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Kenji Fukuzawa - One of the best experts on this subject based on the ideXlab platform.
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Increased production of bioactive lysophosphatidic acid by serum Lysophospholipase D in human pregnancy
Biology of reproduction, 2002Co-Authors: Akira Tokumura, Yumi Kanaya, Maki Miyake, Shuji Yamano, Minoru Irahara, Kenji FukuzawaAbstract:Lysophosphatidic acid (LPA) is a prototype of the lysophospholipid mediator family and has multiple effects in the female reproductive system. Although several metabolic routes have been reported for intracellular formation of LPA, a unique route involving Lysophospholipase D, an extracellular enzyme that produces LPA in blood and body fluids, is particularly intriguing for its agonistic role. In this study, using an assay with radioactive palmitoyl-lysophosphatidylcholine, we found that Lysophospholipase D activity producing palmitoyl-LPA in human serum gradually increased during pregnancy. Elevated activity of Lysophospholipase D was not caused by changes in levels of their precursors, lysophosphatidylcholines, in nonpregnant women or in pregnant women at different gestational periods. With increasing length of gestation, the elevated activity in pregnant women was found to produce increasing proportions of LPA with a palmitoyl group versus other LPAs. These results suggest that LPA formed by increased activity of Lysophospholipase D in blood might participate in maintenance of pregnancy.
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identification of human plasma Lysophospholipase d a lysophosphatidic acid producing enzyme as autotaxin a multifunctional phosphodiesterase
Journal of Biological Chemistry, 2002Co-Authors: Akira Tokumura, Kentaro Kogure, Eiji Majima, Yuko Kariya, Kyoko Tominaga, Katsuhiko Yasuda, Kenji FukuzawaAbstract:Abstract We purified human plasma Lysophospholipase D that produces physiologically active lysophosphatidic acid and showed that it is a soluble form of autotaxin, an ecto-nucleotide pyrophosphatase/phosphodiesterase, originally found as a tumor cell motility-stimulating factor. Its lower K mvalue for a lysophosphatidylcholine than that for a synthetic substrate of nucleotide suggests that lysophosphatidylcholine is a more likely physiological substrate for autotaxin and that its predicted physiological and pathophysiological functions could be mediated by its activity to produce lysophosphate acid, an intercellular mediator. Recombinant autotaxin was found to have Lysophospholipase D activity; its substrate specificity and metal ion requirement were the same as those of the purified plasma enzyme. The activity of Lysophospholipase D for exogenous lysophosphatidylcholine in human serum was found to increase in normal pregnant women at the third trimester of pregnancy and to a higher extent in patients in threatened preterm delivery, suggesting its roles in induction of parturition.
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lack of significant differences in the corrected activity of Lysophospholipase d producer of phospholipid mediator lysophosphatidic acid in incubated serum from women with and without ovarian tumors
Cancer, 2002Co-Authors: Akira Tokumura, B Kyoko S Tominaga, M Katsuhiko D Yasuda, M Hideharu D Kanzaki, Kentaro Kogure, Kenji FukuzawaAbstract:BACKGROUND Several studies have shown that lysophosphatidic acid (LPA), a phospholipidic chemical mediator, is relevant to the pathogenesis of ovarian carcinoma. Higher plasma levels of LPA have been reported in patients with ovarian carcinoma than in healthy patients, and LPA is known to activate ovarian carcinoma cells. To determine the reason for the increased plasma LPA levels in ovarian carcinoma patients, we compared the activities of serum Lysophospholipase D, a novel LPA-producing metallo-enzyme, in healthy volunteers, patients with benign ovarian tumor, and patients with ovarian carcinoma. METHODS Lysophospholipase D activity was assessed by measuring the percentage conversion of [14C]palmitoyl-lysophosphatidylcholine (LPC) added to human serum. The apparent enzyme activities were corrected based on the serum levels of palmitoyl-LPC determined by gas-liquid chromatography after its purification and conversion to fatty acid methyl esters. RESULTS The apparent activity of Lysophospholipase D in serum preparations from four patients with ovarian carcinoma at Stage IV was significantly higher than those from five healthy subjects, five patients with benign ovarian tumors, and fourteen patients with ovarian carcinoma at Stages I (n = 5), II (n = 4), and III (n = 5). The serum levels of LPC, an endogenous substrate of Lysophospholipase D, in ovarian carcinoma patients were less than those in patients with benign ovarian tumors. There were no significant differences in the corrected Lysophospholipase D activity for the LPC levels in healthy women, patients with benign ovarian tumors, and patients with ovarian carcinoma at various stages. CONCLUSIONS The current results suggest that Lysophospholipase D is not associated with the elevated plasma levels of LPA in ovarian carcinoma patients previously reported, although only a limited number of patients were analyzed. Cancer 2002;94:141–51. © 2002 American Cancer Society.
