Regular ArticleA Biphasic Radiometric Assay of Glycogenin Using the Hydrophobic Acceptor n-Dodecyl-β-D-Maltoside
Abstract
Glycogenin is a self-glycosylating protein that catalyzes early glucosyl transfer steps in the biosynthesis of glycogen. In currently used assays of glycogenin activity, the enzyme is incubated with radioactive UDP-glucose, and the labeled reaction product is then isolated by precipitation with trichloracetic acid. A new assay is reported here which is based on the observation that glycogenin is not only self-glycosylating but may also use exogenous alkyl maltosides as substrates. After incubation of the enzyme with n-dodecyl-β-D-maltoside and UDP-[3H]glucose, the radioactivity in the resultant n-dodecyl-β-D-[3H]maltotrioside is determined by any one of the following three procedures, which all rely on the hydrophobic properties conferred on the reaction product by the alkyl aglycone: (i) adsorption of the product to a Sep-Pak C18 cartridge and elution with 70% ethanol; (ii) biphasic liquid scintillation counting in ScintiLene/25% isoamyl alcohol, without isolation of the product, and (iii) precipitation with trichloracetic acid in the presence of carrier protein. The Sep-Pak C18 procedure has the advantage that it allows essentially quantitative isolation of the reaction product, while, under the conditions chosen, only about 50% of the product is precipitated by trichloroacetic acid. For most applications, however, biphasic liquid scintillation counting is the method of choice, since close to 90% of the labeled product is extracted into the organic phase and can be counted directly without interference from the labeled nucleotide sugar which remains in the aqueous phase. In crude enzyme extracts, where nucleotide sugar pyrophosphatase and alkaline phosphatase activities are high, inclusion of 1 mM β-NAD and unlabeled UDP-glucose at a final concentration of 10 μM in the reaction mixtures minimizes the apparent breakdown of the radioactive nucleotide sugar, which results in a high background due to extraction of [3H]glucose into the organic phase. The biphasic procedure is potentially applicable to the assay of any glycosyltransferase, for which a suitable hydrophobic acceptor is available.
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The intramolecular autoglucosylation of monomeric glycogenin
2008, Biochemical and Biophysical Research CommunicationsThe ability of monomeric glycogenin to autoglucosylate by an intramolecular mechanism of reaction is described using non-glucosylated and partially glucosylated recombinant glycogenin. We determined that monomer glycogenin exists in solution at concentration below 0.60–0.85 μM. The specific autoglucosylation rate of non-glucosylated and glucosylated monomeric glycogenin represented 50 and 70% of the specific rate of the corresponding dimeric glycogenin species. The incorporation of a unique sugar unit into the tyrosine hydroxyl group of non-glucosylated glycogenin, analyzed by autoxylosylation, occurred at a lower rate than the incorporation into the glucose hydroxyl group of the glucosylated enzyme. The intramonomer autoglucosylation mechanism here described for the first time, confers to a just synthesized glycogenin molecule the capacity to produce maltosaccharide primer for glycogen synthase, without the need to reach the concentration required for association into the more efficient autoglucosylating dimer. The monomeric and dimeric interconversion determining the different autoglucosylation rate, might serve as a modulation mechanism for the de novo biosynthesis of glycogen at the initial glucose polymerization step.
C-chain-bound glycogenin is released from proteoglycogen by isoamylase and is able to autoglucosylate
2003, Biochemical and Biophysical Research CommunicationsProteoglycogen glycogenin is linked to the glucose residue of the C-chain reducing end of glycogen. We describe for the first time the release by isoamylase and isolation of C-chain-bound glycogenin (C-glycogenin) from proteoglycogen. The treatment of proteoglycogen with α-amylase releases monoglucosylated and diglucosylated glycogenin (a-glycogenin) which is able to autoglucosylate. It had been described that isoamylase splits the glucose–glycogenin linkage of fully autoglucosylated glycogenin previously digested with trypsin, releasing the maltosaccharide moiety. It was also described that carbohydrate-free apo-glycogenin shows higher mobility in SDS–PAGE and twice the autoglucosylation capacity of partly glucosylated glycogenin. On the contrary, we found that the C-glycogenin released from proteoglycogen by isoamylolysis shows lower mobility in SDS–PAGE and about half the autoglucosylation acceptor capacity of the partly glucosylated a-glycogenin. This behavior is consistent with the release of maltosaccharide-bound glycogenin instead of apo-glycogenin. No label was split from auto-[14C]glucosylated C-glycogenin or fully auto-[14C]glucosylated a-glycogenin subjected to isoamylolysis without previous trypsinolysis, thus proving no hydrolysis of the maltosaccharide–tyrosine linkage. The ability of C-glycogenin for autoglucosylation would indicate that the size of the C-chain is lower than the average length of the other glycogen chains.
