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Phospholipid Interactions with Cytochrome P-450 in Reconstituted Vesicles: Preference for Negatively-Charged Phosphatidic Acid



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  • Personal Author:
  • Description:
    Cytochrome P-450 LM2 was reconstituted by the cholate-dialysis method into vesicles containing a mixture of either phosphatidylcholine or phosphatidylethanolamine with up to 50 mol% of phosphatidic acid. Phase transition curves in the presence or absence of cytochrome P-450 were obtained from electron paramagnetic resonance experiments by measuring the partitioning of 2,2,6,6-tetramethylpiperidine-1-oxyl. Protein-free phospholipid vesicles exhibit a phase separation into domains of gel phase enriched in phosphatidic acid in a surrounding fluid matrix containing mainly phosphatidylcholine. The phase transition of the phosphatidic acid domains disappeared following incorporation of cytochrome P-450 into the bilayers. In contrast, in vesicles containing mixtures of egg-phosphatidic acid and dimyristoyl phosphatidylcholine, the phase transition of the domains enriched in dimyristoyl phosphatidylcholine was less sharp than in the corresponding vesicles containing cytochrome P-450. The results of both of these experiments could be explained by a redistribution of the mol fraction of the two phospholipids in the gel phase due to preferential binding of the egg-phosphatidic acid to the cytochrome P-450. For comparison, incorporation of cytochrome P-450 into uncharged vesicles of dimyristoyl phosphatidylcholine and egg-phosphatidylethanolamine did not alter the single symmetric phase transition of the dimyristoyl phosphatidylcholine. In a second set of experiments the effect of reconstituting cytochrome P-450 into egg-phosphatidylcholine/egg-phosphatidylethanolamine was studied by measuring the order parameter of spin-labeled egg-phosphatidic acid or egg-phosphatidylcholine. Incorporation of the protein increased the order parameter of the 5-doxyl spin label from 0.800 to 0.830 +/- 0.005, indicating a reduction of fluidity. However, in the case of 16-doxyl phosphatidic acid spin label, an additional immobilized component was observed in the spectrum. These results suggest strong interactions between phospholipids and cytochrome P-450 with some additional interaction with phosphatidic acid due to its negatively-charged headgroup. [Description provided by NIOSH]
  • Subjects:
  • Keywords:
  • ISSN:
    0006-3002
  • Document Type:
    Text
  • Funding:
    Grant
  • Genre:
    Journal Article
  • Place as Subject:
  • CIO:
    National Institute for Occupational Safety and Health (NIOSH)
  • Topic:
    Workplace Safety & Health
  • Location:
    North America
  • Volume:
    643
  • Issue:
    3
  • NIOSHTIC Number:
    nn:20061156
  • Citation:
    Biochim Biophys Acta 1981 May; 643(3):547-556
  • Federal Fiscal Year:
    1981
  • Performing Organization:
    Stanford University, Stanford, California
  • Peer Reviewed:
    True
  • Start Date:
    19770601
  • Source Full Name:
    Biochimica et Biophysica Acta
  • End Date:
    19801231
  • Collection(s):
  • Main Document Checksum:
    urn:sha-512:046fc6f48bbcd1ece5ff905f32a415dd3e9b1d2ebce24c4524f47f08144005e95033405ef41c7e9b5a94846ce99eff88ed6550aadcec633528109b788a9bde61
  • Download URL:
  • File Type:
    Filetype[PDF - 652.12 KB ]
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