RESUMO
This study reports on the analysis of the lipolytic proteome of cultured human fat cells. We used specific affinity tags to detect and identify the lipolytic and esterolytic enzymes in human subcutaneous (Sc) and visceral (Visc) adipocytes. For this purpose, differentiated fat cells were incubated with a fluorescent suicide inhibitor followed by protein separation using one- or two-dimensional gel electrophoresis. After detection by fluorescence laser scanning, the labeled proteins were tryptically digested and peptides were identified by mass spectrometry. In addition, a biotinylated probe was used for specific enzyme labeling with subsequent avidin affinity isolation of the tagged proteins. Finally, we determined the quantitative differences in protein expression levels between subcutaneous and visceral adipocytes using differential activity-based gel electrophoresis (DABGE). We found that the lipase/esterase patterns of both cell types are very similar, except for some proteins that were only found in Sc cells. Two novel enzyme candidates identified in this study were overexpressed and characterized using biologically relevant glycerolipid substrates in vitro. Both of them showed pronounced hydrolytic activities on hydrophobic acylglycerols and therefore may be considered lipases. The physiological functions of the novel lipolytic proteins in vivo are currently subject to investigation.
Assuntos
Adipócitos/enzimologia , Esterases/metabolismo , Lipase/metabolismo , Proteômica/métodos , Adipócitos/citologia , Adipócitos/metabolismo , Animais , Células COS , Diferenciação Celular/genética , Células Cultivadas , Chlorocebus aethiops , Eletroforese em Gel Bidimensional , Eletroforese em Gel de Poliacrilamida , Esterases/classificação , Esterases/genética , Perfilação da Expressão Gênica , Humanos , Gordura Intra-Abdominal/citologia , Lipase/classificação , Lipase/genética , Lipólise , Masculino , Microscopia de Fluorescência , Proteoma/análise , Proteoma/metabolismo , Gordura Subcutânea/citologia , Adulto JovemRESUMO
Vegetable oils are an essential part of a healthy and balanced diet and have major economic significance. The type of production, whether performed by extraction with solvents, or by mechanical techniques, significantly influences the quality of the oil and its potential field of use. Occasionally, volatile organic substances, which are considered indicative for an oil that has been produced by solvent extraction, are detected in unrefined vegetable oils. This can have a negative impact on high-quality oil mills and their reputation. The goal of the study was to analyse unrefined oils of different raw materials for such compounds. A previously developed and validated method using headspace gas chromatography coupled with a mass spectrometry was used for this task. Another aim was to determine the origin of any solvent residues in the vegetable oils and to find ways to avoid them. Complementary measurements by solid phase micro extraction gas chromatography were conducted to compare the results of the measurements to an orthogonal methodology. Multivariate data analysis was used to find correlations between the spectrum of substances in the oil and other parameters like the producer of the oil or the pattern of fatty acids.
Assuntos
Óleos de Plantas/química , Compostos Orgânicos Voláteis/análise , Cromatografia Gasosa-Espectrometria de Massas , Solventes/químicaRESUMO
PURPOSE: New evidence suggests that the anesthetic effect of parenteral propofol emulsions varies between commercial preparations. We examined and compared different propofol preparations to determine propofol release and binding capacity with regard to plasma lipoproteins and albumin. METHODS: We created a novel assay consisting of microtiter plates coated with either low-density lipoprotein (LDL) or albumin to analyze propofol binding kinetics. Using high performance liquid chromatography (HPLC), we measured propofol release from the oily phase and the corresponding amount of propofol bound to the plates in a time-dependent manner and at equilibrium conditions attained after 30 min of incubation at 37 degrees C. The concentrations of free propofol in the aqueous phase of different propofol preparations - Diprivan, and the generic formulations Propofol "Fresenius" (1% and 2% propofol) and Propofol-Lipuro - were analyzed using ultracentrifugation or dialysis for phase separation. Finally, we investigated the effect of isolated lipoprotein fractions on propofol release. RESULTS: Propofol bound to LDL-coated plates with approximately twofold higher affinity than to albumin-coated plates. No significant differences in total propofol release were observed between preparations. Moreover, similar amounts of free propofol were observed in the aqueous phase of all products tested (1% propofol preparations: 18 microg/ml; 2% propofol preparations: 35 microg/ml), except for the medium-chain and long-chain triglyceride (MCT/LCT) preparation studied, in which the concentration of free propofol was lower. Lipoproteins had no effect on propofol release, except for high-density lipoprotein (HDL), which triggered almost 100% release from the oily phase at HDL concentrations above 1000 microg/ml. CONCLUSIONS: No differences were observed between the binding/release capacity and lipoprotein interactions of any of the propofol preparations tested. We propose that clinical observations of inconsistent propofol activity are related to variations in the lipoprotein profile, enzyme activity or genetic disorders of individual patients, rather than to the propofol preparation itself.
Assuntos
Anestésicos Intravenosos/química , Propofol/química , Anestésicos Intravenosos/metabolismo , Emulsões , Humanos , Cinética , Lipoproteínas LDL/metabolismo , Propofol/metabolismo , Ligação Proteica , Albumina Sérica/metabolismo , SolubilidadeRESUMO
Lipases are responsible for the hydrolysis of acylglycerols and cholesteryl esters in animals, plants, and microorganisms. In this chapter we describe a tool for the concomitant analysis of lipases in complex proteomes. For this purpose, the target enzymes are selectively and covalently labelled with fluorescent suicide inhibitors. Stable lipid-protein complexes are formed that are separated by gel electrophoresis and identified by mass spectrometry.
Assuntos
Tecido Adiposo/química , Corantes Fluorescentes/química , Lipídeos/química , Proteoma , Tecido Adiposo/enzimologia , Alquilação , Animais , Cromatografia Líquida , Eletroforese em Gel de Poliacrilamida , Focalização Isoelétrica , Lipase/metabolismo , Camundongos , Oxirredução , Espectrometria de Massas em TandemRESUMO
The ultimate goal of proteomics is to characterize the function of all proteins in parallel and in the most physiologically relevant settings possible. A step toward this goal has been the introduction of activity-based proteomics. The simultaneous detection of individual protein activities can be facilitated directly in the proteome using specific activity-based probes consisting of a recognition site targeting a certain enzyme species, a properly positioned reactive site which forms a covalent bond with the target and a reporter tag for visualization and/or purification of the covalently bound target. As properties like polarity, size, charge, structure, and chemical reactivity of the reporter tag have a large impact on the reactivity of the probes toward the target enzymes probes suitable for reporter tagging after the enzyme-activity probe-binding event were designed. These probes resemble the natural substrates more closely and are small and hydrophobic enough to cross the membrane of living cells. Here the methodology for detection of lipolytic activities in intact living cells, including synthesis of probe and reporter, labeling procedure, and detection of target enzymes is described.