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 µmol.min−1.mg−1. The enzyme was a heterogeneous dimer of molecular mass 225 kDa 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 (t½ = 3 h). β-Amyloid peptide fragments Aβ17–28 was the better inhibitor for nNOS (Ki = 0.81 µM). After extended incubation of nNOS (96 h) 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 5 nM.min−1. A hydrophobic fragment Aβ17–21 [Leu17 – Val18 – Phe19 – 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.