Tension response in skinned Ca-activated skeletal muscle fibers of the frog to temperature jump following stepwise changes in the fiber length

Joulean temperature jump from 4-7 degrees to 20-25 degrees completed in 0.2 ms was applied to suspended in the air chemically skinned Ca-activated (pCa = 5.5-6) skeletal muscle fibres of the frog 2 ms after stepwise length changes (duration 0.3 ms, amplitudes --6. +3 nm per half sarcomere). The temperature jump induced a biphasic rise of tension, as was described earlier. Neither the time constant of the 2nd slow phase, nor maximum tension after the temperature jump were dependent on the length step amplitude. The amplitude and time constant of the 1st phase (1.2-0.28 ms) decreased after the fibre release. It shows that the 1st phase of the tension rise induced by the temperature jump is due to conformation in cross-bridges attached to thin filaments.     

A spin label study of rat brain membranes. Effects of temperature and divalent cations.

Rat brain myelin, synaptosomal plasma membranes and synaptic vesicles were spin labelled with stearic acid nitroxide derivatives. Their electron spin resonance spectra were studied as a function of temperature and devalent ions (Ca2+ and Mg2+) concentrations. (1) Synaptosomal plasma membranes and synaptic vesicles show identical temperature variations of their order parameter (S = 0.58 at 35 degrees C and S = 0.72 AT 22 DEGREES C). Myelin appears more rigid (S = 0.66 at 35 degrees C and S = 0.76 at 22 degrees C). A discontinuity of the order parameter variation as a function of temperature, is observed between 14.5 degrees C and l9.5 degrees C with the three types of membranes. (2) The hydrophobic core of these membranes is very fluid. No transition temperature is observed. The measured values of the spin label rotation correlation times and rotational activation energies are 2.1 and 2.8 ns at 35 degrees C and 3.1 and 3.6 kcal/mol respectively for synaptosomal plasma membranes and myelin. (3) Ca2+ enhances the membrane rigidity (12+/-0.7% increase of the order parameter at 35 degrees C in the presence of 10(-3) M Ca2+) and increases the transition temperature. At a lower extend, similar effects are observed with Mg2+.     

Effects of divalent cations,temperature, osmotic pressure gradient,and vesicle curvature on phosphatidylserine vesicle fusion

Summary Fusion of phosphatidylserine vesicles induced by divalent cations, temperature and osmotic pressure gradients across the membrane was studied with respect to variations in vesicle size. Vesicle fusion was followed by two different methods: 1) the Tb/DPA fusion assay, whereby the fluorescent intensity upon mixing of the internal aqueous contents of fused lipid vesicles was monitored, and 2) measurement of the changes in turbidity of the vesicle suspension due to vesicle fusion. It was found that the threshold concentration of divalent cations necessary to induce vesicle fusion depended on the size of vesicles; as the diameter of the vesicle increased, the threshold value increased and the extent of fusion became less. For the osmotic pressure-induced vesicle fusion, the larger the diameter of vesicles, the smaller was the osmotic pressure gradient required to induce membrane fusion. Divalent cations, temperature increase and vesicle membrane expansion by osmotic pressure gradient all resulted in increase in surface energy (tension) of the membrane. The degree of membrane fusion correlated with the corresponding surface energy changes of vesicle membranes due to the above fusion-inducing agents. The increase in surface energy of 9.5 dyn/cm from the reference state corresponded to the threshold point of phosphatidylserine membrane fusion. An attempt was made to explain the factors influencing fusion phenomena on the basis of a single unifying theory.     

Lipopolysaccharide bilayer structure: effect of chemotype, core mutations, divalent cations, and temperature.

