An acid phospholipase C from Tetrahymena culture medium (original) (raw)
Applied Microbiology and Biotechnology, 2000
We have described a procedure for the isolation of mutants of Tetrahymena thermophila with hyperscretion of phospholipase A 1 (PLA 1 ). Using random chemical mutagenesis, uniparental cytogamy, genetic crossing and a new, fast and eective screening procedure, four PLA 1 -hypersecretory mutants were isolated. The screening procedure is based on the formation of a halo appearing around cylindrical holes in a lecithincontaining agar plate ®lled with cell-free supernatants. About 3,940 clones were tested with this procedure in primary screening for hypersecretory features, of which 60 putative hypersecretory mutants were isolated, subcloned and tested in a secondary screening. Of these, four selected mutants showed 1.8±2.2 more PLA 1 activity in the cell-free supernatants compared to the wild-type strain CU 438.1. Hypersecretion was only observable for PLA 1 ; no increased activity for two other lysosomal enzymes could be detected. These hypersecretory mutants of T. thermophila can be very useful for increasing the yield of PLA 1 in fermentation processes. This is particularly relevant because, in contrast to other phospholipases, PLA 1 is not available on the commercial market for ®ne chemicals and little is known about the role of PLA 1 in cell signaling and metabolism.
1-O-Alkyl-2-Acetyl-sn-Glycero-3-Phosphate: Phosphohydrolase Activity in Tetrahymena pyriformis
The Journal of Eukaryotic Microbiology, 2000
f f n i v e r s i v of Iounninu, 451 10 loanninu. Greece ABSTRACT. Within the frame of the de novo formation of Platelet-Activating Factor in 7etruhymmu. the occurrence as well as the propertiez of a lipid phosphate phosphohydrolase activity catalyzing the dephosphorylation of I-O-alkyl-2-acetyl-sn-gl~~cero-3-phosphatc was investigated. The activity was distributed in all the membrane fractions of the cell and in the cytosol. It showed preference for acyl~acetyl-.sn-glycero-phosphate as well, and at a much lower level, for dipalmitoyl-glycero-phosphate. Mg2 ' and Caz' caused a dosedependcnt inhibition, while F , EDTA and ECTA had n o effect. The enzymic activity was linear for at least up to 60 min incubation time and up t o 150 kg protein. Microsomal activity exhibited two optimal pH areas, around 7.0 and 9.0, while mitochondrial activity showcd one peak, at pH 7.0. Acyl-GP, acyl-acetyl-GP and alkyl-GP could replace alkyl-acetyl-GP in significant rates, while dipalmitoyl-GP, P-GP, fructose-6-GP, p-nitrophenylphosphate, creatine phosphate or ATP had no effect. Side phospholipasc A' and C activities were also detected. Taking into account the presence of PAF and alkylacetylglycerol in the protozoan as well as the presence ofa dithiothreitolinscnsitive CDP-cho1inc:cholinephosphotransferase activity that converts alkylacetylglycerol to PAF, we suggest that the present phosphohydrolase activity may be involved in the de novo production of PAF within Tetnikpmencr.
Chemistry and Physics of Lipids, 2010
Eukaryotic phosphoinositide-specific phospholipases C (PI-PLC) specifically hydrolyze phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P 2 ], produce the Ca 2؉-mobilizing agent inositol 1,4,5-trisphosphate, and regulate signaling in multicellular organisms. Bacterial PtdIns-specific PLCs, also present in trypanosomes, hydrolyze PtdIns and glycosyl-PtdIns, and they are considered important virulence factors. All unicellular eukaryotes studied so far contain a single PI-PLC-like gene. In this report, we show that ciliates are an exception, since we provide evidence that Tetrahymena species contain two sets of functional genes coding for both bacterial and eukaryotic PLCs. Biochemical characterization revealed two PLC activities that differ in their phosphoinositide substrate utilization, subcellular localization, secretion to extracellular space, and sensitivity to Ca 2؉. One of these activities was identified as a typical membrane-associated PI-PLC activated by low-micromolar Ca 2؉ , modestly activated by GTP␥S in vitro, and inhibited by the compound U73122 [1-(6-{[17-3-methoxyestra-1,3,5(10)-trien-17-yl]amino}hexyl)-1H-pyrrole-2,5-dione]. Importantly, inhibition of PI-PLC in vivo resulted in rapid upregulation of PtdIns(4,5)P 2 levels, suggesting its functional importance in regulating phosphoinositide turnover in Tetrahymena. By in silico and molecular analysis, we identified two PLC genes that exhibit significant similarity to bacterial but not trypanosomal PLC genes and three eukaryotic PI-PLC genes, one of which is a novel inactive PLC similar to proteins identified only in metazoa. Comparative studies of expression patterns and PI-PLC activities in three T. thermophila strains showed a correlation between expression levels and activity, suggesting that the three eukaryotic PI-PLC genes are functionally nonredundant. Our findings imply the presence of a conserved and elaborate PI-PLC-Ins(1,4,5)P 3-Ca 2؉ regulatory axis in ciliates.
