A difference in the affinity labeling of Ca2+, Mg2+-activated ATPases of normal and unc A strains of Escherichia coli by the 2',3'-dialdehyde derivative of adenosine 5'-diphosphate

The 2′,3′-dialdehyde of ADP, obtained by periodate oxidation of ADP, inhibited the hydrolytic activity of the purified Ca²⁺, Mg²⁺-activated ATPase of $\text{Escherichia}\text{coli}$. In the initial stages of the reaction inhibition was due to the reaction of 1 mol inhibitor/active site. When non-specific labelling of amino groups by the dialdehyde was lowered by the simultaneous presence of 15 mM ATP in the reaction mixture, 3 mol “ATP-protectable” binding sites/mol ATPase were found. “ATP-protectable” binding of the dialdehyde was not observed when the hydrolytically inactive ATPase of an $\text{unc A}$ mutant of $\text{E.}\text{coli}$ was used although binding of the inhibitor to non-protected amino groups still occurred. This suggests that the mutant ATPase is unable to bind ATP or that the amino groups with which the dialdehyde reacts in the native enzyme are absent or masked.     

Movement of sodium into human platelets

Addition of ADP induces platelets in plasma to undergo shape change from a disc to a spiny sphere and to develop adhesiveness, i.e. to aggregate. The aggregation of human platelets by ADP is associated with a net uptake of Na⁺. The present experiments demonstrate that the induction of shape change by ADP in acidified or EGTA-treated plasma conditions which inhibit aggregation, is also associated with a movement of Na⁺ into platelets. When ADP-induced platelet shape change and aggregation is inhibited by prostaglandin E₁ Na⁺ uptake is also blocked. Platelets aggregated by epinephrine do not take up Na⁺. In a manner analogous to the effect of ADP, polylysine also induces Na⁺ uptake during aggregation. Vasopressin, in a manner analogous to epinephrine, induces aggregation without Na⁺ uptake. The increase in platelet Na⁺ resulting from ouabain inhibition of Na⁺ efflux induces an increase in the aggregation response to ADP and to epinephrine.     

Grain size,sucrose level and starch accumulation in developing rice grain

Five rices (Oryza sativa L.) differing in final grain size were studied at the midmilky stage to determine if any factor could be identified which might limit rate of starch accumulation. Only UDP glucose pyrophosphorylase activity increased with increasing grain size. Detached rice panicles incubated in liquid medium containing 1% sucrose and 0.1% glutamine, in addition to minerals and vitamins, produced grains similar to those on intact plants. Sucrose level (0–1.5%) in the medium determined the extent of dry matter and starch accumulation and influenced physiological development of the ripening grains. Chemical and enzymic composition of the grain were similar to previously reported levels in grains of intact panicles analysed at regular intervals after anthesis. Addition of 3-P glycerate or K⁺ to the medium did not improve dry matter accumulation in the developing grain.     

Sodium and ouabain induce proliferation of rat thymic lymphocytes via calcium- and magnesium- dependent reactions

When the concentrations of either calcium or of magnesium in the culture medium were increased from the normal 0.6 and 1.0 mM to 1.8 and 2.5 mM respectively mitotic activity of rat thymic lymphocytes increased. Very high (10(-4)M) ouabain concentrations abolished these mitogenic actions whilst lower (10(-7) and 10(-11)M) concentrations had no effect. However in the normal medium these lower concentrations of ouabain were themselves mitogenic. The stimulatory effect of 10(-7)M ouabain was calcium-dependent and oestradiol-blockable and that of 10(-11)M magnesium-dependent and testosterone-blockable. A 10 mM increment in extracellular sodium concentration also stimulated mitosis in these cells in a calcium-dependent manner whilst a 20 mM increment required the presence of magnesium to exert its mitogenic effect. However, when similar osmotic increments were provided by potassium and lithium salts, or sucrose no mitotic stimulation was provoked. Subtle interactions between sodium and the divalent cations are clearly involved in events which lead to mitosis and the steroids oestradiol and testosterone can somehow block these effects.     

