sn-1,2-Diacylglycerol Cholinephosphotransferase from Pig Liver: Mixed Micellar Assay and Kinetic Analysis of the Partially Pure Enzyme

Bru, Roque; Blöchliger, Eveline; Luisi, Pl

doi:10.1006/abbi.1993.1592

Cited by 12 publications

(9 citation statements)

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“…The usefulness of the mixed micellar assay has been demonstrated in the case of diglyceride kinase in octylglucoside/diacylglycerol mixed micelles (27). It has also proved reliable with other enzymes that act or depend on lipids, such as cholinephosphotransferase in Triton X-100/diacylglycerol/phosphatidylcholine (9) and sodium cholate/diacylglycerol (28) diolipin (29). By using C 12 E 8 /linoleic acid, a mixed micellar assay for lipoxygenase activity was designed (16).…”

Section: Resultsmentioning

confidence: 99%

An octaethylene glycol monododecyl ether‐based mixed micellar assay for determining the lipid acyl hydrolase activity of patatin

Jiménez

Escribano

Pérez‐Gilabert

et al. 2001

Lipids

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Patatin was extracted from potato tubers (Solanum tuberosum L. cv. Spunta) and purified to homogeneity by ammonium sulfate salt fractionation and one sole chromatographic step. A spectrophotometric mixed micellar assay for patatin lipid acyl hydrolase (LAH) activity was designed with the detergent octaethylene glycol monododecyl ether (C12E8). Patatin LAH used p-nitrophenyl butyrate (PNP-butyrate) as substrate when solubilized in (C12E8) micelles. In the mixed micellar system, patatin LAH responds to the PNP-butyrate surface concentration expressed as mol% (= [PNP-butyratel x 100/([detergentl critical micellar concentration)) and not to the molarity of PNP-butyrate. The kinetic parameters were determined; Vmax was independent of the mixed micelle concentration, as was Km, when expressed as mol%. However, Km was dependent on C12E8 concentration when expressed in molar concentration. C12E8/PNP-butyrate proved to be a reliable system for assaying patatin LAH activity and is superior to the commonly used Triton X-100 and SDS methods. It permits investigation of the substrate requirements of patatin LAH activity because the concentration-independent Km can be determined both in mol% and as the absolute number of substrate molecules per micelle. In addition, the detergent did not affect the enzyme activity.

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Section: Resultsmentioning

confidence: 99%

An octaethylene glycol monododecyl ether‐based mixed micellar assay for determining the lipid acyl hydrolase activity of patatin

Jiménez

Escribano

Pérez‐Gilabert

et al. 2001

Lipids

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“…The Triton X-100 assay was performed as described (36). The sodium cholate assay was modifed from the assay described by Bru et al (37) and was performed in 50 mM Tris-HCl (pH 8.0), 20 mM MgCl 2 , 20% glycerol, and 10 mol % DAG and 10 mol % PC in 1% sodium cholate using 10 -25 g of membrane protein as the enzyme source. The assay mixture was incubated at 25°C for 5 min to allow for mixed micelle formation and then initiated by the addition of CDP choline or CDP ethanolamine (0.4 mM, 2000 dpm/nmol) and incubated at 25°C for 20 min.…”

Section: Materials-[␣-mentioning

confidence: 99%

Cloning, Genomic Organization, and Characterization of a Human Cholinephosphotransferase

