Interaction of rat liver 3-D-(-)-hydroxybutyrate aopdehydrogenase with phospholipids

The interaction of phospholipids with pure, catalytically inactive rat liver 3-d-(—)-CoA hydroxybutyrate apodehydrogenase (apoHBD) was examined, (a) A relationship could be established between density of packing of phospholipid molecules at the interface and apoHBD activation, namely, the larger the area per polar head, the higher the lipid molar efficiency. In this context, codispersion of lecithins with phospholipids that were inactive or scarcely active per se, such as phosphatidylethanolamine and lysophosphatidylcholine (miristoyl; Iysod₁₄) increased the activating efficiency of lecithins, (b) ApoHBD formed tightly bound, catalytically active complexes with lecithin liposomes and micelles (diC₁₀ + lysoC₁₄; cetylphosphorylcholine), but a phospholipid-water interface was not essential for HBD activity since a molecular dispersion of diheptanoyl lecithin (diC₇) activated apoHBD to a limited extent. ApoHBD formed loosely bound, catalytically inactive complexes with multilayer vesicles, but HBD activity could be restored by sonication or by adding liposome to those complexes. Unlike liposomes and micelles, apoHBD interaction with multilayer vesicles did not involve a hydrophobic contribution, which was apparently necessary for apoHBD activation, (c) LysoC₁₄, did₁₀ + lysoC₁₄, and cetylphosphorylcholine micelles activated apoHBD but diC₇ micelles inhibited the HBD activity of the apoHBD-diC₇ (monomer) complex. The inhibition decreased when the medium ionic strength was increased. Liposomes and diCi₁₀ + lysoC₁₄ micelles activated and stabilized apoHBD much more efficiently than pure lysoC₁₄ or cetylphosphorylcholine micelles, (d) The mode of aggregation of the activating phospholipid strongly affected the kinetics of the HBD reaction. With liposomes the reaction showed an initial lag (or induction) period whose duration varied over a range of 3 to 15 min, depending on the activating phospholipid; with diC₇ monomers and micelles the kinetics was linear throughout, while with multilayer vesicles the lag was virtually infinite since HBD activity was insignificant, (e) Energies of activation for apoHBD-diC₁₄ complexes, either below or above the lecithin gel-to-liquid crystalline transition temperature were not significantly different, in accordance with apoHBD interaction with the proximal end of the hydrocarbon chains, that is, the less subject to phase transitions. With a diC₁₄-substituted mitochondrial preparation, however, no HBD activity was detected below 24 °C (near the gel-to-liquid crystalline transition temperature of diC₁₄), thus indicating that, in the inner membrane, apoHBD interacts with the whole length of the fatty acyl chain and, consequently, is sensitive to phase transition.