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Increased formation of lysophosphatidic acids by Lysophospholipase D in serum of hypercholesterolemic rabbits.
Journal of lipid research, 2002Co-Authors: Akira Tokumura, Yumi Kanaya, Maki Miyake, Masaki Kitahara, Yasuko Yoshioka, Kenji FukuzawaAbstract:Lysophosphatidic acid (LPA) is a biologically ac- tive phospholipid that has been identified as a vasoactive principle in incubated plasma and serum of mammals. Pre- viously, we found that mammalian plasma and serum con- tain a Lysophospholipase D, which hydrolyzes lysophosphati- dylcholines (LPCs) with different fatty acyl groups to the corresponding LPAs during its incubation at 37 8 C. In this study, we examined whether Lysophospholipase D activity and levels of LPCs in rabbit serum were modulated by feed- ing rabbits a high cholesterol diet. Results showed that the serum levels of LPCs increased gradually in animals fed a high cholesterol diet for 12 weeks. We found that the levels of individual LPAs formed on incubation of serum for 24 h increased with an increase in the period of feeding of rab- bits a high cholesterol diet. LPA with a linoleate residue was the most abundant LPA, followed in order by 16:0-, 18:1- and 18:0-LPAs. LPA was found to increase attachment of the monocytic cell line THP-1 to vascular endothelial cells pre-stimulated with tumor necrosis factor- a . These re- sults indicated that increases in the levels of LPAs gener- ated by Lysophospholipase D in the blood of hypercholester- olemic rabbits may be relevant to attachment of monocytes to vascular walls, a key phenomenon observed at an early stage of atherosclerosis. —Tokumura, A., Y. Kanaya, M. Kitahara, M. Miyake, Y. Yoshioka, and K. Fukuzawa. Increased formation of lysophosphatidic acids by Lysophospholipase D in serum of hypercholesterolemic rabbits. J. Lipid Res. 2002. 43: 307-315.
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production of lysophosphatidic acid by Lysophospholipase d in incubated plasma of spontaneously hypertensive rats and wistar kyoto rats
Life Sciences, 1999Co-Authors: Akira Tokumura, Yuko Nishioka, Osamu Yoshimoto, Maki Miyake, Hiroaki Fujimoto, Kenji FukuzawaAbstract:Abstract Lysophosphatidic acid has been identified as a vasopressor principle in incubated mammalian plasma and sera, and shown to be generated extracellulary by Lysophospholipase D-like activity. In this study, we monitored the time course of changes in the major phospholipid fractions during incubation of plasma, and found that polyunsaturated lysophosphatidic acids accumulate more rapidly than saturated lysophosphatidic acids at expense of the corresponding lysophosphatidylcholines. We compared the phospholipase activities for producing bioactive LPA in age-matched spontaeneously hypertensive rats and Wistar Kyoto rats. The Lysophospholipase D activity in rat plasma was found to be independent of strain and age. We suggest that Lysophospholipase D functions in rat for persistent production of bioactive LPA in the circulation throughout life. However, our finding that production of LPA in spontaenously hypertensive rats was not greater than that in Wistar Kyoto rats does not seem to support the idea that increased production of LPA is involved in the pathogenesis of hypertension.
Akira Tokumura - One of the best experts on this subject based on the ideXlab platform.
