ABC  Vol.2 No.4 , November 2012
Purification, characterization, and bioinformatics studies of phosphatidic acid phosphohydrolase from Lagenaria siceraria
ABSTRACT
Phosphatidic acid phosphohydrolase (PAP), EC 3.1.3.4, is the penultimate step in the Kennedy pathway of triacyl glycerol (TAG) synthesis leading to the formation of diacylglycerol (DAG), which is a key intermediate in TAG synthesis. We partially purified a soluble PAP from mid maturing seeds of bottle gourd, Lagenaria siceraria. The steps include both anionic and cationic ion exchanger columns. Catalytic characterization of the partially purified PAP revealed that the optimum pH and temperature for activity were at 5.5?C and 45?C. Under optimum assay condition using dioleoyl phosphatidic acid (DPA) as the substrate, the Vmax and Km were 0.36 ηkat/mg of protein and 200 μM, respectively. For the synthetic substrate, ρ-nitrophenylphosphate, ρ-NPP, the Vmax and Km were 33.0 nkat/mg of protein and 140 μM, respectively. The activity was neither inhibited nor enhanced by the presence of Mg2+ at a concentration range of 0 to 10 mM. Two major protein bands at 42-kDa and 27-kDa were visible in SDS-PAGE after partial purification. Bioinformatics analysis of tryp-sinized protein fractions containing PAP activity showed peptide sequences with sequence homology to various phosphate metabolizing enzymes including cucumber and castor bean purple acid phosphatase, polyphosphate kinase, fructose biphosphate aldolase, and enolase from various dicotyledonous plants including rice, corn, grape, and Arabidopsis lyrata.

Cite this paper
Ullah, A. , Sethumadhavan, K. , Grimm, C. and Shockey, J. (2012) Purification, characterization, and bioinformatics studies of phosphatidic acid phosphohydrolase from Lagenaria siceraria. Advances in Biological Chemistry, 2, 403-410. doi: 10.4236/abc.2012.24050.
References
[1]   Smith, S.W., Weiss, S.B. and Kennedy, E.P. (1957) The enzymatic dephosphorylation of phosphatidic acid. Journal of Biological Chemistry, 228, 915-922.

[2]   Kennedy, E.P. and Weiss, S.B. (1956) The function of cytidine coenzyme in the biosynthesis of phospholipids. Journal of Biological Chemistry, 222, 193-214.

[3]   Kocsis, M.G. and Weselake, R.J. (1996) Phosphatidate phosphatases of mammals, yeast, and higher plants. Lipids, 31, 785-802. Hdoi:10.1007/BF02522974

[4]   Peterfy, M., Phan, J, Xu, P. and Reue, K. (2001) Lipo-dystrophy in the fld mouse results from mutation of a new gene encoding a nuclear protein, lipin. Nature Genetics, 27, 121-124. Hdoi:10.1038/83685H.

[5]   Phan, J. and Reue, K. (2005) Lipin, a lypodystrophy and obesity gene. Cell Metabolism, 1, 73-83. Hdoi:10.1016/j.cmet.2004.12.002

[6]   Han, G.S., Wu, W.I. and Carman, G.M. (2006) The Sac- charomyces cerevisiae lipin homolog is a Mg2+-dependent phosphatidate phosphatase enzyme. The Journal of Biological Chemistry, 281, 9210-9218. Hdoi:10.1074/jbc.M600425200

[7]   Kennedy, E.P. (1961) Biosynthesis of complex lipids. Federation Proceedings, 20, 934-940.

[8]   Stymne, S. and Stobart, A.K. (1987) Triacylglycerol biosynthesis. In: Stumpf, P.K. and Conn, E.E., Eds., The Biochemistry of Plants, Academic Press, Orlando, 175- 214.

[9]   Loukou, A.L., Lognay, G., Barthelemy, J.P., Maesen, P., Baudoin, J.P. and Zoro, B.I. (2011) Effect of harvest time on seed oil and protein contents and compositions in the oleaginous gourd Lagenaria siceraria (Molina) Standl. Journal of the Science of Food and Agriculture, 91, 2073- 2080. Hdoi:10.1002/jsfa.4422

[10]   Akintayo, C.O., Akintayo, E.T., Akinsola, A. and Ziegler, T. (2009) Matrix-Assisted Laser Desorption Ionization time of flight mass spectrometric analysis of some curcurbita oils for triacylglycerol composition. Rivista Italiana Delle Sostanze Grasse, 86, 237-241.

[11]   Heinonen, J.K. and Lahti, R.J. (1981) A new and convenient calorimetric determination of inorganic orthophos- phate and its application to the assay of inorganic pyrophosphates. Analytical Biochemistry, 113, 313-317. Hdoi:10.1016/0003-2697(81)90082-8

[12]   Ullah, A.H.J. and Cummins, B.J. (1987) Purification, N- terminal amino acid sequence and characterization of pH 2.5 optimum acid phosphatase (E.C. 3.1.3.2) from Asper- gillus ficuum. Preparative Biochemistry, 17, 397-422. Hdoi:10.1080/00327488708062504

[13]   Akhtar, M.S., Iqbal, Z., Khan, M.N. and Lateef, M. (2000) Anthelmintic activity of medicinal plants with particular reference to their use in animals in the Indo-Pakistan subcontinent. Small Ruminant Research, 38, 99-107. Hdoi:10.1016/S0921-4488(00)00163-2

[14]   Badifu, G. (1991) Chemical and physical analyses of oils from 4 species of cucurbitaceae. Journal of the American Oil Chemists Society, 68, 428-432. Hdoi:10.1007/BF02663761

[15]   Tsieri, M.M., Niamayoua, R.K., Mampouya, D., Silou, T., Trémolières, A., Héron, S. and Tchapla, A. (2008) Comparative study of fatty acids and triglycerids of Luffa cylindrica versus cucurbitaceae seeds consumed in Congo Brazzaville. Pakistan Journal of Nutrition, 7, 733-740.

