Abstract
Physicochemical techniques were used to investigate the activation of C55-isoprenoid alcohol phosphokinase by synthetic lecithins. Complexes of the enzyme with phospholipids were prepared using a method employing sodium dodecyl sulfate as a protein-solubilizing agent. Circular dichorism and the intrinsic fluorescence of the kinase were used as optical probes of protein conformation with these complexes. No evidence for a major lipid-dependent conformational change in the protein was observed when these complexes were studied under conditions where the lipid mesomorphic transitions occurred. EPR studies of mixtures of synthetic lecithins and the C55-isoprenoid alcohol indicated a correlation between kinase activity and the rotational diffusion rate within the hydrophobic phase. It is concluded that the lipid physical state probably does not affect the enzyme activity by altering the protein conformation but more likely does so by affecting the motion of the molecular participants in the reaction.
Highlights
Physicochemical techniques were used to investigate the activation of C,-isoprenoid alcohol phosphokinase by synthetic lecithins
An electron spin resonance probe was used to measure the viscosity of lipid mixtures of synthetic lecithins and C,-isoprenoid alcohol which were similar to the lipid phase present under assay conditions
Tryptophan fluorescence will monitor a variety of Enzyme activation studies have clearly shown qualitative correlation between the physical state of the lipid and its ability to serve as a kinase activator [1, 2]. One explanation for this correlation is that the conformation of the enzyme is dependent on the physical state of the lipid in which it correlation time of 22-doxylmethylstearate as function of in synthetic lecithins and mixtures of synthetic lecithins with C..-isoprenoid alcohol
Summary
Physicochemical techniques were used to investigate the activation of C,,-isoprenoid alcohol phosphokinase by synthetic lecithins. EPR studies of mixtures of synthetic lecithins and the C,,-isoprenoid alcohol indicated a correlation between kinase activity and the rotational diffusion rate within the hydrophobic phase. In order to investigate the manner in which the physical properties of the lipid phase are coupled to the activity of the membrane enzyme, physical chemical techniques were employed. Circular dichroism and the intrinsic fluorescence of the kinase tryptophans were examined to test the possibility that the protein conformation is a function of the physical state of the lipid in which it is embedded. An electron spin resonance probe was used to measure the viscosity of lipid mixtures of synthetic lecithins and C,,-isoprenoid alcohol which were similar to the lipid phase present under assay conditions
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