| Autor: |
Turner, R S, Suzuki, N, Chyung, A S, Younkin, S G, Lee, V M |
| Zdroj: |
Journal of Biological Chemistry; April 1996, Vol. 271 Issue: 15 p8966-70, 5p |
| Abstrakt: |
Previous studies have demonstrated the presence of amyloid beta (Abeta) in neurons (NT2N) derived from a human embryonal carcinoma cell line (NT2) by steady state metabolic radiolabeling and immunoprecipitation. We show here that Abeta is present intracellularly since trypsin digestion of intact NT2N cells at 4 degrees C did not eliminate the Abeta recovered in cell lysates. To determine whether both Abeta40 and Abeta42 are produced intracellularly, quantitative sandwich enzyme-linked immunosorbent assay (ELISA) was performed using COOH-terminal end-specific anti-Abeta monoclonal antibodies. Sandwich ELISA detected intracellular Abeta40 and A++beta42 in NT2N cell lysates at a ratio of 3:1, whereas secreted Abeta40 and Abeta42 were recovered in medium conditioned by NT2N cells at a ratio of approximately 20:1. Metabolic steady state and pulse-chase labeling studies demonstrated a 2-h delay in the detection of cell-associated Abeta40/Abeta42 in the medium, suggesting that Abeta is generated at a slow rate intracellularly prior to its secretion. Finally, as NT2N cells mature over time in culture, the secretion of Abeta40 and Abeta42 increases more than 5-fold over 7 weeks. This increase in the secretion of Abeta40/Abeta42 in NT2N cells as a function of time may recapitulate a similar phenomenon in the aging brain. |
| Databáze: |
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