Graduate Thesis Or Dissertation
 

Deoxyribose-phosphate aldolase and the biosyntheiss of DNA in the chick embryo

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  • The activity of deoxyribose-phosphate aldolase was determined in chick embryo brains, hearts and livers during the course of development. Assays were made in the direction of synthesis of deoxyribose-5-phosphate. Low levels of activity in the brain and heart remained constant throughout the course of development. Activity in the liver expressed per mg of protein increases up to 14 days, increases more rapidly between 16 and 18 days, after which it levels off until several days after hatching. All activity in the liver is contained in the supernatant after centrifugation at 30,000 x g. Certain conditions for the assay of deoxyribose-phosphate aldolase were evaluated. Although the enzyme seems to be inactivated by ammonium sulfate precipitation, it is stable for several hours at 4°C, but gradually loses activity during prolonged storage at either 4°C or -20°C. Assay of activity of the enzyme is not affected by the action of phosphatase on the product, since deoxyribose and deoxyribose-5-phosphate produce very nearly the same extinction coefficient. Activation by added citrate was shown for the enzyme in heart homogenate, but not in liver homogenate. It is suggested that endogenous levels of citrate in liver tissue are sufficient to provide for maximum enzymatic activity. Inhibition of deoxyribose-phosphate aldolase by diphenylsuccinate reported for rat liver was confirmed for the chick embryo enzyme. Similar inhibition occurred in all three organ homogenates. Complete inhibition was obtained at 10 mM diphenylsuccinate. Hydroxyurea appeared to effect a slight inhibition of the enzyme activity, although an increase in inhibitory effect was not consistent with increased hydroxyurea concentration. Another effect of hydroxyurea is inhibition of the diphenylamine reaction with deoxyribose-5-phosphate, which requires correction of assays made in the presence of hydroxyurea. A third effect of hydroxyurea is a reaction with diphenylamine which produces a blue chromogen with absorption peaks at 430 mμ. and 630 mμ. (instead of the single peak at 600 mil observed in the reaction of deoxyribose-5-phosphate with the diphenylamine reagent). This reaction was obtained when fructose-1, 6-diphosphate was incubated with liver supernatant and the products were treated with diphenylamine. The development of color depends on time of enzymatic incubation, on the concentrations of fructose-1,6-diphosphate and supernatant present in the incubation mixture, and on the concentration of hydroxyurea added either before or after enzymatic incubation. The identity of the product(s) was not determined. The incorporation of label from 1-¹⁴C-deoxyribose into DNA was studied. A maximum of about 0.2% of injected radioactivity was recovered in the nucleic acid extracts from embryonic brains, hearts and livers. Greater incorporation was obtained by injection of deoxyribose into the air space than by injection into the yolk sac. Radioactive DNA was degraded to determine the extent of incorporation of label into carbon-1 of deoxyribose from DNA. Deoxyribose was converted to levulinic acid and levulinic acid into valeric acid. Valerate was then subjected to Schmidt degradation to remove carbon- 1 as carbon dioxide. Up to 75% of deoxyribose was obtained as levulinic acid and up to 50% of levulinic as valeric acid. Specific radioactivity of valerate from DNA samples was lower than that of levulinate, indicating contamination of levulinate with radioactive material that was not carried over into valerate. Schmidt degradation did not result in the trapping of all of the ¹⁴C from valerate samples. However, no radioactivity was detected in most of the residual Schmidt degradation mixtures. There is loss of radioactive material, apparently by evaporation. The lack of radioactivity in the residual solution indicates that the four carbon atoms of valerate left behind as butylammonium ions after decarboxylation are devoid of radioactivity, suggesting that 1-¹⁴C-deoxyribose is incorporated intact into DNA.
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