Phospholipase B from the plasma membrane of Saccharomyces cerevisiae Separation of two forms with different carbohydrate content

Witt, Wolfgang; Schweingruber, M. Ernst; Mertsching, Angelika

doi:10.1016/0005-2760(84)90110-3

Cited by 57 publications

(31 citation statements)

References 19 publications

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“…This is consistent with the hypothetical multimeric structure of cell-associated PLB purified from S. pombe and T delbrueckii [15,16], but unlike that of the water-soluble form of PLB from S. cere isiae [34]. The apparent molecular mass of C. neoformans phospholipase on SDS\PAGE compares with 84 kDa for C. albicans, 100-150 kDa for S. pombe, 145-280 kDa for S. cere isiae, and 140-190 kDa for T. delbrueckii [12,[14][15][16]18], suggesting the possibility of structural differences between PLB enzymes of pathogenic fungi and industrial yeasts.…”

Section: Discussionmentioning

confidence: 99%

“…Studies of fungal phos-pholipases, however, have mainly focused on the characterization of phospholipase B (PLB ; EC 3.1.1.5) from non-pathogenic fungi, including Saccharomyces cere isiae, Torulaspora delbrueckii, Schizosaccharomyces pombe and Penicillium notatum [11][12][13][14][15][16]. In some instances, lysophospholipase (LPL ; EC 3.1.1.5) and lysophospholipase\transacylase (LPTA ; EC 3.1.1.5) activities have also been described [11,13,16], but the biological functions of these phospholipases remain undetermined.…”

Section: Introductionmentioning

confidence: 99%

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Purification and characterization of secretory phospholipase B, lysophospholipase and lysophospholipase/transacylase from a virulent strain of the pathogenic fungus Cryptococcus neoformans

Chen

Wright

GOLDING³

et al. 2000

Biochemical Journal

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Infection caused by the fungus Cryptococcus neoformans is potentially fatal. A highly active extracellular phospholipase, demonstrating phospholipase B (PLB), lysophospholipase (LPL) and lysophospholipase\transacylase (LPTA) activities, was purified to homogeneity from C. neoformans using (NH % ) # SO % fractionation, and hydrophobic-interaction, anion-exchange and gel-filtration chromatography. All three enzyme activities copurified as a single protein with an apparent molecular mass of 70-90 kDa by SDS\PAGE and 160-180 kDa by gel filtration. The ratio of the three activities remained constant after each purification step. The amino acid composition, as well as the sequences of the N-terminus and of five internal peptide fragments were novel. The protein was an acidic glycoprotein containing Nlinked carbohydrate moieties, with pI values of 5.5 and 3.5. The apparent V max values for PLB and LPL activities were 12.3 and 870 µmol\min per mg of protein respectively ; the corresponding

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Purification and characterization of secretory phospholipase B, lysophospholipase and lysophospholipase/transacylase from a virulent strain of the pathogenic fungus Cryptococcus neoformans

Chen

Wright

GOLDING³

et al. 2000

Biochemical Journal

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“…The location of these enzymes within the cell (cytoplasmic, organellar, membrane-bound, cell wall or periplasmic space) is unknown, but the data in Table 3 indicate that PLB, LPL and LPTA activities are released spontaneously over time, or immediately by mechanical disruption. Intracellular pools of enzyme activity have also been noted in Saccharomyces cerevisiae [29] and C. albicans [14], although the amount of total activity which is intracellular was not reported for these yeasts. The ratio of LPL/PLB activity in the supernate of C. neoformans cells disrupted immediately after harvesting was greater than that obtained after the cells were allowed to secrete the enzymes overnight (Table 3), suggesting that separate intracellular locations may exist for LPL and PLB.…”

Section: Discussionmentioning

confidence: 99%

Biochemical and Functional Characterisation of Secreted Phospholipase Activities from Cryptococcus Neoformans in their Naturally Occurring State

Santangelo

Nouri-Sorkhabi

Sorrell

et al. 1999

Journal of Medical Microbiology

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A recent study demonstrated that phospholipase B (PLB), lysophospholipase (LPL) and lysophopholipase transacylase (LPTA) are secreted by Cryptococcus neoformans var. neoformans and showed that the amount of enzyme production correlated with virulence in mice. The present study characterised the extracellular enzyme activities further by radiometric assays and 31 P nuclear magnetic resonance spectroscopy (NMR). All three enzymes were most active between 25 and 40OC. Bovine lung surfactant and its major lipid components, disaturated phosphatidylcholine and phosphatidylglycerol, were the optimal substrates for PLB. Lysophosphatidylcholine was the favoured substrate for LPL and LPTA. PLB and LPL/LPTA were differentially affected by Triton X-100, and palmitoyl carnitine was a potent inhibitor of the three phospholipases. LPL and PLB activities were inhibited by dithiothreitol; N-ethylmaleimide inhibited LPL and LPTA activities. None of the enzymes was inhibited by N-bromosuccinimide or p-bromophenacyl bromide. Cellular disruption experiments indicated that > 85% of the phospholipase activities were cell-associated, with LPL and LPTA being more easily released than PLB. At pH 5.5 and 7.0, the heat-inactivated secreted enzyme preparations decreased the viability of human neutrophils. This effect was attenuated by active supernates. The relative activities of the PLB, LPL and LPTA in the environment of neutrophils are likely to determine the fate of these cells in vivo. Both phospholipases and heat-stable substances secreted by C. neoformans at 37°C could contribute to membrane degradation and virulence.

