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생체밖에서 미국흰불나방 지방세포에 의한 저장단백질의 흡수와 축적에 관하여

In vitro Uptake and Accumulation of Purified Storage Proteins into Fat Body Cells from Huphantria cunea Drury

Abstract

This study was carried out to examine in vitro first whether the storage proteins, which the fat bodies of last larvae from Hyphantria cunea secrete into haemolymph, can be uptaked by the fat body cells of prepupa and then how the uptaked storage proteins can be accumulated in the fat body cells, if uptaken. The fat bodies which had been isolated from last instar larvae were cultured in 1 ml of Grace's insect medium containing $50{\mu}l$ of $^{3}H$-leucine (5.0 mCi/mol, Dupont) at $28{\pm}2^{\circ}C$ for 6 hrs. After the homogenates of the cultured fat bodies were centrifuged at 10,000 rpm for 10 minutes, the proteins included in the supernatant were separated by polyacrylamide gel electrophoreses (non-SDS, 6%). The next treatment of the electrophoresed gel was followed by rinsing. A storage protein band of several bands in the rinsed gel was sliced off. With elution of sliced storage protein bands in Tris-glycine buffer, the purification of radioactive storage proteins from fat bodies was finished. After the purified radioactive storage proteins were added in Grace's insect midis containing fat bodies of the prepupae, they were cultured for the randomly following minutes given as 3, 5, 7, 10, 15, 20 and 30 and for the randomly following hours given as 1, 2, 3 and 4 respectively. The double fixations of the cultured fat bodies in aldehyde and $OsO_4$, were followed by preparation of ultrathin sections from Epon-Araldite blocks through dehydration and embedding. The electron microscope autoradiographic treatment of all prepared sections were performed by the dipping method (Kim et al., 1987). The finally prepared specimens were examined with electron microscope. The fat body cells of the prepupa could be found to uptake the storage preteins of the last instar larvae, which were included in the culture medium, mostly by formation of coated vesicles. The in vitro uptake of the storage proteins actively occurred by 30 minutes after the addition of purified storage proteins in the culture medium. After culture for 7 minutes with the storage proteins, the uptaked radioactive storage proteins labelled a number of lysosomal granules. After culture for 20 minutes with the storage proteins, the radioactive storage proteins were finally incorporated and accumulated in lipid droplets and protein granules. The frequency in the fat body cell of radiolabelled lipid droplets occurs approximately 60%, while the frequency, in which the radiolabelled protein granules occurs in a fat body cell, is approximately 40%.

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