Protein and lipid analysis of detergent-resistant membranes isolated from bovine kidney.
Detergent-resistant membranes (DRM) were prepared from bovine kidney cortex. The criterion used to test their purification was the increase in the activity of a GPI membrane-anchored protein, the alkaline phosphatase. Its association with specific proteins and lipids was tested. Two successive Triton X-100 treatments followed by purification on sucrose gradient at 4 degrees C were necessary to obtain DRM with a maximum of alkaline phosphatase activity and a typical protein pattern. A third Triton treatment did not alter this DRM composition. Among the enriched protein, we identified, by mass spectrometry, a microsomal dipeptidase, which was GPI membrane-anchored. Protein-kinase activities, mainly serine-kinase, were enriched during the DRM purification. Using the typical FTIR olefinic =C-H bands of the acyl chains, a global decrease in the unsaturation level of DRM lipids was observed as compared with total membranes. Three main phospholipids were identified in DRM. Their fatty acid compositions were determined by gas chromatography and compared with those of total membranes. The most enriched saturated fatty acid was palmitic acid (+44% for phosphatidylethanolamine, +52% for phosphatidylcholine and +49% for sphingomyelin), agreeing with a selection of specific phospholipids among the saturated ones during the DRM purification.
Bonnin
Stéphanie
S
Laboratoire de Physico-Chimie Biologique, UMR-CNRS 5013, Université Claude Bernard-Lyon I, Bat. 303, 43, Boulevard du 11 novembre 1918, 69622 Villeurbanne cedex, France.
El Kirat
Karim
K
Becchi
Michel
M
Dubois
Madeleine
M
Grangeasse
Christophe
C
Giraud
Claire
C
Prigent
Annie-France
AF
Lagarde
Michel
M
Roux
Bernard
B
Besson
Françoise
F
D000469:Alkaline Phosphatase / Q000378:metabolism; D000818:Animals; D002417:Cattle; D003902:Detergents / Q000494:pharmacology*; D004150:Dipeptidases / Q000032:analysis; D005227:Fatty Acids / Q000032:analysis / Q000737:chemistry; D058851:GPI-Linked Proteins; D007668:Kidney / Q000166:cytology*; D008563:Membrane Lipids / Q000032:analysis* / Q000737:chemistry; D021962:Membrane Microdomains / Q000737:chemistry* / Q000187:drug effects*; D008565:Membrane Proteins / Q000032:analysis* / Q000737:chemistry; D010743:Phospholipids / Q000032:analysis / Q000737:chemistry; D011494:Protein Kinases / Q000032:analysis / Q000378:metabolism; D017550:Spectroscopy, Fourier Transform Infrared
D003902:Detergents; D005227:Fatty Acids; D058851:GPI-Linked Proteins; D008563:Membrane Lipids; D008565:Membrane Proteins; D010743:Phospholipids; D011494:Protein Kinases; D000469:Alkaline Phosphatase; D004150:Dipeptidases; C038937:dipeptidase; C549872:dipeptidase 1