Henneberry

Wistow

McMaster

2000

Journal of Biological Chemistry

106

113

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A cholinephosphotransferase activity catalyzes the final step in the de novo synthesis of phosphatidylcholine via the transfer of a phosphocholine moiety from CDP choline to diacylglycerol. Ethanolaminephosphotransferase activity catalyzes a similar reaction substituting CDP ethanolamine as the phosphobase donor. We report the identification and cloning of a human cDNA (human cholinephosphotransferase (hCPT1)) that codes for a cholinephosphotransferase-specific enzyme. This was demonstrated using in vitro enzyme assays and in vivo measurement of the reconstitution of the phosphatidylcholine and phosphatidylethanolamine biosynthetic pathways in yeast cells devoid of their own endogenous cholinephosphotransferase and ethanolaminephosphotransferase activities. This contrasted with our previously cloned human choline/ethanolaminephosphotransferase cDNA that was demonstrated to code for a dual specificity choline/ethanolaminephosphotransferase. The hCPT1 and human choline/ethanolaminephosphotransferase (hCEPT1) predicted amino acid sequences possessed 60% overall identity and had only one variation in the amino acid residues within the CDP-alcohol phosphotransferase catalytic motif. In vitro assessment of hCPT1 and hCEPT1 derived cholinephosphotransferase activities also revealed differences in diradylglycerol specificities including their capacity to synthesize platelet-activating factor and platelet-activating factor precursor. Expression of the hCPT1 mRNA varied greater than 100-fold between tissues and was most abundant in testis followed by colon, small intestine, heart, prostate, and spleen. This was in marked contrast to the hCEPT1 mRNA, which has been found in similar abundance in all tissues tested to date. Both the hCPT1 and hCEPT1 enzymes were able to reconstitute the synthesis of PC in yeast to levels provided by the endogenous yeast cholinephosphotransferase; however, only hCEPT1-derived activity was able to complement the yeast CPT1 gene in its interaction with SEC14 and affect cell growth.

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“…The use of mixed micelle surface-dilution kinetic assay systems has resulted in the accurate portrayal of acyl group specificity for several integral membrane lipid-metabolizing enzymes (3,5,(24)(25)(26)(27)(28)(29)(30)(31)(32)(33)(34). The mixed micelle method presents the lipid substrate in a uniform matrix and resolves the problem of the differing lipophilic substrates, activators, or inhibitors physically altering the membrane structure in which the enzyme resides (24).…”

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confidence: 99%

PC and PE synthesis: Mixed micellar analysis of the cholinephosphotransferase and ethanolaminephosphotransferase activities of human choline/ethanolamine phosphotransferase 1 (CEPT1)

Wright

McMaster

2002

Lipids

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The human choline/ethanolamine phosphotransferase 1 (CEPT1) gene codes for a dual-specificity enzyme that catalyzes the de novo synthesis of the two major phospholipids through the transfer of a phosphobase from CDP-choline or CDP-ethanolamine to DAG to form PC and PE. We used an expression system devoid of endogenous cholinephosphotransferase and ethanolaminephosphotransferase activities to assess the diradylglycerol specificity of CEPT1. A mixed micellar assay was used to ensure that the diradylglycerols delivered were not affecting the membrane environment in which CEPT1 resides. The CEPT1 enzyme displayed an apparent Km of 36 microM for CDP-choline and 4.2 mol% for di-18:1 DAG with a Vmax of 14.3 nmol min(-1) mg(-1). When CDP-ethanolamine was used as substrate, the apparent Km was 98 microM for CDP-ethanolamine and 4.3 mol% for di-18:1 DAG with a Vmax of 8.2 nmol min(-1) mg(-1). The preferred diradylglycerol substrates used by CEPT1 with CDP-choline as the phosphobase donor were di-18:1 DAG, di-16:1 DAG, and 16:0/18:1 DAG. A major difference between previous emulsion-based assay results and the mixed micelle results was a complete inability to use 16:0(O)/2:0 as a substrate for the de novo synthesis of platelet-activating factor when the mixed micelle assay was used. When CDP-ethanolamine was used as the phosphobase donor, 16:0/18:1 DAG, di-18:1 DAG, and di-16:1 DAG were the preferred substrates. The mixed micelle assay also allowed the lipid activation of CEPT to be measured, and both the cholinephosphotransferase and ethanolaminephosphotransferase activities displayed the unusual property of product activation at 5 mol%, implying that specific lipid activation binding sites exist on CEPT1. The protein kinase C inhibitor chelerythrine and the human DAG kinase inhibitor R59949 both inhibited CEPT1 activity with IC50 values of 40 microM.

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sn-1,2-Diacylglycerol Cholinephosphotransferase from Pig Liver: Mixed Micellar Assay and Kinetic Analysis of the Partially Pure Enzyme

Cited by 12 publications

References 0 publications

An octaethylene glycol monododecyl ether‐based mixed micellar assay for determining the lipid acyl hydrolase activity of patatin

An octaethylene glycol monododecyl ether‐based mixed micellar assay for determining the lipid acyl hydrolase activity of patatin

Cloning, Genomic Organization, and Characterization of a Human Cholinephosphotransferase

PC and PE synthesis: Mixed micellar analysis of the cholinephosphotransferase and ethanolaminephosphotransferase activities of human choline/ethanolamine phosphotransferase 1 (CEPT1)

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