Adenosine triphosphate and phosphocreatine levels in cochlear structures. Use rate and effect of salicylates.

Guinea pigs were injected with various dosages of salicylate for varying time periods. The temporal bones were removed, frozen quickly, freeze-dried, and the cochlea was dissected into essential auditory component parts and subjected to microchemical analysis for phospho-creatine (P-creatine) and adenosine triphosphate (ATP) levels. It was found that high energy phosphates were not decreased by therapeutic or acutely toxic levels of salicylate. Only when chronic intoxication with salicylate was accomplished was there a reduction in ATP and P-creatine. The data presented do not provide support for the widely held view that uncoupling of oxidative phosphorylation or inhibition of enzymes involved in energy generation in the inner ear structures studied (organ of Corti, stria vascularis, Reissner's membrane, modiolar blood vessels, cochlear nerve and spiral ganglion) are the mechanisms by which salicylates cause reversible hearing loss. The study confirms the existence of a P-creatine gradient opposite to the well known glycogen gradient in the organ of Corti (Krzanowski JJ Jr, Matschinsky M: J Histochem 19:321, 1971) and suggests a relatively uniform energy use rate of this tissue for all four turns (20 mmoles of approximately phosphorus used/kg dry weight/min).     

The enhancement of the alkaline Bohr effect of some fish hemoglobins with adenosine triphosphate.

The oxygen equilibria of the hemoglobins of one holostean fish, the spotted gar (Lepisosteus osculatus), and of four teleost fish, the carpsucker (Carpiodes carpio), the small mouth buffalo fish (Ictiobus bubalus), the Rio Grande cichlid (Cichlasoma cyanoguttatum), and the redear sunfish (Lepomis microlophus), have been measured as a function of pH in the presence and absence of ATP. The oxygen equilibria of the teleost hemoglobins in the presence of 200 μm ATP can be superimposed within experimental error upon the data obtained in the absence of ATP by a simple downward shift of the pH scale by 0.5 unit. Thus the effects of proton and ATP binding appear equivalent: Both can be interpreted in terms of a two-state allosteric model in which binding occurs preferentially to the low-affinity T-state. The oxygen affinities of each of the teleost hemoglobins approach asymptotically a maximal value at high pH. Although these high affinities are associated with decreased cooperativity of oxygen binding, as reflected by the Hill coefficient n, the asymptotic value of n never appears lower than 1.2 to 1.4. This indicates that the data cannot be completely described in terms of a single high-affinity R-state in alkaline solution: At least two different conformations are required. The oxygen affinity of the spotted gar hemoglobin, like that of each of the teleost hemoglobins, reaches a maximal value (minimal value of log PO₂ for half-saturation) above pH 8, but unlike teleost hemoglobins, the Hill coefficient reaches maximal values of 2.6 to 2.7 at high pH. The data in the absence of ATP are superimposable on the data in its presence by a downward shift of the pH scale by 0.25 unit. The measurement of the Bohr effect ($\text{Δlog}\text{P}{}_{30}\text{ΔpH}$) in the presence and absence of ATP shows that the Bohr effect in each of the hemoglobins is substantially enhanced by organic phosphates, as it is in mammalian hemoglobins. The extent of the enhancement of the Bohr effect by 200 μm ATP for each of the hemoglobins is approximately the same in the range pH 6.7 to 7.3 (increase in $\text{Δlog}\text{P}{}_{50}\text{ΔpH}\text{~ 0.3}$). This is a direct consequence of the equivalence of the linked-function relationship to the effects of ATP and proton binding on oxygenation.     

Nucleocytoplasmic transport of RNA. The effect of 3''-deoxyadenosine triphosphate on RNA release from isolated nuclei.

The addition of 3'-deoxyadenosine (cordycepin) to cells in culture results in the inhibition of the appearance of mRNA in the cytoplasm through a mechanism thought to involve the inhibition of polyadenylate synthesis. I studied the effect of 3'-deoxyadenosine triphosphate, the physiologically active form of 3'-deoxyadenosine, on RNA release from isolated nuclei. Nuclei were isolated from baby-hamster kidney (BHK) fibroblasts that had been given a short pulse of radioactive uridine or adenosine in the presence of a low concentration of actinomycin D before harvest. RNA release from the isolated nuclei under the appropriate incubation conditions was time-, temperature- and ATP-dependent. 3'-Deoxyadenosine triphosphate inhibited RNA release from the isolated nuclei. However, RNA that was restricted to the nuclei during incubation with the drug could be chased out of the nuclei if the incubation medium was replaced with medium containing only ATP. The chased poly(A)+ (polyadenylated) RNA had shortened poly(A) tracts, indicating that poly(A)+ RNA with shortened poly(A) tracts can be transported out of the nucleus. An experiment was designed to test the effect of 3'-deoxyadenosine triphosphate on the release of poly(A)+ RNA at drug concentrations which caused 33 or 64% inhibition of RNA release. The release of poly(A)+ RNA and poly(A)- RNA (not polyadenylated) was equally inhibited by the drug. Thus, although 3'-deoxyadenosine triphosphate does inhibit release of RNA from the nucleus, it would appear that the drug does so through a mechanism independent of the inhibition of polyadenylation. The process that is inhibited must be one that is common to both poly(A)+ and poly(A)- RNA. The possibility that 3'-deoxyadenosine triphosphate inhibits a reaction at the nuclear membrane or nuclear pore complex is considered.     

