Ouabain receptor interactions in (Na+ + K+)-ATPase preparations. II. Effect of cations and nucleotides on rate constants and dissociation constants

The action of ATP and its analogs as well as the effects of alkali ions were studied in their action on the ouabain receptor. One single ouabain receptor with a dissociation constant ($\text{K}{}_{\mathrm{<mtext>D</mtext>}}$) of 13 nM was found in the presence of ($\text{Mg}{}^{\mathrm{2+}}\text{+ P}{}_{\mathrm{i}}$) and ($\text{Na}{}^{\mathrm{+}}\text{+ Mg}{}^{\mathrm{2+}}\text{+ ATP}$). pH changes below pH 7.4 did not affect the ouabain receptor. Ouabain binding required Mg²⁺, where a curved line in the Scatchard plot appeared. The affinity of the receptor for ouabain was decreased by K⁺ and its congeners, by Na⁺ in the presence of ($\text{Mg}{}^{\mathrm{2+}}\text{+ P}{}_{\mathrm{i}}$), and by ATP analogs (ADP-C-P, ATP-OCH₃). Ca²⁺ antagonized the action of K⁺ on ouabain binding. It was concluded that the ouabain receptor exists in a low affinity (R_α) and a high affinity conformational state (R_β). The equilibrium between both states is influenced by ligands of $\text{(Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}\text{)-ATPase}$. With 3 mM Mg²⁺ a mixture between both conformational states is assumed to exist (curved line in the Scatchard plot).     

ADP binding to (Na + K)-activated ATPase

[¹⁴C]ADP binding to EDTA-washed ox brain cell membranes was increased by Na⁺, but decreased by K⁺, Mg²⁺ and Ca²⁺. Na⁺ abolished the effect of K⁺ on ADP binding by a competitive mechanism, but could not reverse the inhibitory action of Mg²⁺ and Ca²⁺. It is concluded that the cation-induced changes in ADP binding reflect properties of (Na⁺ + K⁺)-activated ATPase.     

The premelting of nucleoprotein: role of non-histone proteins

In native nucleoprotein, the premelting structural changes of DNA are not observed by circular dichroism measurements. In order to determine which protein fraction of chromatin is responsible for the absence of premelting we have examined a series of nucleoproteins depleted of different protein fractions by treatment with sodium chloride or sodium deoxycholate.     

Variance and discriminatory analyses for the classification of wakefulness-dependent EEG patterns]

1. To describe 8 alertness-dependent EEG activity patterns to be observed during wakefulness with eyes closed, mean values and standard deviations of the frequencies 4, ..., 13, 14 to 17, 18 to 22 and 23 to 40 c/sec, of the percentage activity in the theta, alpha and beta bands as well as of the theta and alpha amplitudes were calculated. 2. On the basis of 18 variables significant differences between 7 activity patterns were ascertained by means of univariate and multivariate analyses of variance. 3. In the course of stepwise reduction of variables within the framework of a linear discriminatory analysis, an optimal set of variables was determined for the separation of the patterns comprising the following variables: mean value and standard deviation of the alpha amplitudes and mean value of the theta amplitudes as well as the percent quantity of the frequencies 3, ..., 7, 10, 14 to 17 and 23 to 40 c/sec. 4. By means of a linear regression analysis it was shown that the EEG scoring system can be reflected on an interval scale. In connection with results on the reliability of the methods used the results were discussed.     

Competitive reversals and environment-dependent resource partitioning in Erodium

Summary Effects of drought and varying plant density on the competitive coexistence of two winter annual Erodium species were studied using multiple regression analysis. Significant indications of resource partitioning were detected for interspecific mixtures under spring drought. Competitive superiority also was environment-dependent with E. botrys dominating with drought in autumn, while E. brachycarpum dominated with drought in spring. The results suggest that competitive coexistence in Erodium is promoted by processes both equilibrial (e.g., resource partitioning) and nonequilibrial (e.g. competitive reversals).     

Effect of different light qualities on the ultrastructure, thylakoid membrane composition and assimilation metabolism of Chlorella fusca

Chlorella fusca (Shihira et Krauss) strain C-1.1.10 was grown under three different light qualities (red, white or blue light) in homocontinuous cultures. Under electron microscopy, blue light cultures showed enlarged cells, thinner cell walls and lower starch content than red light cells. Under blue light, the degree of stacking of the thylakoid membranes was significantly lower than under white or red light conditions. Changing the light from blue to red the ratio of exposed to appressed membranes was doubled. Compared to red light cells, blue light cells exhibited higher photosynthetic rates per chlorophyll molecule and contained less chlorophyll per dry weight. Blue light stimulated the content of soluble protein as well as that of soluble carbohydrates. The dry weight productivity per unit time was enhanced under blue light conditions. The thylakoid protein complexes which are generally assumed to be localized in the exposed membranes were found in higher concentrations under blue light than under red light. In blue light, both the Photosystem II/Photosystem I ratio and the ratio of light-harvesting chlorophyll protein to P-700 chlorophyll a -protein were lower than in red light. Blue light cells contained twice the concentration of cytochrome f , which correlates well with their higher photosynthetic capacity. When altering the light quality, the degree of change in the reaction center complexes was much lower than expected given the corresponding degree of change in the ratio of exposed to appressed membranes. These results are discussed in light of the question as to whether the variation in the stoichiometry of the laterally distributed complexes can be explained by changes in the degree of stacking alone.     

