Tris-HCl (pH 8), 240 mM KCl,
20 mM MgCl
, and 1 mM pyruvate or
10 to 100 µL of spent growth medium.
The preassay mixture was incubated at
30 °C for 2 minutes before the reaction
was initiated with NADH (final con-
centration, 25 mM). The oxidation of
NADH was monitored as a decrease in
the absorbance at 340 nM in a quartz
microtiter plate using a SpectraMax
Plus microplate reader (Molecular
cytochrome c reduction
Mapping of functional domains in
p47phox involved in the activation of
NADPH oxidase by "peptide walking"
J. Biol. Chem. 273: 1543544 (1998).
Igor Morozov, Ofra Lotan, Gili Joseph, Yara Gorzalc-
zany, and Edgar Pick.
The Julius Friedrich Cohnheim-Minerva Center for
Phagocyte Research, Department of Human Microbiol-
ogy, Sackler School of Medicine, Tel Aviv University,
Tel Aviv 69978, Israel.
Methods: cell-free NADPH oxidase
assay. The superoxide generating
NADPH oxidase of phagocytes consists,
in resting cells, of a membrane-associ-
ated electron transporting flavocyto-
chrome (cytochrome b559) and four
cytosolic proteins as follows: p47phox,
p67phox, p40phox, and the small
GTPase, Rac(1 or 2). O
was initiated by the addition of
NADPH and quantified by following
the rate of cytochrome c reduction, at
550 nm, in a kinetic assay performed in
a SpectraMax 340 microplate reader
(Molecular Devices Corp.), using Soft-
Max Pro software.
Evidence for peroxisome proliferator-
activated receptor (PPAR)-indepen-
dent peroxisome proliferation: effects
of PPAR-specific agonists in PPAR-
Mol. Pharmacol. 58: 470-76 (2000).
John G. DeLuca
, Thomas W. Doebber
, Linda J.
, Ramon K. Kemp
, Sylvain Molon-Noblot
Soumya P. Sahoo
, John Ventre
, Margaret S. Wu
frey M. Peters
, Frank J. Gonzalez
, and David E.
Department of Molecular Endocrinology, Merck
Research Laboratories, Rahway, New Jersey.
Department of Safety Assessment/Genetic and Cellu-
lar Toxicology, Merck Research Laboratories, West
Laboratory of Metabolism, National Cancer Institute,
National Institutes of Health, Bethesda, Maryland.
Methods: measurement of CYP4A lev-
els. CYP4A protein levels were assessed
in whole liver homogenate, and samples
were solubilized in 0.5% sodium cho-
late/Triton X-100, using rabbit anti-rat
CYP4A1 as the primary antibody (Gen-
test Corp., Waltham, MA) and goat
anti-rabbit Ig, horseradish peroxidase-
conjugated, as the secondary antibody
(Pierce Chemical Co., Rockford, IL).
Solubilized samples were diluted in
0.05 M carbonate/bicarbonate buffer,
pH 9.6, to give approximately 20 ng of
protein/50 µL. This was adsorbed to 96-
well Maxisorp titer plates (Nalge Nunc