Purification and characterization of glutatathione S-transferase cloned from pseudomonas sp. DJ77 = Pseudomonas sp. DJ77에서 클로닝한 glutathione S-transferase의 분리 및 특성 규명

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A new bacterial glutathione S-transferase phnC gene cloned from Pseudomonas sp. DJ77 was expressed in E. coli. The gene product PhnC was purified by glutathione-affinity chromatography. The results of SDS-polyacrylamide gel electrophoresis and gel permeation chromatography suggested that PhnC was a homo-dimeric protein, Mr of which is 42 kDa. The isoelectric point was 5.8 by isoelectric focusing. These Mr and pI values were similar to Mr (43200) and pI (5.5) predicted from DNA sequence of phnC. Furthermore, nine N-terminal amino acid residues determined by amino acid sequencer were the same as those predicted from DNA sequence. PhnC showed glutathione S-transferase activity toward glutathione and 1-chloro-2,4-dinitrobenzene (CDNB) and the specific activity was 10.6 $\mu$ mol/min/mg. The specific activity was higher than that of any other known bacterial glutathione S-transferase. PhnC was stable below $35^\circ C$ and between pH 6 and pH 9. The highest activity was shown at pH 8.0. An analysis of circular dichroism spectra of PhnC showed that the portions of $\alpha$-helix, $\beta$-sheet, $\beta$-turn and random coil structures in PhnC were 6, 13, 22, 49\%, respectively. The portions of secondary structures were somewhat different from those of known bacterial and mammalian glutathione S-transferases. Computer modelling of the inital-rate using nonlinear least-squares regression analysis favored a rapid-equilibrium ordered bi-bi mechanism with CDNB binding first, which was different from the kinetic mechanisms of mammalian GSTs.
Advisors
Chung, An-Sik정안식
Description
한국과학기술원 : 생명과학과,
Publisher
한국과학기술원
Issue Date
1995
Identifier
98694/325007 / 000933459
Language
eng
Description

학위논문(석사) - 한국과학기술원 : 생명과학과, 1995.2, [ iii, 45 p. ]

Keywords

단백질 분리

URI
http://hdl.handle.net/10203/28444
Link
http://library.kaist.ac.kr/search/detail/view.do?bibCtrlNo=98694&flag=dissertation
Appears in Collection
BS-Theses_Master(석사논문)
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