Yasushi Makino

Purification of Glycogen Debranching Enzyme from Porcine Brain: Evidence for Glycogen Catabolism in the Brain

Amylo-1,6-glucosidase from porcine brain was purified to homogeneity by ammonium sulfate fractionation, followed by sequential steps of liquid chromatography on DEAE-Sephacel, Sephacryl S-300, and Super Q. The purified enzyme had both maltooligosaccharide transferase and amylo-1,6-glucosidase activities within a single polypeptide chain, and the combination of these two activities removed the branches of phosphorylase limit dextrin. Based on these results, the purified enzyme was identified as a glycogen debranching enzyme (GDE). The molecular weight of the brain GDE was 170,000 by gel-filtration and 165,000 by reducing SDS–PAGE. The pH profile of maltooligosaccharide transferase activity coincided with that of the amylo-1,6-glucosidase activity (pH optimum at 6.0). The existence of GDE as well as glycogen phosphorylase in the brain explains brain glycogenolysis fully and supports the hypothesis that glycogen is a significant source of energy in this organ.

Sensitive Assay of Glycogen Phosphorylase Activity by Analysing the Chain-Lengthening Action on a Fluologenic Maltooligosaccharide Derivative

The action of glycogen phosphorylase (GP) is essentially reversible, although GP is generally classified as a glycogen-degrading enzyme. In this study, we developed a highly sensitive and convenient assay for GP activity by analysing its chain-lengthening action on a fluorogenic maltooligosaccharide derivative in a glucose-1-phosphate-rich medium. Characterization of the substrate specificity of GP using pyridylaminated (PA-) maltooligosaccharides of various sizes revealed that a maltotetraosyl (Glc(4)) residue comprising the non-reducing-end of a PA-maltooligosaccharide is indispensable for the chain-lengthening action of GP, and PA-maltohexaose is the most suitable substrate for the purpose of this study. By using a high-performance liquid chromatograph equipped with a fluorescence spectrophotometer, PA-maltoheptaose produced by the chain elongation of PA-maltohexaose could be isolated and quantified at 10 fmol. This method was used to measure the GP activities of crude and purified GP preparations, and was demonstrated to have about 1,000 times greater sensitivity than the spectrophotometric orthophosphate assay.

Properties and functions of the storage sites of glycogen phosphorylase.

Glycogen phosphorylase (GP) is biologically active as a dimer of identical subunits. Each subunit has two distinct maltooligosaccharide binding sites: a storage site and a catalytic site. Our characterization of the properties of these sites suggested that GP activity consists of two activities: (i) binding to the glycogen molecule and (ii) phosphorolysis of the non-reducing-end glucose residues. Activity (i) is mainly due to the activities of the two storage sites, which depended on the ionic strength of the medium and were directly inhibited by cyclodextrins (CDs). Activity (i) is of benefit to GP because a high concentration of non-reducing-end glucose residues is localized on the surface of the glycogen molecule. Activity (ii), the total activity of the two catalytic sites, exhibited relatively little ionic strength dependence. Because the combined activity of (i) and (ii) is deduced using glycogen as an assay substrate, the sole activity of (ii) must be measured using small maltooligosyl-substrates. By using a very low concentration of pyridylaminated maltohexaose, we demonstrated that the GP catalytic sites are active even in the presence of CDs, and that the actions of the catalytic site and the storage site are independent of each other.

Inspection of the Activator Binding Site for 4-α-Glucanotransferase in Porcine Liver Glycogen Debranching Enzyme with Fluorogenic Dextrins

Recently, we found that alpha-, beta- and gamma-cyclodextrins accelerated the 4-alpha-glucanotransferase action of porcine liver glycogen debranching enzyme (GDE) on Glcalpha1-4Glcalpha1-4Glcalpha1-4(Glcalpha1-4Glcalpha1-4Glcalpha1-4Glcalpha1-6)Glcalpha1-4Glcalpha1-4Glcalpha1-4Glcalpha1-4GlcPA (B5/84), and proposed the presence of an activator binding site in the GDE molecule. In liver cells, the structures of alpha-glucans proximal to the site GDE acts are not cyclodextrins, but glycogen and its degradation products. To estimate the structural characteristics of intrinsic activators and to inspect the features of the activator binding site, we examined the effects of four fluorogenic dextrins, (Glcalpha1-6)(m)Glcalpha1-4(Glcalpha1-4)(n)GlcPA (B5/51, m = 1, n = 3; B6/61, m = 1, n = 4; B7/71, m = 1, n = 5; G6PA, m = 0, n = 4), on the debranching of B5/84 by porcine liver GDE. The GDE 4-alpha-glucanotransferase removed the maltotriosyl residue from the maltotetraosyl branch of B5/84, producing Glcalpha1-4Glcalpha1-4Glcalpha1-4(Glcalpha1-6)Glcalpha1-4Glcalpha1-4Glcalpha1-4Glcalpha1-4GlcPA (B5/81). In the presence of G6PA, the removed maltotriosyl residue was transferred to G6PA to give Glcalpha1-4Glcalpha1-4Glcalpha1-4Glcalpha1-4Glcalpha1-4Glcalpha1-4Glcalpha1-4Glcalpha1-4GlcPA (G9PA). In the absence of G6PA, the removed maltotriosyl residue was transferred to water. B7/71, B6/61 and B5/51 did not undergo any changes by the GDE, but they accelerated the action of the 4-alpha-glucanotransferase in removing the maltotriosyl residue. Of the four fluorogenic dextrins examined, B6/61 most strongly accelerated the 4-alpha-glucanotransferase action. The activator binding site is likely to be a space that accommodates the structure of Glcalpha1-6Glcalpha1-4Glcalpha1-4Glcalpha1-4Glcalpha1-4Glc.

