Compared to normal cells, many tumor cells have to subsist in a hypoxic intratumoral environment that has an unstable supply of oxygen and nutrients including glucose. How tumor cells may survive the metabolic stress arising from tumor hypoxia is not yet fully understood. Recent studies revealed that tumor cells are able to accumulate large quantities of intracellular glycogen. Whether glycogen would serve as fuel reserve in hypoxic tumor cells is presently not clear. This question is being addressed in this study. When HeLa, HT29, HEK293 and HepG2 cells were incubated under hypoxic condition in the absence of glucose, the steady state intracellular glycogen level dropped by more than 50% in 3 hours. The specific pharmacological inhibition of the liver isoform glycogen phosphorylase (PYGL) (CAS 648926-15-2) partially inhibited hypoxia-induced glycogen degradation. More complete inhibition was achieved by combined incubation using the pharmacological inhibitor and 2-deoxyglucose. Inhibition of glycogen degradation resulted in decrease in hypoxia-induced lactate formation, supporting the idea that glycogen serves as a fuel reserve in hypoxic cancer cells. Inhibition of autophagy or alpha-glucosidase failed to prevent glycogen degradation in hypoxic condition, suggesting that cytosolic glycogen phosphorylase is the major enzyme involved in glycogen degradation. The mRNA, protein and phosphorylation levels of glycogen phosphorylase were unaltered by hypoxia. The siRNA-mediated knockdown of the brain form of glycogen phosphorylase (PYGB) resulted in markedly greater inhibition of glycogen degradation than did the knockdown of PYGL. Whereas the enzyme activity of PYGB can be markedly stimulated by AMP, the activity of PYGL is only slightly stimulated in the presence of AMP. The relative proportion of AMP-sensitive and AMP-insensitive GP activity is little affected by acute hypoxia. In conclusion, direct evidence is provided in this study that glycogen may serve as an intracellular fuel reserve in tumor cells. The involvement of the brain form of glycogen phosphorylase is for the first time demonstrated to be involved in the mobilization of this fuel reserve in tumor cells. / published_or_final_version / Biochemistry / Master / Master of Philosophy

Thesis (Ph.D.)--Murdoch University, 2006. / Thesis submitted to the Division of Health Sciences. Includes bibliographical references.

Much work has been carried out on glycogen phosphorylase over the last seventy years. Interest has persisted due not only to the usefulness of phosphorylase as a model system of allostery, but also due to the connection to the disease state in type II diabetes. The bulk of research consists of structural studies utilizing the wild-type enzyme from rabbit muscle. In this study we have employed linkage analysis in combination with structural perturbations via site-directed mutagenesis to test kinetic models of activation of phosphorylase b by AMP, and to examine the roles of the N-terminus, the acidic patch, ?-helix 1 and the 280?s loop in activation by AMP. Experiments have been carried out on purified glycogen phosphorylase b variants to determine the effects of perturbations in vitro. The kinetic models of activation by AMP are found to be a relatively accurate description of kinetic behavior of wild-type phosphorylase b, but are found to be technically incorrect with respect to the absolute requirements of two equivalents of AMP to be bound prior to catalysis. Phosphorylase b demonstrates activity in the absence of AMP, though only at high concentrations of phosphate, and a hybrid phosphorylase b with only a single functional AMP binding sight shows slight activation. The truncate ?2-17 shows weakened binding to AMP and phosphate in the apo enzyme, but maintains activation by AMP to an affinity similar to that of wild-type, indicating that the N-terminus is not required for activation by AMP, but has a role in establishing the affinity for both AMP and phosphate in the apo enzyme. Perturbations of the acidic patch indicate that interactions between the acidic patch and the N-terminus enhance the affinities in the apo enzyme, suggesting that the structures of the N-terminus at the acidic patch may represent an active form of the enzyme. ?-helix 1 is found to have a role in homotropic cooperativity in phosphorylase b, but not in heterotropic activation by AMP, while the 280?s loop is confirmed to have a role in the heterotropic coupling between AMP and phosphate. Based on the findings in this study an alternate structural model of activation by AMP involving ?-helix 8 is proposed.

