Δ6- and Δ5-desaturase activities in the human fetal liver: kinetic aspects

Annie Rodriguez,Pierre Sarda,Catherine Nessmann,Pierre Boulot,Claude Louis Leger,Bernard Descomps

doi:10.1016/s0022-2275(20)32170-2

Annie Rodriguez, Pierre Sarda + Show 4 more

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https://doi.org/10.1016/s0022-2275(20)32170-2

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Abstract

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.

Highlights

Abstract ⌬6- and ⌬5-desaturase activities were studied in human fetal liver microsomes obtained after legally approved therapeutic abortion
Though research about the regulation of biosynthesis and cell trafficking of C24 polyunsaturated fatty acid (PUFA) are in progress both in n–3 [4] and n–6 series [5] very little is known about the kinetics of C24 substrate conversion
The three enzyme activities deviated from Michaelis-Menten kinetics at high substrate concentration. ⌬6-desaturase activity increased up to 6.5 pmolиminϪ1иmgϪ1 within a linoleic acid concentration range of 20–30 ␮m (Fig. 1), decreased for higher substrate concentrations (Ϫ35% at 57.2 ␮m)

Summary

Introduction

Abstract ⌬6- and ⌬5-desaturase activities were studied in human fetal liver microsomes obtained after legally approved therapeutic abortion. Beyond 30, 20, and 60 ␮m 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. Comparison of concentrations of the respective endogenous substrates and the kinetic constants of each enzyme suggested that the higher ⌬6-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. ⌬6- and ⌬5-desaturase activities in the human fetal liver: kinetic aspects. A preliminary in vitro study showed that ⌬5- and ⌬6-desaturase activities could be detected, but were lower than in human adults or animals, especially rodents [7]. PUFAs are supplied to the fetus through the placenta or by fetal synthesis, essen-

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