Sensitive, nonradioactive assay of phosphorylase kinase through measurement of enhanced phosphorylase activity towards fluorogenic dextrin

Glycogen phosphorylase (GP) exists in two interconvertible forms, GPa (phosphorylated form, high activity) and GPb (nonphosphorylated form, low activity). Phosphorylase kinase (PhK) catalyses the phosphorylation of GPb and plays a key role in the cascade system for regulating glycogen metabolism. In this study, we developed a highly sensitive and nonradioactive assay for PhK activity by measuring the enhanced GP activity towards a pyridylaminated maltohexaose. The enhanced GP activity (ΔA) was calculated by the following formula: ΔA = A(+) - A(0), where A(+) and A(0) represent the GP activities of the PhK-treated and PhK-nontreated samples, respectively. Using a high-performance liquid chromatograph equipped with a fluorescence spectrophotometer, the product of GP activity could be isolated and quantified at 10 fmol. This method does not require the use of any radioactive compounds and only 1 µg of GPb per sample was needed to obtain A(+) and A(0) values. The remarkable reduction in GPb concentration enabled us to discuss an interesting new role for glycogen in PhK activity.

A new interpretation of sulfate activation of rabbit muscle glycogen phosphorylase

It is widely known that sulfate ion at high concentration serves like an allosteric activator of glycogen phosphorylase (GP). Based on the crystallographic studies on GP, it has been assumed that the sulfate ion is bound close to the phosphorylatable Ser14 site of nonactivated GP, causing a conformational change to catalytically-active GP. However, there are also reports that sulfate ion inhibits allosterically-activated GP by preventing the phosphate substrate from attaching to the catalytic site. In the present study, using a high concentration of sulfate ion, significant enhancement of GP activity was observed when macromolecular glycogen was used as substrate but not when smaller maltohexaose was used. In glycogen solution, nonreducing-end glucose residues are localized on the surface of glycogen and are not distributed homogenously in the solution. Using cyclodextrin-immobilized column chromatography, we found that sulfate at high concentration promoted GP–dextrin binding through the dextrin-binding site (DBS) located away from the catalytic site. This result is consistent with the properties of the DBSs found in glycogen-debranching enzyme and β-amylase. Therefore, we propose a new interpretation of the sulfate activation of GP, wherein sulfate ions at high concentration promote glycogen-binding to the DBS directly, and glycogen-binding to the catalytic site indirectly. Our findings were successfully applied to the affinity purification of porcine brain GP.

Fragmentation of Oligosaccharides from Sodium Adduct Molecules Depends on the Position of N -Acetyl Hexosamine Residue in Their Sequences in Mass Spectrometry

Six different sequences of hexasaccharides, pyridylaminated malto-hexaoses containing one N-acetyl hexosamine (HexNAc) residue, were analyzed using matrix-assisted laser desorption/ionization (MALDI) tandem time-of-flight (TOF) mass spectrometry (MS). Based on the product ion spectra of sodium adducts [M+Na]+, the chemical species of the observed product ions contained a HexNAc residue and had high ion abundance, indicating that the HexNAc residue had a higher affinity to sodium atom than glucopyranose. The acetamide group coordinated easily to sodium atom. This general rule of product ion generation was useful to predict the structure of the oligosaccharides based on the MS/MS product ion spectra.

Discrimination of porcine glycogen debranching enzyme isozymes by the ratios of their 4-α-glucanotransferase and amylo-α-1,6-glucosidase activities

Glycogen debranching enzyme (GDE) is a single-chain protein containing distinct active sites that exhibit 4-alpha-glucanotransferase and amylo-alpha-1,6-glucosidase activities. The ratios of these two activities in porcine liver and muscle GDEs were compared using a set of homologous fluorogenic branched dextrins. For quantifying 4-alpha-glucanotransferase activity, 6(3)-O-alpha-maltotetraosyl-PA-maltooctaose (B3/84), 6(4)-O-alpha-maltotetraosyl-PA-maltooctaose (B4/84), 6(5)-O-alpha-maltotetraosyl-PA-maltooctaose (B5/84) and 6(6)-O-alpha-maltotetraosyl-PA-maltooctaose (B6/84) were used as substrates and maltohexaose (G6) as the acceptor. The substrate for amylo-alpha-1,6-glucosidase activity was 6(3)-O-alpha-glucosyl-PA-maltotetraose (B3/41). HPLC analysis of the fluorogenic branched dextrin digests in the presence of G6 revealed that GDE 4-alpha-glucanotransferases produce the corresponding 6-O-alpha-glucosyl-PA-maltooctaose (GG8PA) and maltononaose (G9). The ratios of the 4-alpha-glucanotransferase activity to amylo-alpha-1,6-glucosidase activity, for the liver and muscle enzymes were respectively 0.240 and 0.0840 for B3/84, 0.204 and 0.0788 for B4/84, 0.145 and 0.0592 for B5/84, and 0.109 and 0.0458 for B6/84. These data clearly indicate that porcine liver and muscle GDEs are different from each other. The ratios of porcine brain GDE were 0.155, 0.131, 0.0990 and 0.0745 for B3/84, B4/84, B5/84 and B6/84, respectively. These results indicate that porcine brain GDE is also unique from liver and muscle enzymes, suggesting that it is either a third enzyme, or a mixture of 45% liver and 55% muscle GDEs.