Abstract
A striking homology is observed between the regions 70-83 and 361-374 of the sequence of bovine arrestin and the calcium-binding loops of calmodulin and troponin C. However, the predicted alpha-helices flanking the calcium-binding site in calmodulin and troponin C are not present in arrestin. Direct measurements therefore were made in order to assess whether arrestin can bind calcium. We found that arrestin does not bind Ca2+ at physiological ionic strength, as determined by equilibrium dialysis, gel filtration, and fluorescence spectroscopy. Rapid and quantitative precipitation of arrestin occurs with Tb3+. The precipitation is reversed by EDTA and blocked by Mg2+ but not by Ca2+. Prompted by several reports, we also investigated whether nucleotides bind to arrestin. Neither ATP nor GTP binds under the conditions tested. Binding of arrestin to photolyzed, phosphorylated rhodopsin also does not influence the binding of calcium or nucleotides.
Highlights
A striking homology is observed between the regions arrestin
The predicted a-helices flankingthe Inthe present work, we have tested ligands which are calcium-binding site incalmodulin and troponin C are not present in arrestin.Direct measurementstherefore were made in order to assess whether arrestin can bind calcium
We found that arrestin does not bind Ca2+at known to be involved in phototransduction to determine whether they bind to arrestin or modulate its interactionwith photolyzed rhodopsin
Summary
The arrestin-containing extract was added to phosphorylated rhodopsin which was prepared from rod outer segments. Rhodopsin, but only whenrhodopsin has been phosphorylated Rod outer segments were prepared from frozen retinas [15].Rod (reviewed in Ref. 2). We showed that arrestin appears to act during phototransduction by blocking rapid dephosphorylabuffer, pH 7.2,containing 2 mM MgCl,, 0.1 mM EDTA, 3 mM ATP, 1 mM GTP, and pg each of aprotinin, leupeptin, benzamidine, and pepstatin. The complex of arrestin with phosphorylated rhodopsin was collected by centrifugation (20min, 48,000X g ) , and the supernatantwas discarded. The supernatant was dialyzedagainst threechanges of 10mM BTP buffer, pH 7.5,over a 16-hperiod Material prepared in this way contains about 70-80% arrestin as judged by SDS-polyacrylamidegel electroaction between phosphorylated, photolyzed rhodopsin and phoresis; it contains small particles of membranes that are not removed by ultracentrifugation. Arrestin (95-100% pure; yield, about 8-12 mg) was dialyzed against the appropriate buffer containing 100 mM KC1
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