Delta 6-desaturase: improved methodology and analysis of the kinetics in a multi-enzyme system.
A new method of assay for the delta 6-desaturation of linoleic acid was developed. This method, which uses HPLC for separation of the fatty acid substrate and product, exhibited a lower coefficient of variation (0.3%) than the reported TLC method (3.5%), and avoided the step of methylation of the saponified fatty acid substrate and product. Using this new method of assay, the kinetics of the delta 6-desaturase in a multi-enzyme system were analysed. A number of factors that could have striking effects on desaturase kinetics were investigated, including the effect of (i) endogenous microsomal linoleic acid on total substrate concentration, and (ii) the pre-reaction catalysed by acyl-CoA synthetase and competing reactions catalysed by lysophospholipid acyltransferase and acyl-CoA hydrolase. Endogenous free linoleate in the hepatic microsomes was found to be 2.9 +/- 1.0 microM (0.5 mg microsomal protein/ml), which was comparable to added substrate concentrations (1.8 to 7.9 microM). The kinetics of the delta 6-desaturase were dissected from the kinetics of the above mentioned pre-reaction and competing reactions through a combination of experimental approaches and computer modeling. From computer modeling, a Km and Vmax of 1.5 microM and 0.63 nmol/min were calculated for the delta 6-desaturase, compared to Km and Vmax of 10.7 microM and 0.08 nmol/min calculated directly from data uncorrected for endogenous substrate. It was concluded that lysophospholipid acyltransferase, acyl-CoA synthetase and endogenous linoleic acid significantly affect the kinetic measurements of hepatic microsomal delta 6-desaturase. These results have implications for kinetic analyses of all desaturates in microsomal systems.
Ivanetich
K M
KM
Biomolecular Resource Center, University of California at San Francisco 94143-0541, USA.
Bradshaw
J J
JJ
Ziman
M R
MR
D008243:1-Acylglycerophosphocholine O-Acyltransferase / Q000378:metabolism; D000214:Acyl Coenzyme A / Q000378:metabolism; D000818:Animals; D002851:Chromatography, High Pressure Liquid; D002855:Chromatography, Thin Layer; D003066:Coenzyme A Ligases / Q000378:metabolism; D003198:Computer Simulation; D004789:Enzyme Activation; D044943:Fatty Acid Desaturases / Q000032:analysis / Q000378:metabolism*; D005227:Fatty Acids / Q000032:analysis / Q000378:metabolism; D007700:Kinetics; D019787:Linoleic Acid; D008041:Linoleic Acids / Q000378:metabolism* / Q000494:pharmacology; D050602:Linoleoyl-CoA Desaturase; D008297:Male; D008862:Microsomes, Liver / Q000201:enzymology*; D008956:Models, Chemical; D051381:Rats; D011919:Rats, Inbred Strains; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins; D000098859:Long-Chain-Fatty-Acid-CoA Ligase
D000214:Acyl Coenzyme A; D005227:Fatty Acids; D008041:Linoleic Acids; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins; D019787:Linoleic Acid; D044943:Fatty Acid Desaturases; D050602:Linoleoyl-CoA Desaturase; D008243:1-Acylglycerophosphocholine O-Acyltransferase; D003066:Coenzyme A Ligases; C090799:FAA2 protein, S cerevisiae; D000098859:Long-Chain-Fatty-Acid-CoA Ligase