Growth of -Amyloid(1-40) Protofibrils by Monomer Elongation and Lateral Association. Characterization of Disti
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- 作者：Michael R. Nichols ; Melissa A. Moss ; Dana Kim Reed ; Wen-Lang Lin ; Rajendrani Mukhopadhyay ; Jan H. Hoh ; and Terrone L. Rosenberry
- 刊名：Biochemistry
- 出版年：2002
- 出版时间：May 14, 2002
- 年：2002
- 卷：41
- 期：19
- 页码：6115 - 6127
- 全文大小：225K
- 年卷期：v.41,no.19(May 14, 2002)
- ISSN：1520-4995
文摘
Amyloid plaques in brain tissue are a hallmark of Alzheimer's disease. Primary componentsof these plaques are 40- and 42-residue peptides, denoted A
(1-40) and A(1-42), that are derived byproteolysis of cellular amyloid precursor protein. Synthetic A(1-40) and A(1-42) form amyloid fibrilsin vitro that share many features with the amyloid in plaques. Soluble intermediates in A fibrillogenesis,termed protofibrils, have been identified previously, and here we describe the in vitro formation andisolation of A(1-40) protofibrils by size exclusion chromatography. In some experiments, the A(1-40) was radiomethylated to better quantify various A species. Mechanistic studies clarified two separatemodes 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-HClbuffers were directed along the elongation pathway by addition of A(1-40) monomer or along theassociation pathway by addition of NaCl. Multi-angle light scattering analysis revealed that protofibrilswith initial molecular masses Mw of (7-30) × 103 kDa grew to Mw values of up to 250 × 103 kDa bythese two growth processes. However, the mass per unit length of the associated protofibrils was about2-3 times that of the elongated protofibrils. Rate constants for further elongation by monomer depositionwith the elongated, associated, and initial protofibril pools were identical when equal number concentrationsof original protofibrils were compared, indicating that the original number of protofibril ends had notbeen altered by the elongation or association processes. Atomic force microscopy revealed heterogeneousinitial protofibrils that became more rodlike following the elongation reaction. Our data indicate thatprotofibril elongation in the absence of NaCl results from monomer deposition only at the ends ofprotofibrils and proceeds without an increase in protofibril diameter. In contrast, protofibril associationoccurs in the absence of monomer when NaCl is introduced, but this association involves lateral interactionsthat result in a relatively disordered fibril structure.
(1-40) and A(1-42), that are derived byproteolysis of cellular amyloid precursor protein. Synthetic A(1-40) and A(1-42) form amyloid fibrilsin vitro that share many features with the amyloid in plaques. Soluble intermediates in A fibrillogenesis,termed protofibrils, have been identified previously, and here we describe the in vitro formation andisolation of A(1-40) protofibrils by size exclusion chromatography. In some experiments, the A(1-40) was radiomethylated to better quantify various A species. Mechanistic studies clarified two separatemodes 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-HClbuffers were directed along the elongation pathway by addition of A(1-40) monomer or along theassociation pathway by addition of NaCl. Multi-angle light scattering analysis revealed that protofibrilswith initial molecular masses Mw of (7-30) × 103 kDa grew to Mw values of up to 250 × 103 kDa bythese two growth processes. However, the mass per unit length of the associated protofibrils was about2-3 times that of the elongated protofibrils. Rate constants for further elongation by monomer depositionwith the elongated, associated, and initial protofibril pools were identical when equal number concentrationsof original protofibrils were compared, indicating that the original number of protofibril ends had notbeen altered by the elongation or association processes. Atomic force microscopy revealed heterogeneousinitial protofibrils that became more rodlike following the elongation reaction. Our data indicate thatprotofibril elongation in the absence of NaCl results from monomer deposition only at the ends ofprotofibrils and proceeds without an increase in protofibril diameter. In contrast, protofibril associationoccurs in the absence of monomer when NaCl is introduced, but this association involves lateral interactionsthat result in a relatively disordered fibril structure.