Detection of a soluble form of BACE-1 in human cerebrospinal fluid by a sensitive activity assay
article
Background: Formation of deposits of the insoluble amyloid β-peptide is believed to be causally related with neurodegeneration in Alzheimer disease (AD). The β-peptide originates from a larger amyloid precursor protein (APP) by the action of proteolytic enzymes. The first proteolytic event leading to amyloid formation is the cleavage of APP by the membrane-bound aspartyl protease BACE-1, also known as memapsin-2. Inhibition of BACE-1 is thought to be a therapeutic approach to AD. Measuring BACE-1 activity in biological samples would be useful to elucidate the mechanism of AD and for development of AD drugs. Methods: We developed a sensitive and specific activity assay for BACE-1. The assay is based on a genetically engineered proenzyme that is specifically activated by BACE-1. The resulting active enzyme is measured with a chromogenic substrate. The use of 2 coupled reactions produces a detection limit as low as 0.4 pmol/L. Results: The assay detected BACE-1 activity in extracts of human brain tissue as well as, unexpectedly, in human cerebrospinal fluid (CSF). Gel electrophoresis and Western blotting identified the BACE-1 present in CSF as a truncated soluble form of the originally membrane-bound BACE-1. Conclusion: Detection of the soluble form of BACE-1 in CSF, a relatively easily accessible biological fluid, may be useful for monitoring the effects of drug candidates in vivo and may have diagnostic or prognostic applications. © 2006 American Association for Clinical Chemistry. Chemicals / CAS: amyloid beta protein, 109770-29-8; aspartic proteinase, 78169-47-8; cathepsin D, 9025-26-7; renin, 61506-93-2, 9015-94-5; Amyloid Precursor Protein Secretases, EC 3.4.-; Aspartic Endopeptidases, EC 3.4.23.-; BACE1 protein, human, EC 3.4.23.46; CASP3 protein, human, EC 3.4.22.-; Caspase 3, EC 3.4.22.-; Caspases, EC 3.4.22.-; Endopeptidases, EC 3.4.-; Protein Precursors; Tissue Extracts
Topics
Biomedical Researchamyloid beta proteinamyloid precursor proteinaspartic proteinasebeta amyloid converting enzyme 1beta secretasecathepsin Dcathepsin Echromogenic substrateenzyme precursorreninunclassified drugAlzheimer diseasebody fluidbrain tissuecerebrospinal fluid analysisdiagnostic valueenzyme activationenzyme assaygel electrophoresisgenetic engineeringhuman tissuemonitoringnerve degenerationprognosisprotein cerebrospinal fluid levelprotein degradationsensitivity and specificityWestern blottingAmyloid Precursor Protein SecretasesAspartic EndopeptidasesBrainCaspase 3CaspasesCross ReactionsEndopeptidasesHumansImmunoassayProtein PrecursorsSensitivity and SpecificitySolubilityTissue Extracts
TNO Identifier
239287
ISSN
00099147
Source
Clinical Chemistry, 52(6), pp. 1168-1174.
Pages
1168-1174
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