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Effects of pCMPS Modification of an Intramembrane Cysteine and Oxidative Crosslinking of Transmembrane Domains on the Human NTPDase 2 Activity
Author(s) -
Chiang WeiChieh,
Knowles Aileen F.
Publication year - 2008
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.22.1_supplement.611.27
Subject(s) - cysteine , chemistry , transmembrane domain , transmembrane protein , extracellular , biochemistry , oxidative phosphorylation , residue (chemistry) , enzyme , serine , mutant , biophysics , membrane , biology , receptor , gene
Human NTPDase 2 is a cell surface glycoprotein that is anchored to the membrane by two transmembrane domains (TMDs) near the N‐ and C‐termini, while the active site resides in the extracellular domain. The enzyme is inactivated by low concentrations of detergents, higher temperatures, and its substrate, and is inhibited by p‐chloromercuripheylsulfonate (pCMPS). The mutant in which the only free cysteine residue is replaced by serine (C26S) is not inhibited by pCMPS, indicating that C26 is the target of pCMPS modification. Reaction of pCMPS with C26, which resides in the N‐terminal TMD, may disrupt TMD interaction that has been shown to regulate NTPDase 2 activity. We also generated mutants containing a single cysteine residue in the C‐terminal TMD with or without C26 to investigate the effects of cross‐linking on enzyme activity. Inter‐ and intramolecular oxidative cross‐linking of the cysteine residues in the TMDs result in significant decrease of the human NTPDase 2 activity. Therefore both TMD interaction and mobility are critical for maximal enzyme activity. However, immobilization of the TMD by intramolecular disulfide bond formation reduces the inactivation of the residual activity of human NTPDase 2 by detergent, higher temperatures and substrate. (Supported by the California Metabolic Research Foundation)