The 45- and 104-kDa forms of phosphatidate phosphatase from Saccharomyces cerevisiae are regulated differentially by phosphorylation via cAMP-dependent protein kinase.

Evidence is presented that demonstrated that the 45- and 104-kDa forms of phosphatidate phosphatase from Saccharomyces cerevisiae (Morlock, K. R., McLaughlin, J. J., Lin, Y.-P., and Carman, G. M. (1991) J. Biol. Chem. 266, 3586-3593) were regulated differentially by phosphorylation. Purified 45-kDa phosphatidate phosphatase was phosphorylated by cAMP-dependent protein kinase whereas purified 104-kDa phosphatidate phosphatase was not phosphorylated. cAMP-dependent protein kinase catalyzed the phosphorylation of pure 45-kDa phosphatidate phosphatase at a serine residue which resulted in a stimulation (2.4-fold) of phosphatidate phosphatase activity. Alkaline phosphatase catalyzed the dephosphorylation of pure 45-kDa phosphatidate phosphatase which resulted in an inhibition (1.3-fold) of phosphatidate phosphatase activity. Results of studies using mutants (bcy1 and cyr1) defective in cAMP-dependent protein kinase activity corroborated the results of the phosphorylation studies using pure preparations of phosphatidate phosphatase. The 45-kDa phosphatidate phosphatase phosphorylated in vitro and in vivo had phosphopeptides in common. The activation of the GAL10-RAS2val19 allele in mutant cells resulted in an increase in the synthesis of diacylglycerols and triacylglycerols. These results were consistent with the phosphorylation and activation of 45-kDa phosphatidate phosphatase by cAMP-dependent protein kinase in vivo.     

Regulation of phosphatidate phosphatase activity by inositol in Saccharomyces cerevisiae.

Regulation of phosphatidate phosphatase (EC 3.1.34) activity was examined in Saccharomyces cerevisiae cells supplemented with phospholipid precursors. Addition of inositol to the growth medium of wild-type cells resulted in a twofold increase in phosphatidate phosphatase activity. The increase in phosphatidate phosphatase activity was not due to soluble effector molecules, and inositol did not have a direct effect on enzyme activity. The phosphatidate phosphatase activity associated with the mitochondrial, microsomal, and cytosolic fractions of the cell was regulated by inositol in the same manner. Cells supplemented with inositol had elevated phospholipid levels and reduced triacylglycerol levels compared with unsupplemented cells. Serine, ethanolamine, and choline did not significantly affect the phosphatidate phosphatase activity of cells grown in the absence or presence of inositol. Enzyme activity was not regulated in inositol biosynthesis regulatory mutants, suggesting that regulation by inositol is coupled to regulation of inositol biosynthesis. Phosphatidate phosphatase activity was pleiotropically expressed in structural gene mutants defective in phospholipid biosynthesis. These results suggested that phosphatidate phosphatase was regulated by inositol at a genetic level.     

Identification and isolation of a 75-kDa inositol polyphosphate-5-phosphatase from human platelets

We have identified, isolated, and characterized a second inositol polyphosphate-5-phosphatase enzyme from the soluble fraction of human platelets. The enzyme hydrolyzes inositol 1,4,5-trisphosphate (Ins (1,4,5)P3) to inositol 1,4-bisphosphate (Ins(1,4)P2) with an apparent Km of 24 microM and a Vmax of 25 mumol of Ins(1,4,5)P3 hydrolyzed/min/mg of protein. The enzyme hydrolyzes inositol (1,3,4,5)-tetrakisphosphate (Ins(1,3,4,5)P4) at a rate of 1.3 mumol of Ins(1,3,4,5)P4 hydrolyzed/min/mg of protein with an apparent Km of 7.5 microM. The enzyme also hydrolyzes inositol 1,2-cyclic 4,5-trisphosphate (cIns(1:2,4,5)P3) and Ins(4,5)P2. We purified this enzyme 2,200-fold from human platelets. The enzyme has a molecular mass of 75,000 as determined by both sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by gel filtration chromatography. The enzyme requires magnesium ions for activity and is not inhibited by calcium ions. The 75-kDa inositol polyphosphate-5-phosphatase enzyme differs from the previously identified platelet inositol polyphosphate-5-phosphatase as follows: molecular size (75 kDa versus 45 kDa), affinity for Ins(1,3,4,5)P4 (Km 7.5 microM versus 0.5 microM), Km for Ins(1,4,5)P3 (24 microM versus 7.5 microM), regulation by protein kinase C, wherein the 45-kDa enzyme is phosphorylated and activated while the 75-kDa enzyme is not. The 75-kDa enzyme is inhibited by lower concentrations of phosphate (IC50 2 mM versus 16 mM for the 45-kDa enzyme) and is less inhibited by Ins(1,4)P2 than is the 45-kDa enzyme. The levels of inositol phosphates that act in calcium signalling are likely to be regulated by the interplay of these two enzymes both found in the same cell.     