Much work has been carried out on glycogen phosphorylase over the last seventy years. Interest has persisted due not only to the usefulness of phosphorylase as a model system of allostery, but also due to the connection to the disease state in type II diabetes. The bulk of research consists of structural studies utilizing the wild-type enzyme from rabbit muscle. In this study we have employed linkage analysis in combination with structural perturbations via site-directed mutagenesis to test kinetic models of activation of phosphorylase b by AMP, and to examine the roles of the N-terminus, the acidic patch, ?-helix 1 and the 280?s loop in activation by AMP. Experiments have been carried out on purified glycogen phosphorylase b variants to determine the effects of perturbations in vitro. The kinetic models of activation by AMP are found to be a relatively accurate description of kinetic behavior of wild-type phosphorylase b, but are found to be technically incorrect with respect to the absolute requirements of two equivalents of AMP to be bound prior to catalysis. Phosphorylase b demonstrates activity in the absence of AMP, though only at high concentrations of phosphate, and a hybrid phosphorylase b with only a single functional AMP binding sight shows slight activation. The truncate ?2-17 shows weakened binding to AMP and phosphate in the apo enzyme, but maintains activation by AMP to an affinity similar to that of wild-type, indicating that the N-terminus is not required for activation by AMP, but has a role in establishing the affinity for both AMP and phosphate in the apo enzyme. Perturbations of the acidic patch indicate that interactions between the acidic patch and the N-terminus enhance the affinities in the apo enzyme, suggesting that the structures of the N-terminus at the acidic patch may represent an active form of the enzyme. ?-helix 1 is found to have a role in homotropic cooperativity in phosphorylase b, but not in heterotropic activation by AMP, while the 280?s loop is confirmed to have a role in the heterotropic coupling between AMP and phosphate. Based on the findings in this study an alternate structural model of activation by AMP involving ?-helix 8 is proposed.

Thesis (Ph. D.)--Virginia Polytechnic Institute and State University, 1992. / Vita. Abstract. Includes bibliographical references (leaves 106-114). Also available via the Internet.

The structure of rabbit muscle glycogen phosphorylase b, an important regulatory enzyme in glycogen metabolism, has been studied by X-ray crystallographic techniques. This work was carried out as part of a group project, and the crystal structure of the enzyme had already been solved to 3 Å resolution, using the technique of Multiple Isomorphous Replacement. A search for additional heavy atom isomorphous derivatives was carried out, and photographic data to 3 Å resolution were collected for a further ethylmercurythiosalicylate derivative, using a screenless oscillation camera. The collection and reduction of this data, and the refinement of the heavy atom positions is described. The inclusion of this data allowed a new electron density map (with figure of merit = O.63) to be calculated, which enabled previously ambiguous areas in the electron density to be interpreted. Data to 2 Å resolution have been collected on an oscillation camera, using a synchrotron radiation source and cylindrical film cassettes. An intensity gain of up to 13O times, compared to a GX6 rotating anode source, was obtained with the synchrotron radiation source. A reduction in radiation damage, was also observed. The collection and reduction of the 2 Å data is described. The final overall merging R-factor was 15%. Some systematic errors remain in the data, and possible sources of these errors are discussed, and improvements to the data processing procedure suggested. The 2 Å data were empirically scaled to the 3 Å data and have been used in the first stages of the refinement of the phosphorylase b structure. The contribution of the crystallographic results towards an understanding of phosphorylase b as an allosteric protein is discussed.

This document only includes an excerpt of the corresponding thesis or dissertation. To request a digital scan of the full text, please contact the Ruth Lilly Medical Library's Interlibrary Loan Department (rlmlill@iu.edu).

Thesis (MSc (Genetics. Plant Biotechnology)--University of Stellenbosch, 2009. / ENGLISH ABSTRACT: Glycogen was isolated from E. coli and analysed for the amount of phosphate present within it. It was confirmed that a significant proportion of the glucose residues were phosphorylated at the C6 position. This glycogen phosphate was found also in both glgb- (glycogen branching enzyme) and glgp- (glycogen phosphorylase enzyme) mutants, demonstrating that a mechanism for phosphate incorporation that does not involve GlgP alone, and which is capable of incorporating phosphate into linear glucans could exist. The degree of phosphorylation depended on the amount of phosphate present in the media, which less being incorporated in media where phosphate was reduced. Screening for glycogen phosphorylating genes using a E. coli genomic library in a functional expression system identified the malP gene as a possible candidate for incorporation of the phosphate at the C6 position. There was no difference, however, between the glycogen phosphate content of the mutant and wild type. Efforts were made to construct a malp-/glgp- double mutant, but these were unsuccessful. In addition the influence of plants and human proteins on yeast glycogen metabolism was also investigated. These proteins have been demonstrated to have an effect on starch or glycogen in humans, plant and E. coli, but the data from this study indicated that this was not the case in yeast. / AFRIKAANSE OPSOMMING: Glikogeen, wat geisoleer was uit E.coli was geanaliseer vir fosfaat inhoud daarin. Daar was gevind dat `n beduidende proporsie van die glukose residue gefosforileerd was op die C6 posisie. Hierdie gefosforileerde glikogeen was ook gevind in glg- (glikogeen vertakkingsensieme) en glgp- (glikogeen fosforileringsensieme) mutante wat daarop dui dat `n meganisme vir fosforilering bestaan was nie slegs aangewese is op die aktiwiteit van GlgP nie, en om fosfaat te inkorporeer in linêre glukane. Die graad van fosforilering was ook afhanklik van die hoeveelheid fosfaat teenwoordig in die medium, met gevolglik minder wat geinkorporeer kan word in medium waar fosfaat verminderd was. Seleksie-gebaseerde ondersoeking vir fosforileringsensieme van glikogeen deur gebruik te maak van E. coli genomiese biblioteke in `n funksionele uitdrukkingssisteem het die malP geen geidentifiseer as een van die moontlike kandidate wat verantwoordelik kan wees vir inkorporering van fosfaat in the C6 posisie. Daar was egter geen verskil in die fosfaat inhoud van glikogeen tussen die wilde tipe en die mutante. Pogings wat aangewend is om `n malp-/glgpdubbel mutant te konstrueer was onsuksesvol. Verder is die invloed van plant en mens proteine op gis glikogeen ook bestudeer. Vroeër is aangetoon dat hierdie proteine `n invloed op stysel en glikogeen het in mense, plante en E. coli, maar data van hierdie studie toon aan dat dit nie die geval in gis is nie.

