Thermodynamics of the disproportionation of adenosine 5'-diphosphate to adenosine 5'-triphosphate and adenosine 5'-monophosphate. I. Equilibrium model

The thermodynamic treatment of the disproportionation reaction of adenosine 5′-diphosphate to adenosine 5′-triphosphate and adenosine 5′-monophosphate is discussed in terms of an equilibrium model which includes the effects of the multiplicity of ionic and metal bound species and the presence of long range electrostatic and short range repulsive interactions. Calculated quantities include equilibrium constants, enthalpies, heat capacities, entropies, and the stoichiometry of the overall reaction. The matter of how these calculations can be made self-consistent with respect to both calculated values of the ionic strength and the molality of the free magnesium ion is discussed. The thermodynamic data involving proton and magnesium-ion binding data for the nucleotides involved in this reaction have been evaluated.     

Calcium-dependent activation and deactivation of rod outer segment phosphodiesterase is calmodulin-independent

ATP-dependent activation and deactivation of retinal rod outer segment phosphodiesterase is affected by calcium [Kawamura, S. and Bownds, M. D., J. Gen. Physiol. 77:571-591(1981)]. Our data demonstrate that although calmodulin has been found in rod outer segments [Liu, Y. P. and Schwartz, H., Biochim. Biophys. Acta 526:186-193(1978); Kohnken, R. E. et al, J. Biol. Chem. 256:12517-12522(1981)], this protein is not involved in calcium-dependent phosphodiesterase activation at light levels at which calcium clearly affects this enzyme's activity. Furthermore, calmodulin does not mediate the calcium-dependent deactivation of phosphodiesterase.     

The mode of action of cytotoxic and antitumor 1-nitroacridines. I. The 1-nitroacridines do not exert their cytotoxic effects by physicochemical binding with DNA

The mode of action of cytotoxic and antitumor 1-nitroacridines and their isomeric derivatives was studied by comparing their effects in cell-free systems and towards cultured tumor HeLa cells, assuming that the nitroacridines considered exert cytotoxic effects by physicochemical binding with the DNA. All the nitroacridines impaired biosyntheses of DNA, RNA and protein in cultured HeLa cells and a causal relationship between nitroacridine inhibition of macromolecular biosyntheses and lethal effects of the agents appears likely. In cell-free systems, the nitroacridines bound with two independent sites on the DNA, forming complexes with enhanced resistance to DNA strand separation upon melting and inhibited the DNA polymerase reaction by altering activity of template and/or of enzyme. The 1-nitroacridines were poorly effective in cell-free systems and were the most potent inhibitors toward the growth of HeLa cells among the derivatives studied. It is concluded that the primary events responsible for cytotoxic effects of antitumor 1-nitroacridines and of their isomeric derivatives are different. The metabolic activation of 1-nitroacridines to more reactive intermediates which will attach to and alter the structure and/or function of DNA of sensitive cells is suggested.     

Differences in macromolecular binding between cyclic AMP and its dibutyryl derivative in vitro

Rat liver cytosol binds ³H-cAMP and ³H-DBcAMP $\text{in vitro}$. Fractionation of bound radioactivity by DEAE-Sephadex chromatography shows that ³H-cAMP is associated with a different cytosolic protein than is ³H-DBcAMP. The pI's of the cAMP-protein and the ³H-DBcAMP-protein complexes are 6.7 and 3.9, respectively. Competition studies between ³H-cAMP and its structural analogues have shown the following order of effectiveness in competing for binding sites in rat liver cytosol: cAMP > N⁶-MBcAMP > O²′-MBcAMP. No inhibition of ³H-cAMP binding was observed with 5′-AMP, adenosine, cGMP or DBcAMP. $\text{In vitro}$ binding experiments with rat serum has shown that only ³H-DBcAMP binds to any significant extent.     

Activation of Cl− Currents in Cultured Rat Retinal Pigment Epithelial Cells by Intracellular Applications of Inositol-1,4,5-triphosphate: Differences Between Rats with Retinal Dystrophy (RCS) and Normal Rats

Using the whole-cell configuration of the patch-clamp technique, we studied the conditions necessary for the activation of Cl⁻-currents in retinal pigment epithelial (RPE) cells from rats with retinal dystrophy (RCS) and nondystrophic control rats. In RPE cells from both rat strains, intracellular application of 10 μm inositol-1,4,5-triphosphate (IP3) via the patch pipette led to a sustained activation of voltage-dependent Cl⁻ currents, blockable by 1 mm 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS). IP3 activated Cl⁻ currents in the presence of a high concentration of the calcium chelator BAPTA (10 mm) in the pipette solution, but failed to do so when extracellular calcium was removed. Intracellular application of 10⁻⁵ m Ca²⁺ via the patch pipette also led to a transient activation of Cl⁻ currents. When the cells were preincubated in a bath solution containing thapsigargin (1 μm) for 5 min before breaking into the whole-cell configuration, IP3 failed to activate voltage-dependent currents. Thus, IP3 led to release of Ca²⁺ from cytosolic calcium stores. This in turn activated an influx of extracellular calcium into the submembranal space by a mechanism as yet unknown, leading to an activation of calcium-dependent chloride currents. In RPE cells from RCS rats, which show an increased membrane conductance for calcium compared to normal rats, we observed an accelerated speed of Cl⁻-current activation induced by IP3 which could be reduced by nifedipine (1 μm). Thus, the increased membrane conductance to calcium in RPE cells from RCS rats changes the response of the cell to the second messenger IP3. Received: 17 July 1995/Revised: 31 January 1996     

