Purification and characterization of the constitutive nitric oxide synthase from human placenta.
Human endothelial nitric oxide synthase (NOS) mRNA was detected in human placenta. In contrast, mRNAs for human neuronal NOS or for human inducible NOS were not detected in placenta. Subsequently, NOS was purified over 3800-fold from placental extract to greater than 80% homogeneity. A single band with an apparent molecular weight of 135 kDa was identified by [125I] calmodulin binding to proteins in a sodium dodecyl sulfate-polyacrylamide gel, which is consistent with the predicted size of the endothelial NOS. Furthermore, the sequence of eight internal peptides derived from this 135-kDa protein was identical to the published sequence of human endothelial NOS. As has been shown for all constitutive NOS isozymes, the purified NOS was absolutely dependent on calcium 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 placental NOS is the constitutive NOS isozyme from endothelial tissue.
Garvey
E P
EP
Wellcome Research Laboratory, Research Triangle Park, North Carolina 27709.
Tuttle
J V
JV
Covington
K
K
Merrill
B M
BM
Wood
E R
ER
Baylis
S A
SA
Charles
I G
IG
D000594:Amino Acid Oxidoreductases / Q000096:biosynthesis / Q000302:isolation & purification* / Q000378:metabolism*; D000595:Amino Acid Sequence; D001483:Base Sequence; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D004730:Endothelium, Vascular / Q000201:enzymology*; D005260:Female; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009474:Neurons / Q000201:enzymology; D019001:Nitric Oxide Synthase; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D010920:Placenta / Q000201:enzymology*; D016133:Polymerase Chain Reaction; D011247:Pregnancy; D012333:RNA, Messenger / Q000032:analysis / Q000096:biosynthesis; D014471:Umbilical Veins
D017931:DNA Primers; D010446:Peptide Fragments; D012333:RNA, Messenger; D019001:Nitric Oxide Synthase; D000594:Amino Acid Oxidoreductases