Evidence for the glycoprotein nature of retina glycogen

Incubation of a bovine retina membrane preparation with micromolar amounts of UDP‐[¹⁴C]glucose resulted in the incorporation of [¹⁴C]glucose into endogenous (1→4)‐α‐glucan, insoluble in trichloroacetic acid, and acidsoluble ethanol‐insoluble glycogen. The trichloroacetic‐acid‐insoluble glucan fraction of retina migrated in 2.6–3% acrylamide gels when subjected to sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS‐PAGE) and was rendered acidsoluble by digestion with pronase. The solubility of the acid‐insoluble glucan in acidified organic solvent was different from that of amylose or glycogen and similar to membrane proteins and glycoproteins. The glycogen fraction of retina contained 1.5–2.0 μ protein/100 μ glucose. When this fraction was analyzed by SDS‐PAGE only one band, which moved near the top of 3% acrylamide gels, was stained with periodic acid Schiff reagent and Coomassie blue. The protein nature of the Coomassie‐blue‐stainable material was demonstrated by iodination of the glycogen fraction with [¹³¹I]iodide and identification of labeled monoiodotyrosine and diiodotyrosine. The bulk of the label comigrated with carbohydrate near the top of gels in SDS‐PAGE and treatmet with α‐amylse decreased the molecular size of both labeled and stainable material. Physical dissociative conditions (7.5 M urea/0.83% mercaptoethanol) and the following chemical tratments failed to dissociate the iodinated protein from glycogen: (a) 0.1 M NaOH/0.1 M NaBH₄ at room temperature for 24h; (b) 1 M HCl in methanol at 50°C for 10 min; (c) trifluoroacetic acid at 50°C for 6 min. ¹³¹I‐labeled glycogenpeptide was isolated after ¹³¹I‐labeled protein‐bound glycogen had been subjected to digestion with papain/pronase and passed through a Sepharose column. The results suggest that at least part of glycogen in bovine retina is firmly combined to protein as a single proteoglycogen molecule. Furthermore some of the proteoglycogen might be present as a trichloroacetic‐acid‐precipitable proteoglucan owing to its lower glucose content.