碩士 / 國立臺灣大學 / 生化學研究所 / 82 / Deoxyribonuclease ( DNase ) of tilapia fish, Oreochromis
mossambicus was purified from the crude extract of the
hepatopancreatic tissue. The purification scheme involved an
ammonium sulfate precipitation step and four chromatographic
steps including DEAE-cellulose, Sephadex G-100, hydroxyapatite
and phenyl-Sepharose. The purified preparation of this enzyme
was homogeneous, evidenced by a single protein band on sodium
dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).
The Mr of the fish DNase is 28 kD based on SDS-PAGE and gel
filtration. The enzyme (pI = 6.0) displays no isoenzyme
heterogeneity as determined by isoelectric focusing on thin
layer polyacrylamide gel. The fish DNase can be bound to a Con
A column and subsequently eluted with glucose, indicating a
glycoprotein. Fish DNase is sensitive to IAA inactivation. and
labile above 60℃. The fish enzyme is a DNase Ⅰ - type enzyme
because it has an alkaline pH activity optimum and reguires for
divalent cation. In contrast to bovine DNase, without Ca2+,
fish DNase resists to trypsin or MSH inactivation at pH 8.0.
The fish DNase-catalyzed pBR322 DNA hydrolysis in addition to
single cuts, a significant amount of double cuts with Mn2+ as
activator but no double cuts with Mg2+ plus Ca2+ or only Mg2+
as activator. The HPLC map of the tryptic digest of the reduced
and S- carboxymethylated fish DNase showed 22 principal
peptides. chymotryptic and CNBr peptides isolated provided
certain for establishing the order in the intact polypeptide
for the 22 major tryptic peptides.The complete amino acid
sequence of fish DNase is derived based partially on the
homology with the bovine DNase sequence. The fish sequence,
ascompared with the bovine sequence, shows two single amino
acid deletions. Both bovine and fish enzymes have one
carbohydrate side chain attached to Asn-18. In contrast to two
disulfides in bovine DNase, there is only one disulfide in fish
DNase.
| Identifer | oai:union.ndltd.org:TW/082NTU00104014 |
| Date | January 1994 |
| Creators | Hsiao, Yi-Min, 蕭懿民 |
| Contributors | Liao, Ta-Hsiu, 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 | 81 |
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