The characterization of plasma membrane Ca2+-ATPase in rich sphingomyelin-cholesterol domains.
According to the raft hypothesis, sphingolipid-cholesterol (CHOL) microdomains are involved in numerous cellular functions. Here, we have prepared liposomes to simulate the lipid composition of rafts/caveolae using phosphatidylchone, sphingomyelin (SPM)-CHOL in vitro. Experiments of both 1,6-diphenyl-1,3,5-hexatriene and merocyanine-540 fluorescence showed that a phase transition from l(d) to l(o) can be observed clearly. In particular, we investigated the behavior of a membrane protein, plasma membrane Ca(2+)-ATPase (PMCA), in lipid rafts (l(o) phase). Three complementary approaches to characterize the physical appearance of PMCA were employed in the present study. Tryptophan intrinsic fluorescence increase, fluorescence quenching by both acrylamid and hypocrellin B decrease, and MIANS fluorescence decrease, indicate that the conformation of PMCA embedded in lipid l(o) phase is more compact than in lipid l(d) phase. Also, our results showed that PMCA activity decreased with the increase of SPM-CHOL content, in other words, with the increase of l(o) phase. This suggests that the specific domains containing high SPM-CHOL concentration are not a favorable place for PMCA activity. Finally, a possible explanation about PMCA molecules concentrated in caveolae/rafts was discussed.
Pang
Yuhong
Y
National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Chaoyang District, Beijing, China.
Zhu
Hua
H
Wu
Ping
P
Chen
Jianwen
J
D020106:Acrylamide / Q000494:pharmacology; D000252:Calcium-Transporting ATPases / Q000378:metabolism*; D002462:Cell Membrane / Q000737:chemistry / Q000201:enzymology* / Q000378:metabolism*; D002784:Cholesterol / Q000378:metabolism*; D005453:Fluorescence; D044367:Phase Transition; D010713:Phosphatidylcholines / Q000378:metabolism; D011510:Proteolipids / Q000737:chemistry* / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013109:Sphingomyelins / Q000378:metabolism*; D014364:Tryptophan / Q000378:metabolism
D010713:Phosphatidylcholines; D011510:Proteolipids; D013109:Sphingomyelins; C020485:proteoliposomes; D020106:Acrylamide; D014364:Tryptophan; D002784:Cholesterol; D000252:Calcium-Transporting ATPases