Glycogen Debranching Enzyme Research Articles

Motor unit firing rates in orbicularis oculi muscle after Bell's palsy

We report a 25-year-old man with glycogenosis III who presented with a progressive 2 year history of fatigue, hand stiffness and cramping. The glycogenoses are a group of rare metabolic disorders which develop as a result of deficiencies in various enzymes involved in the metabolism of glycogen. Some, but not all, glycogenoses, may result in skeletal muscle pathology. Among those that result in vacuolar myopathic changes, glycogen storage disease III or debrancher enzyme deficiency, an autosomal recessive condition, is less commonly encountered than acid maltase (Type II) and myophosphorylase (Type V) deficiencies. Many patients with debrancher enzyme deficiency also have liver involvement. The neurological examination of our patient showed mild proximal limb weakness and decreased reflexes. He had elevated creatine kinase and aldolase levels. He also demonstrated some elevations in his liver function tests, suggesting possible liver involvement. A skeletal muscle biopsy demonstrated vacuolar myopathic changes (acid phosphatase negative) accompanied by focal endomysial fibrosis and chronic inflammation. An ultrastructural examination showed that his vacuoles were filled with glycogen material. An enzyme assay of skeletal muscle tissue showed a significant decrease in debrancher enzyme activity (11% of normal). We review the typical clinical presentation of patients with glycogenosis III and discuss the differential diagnoses of glycogenosis III versus the other glycogenoses resulting in vacuolar myopathy.

Alpha-amylase and Starch Degradation in Kiwifruit

Alpha-amylase activities and activity gel profiles were measured in kiwifruit Actinidia deliciosa var. deliciosa ([A. Chev.], C. F. Liang and Ferguson cv. Hayward) during fruit development and postharvest ripening. As fruit developed, alpha-amylase activity increased on a per fruit basis, mirroring the increase in starch content in the fruit. Alpha-amylase activity increased approximately 2-fold between 134 days after anthesis (DAA), (when fruit starch content is at its maximum before beginning to decline) and harvest. During postharvest ripening with and without ethylene treatment at 20 °C, or during postharvest storage at 0 °C, enzyme activity remained similar to harvest values until fruit were ripe and most of the starch converted to sugars. At this point activity decreased 4–5-fold. Several amylolytic bands as well as starch phosphorylase and debranching enzyme were visible in starch activity gels. Starch phosphorylase was lower in ripe fruit, as was the intensity of two amylolytic bands. None of the other amylolytic bands or the deb ranching enzyme changed in response to fruit development or ripening. The same results occurred if gels were incubated at pH 6.1 or pH 8.0. These results are discussed in relation to the initiation of net starch degradation at the commencement of fruit ripening.

Novel solid-phase synthesis of branched oligoribonucleotides, including a substrate for the RNA debranching enzyme

An effective new route for synthesizing branched oligoribonucleotides in the solid phase in the 5′ to 3′ direction has been developed. This required the synthesis of reversed monomers, viz. protected nucleoside 5′-phosphoramidites bearing 2′-O-Fpmp and (3′-O-pixy) protecting groups as well as special branch-point monomers, viz. protected nucleoside 5′-phosphoramidites bearing either 2′, 3′-O-dipixyl protection in the case of adenosine, cytidine and uridine, or 2′,3′-O-dilaevulinyl protection in the case of guanosine. These monomers are assembled on commercial synthesizers into branched oligoribonucleotides in high yield, the crude products are readily purified by reversedphase HPLC whilst still partially protected, and the fully deprotected products are conveniently analysed by electrospray mass spectrometry. Moreover, the branched oligoribonucleotides can be recognised and cleaved by a specific 2′–5′ phosphodiesterase present in mammalian cell nuclei. We expect that this will prove valuable for future biochemical and biological studies on the properties of branched RNA molecules and the protein factors and enzymes that interact with branched RNA substrates.

Starch- and glycogen-debranching and branching enzymes: Prediction of structural features of the catalytic (β/α)8-barrel domain and evolutionary relationship to other amylolytic enzymes

Sequence alignment and structure prediction are used to locate catalytic alpha-amylase-type (beta/alpha)8-barrel domains and the positions of their beta-strands and alpha-helices in isoamylase, pullulanase, neopullulanase, alpha-amylase-pullulanase, dextran glucosidase, branching enzyme, and glycogen branching enzymes--all enzymes involved in hydrolysis or synthesis of alpha-1,6-glucosidic linkages in starch and related polysaccharides. This has allowed identification of the transferase active site of the glycogen debranching enzyme and the locations of beta-->alpha loops making up the active sites of all enzymes studied. Activity and specificity of the enzymes are discussed in terms of conserved amino acid residues and loop variations. An evolutionary distance tree of 47 amylolytic and related enzymes is built on 37 residues representing the four best conserved beta-strands of the barrel. It exhibits clusters of enzymes close in specificity, with the branching and glycogen debranching enzymes being the most distantly related.

