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Specificity and kinetic studies of deoxyribonucleases from the intestinal mucosa of the rat Lee, Cheuk Yu


The properties of the deoxyribonuclease activity in the intestinal mucosa of the rat have been studied. Two DNases were found in a cell-free extract prepared by homogenizing the mucosal tissue in Krebs-Ringer phosphate buffer and then centrifuging the homogenate at 105,000 x g for 60 mins. Resolution and partial purification of these two enzymes were achieved by ion-exchange chromatography on DEAE-cellulose and partition on hydroxylapatite of the cell-free extract or an acetone powder preparation of the enzymes. One of the enzymes was identified as DNase I by its optimum pH (6.5 to 6.8), its requirements for bivalent metals and by its reaction to known DNase I inhibitors such as EDTA, citrate and arsenate. It was also found to be active toward native DNA, and, to a lesser degree, toward heated DNA. The second DNase was shown to be qualitatively different from DNase I. It has an acidic optimum pH (3.5-4.0), does not require activation by bivalent cations and is not inhibited by EDTA, citrate or arsenate. This second DNase activity is therefore of the DNase II type. The linkage specificity of the two enzymes was studied by isolating the products of the reaction and examining them with respect to chain length, base composition of the mononucleotides, relative frequencies of the dinucleotides and base frequencies at the ends of the oligonucleotides. The hydrolysis reaction was carried out under a variety of conditions. When Mg++ was used as the activating ion and native DNA as substrate, DNase I was found to show a preference for the linkages pApC, pApT and pGpT. But when Mn++ was the activator, or when heated DNA was used as substrate in the presence of Mg++, DNase I did not show any significant order of specificity. With regard to DNase II, the enzyme was found to attack native DNA preferentially at the ApCp, GpCp and GpTp bonds. The mechanism of metal activation of intestinal DNase I was also studied. Preliminary experiments showed that the enzyme was inhibited by high concentrations of both metals and DNA. In addition, optimal activation was found to depend on the molar ratio of metal ion to DNA phosphorus. These experiments suggested that the metal-DNA complex is probably the true substrate. Consequently, a rate equation in terms of the metallosubstrate concentration was developed based on the assumption that intestinal DNase I can combine individually and simultaneously with free metal, free DNA and the metal-DNA complex. Data obtained from initial velocity studies carried out at varying concentrations of metal and DNA fitted this rate equation well. On this basis, it was suggested that the enzyme, the metal activator and the DNA substrate combine to form a ternary complex which then dissociates to give the products.

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