Pancreastatin activates pertussis toxin-sensitive guanylate cyclase and pertussis toxin-insensitive phospholipase C in rat liver membranes

We have recently found the calcium dependent glycogenolytic effect of pancreastatin on rat hepatocytes and the mobilization of intracellular calcium. To further investigate the mechanism of action of pancreastatin on liver we have studied its effect on guanylate cyclase, adenylate cyclase, and phospholipase C, and we have explored the possible involvement of GTP binding proteins by measuring GTPase activity as well as the effect of pertussis toxin treatment of plasma liver membranes on the pancreastatin stimulated GTPase activity and the production of cyclic GMP and myo-inositol 1,4,5-triphosphate. Pancreastatin stimulated GTPase activity of rat liver membranes about 25% over basal. The concentration dependency curve showed that maximal stimulation was achieved at 10^?7 M pancreastatin (EC₅₀ = 3 nM). This stimulation was partially inhibited by treatment of the membranes with pertussis toxin. The effect of pancreastatin on guanylate cyclase and phospholipase C were examined by measuring the production of cyclic GMP and myo-inositol 1,4,5-triphosphate respectively. Pancreastatin increased the basal activity of guanylate cyclase to a maximum of 2.5-fold the unstimulated activity at 30°C, in a time- and dose-dependent manner, reaching the maximal stimulation above control with 10^?7 M pancreastatin at 10 min (EC₅₀ = 0.6 nM). This effect was completely abolished when rat liver membranes had been ADP-ribosylated with pertussis toxin. On the other hand, adenylate cyclase activity was not affected by pancreastatin. Phospholipase C activity of rat liver membranes was rapidly stimulated (within 2–5 min) at 30°C by 10^?7 M pancreastatin, reaching a maximum at 15 min. The dose response curve showed that with 10^?7 M pancreastatin, maximal stimulation was obtained (EC₅₀ = 3 nM). GTP (10^?5 M) stimulated the membrane-bound phospholipase C as expected. However, the incubation of rat liver membranes with GTP partially inhibited the stimulation of phospholipase C activity produced by pancreastatin, whereas GTP enhanced the activation of phospholipase C by vasopressin. This inhibition by GTP was dose dependent and 10^?5 M GTP obtained the maximal inhibition (about 40%). the inhibitory effect of GTP on the stimulatory effect of pancreastatin on phospholipase C activity was completely abolished when rat liver membranes had previously been ADP-ribosylated with pertussis toxin. The presence of 8-Br-cGMP mimics the effect of GTP, whereas GMP-PNP increased both basal and pancreastatin-stimulated phospholipase C, suggesting a role of the cyclic GMP as a feed-back regulator of the synthesis of myo-inositol 1,4,5-triphosphate. However, the pretreatment of membranes with pertussis toxin did not modify the production of myo-Inositol 1,4,5-triphosphate stimulated by pancreastatin. In conclusion, pancreastatin activates guanylate cyclase activity and phospholipase C involving different pathways, pertussis toxin-sensitive, and -insensitive, respectively. © 1994 Wiley-Liss, Inc.     

Changes of inositol phosphates during decapitation-induced lateral bud break of Malus domestica

An increase in phosphatidylcholine (PC), phosphatidyl-ethanolamine (PE), phosphatidylglycerol (PG), and phosphatidylinositol (PI) occurred during bud break induced by decapitation. Inositol-1-phosphate [Ins(l)P₁], inositol-1,4-bisphosphate [Ins(1,4)P₂], and inositol-1,4,5-triphosphate [Ins(1,4,5)P₃] were found in apple buds and increased progressively following decapitation. Ins(1)P₁ and Ins(1,4)P₂ peaked 48 h after decapitation and Ins(1,4,5)P₃ peaked 72 h after decapitation during the metabolic transition when buds emerged from dormancy. Ins(1,4)P₂ and Ins(1,4,5)P₃ levels declined there after. The lateral buds on shoots with intact terminals and decapitated shoots treated with indole-3-acetic acid (IAA) in the terminals tip remained dormant and there were no significant changes in phospholipid and inositol phosphate contents.