RESUMO
The formation of malonyl-CoA is catalyzed by acetyl-CoA carboxylase (ACC), the rate-limiting enzyme of de novo fatty acid synthesis. Monitoring the changes of malonyl-CoA concentration in the brain in response to treatments such as pharmaceutical intervention (via ACC inhibitors) or different dietary conditions (such as varied feeding regimes) is of great interest and could help increase the understanding of how this molecule contributes to feeding behavior and overall energy balance. We have developed a sensitive analytical method for the determination of malonyl-CoA levels in rat brain tissue. The assay involved removal of tissue lipids by liquid-liquid extraction followed by LC/MS/MS analysis of the aqueous layer for malonyl-CoA. The method was sensitive enough (limit of quantitation = 50 ng/mL, or approximately 0.018 nmol/g brain tissue) to determine malonyl-CoA in individual rat brain preparations. The assay performance was sufficiently rugged to support drug discovery screening efforts and provided an additional analytical tool for monitoring brain malonyl-CoA levels.
Assuntos
Química Encefálica , Fracionamento Químico/métodos , Cromatografia Líquida/métodos , Malonil Coenzima A/análise , Espectrometria de Massas em Tandem/métodos , Animais , Malonil Coenzima A/isolamento & purificação , RatosRESUMO
A highly oxygenated phenethyl derivative ustethylin A was isolated from Aspergillus ustus. Gene deletion, isotope labeling, and heterologous expression proved that the phenethyl core structure is assembled from malonyl-CoA by a polyketide synthase harboring a methyltransferase domain. Propionate was converted via acetyl-CoA to malonyl-CoA and incorporated into the molecule. Modifications on the core structure by three different oxidoreductases and one O-methyltransferase lead to the final product, ustethylin A.
Assuntos
Acetilcoenzima A/química , Aspergillus/química , Malonil Coenzima A/química , Metiltransferases/metabolismo , Oxirredutases/metabolismo , Policetídeo Sintases/metabolismo , Acetilcoenzima A/metabolismo , Malonil Coenzima A/isolamento & purificação , Malonil Coenzima A/metabolismo , Metiltransferases/química , Estrutura Molecular , Oxirredutases/química , Policetídeo Sintases/químicaRESUMO
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.
Assuntos
Acil Coenzima A/isolamento & purificação , Fígado/química , Extração em Fase Sólida/métodos , 2-Propanol/química , Acetonitrilas/química , Acil Coenzima A/química , Animais , Ésteres , Malonil Coenzima A/química , Malonil Coenzima A/isolamento & purificação , Palmitoil Coenzima A/química , Palmitoil Coenzima A/isolamento & purificação , Fosfatos/química , Compostos de Potássio/química , Prótons , Ratos , Reprodutibilidade dos TestesAssuntos
Carboxiliases/isolamento & purificação , Mitocôndrias Hepáticas/enzimologia , Mycobacterium tuberculosis/enzimologia , Glândulas Sebáceas/enzimologia , Animais , Carboxiliases/análise , Gansos , Masculino , Malonil Coenzima A/análise , Malonil Coenzima A/isolamento & purificação , Métodos , RatosRESUMO
A method for the synthesis of [14C]malonyl-Coenzyme A starting with 10 mumol of [14C]malonate is reported. The synthesis is accomplished with yields of 48 +/- 4% (1 sigma, n = 6) using a procedure which does not require the isolation or purification of any intermediates.
Assuntos
Malonil Coenzima A/síntese química , Radioisótopos de Carbono , Cromatografia em Papel , Ácido Graxo Sintases/metabolismo , Malonatos , Malonil Coenzima A/isolamento & purificação , Especificidade por SubstratoRESUMO
Two isomers of malonyl-coenzyme A (malonyl-CoA) were detected in a commercial preparation of malonyl-CoA. These compounds were separated by preparative high-performance liquid chromatography (HPLC) and characterized by HPLC/ultraviolet (UV)/mass spectrometry. Both compounds had a UV absorbance maximum at 259-260 nm. Both compounds underwent negative electrospray ionization to produce a [M-H](-)quasi-molecular ion at m/z 852 and both compounds underwent collision-induced dissociation to produce a characteristic fragment at m/z 808, all consistent with the structure of malonyl-CoA. Nuclear magnetic resonance spectrometry showed that the two chromatographically distinguishable malonyl-CoAs are structural isomers: the major component is the naturally occurring malonyl-CoA and the contaminant is 3'-dephospho- 2'-phospho-coenzyme A.
Assuntos
Malonil Coenzima A/isolamento & purificação , Cromatografia Líquida de Alta Pressão/métodos , Coenzima A/química , Coenzima A/isolamento & purificação , Isomerismo , Espectroscopia de Ressonância Magnética/métodos , Malonil Coenzima A/química , Espectrometria de Massas por Ionização por Electrospray/métodos , Espectrofotometria Ultravioleta/métodosRESUMO
A semi-preparative enzymic synthesis of [1-14C]malonyl-CoA from [1-14C]acetate and bicarbonate, and of [3-14C]malonyl-CoA from Na2(14)CO3 and acetate, was achieved by using chloroplasts rapidly isolated from 7-8-day-old pea shoots. Around 70% of the [1-14C]acetate was converted into malonyl-CoA in 2-3 h, and the specific radioactivity of [3-14C]malonyl-CoA synthesized in the system was 25-30 Ci/mol. Reactions were monitored and labelled products were purified by h.p.l.c.