| Budget Amount *help |
¥1,800,000 (Direct Cost: ¥1,800,000)
Fiscal Year 1993: ¥400,000 (Direct Cost: ¥400,000)
Fiscal Year 1992: ¥1,400,000 (Direct Cost: ¥1,400,000)
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| Research Abstract |
In order to understand formation and maturation of autophagic vacuoles, I have characterized autophagic vacuolar membranes isolated from leupeptin-administered rat liver. In the first fiscal year of this research, I found that the isolated vacuolar membrance could be separated into two fractions (AL-L and AL-H) by C_aCl_2 treatment. AL-L closely resembles lysosomal membrane markers of lysosomas (1gp120), ER (cytochrome P450), and emdosomes (mannose 6-phosphate receptor) and is probably derived from vacuoles at earlier stages of the maturation. It is therefore interesting to see whether or not AL-H possesses some other markers specific for nascent autophagic vacuoles. In the second first year, I first tried to separate AL-H membrane proteins by two dimensional SDSPAGE.In immunoblotting analyzes, I found that antibodies raised against AL-H recognize two separate membrane proteins with molecular weights around 70k. The major one having more acidic pl values is a glycoprotein. The protein is unique to AL-H and hence might be a specific marker fo nascent autophagic vacuolar membrane. Amino acid sequence analysis on the 70k is now under progress. In immunoblotting analyzes using group specific antibodies to several types of cytochrome P450 isozymes, AL-H was found to prossess 2B, 2C and 2E isonzymes. However, sequence analysis revealed that the major vacuolar cytochrome P450 is different from cytochrome P45- 2C-11, the mofor P450 isozyme in ER.Thus, the data suggest possible enrollment of a specific P450 isozyme in autophagic vacuolar membranes. In conclusion, further investigation in to Al-H 2ill provide not only valuable information to characterize nascent autophagic vaculoles but also clues with which to study fusion mechanism between autophagic vacuoles and lysosomes.
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