Differences between glycogen biogenesis in fast- and slow-twitch rabbit muscle.

Skeletal muscle glycogen is an essential energy substrate for muscular activity. The biochemical properties of the enzymes involved in de novo synthesis of glycogen were analysed in two types of rabbit skeletal muscle fiber (fast- and slow-twitch). Glycogen concentration was higher in fast-twitch muscle than in slow-twitch muscle, but the latter contained many more small intermediate-acceptor molecules that could act as glycogen synthase substrates. The enzymes involved in de novo synthesis of glycogen in fast-twitch muscle were strongly stimulated by Glc-6-P, but those in slow-twitch muscle were not.

Rat liver genesine: a biochemical approach.

Genesine, the naked autoglucosylating protein involved in glycogen biosynthesis, was partially purified from rat liver and some of its biochemical properties were characterized. Its activity was strongly activated by Mn2+ and two-pH optimums were obtained. UDP-14C-Glc was the preferred glucosyl donor substrate, but also UDP-14C-Xyl was. It was additionally found that more than one glucose was transferred to the protein or to that alpha1,4 glucan already linked to the protein from UDP-Glc. Glucose, maltose, xylose and UDP were inhibitors of genesine activity.

Glycogen brain branching enzyme.

Rat brain glycogen branching enzyme was partially purified in order to elucidate its mechanism of action. The alpha1,4-alpha1,6-glucan polysaccharide was synthesized using rat brain branching enzyme under two different elongation conditions: Glc-1-P and phosphorylase or UDP-Glc and glycogen synthase. The products obtained demonstrated that the cpolysaccharides synthesized (pattern of the spectra obtained in the presence of Krisman's reagent, lambda max, parameter A and R, % beta-amylolysis and degree of branching) under different incubation times are nearly constant. These results imply that the degree of branching of a polysaccharide depends only on the enzyme specificity.

Progress in the understanding of glycogen biogenesis in rat heart.

The sequence of glucosylation steps from "genesine", the naked initiation protein, to rat heart glycogen are described. During a pulse experiment with UDP-14C-glucose the radiolabelled 14C-glucosylated protein band of 38 and 42 kDa appeared first. Mn+2 stimulates the first transfer of glucoses to "genesine" and the 38 kDa and 42 kDa protein bands appear. Although further growth is inhibited by Mn+2, this inhibition is reversed by PMSF+Glc6P. In the absence of Mn+2, a major 14C-glucosylated protein band of 60 kDa and also a faint one in the location of 42 kDa appeared. Designing the synthetic and degradative processes it is possible to go from the 42 kDa 14C-glucosylated protein band through species of higher mw to glycogen and back to the 42 kDa one. In the "genesine" autoglucosylating process involved in the initiation of rat heart biogenesis, several dissimilar activities had to be distinguished. The first glycogen initiator 1 (GI1) is that with an activity stimulated by Mn+2 which transfers one or two glucoses. The other glycogen initiator 2 (GI2) is inhibited by Mn+2 and in its absence produces a glucosylated protein band of 60 kDa. Finally a third one, stimulated by Glc6P, we named it Elongator 1 (E1), which in the presence of mumolar concentration of UDPGlc originates a family of glucosylated protein bands almost from 42 kDa to 200 kDa.

Differential action of branching enzymes according to its source.

Different branching enzymes were partially purified in order to get some insight into their mechanism of action. Each branching enzyme is able to synthesize in vitro at any moment, even during very short incubation times, only one type of alpha 1,4-alpha 1,6 glucopolysaccharide, which possesses unique and specific properties (lambda max; A; R;% of beta-amylolysis;% alpha 1,6). Between two branching points, each branching enzyme will leave, in average, a constant and specific number of glucoses alpha 1,4-linked, which is characteristic of that particular enzyme. Consequently the structure of the polysaccharide will keep the same independently from the moment of synthesis.

Further studies on the "de novo" process of alpha 1,4-alpha 1,6 glucopolysaccharides--corn starch biogenesis.

