An acid phospholipase C from Tetrahymena culture medium (original) (raw)
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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.
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.
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.
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.
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
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.
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.
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.
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.