Effect of novel specific myosin light chain kinase inhibitors on Ca2+-activated Mg2+-ATPase of chicken gizzard actomyosin.

Vascular relaxing agents such as N-(6-aminohexyl)-5-chloro-l-naphthalenesulfonamide (W-7), N²-dansyl-L-arginine-4-t-butyl-piperidine amide (No. 233), prenylamine and chlorpromazine that interact with Ca²⁺-regulated modulator protein of cyclic nucleotide phosphodiesterase inhibited Ca²⁺-dependent phosphorylation of chicken gizzard myosin light chain. Inhibition by the agents of myosin light chain phosphorylation resulted in inhibition of calcium activated, magnesium dependent adenosine triphosphatase of the gizzard actomyosin. The specificity of these agents for inhibition of light chain phosphorylation was shown by negative effect of these agents on ATPase activity of gizzard actomyosin in the phosphorylated form. Results suggest that the agents provide useful tool for the study on the Ca²⁺-sensitive regulatory mechanism of modulator-related enzyme systems.     

Ouabain-insensitivity of highly active Na+, K+-dependent adenosinetriphosphatase from rat kidney.

Highly purified preparations of Na⁺+K⁺-dependent adenosinetriphosphatase were isolated from rat kidney by two different procedures. The I₅₀ values for ouabain inhibition of the rat kidney enzyme at various stages of purification were determined to be essentially the same for all fractions tested (0.7 to 1.0 × 10^?4M). These results suggest that the marked insensitivity of the rat enzyme to inhibition by cardiac glycosides is due to the primary structure of the enzyme, and not to some other component in the tissue.     

Effects of light intensity on membrane differentiation in Rhodopseudomonas capsulata

Cells of Rhodopseudomonas capsulata, strain 37b4, leu^?, precultivated anaerobically under low light intensity, were exposed to high light intensity (2000 W · m^?2). The cells grew with a mass doubling time of 3 h. The synthesis of bacteriochlorophyll (BChl) began after two doublings of cell mass. Reaction center and light-harvesting BChl I (B-875) were the main constituents of the photosynthetic apparatus incorporated into the membrane. The size of the photosynthetic unit (total BChl/reaction center) decreased and light-harvesting BChl I became the dominating BChl species. Concomitant with the appearance of the different spectral forms of BChl the respective proteins were incorporated into the membrane, i.e. the three reaction center polypeptides, the polypeptide associated with light-harvesting BChl I, the two polypeptides associated with BChl II. A polypeptide of an apparent molecular weight of 45 000 was also incorporated. A lowering of the light intensity to 7 W · m^?2 resulted in a lag phase of growth for 6 h. Afterwards, the time for doubling of cell mass was 11 h. The concentration of all three BChl complexes (reaction center, light-harvesting BChl I and II complexes)/cell and per membrane protein increased immediately. Also the size of the photosynthetic unit and the amount of intracytoplasmic membranes/cell increased.The activities of photophosphorylation, succinate dehydrogenase, NADH dehydrogenase and NADH oxidation (respiratory chain)/membrane protein are higher in membrane preparations isolated from cells grown at high light intensities than in such preparations from cells grown at low light intensities.     

Aerobic hydrogenase activity in Anacystis Nidulans The oxyhydrogen reaction

1. The oxyhydrogen reaction of Anacystis nidulans was studied manometrically and polarographically in whole cells and in cell-free preparations; the activity was found to be associated with the particulate fraction.2. Besides O₂, the isolated membranes reduced artificial electron acceptors of positive redox potential; the reactions were unaffected by O₂ levels <10–15%; aerobically the artificial acceptors were reduced simultaneously with O₂.3. H₂-supported O₂ uptake was inhibited by CO, KCN and 2-n-heptyl-8-hydroxyquinoline-N-oxide. Inhibition by CO was partly reversed by strong light. Uncouplers stimulated the oxyhydrogen reaction.4. The kinetic properties of O₂ uptake by isolated membranes were the same in presence of H₂ and of other respiratory substrates.5. Low rates of H₂ evolution by the membrane preparations were found in presence of dithionite; methyl viologen stimulated the reaction.6. The results indicate that under certain growth conditions Anacystis synthesizes a membrane-bound hydrogenase which appears to be involved in phosphorylative electron flow from H₂ to O₂ through the respiratory chain.     

