PART I GLYCOGEN PHOSPHORYLASE IN BRAIN, ILEUM AND UTERUS In recent years considerable attention has been devoted to the nature of the glycogen phosphorylase enzyme system in skeletal muscle, cardiac muscle and liver. It has been established that those factors which affect the action of this enzyme constitute a major metabolic control mechanism. Considerably less attention has been paid to this enzyme system in tissues which are known to contain low glycogen, stores. The present study constitutes a preliminary examination of glycogen phosphorylase in brain, uterus and ileum. It has been found that brain contains considerably higher phosphorylase activity than liver and is comparable to heart. Uterus and ileum contain enzyme levels comparable to that of liver. The data suggests that the enzyme exists in two forms in these three tissues, one active in the presence of adenosine-5'-monophosphate, the other active in the absence of this nucleotide. The enzyme in each tissue is thus very similar to the skeletal muscle enzyme, but different from that in liver. Preliminary evidence was also obtained which indicates that phosphorylase activating and inactivating enzymes were present. Levels of active phosphorylase were increased in brain and uterus in the presence of epinephrine. The intestinal smooth muscle enzyme failed to respond to epinephrine. The evidence, although preliminary, is consistent with the idea that catecholamine-induced muscle contraction is associated with phosphorylase activation. PART II SYNTHESIS AND ENZYMATIC DEGRADATION OF SEVERAL DE0XYRIB0NUCLE0SIDE-3',5’-MONOPHOSPHATES It is known, that the phosphorylase activating action of epinephrine is mediated through adenosine-3',5'-monophosphate. The metabolism of this important nucleotide is attracting widespread attention. In order to study its seemingly manifold actions, the need has arisen for structural analogues of the compound. Several ribonucleoside-3',5'-monophosphates have already been prepared. In the present work several deoxyribonucleoside-3’,5'-monophosphates were synthesized. These included the nucleoside-3’,5'-monophosphates of deoxyadenosine, deoxyinosine, and deoxyuridine. These compounds were shown to be hydrolysed by a phosphodiesterase from brain which is specific for nucleoside-3',5'-monophosphates. The product of the hydrolysis, in each case, was identified as the corresponding deoxyribonucleoside-5'-phosphate. / Medicine, Faculty of / Anesthesiology, Pharmacology and Therapeutics, Department of / Graduate

No description available.

During the purification of the enzyme glycogen synthase from the muscle of the nematode Ascaris suum, approximately 70% of the glycogen synthase activity can be separated from the bulk of cellular glycogen by centrifugation for 60 min at 105,000 x . The glycogen synthase in the supernatant fraction has an Mr of 1.2 x 106 as determined by Sepharose 4B gel filtration chromatography. The glycogen synthase in this high molecular weight complex (glycogen primer complex) can be further purified by ConA-Sepharose affinity chromatography; the enzyme activity was eluted with 100 .mM a-methylmannoside. The glycogen synthase in glycogen'primer complex is predominately in the glucose 6-phosphatedependent form. The glycogen primer complex can catalyze the transfer of glucosyl units from UDP-glucose to an endogenous acceptor in the absence of exogenous glycogen. Analysis by SDS-PAGE showed three proteins (Mr 140,000, 78,000 and 34,000) and a carbohydrate polymer. The carbohydrate polymer can be partially digested with a-amylase. The glycogen primer complex was further digested by acid hydrolysis, and upon descending paper chromatography analysis, eight different carbohydrates were isolated, two of which were tentatively identified as glucose and sialic acid. The [14 C]-autoradiograph showed that in vitro synthesis of a glycogen-like polysaccharide occurred on this carbohydrate polymer. Polyclonal antibodies have been made to the glycogen primer complex, and Western Blot analysis indicated that all three proteins of the glycogen primer complex were antigenic. Collectively, the data indicate that a glycogen-like polysaccharide is synthesized from a carbohydrate-associated protein primer in the muscle of this worm.

