Long‐chain Fatty Acid:CoA Ligase in Rat Brain <i>in vitro:</i> A Comparison of Activities with Oleic and <i>cis</i>‐Vaccenic Acids

Murphy, Mary; Spence, Matthew W.

doi:10.1111/j.1471-4159.1980.tb06606.x

Cited by 30 publications

(8 citation statements)

References 30 publications

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“…Triton X-100 was subsequently used only where enhancement had been observed. Microsomes showed little if any enrichment in activity over homogenate for either substrate; this result differed from previous reports [Murphy and Spence, 1980;Reddy and Bazan, 1985a,b], possibly owing to somewhat different assay conditions. The pH profiles for myelin and microsomes were similar, but showed an optimum at 8.0 with oleate ( Fig.…”

Section: Resultscontrasting

confidence: 83%

“…Myelin showed 11 % of microsomal activity with oleate as substrate and 15% with arachidonate. The effects of two detergents were tested and Triton X-100 was found to [Murphy and Spence, 1980;Reddy and Bazan, 1985a,b], possibly owing to somewhat different assay conditions. The pH profiles for myelin and microsomes were similar, but showed an optimum at 8.0 with oleate ( Fig.…”

Section: Resultsmentioning

confidence: 99%

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Long‐chain acyl‐coenzyme a synthetase in rat brain myelin

Vaswani

Ledeen

1987

J of Neuroscience Research

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Long-chain acyl-CoA synthetase (EC 6.2.1.3), an enzyme(s) that activates fatty acids prior to incorporation into phospholipids and other substances, has been detected in highly purified myelin from rat brain stem. The high levels relative to microsomes (11% and 15% for oleate and arachidonate, respectively) tended to preclude contamination by the latter membrane as the source of activity. Additional evidence came from sequential purification and mixing experiments. Km values were not appreciably different for the two substrates with the two membranes, but Vmax values were approximately 2-4-fold greater for arachidonate in both membranes. Triton X-100 increased activity somewhat in myelin but not in microsomes; with arachidonate as substrate it reduced activity in the latter. Heat inactivation studies and pH profiles suggested the presence of two different enzymes, as previously shown for other tissues.

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Section: Resultscontrasting

confidence: 83%

Section: Resultsmentioning

confidence: 99%

Long‐chain acyl‐coenzyme a synthetase in rat brain myelin

Vaswani

Ledeen

1987

J of Neuroscience Research

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“…No striking differences were observed between apparent K, for palmitic acid in both microvessel and forebrain homogenates. Apparent K, values vaned within 4.0-5.8 pM and were two-to eightfold lower than those reported by Reddy and Bazan (1983) for arachidonic acid in rat brain microsomes and by Murphy and Spence (1980) for oleic acid in rat brain microsomes and homogenate. The presence of endogenous unlabelled fatty acids cannot be entirely ruled out and might account for a substantial isotopic dilution of the radiolabelled substrate added, leading to the underestimation of K, values.…”

Section: Discussioncontrasting

confidence: 56%

Arachidonoyl‐Coenzyme A Synthetase and Nonspecific AcylCoenzyme A Synthetase Activities in Purified Rat Brain Microvessels

Morand

Carré

Homayoun

et al. 1987

Journal of Neurochemistry

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Purified rat brain microvessels were prepared to demonstrate the occurrence of acyl-CoA (EC 6.2.1.3) synthesis activity in the microvasculature of rat brain. Both arachidonoyl-CoA and palmitoyl-CoA synthesis activities showed an absolute requirement for ATP and CoA. This activity was strongly enhanced by magnesium chloride and inhibited by EDTA. The apparent Km values for acyl-CoA synthesis by purified rat brain microvessels were 4.0 microM and 5.8 microM for palmitic acid and arachidonic acid, respectively. The apparent Vmax values were 1.0 and 1.5 nmol X min-1 X mg protein-1 for palmitic acid and arachidonic acid, respectively. Cross-competition experiments showed inhibition of radiolabelled arachidonoyl-CoA formation by 15 microM unlabelled arachidonic acid, with a Ki of 7.1 microM, as well as by unlabelled docosahexaenoic acid, with a Ki of 8.0 microM. Unlabelled palmitic acid and arachidic acid had no inhibitory effect on arachidonoyl-CoA synthesis. In comparison, radiolabelled palmitoyl-CoA formation was inhibited competitively by 15 microM unlabelled palmitic acid, with a Ki of 5.0 microM and to a much lesser extent by arachidonic acid (Ki, 23 microM). The Vmax of palmitoyl-CoA formation obtained on incubation in the presence of the latter fatty acids was not changed. Unlabelled arachidic acid and docosahexaenoic acid had no inhibitory effect on palmitoyl-CoA synthesis. Both arachidonoyl-CoA and palmitoyl-CoA synthesis activities were thermolabile. Arachidonoyl-CoA formation was inhibited by 75% after 7 min at 40 degrees C whereas a 3-min heating treatment was sufficient to produce the same relative inhibition of palmitoyl-CoA synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)

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“…Acid:CoA ligase activity was measured by two related methods. Initial experiments with each enzyme preparation were carried out using a double-label assay (Murphy and Spence, 1980) to ensure that the activity being measured was due to activation of only the added substrates. Subsequent studies were performed using a modified procedure based on the formation of fatty acyl-$H]CoA.…”

Section: Assay F O R Acid:coa Ligase Activitymentioning

confidence: 99%

“…Fatty-acid-poor BSA (20 pg) was added and the mixture chilled on ice for at least 5 min. Tube contents were filtered through Millipore filters as described previously (Murphy and Spence, 1980). Filters and adhering material (including the fatty acyl-CoA) were suspended in Aquasol-2 (New England Nuclear Corp., Lachine, Quebec) and radioactivity measured.…”

Section: Assay F O R Acid:coa Ligase Activitymentioning

confidence: 99%

Acid:Coenzyme A Ligase in Brain: Fatty Acid Specificity in Cellular and Subcellular Fractions

Murphy¹,

Spence²

1982

Journal of Neurochemistry

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We measured long-chain fatty acid: coenzyme A (CoA) ligase (EC 6.2.1.3) activity with four fatty acids in brain homogenates, and cellular and subcellular fractions to determine whether there are differences in activity that could be correlated with differences in fatty acid composition and metabolism. In rat brain homogenates, ligase activity varied appreciably with the four acids, with 18:2 greater than 18:1 greater than 16:0 greater than 22:1 (nmol acyl-CoA formed/min/mg protein: 1.46, 1.20, 0.96, and 0.57, respectively). This order was similar under all incubation conditions tested, including variable pH and fatty acid concentrations. The relative specific activities (RSA, 16:0 = 1.0) with the four substrates were similar in rat brain homogenate, mitochondria, and microsomes, with the highest specific activities in the latter fraction. The RSA were also similar in ox brain homogenates, in rabbit brain microsomes prepared from gray and white matter, in neurons isolated from rat brain, and in cultured neuroblastoma cells. Rat liver homogenates had a significantly different pattern of RSA. These results indicate that the ligase(s) has a preference for certain fatty acids, but suggest that the major control of fatty acid composition and metabolism is a function of subsequent metabolic steps.

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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

Cited by 30 publications

References 30 publications

Long‐chain acyl‐coenzyme a synthetase in rat brain myelin

Long‐chain acyl‐coenzyme a synthetase in rat brain myelin

Arachidonoyl‐Coenzyme A Synthetase and Nonspecific AcylCoenzyme A Synthetase Activities in Purified Rat Brain Microvessels

Acid:Coenzyme A Ligase in Brain: Fatty Acid Specificity in Cellular and Subcellular Fractions

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