Starch biogenesis in corn endosperm from Flint, Sugary, Waxy, as a function of the grain filling/period was studied. We have differentially identified the initiation from the elongation process. After incubating under unprimed conditions, two glucose radiolabelled protein bands of 39,5 and 36 kDa were obtained. UDP(14C)Glc was the preferred glucosyl donor but also ADP(14C)Glc was. It was additionally found that more than one glucose was transferred to the protein or to the alpha 1,4-glucan linked to protein from UDPGlc. These results were supported by the fact that the glucosylated protein from UDPGlc liberates maltooligosaccharides after alpha- or beta-amylase treatment. The elongation activity in the first steps related to the glucan linked to protein is different from starch synthase. Therefore, we are proposing a model for starch biogenesis where two new transglucosylating enzyme activities are necessary to prepare the primer for starch synthase.

Further studies on the primer formation for glycogen biosynthesis in rat heart.

Using selected incubation conditions we have identified intermediate steps, between the first glucose transferred to protein and the appropriate substrate for glycogen synthase. Mn2+ stimulates the addition of the first, and probably, the second glucose molecule to the acceptor protein but inhibits further elongation. In the presence of Mn2+ only one radioglucosylated protein band of M(r) 42 kDa was evident. In the absence of Mn2+, two bands of 60.7 and 64.6 kDa were obtained indicating elongation of the glucan chains. After Glc6P addition a family of glucosylated proteins with higher M(r) was obtained, as reported previously. Mn2+ inhibition of the second step, is reversed by PMSF+Glc6P addition. Under these conditions a family of radioglucosylated protein bands with M(r) far in excess of 42 kDa, similar to that obtained without Mn2+, was obtained. Therefore, two different transglucosylating activities were necessary, at least, to prepare the appropriate substrate for glycogen synthase. Based on these observations the model we proposed earlier for glycogen biogenesis is modified. The original "Glycogen Initiator" implies at present two enzymatic activities, Glycogen Initiator 1 (activated by Mn2+) and Glycogen Initiator 2 (inhibited by Mn2+).

Evidence for the presence of glycogen in rat thymus.

Chemical and biochemical analysis of the polysaccharide, present in rat thymus, indicate that it consists of glucose units alpha-1,4 and alpha-1,6 linked. Electron microscopy reveals the presence of a polysaccharide, similar to the beta-glycogen particles observed in liver and muscle with an average diameter of 20-30 nm. They are located in the cytoplasmic area of T-cells from the cortical region of the thymus. Enzymatic analysis indicates that the beta-particles contain a highly branched glucan with short external chains. Some of the enzymes of glycogen metabolism: synthase, phosphorylase and branching were for the first time partially purified from rat thymus and some of their properties were studied. Therefore, glycogen appeared to be synthesized in rat thymus.

Comparative study of nuclear and cytoplasmic glycogen isolated from mutant HD33 ascites cells.

Mutant cells of the HD33 subline of the Ehrlich-Lettré ascites tumor synthesize and store glycogen mainly intranuclearly, when growing in vivo, and exclusively in the cytoplasm, when permanently cultivated as a suspension cell strain. To investigate whether there exist differences between glycogen of nuclear and cytoplasmic origin, the ultrastructure and the biophysical and biochemical properties of glycogen from in vivo and in vitro grown HD33 ascites cells were compared. Pronounced heterogeneity and differences in glycogen particle ultrastructure were evident in situ and after isolation of the native, high-molecular polysaccharide. Nuclear glycogen contains a fraction of heavier molecules (up to 2 X 10(9)) and larger particles (up to 340 nm) which could not be found in the cytoplasmic preparations, which contained only particles smaller than 140 nm. The subparticles of beta-type are similar in both nuclear and cytoplasmic glycogen. The absorption spectra and glucose analysis after degradation with phosphorylase and debranching enzyme indicate that nuclear glycogen has a higher degree of branching, associated with a decrease in the average chain length between the branching points, and shorter external polyglucosidic chains than cytoplasmic glycogen. This is the first report about the analysis and properties of isolated nuclear glycogen.

The degree of branching in (alpha 1,4)-(alpha 1,6)-linked glucopolysaccharides is dependent on intrinsic properties of the branching enzymes.

1. Branching enzymes from rat and rabbit liver, as well as from potato and maize were prepared. They were almost free from contaminating glucan-degrading enzymes. 2. In 'sweet corn' maize, two separate fractions with (alpha 1,4)glucan: (alpha 1,4)glucan alpha 6-glycosyltransferase activities were obtained. One of them synthesized amylopectin, the branched component of starch, in the presence of phosphorylase and Glc1P, while the other fraction synthesized phytoglycogen. Furthermore, in a maize variety which does not accumulate phytoglycogen, only one fraction of branching activity was found, that formed amylopectin under the above-mentioned conditions. 3. Comparative analyses performed with native (alpha 1,4)-(alpha 1,6)glucopolysaccharides, and those synthesized in vitro with the branching enzyme from the same tissue, demonstrated a close similarity between both glucans. 4. It may be concluded that the branching enzyme is responsible for the specific degree of (alpha 1,6) branch linkages found in the native polysaccharide.

