Δ6- and Δ5-desaturase activities in the human fetal liver: kinetic aspects
Δ6- and Δ5-desaturase activities were studied in human fetal liver microsomes obtained after legally approved therapeutic abortion. Enzyme activities were measured by a radiochemical method using reverse-phase high performance liquid chromatography (HPLC). Free and phospholipid fatty acids were asse...
| Main Authors: | , , , , , |
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| Format: | Article |
| Language: | English |
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Elsevier
1998-09-01
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| Series: | Journal of Lipid Research |
| Subjects: | |
| Online Access: | http://www.sciencedirect.com/science/article/pii/S0022227520321702 |
| _version_ | 1818451712727318528 |
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| author | Annie Rodriguez Pierre Sarda Catherine Nessmann Pierre Boulot Claude Louis Leger Bernard Descomps |
| author_facet | Annie Rodriguez Pierre Sarda Catherine Nessmann Pierre Boulot Claude Louis Leger Bernard Descomps |
| author_sort | Annie Rodriguez |
| collection | DOAJ |
| description | Δ6- and Δ5-desaturase activities were studied in human fetal liver microsomes obtained after legally approved therapeutic abortion. Enzyme activities were measured by a radiochemical method using reverse-phase high performance liquid chromatography (HPLC). Free and phospholipid fatty acids were assessed in each liver sample by a combination of thin-layer chromatography (TLC) and gas–liquid chromatography (GLC) procedures. The kinetic measurements showed higher Δ6-desaturase activity for the n–3 series than for the n–6 series. Apparent Km of 6.5, 3.9, and 24.5 μm and Vm of 7.5, 9.1, and 24.4 pmol·min-1·mg-1 were obtained, respectively, for 18:2n–6 Δ6-, 20:3n–6 Δ5-, and 18:3n–3 Δ6-desaturases. Beyond 30, 20, and 60 μm of 18:2n–6, 20:3n–6, and 18:3n–3 concentration, respectively, the enzyme activity deviated from Michaelis-Menten kinetics, suggesting an inhibition by excess substrate which is unlikely to occur in vivo as endogenous substrate concentration is much lower. We observed a breakdown in linearity between desaturase activity and microsomal protein concentration beyond 4–5 mg microsomal protein, whatever the enzyme or substrate. Both this phenomenon and the inhibition due to excess substrate should be taken into account in the determination of Δ6- and Δ5-desaturase activities. Comparison of concentrations of the respective endogenous substrates and the kinetic constants of each enzyme suggested that the higher Δ6-desaturase activity observed for the n–3 series than for the n–6 series is not physiologically relevant in human fetal liver.—Rodriguez, A., P. Sarda, C. Nessmann, P. Boulot, C. L. Leger, and B. Descomps. Δ6- and Δ5-desaturase activities in the human fetal liver: kinetic aspects. |
| first_indexed | 2024-12-14T21:11:34Z |
| format | Article |
| id | doaj.art-636613576ef64ef997b666cbffbb264f |
| institution | Directory Open Access Journal |
| issn | 0022-2275 |
| language | English |
| last_indexed | 2024-12-14T21:11:34Z |
| publishDate | 1998-09-01 |
| publisher | Elsevier |
| record_format | Article |
| series | Journal of Lipid Research |
| spelling | doaj.art-636613576ef64ef997b666cbffbb264f2022-12-21T22:47:13ZengElsevierJournal of Lipid Research0022-22751998-09-0139918251832Δ6- and Δ5-desaturase activities in the human fetal liver: kinetic aspectsAnnie Rodriguez0Pierre Sarda1Catherine Nessmann2Pierre Boulot3Claude Louis Leger4Bernard Descomps5Laboratoire Biologie et Biochimie des Lipides EA DRED 2033, Faculté de Médecine, Institut de Biologie Boulevard Henri IV, 34060 Montpellier, FranceService Pédiatrie II, Hôpital Arnaud de Villeneuve, CHU de Montpellier, FranceService de Biologie du Développement et de la Reproduction, Hôpital Robert Debré, Paris, FranceService de Gynécologie Obstétrique, Hôpital Arnaud de Villeneuve, CHU de Montpellier, FranceLaboratoire Biologie et Biochimie des Lipides EA DRED 2033, Faculté de Médecine, Institut de Biologie Boulevard Henri IV, 34060 Montpellier, FranceTo whom correspondence should be addressed.; Laboratoire Biologie et Biochimie des Lipides EA DRED 2033, Faculté de Médecine, Institut de Biologie Boulevard Henri IV, 34060 Montpellier, FranceΔ6- and Δ5-desaturase activities were studied in human fetal liver microsomes obtained after legally approved therapeutic abortion. Enzyme activities were measured by a radiochemical method using reverse-phase high performance liquid chromatography (HPLC). Free and phospholipid fatty acids were assessed in each liver sample by a combination of thin-layer chromatography (TLC) and gas–liquid chromatography (GLC) procedures. The kinetic measurements showed higher Δ6-desaturase activity for the n–3 series than for the n–6 series. Apparent Km of 6.5, 3.9, and 24.5 μm and Vm of 7.5, 9.1, and 24.4 pmol·min-1·mg-1 were obtained, respectively, for 18:2n–6 Δ6-, 20:3n–6 Δ5-, and 18:3n–3 Δ6-desaturases. Beyond 30, 20, and 60 μm of 18:2n–6, 20:3n–6, and 18:3n–3 concentration, respectively, the enzyme activity deviated from Michaelis-Menten kinetics, suggesting an inhibition by excess substrate which is unlikely to occur in vivo as endogenous substrate concentration is much lower. We observed a breakdown in linearity between desaturase activity and microsomal protein concentration beyond 4–5 mg microsomal protein, whatever the enzyme or substrate. Both this phenomenon and the inhibition due to excess substrate should be taken into account in the determination of Δ6- and Δ5-desaturase activities. Comparison of concentrations of the respective endogenous substrates and the kinetic constants of each enzyme suggested that the higher Δ6-desaturase activity observed for the n–3 series than for the n–6 series is not physiologically relevant in human fetal liver.—Rodriguez, A., P. Sarda, C. Nessmann, P. Boulot, C. L. Leger, and B. Descomps. Δ6- and Δ5-desaturase activities in the human fetal liver: kinetic aspects.http://www.sciencedirect.com/science/article/pii/S0022227520321702kineticdesaturase activityhuman fetuslivermicrosomesendogenous substrate |
| spellingShingle | Annie Rodriguez Pierre Sarda Catherine Nessmann Pierre Boulot Claude Louis Leger Bernard Descomps Δ6- and Δ5-desaturase activities in the human fetal liver: kinetic aspects Journal of Lipid Research kinetic desaturase activity human fetus liver microsomes endogenous substrate |
| title | Δ6- and Δ5-desaturase activities in the human fetal liver: kinetic aspects |
| title_full | Δ6- and Δ5-desaturase activities in the human fetal liver: kinetic aspects |
| title_fullStr | Δ6- and Δ5-desaturase activities in the human fetal liver: kinetic aspects |
| title_full_unstemmed | Δ6- and Δ5-desaturase activities in the human fetal liver: kinetic aspects |
| title_short | Δ6- and Δ5-desaturase activities in the human fetal liver: kinetic aspects |
| title_sort | δ6 and δ5 desaturase activities in the human fetal liver kinetic aspects |
| topic | kinetic desaturase activity human fetus liver microsomes endogenous substrate |
| url | http://www.sciencedirect.com/science/article/pii/S0022227520321702 |
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