Evidence for Histidine Residues on Plasma Membrane Phosphatidate Phosphohydrolase from Rat Liver

Document Type: Original Article

Authors

1 Department of Biochemistry, Medical School, Ilam University of Medical Sciences, Ilam, Iran.

2 Department of Biochemistry, Isfahan University of Medical Sciences, Isfahan, Iran.

Abstract

Objective(s)
Phosphatidate phosphohydrolase (PAP) catalyzes the dephosphorylation of phosphatidic acid to yield Pi and  diacylglycerol. Two different forms of PAP in rat hepatocyte have been reported. PAP1 is located in cytosolic and microsomal fractions and participates in the synthesis of triacylglycerols, phosphatidylcholine, and phosphatidylethanolamine, whereas the other form of phosphatidate phosphohydrolase (PAP2) is primarily involved in lipid signaling pathways. In rat liver,PAP2 has two isoforms; one PAP2a and another PAP2b. In this study, essentialhistidine residues were investigatedin native form of rat purified PAP2b withdiethylpyrocarbonate.
Materials and Methods
PAP2b purified from rat liver plasma membrane by solubilizing with n-octyle glucoside and several chromatography steps. Gel electrophoresis (SDS-PAGE) performed on purified enzyme in order to evaluate its purity and to measure the molecular weight of the enzyme subunit. The enzyme inactivated with diethylpyrocarbonate (DEPC) and the number of moles of histidine residues modified per mol of enzyme determined.
Results
The specific activity of purified enzyme was 7350mU/mg protein and it showed only a single band on SDS-PAGE with a MW of about 33.8 kDa. The PAP2b inactivated by DEPC. The maximum 6 moles of histidine residues modified per mole of PAP2b, when about 90% of enzyme activity is lost with DEPC.
Conclusion
The data showed that the incubation of PAP2b by DEPC can inhibit enzyme activity.  Our findings also, revealed the presence of essential histidines in the structure of PAP2b which involve in its activity. This enzyme is likely to have a similar hydrolysis catalytic mechanism as its super family through a phosphohistidine intermediate.
 

Keywords


1. Carman GM, Han GS. Roles of phosphatidate phosphatase enzymes in lipid metabolism. Trends Biochem Sci 2006; 31:694-699.

2. Roberts R, Sciorra VA, Morris AJ. Human type 2 phosphatidic acid phosphohydrolases. J Biol Chem 1998; 273:22059-22067.

3.  Smith SW, Weiss SB, Kennedy EP. Enzymatic dephosphorylation of phosphatidic acid. J  Biol  Chem  1957; 228:915-922.

4. Kanoh H, Imai SI, Yamada K, Sakane F.Purification and properties of phosphatidic acid phosphatase from porcine thymus membrane. J Biol Chem 1992; 267:25309- 25314.

5. Fleming IN,Yeaman SJ. Purification and characterization of N-ethylmaleimide-insensitve phosphatidic acid phosphohydrolase (PAP2) from rat liver.  Biochem J 1995; 308:983-989.

6.  Petersen KF, Shulman GI. Etiology of insulin resistance.Am J Med2006; 119:S10-6.

7.  Reue K, Phan J.Metabolic consequences of lipodystrophy in mouse models. Curr Opin Clin Nutr Metab Care2006; 9:436–441.

8. Hegele RA, Pollex RL.Genetic and physiological insights into the metabolic syndrome. Am J Physiol Regul Integr Comp Physiol2005; 289:R663–R669.

9. Nanjundan M, Possmayer F. Pulmonary phosphatidic acid phosphatase and lipid phosphate phosphohydrolase. Am J Physiol Lung Cell Mol  Physiol  2003; 284:L1–L23.

10. Jamal Z, Martin A, Munoz AG, Brindley DN. Plasma membrane fractions from rat liver contain a phosphatidate phosphohydrolase distinct from that in the endoplasmic reticulum and cytosol. J Biol Chem 1991; 266: 2988-2996.

11. Dillon  AD, Chen  X, Zeimetz  GM, Wu WI, Waggoner DW, Dewald J, et al. Mammalian Mg2+ independent phosphatidate phosphatase (PAP2) displays diacylglycerol pyrophosphatase activity. J Biol Chem 1997; 272:10361-10366.

12. Brindley DN. Lipid phosphate phosphatases and related proteins: signaling functions in development, cell division, and cancer.J Cell Biochem 2004; 92:900–912.

13. Sciorra VA, Morris AJ. Roles for lipid phosphate phosphatases in regulation of cellular signaling. Biochim Biophys Acta2002; 1582:45-51.

14. Kanoh H, Kai M,Wada I. Molecular characterization of the type 2 phosphatidic acid phosphatase. Chem Phys Lipids  1999; 98:119-126.

15. Donkor J, Sariahmetoglu M, Dewald J, Brindley DN, Reue K. Three mammalian lipins act as phosphatidate phosphatases with distinct tissue expression patterns. J Biol Chem2007; 282:3450-3457.

