title: The effect of divalent cations on bovine retinal NOS activity.
abstract: The divalent cation requirements of NOS activity in bovine retina homogenate supernatant were investigated. Supernatants were assayed under standard conditions (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 depleted of di- and trivalent cations by passing it over a cation-exchange column (Chelex 100). Surprisingly, NOS activity was 50-100% higher in this preparation. However, addition of either EDTA (33 microM) or EGTA (1 mM) almost fully inhibited NOS activity, suggesting a requirement for residual divalent metal cation(s). Phenanthroline or iminodiacetic acid at low concentrations had little effect on activity, suggesting no requirement for Fe2+, Zn2+ or Cu2+. Ca2+ had a moderate stimulatory effect, with an optimum activity around 0.01 mM. Mg2+ or Mn2+ had little effect at concentrations < 0.25 mM. However, in the presence of EDTA, Mn2+ or Ca2+ markedly stimulated NOS activity 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 La3+, dose-dependently inhibited NOS activity. We propose that retinal NOS requires low concentrations of naturally occurring divalent metal ions, most probably Ca2+, for optimal activity and is inhibited by high di- and trivalent metal concentrations, probably by competition with Ca2+.
authors.affiliation: Department of Ophthalmology, Tel Aviv Sourasky Medical Center and the Sackler Faculty of Medicine, Israel.
authors.forename: O
authors.initials: O
authors.lastname: Geyer
authors.forename: S M
authors.initials: SM
authors.lastname: Podos
authors.forename: Y
authors.initials: Y
authors.lastname: Oron
authors.forename: T W
authors.initials: TW
authors.lastname: Mittag
mesh_terms: D000818:Animals; D002413:Cations, Divalent / Q000494:pharmacology; D002417:Cattle; D004305:Dose-Response Relationship, Drug; D005260:Female; D066298:In Vitro Techniques; D008297:Male; D008670:Metals / Q000494:pharmacology*; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D012160:Retina / Q000187:drug effects* / Q000201:enzymology
chemical_list: D002413:Cations, Divalent; D008670:Metals; D019001:Nitric Oxide Synthase