Isolation of DNase II from bovine small intestine by chromatography on DEAE cellulose of a 105,000 xg supernatant solution prepared from an homogenate of the mucosa in Krebs Ringer phosphate buffer appeared to yield two activities, a major activity which was eluted from the column with 20 mM phosphate buffer and a minor activity which was eluted with a potassium chloride gradient. The two DNase II activities differed in their response to increasing ionic strength, pH, sulfate ion concentration and temperature for the hydrolysis of DNA. The major activity degraded native DNA more rapidly than denatured DBA whereas the minor activity degraded both at the same rate. Previous investigators have reported the presence of two DNases lis with different properties in other tissues. In bovine intestinal DNase II, the minor activity, upon rechromatography on DEAE cellulose, eluted in the same position as the major DNase II and it was concluded; that the appearance of the minor DNase II activity was an artifact of the chromatography. It is likely that a small quantity of DNase II was bound to endogenous DNA on the DEAE cellulose column in the 20 mM phosphate buffer and later eluted from the column along with some of the DNA. with the potassium chloride gradient. DNA present in the minor DNase II preparation probably caused the apparent differences in properties of the two DNase lis by interfering in the enzymic reactions.
Intestinal DNase II was partially purified by ion exchange chromatography and gel filtration and had properties similar to DNase lis from other tissues. The enzyme hydrolysed calf thymus DNA endonucleolytically at acid pH in the absence of a divalent metal ion to oligonucleotides with 3'-phosphate and 5'-hydroxyl terminals. The activation energy for the reaction was 19 kcal/mole; that for denaturation of DNase II itself, k3 kcal/mole. Michaelis-Menton kinetics were observed for the reaction of DNase II with Escherichia coli DNA—the Michaelis constant was 2.42 x 10⁻⁷ M DNA-phosphate. The molecular weight of DNase II was estimated to be 41,000 by gel filtration on Sephadex G100.
The early stages of the digestion of DNA by DNase II were investigated by labelling the reaction products with ³²P at their 5'-terminals using polynucleotide kinase and [x-³²P]ATP and at their 3'-terminals using terminal deoxynucleotidyl transferase and [⍺-³²P]ATP.
The mode of cleavage of native DNA by DNase II was determined by comparing the polynucleotide-catalysed incorporation of 32p from
[x-³²P]ATP into native and denatured DNase II reaction products. Since single-strand cleavage of DNA by DNase II released 5'-hydroxyl terminals that were inaccessible to polynucleotide kinase as long as the DNase II reaction products remained double-stranded, incorporation of ³²P into native products was proportional to the number of double-strand cleavages while incorporation of ^P into denatured products was proportional to the number of double-strand cleavages plus single-strand cleavages. It was found that DNase II degraded native DNA primarily by a double-strand cleavage mechanism.
After DNase II catalysed hydrolysis of DNA each of the four bases present in DNA was found at the 5'- and 3'-terminals of the reaction products. Thus DNase II did not have an exclusive preference for one or two bases at either terminal, and likely cleaved a large number of
different base sequences in the DNA.. The most susceptible internucleotide linkage was GpG; the most resistant, CpT. The base specificity at the 5'-terminal changed during the reaction, especially in the initial and terminal phases. In the initial phase the proportion of guanine was elevated and the order of cytosine and adenine was reversed compared to later stages in the reaction. These changes could reflect the presence of a preferred sequence that was selectively degraded and exhausted during the initial phase of the reaction. Different proportions of terminal bases in cleavage products of DNA from diverse species indicated that susceptible sequences occurred with different frequencies in the various DNAs. / Medicine, Faculty of / Biochemistry and Molecular Biology, Department of / Unknown
| Identifer | oai:union.ndltd.org:UBC/oai:circle.library.ubc.ca:2429/21546 |
| Date | January 1978 |
| Creators | Keys, David Stephen |
| Source Sets | University of British Columbia |
| Language | English |
| Detected Language | English |
| Type | Text, Thesis/Dissertation |
| Rights | For non-commercial purposes only, such as research, private study and education. Additional conditions apply, see Terms of Use https://open.library.ubc.ca/terms_of_use. |
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