Available online at www.sciencedirect.com
Analytical Biochemistry 373 (2008) 392–394
Colorimetric determination of pure Mg2+-dependent
Tara Havriluk a,1, Fred Lozy a,1, Symeon Siniossoglou b, George M. Carman a,*
a Department of Food Science and Rutgers Center for Lipid Research, Rutgers University, New Brunswick, NJ 08901, U.S.A.
b Cambridge Institute for Medical Research, University of Cambridge, CB2 2XY Cambridge, U.K.
The malachite green-molybdate reagent was used for a colorimetric assay of pure Mg2+-dependent phosphatidate phosphatase activ-
ity. This enzyme plays a major role in fat metabolism. Enzyme activity was linear with time and protein concentration, and with theconcentration of water-soluble dioctanoyl phosphatidate. The colorimetric assay was used to examine enzyme inhibition by phenylgly-oxal, propranolol, and dimethyl sulfoxide. Pure enzyme and a water-soluble phosphatidate substrate were required for the assay, whichshould be applicable to a well-deﬁned large-scale screen of Mg2+-dependent phosphatidate phosphatise inhibitors (or activators).
Ó 2007 Elsevier Inc. All rights reserved.
Mg2+-dependent phosphatase (PAP1, 3-sn-phos-
A large-scale search of inhibitors (or activators) of PAP1
phatidate phosphohydrolase; EC 22.214.171.124) catalyzes the
activity requires a sensitive and convenient enzymatic assay.
dephosphorylation of PA yielding diacylglycerol and Pi
The radioactive assays currently used to measure
PAP1 generates the diacylglycerol used for the syn-
PAP1 activity are not conducive to a high-throughput
thesis of triacylglycerol and the diacylglycerol used for
screen of potential drugs to control enzyme activity. Aside
the synthesis of phosphatidylethanolamine and phosphati-
from the radioactive nature of these assays, they require a
dylcholine via the Kennedy pathway Recent studies
chloroform–methanol–water phase partition to separate
have identiﬁed human lipin 1 as a PAP1 enzyme . In a
water-soluble 32Pi from chloroform-soluble [32P]PA or the
mouse model, lipin 1 deﬁciency prevents normal adipose
separation (e.g., thin-layer chromatography) of [3H]diacyl-
tissue development that results in lipodystrophy (i.e., loss
glycerol from [3H]PA . We have developed a nonradio-
of body fat) and insulin resistance, whereas excess lipin 1
active colorimetric PAP1 assay based on orthophosphate
promotes obesity and insulin sensitivity That human
analysis using the malachite green–molybdate reagent
lipin 1 is a PAP1, the penultimate enzyme in the pathway to
The reagent forms a colored complex with ortho-
synthesize triacylglycerol from PA, provides a mechanistic
phosphate that can be measured spectrophotometrically.
basis for how lipin 1 regulates fat metabolism in mamma-
This colorimetric assay is not suitable for use with cell ex-
lian cells. Accordingly, PAP1 activity may represent an
tracts or with crude PAP1 preparations because of a high
important pharmaceutical target for the control of body
phosphate background, hence the reason for using the
radioactive assays . However, the colorimetric assaywas suitable for measuring the activity of pure PAP1, andthe assay was amenable to screening enzyme inhibitors.
* Corresponding author. Fax: +1 (732) 932 6776.
Saccharomyces cerevisiae PAH1-encoded PAP1 was
expressed and puriﬁed to homogeneity as described by
1 These authors contributed equally to this work.
O’Hara et al. The yeast enzyme was used as a model
Abbreviations used: PA, phosphatidate; PAP1, Mg2+-dependent PA
PAP1 to develop the colorimetric assay. Pure PAP1
phosphatase; DiC8 PA, dioctanoyl phosphatidate; DiC18 PA, dioleoylphosphatidate; DMSO, dimethyl sulfoxide.
(12 ng) was used in a standard reaction mixture that
0003-2697/$ - see front matter Ó 2007 Elsevier Inc. All rights reserved.
Notes & Tips / Anal. Biochem. 373 (2008) 392–394
Fig. 1. Time course, enzyme concentration, and substrate concentration dependencies of the colorimetric assay on pure PAP1 activity. (A) PAP1 activitywas measured with 0.2 mM DiC8 PA and 12 ng of pure enzyme for the indicated time intervals. (B) PAP1 activity was measured for 20 min with 0.2 mMDiC8 PA and the indicated amounts of pure enzyme. (C), PAP1 activity was measured for 20 min with 12 ng of pure enzyme and the indicated amounts ofDiC8 PA. The lines were the result of a least-squares analysis of the data. The data shown were derived from triplicate determinations ± SD.