The expression of the <I>glycogen phosphorylase- 2</I> gene (<I>gp2</I>) is initiated during early development and regulated by the extracellular morphogens cAMP and Differentiation Inducing Factor (DIF-1) [1-3]. Glycogen phosphorylase- 2 catalyzes the breakdown of glycogen reserves in developing cells to generate glucose precursors required for the synthesis of the end products of differentiation [4-6]. Thus, the expression of <I>gp2</I> is a significant event for cellular differentiation. The sequence of the <I>gp2</I> promoter, like other <I>Dictyostelium</I> promoters, has an AT-rich bias (88%) [7]. Previous deletional analyses of the promoter provided a map of the regions that contained transcriptional regulatory elements. The regions thus identified contained either "TAAAAATGGA" or C-rich repeat sequences [2]. These regions were dissected further by site-directed mutagenesis (SDM) to better define the physical boundaries of the regulatory elements. It was shown that the mutation of either one of the C-rich repeats resulted in a dramatic drop of about 95% in reporter gene levels. These data strongly suggested that both the C-rich repeats of <I>gp2</I> functioned as transcriptional regulatory elements. I have identified and purified a factor called TF2 that demonstrates a high specificity for a C-rich transcriptional regulatory element, the 5' C box. TF2 was first detected with electrophoretic mobility shift assays of DEAE chromatographic fractions of cell-free extracts. The specificity of TF2 for the 5' C box was tested by competition analysis using six other oligonucleotides. Purification of TF2 was achieved by ion-exchange chromatography, DNA affinity chromatography, gel filtration chromatography, and preparative SDS-PAGE. SDS-PAGE analysis indicated an apparent subunit molecular weight of 28 kDa. The apparent molecular weight of the native protein as estimated by gel filtration was about 53 kDa. This suggested that TF2 binds gp2 as a homodimer. A cDNA clone of the tf2 gene was provided by the Japanese <I>Dictyostelium</I> cDNA project. This allowed me to synthesize probes for Southern and Northern blot analyses. Southern blot analysis indicated that there is only one form of the <I>tf2</I> gene. Northern analysis showed little or no expression of <I>tf2</I> in undifferentiated cells. During development <I>tf2</I> expression increases up to a maximum at 8 h, then decreases in later stages. Attempts to disrupt the gene suggest that <I>tf2</I> mutation may be lethal. / Ph. D.

The gene encoding the Dictyostelium replication protein A large subunit (DdRPA1) has been cloned by screening of an EcoR I partial genomic library and a Hind III genomic sub-library. The complete nucleotide sequence, including the promoter region of the gene has been obtained by sequencing. Though the DdRPA1 protein has a size shift during development, 62 kDa in undifferentiated cells and 81 kDa in differentiated cells; they are the products of the same gene. Northern blot analysis revealed that the expression level of the DdRPA1 was constant throughout differentiation and the size of mRNA is the same at all stages, corresponding to a 81 kDa protein. Thus, it seems that the size change between the 62 kDa and 81 kDa is probably due to posttranslational modification, most likely, proteolytic cleavage. The transcription start site for both sizes of DdRPA1 has been identified at 306 bp upstream of the coding sequence by primer extension reaction. A PCR fragment representing 27% of the gene encoding the DdRPA middle size subunit (DdRPA2) has been generated by using the degenerate primers. This PCR fragment has been cloned and sequenced. The mRNA for this subunit corresponds to a protein of about 35 kDa. A decrease of the DdRPA2 mRNA expression level during differentiation was found by comparison between undifferentiated and differentiated cells. In Dictyostelium, replication protein A is a heterotrimeric protein that can bind with specific DNA sequences in a stage-dependent pattern. These DNA sequences were identified as the cis-acting regulatory sites in differentiation-related genes, including the glycogen phosphorylase 2 gene (gp2). Therefore, it is possible that DdRPA is not only a single-stranded DNA binding protein that is used in multiple essential DNA metabolic processes, such as DNA replication, repair and recombination in undifferentiated cells, but also involved in the transcriptional regulation process during differentiation. / Master of Science