Differential response of basal and tetrahydrobiopterin-stimulated activities of placental type III nitric oxide synthase to sodium dodecyl sulphate: relation to dimeric structure.
The major enzyme isoform that synthesizes nitric oxide (NO) in first trimester human placentae is endothelial or type III NO-synthase (NOS III) which exhibits high specific activity in the microsomal fraction. In the present study, we investigated the possible protective and enzyme-stabilizing role of tetrahydropterin (BH4). The anionic detergent, sodium dodecyl sulphate (SDS) and thermal stress (freeze-thaw) were used as non-specific 'subunit-dissociating' agents, and alterations in enzyme activity and subunit structure were investigated. SDS (> or =0.05% w/v) resulted in significant inhibition both of basal and BH4-stimulated activities of NOS III, but the latter responded more sensitively. Preincubation of microsomes with SDS (> or =0.1%, w/v), followed by incubation in an SDS-depleted reaction mixture led to an inhibition of BH4-stimulated enzyme activity, while no change in the basal activity was noted. This indicated that the SDS effect is only fully reversible in the case of basal activity. Considering that basal activity is due to the presence of endogenous BH4 tightly bound to the enzyme, this differential sensitivity of basal and BH4-stimulated enzyme activities to SDS may be related to a putative differential protective effect of BH4 on the two subunits of the NOS III dimer. Western blot analysis revealed that the SDS-induced inhibition of enzyme activity could not be ascribed to disruption of the dimeric structure. This finding confirms the view that SDS may affect NOS III activity without necessarily deteriorating quaternary protein structure. Nevertheless, BH4 is essential in maintaining dimeric structure under denaturing conditions, e.g. SDS treatment and freezing/thawing; it is even able to reverse the dissociation caused by SDS. A model describing the interaction between BH4 and NOS III, and its implications on the physiology and pathology of the human placenta, is discussed.
Tóth
M
M
Department of Medical Chemistry, Molecular Biology and Pathobiochemistry, Semmelweis University of Medicine, Budapest, Hungary.
Kukor
Z
Z
Sahin-Tóth
M
M
D000975:Antioxidants / Q000494:pharmacology*; D001708:Biopterins / Q000031:analogs & derivatives* / Q000494:pharmacology; D019281:Dimerization; D004305:Dose-Response Relationship, Drug; D005260:Female; D005615:Freezing; D006801:Humans; D015151:Immunoblotting; D008861:Microsomes / Q000201:enzymology; D019001:Nitric Oxide Synthase / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*; D010920:Placenta / Q000201:enzymology*; D011247:Pregnancy / Q000378:metabolism*; D012967:Sodium Dodecyl Sulfate / Q000494:pharmacology*
D000975:Antioxidants; D001708:Biopterins; D012967:Sodium Dodecyl Sulfate; D019001:Nitric Oxide Synthase; C003402:sapropterin