Increase in molecular rigidity of the protein conformation of brain Na+-K+-ATPase by modification with 4-hydroxy-2-nonenal.

The effect of lipid peroxidation product 4-hydroxy-2-nonenal (HNE) on the protein conformation of porcine cerebral cortex Na(+)-K(+)-ATPase was examined in term of the intrinsic tryptophanyl fluorescence measurement. Treatment of ATPase with HNE resulted in a decrease in the fluorescence intensity and an increase in the fluorescence anisotropy in a concentration-dependent manner. The difference in the fluorescence intensity and fluorescence anisotropy observed between the control and HNE-modified ATPase completely disappeared after treatment of the protein with guanidine hydrochloride (1 M). These results suggest that HNE-modification of the Na(+)-K(+)-ATPase induces alterations in the conformation of the enzyme molecule. This interpretation was further supported by a decrease in fluorescence quenching efficiency with acrylamide and sulfhydryl (SH) content. The decrease in quenching efficiency suggests that the proximity of the quencher molecule to the fluorophores located in the enzyme is suppressed. Modification of the enzyme with N-ethylmaleimide (NEM) also resulted in a decrease in quenching efficiency with the loss of SH groups. Furthermore, a good relationship between the SH content and these fluorescence parameters (fluorescence anisotropy and quenching efficiency) were observed. On the other hand, treatment of the Na(+)-K(+)-ATPase with other aldehydes such as malondialdehyde (MDA), 1-hexanal and nonanal did not affect either the quenching efficiency or SH content. Based on these results, the possibility of alterations in the physical properties of the Na(+)-K(+)-ATPase associated with modification by HNE has been discussed.

Contribution of lipid dynamics on the inhibition of bovine brain synaptosomal Na+-K+-ATPase activity induced by 4-hydroxy-2-nonenal.

The effects of lipid hydroperoxide degradation products, such as 4-hydroxy-2-nonenal (HNE) and malondialdehyde (MDA), on bovine brain synaptosomal ATPase activities and their membrane lipid organization were examined. When the synaptosomes were treated with HNE, this resulted in the decrease of Na(+)-K(+)-ATPase activity with the loss of sulfhydryl (SH) groups in the membrane proteins. In contrast, MDA treatment of the synaptosomes did not induce an appreciable decrease in the ATPase activity or a loss of SH groups. The decreases in ATPase activity and SH content by treatment with HNE were also observed, as a Na+-K+-ATPase preparation was used in place of the synaptosomes. On the other hand, HNE had very little effect on synaptosomal Ca2+- and Mg2+-ATPase activities. The results of the kinetic analysis of the Na+-K+-ATPase activity indicated that the decrease in the activity by HNE-modification is due to a decreased affinity for the substrate. ATP completely protected the ATPase from the HNE attack. Modification of the synaptosomes with HNE caused a decrease in the membrane lipid fluidity near the lipid/water interface, not the lipid layer interior. In addition, it was found that there is a good relationship between the lipid fluidity and the Na+-K+-ATPase activity under the presence of various concentrations of HNE, suggesting that the lipid dynamics are closely related to HNE-induced inhibition of the ATPase activity. On the other hand, MDA did not induce change in the membrane lipid fluidity. HNE and MDA are mainly incorporated into the lipid and protein fractions in the synaptosomal membranes, respectively. Based on these results, we proposed a possible mechanism of HNE-induced inhibition of synaptosomal Na+-K+-ATPase activity associated with alterations in the membrane lipid organization.