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RISS 인기검색어
- 검색키워드
- 저자 : 고은희 ( Hay Young Lee (검색결과 2 건)
- 무료
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Purification and Characterization of the Active Site of Phospholipase D
이혜영,최명언,고은희,Lee, Hay-Young,Choi, Myung-Un,Koh, Eun-Hie 생화학분자생물학회 1989 한국생화학회지 Vol.22 No.4
양배추의 phospholipase D(PLD)는 아세톤 침전물로부터 10%의 회수율로 정제하였다. 정제방법은 Sephadex G-200 젤 여과법, DEAE-셀루로즈 크로마토그래피, ${\gamma}$-aminopropane agarose hydrophobic 크로마토그래피법을 순서대로 사용하였다. PLD의 순도는 SOS-PAGE로 확인되었고 분자량은 89,000으로 측정되었다. 활성도는 MES 완충용액 pH 6.3에서 $428{\mu}mol/min/mg$ 이었다. PLD의 열에 대한 안정성은 활용도에 필요한 칼슘이온과는 무관한 것으로 나타났다. Active site의 특성을 조사하기 위하여 N-ethylmaleimide, p-cholromercuribenzoate, p-bromophenacylbromide 및 diethylpyrocarbonate 등의 화학적 변형시약을 검토하였다. 효소의 활동도는 cysteine 변형시약으로 완전히 없어졌으며 active site에는 적어도 한 개의 histidine 기가 있을 것으로 밝혀졌다. Phospholipase D has been partially purified from an acetone powder of savoy cabbage in an overall yield of 10%. The purification involves gel filtration on Sephadex G-200, ion-exchange chromatography on DEAE-cellulose, and hydrophobic affinity chromatography using ${\gamma}$-aminopropane agarose gel. The purity of about 80% and molecular weight of 89,000 were estimated on the basis of gel electrophoresis. The specific activities of the partially purified enzyme was $428{\mu}mole/min/mg$ protein in MES buffer, pH 6.3, containing 4 mM phosphatidylcholine, 2 mM SDS and 10 mM $CaCl_2$ at $37^{\circ}C$. Calcium ion is essential to the enzyme activity but is not involved in the heat stability of the enzyme. To characterize the active site of the enzyme, the following chemicals were examined; N-ethylmaleimide, p-chloromercuribenzoate, p-bromophenacylbromide, and diethylpyrocarbonate. The enzymatic activity was lost completely by cysteine group modifying reagents and it was found that there was at least one histidine group in the active site.
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이혜영,최명언,고은희 ( Hay Young Lee,Myung Un Choi,Eun Hie Koh ) 생화학분자생물학회 1989 BMB Reports Vol.22 No.4
Phospholipase D has been partially purified from an acetone powder of savoy cabbage in an overall yield of 10%. The purification involves gel filtration on Sephadex G-200, ion-exchange chromatography on DEAE-cellulose, and hydrophobic affinity chromatography using γ-aminopropane agarose gel. The purity of about 80% and molecular weight of 89,000 were estimated on the basis of gel electrophoresis. The specific activities of the partially purified enzyme was 428 μmole/min/㎎ protein in MES buffer, pH 6.3, containing 4 mM phosphatidylcholine, 2 mM SDS and 10 mM CaCl₂ at 37℃. Calcium ion is essential to the enzyme activity but is not involved in the heat stability of the enzyme. To characterize the active site of the enzyme, the following chemicals were examined; N-ethylmaleimide, p-chloromercuribenzoate, p-bromophenacylbromide, and diethylpyrocarbonate. The enzymatic activity was lost completely by cysteine group modifying reagents and it was found that there was at least one histidine group in the active site.
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