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Increased production of bioactive lysophosphatidic acid by serum Lysophospholipase D in human pregnancy
Biology of reproduction, 2002Co-Authors: Akira Tokumura, Yumi Kanaya, Maki Miyake, Shuji Yamano, Minoru Irahara, Kenji FukuzawaAbstract:Lysophosphatidic acid (LPA) is a prototype of the lysophospholipid mediator family and has multiple effects in the female reproductive system. Although several metabolic routes have been reported for intracellular formation of LPA, a unique route involving Lysophospholipase D, an extracellular enzyme that produces LPA in blood and body fluids, is particularly intriguing for its agonistic role. In this study, using an assay with radioactive palmitoyl-lysophosphatidylcholine, we found that Lysophospholipase D activity producing palmitoyl-LPA in human serum gradually increased during pregnancy. Elevated activity of Lysophospholipase D was not caused by changes in levels of their precursors, lysophosphatidylcholines, in nonpregnant women or in pregnant women at different gestational periods. With increasing length of gestation, the elevated activity in pregnant women was found to produce increasing proportions of LPA with a palmitoyl group versus other LPAs. These results suggest that LPA formed by increased activity of Lysophospholipase D in blood might participate in maintenance of pregnancy.
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identification of human plasma Lysophospholipase d a lysophosphatidic acid producing enzyme as autotaxin a multifunctional phosphodiesterase
Journal of Biological Chemistry, 2002Co-Authors: Akira Tokumura, Kentaro Kogure, Eiji Majima, Yuko Kariya, Kyoko Tominaga, Katsuhiko Yasuda, Kenji FukuzawaAbstract:Abstract We purified human plasma Lysophospholipase D that produces physiologically active lysophosphatidic acid and showed that it is a soluble form of autotaxin, an ecto-nucleotide pyrophosphatase/phosphodiesterase, originally found as a tumor cell motility-stimulating factor. Its lower K mvalue for a lysophosphatidylcholine than that for a synthetic substrate of nucleotide suggests that lysophosphatidylcholine is a more likely physiological substrate for autotaxin and that its predicted physiological and pathophysiological functions could be mediated by its activity to produce lysophosphate acid, an intercellular mediator. Recombinant autotaxin was found to have Lysophospholipase D activity; its substrate specificity and metal ion requirement were the same as those of the purified plasma enzyme. The activity of Lysophospholipase D for exogenous lysophosphatidylcholine in human serum was found to increase in normal pregnant women at the third trimester of pregnancy and to a higher extent in patients in threatened preterm delivery, suggesting its roles in induction of parturition.
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Physiological and pathophysiological roles of lysophosphatidic acids produced by secretory Lysophospholipase D in body fluids.
Biochimica et Biophysica Acta, 2002Co-Authors: Akira TokumuraAbstract:Recently, a family of phospholipid mediators has received much attention because of its variety of biological activities. Lysophosphatidic acid (LPA) is a central member of the phospholipid autacoid family that exerts diverse effects through binding to and activation of several specific receptors coupled to G-proteins. In accordance with its function as a receptor agonist, there are pathways for extracellular generation of LPA in vivo. One pathway involves a novel Lysophospholipase D activity that was originally found in rat plasma. LPA is also produced in significant amounts after incubation of various plasma-derived body fluids such as human follicular fluid at 25-37 degrees C. In animal models, LPA was shown to stimulate oocyte maturation, embryonic development and transport in the oviduct. An increase in serum Lysophospholipase D activity was observed during pregnancy in human. These results suggest that LPA generated by Lysophospholipase D is likely to play an important role in reproductive biology. LPA produced by Lysophospholipase D activity in body fluids has also been observed under pathophysiological conditions: serum and ascitic fluid from ovarian cancer patients and serum from hypercholesterolemic rabbits. Hence, excess generation of LPA by Lysophospholipase D activity in body fluids has been suggested to be relevant to the pathogenesis of cancer and atherosclerosis.
