Lectin binding and enzymatic deglycosylation of the cGMP-gated channel from bovine rod photoreceptors.
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.
Wohlfart
P
P
Max-Planck-Institut für Biophysik, Abteilung für Molekulare Membranbiologie, Frankfurt am Main, Federal Republic of Germany.
Müller
H
H
Cook
N J
NJ
D000581:Amidohydrolases; D000818:Animals; D002417:Cattle; D003208:Concanavalin A / Q000378:metabolism; D006152:Cyclic GMP / Q000494:pharmacology; D054815:Cyclic Nucleotide-Gated Cation Channels; D004591:Electrophoresis, Polyacrylamide Gel; D005136:Eye Proteins / Q000302:isolation & purification / Q000378:metabolism*; D007473:Ion Channels / Q000378:metabolism*; D007700:Kinetics; D037102:Lectins; D008970:Molecular Weight; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D010786:Photoreceptor Cells / Q000378:metabolism*; D012374:Rod Cell Outer Segment / Q000378:metabolism*; D013816:Thermodynamics
D054815:Cyclic Nucleotide-Gated Cation Channels; D005136:Eye Proteins; D007473:Ion Channels; D037102:Lectins; D003208:Concanavalin A; D000581:Amidohydrolases; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D006152:Cyclic GMP