Differential Effect of Sodium Azide on the Incorporation of Nucleic Acid Precursors into Chinese Hamster Cells in Vitro
Nucleic acid metabolism in Chinese hamster fibroblasts grown in vitro noting effects of concentrations of sodium azide
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Nucleic acid metabolism in Chinese hamster fibroblasts grown in vitro noting effects of concentrations of sodium azide
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Vesicles prepared in a buffer containing ADP, Mg(2+) and Pi synthesized ATP at an initial rate of 2 nmols/min/mg protein after acidification of the bulk medium (pH 8 (right arrow) 4). The intravesicular ATP concentration reached a steady state after about 30 seconds and slowly declined thereafter. ATP synthesis was inhibited by low concentrations of dicyclohexylcarbodiimide and m-chlorophenylhydrazone indicating that synthesis took place in response to the proton gradient. NEM and PCMS, which inhibit vacuolar ATPases and the vacuolar-like ATPases of extreme halophiles, did not affect ATP synthesis, and, in fact, produced higher steady state levels of ATP. This suggested that two ATPase activities were present, one which catalyzed ATP synthesis and one that caused its hydrolysis. Azide, a specific inhibitor of F0F1 ATP Synthases, inhibited halobacterial ATP synthesis. The distribution of acridine orange as imposed by a delta pH demonstrated that azide inhibition was not due to the collapse of the proton gradient due to azide acting as a protonophore. Such an effect was observed, but only at azide concentrations higher than those that inhibited ATP synthesis. These results confirm the earler observations with cells of H. saccharovorum and other extreme halophiles that ATP synthesis is inconsistent with the operation of a vacuolar-like ATPase. Therefore, the observation that a vacuolar-like enzyme is responsible for ATP synthesis (and which serves as the basis for imputing ATP synthesis to the vacuolar-like ATPases of the extreme halophiles, and the Archaea in general) should be taken with some degree of caution.
The proton-translocating ATPases are multimeric enzymes that carry out a multitude of essential functions. Their origin and evolution represent a seminal event in the early evolution of life. Amino acid sequences of the two largest subunits from archaeal ATPases (A-ATPases), vacuolar ATPases (V-ATPases), and FOF1-ATP syntheses (FATPases) suggest these ATPases evolved from an ancestral vacuolar-like ATP syntheses. A necessary consequence of this notion is that the A-ATPases are ATP syntheses. With the possible exception of the A-ATPase from Halobacterium salinarium. no A-ATPase has been demonstrated to synthesize ATP. The evidence for this case is dubious since ATP synthesis occurs only when conditions are distinctively unphysiological. We demonstrated that ATP synthesis in H.saccharovorum is inconsistent with the operation of an A-type ATPase. In order to determine if this phenomenon was unique to H. saccharovorum, ATP synthesis was examined in various extremely halophilic bacteria with the goal of ascertaining if it resembled what occurred in a. saccharovorum, or was consistent with the operation of an A-type ATPase. A-, V-, and F-type ATPases respond singularly to certain inhibitors. Therefore, the effect of these inhibitors on ATP synthesis in several extreme halophiles was determined. Inhibitors that either blocked or collapsed proton-gradients inhibited the steady state synthesis of ATP thus verifying that synthesis took place at the expense of a proton gradient. Azide, an inhibitor of F-ATPases inhibited ATP synthesis. Since the arginine-dependent synthesis of ATP, which occurs by way of substrate-level phosphorylation, was unaffected by azide, it was unlikely that azide acted as an "uncoupler." N -ethylmaleimide and nitrate, which inhibit V- and A-ATPases, either did not inhibit ATP synthesis or resulted in higher steady-state levels of ATP. These results suggest there are two types of proton-motive ATPases in the extreme halophiles (and presumably in other Archaea). One, the V-like enzyme which, provides protons that are subsequently used for solute translocation. The other ATPase is the familiar and ubiquitous F-ATPase that functions as a reversible proton pump and is the ATP Synthase in the extreme halophiles. Thus, while the suggested evolution of the proton -translocating ATPases accounts for the relationship among these ATPases, this scheme does not account for the presence of F-ATPases in the Archaea. Discounting lateral gene transfer, perhaps an F-type ATPase evolved before the eucaryal-archaeal and bacterial bifurcation. The presence of V-type ATPases in the Bacterial Domain is consistent with this suggestion. Finally, it is of interest to note that if an F-type ATPase appeared before the bifurcation, an endosymbiotic event need not be invoked to explain the presence of F-ATPases in the Eucarya.
An electrothermal thruster designed for operation with MIL-grade hydrazine is suitable for operation with propellants having lower freezing points. These propellants are 76% hydrazine - 24% hydrazine azide, Aerozine-50, 50% hydrazine - 50% monomethylydrazine, and a TRW-formulated mixture of 35% hydrazine - 50% monomethylhydrane - 15% ammonia. A steady-state specific impulse of 200 sec was exceeded by all propellants. A pulse-mode value of 175 sec specific impulse was exceeded by the azide blend for pulse widths greater than 50 ms and was met by the carbonaceous propellants for pulse widths greater than 100 ms. Longer residence times were required for the carbonaceous propellants; the original thruster design was modified by increasing the characteristic chamber length and density of screen packing. A substantial amount of thermal energy must be supplied to initiate decomposition of propellants containing unsymmetrical-dimethylhydrazine and monomethylhydrazine. The rate controlling factor appeared to be the endothermic removal of methyl radicals.
A 333 mN electrothermal thruster designed to use MIL-grade hydrazine was demonstrated to be suitable for operation with low freezing point monopropellants containing hydrazine azide, monomethylhydrazine, unsymmetrical-dimethylhydrazine and ammonia. The steady-state specific impulse was greater than 200 sec for all propellants. The pulsed-mode specific impulse for an azide blend exceeded 175 sec for pulse widths greater than 50 msec; propellants containing carbonaceous species delivered 175 sec pulsed-mode specific impulses for pulse widths greater than 100 msec. Longer thrust chamber residence times were required for the carbonaceous propellants; the original thruster design was modified by increasing the characteristic chamber length and screen packing density. Specific recommendations were made for the work required to design and develop flight worthy thrusters, including methods to increase propellant dispersal at injection, thruster geometry changes to reduce holding power levels and methods to initiate the rapid decomposition of the carbonaceous propellants.