A study on peroxidative damage of the porcine intestinal brush-border membranes using a fluorogenic thiol reagent, N-(1-pyrene)maleimide.
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.
Ohyashiki
T
T
Department of Biochemistry, School of Pharmacy, Hokuriku University, Ishikawa, Japan.
Sakata
N
N
Kamata
K
K
Matsui
K
K
D000818:Animals; D004591:Electrophoresis, Polyacrylamide Gel; D005454:Fluorescence Polarization; D005456:Fluorescent Dyes; D007422:Intestines / Q000187:drug effects*; D015227:Lipid Peroxidation; D008301:Maleimides / Q000633:toxicity*; D008563:Membrane Lipids / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism; D008871:Microvilli / Q000187:drug effects*; D013552:Swine; D013696:Temperature
D005456:Fluorescent Dyes; D008301:Maleimides; D008563:Membrane Lipids; D008565:Membrane Proteins; C003944:N-(3-pyrene)maleimide