title: Effect of glycosylation at Asn302 of pro-urokinase on its stability in culture supernatant.
abstract: OBJECTIVE
  To investigate the effect of glycosylation at Asn302 of pro-urokinase (pro-UK) on the stability in culture supernatant.
  
  
  METHODS
  Nonglycosylated pro-UK was constructed by site-directed mutagenesis of Asn302 to Ala302. The pro-UK mutant and native pro-UK were transfected into dhfr(-)-CHO cells, and serum-free culture supernatant was harvested and incubated at 4 degrees C and 37 degrees C, respectively. The pro-UK activity in culture supernatant was measured by the optical density (OD) increase with time (12 hours) at 405 nm. Without thermolysin activation, the percentage of single chain pro-UK was measured.
  
  
  RESULTS
  After 48 hours of incubation at 4 degrees C, the activities of pro-UK mutant and native pro-UK decreased 3.7% and 2.9% respectively, and at 37 degrees C decreased 37.9% and 23.5%, respectively. The total activity of native pro-UK was significantly higher than that of nonglycosylated mutant at 37 degrees C. The single-chain percentage of native pro-UK was higher than that of nonglycosylated mutant at both 4 degrees C and 37 degrees C.
  
  
  CONCLUSIONS
  Higher temperature increases the proteolysis of pro-UK. The glycosylation site on Asn302 is beneficial to pro-UK stability in culture supernatant.
authors.affiliation: Department of Cardiology, General Hospital of Chinese People's Liberation Army, Beijing 100853. dryangb@yahoo.com.cn
authors.forename: Bo
authors.initials: B
authors.lastname: Yang
authors.forename: Tian-De
authors.initials: TD
authors.lastname: Li
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D001216:Asparagine / Q000737:chemistry; D001483:Base Sequence; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D003470:Culture Media; D017931:DNA Primers / Q000235:genetics; D004795:Enzyme Stability; D006031:Glycosylation; D006801:Humans; D066298:In Vitro Techniques; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014568:Urokinase-Type Plasminogen Activator / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
chemical_list: D003470:Culture Media; D017931:DNA Primers; D011994:Recombinant Proteins; D001216:Asparagine; D014568:Urokinase-Type Plasminogen Activator; C067551:saruplase