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3894
Weinreb et al.
Chaperone-like Activity
The chaperone-like activity of -crystallin from control and
UV-irradiated lenses was determined by the heat-induced aggregation assay of L-crystallin at 60C.3 The proteins were
dissolved in a solution of 20 mM sodium phosphate, 100 mM
Na2SO4, 10 mM EDTA, at pH 6.9. The assay was performed at
a concentration of 0.25 mg/ml substrate protein and 0.05
mg/ml -crystallin.
Electron Microscopy
Protein samples from the heating assay were also analyzed by
electron transmission microscopy. Samples were negatively
stained with uranyl acetate (1% v/v). The in vitro filament
formation of -crystallin from control and UV-treated lenses
with L-crystallin was followed by immunoelectron microscopy using antibodies against vimentin, A-, B-, and L-crystallin. The grids were examined with a transmission electron
microscope (Jeol TEM1210, Tokyo, Japan) using 70 to 80 kV.
RESULTS
Electron Microscopy
FIGURE 1. Electron micrographs of (A) -crystallin obtained from control lenses (bar, 200 nm); (B) -crystallin from UV-Atreated lenses
(bar, 200 nm); (C) L-crystallin obtained from control lenses separately
heated at 60C (bar, 500 nm). Complexes were visualized by negative
staining with uranyl acetate.
METHODS
UV-A Irradiation
Lenses, excised from 2- to 4-year-old bovine eyes, were irradiated in special organ culture glass vessels described previously
by Dovrat and Weinreb.15 Briefly, a 400 W UV lamp (Vilber,
Lourmat Cedex, France) contained a filter that provided radiation of 33 J/cm2 for 75 minutes at 365 nm.
Fractionation of Crystallins
Lenses were dissected under a binocular stereomicroscope.
The lens cortex was homogenized in 100 mM Tris buffer at pH
7.5 and spun at 4C at 13,000g for 30 minutes. The supernatant
comprises the water-soluble lens fraction. Separation of this
fraction into -, H-, L-, and -crystallin was carried out by gel
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published electron micrographs of nonheated -crystallin revealed no detectable morphologic differences.18 Normal
-crystallin consists of molecules having an apparent spherical
structure, with a diameter of approximately 17 nm. Likewise
-crystallin obtained from irradiated lenses shows a similar
shape, albeit the size is somewhat smaller (Fig. 1B). After
incubation at 60C, L-crystallin (Fig. 1C) lost its irregular
spherical shape when compared with nonheated -crystallin as
described earlier.18
The in vitro filament formation was also examined by
immunoelectron microscopy (Fig. 2). AntiB- and antiLcrystallin labeling yielded identical results (not shown). The
formation of filament-like structures can be observed after the
heating assay at 60C using 0.05 mg -crystallin obtained from
control lenses with 0.25 mg L-crystallin. The identical experiment with -crystallin from irradiated lenses (Figs. 3A, 3B)
showed more pronounced filament-like structures compared
with the control. The results show that UV-A irradiation promotes the filament-like formation. Experiments with anti-vimentin were negative showing that no intermediate filament
component was involved in the crystallin hybrid formation.
Chaperone-like Activity
The chaperone-like activity determined with the aid of the
protein scattering at 360 nm is depicted in Figure 4. It can
be seen that this property of the water-soluble -crystallin
was affected by UV-A light. Compared with controls (curve
II), -crystallin derived from UV-Airradiated lenses revealed
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Weinreb et al.
FIGURE 5. Fluorescence determination using Thioflavin T (ThT) interaction with samples obtained from the heating assay, measured at
excitation of 450 nm and emission of 482 nm. ThT concentration was
250 nM. The reaction buffer contained 50 mM glycine-NaOH at pH 6.0.
Bars, SD (in four experiments).
CONCLUSION
Previously Slingsby et al.22 suggested a new model for crystallin
assembly in lens fiber cells. In the highly hydrated solution-like
region of the lens, it is envisaged that weak interaction between subunits such as those of -crystallin will occur, forming
elements of a network with dynamic branching. An open gel
structure would maintain proteinprotein interactions at a
high concentration, covering the more prominent hydrophobic regions and preventing random aggregation of subunits. This may possibly explain the present observation that
(heated) L-crystallin assembles with -crystallin, resulting in
filament-like structures. It cannot be excluded that one or more
of UV-Aprovoked alterations23 are related to the ability of
water-soluble -crystallin to form filaments in vitro more effi-
Acknowledgments
We thank Wilfried W. de Jong for fruitful discussions and Lucio
Benedetti for advice concerning electron microscopy.
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