Purification of rabbit skeletal muscle proteoglycogen: studies on the glucosyltransferase activity of polysaccharide-free and -bound glycogenin

Abstract

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.

Mechanisms of Monomeric and Dimeric Glycogenin Autoglucosylation

“…14 C autoglucosylation was measured by precipitation, washing the labeled protein with trichloroacetic acid, and radioactivity counting as described (22). Unless indicated otherwise, the results represent the mean values of three independent experiments, and error bars show S.D.…”

The size of the C-chain maltosaccharide of glycogen: evidence for the presence of only a single branch

“…Purified proteoglycogen was prepared from rabbit skeletal muscle as described previously (Carrizo et al, 1997). Briefly, the muscle was homogenized, the homogenate centrifuged at 4800 × g and proteoglycogen and membrane vesicles collected by centrifugation of the supernatant at 75,000 × g. The loose vesicles layer covering the proteoglycogen pellet was poured off, the pellet was dissolved in a 1.1% mixture of Triton X-114/Triton X-45 (86.5/13.5) at 4°C, and proteoglycogen was partitioned in the aqueous phase after raising the temperature of the solution to 14°C.…”

“…We described a procedure for the isolation of native proteoglycogen from rabbit muscle and of polysaccharide-free glycogenin after exhaustive digestion of proteoglycogen with α-amylase (Carrizo et al, 1997). We also reported the release and isolation of C-chain-bound glycogenin (c-glycogenin) from isoamylolyzed muscle proteoglycogen; the c-glycogenin species was able to autoglucosylate, indicating that it was linked to a maltosaccharide capable of being elongated by autoglucosylation (Romero and Curtino, 2003).…”

Inactivation and Thermal Stabilization of Glycogenin by Linked Glycogen

“…Glycogenin can autoglucosylate and transglucosylate exogenous acceptors (5, 9 -11). Analysing the course of the simultaneous autoglucosylation of glycogenin and transglucosylation of dodecyl-␤-maltoside (DBM), we found that the transglucosylation reaction continued even when the acceptor capacity for autoglucosylation was exhausted (5). This result led us to consider the possibility that under the glycogen-bound form, the enzyme could transglucosylate DBM.…”

“…Proteoglycogen and glycogenin. Proteoglycogen having polysaccharide-bound glycogenin as the only protein constituent was prepared from rabbit skeletal muscle as described previously (5). Glycogenin was released from proteoglycogen (150 g in glycogenin) by amylolysis with 3 g of ␣-amylase (purified as indicated before (12)) for 5 h at 25°C, in 1.0 ml of 3 mM Tris-acetate, pH 7.5, containing 0.06% sodium azide and 0.03 mM each of leupeptin, pepstatin and APMSF.…”