Sidedness of ceramide-phosphoethanolamine synthesis on rat liver plasma membrane.
Phosphatidylethanolamine:ceramide-ethanolaminephosphotransferase catalyzes the synthesis of ceramide-ethanolamine, a sphingomyelin analogue. Its transverse localization in rat liver plasma membrane was studied by treating intact and deoxycholate- or Triton X-100-disrupted membrane vesicles with trypsin or bacterial protease. The latency of ATPase was preserved during protease treatment; its value was 80% in the membrane vesicles obtained by sucrose gradient procedure alone and 91.2% in the vesicles isolated after sucrose gradient plus two-phase partitioning. This suggested that membrane integrity was not altered and that 90% of the vesicles were right-side out. When the sucrose gradient was followed by the two-phase procedure, 62% of phosphatidylethanolamine:ceramide-ethanolamine-phosphotransferase was accessible to the protease action, but only 45% in vesicles obtained by sucrose gradient alone. Our results suggest that at least a sizable portion of the active center of the enzyme responsible of biosynthesis of ceramide-phosphoethanolamine is located on the external side of liver plasma membrane and that the other is embedded in the membrane interior and is not accessible to trypsin, even in the presence of detergent.
Maurice
A
A
Université de Bordeaux II, Laboratoire de Biochimie, France.
Malgat
M
M
Baraud
J
J
D000251:Adenosine Triphosphatases / Q000378:metabolism; D000818:Animals; D002458:Cell Fractionation; D002462:Cell Membrane / Q000201:enzymology* / Q000378:metabolism; D002499:Centrifugation, Density Gradient; D066298:In Vitro Techniques; D008099:Liver / Q000201:enzymology / Q000648:ultrastructure; D009439:Neuraminidase / Q000378:metabolism; D010447:Peptide Hydrolases / Q000494:pharmacology; D051381:Rats; D011919:Rats, Inbred Strains; D013109:Sphingomyelins / Q000096:biosynthesis*; D014357:Trypsin / Q000494:pharmacology
D013109:Sphingomyelins; C060281:ceramide phosphoethanolamine; D009439:Neuraminidase; D010447:Peptide Hydrolases; D014357:Trypsin; D000251:Adenosine Triphosphatases