With age, proteins in the human lens become increasingly associated with cell membranes.
12,18,19 The purpose of this study was to examine this phenomenon in specific regions of the lens as a function of age, with particular attention to age-related changes in the barrier and outer cortex. A modification of the sucrose density gradient protocol described by Chandrasekar and Cendalla
18 was used to separate fractions based on their density.
Each lens was dissected reproducibly into four regions by the use of trephines (see
Fig. 1). The nucleus of the lens was divided into two parts, with a 4.5-mm equatorial diameter (core region) and a 6-mm equatorial diameter (inner region) that correspond to the fetal lens and the infantile lens, respectively, and were examined in a previous study.
12 The cortex was dissected in two using a trephine with a diameter of 8 mm. Lens tissue between 6 and 8 mm was defined as the barrier region, which is the area where an age-related impediment to diffusion occurs.
13,14 The cortical tissue outside 8 mm was referred to as the outer region and is composed of newly synthesized lens tissue.
1 All four regions were examined by sucrose density gradient centrifugation.
Considerable changes were apparent when density gradients of the same regions from a young lens (34 years) and an older lens (74 years) were compared (
Fig. 1). In the young lens, all four regions were similar. In the older lens, two distinct regional density patterns were observed. The core and inner regions displayed similar patterns, with large amounts of protein detected at two dense (SG1 and SG2) interfaces. By comparison, the barrier and outer cortical regions were different, displaying fewer changes in overall protein density and, notably, no SG1 band in either region.
Previously, protein density changes in two nuclear tissues (core and inner regions) of the lens were found to be caused by the membrane binding of proteins.
12 In the present study, the two cortical regions were examined to determine whether similar phenomena occurred.
Figure 2 shows the patterns of protein density in the barrier (
Fig. 2a) and the outer cortex (
Fig. 2b) of the lens with age, as observed by sucrose density centrifugation. Before age 30, most protein in each lens region was located in two interfaces, SG6 and SG5 (
Figs. 3a,
3b), which correspond to water-soluble protein (WSP) and “native” membranes, respectively.
12 Between ages 30 and 45, the SG6 interface became more dispersed, and a new band between SG5 and SG6 (SG6a) appeared (
Fig. 3c). Examination of SG6a by size-exclusion HPLC revealed it was composed primarily of high molecular weight protein complexes.
12 Coinciding with the appearance of SG6a (
Fig. 3c), a slight increase in staining was observed at the SG5 interface (
Fig. 3b), suggesting an increased protein content of the membranes.
After age 40, other changes in protein density were observed in the barrier region. Protein at the SG4 and SG3 interfaces was detected (
Figs. 3d,
3e), and, as the amount of protein in these interfaces increased with age, there was a corresponding loss of protein from SG6 (
Fig. 3a).
After ages 45 to 50, protein was detected at the SG2 interface, increasing linearly with age (
Fig. 3f). In marked contrast to the core and inner regions, there was little, or no, SG1 in any of the lenses examined (
Fig. 2). Trends in the outer cortex mirrored those in the barrier region, though with an approximately 10-year lag period.