Biochemical Characterization of the -Secretase Activity That Produces 
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- 作者：Lili Zhang ; Lixin Song ; Giuseppe Terracina ; Yanhui Liu ; Birendra Pramanik ; and Eric Parker
- 刊名：Biochemistry
- 出版年：2001
- 出版时间：April 24, 2001
- 年：2001
- 卷：40
- 期：16
- 页码：5049 - 5055
- 全文大小：149K
- 年卷期：v.40,no.16(April 24, 2001)
- ISSN：1520-4995
文摘
Recent studies of 
-secretase have pointed out that it may be comprised of a multisubunitcomplex with presenilin 1 and presenilin 2 as central components. Elucidation of the biochemical mechanismof this enzymatic activity will provide important information for developing -secretase inhibitors inAlzheimer's disease therapy. Here we describe the biochemical characterization of -secretase activitiesusing a sensitive, membrane-based assay system. Membranes were isolated from 293 cells expressingC99, the substrate of -secretase. Upon incubation at 37 
C, C99 is cleaved by the endogenous -secretase,and A
peptides are liberated. A40 and A42 -secretase activities are very similar in terms of theirkinetic profiles and pH dependence, supporting the notion that a single enzyme is involved in both A40and A42 production. Pepstatin A inhibited A40 and A42 -secretase activities with similar potency.Peptide difluoroketone and peptide aldehyde inhibitors inhibited A40 production in a dose-dependentfashion, enhanced A42 production at low concentrations, and inhibited A42 production at highconcentrations. Although the selective increase of A42 by low concentrations of peptide difluoroketoneand peptide aldehyde inhibitors has been reported in intact cells, the finding that this phenomenon occursin a membrane-based assay system suggests that these compounds increase A42 by a direct effect on-secretase. The ability of these compounds to increase A42 production may reflect allosteric modulationof the -secretase complex by a mechanism related to that responsible for the increase of A42 productionby mutations in presenilins.
-secretase have pointed out that it may be comprised of a multisubunitcomplex with presenilin 1 and presenilin 2 as central components. Elucidation of the biochemical mechanismof this enzymatic activity will provide important information for developing -secretase inhibitors inAlzheimer's disease therapy. Here we describe the biochemical characterization of -secretase activitiesusing a sensitive, membrane-based assay system. Membranes were isolated from 293 cells expressingC99, the substrate of -secretase. Upon incubation at 37 C, C99 is cleaved by the endogenous -secretase,and A peptides are liberated. A40 and A42 -secretase activities are very similar in terms of theirkinetic profiles and pH dependence, supporting the notion that a single enzyme is involved in both A40and A42 production. Pepstatin A inhibited A40 and A42 -secretase activities with similar potency.Peptide difluoroketone and peptide aldehyde inhibitors inhibited A40 production in a dose-dependentfashion, enhanced A42 production at low concentrations, and inhibited A42 production at highconcentrations. Although the selective increase of A42 by low concentrations of peptide difluoroketoneand peptide aldehyde inhibitors has been reported in intact cells, the finding that this phenomenon occursin a membrane-based assay system suggests that these compounds increase A42 by a direct effect on-secretase. The ability of these compounds to increase A42 production may reflect allosteric modulationof the -secretase complex by a mechanism related to that responsible for the increase of A42 productionby mutations in presenilins.