Combined effect of cytochalasin a and adenosine triphosphate onPhysarum polycephalum

Summary Physarum polycephalum microplasmodia exposed to 1.6×10^–5 M cytochalasin A evidenced intracellular cytoplasmic condensation, slow contraction, and eventual breaks at discrete surface areas, within one hour. Other cytochalasins tested (CB or CD) did not substitute for CA. CA effects on plasmodia were not abolished by immediate washing or media replacement. In nutrient medium, CA plus ATP (375 M) produced within minutes herniation (blebbing) and plasmodial disruption. The order of addition of reagents was important; ATP added simultaneously with or prior to CA stimulated the phenomenon, whereas initial addition of CA resulted in no such dynamic response. Several other nucleotides (e.g., AMP, cAMP) could substitute for ATP; however, such changes were not observed with 5-adenylylimidodiphosphate. Blebbing was not abolished in the presence of 2,4-dinitrophenol. In minimal medium, it was best stimulated by simultaneous addition of Ca⁺⁺ and Mg⁺⁺. Preincubation of CA with L-cysteine or with -mercaptoethanol negates its individual or nucleotide-combined effects. Yet, 10^–5 M ethacrynic acid, a sulfhydryl-reactive liposoluble drug, in the presence of ATP does not mimic the blebbing response. These observed effects, which take place at or near the plasmodial surface, presumably reflect acceleration of normal contractile processes inPhysarum. Abbreviations CA cytochalasin A - CB cytochalasin B - CD cytochalasin D - AMP adenosine 5-monophosphate - ADP adenosine 5-diphosphate - ATP adenosine 5-triphosphate - di-butyryl-cAMP di-butyryl-cyclic adenosine 35-monophosphate - di-butyrylcGMP di-butyryl-cyclic guanosine 35-monophasphate. This work was supported by a grant (AI-11902) from the U.S. Public Health Service.     

Human deoxyuridine triphosphate nucleotidohydrolase. Purification and characterization of the deoxyuridine triphosphate nucleotidohydrolase from acute lymphocytic leukemia.

A new fast assay procedure for increasing deoxyuridine triphosphate nucleotidohydrolase activity was developed. With this assay procedure, this enzyme derived from blast cells of patients with acute lymphocytic leukemia was purified at least 1218-fold. The molecular weight was estimated by gel filtration to be 43,000. The enzyme exhibited optimal activity over a pH range of 7 to 8 and the activation energy was estimated to be 6.5 kcal/mol at pH 7.5. While the enzyme had activity in the absence of added divalent cations, the activity could be inhibited by EDTA but not by phenanthroline. The inhibition caused by EDTA could be reversed by Mg2+ or Zn2+. The enzyme had maximal activity in the presence of Mg2+ (40 muM) and Mg2+ (4 mM) stabilized the enzyme at 37 degrees C. Cupric ion (0.5 mM) inhibited (50%) enzyme activity in the presence or absence of Mg2+. The substrate for the enzyme was dUTP and the apparent Km was 1 muM. No other deoxyribonucleoside or ribonucleoside triphosphate served as a substrate for the enzyme.     

Specific enzymatic determinations of adenosine triphosphate.

The well-known soluble kinases are not specific for ATP (1). All these enzymes convert ATP as well as GTP, ITP, CTP, and UTP, although at different rates. The only exception is adenylate kinase (1). However, with this enzyme, a direct determination of ATP in tissue extracts which contain both the di- and mononucleotides is not possible.Phosphoglycerate kinase from various sources is specific for ATP, GTP, and ITP and does not react with the pyrimidine nucleotides (2), Now, however, it was found that phosphoglycerate kinase from the blue alga Spirulina platensis does not convert GTP and ITP. With this enzyme, therefore, it is possible to specifically determine ATP in tissue extracts or in mixtures of nucleotides. In the same test, GTP and ITP can be determined by adding phosphoglycerate kinase from yeast or from other sources (2).     

Computer-assisted analysis of adenosine triphosphate data.

A computer program has been written to assist in the analysis of adenosine 5'-triphosphate data. The program is designed to calculate a dilution curve and to correct sample and adenosine 5'-triphosphate standard data for background and dilution effects. In addition, basic statistical parameters and estimates of biomass carbon are also calculated for each group of samples and printed in a convenient format. The versatility of the program to analyze data from both qauatic and terrestrial samples is noted as well as its potential use with various types of instrumentation and extraction techniques.