Abstract
Abstract The acid deoxyribonucleases in homogenates of human gastric mucosa and cervix uteri were purified extensively by a procedure including DEAE-cellulose and phosphocellulose chromatographies, gel filtration, and isoelectric focusing. The enzymes in the two preparations had similar properties. The active protein was resolved into at least two forms with different isoelectric points, as judged by the formation of acid-soluble fragments. The major component of the activity had an isoelectric point at pH 7.02, and the minor component at pH 6.86. However, other properties of these two forms were similar. The final preparations of the gastric and uterine acid DNases were free of other types of DNase (DNases I, III, and IV), acid and alkaline phosphatases, acid and basic RNases, and nonspecific phosphodiesterase. Both enzymes had a molecular weight of approximately 38,000, as determined by gel filtration on Sephadex G-100, and showed maximum activity at pH 5.2 to 5.5 in 33 mm acetate buffer. They did not require divalent cations for activity, and hydrolyzed native, double-stranded DNA about 10 to 20 times faster than heat-denatured DNA. They did not act on RNA or calcium bis(p-nitrophenyl) phosphate, indicating that they did not contain intrinsic phosphodiesterase activity. The products formed from native DNA were 3'-phosphoryl- and 5'-hydroxyl-terminated oligonucleotides. The average chain length of the limit digests with these enzymes was approximately 10 and the predominant species were longer than hexanucleotides.
Highlights
Acid DNase activity was scarcely detectable in the muscular layers of the two organs
Purified preparations of acid DNase were obtained from homogenates of human gastric mucosa and cervix uteri as described in this paper
Bernardi and Griffe [34] observed that a highly purified preparation of hog spleen DNase II catalyzed the slow hydrolysis of p-nitrophenol from calcium bis(p-nitrophenyl) phosphate and suggested that the phosphodiesterase activity was an intrinsic property of the acid DNase molecule
Summary
Assay of Acid DNase-Production of acid-soluble radioactivity from [32P]11NA was routinely determined in reaction mixture (0.3 ml) containing 10 pmoles of sodium acetate buffer, pH 5.4, 0.6 Mmole of EDTA, 10 nmoles of E’. coli [32P]DNA, and 0.5 to 5 units of enzyme.Twenty micrograms of bovine plasma albumin (in freshly prepared aqueous solution) [24] were added to the medium, when the enzyme preparation had a specific act,ivity of more than 1000. Assay of Acid DNase-Production of acid-soluble radioactivity from [32P]11NA was routinely determined in reaction mixture (0.3 ml) containing 10 pmoles of sodium acetate buffer, pH 5.4, 0.6 Mmole of EDTA, 10 nmoles of E’. Coli [32P]DNA, and 0.5 to 5 units of enzyme. The mixture was incubated for 10 min at 35”, the acid-soluble fraction was obtained by adding cold perchloric acid, and the radioactivity of an aliquot was determined as described previously [21]. The acid-soluble fraction from control mixture without enzyme contained less than 0.5yo of the added radioactivity. One unit of acid DNase activity is defined as the amount of enzyme catalyzing the production of 1 nmole of acidsoluble 3*P in 10 min under the above conditions, Assay of Other Enzyme Activities-I>Nase
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