contained 50 mM Tris–HCl buﬀer (pH 7.5), 1 mM MgCl2,
assay. Whereas these values were close, it is diﬃcult to
and 0.2 mM DiC8 PA (Avanti Polar Lipids) in a total vol-
make a comparison of an enzyme activity measured with
ume of 0.1 ml. Unless otherwise indicated, all enzyme as-
a water-soluble substrate with that measured with a deter-
says were conducted in triplicate for 20 min at 30 °C. The
malachite green–molybdate reagent was prepared
The suitability of the colorimetric assay for screening for
as described by Mahuren et al. . The reaction was ter-
PAP1 inhibitors was tested with two known inhibitors of
minated by the addition of 200 ll of the malachite green–
the enzyme, phenylglyoxal and propranolol . Phe-
molybdate reagent; 30 ll of 1% polyvinyl alcohol was then
nylglyoxal is an arginine reactive compound , whereas
added to the reaction to stabilize the color complex .
propranolol is thought to interact with the Mg2+ binding
The reaction mixture was vortexed brieﬂy and the absor-
site of the enzyme . Phenylglyoxal (and pro-
bance of the solution was measured with a spectrophotom-
pranolol inhibited PAP1 activity in dose-depen-
eter at 660 nm. The color was stable for at least 1 h. The
dent manners with IC50 values of 1.3 and 0.2 mM,
amount of orthophosphate formed was quantiﬁed from a
respectively. These values were in the same range deter-
standard curve using 0.5–4 nmol of potassium phosphate.
mined for PAP1 activity measured by the radioactive assay
The enzyme reactions and standard curve were performed
in new plastic test tubes. This obviated the concern of
Some enzyme inhibitors are not soluble in aqueous buf-
interfering phosphates from tubes that have been washed
fers and are commonly solubilized in DMSO. Accordingly,
with detergent Statistical analyses were performed
the eﬀect of DMSO on PAP1 activity was tested using the
colorimetric assay. The addition of DMSO to the reaction
The PAP1 colorimetric assay was linear with respect to
mixture resulted in a dose-dependent inhibition of PAP1
time and enzyme concentration (B), indicat-
activity (C). A 1% concentration of DMSO is com-
ing that the enzyme followed zero order kinetics under
monly used for screens of water-insoluble inhibitors, and
these reaction conditions. In addition, PAP1 activity was
only 25% of PAP1 activity was lost using that concentra-
linear with respect to the DiC8 PA substrate at concentra-
tion (C). Thus, a signiﬁcant amount of PAP1 activity
tions of 0.05–0.8 mM Indeed, the analysis of
would still be present in a control reaction when potential
potential inhibitors would be best carried out at a low
inhibitors were solubilized in 1% DMSO.
substrate concentration at or below (e.g., <1 mM) the Km
Detergents (e.g., Triton X-100 and Tween 20) that were
used to solubilize water-insoluble DiC18 PA caused a
For comparison, PAP1 activity was measured by follow-
high background color. This problem was solved by using
ing the release of 32Pi from chloroform-soluble [32P]DiC18
water-soluble DiC8 PA as substrate. That pure PAP1 was a
PA (10,000 cpm/nmol) as described by Carman and Lin .
requirement for the colorimetric assay might be considered
In this assay, 0.2 mM [32P]DiC18PA was solubilized with
a major limitation. However, this limitation is also a major
2 mM Triton X-100 to give a surface concentration of
beneﬁt because the screen for inhibitors (or activators)
9 mol % . The speciﬁc activity (5.6 ± 0.6 lmol/min/mg)
should be carried out under well-deﬁned conditions that
of the pure PAP1 enzyme determined with the colorimetric
are free from other reactions that might generate ortho-
assay was in good agreement with the speciﬁc activity
phosphate and interfere with the interpretation of results.
(5.2 ± 0.1 lmol/min/mg) determined with the radioactive
Obtaining large quantities of pure PAP1 enzyme is facili-tated by the overexpression and puriﬁcation of yeast
and human proteins . As advertised by commercial ven-
The malachite green–molybdate reagent (PiBlue) commercially pre-
dors of the malachite green–molybdate reagent, the PAP1
pared by BioAssay Systems worked equally as well as the reagent preparedin the laboratory.
colorimetric assay was applicability to a 96-well format
Notes & Tips / Anal. Biochem. 373 (2008) 392–394
Fig. 2. Eﬀects of phenylglyoxal, propranolol, and DMSO on PAP1 activity measured with the colorimetric assay. PAP1 activity was measured understandard conditions in the presence of the indicated concentrations of phenylglyoxal (A), propranolol (B), and DMSO (C). The data shown were derivedfrom triplicate determinations ± SD.
(data not shown), which should facilitate a large-scale
 G.M. Carman, Y.-P. Lin, Phosphatidate phosphatase from yeast,
screen of PAP1 inhibitors (or activators).
Methods Enzymol. 197 (1991) 548–553.
 A. Martin, A. Gomez-Munoz, Z. Jamal, D.N. Brindley, Character-
ization and assay of phosphatidate phosphatase, Methods Enzymol.
