Activation and dimerization of type III nitric oxide synthase by submicromolar concentrations of tetrahydrobiopterin in microsomal preparations from human primordial placenta.
We have found in a previous study that 5-50 microM tetrahydrobiopterin (BH4) stimulated 1.2-2.5-fold Ca(2+)-dependent nitric-oxide synthase (NOS) activity in homogenates prepared from primordial human placentae. Now we report on the dramatic, about sixfold, activating effect of BH4 on this activity measured in microsomal preparations. Firstly, both in the absence and presence of BH4, arginine bound to kinetically homogeneous sites, with no significant change between the apparent KM values for arginine (3.12 +/- 1.99 microM and 2.06 +/- 1.13 microM in the absence and presence of 50 microM BH4 respectively, mean +/- s.d., n = 3). On the other hand, the Vmax values measured in different pools of placenta tissue varied between 2.5-7.55 (no BH4 added) and 13.3-58.5 (with BH4 added) pmol/min/mg protein. Secondly, the microsomal preparations responded sensitively to BH4 addition. A dose-response study indicated that as low as 79 nM final BH4 concentration stimulated NOS activity half-maximally, and 1 microM BH4 resulted in an almost maximal effect. Thirdly, immunoblot analysis combined with laser densitometric evaluation demonstrated that BH4 efficiently promoted the aggregation of microsomal NOS type III isozyme into a protein having the characteristics (electrophoretic mobility, resistance of SDS) of the dimeric form. Half-maximal dimerizing activity was reached at 148 +/- 33 nM BH4 (mean +/- s.d., n = 3), whereas 1 microM BH4 led to almost, maximal aggregation of monomers. This is the first time that BH4-induced dimerization of a NOS type III isoform has been demonstrated. Considering that human placenta predominantly expresses NOS type III isoform and BH4 concentration in this tissue is 207 +/- 87 nM, the present results strongly suggest that the dimerizing effect of BH4 is a crucial physiological mechanism for the assembly of active Ca(2+)-dependent NOS in the human primordial placenta.
Tóth
M
M
Department of Medical Chemistry, Molecular Biology and Pathobiochemistry, Budapest, Hungary.
Kukor
Z
Z
Sahin-Tóth
M
M
D001708:Biopterins / Q000031:analogs & derivatives* / Q000494:pharmacology; D002478:Cells, Cultured; D019281:Dimerization; D005260:Female; D006801:Humans; D015151:Immunoblotting; D008861:Microsomes / Q000187:drug effects / Q000201:enzymology*; D019001:Nitric Oxide Synthase / Q000378:metabolism*; D010920:Placenta / Q000187:drug effects / Q000201:enzymology* / Q000648:ultrastructure; D011247:Pregnancy
D001708:Biopterins; D019001:Nitric Oxide Synthase; C003402:sapropterin