| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
|
First published online February 24, 2002; 10.1104/pp.010654 Plant Physiol, March 2002, Vol. 128, pp. 988-996 Isolation of Lysophosphatidic Acid Phosphatase from Developing Peanut Cotyledons1Department of Biochemistry, Indian Institute of Science, Bangalore 560012, India
The soluble fraction of immature peanut (Arachis
hypogaea) was capable of dephosphorylating
[3H]lysophosphatidic acid (LPA) to generate
monoacylglycerol (MAG). The enzyme responsible for the generation of
MAG, LPA phosphatase, has been identified in plants and purified by
successive chromatography separations on octyl-Sepharose, Blue
Sepharose, Superdex-75, and heparin-agarose to apparent homogeneity
from developing peanuts. This enzyme was purified 5,048-fold to a final
specific activity of 858 nmol min
Biosynthesis of lysophosphatidic
acid (LPA) occurs by the acylation of glycerol-3-phosphate (G3P) by G3P
acyltransferase, which is present in both the soluble and membrane
fractions (Bell and Coleman, 1980 There are three ways of metabolism of LPA. LPA is (a) metabolized by
LPA phosphatase to form MAG, (b) acylated to PA by LPA acyltransferase,
and (c) hydrolyzed by LPA phospholipase to G3P. LPA phosphatase has
been identified, purified from bovine brain, and cloned from animal
systems (Xie and Low, 1994
Examination of Different Tissues for LPA Phosphatase Activity Hydrolysis of [32P]LPA was studied in the soluble and the particulate fractions from the tissues of leaf and immature seeds of peanut (Arachis hypogaea) and castor (Ricinus communis; Table I). LPA phosphatase was found mostly in the soluble fraction in all of the plant tissues studied. The immature seed soluble fraction showed a 144- to 198-fold higher total activity than the corresponding leaf tissues. These results suggest the presence of LPA phosphatase in plant tissues. Among the plant tissues examined, peanut immature seed showed the highest specific activity. Immature peanut cotyledons were used for further studies.
Formation of MAGs, Diacylglycerols (DAGs), and TAGs from [3H]LPA and [14C]PA TAG biosynthetic activity was higher in 150,000g pellet than in the supernatant, whereas no significant formation of MAG was observed from PA. Incubation of the soluble fraction with [3H]LPA resulted in the formation of MAGs, DAGs, and TAGs (Table II) and the incorporation was linear with time (Fig. 1A) and protein amounts (Fig. 1C). MAG formation from LPA was about 10-fold higher in the soluble fraction as compared with the membranes. The incorporation of [3H]LPA into MAGs, DAGs, and TAGs in the membrane fraction was also linear with time (Fig. 1B) and protein amounts (Fig. 1D). When the soluble fraction was incubated with [14C]PA, the amount of label in DAG and TAG was less compared with the membranes. These results on the incorporation of [3H]LPA and [14C]PA revealed that only LPA is involved in generation of MAG. When the soluble fraction was incubated with either [3H]trioleoyl-sn-glycerol or [3H]phosphatidylcholine, there was no formation of labeled MAG ruling out the hydrolysis and transacylation of fatty acid (data not shown).
