Effects of N-glycosylation on in vitro activity of Bowes melanoma and human colon fibroblast derived tissue plasminogen activator.
Tissue-type plasminogen activator (t-PA), when isolated from human colon fibroblast (hcf) cells, is N-glycosylated differently than when isolated from the Bowes melanoma (m) cell line (Parekh et al., 1988). Both hcf- and m-t-PA can be separated into type I t-PA (with three occupied N-glycosylation sequons, at Asn-117, -184, and -448) and type II t-PA (with two occupied sequons, at Asn-117 and -448). Oligosaccharide analysis of each of these types of t-PA indicates that hcf-t-PA and m-t-PA have no glycoforms in common, despite having the same primary amino acid sequence. We have therefore compared in vitro the enzymatic activities and fibrin binding of type I and type II hcf- and m-t-PA with those of aglycosyl t-PA isolated from tunicamycin-treated cells. Plasminogen activation kinetics were determined by using an indirect amidolytic assay with Glu-plasminogen and a chromogenic plasmin substrate. In the absence of stimulator, there was little difference in activity between type I and type II t-PA, but the activity of aglycosyl t-PA was 2-4-fold higher than that of the corresponding glycosylated t-PA. In the presence of a fibrinogen fragment stimulator, the Kcat value of type II t-PA was approximately 5-fold that of type I t-PA from the same cell line, while the Km values for activation of Glu-plasminogen were similar (0.13-0.18 microM). The stimulated activity of glycosyl t-PA was similar to that of type II t-PA.(ABSTRACT TRUNCATED AT 250 WORDS)
Wittwer
A J
AJ
Department of Cell Culture and Biochemistry, Monsanto Company, St. Louis, Missouri 63167.
Howard
S C
SC
Carr
L S
LS
Harakas
N K
NK
Feder
J
J
Parekh
R B
RB
Rudd
P M
PM
Dwek
R A
RA
Rademacher
T W
TW
D002478:Cells, Cultured; D003106:Colon / Q000166:cytology; D004789:Enzyme Activation / Q000187:drug effects; D005337:Fibrin; D005347:Fibroblasts; D006031:Glycosylation; D006801:Humans; D008545:Melanoma / Q000473:pathology; D010958:Plasminogen / Q000378:metabolism; D010959:Tissue Plasminogen Activator / Q000378:metabolism*; D014407:Tumor Cells, Cultured; D014415:Tunicamycin / Q000494:pharmacology
D014415:Tunicamycin; D005337:Fibrin; D010958:Plasminogen; D010959:Tissue Plasminogen Activator