Skip to main content
Download PDF
  • Published:

Use of an anaerobic chamber environment for the assay of endogenous cellular protein-tyrosine phosphatase activities

Biological Procedures Online volume 4, pages 1–9 (2002)Cite this article

Abstract

Protein-tyrosine phosphatases (PTPases) have a catalytic cysteine residue whose reduced state is integral to the reaction mechanism. Since exposure to air can artifactually oxidize this highly reactive thiol, PTPase assays have typically used potent reducing agents to reactivate the enzymes present; however, this approach does not allow for the measurement of the endogenous PTPase activity directly isolated from the in vivo cellular environment. Here we provide a method for using an anaerobic chamber to preserve the activity of the total PTPase complement in a tissue lysate or of an immunoprecipitated PTPase homolog to characterize their endogenous activation state. Comparison with a sample treated with biochemical reducing agents allows the determination of the activatable (reducible) fraction of the endogenous PTPase pool.

References

  1. Tonks NK, Neel BG. Combinatorial control of the specificity of protein tyrosine phosphatases Curr. Opin. Cell Biol. 2001;13:182–195.

    Article  PubMed  CAS  Google Scholar 

  2. Zhang ZY. Protein-tyrosine phosphatases — biological function, structural characteristics, and mechanism of catalysis Crit. Revs. Bioch. Mol. Biol. 1998;33:1–52.

    Article  Google Scholar 

  3. Den JM, Dixon JE. Protein tyrosine phosphatases — mechanisms of catalysis and regulation Curr. Opin. Chem. Biol. 1998;2:633–641.

    Article  Google Scholar 

  4. Barford D, Das AK, Egloff MP. The structure and mechanism of protein phosphatases — insights into catalysis and regulation Ann. Rev. Biophys. Biomol. Struct. 1998;27:133–164.

    Article  CAS  Google Scholar 

  5. Lohse DL, Denu JM, Santoro N, Dixon JE. Roles of aspartic acid 181 and serine-222 in intermediate formation and hydrolysis of the mammalian protein-tyrosine-phosphatase ptp1 Biochemistry 1997;36:4568–4575.

    Article  PubMed  CAS  Google Scholar 

  6. Zhu L, Zilbering A, Wu X, Mahadev K, Joseph JI, Jabbour S, Deeb W, Goldstein BJ. Use of an anaerobic environment to preserve the endogenous activity of protein-tyrosine phosphatases isolated from intact cells Published online as The FASEB Journal express article 10.1096/fj.00-0795fje. FASEB J. 2001;15:1637–1639.

    PubMed  CAS  Google Scholar 

  7. Pot DA, Woodford TA, Remboutsika E, Haun RS, Dixon JE. Cloning, bacterial expression, purification, and characterization of the cytoplasmic domain of rat LAR, a receptor-like protein tyrosine phosphatase J. Biol. Chem. 1991;266:19688–19696.

    PubMed  CAS  Google Scholar 

  8. Hashimoto N, Feener EP, Zhang WR, Goldstein BJ. Insulin receptor protein-tyrosine phosphatases — Leukocyte common antigen-related phosphatase rapidly deactivates the insulin receptor kinase by preferential dephosphorylation of the receptor regulatory domain J. Biol. Chem. 1992;267:13811–13814.

    PubMed  CAS  Google Scholar 

  9. Pike LJ, Kuenzel EA, Casnellie JE, Krebs EG. A comparison of the insulin- and epidermal growth factor-stimulated protein kinases from human placenta J. Biol. Chem. 1984;259:9913–9921.

    PubMed  CAS  Google Scholar 

  10. Tonks NK, Diltz CD, Fischer EH. Purification and assay of CD45: An integral membrane proteintyrosine phosphatase Meth. Enzymol. 1991;201:442–451.

    Article  PubMed  CAS  Google Scholar 

  11. Streuli M, Krueger NX, Thai T, Tang M, Saito H. Distinct functional roles of the two intracellular phosphatase like domains of the receptor-linked protein tyrosine phosphatases LCA and LAR EMBO J. 1990;9:2399–2407.