Generation of MAG MAG could be formed either by the direct dephosphorylation of LPA
or by the deacylation followed by dephosphorylation of PA. To find out
the route by which MAG was synthesized, the soluble fraction was
treated with various concentrations of NaF to inhibit phosphatase
activity and the formation of MAG from [3H]LPA
was measured. As shown in Figure 2A,
there was about a 33% reduction in the formation of MAG in the
presence of 20 mM NaF, but lower concentrations of NaF
altered the formation of MAG to a lesser extent (17%) suggesting the
presence of NaF-insensitive phosphatase in the soluble fraction. On the
contrary, the membrane-bound PA phosphatase was sensitive to NaF
(Tumaney et al., 2001
Purification of LPA Phosphatase LPA phosphatase activity was found to be high in the soluble fraction and attempts were made to purify the LPA phosphatase using [3H]LPA as a substrate. Solid ammonium sulfate was added to the supernatant obtained after ultracentrifugation to bring to 1 M and then loaded onto an octyl-Sepharose column. After application to the matrix, the column was washed with buffer A containing 1 M ammonium sulfate, and the enzyme was eluted in the absence of salt. Fractions were collected, and the A280 and the LPA phosphatase activity were measured (Fig. 3A). This step was effective and resulted in a 33-fold purification (Table III). The active fractions from the octyl-Sepharose column were loaded onto a Blue Sepharose column. The enzyme did not bind to the matrix, but this step gave a 3.5-fold purification with 90% recovery of activity. The active unbound fraction from the Blue Sepharose was concentrated, the salt concentration was adjusted to 250 mM KCl and applied onto a preparative Superdex 75 FPLC column. The analysis of column fractions revealed that the most of the LPA phosphatase activity eluted between 31 and 33 fractions (Fig. 3B). The active fractions were pooled and dialyzed against buffer A. The dialyzed sample was applied onto a heparin-agarose column as the final step and the enzyme eluted with 0.5 M NaCl.
The overall purification of LPA phosphatase is summarized in Table III.
The enzyme was purified to 5,048-fold with a 16% yield and the
specific activity was 0.86 µmol MAG formed
min
To confirm the 39 kD is LPA phosphatase, the final preparation was subjected to 12% (w/v) denaturing PAGE without boiling the sample. The gel was cut into 0.5-cm segments, and the protein was eluted from each slice. The formation of MAG from [3H]LPA was measured, and it was found that the activity associated with the area of the gel corresponding to the 39-kD polypeptide (data not shown). The recovery of LPA phosphatase activity from SDS-PAGE was 9% ± 2% of that applied. These results demonstrated that the purified protein is indeed LPA phosphatase. Characteristics of Peanut LPA Phosphatase LPA phosphatase activity was linear with time and protein, and the pH optimum of the enzyme was found to be 7.0. To investigate the substrate specificity of LPA phosphatase, phosphate-containing compounds such as PA, G3P, and p-nitrophenyl phosphate were used. The purified enzyme hydrolyzed LPA specifically and did not hydrolyze other substrates (data not shown). Substrate Specificity of LPA Phosphatase The rate of reaction increased linearly with increasing concentration of LPA (oleoyl) and reached the maximum at 50 µM. The initial rate data showed that the enzyme followed Michaelis-Menten kinetics. The specificity of LPA phosphatase was studied by providing LPA with varying chain lengths and unsaturation as substrates. The LPA phosphatase activity was the highest for oleoyl-LPA and relatively lower for palmitoyl-LPA. Based on the Lineweaver-Burk plot, apparent Vmax and Km values for LPA were obtained under the standard assay conditions (Table IV). The apparent Km values for LPA (oleoyl), LPA (stearoyl), and LPA (palmitoyl) were 28.6, 39.3, and 47.9 µM, respectively (Fig. 5). The saturated fatty acid containing LPA had lower Vmax and higher Km values as compared with oleoyl containing LPA.
Effect of Detergents and Cations on LPA Phosphatase Activity The influence of various detergents on the LPA phosphatase activity was studied (Fig. 6A). In presence of 1 mM Triton X-100, there was a 2-fold increase in the enzyme activity. At 10 mM concentration of Triton X-100 or octylglucoside the enzyme showed a 2- to 3-fold increase in activity. At the same concentration, deoxycholate and 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonic acid (CHAPS) showed a 50% inhibition of the enzyme activity. At a 12 mM concentration of CHAPS, the enzyme lost its activity completely. The effects of cations on the enzyme activity were determined in the presence of 0.1% (w/v) Triton X-100. At 8 mM concentration, Ca2+, Mn2+, and Zn2+ inhibited 40% to 50% of the LPA phosphatase activity, whereas no significant effect was observed with Mg2+ (Fig. 6B).
