Abstract
Purpose :
An eye lens is an onion-like organ that consists of layers of fiber cells. Fiber cell membranes are fluid phase membranes that contain domains and interface regions of continually changing fluidities between these domains. As the eye-lens fiber cells mature the membranes become more protein, cholesterol, and sphingolipid rich. That enrichment affects rotational diffusion (RD) of lipids and the oxygen transport parameter (OTP) within a membrane. The purpose of this research was to develop a new method to assess fiber cell membranes' RD and OTP. This method allowed us to investigate the effects of integral and peripheral membrane proteins on RD and OTP without prior assumptions regarding membrane organization.
Methods :
The intact membranes isolated from porcine eye-lenses, and lens lipid membranes (LLM) that were prepared as multilamellar liposomes using the total lipid extract of the intact membranes, were labeled with a cholesterol analog spin-label (ASL). Saturation recovery electron paramagnetic resonance signals obtained from titration of these membranes with air were fitted with the stretched exponential function. Two fitting parameters obtained from this analysis are the characteristic spin-lattice relaxation rate of spin labels (T1strobs-1) and the heterogeneity parameter βobs. Plots of these parameters versus air fraction allowed to separate RDs of lipids and OTPs within these membranes.
Results :
The comparison of the distributions of T1-1s of ASL in LLMs and intact membranes indicates that at least 81 % of the lipids in the intact membranes have slower rotational diffusion than those in LLMs. The comparison of the oxygen transport parameter indicates that at least 66 % of the lipids in the intact membranes have slower oxygen collision rates than in LLMs.
Conclusions :
We were able to separate two independent physical properties of the lipid bilayer portion of intact membranes, namely, the rotational diffusion of lipids and the oxygen transport parameter. Properties of LLMs represent the average properties of bulk lipids in intact membranes. The results indicate that we are able to see inhomogeneities in bulk lipids using molecular oxygen as a probe molecule.
Acknowledgments: This work was supported by the Grant EY015526 from the National Institutes of Health, USA.
This is a 2020 ARVO Annual Meeting abstract.