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lack of significant differences in the corrected activity of Lysophospholipase d producer of phospholipid mediator lysophosphatidic acid in incubated serum from women with and without ovarian tumors
Cancer, 2002Co-Authors: Akira Tokumura, B Kyoko S Tominaga, M Katsuhiko D Yasuda, M Hideharu D Kanzaki, Kentaro Kogure, Kenji FukuzawaAbstract:BACKGROUND Several studies have shown that lysophosphatidic acid (LPA), a phospholipidic chemical mediator, is relevant to the pathogenesis of ovarian carcinoma. Higher plasma levels of LPA have been reported in patients with ovarian carcinoma than in healthy patients, and LPA is known to activate ovarian carcinoma cells. To determine the reason for the increased plasma LPA levels in ovarian carcinoma patients, we compared the activities of serum Lysophospholipase D, a novel LPA-producing metallo-enzyme, in healthy volunteers, patients with benign ovarian tumor, and patients with ovarian carcinoma. METHODS Lysophospholipase D activity was assessed by measuring the percentage conversion of [14C]palmitoyl-lysophosphatidylcholine (LPC) added to human serum. The apparent enzyme activities were corrected based on the serum levels of palmitoyl-LPC determined by gas-liquid chromatography after its purification and conversion to fatty acid methyl esters. RESULTS The apparent activity of Lysophospholipase D in serum preparations from four patients with ovarian carcinoma at Stage IV was significantly higher than those from five healthy subjects, five patients with benign ovarian tumors, and fourteen patients with ovarian carcinoma at Stages I (n = 5), II (n = 4), and III (n = 5). The serum levels of LPC, an endogenous substrate of Lysophospholipase D, in ovarian carcinoma patients were less than those in patients with benign ovarian tumors. There were no significant differences in the corrected Lysophospholipase D activity for the LPC levels in healthy women, patients with benign ovarian tumors, and patients with ovarian carcinoma at various stages. CONCLUSIONS The current results suggest that Lysophospholipase D is not associated with the elevated plasma levels of LPA in ovarian carcinoma patients previously reported, although only a limited number of patients were analyzed. Cancer 2002;94:141–51. © 2002 American Cancer Society.
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Increased formation of lysophosphatidic acids by Lysophospholipase D in serum of hypercholesterolemic rabbits.
Journal of lipid research, 2002Co-Authors: Akira Tokumura, Yumi Kanaya, Maki Miyake, Masaki Kitahara, Yasuko Yoshioka, Kenji FukuzawaAbstract:Lysophosphatidic acid (LPA) is a biologically ac- tive phospholipid that has been identified as a vasoactive principle in incubated plasma and serum of mammals. Pre- viously, we found that mammalian plasma and serum con- tain a Lysophospholipase D, which hydrolyzes lysophosphati- dylcholines (LPCs) with different fatty acyl groups to the corresponding LPAs during its incubation at 37 8 C. In this study, we examined whether Lysophospholipase D activity and levels of LPCs in rabbit serum were modulated by feed- ing rabbits a high cholesterol diet. Results showed that the serum levels of LPCs increased gradually in animals fed a high cholesterol diet for 12 weeks. We found that the levels of individual LPAs formed on incubation of serum for 24 h increased with an increase in the period of feeding of rab- bits a high cholesterol diet. LPA with a linoleate residue was the most abundant LPA, followed in order by 16:0-, 18:1- and 18:0-LPAs. LPA was found to increase attachment of the monocytic cell line THP-1 to vascular endothelial cells pre-stimulated with tumor necrosis factor- a . These re- sults indicated that increases in the levels of LPAs gener- ated by Lysophospholipase D in the blood of hypercholester- olemic rabbits may be relevant to attachment of monocytes to vascular walls, a key phenomenon observed at an early stage of atherosclerosis. —Tokumura, A., Y. Kanaya, M. Kitahara, M. Miyake, Y. Yoshioka, and K. Fukuzawa. Increased formation of lysophosphatidic acids by Lysophospholipase D in serum of hypercholesterolemic rabbits. J. Lipid Res. 2002. 43: 307-315.
Edward A. Dennis - One of the best experts on this subject based on the ideXlab platform.
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subcellular localization and pkc dependent regulation of the human Lysophospholipase a acyl protein thioesterase in wish cells
Biochimica et Biophysica Acta, 2000Co-Authors: Aijun Wang, Christina A Johnson, Ying Jones, Mark H Ellisman, Edward A. DennisAbstract:Abstract Lysophospholipases play essential roles in keeping their multi-functional substrates, the lysophospholipids, at safe levels. Recently, a 25 kDa human Lysophospholipase A (hLysoPLA I) that is highly conserved among rat, mouse, human and rabbit has been cloned, expressed and characterized and appears to hydrolyze only lysophospholipids among the various lipid substrates. Interestingly, this enzyme also displays acyl-protein thioesterase activity towards a G protein α subunit. To target the subcellular location of this hLysoPLA I, we have carried out immunocytochemical studies and report here that hLysoPLA I appears to be associated with the endoplasmic reticulum (ER) and nuclear envelope in human amnionic WISH cells and not the plasma membrane. In addition, we found that the hLysoPLA I can be up-regulated by phorbol 12-myristate 13-acetate (PMA) stimulation, a process in which phospholipase A2 is activated and lysophospholipids are generated in WISH cells. Furthermore, the PMA-induced hLysoPLA I expression can be blocked by the protein kinase C (PKC) inhibitor Go6976. The regulated expression of the LysoPLA/acyl-protein thioesterase by PKC may have important implications for signal transduction processes.
