ABSTRACT
A novel procedure for the quantitative isolation and purification of acyl-coenzyme A esters is presented. The procedure involves two steps: (1) tissue extraction using acetonitrile/2-propanol (3+1, v+v) followed by 0.1M potassium phosphate, pH 6.7, and (2) purification using 2-(2-pyridyl)ethyl-functionalized silica gel. Recoveries determined by adding radiolabeled acetyl-, malonyl-, octanoyl-, oleoyl-, palmitoyl-, or arachidonyl-coenzyme A to powdered rat liver varied 93-104% for tissue extraction and 83-90% for solid-phase extraction. The procedure described allows for isolation and purification, with high recoveries, of acyl-coenzyme A esters differing widely in chain length and saturation.
Subject(s)
Acyl Coenzyme A/isolation & purification , Liver/chemistry , Solid Phase Extraction/methods , 2-Propanol/chemistry , Acetonitriles/chemistry , Acyl Coenzyme A/chemistry , Animals , Esters , Malonyl Coenzyme A/chemistry , Malonyl Coenzyme A/isolation & purification , Palmitoyl Coenzyme A/chemistry , Palmitoyl Coenzyme A/isolation & purification , Phosphates/chemistry , Potassium Compounds/chemistry , Protons , Rats , Reproducibility of ResultsABSTRACT
Long-chain acyl-CoA's are important intermediates in fatty acid oxidation and phospholipid metabolism. For quantitative analysis of brain acyl-CoA's, and to avoid decomposition due to high brain acyl-CoA hydrolase activity, a fast and efficient analytical method was developed for isolation and determination of acyl-CoA's. The analysis includes solid-phase extraction by an oligonucleotide purification cartridge and HPLC measurements using a synthetic internal standard. Estimates of concentration in rat brain are oleoyl-CoA (11.0 nmol/g), palmitoyl-CoA (6.0 nmol/g), stearoyl-CoA (4.0 nmol/g), and linoleoyl- and arachidonoyl-CoA (2.0 nmol/g) for a total concentration of 23 nmol/g brain.
Subject(s)
Acyl Coenzyme A/analysis , Acyl Coenzyme A/isolation & purification , Brain Chemistry , Animals , Chromatography, High Pressure Liquid/methods , Palmitoyl Coenzyme A/analysis , Palmitoyl Coenzyme A/isolation & purification , Rats , Rats, Sprague-DawleyABSTRACT
Two tandem cysteine residues in the carboxyl-terminal region of rhodopsin have been shown to be covalently linked to palmitate via thioester bonds (Ovchinnikov, Y. A., et al. (1988) FEBS Lett. 230, 1-5). We have synthesized a fluorescent analogue of palmitoyl coenzyme A (16-(9-anthroyloxy)hexadecanoyl coenzyme A ester) and incorporated the fluorescent derivative of palmitate into the protein in high yield (> 40%) through pretreatment of bovine rod outer segments with 1 M hydroxylamine and subsequent incubation with the fluorescent label. Covalent incorporation of label into protein was demonstrated by SDS-polyacrylamide gel electrophoresis. Proteolytic digestion of labeled rhodopsin in the disc membrane with papain and thermolysin verified the C-terminal location of the label. Treatment of SDS-solubilized, labeled rod outer segments with 10% beta-mercaptoethanol provided evidence that partial depalmitoylation may induce the formation of rhodopsin aggregates. Labeled, unbleached rhodopsin was purified by chromatography over hydroxyapatite and concanavalin A-agarose and reconstituted into dimyristoylphosphatidylcholine vesicles. SDS gels of the rhodopsin vesicle preparation verified that all unbound fluorescent label had been removed and that the thioester bond linking probe to protein was not labile.
Subject(s)
Palmitic Acids/metabolism , Rhodopsin/metabolism , Animals , Cattle , Fluorescent Dyes , Hydrolysis , Palmitic Acid , Palmitoyl Coenzyme A/chemical synthesis , Palmitoyl Coenzyme A/isolation & purification , Palmitoyl Coenzyme A/metabolism , Rhodopsin/chemistry , Rhodopsin/isolation & purification , Rod Cell Outer Segment/metabolismABSTRACT
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.
