The effect if divalent cations on bovine retinal NOS activity

The divalent cation requirements of NOS activity in bovine retina homogenat e supernatant were investigated. Supernatants were assayed under standard c onditions (in mM: EDTA 0.45, Ca2+ 0.25, Mg2+ 4.0). In order to investigate the enzyme's dependence on divalent cations, the tissue homogenate was depl eted of di- and trivalent cations by passing it over a cation-exchange colu mn (Chelex 100). Surprisingly, NOS activity was 50-100% higher in this prep aration. However, addition of either EDTA (33 mu M) or EGTA (1 mM) almost f ully inhibited NOS activity, suggesting a requirement for residual divalent metal cation(s). Phenanthroline or iminodiacetic acid at low concentration s had little effect on activity, suggesting no requirement for Fe2+, Zn2+ o r Cu2+. Ca2+ had a moderate stimulatory effect, with an optimum activity ar ound 0.01 mM. Mg2+ or Mn2+ had little effect at concentrations < 0.25 mM. H owever, in the presence of EDTA, Mn2+ or Ca2+ markedly stimulated NOS activ ity with the optimum at 0.1 mM. At high concentrations (> 0.1-0.2 mM), all divalent cations tested (Ba2+, Zn2+, Co2+, Mn2+, Mg2+, Ca2+), as well as La 3+, dose-dependently inhibited NOS activity. We propose that retinal NOS re quires low concentrations of naturally occurring divalent metal ions, most probably Ca2+, for optimal activity and is inhibited by high di- and trival ent metal concentrations, probably by competition with Ca2+.