International Journal of Biochemistry, 1983
I. Culture supernatants from late log phase cultures of Altc~~~~~o~~tr.s c,.spr/icl,ltr hydrolyrcd phosphatidylinositol to glycerylphosphorylinositol and free fatty acid. No Iysophosphatidylirl~~sitol \\as detected. 2. The phospholipasc activity degraded up to 50",, of the substrate and displayed a broad pH optimum from 6.5-8.5. 3. The activity was slightly stimulated in the presence of either Mg' + or Ca' I. hut MXS not inhibited h) EDTA. 4. The apparent K, for phosphatidylinositol was 1.5 mM. 5. Culture sunernatants also contained deacvlating activity which relcascd fatty acid from phosphatl
Expression in yeast of a novel phospholipase A1 cDNA from Arabidopsis thaliana
European Journal of Biochemistry, 2004
During a search for cDNAs encoding plant sterol acyltransferases, we isolated four full-length cDNAs from Arabidopsis thaliana that encode proteins with substantial identity with animal lecithin : cholesterol acyltransferases (LCATs). The expression of one of these cDNAs, AtLCAT3 (At3g03310), in various yeast strains resulted in the doubling of the triacylglycerol content. Furthermore, a complete lipid analysis of the transformed wild-type yeast showed that its phospholipid content was lower than that of the control (void plasmid-transformed) yeast whereas lysophospholipids and free fatty acids increased. When microsomes from the AtLCAT3-transformed yeast were incubated with di-[1-14 C]oleyl phosphatidylcholine, both the lysophospholipid and free fatty acid fractions were highly and similarly labelled, whereas the same incubation with microsomes from the control yeast produced a negligible labelling of these fractions. Moreover when microsomes from AtLCAT3transformed yeast were incubated with either sn-1-or sn-2-[1-14 C]acyl phosphatidylcholine, the distribution of the labelling between the free fatty acid and the lysophosphatidylcholine fractions strongly suggested a phospholipase A1 activity for AtLCAT3. The sn-1 specificity of this phospholipase was confirmed by gas chromatography analysis of the hydrolysis of 1-myristoyl, 2-oleyl phosphatidylcholine. Phosphatidylethanolamine and phosphatidic acid were shown to be also hydrolysed by AtLCAT3, although less efficiently than phosphatidylcholine. Lysophospatidylcholine was a weak substrate whereas tripalmitoylglycerol and cholesteryl oleate were not hydrolysed at all. This novel A. thaliana phospholipase A1 shows optimal activity at pH 6-6.5 and 60-65°C and appears to be unaffected by Ca 2+ . Its sequence is unrelated to all other known phospholipases. Further studies are in progress to elucidate its physiological role.
Biochimica et Biophysica Acta (BBA) - Biomembranes, 1995
The study of phospholipase activities associated with the tonoplast of Acer pseudoplatanus was performed in vitro with sn-2-[14C]acylphosphatidylcholine (PC) as a substrate. The hydrolysis of radiolabelled PC into [14C]phosphatidic acid and [14C]lyso-PC demonstrated the presence of phospholipase D and A 1 activities, respectively, associated with the tonoplast of Acer pseudoplatanus. The vacuolar sap did not show any significant phospholipase activity. In a second step, the properties of the phospholipase A 1 activity was studied using tonoplast endogenous PC labelled in vivo with [lgc]choline as a substrate. The phospholipase A 1 showed an optimal activity at pH about 6-6.5, did not necessarily require divalent cations, but was stimulated by Mg 2+ and particularly by Ca 2+. This work presents the first evidence for the presence of phospholipases A 1 in plant cells.