Lipopolysaccharide (LPS), the primary lipid on the surface of Gram-negative bacteria, is thought to act as a protective and permeability barrier. X-ray diffraction analysis of osmotically stressed LPS multilayers was used to determine the structure and interactive properties of LPSs from strains containing the minimum number of sugars necessary for bacterial survival (Re chemotype) to the maximum number of sugars found in rough bacteria (Ra chemotype). At 20 degrees C in the absence of divalent cations, LPS suspensions gave a sharp wide-angle reflection at 4.23 A and a broad low-angle band centered at 50-68 A depending on the chemotype, indicating the presence of gel phase bilayers separated by large fluid spaces. As osmotic pressure was applied, the apposing bilayers were squeezed together and lamellar diffraction at 6 A resolution was obtained. At low applied pressures (<10(6) dyn/cm2), the total repulsive pressure between bilayers could be explained by electrostatic double layer theory. At higher applied pressures, there was a sharp upward break in each pressure-distance relation, indicating the presence of a hydrophilic steric barrier whose range depended strongly on the LPS chemotype. The positions of these upward breaks, along with electron density profiles, showed that the sugar core width systematically increased from 10 A for the Re chemotype to 27 A for the Ra chemotype. In excess buffer, the addition of divalent cations brought the bilayers into steric contact. Electron density profiles were used to determine the locations of cation binding sites and polar substituents on the LPS oligosaccharide core. The area per hydrocarbon chain was approximately 26 A2 in liquid-crystalline LPS bilayers, an indication of an acyl chain packing that is much tighter than that found in bilayers composed of typical membrane lipids. This unusually tight packing could be a critical factor in the permeability barrier provided by LPS.     

Kinetics of promoter search by Escherichia coli RNA polymerase. Effects of monovalent and divalent cations and temperature

The rapid mixing/photocross-linking technique developed in our laboratory has been employed in the study of the mechanism of promoter binding by Escherichia coli RNA polymerase (RPase). We have previously reported on the quantitation of the one-dimensional diffusion coefficient (D1) for RPase along the DNA template (Singer, P. T., and Wu, C.-W. (1987) J. Biol. Chem. 262, 14178-14189). In this paper, we describe the effect of salt concentration and temperature on the kinetics of promoter search by RPase using plasmid pAR1319 DNA, which contains the A2 early promoter from bacteriophage T7, as template. Over a range of KCl concentrations from 25 to 200 mM, the apparent bimolecular rate constant (ka) for the association of RPase with the A2 promoter on this DNA template varied approximately 2-fold, achieving a maximal value between 100 and 125 mM KCl. More significantly, the transient distribution of RPase among nonspecific DNA binding sites changed markedly as a function of salt concentration, indicative of gross changes in the average number of base pairs covered by sliding during a nonspecific lifetime. Using the mathematical treatment outlined in our earlier report, the nonspecific dissociation rate constant (koff) was calculated from the binding curves for the nonspecific as well as promoter-containing DNA. The observed variations in ka as a function of monovalent cation concentration ([M+]) were due primarily to changes in koff, as D1 was found to be essentially independent of [M+]. Interestingly, D1 decreased by one-third as the concentration of magnesium was lowered from 10 to 1 mM. In addition, the dependence of koff (and consequently the nonspecific equilibrium association constant, keq) on [M+] agreed qualitatively with the results of deHaseth et al. (deHaseth, P.L., Lohman, T. M., Burgess, R. R., and Record, M. T., Jr. (1977) Biochemistry 17, 1612-1622), though we consistently measure a weaker Keq. The association rate constant was also measured between 4 and 37 degrees C, and was found to vary approximately 2-fold over that range. An activation energy for the bimolecular association of RPase to the A2 promoter was calculated to be 2.2 +/- 0.4 kcal/mol, while the activation energy for one-dimensional diffusion was 4.7 +/- 0.8 kcal/mol.     

The effects of temperature, local anaesthetics, pH, divalent cations, and group-specific reagents on repriming and repolarization-induced contractures in frog skeletal muscle.

