Changes of isolated cardiac muscle function in response to extracellular sodium reduction.

The effects of low extracellular sodium concentration [( Na+]0, 76 mmol/l) on force of contraction, transmembrane action potentials and on calcium, sodium and potassium contents were studied in guinea-pig heart muscle using sucrose or lithium as substitutes for sodium. In papillary muscle, the positive inotropic response to low [Na+]0 was accompanied by shortening in action potential duration, in atrial muscle, prolongation at 90% of repolarization was observed. After 30 min. in low [Na+]0 solution (Ca2+ 1.8 mmol/l), the net uptake of calcium in left atria was larger in sucrose- than in lithium-substituted solution, i.e. 0.45 and 0.2 mmol Ca2+/kg wet weight, respectively. The net sodium content decreased monophasically; but the potassium content did not change consistently. The uptake of lithium by the atria was at least partially compensated for by a loss in potassium and by a transient decrease in sodium content. Increase in stimulation frequency from 0.1 to 1 Hz accelerated the time course of change by a factor of 2. In low [Na+]0, post-rest adaptation of twitch tension appeared abolished in atria because of high amplitude contractions. The recovery pattern of post-rest twitch amplitude was greatly accelerated in papillary muscle. In conclusion, low [Na+]0 elevates the cellular calcium content; this extra calcium is probably located in cellular stores that are involved in the regulation of twitch amplitude.

The core subunit structure in RNA polymerase holoenzyme determined by neutron small-angle scattering.

The core subunit arrangement of alpha 2-beta-beta' within DNA-dependent RNA polymerase holoenzyme alpha 2 beta beta' sigma from Escherichia coli was investigated by neutron small-angle scattering using label triangulation. The quaternary structure of multisubunit biomolecules can be studied by this new method if total reconstitution works in a quantitative way and if extensive replacement of C-bound hydrogen (H) by deuterium (2H) is possible. A substitution of the selected subunits by their fully deuterated analogues was used for the analysis of the overall shapes of the core subunits, alpha 2, beta and beta' in situ and for the determination of the intersubunit centre-to-centre distances. The contrast between the buffer and the remaining 'hydrogenated' enzyme vanishes if the buffer contains 42% 2H2O (matching of scattering length densities). The isotopic hybridization of the enzyme fulfils the conditions of isomorphous replacement as required: molecular functions, like enzyme activity, were completely preserved. The orientations of the core subunits within the holoenzyme were derived by comparing theoretical and experimental pair distance distribution functions, P(r), obtained from the scattering intensity differences of the pair-labelled (e.g. both beta and beta' labelled) and both mono-labelled molecules by direct Fourier transformations. Additional, the subunit shapes were refined by P(r) analyses. The arrangement of the stable core structure within the holoenzyme, which contains sigma as a dissociable factor, is presented in a three-dimensional model.

The subunit positions within RNA polymerase holoenzyme determined by triangulation of centre-to-centre distances.

The complete 'centre-of-subunit structure' of the multisubunit enzyme DNA-dependent RNA polymerase was determined by triangulation of the subunit positions using the intersubunit distances calculated from scattering difference measurements and from the corresponding radii of gyration R. In addition to the centre-to-centre distances d between the core subunits alpha 2, beta and beta' presented in the preceding paper, the values of d between initiation factor sigma and alpha 2 (8.4 +/- 1.6 nm), beta (4.4 +/- 2.2 nm) and beta' (10.7 +/- 1.5 nm) were derived from R of sigma (4.1 +/- 0.3 nm) in situ and of the pairs alpha 2--sigma (6.1 +/- 0.4 nm), beta--sigma (5.6 +/- 0.3 nm) and beta'--sigma (7.5 +/- 0.4 nm) within the holoenzyme (alpha 2 beta beta' sigma). The structural parameters of the subunits within their molecular complex are accessible for neutron small-angle scattering measurements using labelling of the different subunits (deuteration), total reconstitution of isotopic hybrids, scattering length density matching of 'hydrogenated' molecular parts and extended exposure times because of weak scattering effects. The overall shape of sigma bound to core enzyme (alpha 2 beta beta') proved to be identical (within experimental resolution) with sigma in the isolated state measured recently by X-ray small-angle scattering. The refined shape of isolated sigma was reduced to an ellipsoid which was orientated with respect to the core structure (alpha 2--beta--beta') in a 'space-filling' way around the position of the sigma centre obtained by triangulation. The complete subunit arrangement of holoenzyme is shown in a three-dimensional model.

The conformer nature of the multiple forms of beef liver catalase as obtained by biochemical and small-angle X-ray scattering experiments. A model for the quaternary structure of the beef liver catalase molecule.

Two of the five multiple forms of beef liver catalase have been extensively studied using biochemical and biophysical analysis techniques. The two molecules, cat I and cat V, have different isoelectric points (pH 6.55 and 5.6), different surface charges (25.8 and 32.7 elementary charges) and display different numbers of primary amino groups on their surfaces. The numbers of tyrosine residues on the surfaces of the two molecules are also different (16 and 10 at pH 10). Since the two forms of catalase can be interconverted, the described changes may be caused by conformational changes of the four protein subunits within the molecule. This mobility of the polypeptide chains is also demonstrated by the different absorption spectra below 390 nm. Using small-angle X-ray scattering, the radii and the volumes of the two catalase forms were shown to be different (cat 1 is smaller than cat V). All five multiple forms of beef liver catalase are concormers of the molecule. A model for the quaternary sturcture of the beef liver catalase are conformers of the molecule. A model for the quaternary structure of the beef liver catalase molecule is suggested. It consists of a regular configuration of four prolate rotational ellipsoids (semiaxes: a = 52 A, b = c = 21 A) in close contact in which the nearest neighbour subunits are shifted by 37 A parallel to each other. Thus the height of the complete molecule is 141 A and the diameter 94 A.

Neutron scattering measurements with the label triangulation method on the 50 S subunit of E. coli ribosomes.

In the E. coli 50 S ribosomal subunit, proteins L7/L12 and L10 were deuterated by partial reconstitution. The distance between L7/L12 and L10 was measured by the label triangulation method and was found to be approximately 100 A or, with low probability, 60 to 70 A, depending on the concentration.