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Long‐chain Fatty Acid:CoA Ligase in Rat Brain in vitro: A Comparison of Activities with Oleic and cis ‐Vaccenic Acids
Author(s) -
Murphy M. G.,
Spence M. W.
Publication year - 1980
Publication title -
journal of neurochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.75
H-Index - 229
eISSN - 1471-4159
pISSN - 0022-3042
DOI - 10.1111/j.1471-4159.1980.tb06606.x
Subject(s) - oleic acid , fatty acid , coenzyme a , biochemistry , chemistry , microsome , chromatography , in vitro , stereochemistry , enzyme , reductase
Long‐chain fatty acid:CoA ligase (EC 6.2.1.3) has been examined in vitro with brain preparations from 12‐ to 16‐day‐old rats using oleic (18: 1[ n‐9 ]) and cis ‐vaccenic (18: 1[ n‐7 ]) acids. A novel assay system, which permits measurement of product as [ 14 C]fatty acyl‐[ 3 H]CoA, [ 14 C]fatty acyl‐CoA, or fatty acyl‐[ 3 H]CoA, was used. With the double‐label assay, reaction conditions were established that gave a molar ratio of [ 3 H]CoA to [ 14 C]fatty acid in the product of 1.06 ± 0.12 (mean ± s.d ., n = 14). This indicates that the method is specific for the fatty acid added to the incubation mixture. The assay is sensitive to the formation of approximately 0.02 nmol fatty acyl‐CoA. With oleic acid, specific activity of the ligase was highest in a microsomal fraction (3.91 ± 0.55 nmol oleoyl‐CoA formed/min/mg protein; n = 16). Optimal activity was obtained at concentrations of fatty acid, CoA, ATP and Mg 2+ of 50 μM, 50 μM, 10 m m , and 8 m m , respectively. The apparent K m s (approximate) for oleic acid and coenzyme A were 20 and 6 μM, respectively. With cis ‐vaccenic acid (18:1[ n‐7 ]), a naturally occurring isomer of oleate, the maximum reaction rate was higher than that with oleate, as were the apparent K m s for both fatty acid (105 μM) and coenzyme A (23 μM).