Lipolytic and esterolytic activity-based profiling of murine liver.
In lipid metabolism, the liver acts as a buffer for transient energy fluctuations. It temporarily stores fatty acids as triacylglycerol and secretes them as very low density lipoprotein into the circulation when the period of maximum lipid load has passed. The lipolytic enzymes responsible for mobilization of internal lipid stores in the liver have not been identified yet. We introduced active site-directed chemical probes for lipolytic activity profiling in complex mixtures, known as activity-based proteomics, and employed it for global analysis and functional annotation of lipolytic proteins in mouse adipose tissue. Here we report the combined application of two approaches using fluorescent and biotinylated probes for discovery and discrimination of lipolytic and esterolytic enzymes in mouse liver subproteomes. Proteomes labeled with the fluorescent probes were analyzed by 2-DE while proteomes labeled with the biotinylated probe were subjected to avidin-affinity isolation. Of 37 totally identified proteins, 15 were detected using both approaches while 14 and 8 were solely identified by 2-DE and avidin-affinity isolation, respectively. Moreover, 12 enzymes were classified as potential lipases and/or cholesteryl esterases by their reaction with probes specific for the respective activities directly in their proteomes.
Birner-Gruenberger
Ruth
R
Center for Medical Research, Medical University of Graz, Graz, Austria.
Susani-Etzerodt
Heidrun
H
Kollroser
Manfred
M
Rechberger
Gerald N
GN
Hermetter
Albin
A
D000818:Animals; D001393:Azoles; D019921:Biotinylation; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D004950:Esterases / Q000378:metabolism*; D005456:Fluorescent Dyes; D008066:Lipolysis / Q000502:physiology*; D008099:Liver / Q000378:metabolism*; D051379:Mice; D009578:Nitrobenzenes; D020543:Proteome / Q000032:analysis*
C510797:7-nitrobenz-2-oxa-1,3-diazol-4-yl; D001393:Azoles; D005456:Fluorescent Dyes; D009578:Nitrobenzenes; D020543:Proteome; D004950:Esterases