Next, ROS phosphorylation of anti-recoverin antibody–treated rat eyes
was examined. In our previous study, we found that anti-recoverin
antibody was incorporated within the inner parts of the retina at 3
hours, and then antibody localization shifted toward the outer parts of
the retina at 12 hours after administration. Between 12 and 24 hours,
the antibody accumulated within both the ONL and photoreceptor layer,
and thereafter the antibody disappeared slowly from the retina within 6
days.
16 Therefore, eyes were enucleated 36 hours after
administration of either preimmune rabbit IgG or anti-recoverin
antibody and incubated on ice for 1 hour in the dark followed by
separation of ROS by the sucrose gradient centrifugation method. ROS
was then homogenized and incubated with 0.5 mM[γ
-
32P] ATP in 100 mM Na-phosphate buffer (pH
7.2) containing 5 mM MgCl
2 in the presence or
absence of Ca
2+ for 10 minutes at 30°C under
150-W lamp illumination. Phosphorylated ROS was analyzed by SDS-PAGE in
which the radioactivity of the corresponding rhodopsin band was counted
(
Fig. 2A ). Intravitreal treatment with anti-recoverin antibody did not affect
the protein concentrations of rhodopsin, rhodopsin kinase, and other
proteins
(Fig. 2A) . However, rhodopsin phosphorylation levels of
anti-recoverin IgG–treated retinas were significantly higher than
those of control in the presence of Ca
2+, in both
the 36-hour (
Fig 2B left, open columns) and 3-week preparations (
Fig. 2B left, shaded columns; conditions A and D). This enhancement of
rhodopsin phosphorylation by anti-recoverin antibody was not
significantly changed by the co-injection of anti-hsc 70 serum (
Fig. 2B , left panel, condition E). Intravitreous injection of anti-hsc 70
serum alone had no effect on rhodopsin phosphorylation (
Fig. 2B , left
panel, condition C). To exclude the possibility that nonincorporated
anti-recoverin antibody outside the cell affected rhodopsin
phosphorylation, retinas dissected from eyes intravitreously injected
with PBS were mixed with anti-recoverin antibody in the ROS preparation
and subjected to rhodopsin phosphorylation analysis (
Fig. 2B , condition
B). No effects of the presence of anti-recoverin IgG on the levels of
rhodopsin phosphorylation were found in the ROS preparation.