Characteristics of $G_{418}$-sensitive mitochondrial ATPase/ATP synthase from pleurotus florida

사철느타리버섯 중 $G_{418}$-sensitive 미토콘드리아성 ATPase/ATP synthase의 특성

The mitochondrion was purified at 44% sucrose layer from pleurotus florida by using ultracentrifuge and sucrose density gradient method. Optimum pH and temperature of ATPase and ATP synthase were pH 7.4, $60^{\circ}C$ and pH 7.5, $57^{\circ}C$ respectively, also their Km values were determined as 11.6mM and 8.4mM. ATPase was activated at 5~6mM ATP substrate concentration, then ATP synthase was 5~10mM range ADP. ATPase/ATP synthase were $Mg^{2+}$-dependent enzyme, partially inhibited by their substrate, and then showed an none competitive inhibition pattern by $G_{418}$. Amino acid composition of ATPase/ATP synthase was as follows, hydrophobic amino acid residue was 50.5%, small residue, 56.1%, hydrogen bonding residue, 43.7% and helix breaking residue, 55.2%. Phosphatidyl choline, phosphatidyl ethanolamine and phosphatidyl glycerol were contained but not phosphatidyl inositol and phosphatidyl serine. Palmitate(51.31%), stearate(18.32%) and unsaturated fatty acids($C_{18:1}$, $C_{18:2}$ and $C_{16:1}$) were predominated.

형질 전환된 사철느타리버섯으로부터 초원심분리 및 설탕밀도 기울기법으로 44% 층에서 미토콘드리아를 분리정제하였다. ATPase와 ATP synthase의 최적 활성조건은 각각 pH 7.4, $60^{\circ}C$ 및 pH 7.5, $57^{\circ}C$였고, km값은 11.6mM과 8.4mM였다. ATPase는 기질농도 5~6mM의 ATP에서, ATP synthase는 5~10mM ADP 농도에서 활성이 높으며, 그 이상의 농도에서는 기질 저해를 받았다. ATPase/ATP synthase 모두 $Mg^{2+}$ 의존성 효소로 $G_{418}$으로 비경쟁적인 저해를 받았다. 효소의 아미노산 분석결과 hydrophobic 아미노산 잔기는 50.5%, small 아미노산 잔기는 56.1%, hydrogen bonding 아미노산 잔기는 43.7%, helix breaking 아미노산 잔기는 55.2%였다. 인지질을 분석한 결과 phosphatidyl glycerol, phosphatidyl choline 및 phosphatidyl ethanolamine으로 구성되었고 phosphatidyl serine과 phosphatidyl inositol은 전혀 없었다. 포화 지방산은 palmitate(51.31%)와 stearate(18.32%)의 함량이 많았고, 불포화 지방산($C_{18:1}$, $C_{18:2}$ 및 $C_{16:1}$)의 함량도 많았다.