Doi:10.1016/j.ab.2007.08.037

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 3.1.3.4) 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.
2 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.
doi:10.1016/j.ab.2007.08.037 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.
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