title: Endothelial plasminogen activator inhibitor (PAI): a new member of the Serpin gene family.
abstract: A human endothelial cDNA expression library, based on the Escherichia coli plasmid pUC9, was screened with a heterologous antibody raised against purified bovine aortic endothelial plasminogen activator inhibitor (PAI). A synthetic oligonucleotide, derived from a partial PAI cDNA expression clone, was used to select a full-length PAI cDNA, the size of which coincides with the length of PAI mRNA (approximately 2350 nucleotides) as determined by Northern blot analysis. The authenticity of full-length PAI cDNA is demonstrated by the expression of biologically active PAI both in lysates of transformed E. coli cells and in conditioned media of mouse Ltk- cells, transfected with PAI cDNA inserted into vector pSV2. Analysis of the de novo synthesized anti-plasminogen activator activity, employing reverse fibrin autography, shows that transfected mouse Ltk- cells synthesize a polypeptide with a mol. wt identical to that of the native PAI glycoprotein (Mr 52,000), whereas in E. coli an unglycosylated, active product with a mol. wt of 43,000 is made. The amino acid sequence, derived from the determined nucleotide sequence, shows that pre-PAI consists of 402 amino acids. It is proposed that the mature PAI is preceded by a signal peptide of 23 amino acid residues. The amino acid sequence of mature PAI includes three potential asparagine-linked glycosylation sites and lacks cysteine residues. The predicted amino acid sequence reveals significant homology with members of the serine protease inhibitor (Serpin) family, e.g. alpha 1-proteinase inhibitor and antithrombin III.(ABSTRACT TRUNCATED AT 250 WORDS)
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D002478:Cells, Cultured; D003001:Cloning, Molecular; D004247:DNA / Q000378:metabolism; D004727:Endothelium / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D005796:Genes; D006023:Glycoproteins / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D010957:Plasmids; D015849:Plasminogen Inactivators; D011061:Poly A / Q000302:isolation & purification; D011480:Protease Inhibitors / Q000235:genetics*; D012313:RNA / Q000302:isolation & purification; D012333:RNA, Messenger
chemical_list: D006023:Glycoproteins; D015849:Plasminogen Inactivators; D011480:Protease Inhibitors; D012333:RNA, Messenger; D011061:Poly A; D012313:RNA; D004247:DNA