Nutrition therapy for hepatic glycogen storage diseases

Hepatic glycogen storage diseases (GSD) are a group of rare genetic disorders in which glycogen cannot be metabolized to glucose in the liver because of one of a number of possible enzyme deficiencies along the glycogenolytic pathway. Patients with GSD are usually diagnosed in infancy or early childhood with hypoglycemia, hepatomegaly, poor physical growth, and a deranged biochemical profile. Dietary therapies have been devised to use the available alternative metabolic pathways to compensate for disturbed glycogenolysis in GSD I (glucose-6-phosphatase deficiency), GSD III (debrancher enzyme deficiency), GSD VI (phosphorylase deficiency, which is less common), GSD IX (phosphorylase kinase deficiency), and GSD IV (brancher enzyme deficiency). In GSD I, glucose-6-phosphate cannot be dephosphorylated to free glucose. Managing this condition entails overnight continuous gastric high-carbohydrate feedings; frequent daytime feedings with energy distributed as 65% carbohydrate, 10% to 15% protein, and 25% fat; and supplements of uncooked cornstarch. In GSD III, though glycogenolysis is impeded, gluconeogenesis is enhanced to help maintain endogenous glucose production. In contrast to treatment for GSD I, advocated treatment for GSD III comprises frequent high-protein feedings during the day and a high-protein snack at night; energy is distributed as 45% carbohydrate, 25% protein, and 30% fat. Patients with GSD IV, VI, and IX have benefited from high-protein diets similar to that recommended for patients with GSD III.

Assignment of the human glycogen debrancher gene to chromosome 1p21

Glycogen debranching enzyme is a monomeric protein containing two independent catalytic activities of glycantransferase and glucosidase that are both required for glycogen degradation. Its deficiency causes type III glycogen storage disease. A majority of the patients with this disease have deficient enzyme activity in both liver and muscle (type IIIa) but ∼15% of them lack enzyme activity only in the liver (type IIIb); however, the enzyme is a monomer and appears to be identical in all the tissues. The cDNA coding for the complete human muscle debranching enzyme has recently been isolated. Using the cDNA clones, the debrancher gene was localized to human chromosome 1 by somatic cell hybrid analysis. Regional assignment to chromosome band 1p21 was determined by in situ hybridization. Mapping of the debrancher gene to a single chromosome site is consistent with our hypotheses that a single gene encodes both liver and muscle debrancher protein.

Glycogen storage disease type III (glycogen debranching enzyme deficiency): correlation of biochemical defects with myopathy and cardiomyopathy.

To determine whether a specific subtype of glycogen storage disease type III is associated with myopathy and cardiomyopathy. Case series. Three referral medical centers. All patients with glycogen storage disease type III who were followed in 1990 and for whom both immunoblot analysis and clinical data were available. Evaluation for myopathy and cardiomyopathy included determinations of serum creatine kinase activity; muscle strength testing; ischemic exercise testing; nerve conduction studies; and electromyographic, electrocardiographic, and echocardiographic studies. Three patients with deficient debranching enzyme activity and deficient immunoreactive material in liver but normal debranching enzyme activity in muscle (glycogen storage disease IIIb) had no clinical evidence of myopathy or cardiomyopathy. Serum creatine kinase activity, muscle strength, ischemic exercise testing, electrocardiograms, and echocardiograms were normal in these patients. These studies and electromyograms were abnormal in seven patients with total debranching enzyme deficiency and an absence of immunoreactive material in both liver and muscle (glycogen storage disease IIIa) and in three patients who had debranching enzyme transferase deficiency but normal glucosidase activity in both liver and muscle (glycogen storage disease IIId). All 10 of these patients had progressive myopathy, and 6 had progressive cardiomyopathy. Clinical features of glycogen storage disease type III correlate with the particular biochemical defect seen with the disorder. Assessments of debranching enzyme or debranching enzyme transferase activity in muscle can be used to predict whether patients with glycogen storage disease type III will develop myopathy and cardiomyopathy.

Properties of yeast debranching enzyme and its specificity toward branched cyclodextrins.