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a specific human Lysophospholipase cdna cloning tissue distribution and kinetic characterization
Biochimica et Biophysica Acta, 1999Co-Authors: Aijun Wang, Hsiuchiung Yang, Peter Friedman, Christina A Johnson, Edward A. DennisAbstract:Abstract Lysophospholipases are critical enzymes that act on biological membranes to regulate the multifunctional lysophospholipids; increased levels of lysophospholipids are associated with a host of diseases. Herein we report the cDNA cloning of a human brain 25 kDa lysophospholipid-specific Lysophospholipase (hLysoPLA). The enzyme (at both mRNA and protein levels) is widely distributed in tissues, but with quite different abundances. The hLysoPLA hydrolyzes lysophosphatidylcholine in both monomeric and micellar forms, and exhibits apparent cooperativity and surface dilution kinetics, but not interfacial activation. Detailed kinetic analysis indicates that the hLysoPLA binds first to the micellar surface and then to the substrate presented on the surface. The kinetic parameters associated with this surface dilution kinetic model are reported, and it is concluded that hLysoPLA has a single substrate binding site and a surface recognition site. The apparent cooperativity observed is likely due to the change of substrate presentation. In contrast to many non-specific lipolytic enzymes that exhibit Lysophospholipase activity, hLysoPLA hydrolyzes only lysophospholipids and has no other significant enzymatic activity. Of special interest, hLysoPLA does not act on plasmenylcholine. Of the several inhibitors tested, only methyl arachidonyl fluorophosphonate (MAFP) potently and irreversibly inhibits the enzymatic activity. The inhibition by MAFP is consistent with the catalytic mechanism proposed for the enzyme – a serine hydrolase with a catalytic triad composed of Ser-119, Asp-174 and His-208.
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Lysophospholipases I and II from P388D1 macrophage-like cell line.
Methods in enzymology, 1991Co-Authors: Ying Yi Zhang, Raymond A. Deems, Edward A. DennisAbstract:Publisher Summary This chapter presents the purification and characterization process of Lysophospholipases I and II from the P388D 1 macrophage-like cell line. Lysophospholipase hydrolyzes the fatty acid ester bond of lysophospholipids, liberating water-soluble glycerol phosphatides and fatty acids. Because of the low levels of Lysophospholipase activity found in cells, a radioactive assay is the best method of determining their activity. The assay employs lysophosphatidylcholine (lyso-PC) that is radiolabeled in the fatty acid chain. Palmitoyllsyo-PC, the substrate used in the standard assay, forms micelles in aqueous solutions and has a critical micelle concentration (CMC) of 7 μM. Two purification schemes of Lysophospholipase I are presented, where first is designed to maximize the yield of Lysophospholipase I, and the second is required to purify Lysophospholipase II. Although Lysophospholipase I can also be obtained from the second method, its yield is much lower than in the first. The purification of Lysophospholipase II is carried out separately, using the same columns but different conditions. The addition of two precipitation steps at the beginning of the purification greatly enhances the efficiency of the diethylaminoethyl (DEAE)-Sephacel column. The first is a streptomycin precipitation that removes nucleic acid and the second is an ammonium sulfate precipitation used to concentrate the sample.
M. A. Clark - One of the best experts on this subject based on the ideXlab platform.
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Purification of a lysophosphatidic acid-hydrolysing Lysophospholipase from rat brain.
