Isolation and biochemical characterisation of lipid rafts from Atlantic cod (Gadus morhua) intestinal enterocytes.
Lipid rafts are glycosphingolipid/cholesterol-enriched membrane microdomains that have been extensively studied during the past two decades. Our aim was to isolate and perform biochemical characterization of lipid rafts from the intestinal brush border membrane (BBM) of Atlantic cod (Gadus morhua) to confirm their existence in a cold-water species and compare their characteristics with lipid rafts from other species in terms of lipid and protein content. To validate the isolation process, we assayed marker enzymes for subcellular organelles, including alkaline phosphatase (AP) and leucine aminopeptidase (LAP), both well-known marker enzymes for BBM and lipid rafts. All biochemical methods showed enrichment of AP in both the BBM and lipid raft fractions. Proteomic studies were performed by MALDI-TOF mass spectrometry using trypsin digested SDS-PAGE samples. Various proteins were associated with the cod intestinal lipid raft preparation such as aminopeptidase-N, prohibitin, and beta-actin. Lipid analysis with (31)P NMR and thin layer chromatography on BBMs and lipid rafts samples gave higher content of sphingomyelin than previously reported in the BBM and lower content of phosphatidylcholine. Furthermore, sphingomyelin was highly dominant in the lipid rafts together with cholesterol. The existence of lipid rafts containing previously reported lipid raft characteristics from the cod intestine has, therefore, been confirmed in a ray-finned fish for the first time to the best of our knowledge.
Department of Biochemistry, Science Institute, University of Iceland, Dunhagi 3, 107 Reykjavik, Iceland.
Gudjón Andri
GA
Gylfason
Erna
E
Knútsdóttir
Bjarni
B
Asgeirsson
D000469:Alkaline Phosphatase / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D002458:Cell Fractionation; D002855:Chromatography, Thin Layer; D020895:Enterocytes / Q000737:chemistry / Q000166:cytology / Q000378:metabolism*; D029941:Fish Proteins / Q000032:analysis / Q000302:isolation & purification; D048389:Gadus morhua / Q000378:metabolism*; D007422:Intestines / Q000166:cytology; D007931:Leucyl Aminopeptidase / Q000378:metabolism; D008055:Lipids / Q000032:analysis / Q000302:isolation & purification; D009682:Magnetic Resonance Spectroscopy; D021962:Membrane Microdomains / Q000737:chemistry / Q000201:enzymology / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D008871:Microvilli / Q000737:chemistry / Q000201:enzymology / Q000378:metabolism*; D008969:Molecular Sequence Data; D020543:Proteome / Q000032:analysis / Q000302:isolation & purification; D040901:Proteomics / Q000379:methods; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D029941:Fish Proteins; D008055:Lipids; D020543:Proteome; D000469:Alkaline Phosphatase; D007931:Leucyl Aminopeptidase