Studies on deoxyribonucleic acid-dependent ribonucleic acid polymerase from Escherichia coli. Variations of the enzyme activity during growth

1. RNA polymerase activity of Escherichia coli extracts prepared from cells in exponential and stationary phases of growth, when measured in the presence and absence of external template, showed significant qualitative differences. 2. In both extracts, polymerase activity was higher when assayed with external template, suggesting the presence of a pool of enzyme not bound to cellular DNA. 3. In the crude extract, the fraction of enzyme bound to cellular DNA is higher during the exponential phase of growth. 4. A method is described for the purification of enzyme molecules not tightly bound to cellular DNA from exponential- and stationary-phase cultures. 5. Purified enzyme preparations showed differences in template requirement and subunit composition. 6. On phosphocellulose chromatography of stationary-phase enzyme, a major portion of polymerase activity eluted from the column with 0.25m-KCl. In the case of exponential-phase enzyme, polymerase activity eluted from a phosphocellulose column mainly with 0.35m-KCl. 7. Enzyme assays done with excess of bacteriophage T(4) DNA showed a strong inhibition of stationary-phase enzyme by this template. The exponential-phase enzyme was only slightly inhibited by excess of bacteriophage T(4) DNA.     

On the enzyme histochemistry of the renal interstitial cells

Summary A histochemical study of the renal interstitial cells demonstrates the absence of two of the principal enzymes involved in the citric acid cycle, the cells thus seem to lack the ability to break down fatty acids. On the other hand the renal interstitial cells demonstrate enzyme activity which could be indirect evidence of fatty acid synthesis. A pronounced nodular acid phosphatase activity and unspecific esterase activity were noticed as well as an uniform cytoplasmic esterase activity.This work was supported by a grant from the Danish State Research Foundation.     

Formation of non-beta 6.3-helical gramicidin channels between sequence-substituted gramicidin analogues.

Using the linear gramicidins as an example, we have previously shown how the statistical properties of heterodimeric (hybrid) channels (formed between the parent [Val1]gramicidin A (gA) and a sequence-altered analogue) can be used to assess whether the analogue forms channels that are structurally equivalent to the parent channels (Durkin, J. T., R. E. Koeppe II, and O. S. Andersen. 1990. J. Mol. Biol. 211:221-234). Generally, the gramicidins are tolerant of amino acid sequence alterations. We report here an exception. The optically reversed analogue, gramicidin M- (gM-) (Heitz, F., G. Spach, and Y. Trudelle. 1982. Biophys. J. 40:87-89), forms channels that are the mirror-image of [Val1]gA channels; gM- should thus form no hybrid channels with analogues having the same helix sense as [Val1]gA. Surprisingly, however, gM- forms hybrid channels with the shortened analogues des-Val1-[Ala2]gA and des-Val1-gC, but these channels differ fundamentally from the parent channels: (a) the appearance rate of these heterodimers is only approximately 1/10 of that predicted from the random assortment of monomers into conducting dimers, indicating the existence of an energy barrier to their formation (e.g., monomer refolding into a new channel-forming conformation); and (b), once formed, the hybrid channels are stabilized approximately 1,000-fold relative to the parent channels. The increased stability suggests a structure that is joined by many hydrogen bonds, such as one of the double-stranded helical dimers shown to be adopted by gramicidins in organic solvents (Veatch, W. R., E. T. Fossel, and E. R. Blout. 1974. Biochemistry. 13:5249-5256).     

Molecular and channel-forming characteristics of gramicidin K's: a family of naturally occurring acylated gramicidins.

The gramicidin K family is a set of naturally occurring acylated linear peptides in which a fatty acid is esterified to the ethanolamine hydroxyl of either gramicidin A or C, and possibly also to gramicidin B (Koeppe, R. E., II, Paczkowski, J. A., & Whaley, W. L. (1985) Biochemistry 24, 2822-2826). These acylated gramicidins form membrane-spanning channels in planar lipid bilayers and therefore constitute a model system with which to study the structural and functional consequences of acylation on membrane proteins. This paper serves to characterize further the channels formed by acylated gramicidins A and C and to demonstrate that these channels are structurally equivalent to the channels formed by the standard gramicidins. We also present additional evidence for the ester linkage in the natural acylated gramicidins A and C and identify the fatty acyl chains.     

Cyanidin 3-[6-(p-coumaroyl)-2-(xylosyl)-glucoside]-5-glucoside and other anthocyanins from fruits of Sambucus canadensis.

From the fruits of Sambucus canadensis four anthocyanin glycosides have been isolated by successive application of an ion-exchange resin, droplet-counter chromatography and gel filtration. The structure of the novel, major (69.8%) pigment, cyanidin 3-O-[6-O-(E-p-coumaroyl-2-O-(beta-D-xylopyranosyl)-beta-D- glucopyranoside]-5-O-beta-D-glucopyranoside, was determined by means of chemical degradation, chromatography and spectroscopy, especially homo- and heteronuclear two-dimensional NMR techniques. The other anthocyanins were identified as cyanidin 3-sambubioside-5-glucoside (22.7%), cyanidin 3-sambubioside (2.3%) and cyanidin 3-glucoside (2.1%).     

Steady-state gating of batrachotoxin-modified sodium channels. Variability and electrolyte-dependent modulation.

The steady-state gating of individual batrachotoxin-modified sodium channels in neutral phospholipid bilayers exhibits spontaneous, reversible changes in channel activation, such that the midpoint potential (Va) for the gating curves may change, by 30 mV or more, with or without a change in the apparent gating valence (za). Consequently, estimates for Va and, in particular, za from ensemble-averaged gating curves differ from the average values for Va and za from single-channel gating curves. In addition to these spontaneous variations, the average Va shifts systematically as a function of [NaCl] (being -109, -88, and -75 mV at 0.1, 0.5, and 1.0 M NaCl), with no systematic variation in the average za (approximately 3.7). The [NaCl]-dependent shifts in Va were interpreted in terms of screening of fixed charges near the channels' gating machinery. Estimates for the extracellular and intracellular apparent charge densities (sigma e = -0.7 and sigma i = -0.08 e/nm2) were obtained from experiments in symmetrical and asymmetrical NaCl solutions using the Gouy-Chapman theory. In 0.1 M NaCl the extracellular and intracellular surface potentials are estimated to be -94 and -17 mV, respectively. The intrinsic midpoint potential, corrected for the surface potentials, is thus about -30 mV, and the standard free energy of activation is approximately -12 kJ/mol. In symmetrical 0.1 M NaCl, addition of 0.005 M Ba2+ to the extracellular solution produced a 17-mV depolarizing shift in Va and a slight reduction in za. The shift is consistent with predictions using the Gouy-Chapman theory and the above estimate for sigma e. Subsequent addition of 0.005 M Ba2+ to the intracellular solution produced a approximately 5-mV hyperpolarizing shift in the ensemble-averaged gating curve and reduced za by approximately 1. This Ba(2+)-induced shift is threefold larger than predicted, which together with the reduction in za implies that Ba2+ may bind at the intracellular channel surface.