Biomedical subjects

J Lomako

Publications and source records attributed to J Lomako.

At least 19 recordsLinked to original sources

PubMed1996

The human intron-containing gene for glycogenin maps to chromosome 3, band q24.

Glycogenin is the autocatalytic, self-glucosylating primer for glycogen synthesis, providing the anchor on which the macromolecule is constructed. We have sequenced the cDNA coding for human muscle glycogenin and have deduced the corresponding amino acid sequence. By means of the polymerase chain reaction and fluorescence in situ hybridization, we have found the chromosomal location of the gene coding for glycogenin. This is localized to human chromosome 3, band q24.

Amino Acid Sequence↗

PubMed1995

beta-Glucosylarginine: a new glucose-protein bond in a self-glucosylating protein from sweet corn.

In the search for a protein primer for starch synthesis, an autocatalytic self-glucosylating protein has been isolated from sweet corn. Several tryptic peptides were obtained from the [14C]glucosylated protein and were sequenced, corresponding to over 40% of the estimated total sequence (molecular mass 42 kDa). There is no homology with the amino acid sequence of the autocatalytic glycogen primer, glycogenin, nor in respect of the nature of the union between the autocatalytically added glucose and the protein, which, in the case of the corn protein, now named amylogenin, is a novel glucose-protein bond, a single beta-glucose residue joined to an arginine residue.

Amino Acid Sequence↗

PubMed1995

Glycogen metabolism in quail embryo muscle. The role of the glycogenin primer and the intermediate proglycogen.

Cultured quail embryo muscle has proven to be an excellent model system for studying the synthesis of macromolecular glycogen from, and its degradation to, glycogenin, the autocatalytic, self-glucosylating primer for glycogen synthesis. We recently demonstrated that proglycogen, a low-M(r) form of glycogen, is an intermediate in the synthesis. Here we show that proglycogen also functions as an intermediate in macroglycogen degradation and, in one set of circumstances, represents an arrest point in glycogen breakdown, which does not continue to glycogenin. We suggest that in the nutritionally dependent turnover of glycogen in tissues, the molecules cycle between proglycogen and macromolecular glycogen and are not normally degraded to glycogenin. Nevertheless, when this does happen, the released glycogenin is active, capable of re-initiating glycogen synthesis. Under culture conditions where the conversion of proglycogen into glycogenin does take place, the intermediates lying between form a discrete rather than a continuous series, suggestive of a cluster structure for proglycogen and indicating that breakdown is stepwise. Evidence of post-translational modification of glycogenin was obtained by the finding that, in glycogen from cultured muscle, glycogenin is phosphorylated.

Animals↗

PubMed1995

Catalytic activities of glycogenin additional to autocatalytic self-glucosylation.

Glycogenin is the autocatalytic, self-glucosylating protein that initiates glycogen synthesis in muscle and other tissues. We have sequenced the cDNA for rabbit muscle glycogenin and expressed and purified the protein in high yield as well as two mutant proteins in which Phe or Thr replaces Tyr-194, the site of glucosylation. While the wild-type protein can self-glucosylate, the mutants cannot, but all three utilize alternative acceptors by intermolecular glucose transfer for which the mutants have altered specificity. Tyr-194 is therefore not essential for the catalytic activity of glycogenin. All three proteins also hydrolyze UDP-glucose to glucose at rates comparable with the rate of self-glucosylation. The hydrolysis is competitive with glucose transfer to p-nitrophenyl alpha-maltoside. Self-glucosylation, glucosylation of other acceptors, and hydrolysis all appear to be catalyzed by the same active center. In the absence of peptidase inhibitors, the homogenous recombinant proteins of M(r) 37,000 break down to equally active species having M(r) 32,000. The kinetics of self-glucosylation catalyzed by the wild-type enzyme suggest that the reaction could be intermolecular rather than, as previously reported, intramolecular. The wild-type recombinant enzyme and native muscle glycogenin, which is phosphorylated, are inhibited quite differently by ATP at physiological concentration.

Amino Acid Sequence↗

PubMed1995

New and specific nucleoside diphosphate glucose substrates for glycogenin.

