Shedding and isolation of the delta 6-desaturase system from rat liver microsomes by application of high hydrostatic pressure.
A procedure using high hydrostatic pressure without detergent has been applied in this study to subfractionate the components of the delta 6-desaturase system from the rat liver endoplasmic reticulum. Microsomes were suspended in a buffer containing liposomes made of phosphatidylcholine. The mixture was placed in a sealed pressure bomb and subjected to hydrostatic pressure of up to 1500 bars. Under these conditions the total desaturase activity was found in the liposomal fraction thus indicating that the three components of the desaturase system, namely NADH-cytochrome b5 reductase, cytochrome b5 and the delta 6-desaturase co-extracted. Size chromatography and FPLC of the released proteins followed by SDS-PAGE confirmed the independent release of the three components corresponding to the delta 6-desaturase, system. delta 6-Desaturase activity could be fully regenerated by mixing the aqueous dispersions of the three components without further purification. Our results indicate that these components are physically facing a similar lipid environment in the microsomal membrane.
Leikin
A
A
Department of Membrane Research and Biophysics, Weizmann Institute of Science, Rehovot, Israel.
Shinitzky
M
M
D000818:Animals; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D003579:Cytochrome Reductases / Q000302:isolation & purification; D042966:Cytochrome-B(5) Reductase; D015786:Cytochromes b5 / Q000302:isolation & purification; D004591:Electrophoresis, Polyacrylamide Gel; D044943:Fatty Acid Desaturases / Q000302:isolation & purification* / Q000378:metabolism; D006874:Hydrostatic Pressure; D050602:Linoleoyl-CoA Desaturase; D008081:Liposomes / Q000378:metabolism; D008297:Male; D008862:Microsomes, Liver / Q000201:enzymology*; D010713:Phosphatidylcholines; D051381:Rats; D017208:Rats, Wistar
D008081:Liposomes; D010713:Phosphatidylcholines; D015786:Cytochromes b5; D044943:Fatty Acid Desaturases; D050602:Linoleoyl-CoA Desaturase; D003579:Cytochrome Reductases; D042966:Cytochrome-B(5) Reductase