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J Hugh McDowell, W Clay Smith, Susan Bolch, Donald Dugger; Arrestin inhibits AldolaseC activity. Invest. Ophthalmol. Vis. Sci. 2013;54(15):707.
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In a previous study, we showed that arrestin in a retinal homogenate was crosslinked to enolase1 using a water-soluble crosslinker. Further study showed that arrestin enhanced the activity of enolase1. Here we followed the same crosslinking protocol but used a lipid soluble crosslinker to identify other binding partners for arrestin that may be involved with the light-induced translocation of arrestin.
A bovine retinal homogenate was crosslinked using disuccinimidyl glutarate, a lipid soluble crosslinker. The majority of the crosslinked arrestin products were found in the supernatant from the retinal homogenate as well as in the rod outer segments prepared from the retinal homogenate. After following arrestin purification protocols, SDS-PAGE gels were run, stained, and immunoblots were developed with anti-arrestin antibodies. Coomassie-stained bands that were also reactive with the anti-arrestin antibody were submitted to mass spectrometric analysis to identify the proteins in the band. His(6)-tagged arrestin was expressed in yeast as previously described. AldolaseC was heterologously expressed in bacteria with a His(6) tag. AldolaseC activity was determined following published procedures using fructose-1,6-bisphosphate and hydrazine sulfate in the presence and absence of his-tagged arrestin.
A gel band at approximately 125kD was observed in the purified sample that was stained with Coomassie and was also detected in an immunoblot developed with an anti-arrestin antibody. AldolaseC and arrestin were found to be the principal components of this band. In an assay of aldolase enzymatic activity, the presence of arrestin resulted in a small, but significant reduction in the activity of aldolaseC.
AldolaseC was found to be an interaction partner for arrestin in bovine retinas. Mass spectrum analysis demonstrated that both aldolaseC and arrestin were present in an approximately 125 kD gel band. The presence of arrestin has a small inhibitory effect on the activity of aldolaseC. The binding of arrestin to aldolaseC appears to be weak as additional experiments intended to show binding failed to do so (data not shown). This relatively weak interaction suggests that aldolaseC may not have a significant role in the light-induced translocation of arrestin, but that arrestin may have a role in regulating glycolysis in the rod inner segment.
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