Nuclear magnetic resonance investigation of the state of water in protein solution.

The line width of the NMR signal of water protons in solutions of native actomyosin and actomyosin denatured by heat, acetone or urea was measured over the temperature range from -10 degrees to below the freezing point. The line widths of the water band which increased exponentially with decreasing temperature were compared with each other and also with those of the corresponding control solution without actomyosin. The line broadening observed for native actomyosin solution on lowering the temperature was significantly smaller than that for heat-denatured actomyosin solution. This difference implies that this signal is sensitive to conformational perturbations of the protein. In addition, the temperature dependence of the line width for heat-, acetone-, or urea-denatured actomyosin solution was similar to that for the corresponding control solution. These phenomena can be interpreted in terms of the state of water associated with the hydrophobic and hydrophilic residues. Similar NMR studies of actomyosin solution containing dimethyl sulfoxide (DMSO) or dimethylformamide (DMF) showed that DMSO and DMF prevent the formation of ice crystals until about -70 degrees, suggesting that the cryoprotective effects of DMSO and DMF are due to the change in the state of water described above. These differences in temperature dependence between the sample and control solutions are well-correlated with the viscosity of the solution. This correlation is useful for elucidation of the mechanism of the protein denaturation.

Effects of changes of ionic environment on the negative after-potential of the spike in rat uterine muscle.

1. The spontaneous activity of the smooth muscle of rat uterus consists of bursts of spikes, each spike being followed by a negative after-potential. The effect of changes in ionic environment on the negative after-potential was investigated at various stages of pregnancy and in the non-pregnant condition.2. The amplitude of the negative after-potential was the same in spontaneously generated and electrically evoked spikes. During repetitive discharge, whether spontaneous or in response to depolarizing current application, the amplitude of the after-potential was smallest in the first spike of a burst and it increased gradually with repetition of discharge.3. The decay of the negative after-potential was slower than the passive return of the membrane potential to its resting level.4. The amplitude of the negative after-potential was larger in non-pregnant uterus than during late pregnancy.5. In pregnant uterus, the replacement of the Cl in the medium with benzene sulphonate transiently augmented the negative after-potential and then gradually reduced it. Eventually, the negative after-potential disappeared and, instead, a positive after-potential was observed. This conversion took place without a noticeable change in the resting potential or in the initial falling phase of the action potential itself. Replacement of Cl with NO(3) had no appreciable effect on the negative after-potential.6. In non-pregnant uterus, the conversion of the negative to a positive after-potential was never observed. However, in Cl-deficient solution the size and duration of the negative after-potential were reduced.7. In Cl-deficient solution (benzene sulphonate substitution), the decay of the electrotonic potential following the break of cathodal current became faster than that in normal solution. On the other hand the development of the anodic electrotonic potential became slower.8. Replacement of the NaCl in the medium with sucrose converted the negative after-potential to a positive after-potential. On the other hand, reduction of Na only by replacement of NaCl with Tris-Cl had no noticeable effect on the negative after-potential.9. It is concluded that the negative after-potential of the spike in rat uterine muscle is largely due to an increase of Cl conductance of the membrane.

An estimate of the proportion of the resting membrane conductance of the smooth muscle of guinea-pog taenia coli attributable to chloride.

1. The contribution of Cl and K to the steady-state membrane conductance of the smooth muscle of the guinea-pig taenia coli has been investigated by measuring the increase in membrane resistance in Cl-deficient and K-deficient solutions. A large external electrode was used for passing current and the voltage change was recorded with an intracellular electrode. The experiments were carried out in hypertonic solution in order to suppress spontaneous activity.2. When the tissue was exposed to K-deficient solution, the amplitude and time course of the electrotonic potential was increased and the membrane was hyperpolarized.3. When the tissue was exposed to Cl-deficient solutions in which Cl was replaced with an impermeant anion, benzene sulphonate, the size and time course of the electrotonic potential was increased whereas the membrane potential, after 20-30 min equilibration, was unchanged. The magnitude of the increase of the electrotonic potential depended on the substituted amount of Cl.4. When the tissue was exposed to low K the relation between the membrane conductance and the remaining external K concentration was the same in the presence of the normal Cl concentration and in Cl-deficient solution (7 mM-Cl). It was therefore concluded that any effect of benzene sulphonate on the K conductance is negligible.5. The effects of Cl or K deficiency on the electrotonic potential indicate that Cl and K ions account for most of the membrane conductance and that the contribution by Cl is about 40%.6. The ratio of P(Cl)/P(K) was calculated from the constant field theory on the assumption that the contribution of ions other than K and Cl to the membrane conductance is negligibly small. The mean value was 0.40.