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Lectin binding and enzymatic deglycosylation of the cGMP-gated channel from bovine rod photoreceptors

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Wohlfahrt,  Paulus
Department of Molecular Membrane Biology, Max Planck Institute of Biophysics, Max Planck Society;

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Müller,  Heidi
Department of Molecular Membrane Biology, Max Planck Institute of Biophysics, Max Planck Society;

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Cook,  Neil J.
Department of Molecular Membrane Biology, Max Planck Institute of Biophysics, Max Planck Society;

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Citation

Wohlfahrt, P., Müller, H., & Cook, N. J. (1989). Lectin binding and enzymatic deglycosylation of the cGMP-gated channel from bovine rod photoreceptors. The Journal of Biological Chemistry, 264(35), 20934-20939. doi:10.1016/S0021-9258(19)30026-2.


Cite as: http://hdl.handle.net/21.11116/0000-0007-835F-B
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.