Tissue-type plasminogen activator (t-PA) is synthesized in mammalian cells as a mixture of two forms that differ in their extent of N-linked glycosylation. We have investigated the mechanism underlying this variation in glycosylation, using a cell-free system that consists of a rabbit reticulocyte lysate optimized for the formation of disulphide bonds and supplemented with dog pancreas microsomal membranes. Molecules of human t-PA synthesized in vitro are enzymically active and responsive to natural activators and inhibitors, and are glycosylated in a pattern identical with that of the protein produced in vivo. This demonstrates that t-PA synthesized in vitro folds into the same conformation as the protein synthesized in vivo. We show that the extent of glycosylation of individual t-PA molecules is dependent on the state of folding of the polypeptide chain, since the probability of addition of an oligosaccharide side chain at Asn-184 is decreased under conditions that promote the formation of enzymically active molecules. This variation in glycosylation is independent of the rate of protein synthesis.
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August 1992
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Research Article|
August 15 1992
Cell-free synthesis of enzymically active tissue-type plasminogen activator. Protein folding determines the extent of N-linked glycosylation
N J Bulleid;
N J Bulleid
*Department of Biochemistry and Molecular Biology, University of Manchester, Oxford Road, Manchester M13 9PT, U.K.
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R S Bassel-Duby;
R S Bassel-Duby
‡Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75235, U.S.A.
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R B Freedman;
R B Freedman
§The Biological Laboratory, The University, Canterbury, Kent CT2 7NJ, U.K.
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J F Sambrook;
J F Sambrook
∥Department of Biochemistry, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75235, U.S.A.
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M J H Gething
M J H Gething
‡Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75235, U.S.A.
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Publisher: Portland Press Ltd
Online ISSN: 1470-8728
Print ISSN: 0264-6021
© 1992 The Biochemical Society, London
1992
Biochem J (1992) 286 (1): 275–280.
Citation
N J Bulleid, R S Bassel-Duby, R B Freedman, J F Sambrook, M J H Gething; Cell-free synthesis of enzymically active tissue-type plasminogen activator. Protein folding determines the extent of N-linked glycosylation. Biochem J 15 August 1992; 286 (1): 275–280. doi: https://doi.org/10.1042/bj2860275
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