Debranching enzyme was purified from Saccharomyces cerevisiae by DEAE-cellulose, omega-aminobutyl agarose and hydroxyapatite column chromatography. The activity of the eluent was monitored by the iodine-staining method which detects both the direct and indirect debranching enzymes. The elution profiles at every step showed a single peak with no shoulder. The crude and the purified enzyme preparations gave a single activity band with the same mobility on PAGE. The crude product produced 80% glucose compared to reducing sugar from glycogen-phosphorylase-limited dextrin while the partially purified and purified preparations produced 100% glucose. The activity of the purified enzyme was characterized and compared with that of the rabbit muscle enzyme by using various branched cyclodextrins as substrates. Both enzymes hydrolyzed 6-O-alpha-D-glucosyl cyclodextrins to glucose and cyclodextrins, but did not act on 6-O-alpha-maltosyl cyclomaltoheptaose. The yeast enzyme gave rise to glucose as a sole reducing sugar from 6-O-alpha-maltotriosyl cyclomaltoheptaose and 6-O-alpha-maltotetraosyl cyclomaltoheptaose, indicating that maltosyl and maltotriosyl transfers, respectively, had occurred, prior to the action of amylo-1,6-glucosidase. 6-O-alpha-D-Glucosyl cyclomaltoheptaose and 6-O-alpha-D-glucosyl cyclomalto-octaose, respectively, were better substrates than glycogen-phosphorylase-limited dextrin for the yeast and muscle enzymes. The yeast enzyme released glucose at a similar rate from 6-O-alpha-maltotriosyl cyclomaltoheptaose as from 6-O-alpha-maltotetraosyl cyclomaltoheptaose, but considerably lower rates than that from limit dextrin. The yeast debranching enzyme appears to be exclusively oligo-1,4----1,4-glucantransferase-amylo-1,6-glucosidase and does not have isoamylase.

Molecular cloning and nucleotide sequence of cDNA encoding human muscle glycogen debranching enzyme.

cDNA comprising the entire length of the human muscle glycogen debranching enzyme was cloned and its nucleotide sequence determined. The debrancher mRNA includes a 4545-base pair coding region and a 2371-base pair 3'-nontranslated region. The calculated molecular mass of the debrancher protein derived from cDNA sequence is 172,614 daltons, consistent with the estimated size of purified protein (Mr 165,000 +/- 500). A partial amino acid sequence (13 internal tryptic peptides with a total of 213 residues) determined on peptides derived from purified porcine muscle debrancher protein confirmed the identity of the cDNA clone. Comparison of the amino acid sequence predicted from the human glycogen debrancher cDNA with the partial protein sequence of the porcine debrancher revealed a high degree (88%) of interspecies sequence identity. RNA blot analysis showed that debrancher mRNA in human muscle, lymphoblastoid cells, and in porcine muscle are all similar in size (approximately 7 kilobases). Two patients with inherited debrancher deficiency had a reduced level of debrancher mRNA, whereas two other patients had no detectable abnormality in RNA blots. The isolation of the debrancher cDNA and determination of its primary structure is an important step toward defining the structure-function relationship of this multifunctional enzyme and in understanding the molecular basis of the type III glycogen storage disease.

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 α-maltotrioside and higher homologs also act as substrates. The splitting of the glycogen-glycogenin bond by isoamylase indicates the α-anomeric configuration of the terminal d-glucose unit. It also provides a means of preparing apoglycogenin. 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.

Glycogen debranching enzyme deficiency: Long‐term study of serum enzyme activities and clinical features

In glycogen storage disease type III (glycogen debranching enzyme (DE) deficiency), the activities of serum alanine aminotransferase, aspartate aminotransferase and lactate dehydrogenase may be strikingly elevated during childhood but are low during adult life. To determine the pattern of the elevated serum enzyme activities in relationship to diet, the biochemical subtype and clinical symptoms, 13 patients with DE deficiency were studied. Activities of serum aspartate and alanine transaminases, lactate dehydrogenase, and alkaline phosphatase were markedly elevated during infancy. Continued elevation of enzyme activities during childhood appeared to be related to DE deficiency in liver, but unrelated to DE deficiency in muscle. Activity elevations correlated inconsistently with diet and poorly with childhood growth rate or the presence of hypoglycaemia. The serum enzyme activities declined around puberty concomitantly with a decrease in liver size. Although periportal fibrosis and micronodular cirrhosis indicated the presence of hepatocellular damage during childhood, the decline in serum enzyme activities with age and the absence of overt hepatic dysfunction suggest that the fibrotic process may not always progress.

