저밀도 지방단백질 수용체(Low Density Lipoprotein Receptor)의 생합성에 관한 연구 : (II) Ginsenoside의 간 Cholesterol 대사촉진작용

주충노,최주영,이용우,Joo, Chung-No,Choi, Joo-Young,Lee, Yong-Woo 생화학분자생물학회 1987 한국생화학회지 Vol.20 No.4

It was demonstrated that liver cholesterol level of high cholesterol diet and ginsenoside administered rat (test group) was considerably lower than that of control group (high cholesterol diet fed rat). Acyl-coenzyme A: cholesterol acyltransferase(ACAT) activity of the test group was found stimulated. The effect of ginsenoside on the biosynthesis of bile acid from cholesterol by rat liver homogenate was also investigated in vitro and found that ginsenoside stimulated the biosyntehsis. It was also observed the effects several amphiphiles such as sodium dodecyl sulfate. Triton X-100, Lubrol-wx as well as ginsenoside mixture or purified ginsenosides on ACAT and found that all of them stimulated the ACAT activity considerably when they were present in an adequate amount respectively suggesting that nonspecific enzyme stimulating effect of the ginsenoside might be due to the surface activity of ginsenoside. From the above experimental results, it seemed that hypocholesterolemic activity of ginsenosides might be due to the lowered liver cholesterol level by enhanced ACAT activity as well as rapid bile acid biosynthesis from cholesterol. Low cholesterol level might also prevent the LDL receptor biosynthesis inhibition induced by high cholesterol diet resulting in a rapid removal of raised palsma LDL level. Ginsenoside의 고cholesterol혈증 억제작용메카니즘을 규명하기 위해 cholesterol 대사에 미치는 ginsenoside혼합물과 정제된 ginsenoside $Rb_1$, $-Rb_2$, -Re, $-Rg_1$ 의 영향을 조사하였다. 고cholesterol식이와 ginsenoside을 함께 투여한 시험군의 간cholesterol농도는 고cholesterol식이만으로 사육한 대조군에 비하여 현저히 낮았으며, acyl-coenzyme A: cholesterol acyltransferase(ACAT)활성은 시험군의 경우 대조군보다 불과 10% 정도 증가하였으나, 간세포의 미크로좀분획을 이용한 ACAT활성에 미치는 ginsenoside의 in vitro 영향은 ginsenoside의 농도가 $1{\times}10^{-4}{\sim}10^{-5}%$ 일 때 크게 증가하였다. 또한 간 파쇄액을 효소원으로 사용하여 cholesterol로부터의 담즙산 합성에 미치는 gisenoside의 영향을 in vitro에서 관찰한 결과 반응 혼합물에서의 ginsenoside의 농도가 $1{\times}10^{-3}%{\sim}10^{-4}%$ 일때 담즙산 합성이 현저히 증가하였다. 이상의 실험결과로부터 ginsenoside는 간에서의 cholesterol 대사를 촉진하여 간세포내 cholesterol농도를 효과적으로 낮춤으로써 cholesterol로 인한 LDL수용체합성억제를 완화하므로써 고cholesterol식이로 인한 고cholesterol혈증유발을 억제하는 것으로 생각된다.

( 2 ) Ginsenoside 의 간 Cholesterol 대사 촉진작용

주충노,최주영,이용우 ( Joo Young Choi,Yong Woo Lee,Chung No Joo ) 생화학분자생물학회 1987 BMB Reports Vol.20 No.4

It was demonstrated that liver cholesterol level of high cholesterol diet and ginsenoside administered rat (test group) was considerably lower than that of control group (high cholesterol diet fed rat). Acyl-coenzyme A: cholesterol acyltransferase(ACAT) activity of the test group was found stimulated. The effect of ginsenoside on the biosynthesis of bile acid from cholesterol by rat liver homogenate was also investigated in vitro and found that ginsenoside stimulated the biosyntehsis. It was also observed the effects several amphiphiles such as sodium dodecyl sulfate, Triton X-100, Lubrol-wx as well as ginsenoside mixture or purified ginsenosides on ACAT and found that all of them stimulated the ACAT activity considerably when they were present in an adequate amount respectively suggesting that nonspecific enzyme stimulating effect of the ginsenoside might be due to the surface activity of ginsenoside. From the above experimental results, it seemed that hypocholesterolemic activity of ginsenosides might be due to the lowered liver cholesterol level by enhanced ACAT activity as well as rapid bile acid biosynthesis from cholesterol. Low cholesterol level might also prevent the LDL receptor biosynthesis inhibition induced by high cholesterol diet resulting in a rapid removal of raised palsma LDL level.

쥐의 간 미토콘드리아 Aldehyde Dehydrogenase 의 분포와 특성에 관한 연구

최주영,주충노 ( Joo Young Choi,Chung No Joo ) 생화학분자생물학회 1992 BMB Reports Vol.25 No.5

Separation of inner membrane, outer membrane and matrix were accomplished by treating rat liver mitochondria preparation with digitonin followed by differential centrifugation. Mitochondrial ALDH isozymes were founded to be located in outer membrane as well as matrix fraction. Soluble low K_m ALDH in matrix had a very low K_m ($lt;10^(-6)M) for acetaldehyde, but the K_m of soluble high K_m ALDH in the matrix was 9.8 × 10^(-4) M, outer membrane bound ALDH showed a relatively high K_m, 1.4 × 10^(-2)M. The outer membrane bound ALDH was purified and characterized. Optimal pH was 9.5 and pI was 6.1. Subunit molecular weight was determined by SDS-PAGE and found to be 55,000. These results suggest that the soluble low K_m ALDH might be primarily response for the oxidation of acetaldehyde in rat liver, but the outer membrane bound ALDH will serve as barrier when the concentration of acetaldehyde in cytosol is high.