A (1----4)-alpha-D-glucan-protein involved in liver glycogen biosynthesis.

A macromolecular (1----4)-alpha-D-[14C]glucan-protein complex was synthesized with a rat liver preparation and uridine diphosphate D-[14C]glucose. The size of the complex is contributed by both the protein and the (1----4)-alpha-D-glucosyl-oligomer components. Iodoacetamide treatment did not change the migration properties on Bio-Gel A-50m. Therefore, disulfide bonds linking glucan-protein subunits seem not to be involved. The [14C]glucan-protein, precipitated by diluted trichloroacetic acid, was digested by alpha-amylase, phosphorylase a, and proteases. The extent of proteolysis was greater for a complex having fewer D-glucose units incorporated. After proteolytic digestion of that complex, the labeled fragments behaved on electrophoresis, and ion-exchange and gel chromatography as [14C]glucosylated peptides. These findings support previous conclusions that the primer for liver glycogen synthesis is a protein on which glycogen is built up by covalent attachment.

A method for the direct measurement of glycogen synthase activity on gels after polyacrylamide gel electrophoresis.

A method for the detection of glycogen synthase activity after nondenaturing polyacrylamide gel electrophoresis is described. After the electrophoretical run, the gels were incubated in situ with UDP-glucose and glycogen. Labeled or unlabeled UDP-glucose could be used, since similar activity patterns were obtained by autoradiography or iodine staining of the gels. The method here described offers several advantages in terms of speed, sensitivity, and economy when compared with other procedures.

The initiation of glycogen biosynthesis in rat heart alpha-1,4 glucans tightly associated with glycogen synthase.

A partially purified glycogen synthase from rat cardiac muscle transferred glucosyl residues from UDP-[14C]glucose to an endogenous protein acceptor in the absence of added primer. After native gel electrophoresis of the enzyme preparation, unprimed activity was detected. Primer-dependent and independent activities were found in the same position. After denaturing gel electrophoresis of the reaction products, radioactivity comigrated with protein. Pulse-chase experiments showed that the size of the reaction products increased as a function of time. These products were degraded by amyloglucosidase, thus suggesting that glycogen-like molecules had grown on the protein acceptor. The activity of the enzyme was markedly reduced upon preincubation with alpha-amylase. Therefore, preformed protein-bound alpha-1,4-glucans were acting as primers. The glucoprotein acceptor may be a protein strongly associated with glycogen synthase, or alternatively, the enzyme itself.

The initiation of glycogen biosynthesis in rat heart. Studies with a purified preparation.

Two fractions of glycogen synthase were isolated from rat cardiac muscle on the basis of a different affinity for DEAE-cellulose and omega-aminobutyl-agarose. One of these fractions was able to transfer glucosyl residues from UDP-glucose not only to glycogen (GS-1 activity) but also to an endogenous acceptor. The latter reaction (GS-2 activity) occurred in the absence of added glycogen, and its reaction product was insoluble in trichloroacetic acid. This compound was degraded by amylolytic enzymes, thus showing that the product synthesized on the endogenous acceptor was an alpha 1,4-glucan. After incubation with alpha-amylase-free proteolytic enzyme, the compound was rendered trichloroacetic acid-soluble. Polyacrylamide gel electrophoresis, under both native and denaturing conditions, showed that GS-2 reaction products moved electrophoretically associated to protein. Our results give further evidence for the association between an alpha 1,4-glucan and protein, which we postulate is related to the initiation of glycogen biosynthesis.

Branching enzyme assay: selective quantitation of the alpha 1,6-linked glucosyl residues involved in the branching points.

