Functional properties of carbohydrate-depleted tissue plasminogen activator.
In order to evaluate the importance of the carbohydrate moiety of human tissue plasminogen activator (TPA), human melanoma (Bowes) cells were treated with a glycosylation inhibitor, tunicamycin (TM), and cellular fractions were assayed for fibrinolytic activity. Where glycosylation was inhibited by 90% and protein synthesis by 30%, TPA specific activity measured by fibrinolytic assays decreased 6-10-fold in the tissue culture medium and cell cytosol with a concomitant 2-fold increase in the 100000g microsomal pellet. In addition, TPA purified to apparent homogeneity was treated with endo-beta-N-acetylglucosaminidase H (Endo-H), producing a fraction that in contrast to native TPA did not adsorb to concanavalin A-Sepharose (Con A-Sepharose). This fraction represented TPA from which 85-90% of N-linked carbohydrate residues had been removed. Native TPA effectively activated plasminogen in the presence of fibrin (Km = 1 microM, kcat = 0.09 s-1) whereas saturation of the enzyme was not achieved at 100 microM plasminogen in the absence of fibrin. Glycosidase-treated and native TPA activated plasminogen at identical high rates in the presence and at identical negligible rates in the absence of fibrin. These studies indicate that the inhibition of glycosylation of TPA results in the inhibition of secretion of the molecule as has been observed for some other glycoproteins. The enzymatic removal of N-linked carbohydrate from purified TPA does not change its unique fibrin-directed properties.
D000118:Acetylglucosaminidase / Q000378:metabolism; D002241:Carbohydrates / Q000502:physiology*; D002460:Cell Line; D002846:Chromatography, Affinity; D004789:Enzyme Activation; D005337:Fibrin / Q000378:metabolism; D005944:Glucosamine / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; D008545:Melanoma / Q000378:metabolism; D010958:Plasminogen / Q000378:metabolism; D010960:Plasminogen Activators / Q000502:physiology*; D014415:Tunicamycin / Q000494:pharmacology
D002241:Carbohydrates; D014415:Tunicamycin; D005337:Fibrin; D010958:Plasminogen; D000118:Acetylglucosaminidase; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; D010960:Plasminogen Activators; D005944:Glucosamine