Pyridoxine-deficient rats are known to exhibit little, if any, weight gain; they also have decreased fat stores in comparison with their pair-fed controls. The defect in energy metabolism responsible for this phenomenon is not well understood at present. This study was undertaken to investigate some aspects of glycogenesis and glycogenolysis in order to add to the present information on energy metabolism in the pyridoxine deficiency state. Meal-fed animals were used, in order to eliminate differences due to the mode of feeding between the experimental and the pair-fed control animals. Male weanling rats were fed a pyridoxine-deficient diet in one 2-hour daily meal, while the controls were pair-fed. This eliminated differences due to feeding frequency when these groups were compared with each other. Aspartate amino-transferase and alanine aminotransferase activities were assayed in liver and erythrocytes in order to verify the presence of a pyridoxine deficiency state under the conditions used in this laboratory. The activities of glycogen phosphorylase, the rate-limiting enxyme in glycogenolysis, and glycogen UDP-glucosyltransferase were assayed in liver and muscle. Glycogen storage in these tissues was also measured. Finally, the incorporation of labelled carbon atoms into blood glucose and liver glycogen following intraperitoneal injection of L-alanine-¹⁴C was assayed. Glycogen phosphorylase activity was reduced in pyridoxine-deficient animals. This defect was not accompanied by a concomitant increase in the deposition of glycogen. There was, therefore, the possibility of a decreased ability to form glycogen. Glycogen UDP-glucosyltransferase activity was normal in muscle and elevated in liver indicating, if anything, an unimpaired ability to synethesize glycogen from UDPG. A trend towards a lesser incorporation of labelled carbon atoms into the blood glucose by the pyridoxine-deficient group appeared when the results were expressed as a percent of administered dose per ml. This became statistically significant when the data was expressed in terms of the circulating glucose pool. Although not at a statistically significant level, there was a greater incorporation of labelled carbon atoms into the liver glycogen of the pyridoxine-deficient group. It appeared from these findings that the defect in energy metabolism in pyridoxine deficiency may be the result of a reduced availability of carbon skeletons and occurred prior to the formation of glycogen. Further study in this area is necessary to reveal the exact point at which energy loss occurred. / Land and Food Systems, Faculty of / Graduate

Indiana University-Purdue University Indianapolis (IUPUI) / Glycogen synthase (GS) is the rate limiting enzyme in the synthesis of glycogen. Eukaryotic GS catalyzes the transfer of glucose from UDP-glucose to the non-reducing ends of glycogen and its activity is negatively regulated by phosphorylation and allosterically activated by glucose-6-phosphate (G6P). A highly conserved cluster of six arginine residues on the C-terminal domain controls the responses toward these opposing signals. Previous studies had shown that tetrameric enzyme exists in three conformational states which are linked to specific structural changes in the regulatory helices that carry the cluster of arginines. These helices are found opposite and anti-parallel to one another at one of the subunit interfaces. The binding of G6P beneath the regulatory helices induces large scale conformational changes which open up the catalytic cleft for better substrate access. We solved the crystal structure of the enzyme in its inhibited state and found that the tetrameric and regulatory interfaces are more compacted compared to other states. The structural consequence of the tighter interfaces within the inhibited state of the tetramer is to lower the ability of glycogen chains to access to the catalytic cleft. Based on these observations, we developed a novel regulatory feature in yeast GS by substituting two of its conserved arginine residues on the regulatory helix with cysteines that permits its activity to be controlled by reversible oxidation/reduction of the cysteine residues which mimics the effects of reversible phosphorylation. In addition to defining the structural changes that give rise to the inhibited states, we also used X-ray crystallography to define the mechanism by which the enzyme discriminates between different UDP-sugar donors to be used as substrates in the catalytic mechanism of yeast GS. We found that only donor substrates can adopt the catalytically favorable bent conformation for donor transfer to a growing glycogen chain.

Glycogen synthase, the enzyme that catalyzes the rate-limiting reaction of glycogen syntheses has been purified and characterized from Ascaris suum muscle. Glycogen in the crude extract was digested to release the enzyme, eluted from a DE52 cellulose column and then applied to a Sepharose affinity column. The purified Ascaris enzyme was found to be homologous to the mammalian enzyme with regard to subunit and holoenzyme Mr^3 allosteric activation, substrate affinity and covalent modification. However, the association between Ascaris glycogen synthase and endogenous glycogen differed from that in mammalian systems.

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

No description available.

No description available.

No description available.