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Molecular cloning, characterization and expression of a nitric oxide synthase from porcine pulmonary artery endothelial cells.

Authors
  • Zhang, J
  • Patel, J M
  • Block, E R
Type
Published Article
Journal
Comparative Biochemistry and Physiology Part B Biochemistry and Molecular Biology
Publisher
Elsevier
Publication Date
Apr 01, 1997
Volume
116
Issue
4
Pages
485–491
Identifiers
PMID: 9149402
Source
Medline
License
Unknown

Abstract

The lack of sequence information and clones of porcine pulmonary artery endothelial cell (PAEC) constitutive nitric oxide synthase (ecNOS) cDNA limits comparative analysis between porcine and human PAEC. Therefore, we cloned, characterized and expressed the ecNOS cDNA from porcine PAEC. Two oligonucleotide primers were designed based on the published human ecNOS cDNA sequence and used to clone porcine PAEC ecNOS using 5' and 3' rapid amplification of cDNA ends reverse transcriptase polymerase chain reaction technique. A full-length ecNOS cDNA was cloned and sequenced, representing a protein of 1205 amino acids with a molecular mass of 134 kDa. A mammalian expression vector (pcDNA3) containing this cDNA was transfected into COS-7 cells, and ecNOS activity was detected by monitoring the formation of [3H]-citrulline from [3H]-L-arginine. Expression of ecNOS activity was predominantly associated (> 90%) with the total membrane fraction of these transfected cells. The deduced amino acid sequence of porcine ecNOS cDNA, containing binding sites for NADPH, flavin adenine dinucleotide and bound flavin mononucleotide, shows 94% identity to human ecNOS. The molecular weight of porcine ecNOS mRNA was estimated to be 4.7 kb by Northern blot analysis, similar to human ecNOS mRNA. This suggests that porcine ecNOS is similar to human ecNOS in deduced amino acid sequence and structure.

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