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Purification and characterization of the human protein tyrosine phosphatase, PTPμ, from a baculovirus expression system

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Reversible Protein Phosphorylation in Cell Regulation

Part of the book series: Developments in Molecular and Cellular Biochemistry ((DMCB,volume 11))

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Abstract

The receptor like PTPase, PTPμ, displays structural similarity in its extracellular segment to members of the immunoglobulin superfamily of cell adhesion molecules. The full length form of PTPμ (200 kD) and a construct expressing only the intracellular PTPase domain-containing segment (80 kD) were expressed in the baculovirus/Sf9 cell system, purified and characterized. Full length PTPμ was membrane associated while the truncated form was recovered in the soluble fraction. PTPμ preferentially dephosphorylated a reduced carboxamidomethylated and maleylated derivative of lysozyme (RCML) over other tyrosine phosphorylated substrates such as myelin basic protein (MBP) or the syn thetic peptide EDNDYINASL. The enzymatic properties of the soluble, truncated form of the enzyme were examined in detail. The pH optimum was 7.5. It dephosphorylated RCML with a Km of 400 nM and a Vmax of 725 nmol/min/mg. This form of the enzyme was 2 fold more active than full length PTPμ. Trypsinization of the full length form inhibited activity. Vanadate and molybdate, potent tyrosine phosphatase inhibitors, abolished activity of the enzyme. Zn++ and Mn++ ions, polylysine, poly-glu/tyr, and spermine were also inhibitory. (Mol Cell Biochem 127/128: 131–141, 1993)

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Authors and Affiliations

  1. Cold Spring Harbor Labs, Demerec Building, Cold Spring Harbor, NY, 11724-2208, USA

    Susann M. Brady-Kalnay & Nicholas K. Tonks

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  1. Susann M. Brady-Kalnay
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  2. Nicholas K. Tonks
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Editors and Affiliations

  1. Department of Biochemistry, University of Saskatchewan, Saskatoon, Saskatchewan, Canada, S7N 0W0

    R. L. Khandelwal

  2. Department of Medical Biochemistry, University of Calgary, Health Science Centre, 3330 Hospital Drive N.W., Calgary, Alberta, Canada, TZN 4N1

    J. H. Wang

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© 1993 Kluwer Academic Publishers

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Brady-Kalnay, S.M., Tonks, N.K. (1993). Purification and characterization of the human protein tyrosine phosphatase, PTPμ, from a baculovirus expression system. In: Khandelwal, R.L., Wang, J.H. (eds) Reversible Protein Phosphorylation in Cell Regulation. Developments in Molecular and Cellular Biochemistry, vol 11. Springer, Boston, MA. https://doi.org/10.1007/978-1-4615-2600-1_12

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  • DOI: https://doi.org/10.1007/978-1-4615-2600-1_12

  • Publisher Name: Springer, Boston, MA

  • Print ISBN: 978-0-7923-2637-3

  • Online ISBN: 978-1-4615-2600-1

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