Tetrahydrobiopterin preferentially stimulates activity and promotes subunit aggregation of membrane-bound calcium-dependent nitric oxide synthase in human placenta

Type III nitric oxide synthase (NOS III) is responsible for > 90% of nitric oxide (NO) synthesizing activity in first trimester placentae. Enzyme activity is distributed between cytosolic (30%) and membrane-bound forms (70%), with highest specific activity observed in microsomal fractions. In the present study, the effect of tetrahydrobiopterin (BH4) on subunit structure and activity of microsomal and cytosolic NOS III was compared. As revealed by immunoblot analysis, incubation of microsomal membranes with 50 microM final concentration BH4 for 10 min at 37 degrees C resulted in a striking conversion of monomeric NOS III into a protein having the characteristics (electrophoretic mobility, resistance to sodium dodecyl sulphate) of the homodimeric form. In contrast, BH4 induced significantly less marked changes in the NOS III dimer content of cytosolic fractions. Enzyme activity in microsomes is stimulated approximately 6-fold upon addition of 50 microM BH4, while only a 2-fold activation is detectable in cytosolic fractions. Taken together, the observations suggest that BH4 activates NOS III in the primordial human placenta by promoting its subunit assembly in the membrane, while cytosolic NOS III is relatively insensitive to BH4. Compartment-specific action of BH4 represents a novel mechanism which is implicated in the regulation of placental NOS activity.