Amyloid plaques in brain tissue are a hallmark of Alzheimer's disease. Primary components of these plaques are 40- and 42-residue peptides, denoted A beta(1-40) and A beta(1-42), that are derived by proteolysis of cellular amyloid precursor protein. Synthetic A beta(1-40) and A beta(1-42) form amyloid fibrils in vitro that share many features with the amyloid in plaques. Soluble intermediates in A beta fibrillogenesis, termed protofibrils, have been identified previously, and here we describe the in vitro formation and isolation of A beta(1-40) protofibrils by size exclusion chromatography. In some experiments, the A beta(1-40) was radiomethylated to better quantify various A beta species. Mechanistic studies clarified two separate modes of protofibril growth, elongation by monomer deposition and protofibril-protofibril association, that could be resolved by varying the NaCl concentration. Small isolated protofibrils in dilute Tris-HCl buffers were directed along the elongation pathway by addition of A beta(1-40) monomer or along the association pathway by addition of NaCl. Multi-angle light scattering analysis revealed that protofibrils with initial molecular masses M(w) of (7-30) x 10(3) kDa grew to M(w) values of up to 250 x 10(3) kDa by these two growth processes. However, the mass per unit length of the associated protofibrils was about 2-3 times that of the elongated protofibrils. Rate constants for further elongation by monomer deposition with the elongated, associated, and initial protofibril pools were identical when equal number concentrations of original protofibrils were compared, indicating that the original number of protofibril ends had not been altered by the elongation or association processes. Atomic force microscopy revealed heterogeneous initial protofibrils that became more rodlike following the elongation reaction. Our data indicate that protofibril elongation in the absence of NaCl results from monomer deposition only at the ends of protofibrils and proceeds without an increase in protofibril diameter. In contrast, protofibril association occurs in the absence of monomer when NaCl is introduced, but this association involves lateral interactions that result in a relatively disordered fibril structure.

Deposition of ␤-amyloid (A␤) peptides in the brain is an early and invariant feature of all forms of Alzheimer's disease. As with any secreted protein, the extracellular concentration of A␤ is determined not only by its production but also by its catabolism. A major focus of Alzheimer's research has been the elucidation of the mechanisms responsible for the generation of A␤. Much less, however, is known about the mechanisms responsible for A␤ removal in the brain. In this report, we describe the identification of endothelin-converting enzyme-1 (ECE-1) as a novel A␤-degrading enzyme. We show that treatment of endogenous ECE-expressing cell lines with the metalloprotease inhibitor phosphoramidon causes a 2-3-fold elevation in extracellular A␤ concentration that appears to be due to inhibition of intracellular A␤ degradation. Furthermore, we show that overexpression of ECE-1 in Chinese hamster ovary cells, which lack endogenous ECE activity, reduces extracellular A␤ concentration by up to 90% and that this effect is completely reversed by treatment of the cells with phosphoramidon. Finally, we show that recombinant soluble ECE-1 is capable of hydrolyzing synthetic A␤40 and A␤42 in vitro at multiple sites.