쥐간 Cytosol 에 있는 Dolichol 의 결합인자의 부분정제와 특성

윤교희,shidoji Yoshihiro,takai Katsuji ( Kyo Hie Yoon ) 생화학분자생물학회 1991 BMB Reports Vol.24 No.6

The present study describes the partial purification and characterization of dolicholbinding component(s). [³H]dolichol binding was assessed by a newly developed binding assay of fast flow liquid chromatography on DEAE-Toyopearl 650 M (1 ml). The binding component(s) for dolichol in rat liver cytosol was purified about 60-fold by heat treatment (80℃, 5 min) and DEAE-Toyopearl 650 M column chromatography. [³H]dolichol complex of the partially purified component(s) behaved as an anionic entity on DEAE-Toyopearl and the binding was abolished by Pronase proteolysis. Scatchard analysis revealed that the partially purified binding component(s) possessed a high affinity (K_d=1.25 × 10^(-7) M) and low capacity (242 pmol/㎎ protein) binding site, and also a low affinity and high capacity site(s). By competitive binding assay, the partially purified binding component(s) showed an affinity with dolichol and its derivatives, but not with palmitic acid, oleic acid and retinol. Though not dolichol congeners, squalene, ubiquinone and cholesterol were partially competitive. No lipid binding (carrier) proteins so far described accounted for the dolichol binding activity in our preparation, and the total binding capacity estimated herein comparable to the amounts of dolichol in rat liver cytosol.

Partial Purification and Characterization of Dolichol-Binding Component(s) in Rat Liver Cytosol

윤교희,Yoon, Kyo-Hie,Shidoji, Yoshihiro,Takai, Katsuji 생화학분자생물학회 1991 한국생화학회지 Vol.24 No.6

본 연구에서는 쥐간 cytosol에 있는 dolichol 결합인자의 부분정제를 행하였으며, 부분정제품의 특성을 밝혔다. 결합활성의 측정을 위하여 고속이온교환 크로마토그래피(FFLC)법을 새로이 개발하였다. 이 FFLC법은 30분 이내에 분석이 가능하며, 재현성이 양호하였다. 쥐간 cytosol의 dolichol 결합인자는 $80^{\circ}C$ 5분간의 열처리와 DEAE-Toyopearl 650M column 크로마토그래피에 의해 약 60배로 정제되었다. 부분정제된 결합인자와 $[^3H]$dolichol과의 복합체는 DEAE-Toyopearl column에 흡착되는 음이온 성질을 보였으며, 단백질 분해효소인 Pronase 처리에 의해 그 결합능이 상실되는 점으로 보아 단백질의 가능성을 시사하였다. Scatchard 분석에 의해 부분정제품에는 높은 친화력 $(K_d=1.25{\times}10^{-7}M)$과 낮은 용량(242 pmol/mg protein)의 결합부위와 낮은 친화력과 큰 용량의 결합부위가 있음을 알 수 있었다. 또한 부분정제품의 ligand 결합 특이성을 검토한 결과, 부분정제품은 dolichol과 dolichol 유도체에 대하여 친화력을 보였으며, palmitic acid, oleic acid, retinol에 대하여는 친화력을 보이지 않았다. 한편, squalene, ubiquinone, cholesterol에 대해서도 부분적으로 친화력을 보였다. The present study describes the partial purification and characterization of dolicholbinding component(s). $[^3H]dolichol$ binding was assessed by a newly developed binding assay of fast flow liquid chromatography on DEAE-Toyopearl 650 M (1 ml). The binding component(s) for dolichol in rat liver cytosol was purified about 60-fold by heat treatment ($80^{\circ}C$, 5 min) and DEAE-Toyopearl 650 M column chromatography. $[^3H]dolichol$ complex of the partially purified component(s) behaved as an anionic entity on DEAE-Toyopearl and the binding was abolished by Pronase proteolysis. Scatchard analysis revealed that the partially purified binding component(s) possessed a high affinity $(K_d=1.25{\times}10^{-7}M)$ and low capacity (242 pmol/mg protein) binding site, and also a low affinity and high capacity site(s). By competitive binding assay, the partially purified binding component(s) showed an affinity with dolichol and its derivatives, but not with palmitic acid, oleic acid and retinol. Though not dolichol congeners, squalene, ubiquinone and cholesterol were partially competitive. No lipid binding (carrier) proteins so far described accounted for the dolichol binding activity in our preparation, and the total binding capacity estimated herein comparable to the amounts of dolichol in rat liver cytosol.