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“…The highly glycosylated enzyme of about 220 kDa (73 kDa for the protein part, predicted from the sequence) is enriched in the yeast plasma membrane but was also found in the periplasmic space and in the culture supernatant. The lysophospholipase activity of Plb1p greatly exceeds the activity catalyzing the first step of hydrolysis; thus, lyso-phospholipids do not accumulate as intermediate products of Plb1p activity (2)(3)(4). In addition, this enzyme has transacylase activity, catalyzing the synthesis of phosphatidylcholine (PtdCho) 1 from two molecules of lyso-phosphatidylcholine.…”

mentioning

confidence: 99%

Characterization and Function in Vivo of Two Novel Phospholipases B/Lysophospholipases fromSaccharomyces cerevisiae

Merkel

Fido

Mayr

et al. 1999

Journal of Biological Chemistry

102

118

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The yeast genome contains two genes, designated as PLB2 and PLB3, that are 67% and 62% identical, respectively, to PLB1, which codes for a phospholipase B/lysophospholipase in yeast (Lee, S. K., Patton, J. L., Fido, M., Hines, L. K., Kohlwein, S. D., Paltauf, F., Henry, S. A., and Levin, D. E. (1994) J. Biol. Chem. 269, 19725-19730). Deletion and overexpression studies and in vivo and in vitro activity measurements suggest that both genes indeed code for phospholipases B/lysophospholipases. In cell free extracts of a plb1 plb2 plb3 triple mutant, no phospholipase B activity was detectable. Upon overexpression of PLB2 in a plb1 plb3 mutant background, phospholipase B activity was detectable in the plasma membrane, periplasmic space extracts and the culture supernatant. Similar to Plb1p, Plb2p appears to accept all major phospholipid classes, with a preference for acidic phospholipids including phosphatidylinositol 3,4-bisphosphate and phosphatidic acid. Consistent with a function as an extracellular lysophospholipase, PLB2 overexpression conferred resistance to lyso-phosphatidylcholine. Deletion of Plb2p function had no effect on glycerophosphoinositol or glycerophosphocholine release in vivo, in contrast to a deletion of Plb3p function, which resulted in a 50% reduction of phosphatidylinositol breakdown and glycerophosphoinositol release from the cells. In vitro, Plb3p hydrolyzes only phosphatidylinositol and phosphatidylserine and, to a lesser extent, their lysoanalogs. Plb3p activity in a plb1 plb2 mutant background was observed in periplasmic space extracts. Both Plb3p and Plb2p display transacylase activity in vitro, in the presence or absence, respectively, of detergent.Phospholipases B catalyze the hydrolytic cleavage of both acylester bonds of glycerophospholipids. Products of phospholipase B activity are fatty acids and water-soluble glycerophosphodiesters. In yeast, soluble degradation products are released to some extent into the culture medium, and are thus an indicator of cellular phospholipase B activity. The only acylester-hydrolyzing enzyme from yeast characterized so far at the molecular level is a phospholipase B encoded by the PLB1 gene (1). The highly glycosylated enzyme of about 220 kDa (73 kDa for the protein part, predicted from the sequence) is enriched in the yeast plasma membrane but was also found in the periplasmic space and in the culture supernatant. The lysophospholipase activity of Plb1p greatly exceeds the activity catalyzing the first step of hydrolysis; thus, lyso-phospholipids do not accumulate as intermediate products of Plb1p activity (2-4). In addition, this enzyme has transacylase activity, catalyzing the synthesis of phosphatidylcholine (PtdCho) 1 from two molecules of lyso-phosphatidylcholine. The physiological function of yeast phospholipase B is still unclear; neither disruption of the PLB1 gene nor its overexpression result in detectable growth phenotypes. However, the amount of glycerophosphocholine (GroPCho) and glycerophosphoethanolamine released into the culture su...

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Phospholipase B from the plasma membrane of Saccharomyces cerevisiae Separation of two forms with different carbohydrate content

Cited by 57 publications

References 19 publications

Purification and characterization of secretory phospholipase B, lysophospholipase and lysophospholipase/transacylase from a virulent strain of the pathogenic fungus Cryptococcus neoformans

Purification and characterization of secretory phospholipase B, lysophospholipase and lysophospholipase/transacylase from a virulent strain of the pathogenic fungus Cryptococcus neoformans

Biochemical and Functional Characterisation of Secreted Phospholipase Activities from Cryptococcus Neoformans in their Naturally Occurring State

Characterization and Function in Vivo of Two Novel Phospholipases B/Lysophospholipases fromSaccharomyces cerevisiae

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