Methods previously described for glycogen or amylopectin branching enzymatic activity are insufficiently sensitive and not quantitative. A new, more sensitive, specific, and quantitative one was developed. It is based upon the quantitation of the glucose residues joined by alpha 1,6 bonds introduced by varying amounts of branching enzyme. The procedure involved the synthesis of a polysaccharide from Glc-1-P and phosphorylase in the presence of the sample to be tested. The branched polysaccharide was then purified and the glucoses involved in the branching points were quantitated after degradation with phosphorylase and debranching enzymes. This method appeared to be useful, not only in enzymatic activity determinations but also in the study of the structure of alpha-D-glucans when combined with those of total polysaccharide quantitation, such as iodine and phenol-sulfuric acid.

The initiation of glycogen biosynthesis in rat heart.

A rat heart extract synthesizes glycogen-like material from UDP-glucose in the absence of added primer. Most of this material is precipitable by dilute trichloroacetic acid, in contrast to glycogen synthesized in presence of added glycogen primer. It is postulated that the endogenous initiation of glycogen synthesis occurs by apposition of glucose residues to a protein and that the material so synthesized in vitro corresponds to the protein-bound glycogen reported to exist in rat heart. The ability to label the putative protein primer in vitro with [14C]glucose should assist the isolation of the primer and the elucidation of the nature of the association between the carbohydrate and protein moeities.

Initiation of glycogen biosynthesis in Escherichia coli. Studies of the properties of the enzymes involved.

The properties of the enzymes involved in the initiation of glycogen biosynthesis in Escherichia coli were studied. It was found that the enzymic activities which transfer the glycosyl residues from UDPglucose or ADPglucose for the glucoprotein synthesis had differing stabilities upon storage at 4 degrees C. The small amount of glycogen and the saccharide firmly bound to the membrane preparation, were degraded during the storage period. The activity measured in fresh and in stored preparations gave different time dependence curves. The stored preparation had a lag period which could be due to the transfer of the first glucose units to the protein. Both UDPglucose and ADPglucose : protein glucosyltransferases were affected in different ways by detergents. Based on the results presented, it may be concluded that both enzymatic activities are due to different enzymes. Furthermore, both enzymatic activities are different from that which transfers glucose from ADPglucose to glycogen. The following mechanism for the de novo synthesis is suggested. Glycogen in E. coli could be initiated by two different enzymes which transfer glucose to a protein acceptor either from UDPglucose or ADPglucose. Once the saccharide linked to the protein has reached a certain size it is almost exclusively enlarged by another ADPglucose-dependent enzyme. The participation of branching enzyme will produce a polysaccharide with the characteristics of glycogen.

Glycogen synthesis in the fungus Neurospora crassa.

An enzymic activity, obtained from Neurospora crassa, catalyzing the incorporation of [14C]glucose from ADP-[14C]glucose into a glucan of the glycogen type, is described. The properties of the ADPglucose : glycogen glucosyltransferase as compared with those of the already known UDP glucose : glycogen glucosyltransferase were studied. The radioactive products obtained with UDP-14C]glucose or ADP-[14C]glucose released all the radioactivity as maltose after alpha or beta amylase treatment. Glucose 6-phosphate stimulated the synthetase when UDP-[14C]glucose was the substrate but the stimulation was much greater with ADP-[14C]glucose as glucosyl donor. Glucose 6-phosphate plus EGTA gave maximal stimulation. The system was completely dependent &on the presence of a 'primer' of the alpha 1 leads to 4 glucan type.

[Studies on the initiation of glycogen metabolism in Escherichia coli (author's transl)]. / Estudios sobre la inciación de la biosíntesis de glucogeno en Escherichia coli.

Glycogen biosynthesis was studied in Escherichia coli. An enzyme complex composed of UDP-glucose; protein glucosyltransferase, ADP-glucose: protein glucosyltransferase and ADP-glucose: alpha-1,4 glucan alpha-4-glucosyltransferase was found. Further results revealed that while glycogen concentration remained unchanged, the specific activity of the glucosyltransferase complex increased during the growth phase of the culture. The detergents Lubrol and Brij provoked a decrease of 80% and 20% in the glucose transfer to protein from ADP-glucose and UDP-glucose, respectively. These detergents did not inhibit the glucose incorporation into glycogen by ADP-glucose: alpha-1,4-glucosyltransferase. We postulated that the biosynthesis of glycogen in Escherichia coli could be initiated by two different enzymes which catalyze the transfer of glucose from UDP-glucose or ADP-glucose to an acceptor protien. In a second step, the glucan protein formed is used as primer by the ADP-glucose: alpha-1,4 glucan alpha-1-glucosyltransferase for glycogen formation.