Enzymatic Reconstitution of Brain Membrane and Membrane Opiate Receptors

Zohreh T. Farahbakhsh, David W. Deamer, Nancy M. Lee, Horace H. Loh

Research output: Contribution to journalArticlepeer-review

15 Scopus citations

Abstract

Abstract: A new method using lysophosphatide and acyl‐CoA as detergents has been used to solubilize the rat brain opiate receptor. After solubilization, lysophosphatide and acyl‐CoA can be almost completely removed by an enzymatic reaction that uses an acyltransferase from rat liver microsomes and reconstitutes the solubilized receptor in membranous vesicles. Morphological studies performed with negative staining and freeze‐fracture electron microscopy revealed that the general appearance and intramembrane particle distribution of fracture faces in the reconstituted membrane are similar to those of the native membrane; this indicates that hydrophobic protein components of the original membrane were incorporated during reconstitution. Reconstituted membrane, however, contained higher levels of phosphatidylcholine and lower levels of cholesterol. The activities of the membrane‐bound enzymes Na+, K ‐ATPase and Ca2 ‐, Mg2+‐ATPaxe in the reconstituted system were 24 and 3%, respectively, those of the native membrane. Although binding of opiate ligands to the reconstituted membrane was stereospecific and saturable, higher concentrations of some of the unlabeled ligands were required to inhibit binding of the radiolabeled ligands. These changes in receptor characteristics are likely due to changes in lipid composition, physical state, and/or distribution of the lipids in the reconstituted membrane bilayer. This conclusion is supported by an increase in the affinity of opiate ligands for reconstituted membrane after adjustment of the latter's lipid composition to match more closely that of the original membrane. This was accomplished by treatment with phospholipid exchange protein to remove the excess phosphatidylcholine and by incorporation of cholesterol into the reconstituted membrane.

Original languageEnglish (US)
Pages (from-to)953-962
Number of pages10
JournalJournal of Neurochemistry
Volume46
Issue number3
DOIs
StatePublished - Mar 1986

Keywords

  • Acyltransferase
  • Brain opiate receptor
  • Lipid distribution
  • Membrane solubilization
  • Phospholipid exchange protein
  • Reconstituted membrane

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