Rodriguez A, Sarda P, Nessmann C, Boulot P, Leger C L, Descomps B
Laboratoire Biologie et Biochimie des Lipides EA DRED 2033, Faculté de Médecine, Institut de Biologie Boulevard Henri IV, Montpellier, France.
J Lipid Res. 1998 Sep;39(9):1825-32.
Delta6- and delta5-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 delta6-desaturase activity for the n-3 series than for the n-6 series. Apparent Km of 6.5, 3.9, and 24.5 microM and Vm of 7.5, 9.1, and 24.4 pmol x min(-1) x mg(-1) were obtained, respectively, for 18:2n-6 delta6-, 20:3n-6 delta5-, and 18:3n-3 delta6-desaturases. Beyond 30, 20, and 60 microM 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 delta6- and delta5-desaturase activities. Comparison of concentrations of the respective endogenous substrates and the kinetic constants of each enzyme suggested that the higher delta6-desaturase activity observed for the n-3 series than for the n-6 series is not physiologically relevant in human fetal liver.
在经合法批准的治疗性流产后获取的人胎儿肝脏微粒体中研究了Δ6-和Δ5-去饱和酶活性。酶活性通过使用反相高效液相色谱法（HPLC）的放射化学方法进行测量。通过薄层色谱法（TLC）和气液色谱法（GLC）相结合的方法评估每个肝脏样品中的游离脂肪酸和磷脂脂肪酸。动力学测量表明，n-3系列的Δ6-去饱和酶活性高于n-6系列。对于18:2n-6 Δ6-、20:3n-6 Δ5-和18:3n-3 Δ6-去饱和酶，表观Km分别为6.5、3.9和24.5微摩尔，Vm分别为7.5、9.1和24.4皮摩尔×分钟-1×毫克-1。当18:2n-6、20:3n-6和18:3n-3的浓度分别超过30、20和60微摩尔时，酶活性偏离米氏动力学，表明过量底物存在抑制作用，而由于内源性底物浓度低得多，这种情况在体内不太可能发生。我们观察到，无论酶或底物如何，当微粒体蛋白浓度超过4 - 5毫克时，去饱和酶活性与微粒体蛋白浓度之间的线性关系就会被破坏。在测定Δ6-和Δ5-去饱和酶活性时，应考虑到这种现象以及过量底物引起的抑制作用。各内源性底物浓度与每种酶的动力学常数的比较表明，在人胎儿肝脏中观察到的n-3系列比n-6系列更高的Δ6-去饱和酶活性在生理上并不相关。