Functional Characterization of Putative Non-Specific Phospholipase C (NPC) in Arabidopsis thaliana
2011
Phospholipases are enzymes that hydrolyze phospholipids. In terms of the position of bond hydrolysis, phospholipases are classified into four major types: phospholipase C (PLC), phospholipase D (PLD), phospholipase A1 (PLA1) and phospholipase A2 (PLA2). PLC hydrolyzes phospholipids at the first phosphodiester bond, producing diacylglycerol (DAG) and a phosphorylated head group. Based on substrate specificity, PLC is divided into two distinctively different groups: phosphoinositide-specific phospholipase C (PIPLC) and non-specific phospholipase C (NPC). PI-PLC has been extensively studied and well characterized in animal systems. There are six members of the NPC family in Arabidopsis, designated NPC1 through 6 that bear sequence homology to bacterial phosphatidylcholine hydrolyzing PLC (PC-PLC). However, their function remains largely unknown. This project was undertaken to study the biochemical properties and the physiological function of this family of enzymes in Arabidopsis. Histi...
Some Characteristics of Phospholipase C from Bacillus cereus
European Journal of Biochemistry, 1977
1. The amino acid composition of purified Bacillus cereus phospholipase C is reported. The enzyme contains one methionine residue and two fragments are obtained after cyanogen bromide cleavage. The sequence of the amino-terminal fragment (25 residues) is reported.
Journal of Experimental Botany, 2004
Two cDNAs encoding proteins, PpPLC1 and PpPLC2, with catalytic and C2 domains conserved in plant phosphoinositide-spe-ci®c phospholipase C (PI-PLC) were isolated from Physcomitrella patens. The N domain, which has been identi®ed in Arabidopsis PI-PLCs as an EF hand-like domain, was found in both isoforms, although that in PpPLC2 was a split type. At micromolar Ca 2+ concentrations, PpPLC1 preferentially hydrolysed phosphatidylinositol-4,5-bisphosphate, while PpPLC2 showed no speci®city. Furthermore, at millimolar Ca 2+ , phosphatidylinositol was hydrolysed by PpPLC2, but not by PpPLC1. Thus, PpPLC1 and PpPLC2 are typical and novel types of plant PI-PLC, respectively.
Phosphatidylinositol 4,5-bisphosphate specific phospholipase C in Pharbitis nil membranes
Biologia Plantarum, 1992
Phosphatidylinositol 4,5-bisphosphate specific phospholipase C has been detected in a membrane preparation fromPharbitis nil cotyledons. The enzyme has a pH optimum of 6.8 and activated by calcium ions, deoxycholate, phosphatidylinositol and phosphatidylethanolamine. The enzyme is inhibited to varying degrees by Tween 20, Triton XI00, zinc, copper, cobalt and manganese ions and phosphatidylserine. G-protein activators do not affect the activity ofPharbitis nil phospholipase C. Analysis of the products of the reaction by HPLC shows inositol 1,4,5-trisphosphate from phospholipase C and inositol bisphosphate from inositol-1 and -5 phosphatase activity.
Properties of phospholipase C isolated from rat liver lysosomes
The Journal of biological chemistry, 1980
Phospholipase C (EC 3.1.4.3) has been identified in a soluble, delipidated protein fraction isolated from rat liver lysosomes. Lysosomal phospholipase C is active against all phospholipids tested, including phosphatidylcholine, phosphatidylinositol, phosphatidylglycerol, phosphatidylethanolamine, and phosphatidylserine. It has an acid pH optimum, does not require divalent cations, and is not inhibited by EDTA. With [1-14C]dioleoylphosphatidylcholine as the substrate, 14C-labeled monoglyceride and diglyceride are the reaction products. Monoglyceride is formed rapidly from diglyceride by a lysosomal acid lipase, although some monoglyceride may be formed directly by phospholipase C hydrolysis of lysophosphatidylcholine. The other product, phosphocholine, has been identified by its behavior during Dowex 1-formate anion exchange chromatography. This appears to be the first demonstration in mammalian systems ofa phospholipase C which is active against all phosphoglycerides.
Purification and Characterization of Phospholipase D from Cabbage Leaves
Food Science and Technology Research, 2000
Phospholipase D (PLD) was purified from cabbage leaves. The molecular weight of purified PLD was estimated as approximately 73 and 87 kDa by gel filtration using Superdex 200 HR column and, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), respectively! The transphosphatidylation capacity for phosphatidylcholine of this enzyme reached over 90 % , and the enzyme's hydrolysis efficiency for phosphatidylcholine was five-fold higher than that for phosphatidylglycerol. These findings indicated that the cabbage PLD efficiently transferred phosphatidylcholine to phosphatidylglycerol. On N-terminal amino acid sequence analysis of the band separated by SDS-PAGE, two sequences with differing N-terminus were detected. This N-terminal difference may have been generated by processing during maturation of PLD.