Contractures appear during repolarization of frog toe muscles in media containing perchlorate in place of chloride. These contractures were suppressed or delayed by certain procedures which retard the repriming of K contractures, i.e., by sufficient reduction in temperature or by alkaline pH in solutions lacking divalent cations. They also were greatly reduced without interference with repriming after treatment with a reagent which selectively modifies free amino groups. In the presence of appropriate concentrations of procaine, repriming was markedly impaired with only a small reduction in the amplitude of repolarization-induced contractures. Small contractures were produced during repolarization in chloride solutions in the presence of 10 mM procaine at pH 8.0. None of these procedures affected the changes produced by perchlorate solutions in the potential dependence and the time course of K contractures. The results support the view that activation and inactivation of contraction following depolarization are separate potential dependent processes. Tension appears to develop during repolarization when the reversal of inactivation occurs before the reversal of activation is completed, both steps being necessary to recover the reprimed resting state.     

Effects of Ca2+ and other divalent cations on K+-evoked force production of slow muscle fibers from Rana esculenta and Rana pipiens

Summary Slow muscle fibers were dissected from cruralis muscles of Rana esculenta and Rana pipiens. Isometric contractures were evoked by application of K⁺-rich Ringer's containing Ca²⁺, Ni²⁺, Co²⁺, Mn²⁺ or Mg²⁺. High (7.2 mmol/liter) external Ca²⁺ concentration raised, 0 Ca²⁺ lowered the K⁺ threshold. Replacing Ca²⁺ by Ni²⁺ or Co²⁺ had an effect similar to that of high Ca²⁺ Ringer's. In Mg²⁺ Ringer's the K⁺ concentration-response curve was flattened. These effects were observed already after short exposure times in both species of slow fibers. When Ca²⁺ was removed for long periods of time the slow fibers of R. esculenta lost their contractile response to application of high K⁺ concentrations much more quickly than those of R. pipiens, while the response to caffeine (20 mmol/liter) was maintained. Upon readmission of Ca²⁺ contractile ability was quickly restored in the slow fibers of both R. esculenta and R. pipiens, but the effects of Ni²⁺ (or Co²⁺, Mn²⁺ and Mg²⁺) were much larger in R. esculenta than in R. pipiens slow fibers. It is concluded that divalent cations have two different sites of action in slow muscle fibers. K⁺ threshold seems to be affected through binding to sites at the membrane surface; these sites bind Ni²⁺ and Co²⁺ more firmly than Ca²⁺. The second site is presumably the voltage sensor in the transverse tubular membrane, which controls force production, and where Ca²⁺ is the most effective species of the divalent cations examined.We are grateful to Mrs. S. Pelvay for technical assistance.     

Lanthanide binding to cardiac and skeletal muscle microsomes. Effects of adenosine triphosphate, cations, and ionophores.

Lanthanide (gadolinium, Gd) binding to cardiac and skeletal muscle microsomes was studied, and high- and low-affinity sites were identified. The high-affinity constant was 10⁶ M^?1, and there were 131 and 107 nmol/mg bound to this site in dog heart and rabbit skeletal muscle, respectively. Zn²⁺, Cd²⁺, Al³⁺, and Ca²⁺ (5 mm) inhibited binding, especially of the high-affinity site. Ionophores X537A (10 μm) and A23187 (1–2 μm) increased lanthanide binding and did not cause release. Addition of ATP in low concentration (20–50 μm) increased the binding of Gd without hydrolysis of the ATP. The extra binding induced by ATP was blocked by heating the microsomes and was reversed by [ethylenebis(oxyethylenenitrilo)]tetraacetic acid. High concentrations (10^?4–10^?3, m) of ATP blocked extra Gd binding by competitive chelation. The Ca²⁺-activated ATPase was inhibited by Gd and stimulated by X537A. The Gd did not block the ionophore-stimulated increase in Ca²⁺-ATPase activity. It is postulated that lanthanides bind predominantly to the ionophoric component of the Ca-transport site rather than the hydrolytic site and that ATP may facilitate such binding without being split.