Effect of Fatty Acids, Phospholipids, and Sphingoid Bases on LPA Phosphatase Activity The effects of various lipids on the enzyme activity were determined in the presence of 0.1% (w/v) Triton X-100 (Fig. 7A). Myristic, palmitic, stearic, and oleic acids stimulated the LPA phosphatase activity, whereas dodecanoic acid had no effect. Among the fatty acids examined, palmitic acid showed a 2-fold stimulation at 5 µM, whereas myristic acid showed a 50% increase; and at the same concentration, oleic and stearic acids showed only a 25% increase in enzyme activity. However, the stimulation was not concentration dependent. At higher concentrations of stearic acid (100 µM), the activity was reduced to almost one-half of the original value. Phosphatidylinositol had a 40% stimulating effect at 10 µM on the purified LPA phosphatase activity. Phosphatidylcholine, phosphatidylethanolamine, and PA had no significant effect on the enzyme activity (Fig. 7B). Sphingomylein and lower concentrations (2.5-5 µM) of sphingosine activated the LPA phosphatase activity, whereas concentrations above 10 µM of sphingosine showed an inhibitory effect on the enzyme activity (Fig. 7C). The derivatives of sphingosine, dehydrosphingosine, and dihydrosphingosine showed no significant effect on the enzyme activity.
The biosynthesis of DAG is known to occur by the sequential
acylation of G3P followed by dephosphorylation of PA in the microsomal membranes (Kennedy, 1961 This study also provides evidence for the soluble nature of LPA
phosphatase in plants. First, the activity is associated with 150,000g supernatant. Second, the LPA phosphatase is
purified by successive column chromatographic separations without
detergent from the soluble fraction. It was reported earlier that both
in animal (Bell and Coleman, 1980 The kinetic experiments revealed that the overall catalytic efficiency
for oleoyl-LPA was 2-fold higher than that of saturated fatty acid
containing LPA suggesting that the oleoyl-LPA was the preferred
substrate (Table IV). The characterization of the purified bovine LPA
phosphatase showed a complete inhibition of activity with 2.5 mM NaF (Hiroyama and Takenawa, 1998 The generation of MAG by the LPA phosphatase may serve as a precursor
for TAG biosynthesis in plants. The synthesized MAG is acylated by a
soluble MAG acyltransferase (Tumaney et al., 2001
Materials [9,10-3H(N)]Trioleoylglycerol
(10 Ci mmol Preparation of the Soluble and the Membrane Fractions Frozen immature seeds (100 g) were ground in a prechilled mortar
and pestle with 10 g of acid-washed sand and 250 mL of extraction buffer containing 50 mM Tris-HCl (pH 8.0), 1 mM
EDTA, 10 mM KCl, 1 mM MgCl2, 1 mM Incorporation of [3H]LPA and [14C]PA into MAGs, DAGs, and TAGs In the labeling experiments, 25 to 125 mg of freshly prepared
soluble and membrane fractions were used in 0.1 mL of buffer containing
50 mM Tris-HCl (pH 7.0), 10 mM KCl, 1 mM MgCl2, 1 mM Enzyme Assays Lipid was suspended in 1% (w/v) Triton X-100 and sonicated for 5 min in the sonic bath. The substrate stock solution was diluted 10-fold in the assay. LPA phosphatase assay mixture consisted of 50 mM Tris-HCl (pH 7.0), 50 µM [3H]LPA (220,000 dpm) and 20 to 45 µg enzyme in a total volume of 100 µL. The incubation was carried out at 30°C for indicated time points and stopped by the addition of 50 µL of 6 N HCl followed by 400 µL of chloroform:methanol (1:2, v/v). After lipid extraction, the lower chloroform-soluble materials were separated by thin-layer chromatography on 250-µm silica gel G plates using chloroform:methanol:water (98:2:0.5, v/v) as the solvent system. The lipids were visualized with iodine vapor and spots of MAG were scraped off for determination of radioactivity by liquid scintillation counting. LPA phosphatase was also assayed using [32P]LPA (50 µM; 180,000-270,000 cpm). The assay components and conditions were same as described above, and the amount of [32P]Pi released was measured in the upper phase after lipid extraction. Boiled soluble and membrane fractions were used as a control, and control values were subtracted from the values measured in the presence of active enzyme sources. Purification of LPA Phosphatase All the operations were carried out at 4°C except FPLC purification step that was carried out at ambient temperature. Octyl-Sepharose Chromatography Solid ammonium sulfate was added to bring the developing peanut
