[13] The action of pure phospholipases on native and ghost red cell membranes

Roelofsen, B.; Zwaal, R.F.A.; Woodward, C.B.

doi:10.1016/0076-6879(74)32016-2

Cited by 15 publications

(8 citation statements)

References 13 publications

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“…SM Synthase Activity Is Specific to the Phospholipase C of P. aeruginosa-Other bacterial phospholipases C have been identified, such as sphingomyelinase and PC-specific phospholipase C from B. cereus (15,30) or S. aureus (16). These enzymes show similar substrate preferences (PC or SM) to the P. aeruginosa PlcH, and, therefore, they were evaluated for SM synthase activity.…”

Section: Characterization Of Ph Dependence and Effects Of Cations Onmentioning

confidence: 99%

Purification, Characterization, and Identification of a Sphingomyelin Synthase from Pseudomonas aeruginosa

Luberto

Stonehouse

Collins

et al. 2003

Journal of Biological Chemistry

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Sphingomyelin synthase is the enzyme that synthesizes sphingomyelin (SM) in mammalian cells by transferring a phosphorylcholine moiety from phosphatidylcholine to ceramide. Despite its importance, the gene and/or the protein responsible for this activity has not yet been identified. Here we report the purification, identification, and biochemical characterization of an enzymatic activity that synthesizes SM in Pseudomonas aeruginosa. SM synthase-like activity was found secreted in the culture medium of P. aeruginosa, strains PA01 and PAK, whereas it could not be detected in cultures of Escherichia coli. From the medium of PAK cultures, SM synthase was purified through sequential chromatographic columns. After separation on polyacrylamide-SDS gels and visualization by silver staining, the purified enzyme showed two bands, one of ϳ75 kDa and one of 30 -35 kDa. Interestingly, the highly purified SM synthase preparation also showed neutral sphingomyelinase activity. We therefore investigated whether the protein we purified as SM synthase could actually be the previously identified PlcH, a 78-kDa phospholipase C known to hydrolyze phosphatidylcholine and SM in P. aeruginosa. First, the purified SM synthase preparation contained a 78-kDa protein that reacted with monoclonal antibodies raised against purified PlcH. Second, purified PlcH showed SM synthase activity. Third, using different knockout mutant strains for the PlcH operon, PlcH was found to be necessary for SM synthase activity in P. aeruginosa. Interestingly, SM synthase activity was specific to the Pseudomonas PlcH as other bacterial phospholipases did not display SM synthase activity. Biochemical studies on the Pseudomonas SM synthase confirmed that it is a transferase, similar to the mammalian enzyme, that specifically recognizes the choline head-group and the primary hydroxyl on ceramide. This SM synthase did not have reverse transferase activity. In conclusion, the Pseudomonas PlcH also exerts SM synthase activity; therefore, for the first time, we have identified a structural gene for a SM synthase.

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Section: Characterization Of Ph Dependence and Effects Of Cations Onmentioning

confidence: 99%

Purification, Characterization, and Identification of a Sphingomyelin Synthase from Pseudomonas aeruginosa

Luberto

Stonehouse

Collins

et al. 2003

Journal of Biological Chemistry

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“…Lipids were extracted by the procedure recommended by Roelofsen et al (1974) for phospholipase C-treated membranes. Individual phospholipids were determined after separation in twodimensional t.l.c.…”

Section: Preparation Ofplateletsmentioning

confidence: 99%

Effect of phospholipase C (Bacillus cereus) on freshly isolated and 4-day-stored human platelets

Solberg¹,

Little²,

Holme³

et al. 1984

Biochemical Journal

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Phospholipase C (from Bacillus cereus) was used to study fresh and stored human platelets. Provided that the enzyme was inactivated before lipid extraction, no significant degradation of phospholipid in fresh cells was noted, even when platelets were activated or induced to change shape by ADP, collagen or thrombin. With platelets isolated from concentrates stored for transfusion for 4 days at 22 degrees C, membrane phospholipids were degraded by the enzyme to an extent depending on the pH in the platelet concentrate at day 4 of storage. The extent of phospholipid hydrolysis in platelets correlated well with the extent of release of lactate dehydrogenase during storage, with both being minimal for platelets from concentrates of final pH 6.5-6.9. Under non-lytic conditions, phosphatidylcholine was the phospholipid most degraded (40%), with no significant degradation of phosphatidylserine being detected. Storage does not seem to alter the distribution of phospholipids at the external leaflet of the plasma membrane.

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“…Below 15 vol.% this solvent will not in general influence the native structure of a protein [27]. However, increasing the dioxane concentration may give rise to an unfolding of the protein through the disruption of hydrophobic bonds which play an important role in dictating the tertiary structure of a protein [28,29J. Previous studies [30,31] from this laboratory have shown that phospholipases evoke hemolysis of erythrocytes in the presence of sublytic concentrations of sodium deoxycholate. In this study, it is demonstrated that deoxycholate, isobutanol and dioxan greatly facilitate the hydrolytic action of phospholipases towards the endogenous phosphatidylcholine molecule bound to the exchange protein.…”

Section: Introductionmentioning

confidence: 98%

Action of phospholipases on the phosphatidylcholine exchange protein from beef liver

Kamp

Sprengers

Westerman

et al. 1975

Biochimica et Biophysica Acta (BBA) - Lipids and Lipid Metaboli

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SummaryThe phos~holipases A*, C and D have been used to investigate the localization of phosphatidylcholine in the phosphatidylcholine exchange protein from beef liver. The rate of enzymatic hydrolysis of the protein-bound phosphatidylcholine was found to be very low. Addition of deoxycholate, isobutano1 or dioxane to the native protein, under conditions where delipidation did not occur, greatly enhanced the hydrolytic action of the phospholipases.From these results it is concluded that phosphatidylcholine may be buried in the protein molecule.

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[13] The action of pure phospholipases on native and ghost red cell membranes

Cited by 15 publications

References 13 publications

Purification, Characterization, and Identification of a Sphingomyelin Synthase from Pseudomonas aeruginosa

Purification, Characterization, and Identification of a Sphingomyelin Synthase from Pseudomonas aeruginosa

Effect of phospholipase C (Bacillus cereus) on freshly isolated and 4-day-stored human platelets

Action of phospholipases on the phosphatidylcholine exchange protein from beef liver

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