Increase of the molecular rigidity of the protein conformation in the intestinal brush-border membranes by lipid peroxidation.
The effect of lipid peroxidation on the protein conformation of the porcine intestinal brush-border membranes was studied using a fluorogenic thiol reagent, N-[7-dimethylamino-4-methylcoumarinyl]maleimide (DACM). By a kinetic analysis of the reaction of the membranes with DACM, it was shown that the reaction rate of the SH groups (SHf) of the membrane proteins, whose reaction with the dye is very fast, decreases in proportion to the extent of thiobarbituric acid-reactive substance formation. The difference in the rate of the reaction of the SHf groups for DACM between the control and peroxidized membranes completely disappeared after denaturation of the proteins by treatment with guanidine hydrochloride. The reaction of DACM with the SHf groups of the control membranes accelerated when the temperature was increased with an apparent transition temperature between 25 degrees C and 30 degrees C. On the other hand, no transition was observed in the peroxidized membranes over the temperature range 20-43 degrees C. These results suggest that the conformation around the SHf groups of the proteins in the peroxidized membranes is apparently different from that in the control membranes. A modification of the conformation around the SH groups in the membrane proteins associated with lipid peroxidation was further demonstrated by finding that the quenching efficiency of the fluorescence of the DACM-labeled membranes by Tl+ was markedly decreased after lipid peroxidation. Based on these results, changes in the protein conformation of the porcine intestinal brush-border membranes by lipid peroxidation are discussed.
Ohyashiki
T
T
Department of Physiological Chemistry, School of Pharmacy, Hokuriku University, Ishikawa, Japan.
Ohtsuka
T
T
Mohri
T
T
D000818:Animals; D019791:Guanidine; D006146:Guanidines / Q000494:pharmacology; D007421:Intestine, Small / Q000648:ultrastructure*; D007700:Kinetics; D008054:Lipid Peroxides / Q000378:metabolism*; D008301:Maleimides / Q000378:metabolism; D008315:Malondialdehyde; D008560:Membrane Fluidity; D008563:Membrane Lipids / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism; D008871:Microvilli / Q000378:metabolism; D011487:Protein Conformation; D011489:Protein Denaturation / Q000187:drug effects; D013050:Spectrometry, Fluorescence; D013438:Sulfhydryl Compounds / Q000378:metabolism; D013439:Sulfhydryl Reagents; D013552:Swine; D013696:Temperature
D006146:Guanidines; D008054:Lipid Peroxides; D008301:Maleimides; D008563:Membrane Lipids; D008565:Membrane Proteins; D013438:Sulfhydryl Compounds; D013439:Sulfhydryl Reagents; D008315:Malondialdehyde; C019347:N-(7-dimethylamino-4-methylcoumarinyl)maleimide; D019791:Guanidine