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Journal of Biochemistry Advance Access originally published online on March 24, 2009
Journal of Biochemistry 2009 146(1):141-148; doi:10.1093/jb/mvp054
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© The Authors 2009. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved

Accurate Determination of Carboxyl-Terminal Fragment of Presenilin 1 in Various Tissues from Rat and Cell Lines

Hui Lin Chai and Satoshi Miura*

Radioisotope Research Center, Yokohama City University School of Medicine, 3-9 Fukuura, Kanazawa-ku, Yokohama 236-0004, Japan

*To whom correspondence should be addressed. Tel: +81-45-787-2760, Fax: +81-45-782-1251, E-mail: smiura{at}med.yokohama-cu.ac.jp

Received March 16, 2009; Accepted March 16, 2009


  Abstract

Presenilin 1 (PS1) has been identified as a causative gene for the early-onset of familial Alzheimer's disease, and it is mainly localized in the endoplasmic reticulum and the Golgi membrane as a multiple membrane-spanning protein. In the cell, PS1 is proteolytically processed to a 30-kDa N-terminal fragment and a 20-kDa C-terminal fragment (CTF), both of which exist as a stable high-molecular-weight protein complex, together with other components of {gamma}-secretase. However, as there has been no report about the precise amount of PS1 expressed in mammalian tissues, the aim of this study was to quantitatively determine PS1–CTF amounts in various tissues such as liver, kidney, brain and heart of rat by western blotting using a [35S]-methionine-labelled PS1-CTF as a standard synthesized in a wheat germ cell-free protein synthesizing system. PS1–CTF contents in kidney, liver, brain and heart were 17.0, 6.6, 6.4 and 0.2 fmol/mg protein, respectively. PS1–CTF contents were also determined in cultured cell lines such as HeLa, HEK293 and COS-1.

Key Words: Alzheimer's disease, cell-free protein synthesis, presenilin 1, quantitative analysis, western blotting

Abbreviations: Aβ, amyloid β; AD, Alzheimer's disease; APP, amyloid β precursor protein; CTF, C-terminal fragment; FAD, early-onset familial Alzheimer's disease; GST, glutathione S-transferase; MBP, maltose-binding protein; NTF, N-terminal fragment; PS1, presenilin 1; PMSF, phenylmethylsulphonyl fluoride; PVDF, polyvinylidene difluoride; TL-PS1-CTF, PS1-CTF synthesized in vitro


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