Activation and dimerization of type III nitric oxide synthase by submicromolar concentrations of tetrahydrobiopterin in microsomal preparations from human primordial placenta

M. Tóth, Z. Kukor, M. Sahin-Tóth

Research output: Contribution to journalArticle

13 Citations (Scopus)

Abstract

We have found in a previous study that 5-50 μM tetrahydrobiopterin (BH4) stimulated 1.2-2.5-fold Ca2+-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 apparant KM values for arginine (3.12 ± 1.99 μM and 2.06 ± 1.13 μM in the absence and presence of 50 μM BH4 respectively, mean ± s.d., n = 3). On the other hand, the V(max) 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 μM 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 to SDS) of the dimeric form. Half-maximal dimerizing activity was reached at 148 ± 33 nM BH4 (mean ± s.d., n = 3), whereas 1 μM 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 Ca2+-dependent NOS in the human primordial placenta.

Original languageEnglish
Pages (from-to)189-196
Number of pages8
JournalPlacenta
Volume18
Issue number2-3
DOIs
Publication statusPublished - Jan 1 1997

ASJC Scopus subject areas

  • Reproductive Medicine
  • Obstetrics and Gynaecology
  • Developmental Biology

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