|Budget Amount *help
¥2,100,000 (Direct Cost : ¥2,100,000)
Fiscal Year 1994 : ¥800,000 (Direct Cost : ¥800,000)
Fiscal Year 1993 : ¥1,300,000 (Direct Cost : ¥1,300,000)
The extent in the induction of hepatic CYP2B P-450 with phenobarbital is greatly different in rats and guinea pigs. To answer this difference the binding of protein to the oligonucleotide probe of-89 to-73 bp region (Barbie box) of CYP2B1 gene was compared in both animal species. When the nuclear extracts of liver were used as the protein fraction, all preparations from phenobarbital-pretreated rats showed strong binding to the Barbie box, but many preparations from untreated rats had only weak activities. On the contrary, the binding with liver nuclear extracts of guinea pigs was not increased by phenobarbital treatment. Therefore, the response to phenobarbital treatment on the nuclear protein-probe interaction was markedly different in rats and guinea pigs. Since this difference correlated with the contrast of the inducibility between rat and guinea pig CYP2B P-450 protein, it was suggested that the expression and induction of the CYP2B P-450 is related with the interaction of nuclea
r proteins and Barbie box region of the gene. Alteration of the binding between the probe and rat nuclear protein with chemical effectors, and by heating and protease treatments was also examined. The results showed that 1)Ca2+ and Mg2+ effectively increase the binding ; 2)hemin and Triton X-100 but not phenobarbital and dexamethasone increase the binding ; and 3)nuclear protein resists to the heat-denaturation until 60。C and to the proteolysis with proteases. On the other hand, the former two effects were not seen or the extents were limited in the binding with guinea pig liver nuclear extracts. The guinea pig protein capable of binding to the probe were less stable against heating and were much sensitive against proteases than rat protein. The ability of cytosol to bind Barbie box was compared in rats and guinea pigs, and the results are also reported. A guinea pig protein (BBBP-GP) was purified from liver nuclear extracts by monitoring its binding activity toward DNA probe to electrophoretically homogeneous. The minimum molecular weight was estimated to be 27,000. Rat nuclear protein was also purified though only partially. The analysis of the N-terminal sequence indicated that BBBP-GP is a new protein. This protein interacted with oligonucleotide probe, but the shifted band was stretched.
2.ラット肝核抽出物とBarbie boxとの結合は、未処理動物では微弱であるが、動物を誘導剤フェノバルビタールで前処理すると著しく増大した。対照的に、モルモットでは、動物前処理の有無にかかわらず同程度の結合が認められた。従って、Barbie box-核タンパク質結合のフェノバルビタールに対する応答性はラットとモルモットで大きく異なることが実証され、上記1の推定が支持された。
4.核抽出液中よりBarbie box結合性タンパク質の精製を行い、モルモットより1種(BBBP-GP)を純粋に単離することに成功した。BBBP-GPは電気泳動より算出した最小分子量が約27,000であり、アミノ末端配列の分析結果より新種のタンパク質であることが明かであった。 Less