Inactivation and thermal stabilization of glycogenin by linked glycogen
2001, Biochemical and Biophysical Research CommunicationsGlycogen-free but not glycogen-bound glycogenin transglucosylates dodecyl-β-maltoside. Furthermore, its sugar nucleotide-binding site can be photoaffinity labeled using [β-32P]5-azido-UDP-glucose. Disruption with DMSO of the hydrogen bonds that stabilize the α-helical structure of glycogen restored the photoaffinity labeling of the glycogen-bound enzyme but not its transglucosylation activity. The larger size polysaccharide that linked to glycogenin allowed transglucosylation corresponding to that of PG-200, a proteoglycogen species of Mr 200 kDa. PG-200 showed lower activity and increased activation energy than glycogen-free glycogenin. Heat denaturation of glycogen-free and glycogen-bound glycogenin occurred at 51 and 64°C, respectively. Active glycogenin was recovered after the glycogen-bound form was heated at 60–70°C and immediately cooled. Treatment at 60°C of the glycogen-free enzyme resulted in inactivation. This is the first report describing the inactivation and thermal stabilization of an enzyme by linked polysaccharide.
Manganese sulfate-dependent glycosylation of endogenous glycoproteins in human skeletal muscle is catalyzed by a nonglucose 6-P-dependent glycogen synthase and not glycogenin
1999, Biochimica et Biophysica Acta - General SubjectsGlycogenin, a Mn2+-dependent, self-glucosylating protein, is considered to catalyze the initial glucosyl transfer steps in glycogen biogenesis. To study the physiologic significance of this enzyme, measurements of glycogenin mediated glucose transfer to endogenous trichloroacetic acid precipitable material (protein-bound glycogen, i.e., glycoproteins) in human skeletal muscle were attempted. Although glycogenin protein was detected in muscle extracts, activity was not, even after exercise that resulted in marked glycogen depletion. Instead, a MnSO4-dependent glucose transfer to glycoproteins, inhibited by glycogen and UDP-pyridoxal (which do not affect glycogenin), and unaffected by CDP (a potent inhibitor of glycogenin), was consistently detected. MnSO4-dependent activity increased in concert with glycogen synthase fractional activity after prolonged exercise, and the MnSO4-dependent enzyme stimulated glucosylation of glycoproteins with molecular masses lower than those glucosylated by glucose 6-P-dependent glycogen synthase. Addition of purified glucose 6-P-dependent glycogen synthase to the muscle extract did not affect MnSO4-dependent glucose transfer, whereas glycogen synthase antibody completely abolished MnSO4-dependent activity. It is concluded that: (1) MnSO4-dependent glucose transfer to glycoproteins is catalyzed by a nonglucose 6-P-dependent form of glycogen synthase; (2) MnSO4-dependent glycogen synthase has a greater affinity for low molecular mass glycoproteins and may thus play a more important role than glucose 6-P-dependent glycogen synthase in the initial stages of glycogen biogenesis; and (3) glycogenin is generally inactive in human muscle in vivo.
Self-glucosylation of glycogenin, the initiator of glycogen biosynthesis, involves an inter-subunit reaction
1999, Archives of Biochemistry and BiophysicsGlycogenin is a dimeric self-glucosylating protein involved in the initiation phase of glycogen biosynthesis. As an enzyme, glycogenin has the unusual property of transferring glucose residues from UDP-glucose to itself, forming an α-1,4-glycan of around 10 residues attached to Tyr194. Whether this self-glucosylation reaction is inter- or intramolecular has been debated. We used site-directed mutagenesis of recombinant rabbit muscle glycogenin-1 to address this question. Mutation of highly conserved Lys85 to Gln generated a glycogenin mutant (K85Q) that had only 1–2% of the self-glucosylating activity of wild-type enzyme. Consistent with previous work, mutation of Tyr194 to Phe in a GST-fusion protein yielded a mutant, Y194F, that was catalytically active but incapable of self-glucosylation. The Y194F mutant was able to glucosylate the K85Q mutant. However, there was an initial lag in the self-glucosylation reaction that was abolished by preincubation of the two mutant proteins. The interaction between glycogenin subunits was relatively weak, with a dissociation constant inferred from kinetic experiments of around 2 μM. We propose a model for the glucosylation of K85Q by Y194F in which mixing of the proteins is followed by rate-limiting formation of a species containing both subunit types. The results provide the most direct evidence to date that the self-glucosylation of glycogenin involves an inter-subunit reaction.
Biosynthesis of proteoglycogen: Modulation of glycogenin expression in the developing chicken
1997, Biochemical and Biophysical Research CommunicationsGlycogenin, the autoglucosyltransferase that primes the biosynthesis of proteoglycogen, is found in the polysaccharide linked proteoglycogen form in mammals and chicken. Glycogenin was released from proteoglycogen and its activity was measured, together with that of glycogen synthase as well as glycogen content, in muscle, liver, and brain during chicken development. The specific activity of glycogenin, expressed per protein, increased with development only in muscle and was higher than the specific activities measured in liver and brain at any time. Concomitant with the rise in activity, an enhanced expression of the protein was observed with Western blot. The specific activity of glycogen synthase increased with development in muscle and liver, while glycogen accumulation was noticeable only in liver. The results indicate that the molar concentration of proteoglycogen is higher in muscle than in liver. The high glycogen content of liver may indicate that the size of the polysaccharide moiety of proteoglycogen is larger in liver than in muscle. This is the first report of developmental modulation ofde novobiosynthesis of glycogen at the level of the primer that initiates glucose polymerization.