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KMID : 0578319960060040400
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Molecules and Cells
1996 Volume.6 No. 4 p.400 ~ p.404
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Determination of Met-enkephalin in Pheochromocytoma Cells (PC12) Using Nonisotopic Methods
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Choi, Cheol-Hee
Um, Mee-Jung/Chang, In-Youb/Kim, Jin-Ho/You, Ho-Jin/Kee, Keun-Hong/Kim, Dong-Yoon
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Abstract
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Nonisotopic methods for the determination of biologically active substances have been introduced for safety and environmental reasons. In the present study, reversed-phase high performance liquid chromatography (HPLC) and immunodot blotting methods as alternatives to overcome some problems of radioisotopic methods were used to measure amounts of enkephalin in pheochromocytoma cells (PC12). The purification of enkephalin from cell extract was carried out by using octadesylsilica minicolumn (Sep-Pak). Using HPLC, 4.16 ng of Met-enkephalin was detected in 10^(7) PC12 cells but Leu-enkephalin was not. In order to detect a lower concentration of enkephalin, the immunodot blotting method was used. Spots were visualized using the avidin/biotin peroxidase complex (ABC) technique and nickel ammonium sulfate-glucose oxidase intensification (NASGOI) method. As the result of densitometry, the detection limit of Met-enkephalin of the former method was approximately 3.13 ng and that of the latter one about 0.39 ng. When using the NASGOI method, the cellular content of Met-enkephalin in 10^(7) PC12 cells was 4.69 ng. From the above results, it is thought that nonisotopic methods are to some extent sensitive, reproducible, less costly, safer, and less laborious than the isotopic methods for the determination of Met-enkephalin in PC12 cells.
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