[16]   Pearce, M.L. and Slabas, A.R. (1998) Phosphatidate phosphatase from avocado (Persea americana)—Purification, substrate specificity and possible metabolic implications for the Kennedy pathway and cell signaling in plants. The Plant Journal, 14, 555-564. Hdoi:10.1046/j.1365-313X.1998.00152.x

[17]   Faulkner, A.J., Chen, X., Rush, J., Horazdovsky, B., Waechter, C.J., Carman, G.M. and Sternweis, P.C. (1999) The LPP1 and DPP1 gene products account for most of the isoprenoid phosphate phosphatase activities in Saccharomyces cerevisiae. The Journal of Biological Chemistry, 274, 14831-14837. Hdoi:10.1074/jbc.274.21.14831

[18]   Eastmond, P.J., Quettier, A.L., Kroon, J.T., Craddock, C., Adams, N. and Slabas, A.R. (2010) Phosphatidic acid phosphohydrolase 1 and 2 regulate phospholipid synthesis at the endoplasmic reticulum in Arabidopsis. Plant Cell, 22, 2796-2811. Hdoi:10.1105/tpc.109.071423

[19]   Nakamura, Y., Koizumi, R., Shui, G., Shimojima, M., Wenk, M.R., Ito, T. and Ohta, H. (2009) Arabidopsis lipins mediate eukaryotic pathway of lipid metabolism and cope critically with phosphate starvation. Proceedings of the National Academy of Sciences of the United States of America, 106, 20978-20983. Hdoi:10.1073/pnas.0907173106

[20]   Mietkiewska, E., Siloto, R.M.P., Dewald. J., Shah, S., Brindley, D.N. and Weselake, R.J. (2011) Lipins from plants are phosphatidate phosphatases that restore lipid synthesis in a pah1D mutant strain of Saccharomyces cerevisiae. FEBS Journal, 278, 764-775. Hdoi:10.1111/j.1742-4658.2010.07995.x

[21]   Pierrugues, O., Brutesco, C., Oshiro, J., Gouy, M., Deveaux, Y., Carman, G.M., Thuriaux, P. and Kazmaier, M. (2001) Lipid phosphate phosphatases in Arabidopsis. Regulation of the AtLPP1 gene in response to stress. The Journal of Biological Chemistry, 276, 20300-20308. Hdoi:10.1074/jbc.M009726200

[22]   Katagiri, T., Ishiyama, K., Kato, T., Tabata, S., Kobayashi, M. and Shinozaki, K. (2005) An important role of phosphatidic acid in ABA signaling during germination in Arabidopsis thaliana. Plant Journal, 43, 107-117. Hdoi:10.1111/j.1365-313X.2005.02431.x

[23]   Costa Fran?a, M.G., Matos, A.R., Darcy-Lameta, A., Passaquet, C., Lichtlé, C., Zuily-Fodil, Y. and Pham-Thi, A.T. (2008) Cloning and characterization of drought-stimulated phosphatidic acid phosphatase genes from Vigna unguiculata. Plant Physiology and Biochemistry, 46, 1093-1100. Hdoi:10.1016/j.plaphy.2008.07.004

[24]   Santos-Rosa, H., Leung, J., Grimsey, N., Peak-Chew, S. and Siniossoglou, S. (2005) The yeast lipin Smp2 couples phospholipid biosynthesis to nuclear membrane growth. EMBO Journal, 24, 1931-1941. Hdoi:10.1038/sj.emboj.7600672

[25]   Finck, B.N., Gropler, M.C., Chen, Z., Leone, T.C., Croce, M.A., Harris, T.E., Lawrence Jr., J.C. and Kelly, D.P. (2006) Lipin 1 is an inducible amplifier of the hepatic PGC-1α/PPARα regulatory pathway. Cell Metabolism, 4, 199-210.Hdoi:10.1016/j.cmet.2006.08.005

[26]   Han, G.S., Siniossoglou, S. and Carman, G.M. (2007) The cellular functions of the yeast lipin homolog PAH1p are dependent on its phosphatidate phosphatase activity. The Journal of Biological Chemistry, 282, 37026-37035. Hdoi:10.1074/jbc.M705777200

[27]   Yang, P., Li, X., Shipp, M.J., Shockey, J.M. and Cahoon, E.B. (2010) Mining the bitter melon (Momordica charantia I.) seed transcriptome by 454 analysis of non-normalized and normalized cDNA populations for conjugated fatty acid metabolism-related genes. BMC Plant Biology, 10, 250. Hdoi:10.1186/1471-2229-10-250

 
 
Top