Properties of Pervanadate and Permolybdate
CONNEXIN43, PHOSPHATASE INHIBITION, AND THIOL REACTIVITY AS MODEL SYSTEMS*
- From the Departments of ‡Environmental and Occupational Cancer and ¶Biophysics, Institute for Cancer Research, The Norwegian Radium Hospital, N-0310 Oslo, Norway
Abstract
Pervanadate and permolybdate are irreversible protein-tyrosine phosphatase inhibitors, with IC50values of 0.3 and 20 μm, respectively, in intact cells. Maximal inhibition was obtained within 1 min at higher concentrations of the compounds. They induced prominent changes in the phosphorylation status of the gap junction protein, connexin43. These effects were utilized as model systems to assess the stability and inactivation of the compounds. Although the concentrated stock solutions were relatively stable, the diluted compounds were unstable. The biological activity had decreased to 20–30% after 6 h of incubation in a phosphate buffer, 1 h in phosphate buffer with 10% fetal calf serum, and 1–3 minutes in culture medium. Thiols reacted rapidly with the compounds and inactivated them (initial reaction rates with cysteine: permolybdate > pervanadate > H2O2). Catalase inactivated the compounds, and permolybdate was the more sensitive. The cells inactivated permolybdate faster than pervanadate. Cellular inactivation of permolybdate, and to a lesser degree pervanadate, appeared to be partly dependent on catalase and thiols. However, a general decrease in cellular thiols was not the mediator of the biological effects of pervanadate or permolybdate. Mathematical modeling of the thiol reactivity suggested that monoperoxovanadate at maximum could possess 20% of the biological activity of diperoxovanadate.
Footnotes
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↵* This work was supported by grants from the Blix Family’s Fund for the Advance of Medical Research (to S.-O. M.), Anders Jahre’s Fund (to S.-O. M and Dr. T. Sanner), and funds from the Faculty of Medicine, University of Oslo (to S.-O. M.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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↵§ To whom correspondence should be addressed. Tel.: 47-2293-4705; Fax: 47-2293-5767; E-mail: s.o.mikalsen{at}labmed.uio.no.
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↵1 The abbreviations used are: PTPase, protein-tyrosine phosphatase; Cx43, connexin43; pNPP,para-nitrophenyl phosphate; DTNB, 5,5′-dithio-bis(2-nitrobenzoic acid); NEM, N-ethylmaleimide; PBSS, phosphate-buffered saline supplemented with Ca2+ and Mg2+; HBSS, Hepes-buffered saline supplemented with Ca2+ and Mg2+; DMEM, Dulbecco’s modified Eagle’s medium; FCS, fetal calf serum; DTT, dithiothreitol; PV, pervanadate; PMo, permolybdate; NP, nonphosphorylated.
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↵2 The NIH Image software is obtainable on the Internet by anonymous file transfer protocol fromftp://zippy.nimh.nih.gov.
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↵3 S.-O. Mikalsen, unpublished observations.
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- Received June 25, 1997.
- Revision received February 9, 1998.
- The American Society for Biochemistry and Molecular Biology, Inc.
