Incubation of a bovine retina membrane preparation with micromolar amounts of UDP-['4C]glucose resulted in the incorporation of [14C]glucose into endogenous (1 +4)-a-glucan, insoluble in trichloroacetic acid, and acidsoluble ethanol-insoluble glycogen.1. 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.2. 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.3. The glycogen fraction of retina contained 1.5 -2.0 pg protein/lOO pg 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. 4. The protein nature of the Coomassie-blue-stainable material was demonstrated by iodination of the glycogen fraction with [1311]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 treatment with a-amylse decreased the molecular size of both labeled and stainable material. 5.Physical dissociative conditions (7.5 M ureaj0.83 SDSj0.83 % mercaptoethanol) and the following chemical treatments failed to dissociate the iodinated protein from glycogen: (a) 0.1 M NaOH/O.l M NaBH, at room temperature for 24 h; (b) 1 M HCl in methanol at 50 "C for 10 min; (c) trifluoroacetic acid at 50 "C for 6 min.6. 1311-labeled glycogenpeptide was isolated after 1311-labeled protein-bound glycogen had been subjected to digestion with papain/pronase and passed through a Sepharose column.7. 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-acidprecipitable proteoglucan owing to its lower glucose content.The synthesis of (1 -+4)-a-glucan, bound to protein, has been inferred from results obtained in vitro in several systems such as rat liver [l], Escherichia coli [2], Neurospora crassa [3], potato tuber [4] and bovine retina [5]. The presence of protein linked to glucan was argued from the insolubility in trichloroacetic acid and sensitivity to pronase of the synthesized products. However, unbranched long glucan chains are intrinsically insoluble in trichloroacetic acid [6]. The materials released by pronase were not further analyzed and controls for the possible presence of contamihating amylolytic activity in the protease preparations were not shown.Evidence for protein strongly bound to glucan was claimed after dinitrophenylation of a 'glycogen-peptide' obtained from a trichloroacetic acid-insoluble glucan fraction of liver [7], but no dinitrophenylated amino acid was demonstrated.In the present work the attempt to demonstrate protein firmly combined to (1 +4)-a-...

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Tyrosine-glycogen obtained from retina proteoglycogen by exhaustive proteolytic digestion was radiolabelled with 125I. The 125I-labelled tyrosine-glycogen was degraded by amylolytic digestion to a very small radioactive product, which was identified as iodotyrosine by h.p.l.c. The amylolytic mixture used released glucose and maltose that were alpha-linked to the phenolic hydroxy group of p-nitrophenol. No free iodotyrosine was found before or after the intact [125I]iodotyrosine-glycogen was subjected to two cycles of the Edman degradation procedure. The linkage between protein and glycogen was alkali-stable. Therefore it is concluded that the protein-bound glycogen was O-glycosidically linked to the phenolic hydroxy group of tyrosine. The amino acid has not been heretofore found to be involved in the linkage of carbohydrates to proteins.

Proteoglycogen is the end product in the process of glycogen biogenesis. We have purified rabbit muscle proteoglycogen and studied the glucosyltransferase reactions catalyzed by its protein moiety, glycogenin, free or bound to the polysaccharide. The purification strategy involved dissolution of proteoglycogen and cosedimenting membrane vesicles in a Triton X-114/Triton X-45 mixture followed by partition in the aqueous phase, potassium iodide precipitation of accompanying proteins, and washing by high-speed centrifugation. Glycogenin or a proteoglycogen species of an average molecular mass of 200 kDa was isolated by ion-exchange chromatography after the purified proteoglycogen had been subjected to long or short amylolytic digestion, respectively. Besides autoglucosylation from UDP-glucose, glycogenin was capable of autogalactosylation from UDP-galactose. The autoglucosylation reaction was not inhibited by the simultaneous glucosylation of the exogenous acceptors N-(maltosyl-alpha-1-4-(1-deoxiglucitol))-peptide or n-dodecyl-beta-D-maltoside. The polysaccharide-bound glycogenin species of 200 kDa showed to be active for the glucosylation of exogenous acceptor and represented the isolated proteoglycogen of higher size having glucosyl transferase activity. This is the first description of the isolation of native proteoglycogen and a proteoglycogen species having glucosyltransferase activity.