Characterization of a human recombinant receptor-linked protein tyrosine phosphatase.
The receptor-linked tyrosine phosphatase RPTP alpha from human brain (Kaplan, R., Morse, B., Huebner, K., Croce, C., Howk, R., Ravera, M., Ricca, G., Jaye, M., and Schlessinger, J. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 7000-7004) was expressed in insect cells following infection with recombinant baculovirus. Two major forms of the enzyme, with molecular sizes of 98 kDa and 114 kDa, were detected by immunoblot analysis. This heterogeneity could be ascribed to N-linked glycosylation on the basis of two lines of evidence; namely, blockage of glycosylation with tunicamycin in vivo and removal of carbohydrates by endoglycosidase F in vitro. The 114-kDa form was purified to homogeneity by chromatography on Superose 12 and Mono Q. Compared to the low Mr placenta and T-cell tyrosine phosphatases, RPTP alpha displayed a low optimum pH of 6 and a high Km in the micromolar range toward two artificial substrates (tyrosyl-phosphorylated myelin basic protein and modified lysozyme, respectively). Most effectors had a different and often an opposite influence on phosphatase activity depending on the nature of the substrate and the pH at which the assays were performed. Determination of Km and Vmax values for RPTP alpha suggests that the enzyme could exist in low and high substrate affinity states.
Daum
G
G
Department of Biochemistry SJ-70, University of Washington, Seattle 98195.
Zander
N F
NF
Morse
B
B
Hurwitz
D
D
Schlessinger
J
J
Fischer
E H
EH
D016367:Baculoviridae / Q000235:genetics; D001921:Brain / Q000201:enzymology*; D002413:Cations, Divalent; D002850:Chromatography, Gel; D004247:DNA / Q000235:genetics; D004492:Edetic Acid; D004591:Electrophoresis, Polyacrylamide Gel; D015971:Gene Expression Regulation, Enzymologic; D015967:Gene Expression Regulation, Viral; D005814:Genes, Viral; D006031:Glycosylation; D006493:Heparin / Q000494:pharmacology; D006801:Humans; D008670:Metals / Q000494:pharmacology; D010749:Phosphoprotein Phosphatases / Q000737:chemistry* / Q000235:genetics; D017027:Protein Tyrosine Phosphatases; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D013095:Spermidine / Q000494:pharmacology; D013096:Spermine / Q000494:pharmacology; D013379:Substrate Specificity
D002413:Cations, Divalent; D008670:Metals; D011994:Recombinant Proteins; D013096:Spermine; D006493:Heparin; D004247:DNA; D004492:Edetic Acid; D010749:Phosphoprotein Phosphatases; D017027:Protein Tyrosine Phosphatases; D013095:Spermidine