pH, urea and substrate gradients for the optimization of ultrathin polyacrylamide gel zymograms

The preparation of ultrathin polycrylamide gels with different kinds of gradients (pH, substrates, inhibitors) is described. By using these gels fro contact printing after isolectric with Ampholines or Immobilines and for diffusion tests, the influence of pH or increasing amounts of substrates or inhibitors on enzyme activities is studied. These methods are successfully applied for the optimization of zymogram techniques and for the easy characterization of industrial microbiol enzyme preparations for technological purposes. With buffer-generated pH gradient gels, the pH optimum of all isoenzyme activities is demonstrated by contatc printing; the total amount of esoenzyme acitivities dependent on pH is determined by a diffusion test. Gels with a linear gradient between 0 and 8 M urea are used for isoelectric focusing, diffusion tests and contact printing in order to differentiate the unfolding and denaturing effects of urea on isoenzymes. Alterations in polygalacturonase isoenzyme patterns dependent on urea concentration of denaturation but by the change of chargers. In respect to band sharpness and straightness urea can be added advantagenously up to 2 M without changing the isoelectric points or activities of the isoenzymes. for the reproducibility of zymograms it is interesting to see that different substrate concentrations reveal different isoenzyme patterns.     

Simultaneous extraction and purification of a cell wall-associated peptidase and β-casein specific proteas fromStreptococcus cremoris AC1

Summary A cell wall-associated, β-casein specific protease and a peptidase were purified simulta-neously fromStreptococcus cremoris AC1. The molecular weights are 145,000 daltons for the pro-tease and 36,000 for the peptidase. The protease has a pH optimum at 5.5-6 and a temperature optimum at 40° C. It is activated by 1 mM of Ca⁺⁺ but severely inhibited by higher Ca⁺⁺ concentrations. Further inhibitor studies indicate that the protease is probably a serin protease. The peptidase shows aminopeptidase activity and most effectively hydrolyses L-lysyl-p-nitroanilide, and to a lesser extent IMeucyl-, L-alanyl- and L-alanyl-L-alanyl-p-nitroanilide. No endopeptidase activity could be detected. The peptidase is irreversibly inhibited by EDTA.     

Covalent cross-linking of AcVal-tRNA to Tetrahymena thermophila cytoplasmic ribosomes and two of its 17S rRNA mutants

Tetrahymena thermophila 80S ribosomes have been cross-linked to non-enzymatically bound AcVal-tRNA, presumably at the ribosomal P-site. Like the ribosomes from Escherichia coli, yeast, and Artemia salina, cross-linking is exclusively to C-1609, the equivalent of the E. coli C-1400 residue. Mutation of the RNA from G-1707 to A or from U-1711 to C which results in resistance to paromomycin or hygromycin, respectively, failed to affect the rate, yield, or site of cross-linking. The presence of the antibiotics during cross-linking also was without effect. It is concluded that at these two positions the base changes made do not interfere with the tertiary structure of the decoding site.     

Morphological,Biometric, and Morphogenetic Comparison of Two Closely Related Species,Stylonychia vorax and S. pustulata (Ciliophora: Oxytrichidae)1

Differences in the morphology of Stylonychia vorax Stokes, 1885 and S. pustulata (Müller, 1786) Ehrenberg, 1838 recognizable in vivo are the shape, the ventral cirral pattern, the caudal cirri, and the mode of moving. The dorsal-bristle complexes are distinguishable by the length of dorsal kinety four and the spaces among the pairs of basal bodies. When the ranges of variation of different populations and clones are compared by biometric analyses, S. vorax shows a relatively stable cortical pattern whereas in S. pustulata the cortical elements are regulated depending on the size of the body and the number of adoral membranelles. In S. vorax morphogenesis begins with a proliferation of basal bodies close to the transverse cirri. In contrast, in S. pustulata, the oral primordium appears de novo between the left marginal row and the postoral cirri. All other morphogenetic events are the same for both species. In proters and opisthes the six anlagen of the frontal-ventral-transverse cirri are of different origin and evolve independently. Three anlagen of the opisthe separate from the oral primordium, two originate from the right, and one from the left postoral cirrus. Three anlagen of the proter evolve from the posteriormost cirrus in the frontal area, one from the parental undulating membranes, one from the buccal cirrus, and one from the cirrus below the buccal cirrus. The anlagen one to six generate one, three, three, three, four, and four cirri. The characteristic arrangement of the undulating membranes and the participation of only two postoral cirri in the formation of primordia provide features that distinguish between the often confused genera Oxytricha and Stylonychia.