Myoinositol biosynthesis by Sertoli cells, and levels of myoinositol biosynthetic enzymes in testis and epididymis.

Levels of glucose-6-phosphate cyclase (myoinsitol-1-phosphate synthase, EC 5.5.1.4) and myoinositol-1-phosphate phosphatase (myoinositol-1-phosphatase, EC 3.1.3.25) were determined in extracts of testes from10-, 20-, and 30-day-old rats, and in extracts of Sertoli cells, germinal cells, and epididymides. The specific activity of the cyclase was approximately 1/10th that of the phosphatase in all extracts found to contain either enzyme. Among cells in the testis examined, Sertoli cells had highest levels of enzymes required for inositol biosynthesis from glucose, while spermatocytes and round spermatids did not have detectable activity. Spermatozoa from the epididymis also had no detectable cyclase or phosphatase activity. In contrast, extracts of washed epididymides contained exceedingly high specific activities of these enzymes. Primary cultures of Sertoli cells, maintained in a chemically defined medium without added inositol, released inositol into the medium during three successive 24-h periods. The amounts released were greater in cells stimulated by dibutyryl cyclic AMP. Results were interpreted to indicate that inositol in the fluid of seminiferous tubules most probably originates from Sertoli cells, which synthesize inositol from glucose. Additional inositol in the fluid of epididymal tubules could readily be provided by metabolism of glucose by epididymal epithelial cells     

Partial purification, properties, and subcellulsr distribution of rat liver phosphatidate phosphatase.

Phosphatidate phosphatase (EC 3.1.3.4Y was purified 15- to 20-fold from the soluble fraction of rat liver. The purification procedure involved calcium phosphate gel adsorption and elution, ammonium sulfact precipitation, and molecular-sieve chromatography. For the enzyme assay, and aqueous dispersion of phosphatidate, rather than "membrane-bound" phosphatidate, was used as substrate. The partially purified enzyme depends almost entirely on the presence of Mg2+ for its activity. Morover, the activity of the enzyme is stimulated by phosphatidylcholine. The enzyme exhibits a high substrate specificity for phosphatidate. The apparent Km for phosphatidate is approximately 0.05 mM. The optimum pH is between 7.4 and 7.6. The enzyme is inhibited by fluoride and by p-chloromercuribenzoate. The subcellular distribution of phosphatidate phosphatase in rat liver was studied by assaying the activity of the enzyme in the presence of Mg2+ and phosphatidylcholine. In contrast ot the results of previous studies, most of the enzyme activity was found in the soluble fraction.     

Vacuole membrane topography of the DPP1-encoded diacylglycerol pyrophosphate phosphatase catalytic site from Saccharomyces cerevisiae

The Saccharomyces cerevisiae DPP1-encoded diacylglycerol pyrophosphate phosphatase is a vacuole membrane-associated enzyme that catalyzes the removal of the beta-phosphate from diacylglycerol pyrophosphate to form phosphatidate, and it then removes the phosphate from phosphatidate to form diacylglycerol. The enzyme has six putative transmembrane domains and a hydrophilic region that contains a phosphatase motif required for its catalytic activity. In this work, we examined the topography of diacylglycerol-pyrophosphate phosphatase catalytic site within the transverse plane of the vacuole membrane. Results of protease protection analysis using endoproteinase Lys-C and labeling of cysteine residues using sulfhydryl reagents were consistent with a model where the catalytic site of diacylglycerol-pyrophosphate phosphatase was oriented to the cytosolic face of the vacuole membrane. In addition, diacylglycerol-pyrophosphate phosphatase activity was found with intact vacuoles. The phospholipids diacylglycerol pyrophosphate (0.6 mol %) and phosphatidate (1.4 mol %) were found in the vacuole membrane, and their levels decreased to an undetectable level and by 79%, respectively, when cells were depleted for zinc. The reduced levels of diacylglycerol pyrophosphate and phosphatidate correlated with the induced expression of diacylglycerol-pyrophosphate phosphatase. This work suggested that diacylglycerol pyrophosphate phosphatase functions to regulate the levels of diacylglycerol pyrophosphate and phosphatidate on the cytosolic face of the vacuole membrane.     

