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A study on peroxidative damage of the porcine intestinal brush-border membranes using a fluorogenic thiol reagent,
T Ohyashiki1, N Sakata, K Kamata
1Department of Biochemistry, School of Pharmacy, Hokuriku University, Ishikawa, Japan.
Abstract:
To examine the effects of lipid peroxidation on the protein conformation in the porcine intestinal brush-border membranes, a fluorogenic thiol reagent, N-(1-pyrene)maleimide (NPM) was employed. By treatment of NPM-labeled membranes with 100 microM ascorbic acid/10 microM Fe2+ in the presence of various concentrations of tert-butyl hydroperoxide (t-BuOOH), the fluorescence intensity of the complex decreased with the formation of conjugated diene, depending on the hydroperoxide concentration. The temperature dependence profile of the fluorescence intensity of NPM-labeled control membranes showed a thermal transition of the NPM fluorescence at 27-28 degrees C. The transition phenomenon of the NPM fluorescence in the membranes around this temperature disappeared by treatment of the labeled membranes with 100 microM ascorbic acid/10 microM Fe2+/0.6 mM t-BuOOH. The difference in response of the fluorescence characteristics of the bound NPM for temperature variation between the control and peroxidized membranes was also observed in the quenching efficiency with acrylamide. Measurement of the fluorescence polarization revealed that the harmonic mean of the rotational relaxation times of the bound NPM molecules to the membrane proteins increased from 1.96 to 4.93 microseconds by lipid peroxidation of the membranes. This indicates that the movement of the region containing NPM-labeled SH groups in the membrane proteins is restricted by lipid peroxidation. Treatment of NPM-labeled peroxidized membranes with sodium dodecyl sulfate (SDS) resulted in a restoration of the intensity of the NPM fluorescence to the level of the control ones. In addition, the temperature dependence profile of the fluorescence intensity of NPM-labeled peroxidized membranes in the presence of SDS also showed an appearance of a transition phenomenon around 30 degrees C. The result of SDS-polyacrylamide gel electrophoresis of the peroxidized membranes revealed that high-molecular-weight aggregates of the membrane proteins were not formed by lipid peroxidation. On the basis of these results, changes in the environmental properties around NPM-labeled SH groups in the membrane proteins by lipid peroxidation are discussed.
Insights
Lipid peroxidation alters protein conformation in intestinal membranes by restricting movement around thiol groups. This change is reversible with sodium dodecyl sulfate (SDS) treatment, indicating altered protein environments.
Area of Science:
- Biochemistry
- Membrane Biology
- Protein Chemistry
Background:
- Lipid peroxidation is a process that damages cell membranes.
- Understanding its effects on membrane protein conformation is crucial for cell function.
- Porcine intestinal brush-border membranes serve as a model system.
Purpose of the Study:
- To investigate how lipid peroxidation affects protein conformation in porcine intestinal brush-border membranes.
- To characterize the changes in the environment of thiol groups on membrane proteins.
Main Methods:
- Utilized N-(1-pyrene)maleimide (NPM), a fluorogenic thiol reagent, to label membrane proteins.
- Induced lipid peroxidation using ascorbic acid, Fe2+, and tert-butyl hydroperoxide (t-BuOOH).
- Analyzed fluorescence intensity, temperature dependence, quenching efficiency, fluorescence polarization, and SDS-polyacrylamide gel electrophoresis.
Main Results:
- Lipid peroxidation decreased NPM fluorescence intensity and altered its temperature-dependent profile.
- Rotational relaxation times of NPM-bound proteins increased, indicating restricted movement.
- Sodium dodecyl sulfate (SDS) treatment restored fluorescence intensity and revealed a transition around 30°C.
Conclusions:
- Lipid peroxidation restricts the movement of NPM-labeled SH groups in membrane proteins.
- The environmental properties around these SH groups are significantly altered.
- SDS treatment can reverse some of these conformational changes.