 K. Itaya, M. Ui, A new micromethod for the colorimetric
This work was supported in part by United States Public
determination of inorganic phosphate, Clin. Chim. Acta 14 (1966)
Health Service, National Institutes of Health Grant GM-
 P.P. van Veldhoven, G.P. Mannaerts, Inorganic and organic phos-
28140 (to G.M.C.) and by a Wellcome Trust Career Devel-
phate measurements in the nanomolar range, Anal. Biochem 161
opment Fellowship in Basic Biomedical Science (to S.S.).
We thank Gil-Soo Han for helpful discussions during the
 L. O’Hara, G.S. Han, S. Peak-Chew, N. Grimsey, G.M. Carman, S.
Siniossoglou, Control of Phospholipid Synthesis by Phosphorylationof the Yeast Lipin Pah1p/Smp2p Mg2+ dependent PhosphatidatePhosphatase, J Biol. Chem. 281 (2006) 34537–34548.
 J.D. Mahuren, S.P. Coburn, A. Slominski, J. Wortsman, Micro-
assay of phosphate provides a general method for measuring the
 S.W. Smith, S.B. Weiss, E.P. Kennedy, The enzymatic dephospho-
activity of phosphatases using physiological, nonchromogenic
rylation of phosphatidic acids, J. Biol. Chem. 228 (1957) 915–922.
substrates such as lysophosphatidic acid, Anal. Biochem 298
 D.N. Brindley, Intracellular translocation of phosphatidate phospho-
hydrolase and its possible role in the control of glycerolipid synthesis,
 G.M. Carman, R.A. Deems, E.A. Dennis, Lipid signaling enzymes
Prog. Lipid Res. 23 (1984) 115–133.
and surface dilution kinetics, J. Biol. Chem. 270 (1995) 18711–
 G.M. Carman, Phosphatidate phosphatases and diacylglycerol pyro-
phosphate phosphatases in Saccharomyces cerevisiae and Escherichia
 Z. Jamal, A. Martin, A. Gomez-Munoz, D.N. Brindley, Plasma
coli, Biochim. Biophys. Acta 1348 (1997) 45–55.
membrane fractions from rat liver contain a phosphatidate phospho-
 M.G. Kocsis, R.J. Weselake, Phosphatidate phosphatases of mam-
hydrolase distinct from that in the endoplasmic reticulum and cytosol,
mals, yeast, and higher plants, Lipids 31 (1996) 785–802.
J. Biol. Chem. 266 (1991) 2988–2996.
 M. Nanjundan, F. Possmayer, Pulmonary phosphatidic acid phos-
 K.R. Morlock, J.J. McLaughlin, Y.-P. Lin, G.M. Carman,
phatase and lipid phosphate phosphohydrolase, Am. J Physiol Lung
Phosphatidate phosphatase from Saccharomyces cerevisiae. Isola-
Cell Mol. Physiol 284 (2003) L1–L23.
tion of 45-kDa and 104-kDa forms of the enzyme that are
 G.-S. Han, W.-I. Wu, G.M. Carman, The Saccharomyces cerevisiae
diﬀerentially regulated by inositol, J. Biol. Chem. 266 (1991) 3586–
lipin homolog is a Mg2+-dependent phosphatidate phosphatase
enzyme, J Biol. Chem. 281 (2006) 9210–9218.
 R.M.C. Dawson, D.C. Elliott, W.H. Elliott, K.M. Jones, Biochemical
 M. Peterfy, J. Phan, P. Xu, K. Reue, Lipodystrophy in the ﬂd mouse
Reagents. B. Reagents for Protein Modiﬁcation, Oxford, 1986 (pp.
results from mutation of a new gene encoding a nuclear protein, lipin,
 A.A. Abdel-Latif, J.P. Smith, Studies on the eﬀects of Mg2+ ion and
 J. Phan, K. Reue, Lipin, a lipodystrophy and obesity gene, Cell
propranolol on iris muscle phosphatidate phosphatase, Can. J.
Biochem. Cell. Biol. 62 (1984) 170–177.
Fluid Complications By Frederic W. Grannis, Jr., MD , Lily Lai, MD1 Section of Thoracic Surgery, City of Hope National Medical Center Division of General and Oncologic Surgery, City of Hope National Medical Center MALIGNANT PLEURAL EFFUSION CONTENTS Malignant pleural effusion complicates the care of approximately 150,000 Malignant Pleural people in the United States each year
Nuc .brief & link 2006-02-17 UT 04:30 NFCC Nuclear Fuel Cycle Consulting Exelon Nuclear is launching an initiative across its 10-station nuclear fleet to systematically assess systems that handle tritium and take the necessary actions to minimize the risk of inadvertent discharge of tritium to the environment. The assessments will take place in 2006 and will cover pipes, pumps,