indicating considerable thermal stability in the storage complex. Possible effects on the solvent T₁ due to paramagnetic ions have been considered with the conclusion that they are probably negligible or of minor significance.     

等量分次施氮对冬枣15N和13C利用与分配特性的影响

以4年生盆栽冬枣为试材,采用¹³C、¹⁵N双标记示踪技术,在果实发育期研究了等氮量分次追施氮肥对冬枣植株¹⁵N和¹³C吸收、利用、积累和分配的影响.结果表明: 至果实采收期,冬枣各器官Ndff值(植株器官从肥料中吸收分配到的¹⁵N量对该器官全氮量的贡献率)随追氮次数的增多而显著增大.生殖器官(果实)和营养器官(叶片、枣吊、新生枣头枝和细根)的¹⁵N分配率以4次追氮处理最高,1次追氮处理最低,贮藏器官(主干、多年生枝和粗根)¹⁵N分配率的趋势相反;4次追氮处理¹⁵N利用率分别比1次和2次追氮处理高27.4%和15.5%.追氮次数越多,植株总氮量和¹⁵N吸收量越大;随时间的推移,1次追氮处理土壤¹⁵N丰度和总氮含量持续降低,2次追氮处理呈先升高后降低的趋势,4次追氮处理变化相对最为平稳,至处理后期显著高于其他处理;果实白熟至采收期,叶片叶绿素、氮含量和净光合速率均表现为4次追氮>2次追氮>1次追氮.不同处理¹³C同化物积累与分配不同.4次追氮处理¹³C固定总量分别是1次和2次追氮处理的1.1和1.2倍.增加追氮次数,促进了¹³C同化物向果实和贮藏器官的转移,而减少了向当年生营养器官的分配.综上,果实发育期4次追氮通过保证根层稳定、充足的氮素供应,提高了对氮素的吸收和利用,进而维持了较高的净光合速率,促进并优化了光合同化物的积累和分配,最有利于冬枣树体的生长及产量和品质的提高.     

NMR spin-lattice relaxation times of intracellular Na-23 on rat livers and related lipid peroxidation following CCl4 intoxication

Liver tissues were isolated from rats acutely intoxicated with carbon tetrachloride, and Na-23 NMR signals were analyzed to investigate the T1 relaxation times of intracellular sodium ions under pathological conditions in presence of the paramagnetic shift reagent (dysprosium tripolyphosphate). We studied the significant increase of T1 found in CCl4 treated rats with respect to controls, which was elsewhere demonstrated as being independent of cell necrosis. Evidence is given that neither fat accumulation nor proliferative processes affect the observed T1 lengthening. When T1 relaxation times were measured in the liver of vitamin E treated rats subsequently intoxicated with carbon tetrachloride, a significative shortening of T1 with respect to CCl4-intoxicated rats was observed. These results were discussed in terms of the antioxidant action exerted by vitamin E, taking into account that peroxidation of microsomal lipids is the key factor in the process of carbon tetrachloride induced liver injury. Furthermore, the observed T1 changes were discussed in terms of the interactions of Na+ with cell membranes and/or the occurrence of viscosity changes.     

Effects of prostaglandin E1 on contractility and 45Ca release in rabbit aortic smooth muscle

Concentrations of prostaglandin E₁ (PGE₁; 10^?7 M) that do not elicit tension responses in aortic strips potentiate contractions induced by submaximal concentrations (10^?8 ? 10^?7 M) of norepinephrine (NE) or angiotensin III (Ang III) but not those of high K⁺ depolarization or maximal NE or Ang III concentrations. Higher concentrations of PGE₁ (10^?6 M and above) initiate contractions which are additive with submaximal responses to NE and Ang III but not to K⁺. These same concentrations of PGE₁ also decrease ⁴⁵Ca retention at high affinity La⁺⁺⁺-resistant sites in a manner similar to but not additive with NE and Ang III. Uptake of ⁴⁵Ca at low affinity La⁺⁺⁺-resistant sites (which is increased by high K⁺-depolarization) is not altered by 10^?6 M PGE₁. The effects of PGE₁ are not altered by decreased extracellular Ca⁺⁺ (0.1 mM), decreased temperature, phentolamine or meclofenamate. Thus, PGE₁ does not appear to increase uptake of extracellular Ca⁺⁺ in this smooth muscle tissue. Instead, PGE₁ increases mobilization of Ca⁺⁺ from the same high affinity La⁺⁺⁺-resistant sites affected by Ang III and NE and, in this manner, may increase responses to these two stimulatory agents.     

13C NMR studies on fluorescent probes: 13C chemical shifts and longitudinal relaxation times of n-hydroxy-fatty (n=2,6,9,12) acids and n-(9-anthroyloxy)-stearic (n=6,12) acids

¹³C NMR has been used to confirm the structure of two fluorescent probes, n-(9-anthroyloxy)-stearic acids (n=6,12), and the series of n-hydroxy-fatty acids (n=2,6,9,12) from which the set of fluorescent fatty acids may be synthesised. ¹³C longitudinal relaxation times and correlation times of the individual carbon atoms in 12-hydroxy- and 6- and 12-(9-anthroyloxy)-stearic acids show differences in motional properties between these derivatives and the parent stearic acid in chloroform(d) solution. The correlation times of the substituted carbons in 6-, 9-, and 12-hydroxy-stearic acids are longer than the corresponding carbons in stearic acid. The change in correlation times at the substituted carbons reflects the increase in motion along the acyl chain. Attachment of the bulky anthracene ring causes greater restriction of motion at the substituted carbon atom but the gradient of motion along the chain is preserved. These results are discussed in terms of the types of motion which lead to fluorescence depolarization when the fluorescent fatty acids are used as fluidity probes in biomembranes.