Calcium transport by pigeon erythrocyte membrane vesicles

Membrane vesicles from pigeon erythrocytes show a rapid, ATP-dependent accumulation of ⁴⁵Ca²⁺. Ca²⁺ accumulation ratios greater than or approximately equal to 10⁴ are readily attained. For ATP-dependent Ca²⁺ uptake, V is 1.5 mmol · 1^?1 · min^?1 at 27°C (approx. 0.9 nmol · mg^?1 protein · min^?1), [Ca²⁺]_{$\text{1}\text{2}$} is 0.18 μM, [ATP]_{$\text{1}\text{2}$} is 30–60 μM, the Ca²⁺ uptake rate depends on [Ca²⁺]² and the dependence of uptake rate on ATP concentration implies strong ATP-ATP cooperativity. The Arrhenius activation energy is 19.1 ± 1.4 kcal/mol and the pH optimum is approx. 6.9.     

Effect of heparin on the inhibition of thrombin by alpha 1-proteinase inhibitor.

Heparin interferes with the inhibition of thrombin by α₁-proteinase inhibitor (αPI). The inhibitory effect of heparin is due to its binding to thrombin. Other glycosaminoglycans and carboxyl-modified heparin do not have the same effect as heparin. The results indicate that there are similarities in the structural requirements in heparin, for anticoagulant activity and for the inhibition of αPI interaction with thrombin.     

Rapid cation flux from Torpedo californica membrane vesicles: comparison of acetylcholine receptor enriched and selectively extracted preparations.

Rapid efflux of ²²Na from within closed vesicles derived from $\text{Torpedo}\text{californica}$ electroplax membranes has been studied as an $\text{in}\text{vitro}$ assay of acetylcholine receptor functionality. The most highly purified membrane preparations contained major polypeptides of M.W. 43 and 90 × 10³ daltons in addition to the four peptides characteristic of the acetylcholine receptor (40, 50, 60, 65 × 10³ daltons). Removal of these extra peptides by base extraction did not significantly alter the characteristics of carbamylcholine induced ²²Na efflux: the agonist dose response curve was similar, preequilibration with agonist caused desensitization, the irreversible antagonist α-Bungarotoxin blocked the efflux and the reversible blockade by the neurotoxin perhydrohistrionicotoxin was also retained. The dose response curve for perhydrohistrionicotoxin corresponded closely to its known binding characteristics for base extracted membranes.     

Binding of polycyclic aromatic hydrocarbons to DNA of cells in culture: a rapid method for its analysis using hydroxylapatite column chromatography.

A rapid procedure to study the interaction of carcinogens with DNA in cultured cells has been developed. The cells, which are labeled with 7,12-[3H]dimethylbenz[a] anthracene ([3H]DMBA), are lysed with 0.24 M phosphate buffer (pH 6.8), 1% sodium dodecyl sulfate (SDS), 8 M urea and 0.01 M ethylenediamine-tetraacetate (EDTA) and sonicated. The cell lysates are fractionated on columns of hydroxylapatite. Proteins and RNA are removed with 8 M urea in 0.24 M phosphate buffer (pH 6.8). DMBA-bound DNA is eluted with 0.4 M phosphate buffer (pH 6.8). DMBA-DNA isolated by this procedure is virtually free from proteins and RNA. Thermal stability, ultraviolet spectra and the density of DNA is not altered by DMBA binding. The uptake of DMBA by mouse epidermal cells is rapid and the binding of DMBA to DNA is linear for the first 8 h of exposure. DMBA binds to DNA in all phases of the cell cycle. However, the highest binding occurs immediately following maximum DNA synthesis.