16. Peterfy M, Phan J, Xu P, Reue K. Lipodystrophy in the fld mouse results from mutation of a new gene encoding a nuclear protein, lipin. Nat Genet 2001;27: 121-124.

17. Phan J, Reue K. Lipin, a lipodystrophy and obesity gene. Cell Metab 2005; 1: 73-83.

18. Peterfy M, Phan J, Reue K. Alternatively spliced lipin isoforms exhibit distinct expression pattern, subcellular localization, and role in adipogenesis. J Biol Chem 2005; 280:32883-32889.

19. Roberts R, Sciorra VA, Morris AJ. Human type 2 phosphatidic acid phosphohydrolase a substrate specificity of the type 2a, 2b, and 2c enzymes and cell surface activity of the 2a isoform. J Biol Chem 1998;273:22059-22067.

20. Brindley DN, Waggoner DW. Mammalian lipid phosphate phosphohydrolases. J  Biol  Chem  1998; 273:24281-24284.

21. Pyne S, Long JS, Ktistakis NT, Pyne NJ.Lipid phosphate phosphatases and lipid phosphate signaling. Biochem Soc Trans 2005; 3:1370-1374.

22. Toke DA, Bennett WL, Oshiro J, Wu WI, Voelker DR, Carman GM. Isolation and characterization of the Saccharomyces cerevisiae LPP1 gene encoding a Mg2+-independent phosphatidate phosphatase. J Biol Chem 1999; 273:14331-14338.

23.Carman GM, Wu WI. Lipid phosphate phosphatase from Saccharomyces cerevisiae. Methods Enzymol 2007; 434:305–315.

24. Carman GM, Han GS. Roles of phosphatidate phosphatase enzymes in lipid metabolism. Trends Biochem Sci 2006; 31:694-699.

25. Spiegel S, English D, Milstien S. Sphingosin 1-phosphate signaling providing cell with a sense of direction. Trends Cell Biol 2002; 12:236-242.

26.Ishikawa T, Kai M, Wada D, Kanoh H. Cell surface activities of the human type 2b phosphatidic acid phosphatase1.J Biochem2000; 127:645-651.

27. Haghighi B, Honarjou S. The effects of hydrazine on the phosphatidate phosphohydrolase activity in rat liver. Biochem  Pharmacol1987; 36:1163-1165.

28. Carman GM, Deems RA, Dennis EA.  Lipid signaling enzymes and surface dilution kinetics. J  Biol Chem 1995; 270:18711-18714.

29. Hirs CHW, Timasheff SN. Modification of histidyl residues in protein by diethylpyrocarbonate.  Methods Enzymol 1977; 47:431-441.

30. Haghighi B, Takhshid A. The evidence for essential histidine residue in soluble phosphatidate phosphohydrolase of rat liver. Iran J Sci Tech 1999; 23:109-116.

31. Bradford MMA. Rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein dye binding. Anal Biochem 1976; 72:248-254.

32. Thompson S, Maddy AH. Gel electrophoresis of erythrocyte membrane proteins in red cell membranes, a methodological approach. London, New York: Academic Press; 1982, p. 67-93.

33. Feuerstein N, Cooper HL. Rapid protein phosphorylation induced by phorbol ester in HL-60 cells. J  Biol Chem 1983; 258:10786-10793.

34. Heukeshoven J, Dernick R. Simplified method for silver staining of proteins in polyacrylamide gels and the mechanism of the silver staining.  Electrophoresis 1985; 6:103-112.

35. Siess EA, Hofstetter MM. Identification of phosphatidate phosphohydrolase purified from rat liver membrane on SDS-polyacrylamide gel electrophoresis.  FEBS Lett 1996; 381:169-173.

36. Kia M, Wada I, Imai SI, Ankane FS, Kanoh H.  Identification and cDNA cloning of 35 kDa phosphatidic acid phosphatase (type 2) bound to plasma membranes. J Biol Chem 1996; 271:18931-18938.

37. Hjelmeland LM, Chambach A. Solubilization of functional membrane proteins.Methods Enzymol 1984; 104:305-315.

38. Lundblad R L. Techniques in protein modification, Boca Raton: CRC Press; 1995:105-127.

39. Smith CI, Barber RD, Ferry JG. The role of histidine in the acetate kinase from methanosarcina thermophila. J Biol Chem 2000; 275:33765-33770. 

40. Cousineau J, Meighen E. Chemical modification of bacterial luciferase with ethoxyformic anhydride; Evidence for a histidyl residue. Biochemistry 1976; 15:4992-5000.

41. Burstein Y, Walsh KA, Neurath H. Evidence of an essential histidine residue in Thmolysine.Biochemistry1974; 13:205- 210.

42. Haghighi B, Heidarian E. Evidence for an essential lysine residue on cytosolic phosphatidate phosphohydrolase from rat liver. Iran J Soc Tech 2001; 25:1-8.