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Cholesterol oxidase senses subtle changes in lipid bilayer structure.

Biochemistry (2004-01-21)
Kwang-wook Ahn, Nicole S Sampson
ABSTRACT

We investigated the dependence of cholesterol oxidase catalytic activity and membrane affinity on lipid structure in model membrane bilayers. The binding affinities of cholesterol oxidase to 100-nm unilamellar vesicles composed of mixtures of DOPC or DPPC and cholesterol are not sensitive to cholesterol mole fraction if the phase of the membrane is in a fluid state. When the membrane is in a solid-ordered state, the binding affinity of cholesterol oxidase increases approximately 10-fold. The second-order rate constants (kcat*/Km*) for different lipid mixtures show a 2-fold substrate specificity for cholesterol in the l(d) phase of high cholesterol chemical activity over cholesterol in the l(o) phase. Moreover, the enzyme is 2-fold more specific for cholesterol in the l(o) phase than in the s(o) phase. Likewise, there is 2-fold substrate specificity for the high cholesterol chemical activity l(d) phase over the low chemical activity l(d) phase. The specificities for the l(d) phase of low cholesterol chemical activity and the l(o) phase are the same. These data indicate that the more ordered the lipid cholesterol structure in the bilayer, the lower the catalytic rate. However, under all of the conditions investigated, the enzyme is never saturated with substrate. The enzymatic activity directly reflects the facility with which cholesterol can move out of the membrane, whether changes in cholesterol transfer facility are due to phase changes or more localized changes in packing. We conclude that the activity of cholesterol oxidase is directly and sensitively dependent on the physical properties of the membrane in which its substrate is bound.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Cholesterol Oxidase from microorganisms, lyophilized powder, ≥50 units/mg protein, recombinant, expressed in E. coli
Sigma-Aldrich
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(+)-4-Cholesten-3-one, 98%
Sigma-Aldrich
Cholesterol Oxidase microbial, recombinant, lyophilized powder, ≥10 units/mg protein
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