ATP-levels of germinating seeds in relation to vigor

Determination of seed vigor was attempted by comparing ATP-levels of deteriorating seed to germination percentage and production of dry matter. Immediately after imbibition of any seed lot investigated, a production of ATP took place. This ATP-accumulation invariably reached a plateau after 6 h of imbibition. Two well germinating seed lots of rape, one of cauliflower and one of sugar beet, were artificially aged by means of elevated storage temperature and humidity. Every second week through 16 weeks of deterioration the levels of ATP, ADP and AMP after 7 h of imbibition were compared with the germination percentage. While ADP- and AMP-contents of germinating seed displayed no change (when imbibed 7 h) during the period of artificial aging, seed deterioration was reflected in the ATP-levels long before loss of viability could be detected by the conventional germination test.
When ATP-levels per seed were related to germination percentage throughout the aging, all four seed lots displayed similar patterns although the absolute figures differed. In contrast to the conventional "per seed' basis, however, ATP per gram seed not only displayed similar deterioration patterns, but the absolute values were also of the same magnitude.     

Binding of ADP to beef-heart mitochondrial ATPase (F1)

1. ADP binding to beef-heart mitochondrial ATPase (F₁), in the absence of Mg²⁺, has been determined by separating the free ligand by ultrafiltration and determining it in the filtrate by a specially modified isotachophoretic procedure.2. Since during the binding experiments the ‘tightly’ bound ADP (but not the ATP) dissociates, it is necessary to take this into account in calculating the binding parameters.3. The binding data show that only one tight binding site (K_d about 0.5 μM) for ADP is present.4. It is not possible to calculate from the binding data alone the number of or the dissociation constants for the weak binding sites. It can be concluded, however, that the latter is not less than about 50 μM.     

Kinetics of drug-DNA interaction. Dependence of the binding mechanism on structure of the ligand

Kinetic and equilibrium studies of the binding of several phenanthridines and acridines to DNA have been performed to investigate the physical processes underlying the direct ligand transfer mechanism of drug-DNA interaction· Substitution of the 6-phenyl ring of dimidium with a p-carboxyl residue, or complete removal of either the 6-substituent or the 3-amino group, does not prevent the phenanthridine chromophore from transferring directly between binding sites. Loss of the aromatic ring increases association rate constants three- to ninefold and enhances dissociation rates by factors of up to 12; the rates of direct transfer and dissociation from site 1 are the most perturbed. The presence of a phenyl ring stabilizes the site 1 complex and lowers the binding constant to site 2. Introduction of the p-carboxyl group does not affect the equilibrium distribution of bound forms but produces equivalent increases (2·5-fold) in forward and reverse rate constants for binding to site 1 and for the direct transfer step. The 3-amino group greatly stabilizes the site 1 complex. Its removal accelerates all kinetic processes except for the reverse transfer step; the transfer rate is enhanced 25-fold and binding to site 2 is increased 12-fold. The dissociation rate from site 1 rises by a factor of 45 and that from site 2 by a factor of 5·8.10-Methyl-9-aminoacridine binds via the direct transfer pathway with rate and equilibrium constants similar to those of the 3-desamino derivative of ethidium. This compound provides the first fully characterized example of an acridine that utilizes bimolecular transfer. By contrast, rivanol (6,9-diamino-2-ethoxyacridine) interacts with DNA via a two-step sequential mechanism analogous to that seen with proflavine, yet its intrinsic association constant is three times higher. This results from tighter ‘external’ attachment to the helix, together with a decrease in equilibrium constant for the insertion step, which is markedly slower than that of proflavine. There appears to be a simple relation between the apparent enthalpy of binding and the number of extracyclic amino substituents on the intercalating chromophore.We propose that the two bound forms that participate in direct ligand transfer represent molecules intercalated via one or other of the grooves of DNA, and that the transfer pathway corresponds to exchange of drug between the wide groove of one helix and the narrow groove of another. The ability to form strongly bound complexes at the surface of the helix appears to play a major role in determining the mechanism of ligand binding.