쥐간 Cytosol에 있는 Dolichol 의 결합인자에 관한 연구

윤교희,( Kyo Hie Yoon,Shidoji Yoshihiro,Takai Katsuji ) 생화학분자생물학회 1991 BMB Reports Vol.24 No.6

This paper describes an approach to search cytosolic binding component(s) for dolichol in rat liver. The contents of endogenous dolichol were estimated to be 13.5 ㎍/g liver (132 ng/㎎ protein) in the homogenate, 8.8 ㎍/g liver (692 ng/㎎ protein) in the microsome, and 1.8 ㎍/g liver (19 ng/㎎ protein) in cytosol indicating that about 13% of the hepatic dolichol stayed in the pool of cytosol. With respect to the chain distibution, dolichol-18 and -19 were found predominate in cytosol alike in homogenate. When rat liver cytosol was incubated with [³H]dolichol and applied to a column of Sephacryl S-300, 40% of total radioactivity appeared at the void volume. On the same chromatography of aqueous dispersion of [³H]dolichol as a blank, approximately 10% of the radioactivity appeared was eluted at the void volume indicating that the binding assay with gel filtration was not acceptable. When cytosol labeled with [³H]dolichol was applied to a column of DEAE-Sephacel, a single peak of radioactivity (about 10%) was eluted centered at around 0.3 M NaCl behind a peak of major proteins. On the same ion-exchange chromatography of [³H]dolichol dispersed in the buffer, no radioactivity was found in a linear gradient of NaCl concentration. When cold dolichol in 400-fold molar excess over [³H]dolichol was added upon labeling the cytosol, 75% of [³H]dolichol in 0.3 M NaCl fraction disappered indicating that the binding site(s) are saturable. These results appeared likely that certain acidic and macromolecular component(s) in the cytosol exhibited the specific binding activity with [³H]dolichol.

Search for Dolichol-Binding Component(s) in Rat Liver Cytosol

윤교희,Yoon, Kyo-Hie,Shidoji, Yoshihiro,Takai, Katsuji 생화학분자생물학회 1991 한국생화학회지 Vol.24 No.6

쥐간 dolichol의 함유량을 순상 HPLC로 측정한 결과, homogenate가 132 ng/mg protein, rnicrosome에 692 ng/mg protein, cytosol에는 47 ng/mg protein으로서 cytosol 중의 dolichol 총량은 homogenate의 약 13%에 이르는 것을 확인하였다. Cytosol dolichol의 chain distribution의 패턴은 역상 HPLC로 측정한 결과, homogenate의 dolichol과 유사하였다. Cytosol 에 $[^3H]dolichol$을 첨가하고 Sephacryl S-300 column으로 gel filteration을 행하면, 거의 모든 radioactivity가 void volume에 용출되었다. 그러나 유리형의 $[^3H]dolichol$도 일부 같은 fraction에 용출되는 점으로 보아서 종래의 방법으로는 $[^3H]dolichol$의 결합형과 유리형의 분리가 불완전함을 알 수 있었다. 한편, DEAE-Sephacel을 사용한 이온교환 크로마토그래피로는 결합형 $[^3H]dolichol$이 0.3M NaCl 근방에 용출되고 유리형 $[^3H]dolichol$은 전혀 용출되지 않는 점으로 보아 결합형과의 분리가 완전하였다. 또한, 40배 molar excess의 비표식(cold) dolichol을 공존시켰더니 0.3M 근방에 용출되는 $[^3H]dolichol$ peak는 25%로 감소되었다. 이상의 결과로 보아 cytosol에는 음이온교환수지에 흡착되고, dolichol에 대해 포화형의 결합활성을 나타내는 인자가 존재하고 있음을 알 수 있었다. This paper describes an approach to search cytosolic binding component(s) for dolichol in rat liver. The contents of endogenous dolichol were estimated to be $13.5{\mu}g/g$ liver (132 ng/mg protein) in the homogenate, $8.8{\mu}g/g$ liver (692 ng/mg protein) in the microsome, and $1.8{\mu}g/g$ liver (19 ng/mg protein) in cytosol indicating that about 13% of the hepatic dolichol stayed in the pool of cytosol. With respect to the chain distibution, dolichol-18 and -19 were found predominate in cytosol alike in homogenate. When rat liver cytosol was incubated with $[^3H]dolichol$ and applied to a column of Sephacryl S-300, 40% of total radioactivity appeared at the void volume. On the same chromatography of aqueous dispersion of $[^3H]dolichol$ as a blank, approximately 10% of the radioactivity appeared was eluted at the void volume indicating that the binding assay with gel filtration was not acceptable. When cytosol labeled with $[^3H]dolichol$ was applied to a column of DEAE-Sephacel, a single peak of radioactivity (about 10%) was eluted centered at around 0.3 M NaCl behind a peak of major proteins. On the same ion-exchange chromatography of $[^3H]dolichol$ dispersed in the buffer, no radioactivity was found in a linear gradient of NaCl concentration. When cold dolichol in 400-fold molar excess over $[^3H]dolichol$ was added upon labeling the cytosol, 75% of $[^3H]dolichol$ in 0.3 M NaCl fraction disappered indicating that the binding site(s) are saturable. These results appeared likely that certain acidic and macromolecular component(s) in the cytosol exhibited the specific binding activity with $[^3H]dolichol$.