碩士 / 國立臺灣大學 / 生物化學暨分子生物學研究所 / 90 / Nucleases, in a broad sense, are enzymes capable of hydrolyzing both DNA and RNA. Bovine pancreatic DNase I (EC 3.1.21.1) is the DNase I that has been studied most thoroughly. Although the enzymatic properties of shrimp nuclease are typical of bovine DNase I, its sequence is distinct from those of bovine DNase I and other vertebrates. The protein and cDNA sequences for shrimp nuclease have been determined. Surprisingly, the sequences exhibited no similarity to those of the bovine DNase-I-like proteins. However, in the motif of residues 205-255, a number of identical residues were found when aligned with the conserved active sites of several nucleases.
We improved the recovery of shrimp nuclease during purification. Briefly, the hepatopancreatic tissue of shrimp was homogenized with deionized H2O at the ratio of 1:2, followed by chromatography on the DEAE-cellulose, Sephadex G-100, Phenyl-Sepharose, and two consecutive hydroxyapatite columns. The enzyme was purified to homogeneity as evidenced by a single band of Mr 45 kDa on SDS-PAGE. Shrimp nuclease in a solution containing 10 mM MgCl2 and 5 mM iodoacetate at 37 ℃ was 95 % inactivated in 48 hours. For the identification of the probable active site residues, shrimp nuclease was first modified with iodo[2-14C]acetate. Three major radioactive peptides were then isolated by chromatography on a Superdex Peptide PE7.5/300 column from the thermolytic digest of the labelled enzyme. The identification of the radioactive peptides by amino acid composition analysis revealed that the radio-labelled residues were His211 and Met294 when modification was carried out in the presence of Mg2+. However, Met294 could still be labelled by IAA in the absence of Mg2+, and the activity decreased 30%. Therefore, His211 is considered to be the active site of shrimp nuclease, and Met294 may also be involved in other unknown functions of catalysis.
| Identifer | oai:union.ndltd.org:TW/090NTU01104011 |
| Date | January 2002 |
| Creators | HUAN-TSUNG HSU, 許煥宗 |
| Contributors | TA-HSIU LIAO, 廖大修 |
| Source Sets | National Digital Library of Theses and Dissertations in Taiwan |
| Language | zh-TW |
| Detected Language | English |
| Type | 學位論文 ; thesis |
| Format | 77 |
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