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A single-stranded DNA-dependent ATP y-phosphohydrolase of M I 56000 induced after infection of Escherichia coli cells with bacteriophage T4, probably the ATPase dependent on gene dda of the phage, was isolated. Studies on the enzyme show that in the presence of ATP and Mg2+ ions it is capable of dissociating partially double-stranded fd bacteriophage DNA into the single strands and that some 300Cbenzyme copies are required to unwind the 6400-nucleotides-long DNA. Unwinding is inhibited by reducing the length of the single-stranded portion of DNA to two nucleotides. In addition it can be inhibited by sulfhydryl reagents which block the ATPase or by trapping free enzyme molecules in the assay system. The results suggest that unwinding is initiated near the single-stranded portion of the DNA and is driven by the ATPase. It further appears that the enzyme unwinds by adsorbing to the DNA. Affinity of the enzyme for double-stranded DNA is not detectable by DNA binding assay.Infection of Escherichia coli with bacteriophage T4 leads to the appearance of several DNA-dependent ATPases. The most prominent of these has M , near 50000 and depends on the viral gene ddu [l -31. T4 ddu ATPase is easily identified by means of chromatography. It elutes from DEAE-cellulose in front of most other proteins at < 50mM NaCl and from Sephadex gel columns later than other ATPases in the 20000-MI range of the column (in disagreement with the molecular weight of the protein). Loss of the ATPase by deletion of dda is not lethal to the phage [4,51.Starting from wild-type T4-infected cells we have isolated a DNA-dependent ATPase showing the chromatographic properties characteristic of ddu ATPase. Studies on the purified enzyme demonstrate that in the presence of ATP it is able to separate, catalytically, double-stranded DNA into the single strands. Further Dedicated to Prof. Reinhard W Kaplan on the occasion of his 65th birthday.

(Received Septcmber 1 Deccmbcr 7, 19x2) -EJH 59591. The ability of external ATP to induce calcium uptake in isolated rat liver cells was further characterized. Stimulation of calcium uptake was specific for ATP, other nucleotides or ATP metabolites had no comparable effect. ATP was dephosphorylated while stimulating calcium uptake, but there was no stoichiometry between ATP hydrolysis and calcium uptake nor did dephosphorylation depend on calcium concentration. ATP acted from outside and was dephosphorylated by an ccto-ATPase of the cells.2. In addition to its direct action, ATP enhanccd succinate-dependent calcium uptake in a coopcrativc fashion. This is best explained by different sites of action. ATP increases cell membrane permeability while succinatc stimulates uptake into mitochondria.3. ATP was able to lower Na' and K t gradients and the pH gradient between cells and incubation medium. Increasing calcium concentration countcracted this effect though calcium uptake was then stimulated.4. Succinate alone did not affect monovalent cation gradients but raised the pH gradient. It partially coiinteractcd the ATP effects on these gradients.5. Since catecholamine-like actions of ATP may be mediated by an increase in cytoplasmic calcium concentration, the action of extracellular ATP can be taken as a model t o study the role of calcium as a transmitter of hormone actions. From interdependence between ATP-stimulated and succinatc-stimulated calcium uptake, conclusions can be drawn on the resulting cytoplasmic calcium concentration and its effect on plasma membrane permeability.In a previous study, isolated liver cells proved to be a useful model to study the delicately poised equilibrium of calcium distribution across the various cellular compartments [l]. This was especially true for disclosing the role of cellular mitochondria in this interplay. Respiratory substrates, pH, inorganic phosphate, and the redox grade of pyridine nucleotides were found important in triggering calcium movements at the mitochondria1 site, whereas external ATP and La3+ induced calcium uptake at the plasma membrane site [I. 21.In this report, thc action of added ATP is further characterizcd. The responses of functional parameters such as cation distribution and the pH gradient between cells and incubation medium are compared with those seen after succinate addition. Relations between the different responses and the probablc changes in cytoplasmic calcium concentrations are discussed. This is of interest because external ATP was previously reported to exert a catecholamine-like action on the plasma membrane permeability [3, 41 and to prcvent the stimulatory effect of insulin on hexose transport and oxidation [S -71. As r-adrenergic stimulation generally results in changes in both calcium and potassium distribution (for review see [8]) the study of ATP action might shed light on the relcvance of various cellular parameters in a state similar to that during a-adrenergic action. MATERIALS AND METHODS Radiochmicals.

Responses of isolated perfused rat liver to leukotriene C4 were studied in order to assess the mechanisms involved in leukotriene-mediated liver injury. Infusion of leukotriene C4 (11 and 44 pmoles per min per gm liver weight) into the portal vein resulted in a rise in portal pressure, a decrease in oxygen consumption, an increase in hepatic glucose and lactate efflux and lactate/pyruvate ratio in the perfusate and a small decrease in bile flow. Isoproterenol (1 microM) counteracted the effects of leukotriene C4 on respiration and portal pressure, whereas bile flow and glucose efflux were reversibly stimulated. The same changes were observed upon withdrawal of leukotriene C4. The release of glucose was correlated with the increase in oxygen consumption upon both isoproterenol addition and withdrawal of leukotriene C4. These results are indicative of leukotriene C4-induced microcirculatory redistribution of perfusate flow. Since, in the presence of nitroprusside (50 microM), both the effects of leukotriene C4 and their reversal by isoproterenol were diminished, a vascular site of action can be assumed. Accordingly, the accompanying metabolic responses can be explained by gradual changes in oxygen supply to parts of the liver. Reversibility of the leukotriene C4 effects and lack of short-term impairment of viability of the isolated liver suggest that leukotriene-mediated liver injury is a long-term effect related to events subsequent to microcirculatory changes.