MOLECULAR-CLONING, CHARACTERIZATION AND EXPRESSION A NITRIC-OXIDE SYNTHASE FROM PORCINE PULMONARY-ARTERY ENDOTHELIAL-CELLS

The lack of sequence information and clones of porcine pulmonary arter y endothelial cell (PAEC) constitutive nitric oxide synthase (ecNOS) c DNA limits comparative analysis between porcine and human PAEC. Theref ore, we cloned, characterized and expressed the ecNOS cDNA from porcin e PAEC. Two oligonucleotide primers were designed based on the publish ed human ecNOS cDNA sequence and used to clone porcine PAEC ecNOS usin g 5' and 3' rapid amplification of cDNA ends reverse transcriptase pol ymerase chain reaction technique. A full-length ecNOS cDNA was cloned and sequenced, representing a protein of 1205 amino acids with a molec ular mass of 134 kDa. A mammalian expression vector (pcDNA3) containin g this cDNA was transfected into COS-7 cells, and ecNOS activity was d etected by monitoring the formation of [H-3]-citrulline from [H-3]-L-a rginine. Expression of ecNOS activity was predominantly associated (>9 0%) with the total membrane fraction of these transfected cells. The d educed amino acid sequence of porcine ecNOS cDNA, containing binding s ites for NADPH, flavin adenine dinucleotide and bound flavin mononucle otide, shows 94% identity to human ecNOS. The molecular weight of porc ine ecNOS mRNA was estimated to be 4.7 kb by Northern blot analysis, s imilar to human ecNOS mRNA. This suggests that porcine ecNOS is simila r to human ecNOS in deduced amino acid sequence and structure. (C) 199 7 Elsevier Science Inc.