Differences in phosphatidate hydrolytic activity of human alkaline phosphatase isozymes

Hydrolytic activities of human alkaline phosphatase isozymes were investigated using phosphatidases with various fatty acyl chains (egg phosphatidate and dioleoyl, distearoyl, dipalmitoyl, dimyristoyl and dilauroyl phosphatidates). In the presence of sodium deoxycholate, purified human placental and intestinal alkaline phosphatases hydrolyzed all the phosphatidates examined. The hydrolytic activity was maximal in the presence of 10 g/l sodium deoxycholate. Of the phosphatidates, dilauroyl phosphatidate was the best substrate. Using the same unit of the enzyme, the phosphatidate hydrolytic activity of placental alkaline phosphatase was 2- to 3-times higher than that of the intestinal enzyme. In contrast, liver alkaline phosphatase did not hydrolyze phosphatidates with long fatty acyl chains (C16-18) even in the presence of sodium deoxycholate. The liver enzyme hydrolyzed dimyristoyl and dilauroyl phosphatidates very slowly. These results show that the phosphatidates with long fatty acyl chains were useful to differentiate placental and intestinal alkaline phosphatases from the liver enzyme, and suggest that the former enzymes play a different physiological role from the liver enzyme.     

Isolation of extracellular 28- and 42-kilodalton beta-1,3-glucanases and comparison of three beta-1,3-glucanases produced by Bacillus circulans IAM1165.

Bacillus circulans IAM1165 produces three major extracellular beta-1,3-glucanases (molecular masses, 28, 42, and 91 kDa) during the stationary phase of growth. The 28- and 42-kDa enzymes were purified to homogeneity from the culture supernatant in this study. The properties of these two enzymes were examined, together with those of the 91-kDa enzyme previously isolated. The enzymatic properties of the 28- and 42-kDa beta-1,3-glucanases closely resemble each other. The enzymes belong to a category of endo type 1,3-beta-D-glucan glucanohydrolases. The enzymes were active at pH 4.0 to 7.0. The optimum temperature of the reactions was 60 degrees C when laminarin (a soluble beta-1,3-glucan) was used as the substrate at pH 7.0. The enzymes hydrolyzed barley glucan and lichenan (beta-1,3-1,4-glucans) more effectively than laminarin. Of the three enzymes, the 42-kDa enzyme lysed fungal cell walls the most effectively.     

Characterization of multiple forms of cholesteryl ester hydrolase in the rat testis

Cholesterol ester hydrolase (EC 3.1.1.13) activity from the 104,000 X g supernatant of rat testis was fractionated into 28-kDa, 72-kDa, and 420-kDa molecular mass forms by high performance size exclusion chromatography. The 72-kDa and 420-kDa forms (temperature-labile) were completely inactivated by elevation of temperature from 32 to 37 degrees C. Apparent disaggregation of the 420-kDa form suggested that the 72-kDa and 420-kDa enzymes are monomeric and multimeric forms of the same enzyme. The 28-kDa form was shown to be a different enzyme (temperature-stable) which retained activity at 37 degrees C. In contrast, cholesteryl ester hydrolase activities from 104,000 X g supernatants of liver or adrenal gland were unaffected and increased 4-fold, respectively, by elevation of temperature from 32 to 37 degrees C. Both testicular enzymes exhibited pH optima at about 7.3, and were activated by sodium cholate at concentrations near the critical micellar concentration (0.03-0.07%), but inhibited by higher concentrations. The temperature-labile cholesteryl ester hydrolase exhibited a high specificity for cholesteryl esters of monoenoic fatty acids of 18-24 carbons, especially nervonate (24:1), whereas the temperature-stable cholesteryl ester hydrolase exhibited highest specificity for cholesteryl oleate and arachidonate. Neither enzyme hydrolyzed cholesteryl acetate, myristate, palmitate, linoleate, or docosahexaenoate . Both enzymes reached maximum rates of hydrolysis at 150 microM substrates, with each substrate and at both reaction temperatures. Substrate inhibition was observed at higher concentrations (200 microM). The temperature-labile cholesteryl ester hydrolase was induced 20-fold in hypophysectomized rats by injection of follicle-stimulating hormone (FSH) and was localized in Sertoli cells, the target cells for FSH, but was not induced by luteinizing hormone. The temperature-stable cholesteryl ester hydrolase was induced by both FSH and LH and was found in both Sertoli cells and Leydig cells, the respective target cells for FSH and luteinizing hormone. Neither form of the enzyme was present at detectable levels in the germinal cells. The unique properties, localization, and hormonal regulation of both temperature-labile and temperature-stable cholesterol ester hydrolases suggest important roles for these enzymes in the testis.     

