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 ni tric oxide (NO) synthesizing activity in first trimester placentae, En zyme activity is distributed between cytosolic (30%) and membrane-boun d forms (70%), with highest specific activity observed in microsomal f ractions. In the present study, the effect of tetrahydrobiopterin (BH4 ) on subunit structure and activity of microsomal and cytosolic NOS II I was compared. As revealed by immunoblot analysis, incubation of micr osomal membranes with 50 mu M 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, resis tance to sodium dodecyl sulphate) of the homodimeric form. In contrast , BH4 induced significantly less marked changes in the NOS III dimer c ontent of cytosolic fractions. Enzyme activity in microsomes is stimul ated similar to 6-fold upon addition of 50 mu M BH4, while only a 2-fo ld activation is detectable in cytosolic fractions. Taken together, th e observations suggest that BH4 activates NOS III in the primordial hu man placenta by promoting its subunit assembly in the membrane, while cytosolic NOS III is relatively insensitive to BH4. Compartment-specif ic action of BH4 represents a novel mechanism which is implicated in t he regulation of placental NOS activity.