Two Classes of Starch Debranching Enzymes from Developing Maize Kernels

Summary Based on their substrate specificities, two distinct classes of starch debranching enzymes have been isolated from developing maize kernels. One class of enzyme hydrolyzed pullulan most rapidly followed by β-limit dextrin, amylopectin, and phytoglycogen. This activity was classified as a pullulanase (E.C. # 3.2.1.41). A different class of enzyme debranched amylopectin most rapidly, followed by β-limit dextrin and phytoglycogen. It, however, would not hydrolyze pullulan and was classified as isoamylase (E.C. # 3.2.1.68). Two forms of isoamylase were isolated which differed in relative molecular mass and eluted separately on anion exchange chromatography, but only one form was characterized. Both classes of enzyme activity caused an increase in starch-iodine staining when incubated in an amylopectin solution. The optimal pH for pullulanase activity was pH 5.5, whereas isoamylase had optimal activity at pH 6.0 to 7.5. Both classes of enzyme required 5 mM dithiothreitol or the equivalent sulfhydryl reducing power for activity. We speculate that pullulanase may be specialized to function in conjunction with β-amylase in hydrolyzing exposed α-1,6 branches, whereas isoamylase appears to be more efficient at hydrolyzing less exposed branches in amylopectin.

Changes of a Rice Debranching Enzyme during Seed Formation and Germination

Summary A starch debranching enzyme was purified from rice seeds to be homogeneous in molecular mass and used to raise antiserum. This antiserum reacted specifically with a debranching enzyme isolated from rice seeds, but did not react with those from potato tuber, barley seed or Klebsiella cells. Rice seeds were collected during seed formation and germination, and extracted proteins were analyzed by immunostaining following electrophoresis. The concentration of this enzyme protein estimated by immunostaining was maximal at the mid-maturation stage and was essentially constant during the late-maturation stage; this was contradictory to a decrease in activity. In dry seeds there was little debranching enzyme present in the soluble protein fraction, but there was a rapid increase of it during germination associated with an increase in its activity and a slight change of isoelectric point. The enzyme present in the total protein fraction showed maximum accumulation during the early stage of germination but decreased slightly later. These quantitative results suggest that the latent form of debranching enzyme was converted into an active form during seed germination.

AGL1-AGL6, an Arabidopsis gene family with similarity to floral homeotic and transcription factor genes.

The predicted products of floral homeotic genes, AGAMOUS (AG) from Arabidopsis thaliana and DEFICIENS A (DEF A) from Antirrhinum majus, have been shown previously to share strong sequence similarity with transcription factors from humans (SRF) and yeast (MCM1). The conserved sequence between these proteins is localized within a domain known to be necessary for the DNA binding and for the dimerization of SRF. We have isolated six new genes from A. thaliana, AGL1-AGL6, which also have this conserved sequence motif. On the basis of the sequence comparison between the AG and AGL genes, they can be assigned to two subfamilies of a large gene family. RNA dot blot analysis indicates that five of these genes (AGL1, AGL2, AGL4, AGL5, and AGL6) are preferentially expressed in flowers. In addition, in situ RNA hybridization experiments with AGL1 and AGL2 show that their mRNAs are detected in some floral organs but not in others. Our results suggest that these genes may act to control many steps of Arabidopsis floral morphogenesis. In contrast, the AGL3 gene is expressed in vegetative tissues as well as in flowers, suggesting that it functions in a broader range of tissues. We discuss possible roles of this gene family during the evolution of flowers.

Type III glycogen storage disease: An adult case with mild disease but complete absence of debrancher protein

A 54-yr-old woman who presented with chest pain and elevated serum creatine kinase levels was found to have type III glycogen storage disease. Except for a history of hepatomegaly in childhood, she was healthy and lived a normal life. There was no hypoglycemia, seizure disorder or growth retardation. Muscle weakness was not apparent until the sixth decade. Despite the mild clinical course, debranching enzyme activity was not detectable by biochemical assay, and immunoblot analysis using a polyclonal antibody showed a complete absence of debrancher protein. Thus, mild clinical manifestations in this patient could not be explained by the residual debrancher enzyme and/or activity.

Hormonal control of cardiac lipolysis by glyco(geno)lysis

The importance of the glucose/fatty acid cycle in the control of cardiac lipolysis is emphasized by the following observations. Addition of the glycogen debranching inhibitor deoxynojirimycin or an O 2-vehicle, fluorocarbon F-43, to media perfusing paced, lipid-enriched, Langendorff hearts lower cardiac lactate and glycerol 3-phosphate levels together with inhibition of glucagon-stimulated glycerol (and lactate) release. The absence of fluorocarbon during perfusion of 5 Hz paced langendorff hearts probably results in limited tissue oxygenation, resulting in glycogenolysis and lipolysis. The results indicate hormonal control of cardiac lipolysis by glyco(geno)lysis.