Phosphoinositide metabolism in cultured glioma and neuroblastoma cells: subcellular distribution of enzymes indicate incomplete turnover at the plasma membrane

The hypothesis that the small portion of cellular phosphoinositide participating in signal transduction might be preferentially recycled within the plasma membrane was tested in rat glioma (C6) and murine neuroblastoma (N1E-115) cells. Percoll density gradient centrifugation was used to isolate a purified plasma membrane fraction and the subcellular distribution of all enzymes mediating phosphoinositide turnover was assessed. A small but significant proportion of PtdInsP2-specific phosphodiesterase was located in the plasma membrane but only two of the five enzymes required to replace PtdInsP2 (diacylglycerol kinase and PtdInsP kinase) also were present. CTP:phosphatidate cytidylyltransferase and CMP-phosphatidate:inositol phosphatidyltransferase were located exclusively in a microsomal fraction containing enriched levels of endoplasmic reticulum markers. Thus, diacylglycerol from agonist-stimulated cleavage of PtdInsP2, or phosphatidic acid formed from it, must be transferred to the endoplasmic reticulum for conversion to PtdIns. Plasma membrane also lacked PtdIns kinase. If the soluble PtdIns kinase has access to membrane-bound substrate, PtdIns may be phosphorylated to PtdInsP before or during transport to the plasma membrane. Phosphorylation by the predominantly plasma membrane PtdInsP kinase to form PtdInsP2 completes the cycle. PtdInsP phosphatase was present in all membrane fractions suggesting that PtdInsP can be returned to the PtdIns pool in plasma membrane and elsewhere. PtdInsP2 phosphatase was almost exclusively in the cytosol suggesting that reversible interchange between PtdInsP and PtdInsP2 in the plasma membrane may be modulated by the ability of this phosphatase to act on PtdInsP2 in the membrane. Thus, PtdIns resynthesis in the plasma membrane of these cells does not occur and is not required for phosphoinositide-mediated signal transduction.     

Secretory hydrolases of Entamoeba histolytica

Cells of Entamoeba histolytica grown over a period of four days contained NADP+-dependent alcohol dehydrogenase exclusively inside the cells. No activity of this enzyme could be found in the growth medium after harvesting the cells. Under the same conditions, acid phosphatase, beta-N-acetylglucosaminidase, esterase, alpha-glucosidase, and different amylases of the parasite were found both inside the cells and in the medium. The activities present in the cell homogenate and in the medium before and after growth of the amoebas were partially separated by gel filtration on Sephadex G150 and G75, respectively. The comparison of the elution diagrams revealed that NADP+-dependent alcohol dehydrogenase, acid phosphatase, esterase, and amylases occurred as multiple forms inside the cells. These activities, as well as beta-N-acetylglucosaminidase and alpha-glucosidase, were released into the extracellular environment to a different degree. The enzymes originating from the parasite were identified and distinguished from those of the ingredients of the growth medium according to their molecular mass and pH optimum. Furthermore, the amoebic origin of the secreted enzymes was shown on the basis of their inhibition by antibodies prepared against the supernatant fraction of the homogenate.     

Mitogen-induced tyrosine-phosphorylated 41- and 43-kDa proteins are family members of extracellular signal-regulated kinases/microtubule-associated protein 2 kinases.

Two antipeptide antibodies, one against the peptide corresponding to residues 307-327 (alpha Y91) and one against the peptide corresponding to the C-terminal portion (alpha C92) of the deduced amino acid sequence of the extracellular signal-regulated kinase 1 (ERK1), precipitated two 41-kDa and/or two 43-kDa phospho-proteins from mitogen-stimulated Swiss 3T3 cells. Electrophoretic mobilities on two-dimensional gels of the immunoprecipitated 41- and 43-kDa phosphoproteins were similar to those of the 41- and 43-kDa cytosol proteins, whose increased tyrosine phosphorylation we and others had originally identified in various mitogen-stimulated cells (Cooper, J. A., Sefton, B. M., and Hunter, T. (1984) Mol. Cell. Biol. 4, 30-37; Kohno, M. (1985) J. Biol. Chem. 260, 1771-1779); phosphopeptide map analysis revealed that they were respectively identical molecules. All those phosphoproteins contained phosphotyrosine, and the more acidic forms contained additional phosphothreonine. Immunoprecipitated 41- and 43-kDa phosphoproteins had serine/threonine kinase activity toward myelin basic protein (MBP) and microtuble-associated protein 2 (MAP2). With the combination of two-dimensional gel electrophoresis and the kinase assay in MBP-containing polyacrylamide gels of the alpha Y91 immunoprecipitates, with or without phosphatase 2A treatment, we showed that only their acidic forms were active. These results clearly indicate that 41- and 43-kDa proteins, the increased tyrosine phosphorylation of which is rapidly and commonly induced by mitogen stimulation of fibroblasts, are family members of ERKs/MAP2 kinases and that phosphorylation both on tyrosine and threonine residues is necessary for their activation.     

