Figure 3. 9-cis-Retinal was
released from CRALBP by acidic glycerophospholipids. Cellular
retinaldehyde-binding protein (CRALBP)·9-cis-retinal was
incubated in the dark with NH2OH and small unilamellar
vesicles (SUVs). Release of 9-cis-retinal was followed using the
spectral assay (A and C) and the HPLC assay (B
and D). Incubation of CRALBP·9-cis-retinal with SUVs
composed of 50 mol% phosphatidylcholine (PC) and 50 mol% phosphatidic
acid (PA) produced spectral changes over 18 h consistent with release
of 9-cis-retinal for reaction with NH2OH (A),
and high performance liquid chromatography (HPLC) analysis of an 18 h
sample demonstrated recovery of 9-cis-retinal oximes (B).
Incubation with SUVs composed of 100 mol% PC had little effect on the
absorption spectrum of 9-cis-retinal bound to CRALBP (C),
and minimal amounts of 9-cis-retinal oximes were recovered (D).
The amount of 9-cis-retinal released in A was
approximately 60% of the total bound to the protein. In B and D,
only the oxime regions of the chromatograms are shown. The data shown
in the panels are representative of at least five independent repeats
of the experiment.