Glycogenin, the autocatalytic, self-glucosylating primer for glycogen synthesis by glycogen synthase, is presumed, in vivo, to use UDP-glucose as the source of the glucose residues it adds to itself. When we tested its ability to utilize other nucleoside diphosphate glucoses, it emerged that purine nucleotides are not utilized but two pyrimidine nucleotides are used, in addition to UDP-glucose. These are CDP-glucose and TDP-glucose. CDP-glucose is utilized at 70% of the rate of UDP-glucose. While there is no evidence that CDP-glucose is a natural substrate for glycogenin, it has the advantage over UDP-glucose in that it can be used specifically to detect and assay glycogenin in the presence of glycogen synthase because CDP-glucose, unlike UDP-glucose, is not a substrate for the synthase.

Glucose↗

PubMed1995

A new look at the biogenesis of glycogen.

The discovery of glycogenin as a self-glucosylating protein that primes glycogen synthesis has significantly increased our understanding of the structure and metabolism of this storage polysaccharide. The amount of glycogenin will influence how much glycogen the cell can store. Therefore, the production of active glycogenin primer in the cell has the potential to be the overall rate-limiting process in glycogen formation, capable of overriding the better understood hormonally controlled mechanisms of protein phosphorylation/dephosphorylation that regulate the activities of glycogen synthase and phosphorylase. There are indications that a similar covalent modification control is also being exerted on glycogenin. Glycogenin has the ability to glucosylate molecules other than itself and to hydrolyze UDPglucose. These are independent of self-glucosylation, so that glycogenin, even when it has completed its priming role and become part of the glycogen molecule, retains its catalytic potential. Another new component of glycogen metabolism has been discovered that may have even greater influence on total glycogen stores than does glycogenin. This is proglycogen, a low molecular mass (approximately 400 kDa) form of glycogen that serves as a stable intermediate on the pathways to and from depot glycogen (macroglycogen, mass 10(7) Da, in muscle). It is suggested that glycogen oscillates, according to glucose supply and energy demand, between the macroglycogen and proglycogen, but not usually the glycogenin, forms. The proportion of proglycogen to macroglycogen varies widely between liver, skeletal muscle, and heart, from 3 to 15% to 50% by weight, respectively. On a molar basis, proglycogen is greatly in excess over macroglycogen in muscle and heart, meaning that if the proglycogen in these tissues could be converted into macroglycogen, they could store much more total glycogen. Discovering the factors that regulate the balance between glycogenin, proglycogen, and macroglycogen may have important implications for the understanding and management of noninsulin-dependent diabetes and for exercise physiology.

Animals↗

PubMed1994

Properties of carbohydrate-free recombinant glycogenin expressed in an Escherichia coli mutant lacking UDP-glucose pyrophosphorylase activity.

Glycogenin, the self-glucosylating primer for glycogen synthesis, is expressed in wild-type E. coli as a recombinant protein in an already partly glucosylated form, owing to the presence of its substrate, UDP-glucose. By using an E. coli mutant strain lacking in UDP-glucose pyrophosphorylase activity, we have succeeded in expressing carbohydrate-free glycogenin (apo-glycogenin) in good yield. When provided with UDPxylose, it autocatalytically adds 1 xylose residue. With UDP-glucose, an average of 8 glucose residues are added. However, release of the self-synthesized maltosaccharide chains with isoamylase reveals them to be a mixture. Chains as long as 11 glucose residues (maltoundecaose) are present. The ability of recombinant apo-glycogenin to self-glucosylate is further proof that a separate enzyme is not needed for the addition of the first glucose residue to Tyr-194 of the protein.

Escherichia coli↗

PubMed1994

Tyrosine-194 of glycogenin undergoes autocatalytic glucosylation but is not essential for catalytic function and activity.

Glycogenin is the protein primer for glycogen synthesis. By autocatalytic transglucosylation from UDPglucose, it creates a malto-octaose chain attached to its Tyr-194. It has been uncertain whether the autocatalysis includes the addition of the first glucose residue to Tyr-194. We now show this to be the case. However, we also demonstrate, contrary to a claim by others, that Tyr-194 is not necessary for the catalytic function and activity of glycogenin.

Animals↗

PubMed1994

The role of phosphate in muscle glycogen.