Production of cell-bound and vesicle-associated trypsin-like protease, alkaline phosphatase and N-acetyl-beta-glucosaminidase by Bacteroides gingivalis strain W50

Bacteroides gingivalis strain W50 was grown in batch and continuous culture on complex medium with haemin. In batch culture, cell-bound levels of trypsin-like protease (EC 3.4.21.4), alkaline phosphatase (EC 3.1.3.1) and N-acetyl-beta-glucosaminidase (EC 3.2.1.30) increased during the exponential phase of growth. These enzyme activities were also detected in extracellular vesicles and in extracellular soluble forms in the supernatant fluid, but in lower amounts per unit biomass compared to cell-bound levels. In continuous culture, at high relative growth rates (0.7-0.9 murel), the highest proportions of enzyme activities were cell-bound. In contrast, at low relative growth rates (0.1-0.2 murel), highest enzyme levels were detected in the extracellular vesicle fraction. Levels of extracellular soluble enzymes were always low compared to cell-bound or extracellular vesicle levels, but were highest at low relative growth rates. All three enzymes appeared to be relatively stable in their soluble forms. Vesicle production appeared to be associated with actively growing cells but was influenced by growth rate. The results are consistent with the hypothesis that cell-bound 'periplasmic' enzymes are encapsulated into vesicles which are subsequently released by the cells. Therefore, levels of total extracellular enzyme (extracellular vesicle plus extracellular soluble) may depend on the rate of vesicle formation superimposed on the rates of production of 'periplasmic' enzymes in the cell.     

Soluble rat adipocyte phosphatidate phosphatase activity: characterization and effects of fasting and various lipids.

Phosphatidate phosphatase (phosphatidate phosphohydrolase, EC 3.1.3.4) was present at very high specific activity in the soluble fraction of isolated rat adipocytes. Using phosphatidate in aqueous dispersion 90% of its hydrolysis depended on the presence of Mg2+. Mg2+ appeared to almost saturate the enzyme at 20-40 mM with no indication of an optimum. The substrate concentration was optimum at 1.2 mM and the pH at 6.8. Initial rates were linear for only 4-5 min at optimum conditions. Increasing inhibition occurred at high phosphatidate concentrations. At optimum conditions acid or alkaline phosphatase activity was not measurable. The Mg2+-dependent activity was enhanced by 3-sn-phophatidylcholine and inhibited by albumin, 3-sn-phosphatidyletanolamine, 3-sn-phosphatidylinositol, diacylglycerol, oleoyl-CoA, and oleate. Oleoyl-CoA was the most potent "effector". Fasting for 24, 48 and 72 h decreased the activity both relative to protein and to DNA. The activity thus decreased to about one-third of that of the fed rat during 72 h of fasting. The effects of Mg2+, various lipids, and fasting may indicate that some form of control of glyceride synthesis can be exerted through the soluble phosphatidate phosphatase.     

An Active 32-kDa Cathepsin L Is Secreted Directly from HT 1080 Fibrosarcoma Cells and Not via Lysosomal Exocytosis

