UBC Theses and Dissertations
Studies on the cytoplasmic dna polymerases from the intestinal mucosa of rat Waung, Lucille Yih-Lo
Significant DNA polymerase activity has been found in cytoplasmic preparations from rat intestinal mucosa. The present work involves a partial purification and a study of the general properties of this cytoplasmic enzyme activity. Crude cytoplasmic enzyme was prepared by high-speed centrifugation of the homogenate of washed mucosal scrapings. A strong inhibitor of DNA polymerase was sedimented by the high-speed centrifugation. The bulk of the enzyme activity was unadsorbed on DEAE-cellulose. However, a minor portion of the enzyme was adsorbed, and was eluted with 0.1 M KC1. When crude cytoplasmic enzyme was chromatographed by gel-filtration on Sephadex G-150, a single peak of DNA polymerase activity was detected. By the use of protein markers with known molecular parameters, the molecular weight of the DNA polymerase fraction was estimated to be 101,000. The enzyme required the presence of a DNA template and Mg++ ions. Activity was only slightly enhanced by the addition of dithiothreitol. For maximum activity, the presence of all four deoxy-nucleoside triphosphates were required. Heat-denatured DNA was preferred as primer. The optimum pH for this enzymatic activity was found to be 7.2 in potassium phosphate buffer, and 8.0 in Trisacetate buffer. Time course studies on the enzyme reaction indicated that the reaction was linear with respect to incubation time for at least 30 min. The DNA polymerase activity was stable up to 13 days under temperature conditions of 4°C to -20°C. Glycerol in 20% to 35% (v/v) concentrations was found to have both a stimulatory and a stabilizing effect on the enzyme activity. Ethylene glycol at 20% (v/v) concentration was also found to have a stimulatory effect on the enzyme activity. The enzyme was strongly inhibited in the presence of 0,10 M phosphate ions and activity was drastically reduced in phosphate ion concentrations of 0.20 M and above. The product of the DNA polymerase reaction could be destroyed by DNase, indicating that it was DNA in nature. The purpose of the present work was to determine whether the DNA polymerase activity in the cytoplasmic preparation is actually of cytoplasmic origin, or whether it is due to nuclear contamination. The above results were compared with the results obtained by other workers on the nuclear DNA polymerases. The evidence seems to indicate that the cytoplasmic enzyme activity is not due to nuclear contamination. The nuclear preparation contained several DNA polymerases, while the cytoplasmic preparation contained a single major DNA polymerase activity. This cytoplasmic activity resembled one of the nuclear activities in many respects. The cytoplasmic preparation also contained a minor DNA polymerase activity which may be mitochondrial in origin.
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