As previously shown for rabbit liver glycogen, rabbit muscle glycogen contains a small amount of phosphate ester and there is an intermolecular heterogeneity in phosphate content such that the glycogen may be fractionated on DEAE-cellulose into components differing 10-fold in their phosphate contents. We now know that the phosphate ester is of two types, mono and di. The availability of the phosphomonoester component to hydrolysis by alkaline phosphatase is sterically regulated and is highest in the fraction of highest total phosphate content. The ability of the glycogen fractions to act as primers for glycogen synthase also varies with phosphate content, differing overall by > 2-fold. The priming ability increases with increasing phosphate content. We suggest that the phosphate content of a glycogen molecule may be related to its age and that this may be used as a metabolic marker when studying the turnover of glycogen, also that phosphate may be a signal for transport of glycogen to the lysosome. The phosphodiester grouping may act as a point of branching in glycogen, additional to the recognized interglucosidic branch, and is a candidate for the acid- and alkali-labile bond that has been reported in glycogen.

Alkaline Phosphatase↗

PubMed1993

Glycogen contains phosphodiester groups that can be introduced by UDPglucose: glycogen glucose 1-phosphotransferase.

Rabbit-muscle glycogen contains covalently bound phosphorus, equivalent to 1 phosphate group per 208 glucose residues. This often disputed, minor component was previously thought to represent a phosphomonoester group at C-6 of a glucose residue. Here we show that more than half the phosphorus is present as a phosphodiester, the remainder being monoester. A novel enzyme activity has been found in muscle that can account for the presence of the phosphodiester in glycogen. This is a UDPglucose: glycogen glucose 1-phosphotransferase that positions glucose 1-phosphate on C-6 of glucose residues in glycogen, forming a diester. The phosphomonoester groups present may arise by removal of the glucose residue originally transferred as glucose 1-phosphate.

Animals↗

PubMed1993

Glycogen synthesis in the astrocyte: from glycogenin to proglycogen to glycogen.

The astrocyte of the newborn rat brain has proven to be a versatile system in which to study glycogen biogenesis. We have taken advantage of the rapid stimulation of glycogen synthesis that occurs when glucose is fed to astrocytes, and the marked limitation on this synthesis that occurs in astrocytes previously exposed to ammonium ions. These observations have been related to our earlier reports of the initiation of glycogen synthesis on a protein primer, glycogenin, and the discovery of a low-molecular-weight form of glycogen, proglycogen. The following conclusions have been drawn: 1) In the ammonia-treated astrocytes starved of glucose, free glycogenin is present. 2) When these astrocytes are fed with glucose, proglycogen is synthesized from the glycogenin primer by a glycogen-synthase-like UDPglucose transglucosylase activity (proglycogen synthase) distinct from the well-recognized glycogen synthase, and synthesis stops at this point. 3) Proglycogen is the precursor of macromolecular glycogen, which is synthesized from proglycogen by glycogen synthase when glucose is fed to untreated astrocytes, accounting for the much greater accumulation of total glycogen. 4) The stimulus to proglycogen and macroglycogen synthesis that occurs on feeding glucose to untreated or ammonia-treated astrocytes is the result of the activation of proglycogen synthase, not of glycogen synthase. 5) Therefore, in the synthesis of macromolecular glycogen from glycogenin via proglycogen, the step between glycogenin and proglycogen is rate-limiting. 6) The discovery of additional potential control points in glycogen synthesis, now emerging, may assist the identification of so-far-unexplained aberrations of glycogen metabolism.

Ammonia↗

PubMed1992

The substrate specificity of isoamylase and the preparation of apo-glycogenin.

A new facet of the specificity of the glycogen-debranching enzyme, isoamylase, namely, the hydrolysis of a carbohydrate-amino acid linkage, is described. This bond joins the terminal, reducing-end D-glucose unit of glycogen to the hydroxyl group of tyrosine in glycogenin, the primer protein for glycogen biogenesis. The specificity was further defined by demonstrating that 4-nitrophenyl alpha-maltotrioside and higher homologs also act as substrates. The splitting of the glycogen-glycogenin bond by isoamylase indicates the alpha-anomeric configuration of the terminal D-glucose unit. It also provides a means of preparing apo-glycogenin. Pullulanase, a somewhat similar starch- and glycogen-debranching enzyme, does not split these new isoamylase substrates, permitting the 4-nitrophenyl saccharides to be used in distinguishing between isoamylase and pullulanase.

Glucosyltransferases↗

PubMed1991

Proglycogen: a low-molecular-weight form of muscle glycogen.

