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

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, K(m) and V(max) of 2.6 μM and 996 nmol.min(-1).ml(-1), respectively and was relatively stable at the optimum conditions (t(½) = 3 h). β-Amyloid peptide fragments Aβ(17-28) was the better inhibitor for nNOS (K(i) = 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 Aβ(17-21) [Leu(17) - Val(18) - Phe(19) - Phe(20) - Ala(21)] and glycine zipper motifs within the peptide fragment Aβ(17-35) were critical in binding and in fibrillogenesis confirming that nNOS was amyloidogenic catalyst.