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. 2007 Aug 8;27(32):8628–8635. doi: 10.1523/JNEUROSCI.1549-07.2007

Figure 1.

Figure 1.

ACE converts Aβ1–42 to Aβ1–40. A, We confirmed the specificities of monoclonal anti-Aβ1–40 (1A10) and polyclonal anti-Aβ1–42 antibodies. One microgram each of Aβ1–40 and Aβ1–42 was subjected to SDS-PAGE and then blotted onto a nitrocellulose membrane, and the membrane was probed with anti-Aβ1–40 and anti-Aβ1–42 antibodies. B, Mouse brain homogenate was mixed with or without synthetic Aβ1–42 and incubated at 37°C for 8 h. An anti-Aβ1–40 antibody was used for detecting Aβ1–40 generation. Captopril (1 μm) or enalapril (1 μm) markedly inhibited the generation of Aβ1–40 from Aβ1–42. C, Top, The genotypes of ACE(+/+), ACE(+/−), and ACE(−/−) mice were confirmed by Western blot analysis of the brain homogenate using an anti-ACE polyclonal antibody (AF1513; R & D Systems). Middle, Bottom, Mouse brain homogenate (middle) or serum (bottom) was mixed with 30 μm Aβ1–42 and incubated at 37°C for 8 h. The generation of Aβ1–40 was detected by Western blot analysis. D, Purified human ACE (2 U/ml) was mixed with synthetic Aβ1–42 (30 μm), and the mixture was incubated at 37°C with or without ACE inhibitors. Top, Aβ1–40 was generated in a time-dependent manner. Bottom, EDTA (10 μm), captopril (1 μm), and enalapril (1 μm) completely inhibited the conversion of Aβ1–42 to Aβ1–40. E, MALDI-TOF-MS revealed a new peak with a mass of 4330 (corresponding to that of Aβ1–40) after the incubation of Aβ1–42 with ACE, indicating Aβ1–40 generation. F, Captopril blocked Aβ1–40 generation in the mixture of Aβ1–42 and purified human ACE in a dose-dependent manner. The density of Aβ1–40 bands was measured by densitometry and normalized to the mean of the bands in the case of incubation without captopril. IC50 was estimated to be ∼10 nm. G, Time-dependent alterations in Aβ1–42 and Aβ1–40 levels in the solution of Aβ1–42 (10 μm) alone; Aβ1–42 (10 μm) and ACE (2 U/ml); or Aβ1–42 (10 μm), ACE (2 U/ml), and captopril (1 μm). Each solution was incubated at 37°C for the time indicated. Ten microliters of each solution were collected at different time points and immediately frozen at −80°C until analysis. The levels of Aβ1–42 and Aβ1–40 in each sample were determined by ELISA. Data are the mean ± SEM of three samples.