Association of β-amyloid peptide fragments with neuronal nitric oxide synthase: Implications in the etiology of Alzheimers disease

Abstract

Neuronal nitric oxide synthase (nNOS) was purified on DEAE-Sepharose anion-exchange in a 38% yield, with 3-fold recovery and specific activity of 5 mol.min-1.mg-1. The enzyme was a heterogeneous dimer of molecular mass 225kDa having a temperature and pH optima of 40°C and 6.5, Km and Vmax of 2.6 μM and 996 nmol.min-1.ml-1, respectively and was relatively stable at the optimum conditions (t 1/2=3h). β-Amyloid peptide fragments Aβ17-28 was the better inhibitor for nNOS (Ki=0.81 μM). After extended incubation of nNOS (96h) with each of the peptide fragments, Congo Red, turbidity and thioflavin-T assays detected the presence of soluble and insoluble fibrils that had formed at a rate of 5nM.min-1. A hydrophobic fragment Aβ17-21 [Leu17 - Val18 - Phe 19 -Phe20 -Ala21] and glycine zipper motifs within the peptide fragment Aβ17-35 were critical in binding and in fibrillogenesis confirming that nNOS was amyloidogenic catalyst. © 2012 Informa UK, Ltd.