cotyledon soluble fraction to 1 M followed by
centrifugation. The clear supernatant was loaded onto an
octyl-Sepharose column (4.4 × 15 cm) that had been
pre-equilibrated with 1 M ammonium sulfate in buffer A (50 mM Tris-HCl [pH 8.0], 1 mM EDTA, 100 mM KCl, 1 mM MgCl2, 1 mM Blue Sepharose Chromatography Active fractions from the octyl-Sepharose were combined and applied onto a Blue Sepharose column (4.4 × 12 cm). The LPA phosphatase did not bind to the column. Size-Exclusion Chromatography The unbound fractions from the Blue Sepharose were concentrated
and filtered. The filtrate was applied onto a preparative Superdex 75 FPLC column (HR 26/60) fitted with the BioLogic low-pressure chromatography system (Bio-Rad Hercules, CA). The column was eluted with the same buffer at a flow rate of 5 mL min Heparin-Agarose Chromatography A 2.5-mL column matrix was preequilibrated with buffer A at room temperature, and the active fractions from the size exclusion column were mixed with matrix for 1 h at 4°C. The mixture was then poured into a column, washed with buffer A, and eluted with buffer A containing 0.25, 0.5, and 1 M NaCl. The LPA phosphatase activity was eluted with 0.5 M NaCl. Molecular Mass Determination by Gel Filtration The purified preparation was loaded onto a FPLC HR 10/30 Superdex 75 gel filtration column equilibrated on buffer A containing 0.25 M KCl. The column was calibrated with blue dextran 2000, albumin, ovalbumin, chymotrypsinogen A, and ribonuclease A. The column was eluted with 33 mL of the same buffer, and 0.5-mL fractions were collected. LPA phosphatase activity was assayed, and active fractions 16 to 18 were pooled and dialyzed against buffer A. Reconstitution of LPA Phosphatase Activity The purified preparation was resolved on a 12% (w/v) SDS
polyacrylamide gel (10 × 10 cm) in the presence of
0.1% (w/v) SDS without boiling the sample, and the gel was
resolved under constant current (100 V) at 4°C. After the run, the
resolving gel (7 cm) was progressively cut into 0.5-cm slices, the
protein was eluted from each segment by finely crushing the gel pieces
in 10 mM Tris-HCl (pH 7.0), 1 mM EDTA, 100 mM KCl, 1 mM MgCl2, 1 mM
We thank Dr. P.N. Rangarajan (Indian Institute of Science) for his helpful discussion and support. We are grateful to Dr. D.L. Savithramma (University of Agricultural Sciences, Bangalore, India) for providing us the immature peanut and castor seeds.
Received July 23, 2001; returned for revision September 17, 2001; accepted November 19, 2001. 1 This work was supported by the seed grant from the Indian Institute of Science, Bangalore, India.
2 These authors contributed equally to the paper.
* Corresponding author; e-mail lipid{at}biochem.iisc.ernet.in; fax 91-80-3602627.
Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.010654.
This article has been cited by other articles:
|
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| ASPB Publications | PLANT PHYSIOLOGY | THE PLANT CELL | |
|---|---|---|---|
TOP
ABSTRACT
INTRODUCTION
1 mg
) and the protein (
). B, Unbound
active fractions from Blue Sepharose were pooled, concentrated, and
loaded onto a preparative gel filtration column (Superdex 75).
Fractions (5 mL) were collected and assayed for the LPA phosphatase
activity (
-32P]ATP (3,000 Ci
mmol
80°C until further use. Castor (Ricinus
communis) seedpods were harvested from field grown castor plants at 35 to 44 d after flowering. Endosperm was collected and
used either fresh or stored at 
-mercaptoethanol, 0.1 mM
phenylmethylsulfonyl fluoride, 1 µg mL