Glycogen metabolism during tension generation and maintenance in vascular smooth muscle.

To study the regulation of glycogen utilization in vascular smooth muscle, we measured the content of glycogen and glucose 6-phosphate and the activity of the glycogen phosphorylase and glycogen debrancher enzymes in porcine carotid artery. During active contraction, the rates of glycogen phosphorylase and glycogenolysis were as high as expected. Despite this, glycogen content did not decrease to less than approximately 50% of control levels even after sustained contractions. The activity of glycogen debrancher enzyme was found to be limiting glycogen utilization at this point. Although glycogenolysis is closely coordinated with increases in oxidative metabolism concomitant with active contraction, the maximal level of tension obtained after stimulation was not substantially reduced under conditions where glycogen debrancher enzyme was limiting glycogen utilization. On the other hand, the rate of tension generation was increased in these tissues. Thus glycogen utilization is not necessary for maximal force generation per se, but may influence other muscle contractile properties. Finally, during steady-state tension maintenance, glycogen utilization is likely to be regulated by the intracellular concentrations of metabolic intermediates (glucose, glucose 6-phosphate), as it is in skeletal muscle.

Disorders of Glycogen Metabolism of Muscle

Glycogen is a crucial source of energy in the initial stages of muscle activity and during exercise of high intensity. There are 10 well-defined biochemical defects of glycogen metabolism expressed in muscle and affecting the following enzymes: alpha 1,4 glucosidase (glycogenesis type II), debrancher enzyme (III), brancher enzyme (IV), phosphorylase (V), phosphofructokinase (VII), phosphorylase b kinase (VIII), phosphoglycerate kinase (IX), phosphoglycerate mutase (X), lactate dehydrogenase (XI). These disorders cause two main syndromes: one characterized by exercise intolerance with cramps and myoglobinuria, the other by fixed weakness. However, there are examples of clinical and biochemical heterogeneity for each disease, and molecular genetic analysis is already showing evidence of genetic heterogeneity. Although our understanding of the biochemical errors has progressed considerably, the pathogenesis of symptoms and signs remains incomplete.

Accurate and efficient pre-mRNA splicing in Drosophila cell-free extracts.

Synthetic mRNA precursors from the Drosophila fushi tarazu (ftz) gene were shown to be accurately and efficiently spliced in Drosophila nuclear extracts derived from Kc tissue culture cells or 0- to 12-hr embryos. Splicing the ftz pre-mRNA requires ATP and low levels of Mg2+. The reaction proceeds with a lag of 20-30 min prior to appearance of spliced mRNA and appears to proceed in two steps. The first step is cleavage at the 5' splice site to generate a 5' exon (E1) fragment and an intron-3' exon (IVS-E2) species. The second step involves cleavage at the 3' splice site, ligation of the two exons (E1-E2), and intron (IVS) release. The excised intron (IVS) and intron-3' exon (IVS-E2) exhibit anomalous electrophoretic mobility, suggesting that they contain branched structures. Nuclease analysis using two-dimensional thin-layer chromatography indicates that both the IVS and IVS-E2 species possess branched trinucleotides in which a guanosine residue at the 5' end of the intron is linked in a 2'-5' phosphodiester bond to the 2' hydroxyl group of an adenosine residue in the intron. The site of branchpoint formation was localized by debranching the Drosophila lariat with mammalian (HeLa) cell debranching enzyme and by P1 and T2 nuclease analysis. These findings indicate that nuclear extracts derived from Drosophila cultured cells or embryos can accurately splice mRNA precursors and that the reaction mechanism is the same as has been observed in yeast and mammalian cells. This system provides an initial step toward the biochemical analysis of developmentally regulated pre-mRNA splicing events in Drosophila.

Phosphorus NMR spectroscopy study of muscular enzyme deficiencies involving glycogenolysis and glycolysis.

We used phosphorus NMR spectroscopy to study 16 patients with muscular enzyme deficiencies affecting glycogenolysis and glycolysis. Study of phosphomonoester (Pm) kinetics and intracellular pH during exercise and recovery provided criteria for the distinction of these metabolic myopathies by NMR spectroscopy. The Pm peak was undetectable in patients lacking debrancher enzyme or phosphorylase. By contrast, in phosphofructokinase (PFK) or phosphoglycerate kinase (PGK) deficiency, the Pm peak was larger than that of inorganic phosphate in exercise, whereas it was always smaller in normal subjects. During recovery, the disappearance of Pm was slower in PGK than in PFK deficiency.