Purification and properties of phospholipase A from porcine pancreas
Biochimica et biophysica acta, 1968
Phospholipase A,, purified from the culture filtrate of Corticium centrifugum, was found to possess lysophospholipase 1 activity. The isoelectric point was pH 3.3, and the molecular weight was about 26,800. Both enzyme activities had their pH optimum between 4.0 and 4.5 and their pH stability between 6.0 and 8.0, and were heat-unstable. Both were inhibited by p-diazobenzenesulfonic acid and N-bromosuccinimide. Although ether, 1-propanol and Triton X-100 stimulated phospholipase AI activity, these substances showed an inhibitory effect against lysophospholipase I activity. Phospholipase A, activity was almost completely inhibited by Fe2+, Fe3+ and Al3+ but the inhibition was lessened by the presence of Triton X-100. Lysophospholipase 1 activity was inhibited by Hg2+, Fe3+ and Al3+. The mode of inhibition by Felt against lysophospholipase 1 activity was apparently an uncompetitive type. Phospholipase A1 hydrolyzed various phospholipids, but not triolein.
Evidence for the control of the action of phospholipases A by the physical state of the substrate
Biochemistry, 1984
Abbreviations: diClzPG, 1,2-didodecanoyl-sn-glycero-3-phosphorac-glycerol; diEPG, 1,2-dihexadecyl-sn-glycero-3-phospho-rac-glycerol; diPBPG, 1,2-bis[(pyren-l-yl)butanoyl]-sn-glycero-3-phospho-racglycerol; cmc, critical micellar concentration; ffa, free fatty acid; I,, pyrene excimer emission intensity; I,, pyrene monomer emission intensity; PLA, phospholipase A; PLAI, phospholipase A,; PLA,, phospholipase A,; Tris-HC1, tris(hydroxymethy1)aminomethane hydrochloride; EDTA, ethylenediaminetetraacetic acid.
Secretory Phospholipases A2 in Plants
Frontiers in Plant Science
Secreted phospholipases (sPLA 2 s) in plants are a growing group of enzymes that catalyze the hydrolysis of sn-2 glycerophospholipids to lysophospholipids and free fatty acids. Until today, around only 20 sPLA 2 s were reported from plants. This review discusses the newly acquired information on plant sPLA 2 s including molecular, biochemical, catalytic, and functional aspects. The comparative analysis also includes phylogenetic, evolutionary, and tridimensional structure. The observations with emphasis in Glycine max sPLA 2 are compared with the available data reported for all plants sPLA 2 s and with those described for animals (mainly from pancreatic juice and venoms sources).
The EMBO Journal, 1994
The microorganism Dictyostelium uses extracellular cAMP to induce chemotaxis and cell differentiation. Signals are transduced via surface receptors, which activate G proteins, to effector enzymes. The deduced protein sequence of Dictyostelium discoideum phosphatidylinositol-specific phospholipase C (PLC) shows strong homology with the mammalian PLC-6 isoforms. To study the role of PLC in Dictyostelium, a plcmutant was constructed by disruption of the PLC gene. No basal or stimulated PLC activity could be measured during the whole developmental programme of the plccells. Loss of PLC activity did not result in a visible alteration of growth or development. Further analysis showed that developmental gene regulation, cAMP-mediated chemotaxis and activation of guanylyl and adenylyl cyclase were normal. Although the cells lack PLC activity, inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] was present at only slightly lower concentrations compared with control cells. Mass analysis of inositol phosphates demonstrated the presence of a broad spectrum of inositol phosphates in Dictyostelium, which was unaltered in the plcmutant. Cell labelling experiments with [3H]inositol indicated that [3H]Ins(1,4,5)P3 was formed in a different manner in the mutant than in control cells.
Phosphatidylcholine-specific phospholipase C from Achromobacter xylosoxidans
Acta microbiologica Bulgarica
A new phosphatidylcholine-specific phospholipase C (EC 3.1.4.3.) has been isolated from the culture broth of Achromobacter xylosoxidans. Growth conditions were optimized for maximum production of the enzyme. A temperature-sensitive synthesis was established. Chromatography on CM Sephadex and polyacrylamide gel electrophoresis of the native enzyme revealed a number of isozymes. The water soluble substrate for phospholipase p-nitrophenylphosphorylcholine was not hydrolysed by the enzyme.