Delta6- and delta5-desaturase activities in the human fetal liver: kinetic aspects.
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.
Rodriguez
A
A
Laboratoire Biologie et Biochimie des Lipides EA DRED 2033, Faculté de Médecine, Institut de Biologie Boulevard Henri IV, Montpellier, France.
Sarda
P
P
Nessmann
C
C
Boulot
P
P
Leger
C L
CL
Descomps
B
B
D000090943:Delta-5 Fatty Acid Desaturase; D004791:Enzyme Inhibitors / Q000494:pharmacology; D044943:Fatty Acid Desaturases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D005230:Fatty Acids, Nonesterified / Q000378:metabolism; D005865:Gestational Age; D006801:Humans; D007700:Kinetics; D019787:Linoleic Acid / Q000008:administration & dosage / Q000378:metabolism / Q000494:pharmacology; D050602:Linoleoyl-CoA Desaturase; D008099:Liver / Q000196:embryology* / Q000201:enzymology*; D008862:Microsomes, Liver / Q000201:enzymology; D010743:Phospholipids / Q000378:metabolism
D000090943:Delta-5 Fatty Acid Desaturase; D004791:Enzyme Inhibitors; D005230:Fatty Acids, Nonesterified; D010743:Phospholipids; D019787:Linoleic Acid; D044943:Fatty Acid Desaturases; D050602:Linoleoyl-CoA Desaturase