Measurement of the acyl-CoA intermediates of beta-oxidation by h.p.l.c. with on-line radiochemical and photodiode-array detection. Application to the study of [U-14C]hexadecanoate oxidation by intact rat liver mitochondria.
The quantitative isolation of acyl-CoA esters of chain length C2-C17 from mitochondrial incubations and their analysis by reverse-phase radio-h.p.l.c. is described. Photodiode-array detection was used to characterize 2-enoyl-CoA esters. The chromatographic behaviour of all 27 intermediates of the beta-oxidation of hexadecanoyl-CoA is documented. Only C16, C14 and C12 intermediates were detected in uncoupled mitochondria oxidizing [U-14C]hexadecanoyl-CoA in the presence of fluorocitrate and carnitine, providing evidence for some organization of the enzymes of beta-oxidation [Garland, Shepherd & Yates (1965) Biochem. J. 97, 587-594; Sumegi & Srere (1984) J. Biol. Chem. 259, 8748-8752]. Rotenone increased concentrations of 3-hydroxyacyl-CoA and 2-enoyl-CoA esters and inhibited flux. These experiments provide the first direct unambiguous measurements of acyl-CoA esters in intact respiring rat liver mitochondrial fractions.
Watmough
N J
NJ
Department of Neurology, University of Newcastle upon Tyne, U.K.
Turnbull
D M
DM
Sherratt
H S
HS
Bartlett
K
K
D000214:Acyl Coenzyme A / Q000302:isolation & purification / Q000378:metabolism*; D000818:Animals; D002331:Carnitine / Q000494:pharmacology; D002851:Chromatography, High Pressure Liquid; D008930:Mitochondria, Liver / Q000378:metabolism*; D010084:Oxidation-Reduction; D010169:Palmitic Acids / Q000378:metabolism*; D010171:Palmitoyl Coenzyme A / Q000302:isolation & purification; D051381:Rats; D012402:Rotenone / Q000494:pharmacology
D000214:Acyl Coenzyme A; D010169:Palmitic Acids; D012402:Rotenone; C034681:octanoyl-coenzyme A; D010171:Palmitoyl Coenzyme A; C024343:butyryl-coenzyme A; D002331:Carnitine