The Biochemical journal, 1994Co-Authors: F J Thompson, M. A. ClarkAbstract:A lysophosphatidic acid (LPA)-hydrolysing Lysophospholipase was purified from rat brain and characterized. This membrane-bound Lysophospholipase was solubilized by using n-octyl glucoside and purified by sequential cation, hydrophobic and gel-filtration chromatography. The purified protein has a mass of 80 kDa as assayed by SDS/PAGE. This Lysophospholipase catalysed the hydrolysis of a variety of lysophosphatidic acids, but with different rates, depending on the length and degree of saturation of the sn-1 acyl group (1-oleoyl-LPA approximately 1-stearoyl-LPA > 1-palmitoyl-LPA > 1-myristoyl-LPA). This enzyme had no-measurable catalytic activity when other lysophospholipids, monoacylglycerol or phosphatidic acid were used as substrates. On the basis of its chromatographic properties, substrate specificity and cellular localization, we conclude that this Lysophospholipase differs from those previously purified and speculate that it has an important function in terminating biological responses to LPA.
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The substrate specificities of four different Lysophospholipases as determined by a novel fluorescence assay.
The Biochemical journal, 1994Co-Authors: H S She, D E Garsetti, M R Steiner, R W Egan, M. A. ClarkAbstract:A novel fluorescence assay for quantifying Lysophospholipase activity is described which utilizes a commercially available acrylodated intestinal fatty-acid-binding protein (ADIFAB) and non-radiolabelled substrate. Quantification of enzyme activity is based on the decrease in ADIFAB fluorescence at 432 nm in the presence of nanomolar concentrations of non-esterified ('free') fatty acids. Lysophospholipase activity measured by the ADIFAB assay and a conventional radiometric assay yield comparable results and have comparable levels of sensitivity (approximately 10 pmol/min per ml). The ADIFAB assay has the advantageous features of continuous monitoring of enzyme activity and the availability of a broad range of potential substrates, because non-radiolabelled lysophospholipids can be employed in the assay. The hydrolytic activities of four Lysophospholipases were determined, including a bacterial secreted phospholipase A2/Lysophospholipase, the human-eosinophil-secreted Lysophospholipase, a human intracellular Lysophospholipase (peak 3) isolated from HL-60 cells and a high-molecular-mass cytosolic phospholipase A2/Lysophospholipase from a mouse mammary carcinoma. Each of these enzymes was found to have a distinctive hydrolytic profile as determined by an array of lysophospholipids differing in their polar headgroups and sn-1 fatty-acyl substituents.
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Comparison of six mammalian Lysophospholipases.
Journal of lipid mediators, 1993Co-Authors: D E Garsetti, Marion R. Steiner, F. Holtsberg, L. E. Ozgür, Robert W. Egan, M. A. ClarkAbstract:Lysophospholipases participate in the regulation of the levels of lysophospholipid, compounds with pleiotropic biological effects. Lysophospholipases were purified from a macrophage cell line (WEHI 265.1), a myelocytic leukemia cell line (HL-60) and peripheral blood eosinophils. WEHI 265.1 cells contain three Lysophospholipases 28, 27 and 110 kDa as determined by polyacrylamide gel electrophoresis. The 110 kDa Lysophospholipase also exhibits phospholipase A2 activity and appears to be identical to a previously described 110 kDa phospholipase A2. Similarly, the HL-60 cells have three Lysophospholipases, the largest again a 110 kDa enzyme with phospholipase A2 activity and the smaller are 20 and 21 kDa. The low molecular mass Lysophospholipases have distinctive chromatographic properties and amino acid compositions. However, the two low molecular mass enzymes from a given cell type are not radically different, e.g., 15 of the 20 amino acids of the C-terminal sequences of the HL-60 enzymes are identical. A single Lysophospholipase, approx. 15 kDa, is a major eosinophil protein. This enzyme is different from those described above.
Hiroyuki Sugimoto - One of the best experts on this subject based on the ideXlab platform.
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Thioesterase activity and subcellular localization of acylprotein thioesterase 1/Lysophospholipase 1.
