NADH oxidase activity (NOX) and enlargement of HeLa cells oscillate
with two di¡erent temperature-compensated period lengths of 22 and 24
minutes corresponding to di¡erent NOX forms
Sui Wang
a
, Rhea Pogue
a
, Dorothy M. Morre
¨
a
, D. James Morre
¨
bY
*
a
Department of Foods and Nutrition, Purdue University, West Lafayette, IN, USA
b
Department of Medicinal Chemistry and Molecular Pharmacology, Purdue University, Hansen Life Sciences Research Building,
West Lafayette, IN 47907-1333, USA
Received 26 October 2000; received in revised form 18 April 2001; accepted 19 April 2001
Abstract
NOX proteins are cell surface-associated and growth-related hydroquinone (NADH) oxidases with protein disulfide^thiol
interchange activity. A defining characteristic of NOX proteins is that the two enzymatic activities alternate to generate a
regular period length of about 24 min. HeLa cells exhibit at least two forms of NOX. One is tumor-associated (tNOX) and is
inhibited by putative quinone site inhibitors (e.g., capsaicin or the antitumor sulfonylurea, LY181984). Another is
constitutive (CNOX) and refractory to inhibition. The periodic alternation of activities and drug sensitivity of the NADH
oxidase activity observed with intact HeLa cells was retained in isolated plasma membranes and with the solubilized and
partially purified enzyme. At least two activities were present. One had a period length of 24 min and the other had a period
length of 22 min. The lengths of both the 22 and the 24 min periods were temperature compensated (approximately the same
when measured at 17, 27 or 37³C) whereas the rate of NADH oxidation approximately doubled with each 10³C rise in
temperature. The rate of increase in cell area of HeLa cells when measured by video-enhanced light microscopy also exhibited
a complex period of oscillations reflective of both 22 and 24 min period lengths. The findings demonstrate the presence of a
novel oscillating NOX activity at the surface of cancer cells with a period length of 22 min in addition to the constitutive
NOX of non-cancer cells and tissues with a period length of 24 min. ß 2001 Elsevier Science B.V. All rights reserved.
Keywords: Hydroquinone (NADH) oxidase; Tumor associated hydroquinone (NADH) oxidase ; Oxidation of NADH; Cell enlargement;
Growth; Temperature compensation; Ubiquinone; HeLa cell; Coenzyme Q
1. Introduction
A growth factor- and hormone- or drug-responsive
oxidation of both NADH and hydroquinones cata-
lyzed by a family of proteins referred to as NOX
proteins has been found associated with plasma
membranes of the animal and plant cell surface
[1,2]. With rat hepatomas [3] and with cancer cells
in culture [4], a constitutively activated component of
NADH oxidation was observed which was no longer
0167-4889 / 01 / $ ^ see front matter ß 2001 Elsevier Science B.V. All rights reserved.
PII: S0167-4889(01)00107-0
Abbreviations: HeLa, human cervical carcinoma cell line;
NOX, plasma membrane-located hydroquinone (NADH) oxidase
with protein disul¢de^thiol interchange activity; CNOX, consti-
tutive NOX; tNOX, tumor-associated NOX; LY181984, N-(4-
methylphenylsulfonyl)-NP-(4-chlorophenyl)urea; LY181985, N-
(4-methylphenylsulfonyl)-NP-(4-phenyl)urea; capsaicin, 8-methyl-
N-vanillyl-6-noneamide
* Corresponding author. Fax: +1-765-494-4007;
E-mail: morre@pharmacy.purdue.edu
BBAMCR 14761 13-6-01
Biochimica et Biophysica Acta 1539 (2001) 192^204
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