쥐의 간 시토졸 Aldehyde dehydrogenase 의 정제 및 특성에 관한 연구

최주영,주충노 ( Joo Young Choi,Chung No Joo ) 생화학분자생물학회 1993 BMB Reports Vol.26 No.1

Cytosolic ALDH of rat liver was purified by ammonium sulfate precipitation, DEAE-Sephacel, CM-cellulose ion exchange chromatography, Sephacryl S-300 gel filtration, and 5`-AMP Sepharose 4B affinity chromatography. The molecular weight was determined by Sephacryl S-300 gel filtration and found to be 217,000 dalton. SDS-PAGE showed that it was homotetramer consisting of four identical subunits whose molecular weight was 53,000 dalton. Optimal pH of this enzyme was 9.5 and pI value was 6.75. K_m value of rat liver cytosolic ALDH for acetaldehyde was 1.06×10^(-3) M, but those of indole-3-acetaldeghyde($lt;10^(-6) M) and benzaldehyde (1.59×10^(-5) M) were extremely low. These results suggested that rat liver cytosolic ALDH might be primarily involved in the oxidation of aromatic aldehyde including biogenic aldehyde.

쥐의 간 시토졸 Aldehyde dehydrogenase의 정제 및 특성에 관한 연구

최주영,주충노,Choi, Joo-Young,Joo, Chung-No 생화학분자생물학회 1993 한국생화학회지 Vol.26 No.1

쥐의 간 시토졸 Aldehyde dehydrogenase(ALDH)를 황산 암모늄 처리, DEAE-Sephacel, CM-cellulose, Sephacryl S-300, 5'-AMP Sepharose 4B chromatography 순으로 정제 하고 TSK G3000SW column을 이용하여 HPLC로 분석한 결과 순수한 단일 peak를 얻었고, Sephacryl S-300 chromatography 방법으로 측정된 분자량은 217,000 dalton이었으며 SDS-PAGE를 행한 결과 분자량이 53,000인 subunit로 구성된 homotetramer임이 밝혀졌다. 이 효소의 pI값은 6.75, 최적 pH는 9.5이며 dehydrogenase활성 뿐 아니라 esterase활성도 나타내었다. Acetaldehyde에 대한 $K_m$값은 $1.06{\times}10^{-3}M$이지만 indole 3-acetaldehyde에 대한 $K_m$, 값은 $10^{-6}M$이하, benzaldehyde에 대한 $K_m$값이 $(1.59{\times}10^{-5}M)$로 낮은 값을 나타내는 것으로 보아 시토졸 ALDH는 주로 biogenic aldehyde나 aromatic aldehyde 대사에 관여하는 것으로 생각된다. Cytosolic ALDH of rat liver was purified by ammonium sulfate precipitation, DEAE-Sephacel, CM-cellulose ion exchange chromatography, Sephacryl S-300 gel filtration, and 5'-AMP Sepharose 4B affinity chromatography. The molecular weight was determined by Sephacryl S-300 gel filtration and found to be 217,000 dalton. SDS-PAGE showed that it was homotetramer consisting of four identical subunits whose molecular weight was 53,000 dalton. Optimal pH of this enzyme was 9.5 and pI value was 6.75. $K_m$ value of rat liver cytosolic ALDH for acetaldehyde was $1.06{\times}10^{-3}M$, but those of indole-3-acetaldeghyde(<$10^{-6}M$) and benzaldehyde $(1.59{\times}10^{-5}M)$ were extremely low. These results suggested that rat liver cytosolic ALDH might be primarily involved in the oxidation of aromatic aldehyde including biogenic aldehyde.

쥐의 간 시토졸 Aldehyde dehydrogenase 의 반응 메카니즘에 관한 연구

최주영,주충노 ( Joo Young Choi,Chung No Joo ) 생화학분자생물학회 1993 BMB Reports Vol.26 No.1

Chemical modification experiments suggested that cysteine, serine, glutamate residues might participate in the catalytic role and it was suggested that a probable reaction mechanism of ALDH through substrate activation, chloral hydrate inhibition and disulfiram inhibition behaviors towards ALDH activity.

쥐의 간 시토졸 Aldehyde dehydrogenase의 반응 메카니즘에 관한 연구

최주영,주충노,Choi, Joo-Young,Joo, Chung-No 생화학분자생물학회 1993 한국생화학회지 Vol.26 No.1

정제된 쥐의 간 시토졸 aldehyde dehydrogenase(ALDH)의 활성부위를 화학변형 시약을 이용하여 검색한 결과 cysteine, serine 및 glutamate 잔기가 활성에 중요한 역할을 할 것으로 생각된다. 또한 ALDH의 기절 활성화 특성과 chloral hydrate에 의한 억제양상 및 disulfiram에 의한 억제양상으로부터 ALDH의 반응 메카니즘을 추정하였다. Chemical modification experiments suggested that cysteine, serine, glutamate residues might participate in the catalytic role and it was suggested that a probable reaction mechanism of ALDH through substrate activation, chloral hydrate inhibition and disulfiram inhibition behaviors towards ALDH activity.