Biochimica et biophysica acta, 2009Co-Authors: Tohko Hirano, Hiroyuki Sugimoto, Mikiko Kishi, Ryo Taguchi, Hideru Obinata, Noriyasu Ohshima, Kazuaki Tatei, Takashi IzumiAbstract:Abstract Acylprotein thioesterase 1 (APT1), also known as Lysophospholipase 1, is an important enzyme responsible for depalmitoylation of palmitoyl proteins. To clarify the substrate selectivity and the intracellular function of APT1, we performed kinetic analyses and competition assays using a recombinant human APT1 (hAPT1) and investigated the subcellular localization. For this purpose, an assay for thioesterase activity against a synthetic palmitoyl peptide using liquid chromatography/mass spectrometry was established. The thioesterase activity of hAPT1 was most active at neutral pH, and did not require Ca2+ for its maximum activity. The KM values for thioesterase and Lysophospholipase (against lysophosphatidylcholine) activities were 3.49 and 27.3 μM, and the Vmax values were 27.3 and 1.62 μmol/min/mg, respectively. Thus, hAPT1 revealed much higher thioesterase activity than Lysophospholipase activity. One activity was competitively inhibited by another substrate in the presence of both substrates. Immunocytochemical and Western blot analyses revealed that endogenous and overexpressed hAPT1 were mainly localized in the cytosol, while some signals were detected in the plasma membrane, the nuclear membrane and ER in HEK293 cells. These results suggest that eliminating palmitoylated proteins and lysophospholipids from cytosol is one of the functions of hAPT1.
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Sequence, expression in Escherichia coli, and characterization of Lysophospholipase II.
Biochimica et biophysica acta, 1999Co-Authors: Takashi Toyoda, Hiroyuki Sugimoto, Satoshi YamashitaAbstract:Abstract Here we report the sequence, expression in Escherichia coli cells, and characterization of a new small-form Lysophospholipase named Lysophospholipase II from mouse embryo. The cDNA clone was found and identified among mouse expressed sequence tags in the database search for the homologue of Lysophospholipase I previously cloned from rat liver (H. Sugimoto et al., J. Biol. Chem. 271 (1996) 7705–7711). The predicted amino acids sequence contained 231 residues with a calculated molecular weight of 24 794, and showed 64% identity to that of Lysophospholipase I with the Gly–X–Ser–X–Gly esterase/lipase consensus. The lacZ fusion protein expressed in E. coli cells exhibited Lysophospholipase activity and reacted with antibody raised against previously purified pig gastric Lysophospholipase II (H. Sunaga et al., Biochem. J. 308 (1995) 551–557), but not with antibody against rat liver Lysophospholipase I. The expressed enzyme was purified to a specific activity of 0.15 μmol/min per mg by DEAE–Sepharose A-500 chromatography. The enzyme preferentially utilized zwitterionic lysophospholipids in the order of lysophosphatidylcholine>lysophosphatidylethanolamine, but poorly acidic lysophospholipids, such as lysophosphatidylserine, lysophosphatidylinositol, and lysophosphatidic acid. Not only the 1-acyl isomer, but also the 2-acyl isomer were deacylated. Northern blot analysis and reverse transcription–polymerase chain reaction revealed that Lysophospholipase II transcript as well as Lysophospholipase I transcript was widely distributed in mouse tissues.
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cloning and expression of cdna encoding rat liver 60 kda Lysophospholipase containing an asparaginase like region and ankyrin repeat
Journal of Biological Chemistry, 1998Co-Authors: Hiroyuki Sugimoto, Shoji Odani, Satoshi YamashitaAbstract:Abstract Mammalian tissues contain small form and large form Lysophospholipases. Here we report the cloning, sequence, and expression of cDNA encoding the latter form of Lysophospholipase using antibody raised against the enzyme purified from rat liver supernatant (Sugimoto, H., and Yamashita, S. (1994) J. Biol. Chem. 269, 6252–6258). The 2,539-base pair cDNA encoded 564 amino acid residues with a calculated M r of 60,794. The amino-terminal two-thirds of the deduced amino acid sequence significantly resembled Escherichia coliasparaginase I with the putative asparaginase catalytic triad Thr-Asp-Lys and was followed by leucine zipper motif. The carboxyl-terminal region carried ankyrin repeat. When the cDNA was transfected into HEK293 cells, not only Lysophospholipase activity but also asparaginase and platelet-activating factor acetylhydrolase activities were expressed. Reverse transcription-polymerase chain reaction revealed that the transcript occurred at high levels in liver and kidney but was hardly detectable in lung and heart from which large form Lysophospholipases had been purified, suggesting the presence of multiple forms of large form Lysophospholipase in mammalian tissues.
