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Partial purification and characterization of dnase I from the intestinal mucosa of rat

University of British Columbia

DNase I activity has been found in the small intestine of the rat. This work involves a partial purification and characterization of this enzyme. Crude enzyme extract was prepared by ultracentrifugation of the homogenate of washed mucosal scrapings. The DNase I activity of the crude enzyme preparation was not stable, it could be stabilized by divalent metal Ions, The crude enzyme extract was chromatographed on DEAE cellulose in phosphate buffer and the adsorbed enzyme eluted with a phosphate gradient. The crude enzyme was "shown to contain proteolytic enzymes. The active material was freeze-dried, redissolved freed of phosphate and chromatographed on Sephadex G-100. The active material eluted from the Sephadex column was adsorbed on DEAE cellulose and eluted with a linear gradient of phosphate. This procedure gave a purification of 200-400 fold, relative to the crude enzyme extract. The product was not stable in the absence of Ca⁺⁺ and contained proteolytic enzymes. The molecular weight of the enzyme was estimated as 3.05 x 10⁴ daltons by gel filtration on Sephadex G-100. The properties of the rat enzyme were compared to those of the bovine enzyme. No significant difference was found in molecular-weight, inhibition by Na⁺, K⁺, haemoglobin or iodoacetate, pH optimum, or metal ion requirements. The interactions of Ca⁺⁺ with the DNA-DNase system were explored. In the presence of Mg⁺⁺, Ca⁺⁺ first activates and then inhibits DNase activity.

Text

2011-03-21

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http://hdl.handle.net/2429/32662

Biochemistry and Molecular Biology

University of British Columbia

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