    PubMed  CAS  Google Scholar 

  12. Hunter T. Protein kinases and phosphatases: the yin and yang of protein phosphorylation and signaling Cell 1995;80:225–236.

    Article  PubMed  CAS  Google Scholar 

  13. Barrett WC, DeGnore JP, Keng YF, Zhang ZY, Yim MB, Chock PB. Roles of superoxide radical anion in signal transduction mediated by reversible regulation of protein-tyrosine phosphatase 1B J. Biol. Chem. 1999;274:34543–34546.

    Article  PubMed  CAS  Google Scholar 

  14. Claiborne A, Yeh JI., Mallett TC, Luba J, Crane EJ, Charrier V, Parsonage D. Protein-sulfenic acids: Diverse roles for an unlikely player in enzyme catalysis and redox regulation Biochemistry 1999;38:15407–15416.

    Article  PubMed  CAS  Google Scholar 

  15. Barrett WC, DeGnore JP, Konig S, Fales HM, Keng YF, Zhang ZY, Yim MB, Chock PB. Regulation of PTP1B via glutathionylation of the active site cysteine 215 Biochemistry 1999;38:6699–6705.

    Article  PubMed  CAS  Google Scholar 

  16. Denu JM, Tanner KG. Specific and reversible inactivation of protein tyrosine phosphatases by hydrogen peroxide — evidence for a sulfenic acid intermediate and implications for redox regulation Biochemistry 1998;37:5633–5642.

    Article  PubMed  CAS  Google Scholar 

  17. Lee SR, Kwon KS, Kim SR, Rhee SG. Reversible inactivation of protein-tyrosine phosphatase 1B in A431 cells stimulated with epidermal growth factor J. Biol. Chem. 1998;273:15366–15372.

    Article  PubMed  CAS  Google Scholar 

  18. Goldstein BJ. Protein-tyrosine phosphatases and the regulation of insulin action. In: LeRoith D, Olefsky JM, Taylor SI, editors. Diabetes mellitus: A fundamental and clinical text. 2nd edition., Philadelphia: Lippincott Press; 2000. p. 206–217.

    Google Scholar 

  19. Goldstein BJ. Protein-Tyrosine Phosphatase 1B (PTP1B): A Novel Therapeutic Target for Type 2 Diabetes Mellitus, Obesity and Related States of Insulin Resistance Curr. Drug Targets- Immune, Endocrine Metab. Dis. 2001;1:265–275.

    Article  CAS  Google Scholar 

  20. Mahadev K, Zilbering A, Zhu L, Goldstein BJ. Insulin-stimulated hydrogen peroxide reversibly inhibits protein-tyrosine phosphatase 1B in vivo and enhances the early insulin action cascade J. Biol. Chem. 2001;276:21938–21942.

    Article  PubMed  CAS  Google Scholar 

  21. Mahadev K, Wu X, Zilbering A, Zhu L, Lawrence JTR, Goldstein BJ. Hydrogen peroxide generated during cellular insulin stimulation is integral to activation of the distal insulin signaling cascade in 3T3-L1 adipocytes J. Biol. Chem. 2001;276:48662–48669.

    Article  PubMed  CAS  Google Scholar 

  22. Sullivan DM, Wehr NB, Fergusson MM, Levine RL, Finkel T. Identification of oxidant-sensitive proteins: TNF-alpha induces protein glutathiolation Biochemistry 2000;39:11121–11128.

    Article  PubMed  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Dorrance H. Hamilton Research Laboratories, Division of Endocrinology and Metabolic Diseases, Department of Medicine, Jefferson Medical College, Thomas Jefferson University, 19107, Philadelphia, PA, USA

    Li Zhu & Barry Goldstein M.D., Ph.D. (Director)

Authors
  1. Li Zhu
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Barry Goldstein M.D., Ph.D.
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Barry Goldstein M.D., Ph.D..

Additional information

Published: June 11, 2002.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Zhu, L., Goldstein, B. Use of an anaerobic chamber environment for the assay of endogenous cellular protein-tyrosine phosphatase activities. Biol Proced Online 4, 1–9 (2002). https://doi.org/10.1251/bpo28

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1251/bpo28

Indexing terms

Biological Procedures Online

ISSN: 1480-9222

Contact us