We recently reported that muscle contains a trichloroacetic acid-precipitable component having Mr approx. 400 kDa that can be glucosylated by an endogenous enzyme acting on UDPglucose. This component contains within itself the autocatalytic, self-glucosylating protein glycogenin, the primer for glycogen synthesis. We now report that this substance, to which we give the name proglycogen, is a glycogen-like molecule constituting about 15% of total glycogen. It acts as a very efficient acceptor of glucose residues added from UDPglucose. Further, that the endogenous enzyme that adds the glucose to proglycogen is not the autocatalytic protein but a glycogen synthase-like enzyme. Proglycogen may be an intermediate in the synthesis and degradation of macromolecular glycogen and may exist and be metabolized as a separate entity. Consideration should now be given to the revival of the concept that tissue contains two forms of glycogen. One is proglycogen. The other is the 'classical', macromolecular glycogen. Additionally, proglycogen and glycogen may be glucosylated by different forms of synthase.

Amylases↗

PubMed1990

The nature of the primer for glycogen synthesis in muscle.

We and others have reported that glycogenin, the covalently bound protein found in muscle glycogen, also exists in muscle in a glycogen-free form (Mr 38,000-39.000) that is autocatalytic, undergoes self-glucosylation and acts as a primer for glycogen synthesis. We now report that this entity is not present in a fresh muscle extract. Instead it exists within a pro form of much higher molecular mass which breaks down spontaneously to the Mr 38,000-39,000 form. Such breakdown is accelerated by the addition of alpha-amylase and is prevented by protease inhibitors. Multiple intermediates of the breakdown process have been detected, each capable of undergoing glucosylation.

Animals↗

PubMed1990

Substrate specificity of the autocatalytic protein that primes glycogen synthesis.

The autocatalytic protein that primes muscle-glycogen synthesis, and which glucosylates itself from UDPglucose, is inhibited by maltose. Investigation of the reason for the inhibition led to the finding that the protein will glucosylate substrates other than itself. p-Nitrophenyl alpha-glucoside, alpha-maltoside, alpha-maltotrioside and alpha-maltotetraoside each inhibit self-glucosylation of the protein by acting as alternative acceptor substrates. The alpha-maltoside is the best acceptor. The alpha-maltohexaoside did not act as an acceptor but was an effective inhibitor. These findings help to explain the self-limiting nature of the autocatalytic extension of the maltosaccharide chain of the protein and suggest that protein self-glucosylation may be an intermolecular event. They may also point to the mechanism by which the autocatalytic protein is initially glycosylated.

Animals↗

PubMed1990

Glycogen biogenesis in the adipocyte and mammary adenocarcinoma and the influence of insulin.

3T3 LI P-3 adipocytes and a rat mammary carcinoma each contain a protein (or proteins) of Mr 400 kDa approx. that undergoes glucosylation when incubated with UDPglucose and Mn2+. In each case, the product of glucosylation is a protein-bound maltosaccharide containing up to 14 glucose residues, and therefore capable of priming glycogen synthesis. The proteins bear a formal similarity to the self-glucosylating protein of Mr 37 kDa that primes muscle glycogen synthesis, but do not cross-react with an antibody that cross-reacts with the muscle protein. The extent of protein glucosylation in the adipocyte is rapidly and markedly stimulated when the cells are bathed in insulin, suggesting that this initial step in glycogen biogenesis, protein glucosylation, is under the influence of the hormone.

Adenocarcinoma↗

PubMed1990

The biogenesis of muscle glycogen: regulation of the activity of the autocatalytic primer protein.

Glycogen synthesis in rabbit muscle is initiated by an autocatalytic, self-glucosylating protein (SGP). This creates a maltosaccharide primer on itself that in turn primes glycogen synthesis. Here we describe the powerful allosteric inhibition of autocatalysis by ATP and ADP, sufficient, at the physiological concentration of ATP in muscle, to inhibit autocatalysis. We also examined inhibition of self-glucosylation by analogues of the substrate UDPglucose. One of them, UDPxylose, acts as an alternative substrate and serves to block glucosylation. An improved purification procedure for the SGP is also described.

Allosteric Regulation↗

PubMed1990

The biogenesis of glycogen: nature of the carbohydrate in the protein primer.

Glycogen synthesis in rabbit muscle takes place on an autocatalytic protein that glucosylates itself to form a maltosaccharide that in turn primes the actions of glycogen synthase and branching enzyme to form glycogen. Here we show that in the form in which the protein is isolated from muscle it already contains one molecular proportion of a maltosaccharide ranging in length from 2 to 8 glucose residues. Self-glucosylation consists in the lengthening of all the chains to become malto-octaose.

Animals↗