ACTIVATION AND DIMERIZATION OF TYPE-III NITRIC-OXIDE SYNTHASE BY SUBMICROMOLAR CONCENTRATIONS OF TETRAHYDROBIOPTERIN IN MICROSOMAL PREPARATIONS FROM HUMAN PRIMORDIAL PLACENTA
M. Toth et al., ACTIVATION AND DIMERIZATION OF TYPE-III NITRIC-OXIDE SYNTHASE BY SUBMICROMOLAR CONCENTRATIONS OF TETRAHYDROBIOPTERIN IN MICROSOMAL PREPARATIONS FROM HUMAN PRIMORDIAL PLACENTA, Placenta, 18(2-3), 1997, pp. 189-196
We have found in a previous study that 5-50 mu M tetrahydrobiopterin (
BH4) stimulated 1.2-2.5-fold Ca2+-dependent nitric-oxide synthase (NOS
) activity in homogenates prepared from primordial human placentae. No
w we report on the dramatic, about sixfold, activating effect of BH4 o
n this activity measured in microsomal preparations. Firstly, both in
the absence and presence of BH4, arginine bound to kinetically homogen
eous sites, with no significant change between the apparant K-M values
for arginine (3.12 +/- 1.99 mu M and 2.06 +/- 1.13 mu M in the absenc
e and presence of 50 mu M BH4 respectively, mean +/- s.d., n=3). On th
e 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 res
ponded sensitively to BH4 addition. A dose-response study indicated th
at as low as 79 nM final BH4 concentration stimulated NOS activity hal
f-maximally, and 1 mu M BH4 resulted in an almost maximal effect. Thir
dly, immunoblot analysis combined with laser densitometric evaluation
demonstrated that BH4 efficiently promoted the aggregation of microsom
al NOS type III isozyme into a protein having the characteristics (ele
ctrophoretic mobility, resistance to SDS) of the dimeric form. Half-ma
ximal dimerizing activity was reached at 148 +/- 33 nM BH4 (mean +/- s
.d., n=3), whereas 1 mu M BH4 led to almost, maximal aggregation of mo
nomers. This is the first time that BH4-induced dimerization of a NOS
type III isoform has been demonstrated. Considering that human placent
a predominantly expresses NOS type III isoform and BH4 concentration i
n this tissue is 207 +/- 87 nM, the present results strongly suggest t
hat the dimerizing effect of BH4 is a crucial physiological mechanism
for the assembly of active Ca2+-dependent NOS in the human primordial
placenta. (C) 1997 W. B. Saunders Company Ltd.