Lectin Binding and Enzymatic Deglycosylation of the cGMP-Gated Channel from Bovine Rod Photoreceptors

P Wohlfart,H Müller,N J Cook

doi:10.1016/s0021-9258(19)30026-2

Abstract

In order to investigate the lectin-binding properties of the photoreceptor cGMP-gated channel, solubilized and purified channel protein was incubated with immobilized lectins followed by reconstitution of unbound proteins. Of the lectins tested, only concanavalin A (ConA) was able to specifically sediment channel activity. A 240-kDa protein, which copurifies with the 63-kDa channel protein but does not bind ConA, was also found to be sedimented by the ConA-affinity matrix, thereby implicating that it is associated with the channel complex. Treatment of the purified channel protein with the enzyme glycopeptidase F in the presence of the denaturing detergent sodium dodecyl sulfate resulted in a rapid reduction of the apparent molecular mass by 1.90 kDa, and the abolition of ConA-binding. No intermediate molecular weight species were observed, suggesting that the channel protein is N-glycosylated at one site only. Under nondenaturing conditions, the kinetics of deglycosylation were distinctly two-phased: 50-60% deglycosylation was achieved rapidly; however, prolonged incubation was required to arrive at complete deglycosylation. Reconstitution experiments showed that deglycosylation had no significant effect on the kinetics of channel protein activation by cGMP.

Highlights

In order to investigate the lectin-binding properties tical to those of the channel protein in situ [8], (iii) direct of the photoreceptor cGMP-gated channel, solubilized and purified channel protein was incubated with immobilized lectins followed by reconstitution of unbound proteins
If the channel protein binds to a particular lectin, it should become immobilized during incubation, and reconstitution of the nonprecipitated material after centrifugatiosnhould show no channel activity
We investigated the binding of Ricin communis agglutinin lectin to the cGMPgated channel since it has been previously reported [19, 20] that bovine ROS membranes contain plasma membrane-specific receptors for this lectin

Summary

Introduction

In order to investigate the lectin-binding properties tical to those of the channel protein in situ [8], (iii) direct of the photoreceptor cGMP-gated channel, solubilized and purified channel protein was incubated with immobilized lectins followed by reconstitution of unbound proteins. Experiments showed that deglycosylation had no significant effect on the kinetics of channel protein activation by cGMP.

Results

Conclusion