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Molecular cloning and characterization of a tumor-associated, growth-related, and time-keeping hydroquinone (NADH) oxidase (tNOX) of the HeLa cell surfaceNOX proteins are growth-related cell surface proteins that catalyze both hydroquinone or NADH oxidation and protein disulfide interchange and exhibit prion-like properties. The two enzymatic activities alternate to generate a regular period length of about 24 min. Here we report the expression, cloning, and characterization of a tumor-associated NADH oxidase (tNOX). The cDNA sequence of 1830 bp is located on gene Xq25-26 with an open reading frame encoding 610 amino acids. The activities of the bacterially expressed tNOX oscillate with a period length of 22 min as is characteristic of tNOX activities in situ. The activities are inhibited completely by capsaicin, which represents a defining characteristic of tNOX activity. Functional motifs identified by site-directed mutagenesis within the C-terminal portion of the tNOX protein corresponding to the processed plasma membrane-associated form include quinone (capsaicin), copper and adenine nucleotide binding domains, and two cysteines essential for catalytic activity. Four of the six cysteine to alanine replacements retained enzymatic activity, but the period lengths of the oscillations were increased. A single protein with two alternating enzymatic activities indicative of a time-keeping function is unprecedented in the biochemical literature.
Document ID
20040088418
Document Type
Reprint (Version printed in journal)
External Source(s)
Authors
Chueh, Pin-Ju (Purdue University West Lafayette, Indiana 47907, United States)
Kim, Chinpal
Cho, NaMi
Morre, Dorothy M.
Morre, D. James
Date Acquired
August 21, 2013
Publication Date
March 19, 2002
Publication Information
Publication: Biochemistry
Volume: 41
Issue: 11
ISSN: 0006-2960
Subject Category
Life Sciences (General)
Funding Number(s)
CONTRACT_GRANT: R01 CA75461
CONTRACT_GRANT: P50 AT00477
Distribution Limits
Public
Copyright
Other
Keywords
NASA Discipline Cell Biology
Non-NASA Center