PURIFICATION AND CHARACTERIZATION OF THE CONSTITUTIVE NITRIC-OXIDE SYNTHASE FROM HUMAN PLACENTA

Human endothelial nitric oxide synthase (NOS) mRNA was detected in hum an placenta. In contrast, mRNAs for human neuronal NOS or for human in ducible NOS were not detected in placenta. Subsequently, NOS was purif ied over 3800-fold from placental extract to greater than 80% homogene ity. A single band with an apparent molecular weight of 135 kDa was id entified by [I-125] calmodulin binding to proteins in a sodium dodecyl sulfate-polyacrylamide gel, which is consistent with the predicted si ze of the endothelial NOS. Furthermore, the sequence of eight internal peptides derived from this 135-kDa protein was identical to the publi shed sequence of human endothelial NOS. As has been shown for all cons titutive NOS isozymes, the purified NOS was absolutely dependent on ca lcium and calmodulin. NOS was also purified from human umbilical vein endothelial cells and, on the basis of similar kinetic parameters and dependence upon calcium and calmodulin, appeared to be the same as the purified placental NOS. Together, these data indicate that the placen tal NOS is the constitutive NOS isozyme from endothelial tissue. (C) 1 994 Academic Press, Inc.