Cathepsin L [EC 3.4.22.15] is secreted via lysosomal exocytosis by several types of cancer cells, including prostate and breast cancer cells. We previously reported that human cultured fibrosarcoma (HT 1080) cells secrete cathepsin L into the medium; this secreted cathepsin is 10-times more active than intracellular cathepsin. This increased activity was attributed to the presence of a 32-kDa cathepsin L in the medium. The aim of this study was to examine how this active 32-kDa cathepsin L is secreted into the medium. To this end, we compared the secreted active 32-kDa cathepsin L with lysosomal cathepsin L by using a novel gelatin zymography technique that employs leupeptin. We also examined the glycosylation and phosphorylation status of the proteins by using the enzymes endoglycosidase H [EC 3.2.1.96] and alkaline phosphatase [EC 3.1.3.1]. Strong active bands corresponding to the 32-kDa and 34-kDa cathepsin L forms were detected in the medium and lysosomes, respectively. The cell extract exhibited strong active bands for both forms. Moreover, both forms were adsorbed onto a concanavalin A-agarose column. The core protein domain of both forms had the same molecular mass of 30 kDa. The 32-kDa cathepsin L was phosphorylated, while the 34-kDa lysosomal form was dephosphorylated, perhaps because of the lysosomal marker enzyme, acid phosphatase. These results suggest that the active 32-kDa form does not enter the lysosomes. In conclusion, our results indicate that the active 32-kDa cathepsin L is secreted directly from the HT 1080 cells and not via lysosomal exocytosis.     

The metabolism of tris- and tetraphosphates of inositol by 5-phosphomonoesterase and 3-kinase enzymes

Phospholipase C cleaves phosphatidylinositol 4,5-bisphosphate to form both inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) and inositol 1,2-cyclic 4,5-trisphosphate (cInsP3). The further metabolism of these inositol trisphosphates is determined by two enzymes: a 3-kinase and a 5-phosphomonoesterase. The first enzyme converts Ins(1,4,5)P3 to inositol 1,3,4,5-tetrakisphosphate (InsP4), while the latter forms inositol 1,4-bisphosphate and inositol 1,2-cyclic 4-bisphosphate from Ins(1,4,5)P3 and cInsP3, respectively. The current studies show that the 3-kinase is unable to phosphorylate cInsP3. Also, the 5-phosphomonoesterase hydrolyzes InsP4 with an apparent Km of 0.5-1.0 microM to form inositol 1,3,4-trisphosphate at a maximal velocity approximately 1/30 that for Ins(1,4,5)P3. The apparent affinity of the enzyme for the three substrates is InsP4 greater than Ins(1,4,5)P3 greater than cInsP3; however, the rate at which the phosphatase hydrolyzes these substrates is Ins(1,4,5)P3 greater than cInsP3 greater than InsP4. The 5-phosphomonoesterase and 3-kinase enzymes may control the levels of inositol trisphosphates in stimulated cells. The 3-kinase has a low apparent Km for Ins(1,4,5)P3 as does the 5-phosphomonoesterase for InsP4, implying that the formation and breakdown of InsP4 may proceed when both it and its precursor are present at low levels. Ins(1,4,5)P3 is utilized by both the 3-kinase and 5-phosphomonoesterase, while cInsP3 is utilized relatively poorly only by the 5-phosphomonoesterase. These findings imply that inositol cyclic trisphosphate may be metabolized slowly after its formation in stimulated cells.     

Lipid phosphate phosphatases in Arabidopsis. Regulation of the AtLPP1 gene in response to stress

An Arabidopsis thaliana gene (AtLPP1) was isolated on the basis that it was transiently induced by ionizing radiation. The putative AtLPP1 gene product showed homology to the yeast and mammalian lipid phosphate phosphatase enzymes and possessed a phosphatase signature sequence motif. Heterologous expression and biochemical characterization of the AtLPP1 gene in yeast showed that it encoded an enzyme (AtLpp1p) that exhibited both diacylglycerol pyrophosphate phosphatase and phosphatidate phosphatase activities. Kinetic analysis indicated that diacylglycerol pyrophosphate was the preferred substrate for AtLpp1p in vitro. A second Arabidopsis gene (AtLPP2) was identified based on sequence homology to AtLPP1 that was also heterologously expressed in yeast. The AtLpp2p enzyme also utilized diacylglycerol pyrophosphate and phosphatidate but with no preference for either substrate. The AtLpp1p and AtLpp2p enzymes showed differences in their apparent affinities for diacylglycerol pyrophosphate and phosphatidate as well as other enzymological properties. Northern blot analyses showed that the AtLPP1 gene was preferentially expressed in leaves and roots, whereas the AtLPP2 gene was expressed in all tissues examined. AtLPP1, but not AtLPP2, was regulated in response to various stress conditions. The AtLPP1 gene was transiently induced by genotoxic stress (gamma ray or UV-B) and elicitor treatments with mastoparan and harpin. The regulation of the AtLPP1 gene in response to stress was consistent with the hypothesis that its encoded lipid phosphate phosphatase enzyme may attenuate the signaling functions of phosphatidate and/or diacylglycerol pyrophosphate that form in response to stress in plants.