Abstract
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.
Highlights
U.S A . 87, 7000-7004) was expressed in insect cells ences in the size and structure of the extracellular segments following infection with recombinant baculovirus. of these receptors suggesting that they might play different
Two major forms of the enzyme, with molecular sizes of 98 kDa and 114 kDa, weredetected 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 parts in the regulation of cellular processes
Compared to the low M, placenta and T- similarities with the neural cell adhesion molecules in that cell tyrosine phosphatases, RPTPa displayed a low they contain typeI11 fibronectin andIgG-like domains, while optimum pH of 6 and a high K, in the micromolar HPTPp hasonly type I11 fibronectin repeats [4].Their strucrange toward two artificial substrates
Summary
Length cDNAof RPTPa [10] was cloned into pML2D [13] using a Cloning and TimeCourseofExpression-Thefull length unique XhoI restriction site found within a multiple cloning site inserted between nucleotides 23 and 375 of the vector Digestion of this construct with DraI andBglII resulted in a2.5-kilobasefragment that was ligated into theBamHI siteof the pVL 941 expression vector [14] which had first been treated with Klenow fragment and deoxcDNA of RPTPa was cloned into the pVL941 expression vector as described under "Experimental Procedures." Using the method ofdot-blot hybridization,cells infected by recombinant virus could be detected following co-transfection of ynucleotides to generate blunt ends. Triton X-100 extract (5pg of protein) and the buffer AT, flow rate: 0.5 ml/min, fraction size:0.5 ml). Active frac- indicated fractions of the chromatography on Superose 12 and Mono tions were pooled and dialyzed for 1h against buffer A T containing Q (8pl each) were subjected to SDS-PAGE [26].A, the 7.5% gel was.
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