Characterization of single chain urokinase-type plasminogen activator with a novel amino-acid substitution in the kringle structure.
ECV304 is a cell line established by a spontaneous transformation of endothelial cells of a human umbilical vein. It was shown that ECV304 secretes single chain urokinase-type plasminogen activator (scu-PA). A subclone, ECV304 clone 15, was obtained by acclimatization of parental clone to serum-free medium followed by limiting dilution. The clone was found to produce approximately five times as much scu-PA (approximately 20 IU/10(6) cells per day) as the parental clone after a 40 days' culture. Though the biochemical characteristics of the purified scu-PA were indistinguishable from those of the native scu-PA, it had a lower affinity for fibrin clots under the employed conditions. Molecular cloning of a cDNA encoding the scu-PA has identified a novel substitution from C to T in the nucleotide sequence encoding the kringle structure. The substitution resulted in an alteration from Pro (CCG) to Leu (CTG) at amino-acid position 121, which may be directly or indirectly involved in the decrease in the apparent affinity.
Yoshimoto
M
M
Department of Applied Biology, Research Center, Taisho Pharmaceutical Co., Ltd., Saitama, Japan.
Ushiyama
Y
Y
Sakai
M
M
Tamaki
S
S
Hara
H
H
Takahashi
K
K
Sawasaki
Y
Y
Hanada
K
K
D000595:Amino Acid Sequence; D001483:Base Sequence; D001777:Blood Coagulation; D002461:Cell Line, Transformed; D002999:Clone Cells / Q000201:enzymology; D003001:Cloning, Molecular; D016895:Culture Media, Serum-Free; D018076:DNA, Complementary / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D004730:Endothelium, Vascular / Q000201:enzymology*; D004792:Enzyme Precursors / Q000737:chemistry / Q000235:genetics; D005337:Fibrin / Q000378:metabolism; D006801:Humans; D018082:Kringles / Q000235:genetics*; D008969:Molecular Sequence Data; D017354:Point Mutation / Q000235:genetics; D011485:Protein Binding; D014471:Umbilical Veins; D014568:Urokinase-Type Plasminogen Activator / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
D016895:Culture Media, Serum-Free; D018076:DNA, Complementary; D004792:Enzyme Precursors; D005337:Fibrin; D014568:Urokinase-Type Plasminogen Activator