The catalytic mechanisms for porcine spleen Deoxyribonuclease II using DNA and NPPP as substrates

• Main Authors: Huang, Kao Kuei, 黃國魁
• Other Authors: Liao T.-H.
• Format: Others
• Language: zh-TW
• Published: 1998
• Online Access: http://ndltd.ncl.edu.tw/handle/97128870431496652355

碩士 === 國立臺灣大學 === 生化學研究所 === 86 === Abstract Deoxyribonucleases(DNases) are endonucleases. There are two major types of DNases, one of which, DNase II, is located in lysosomes. Porcin e spleen DNase II is the most studied DNase II. It is composed of three polype ptide chains: α1, α2, β. In order to find the optimal reaction conditions, DNa-se II activities of mouse spleen extract were examined, particularly with respect to the effects of pH and of the type and concentration of added salts. However, DNase II preparation used in these studies was not a fully purified one, one had to consider whether there were other factors in the extract distu rbed the results. We used purified porcine spleen DNase II to study the effect s of ions in this study. The results showed that the hyperchromicity curves of DNA hydrolysis catalyzed by DNase II were different with low and high ionic s trengths and the difference was probably due to DNA conformational changes. Mo reover, the effects of cations and anions on DNase II activity were also inves tigated. Sulfate was the best ion on the DNase II activation and inhibition. We used the synthetic molecule NPPP as substrate to investigate the kinetics of small molecule hydrolyzed by DNase II. The results showed that more than o ne NPPP molecule was bound per molecule of DNase II with co-operativity. When NPPP concentrations were higher than 40 mM, NPPP hydrolysis was inhibited. Ani ons also inhibited NPPP hydrolysis and the inhibitory efficiency varied in the following order: sulfate >> methylsulfate > phosphate. For sulfate, half inhi bition occurred at 10 μM which was equivalent to the concentration required f or half protection of DNase II from iodoacetate inactivation. It may thus be c oncluded that the same sulfate binding site on DNase II is responsible for not only the inhibition of NPPP hydrolysis but also the protection of iodoacetate inactivation.