The presence of acetone in diabetic urine was reported more than a century ago. Since that time considerable effort has been devoted to studies on ketosis and ketogenesis. Recent studies in several laboratories have revealed the precise enzymatic mechanism for the formation of acetoacetate, the primary ketone body. For these studies, investigators have used liver extracts obtained from various species such as beef, dog, rabbit, pigeon and chicken, as well as omasum and rumen epithelium obtained from sheep. A study of acetoacetate formation in extracts of chicken liver is complicated by the presence of a factor which inhibits: in vitro acetoacetate formation in the assay used. This "inhibitor" also depresses acetoacetate formation by beef liver extracts. A study of the nature of this inhibitor action was begun in this laboratory in the hope that an understanding of its action might shed some light on the mechanism of acetoacetate formation. The mechanism of acetoacetate formation was subsequently worked out by independent means. It was still of interest, however, to clarify the nature of the inhibitor factor. This thesis constitutes such a study. The "inhibitor" protein has been purified 300-fold from extracts of fresh chicken liver. The various possible sites of action on the in vitro acetoacetate-forming system have been examined. Preliminary studies revealed that the factor does not act on any of the enzymatic components of acetoacetate formation. Evidence is presented that it inhibits acetoacetate formation through the inactivation of both the free and esterified forms of coenzyme A. The product of its action on coenzyme A has been isolated and characterized by paper and ion-exchange chromatography and chemical analysis as 3'-dephosphocoenzyme A. The inhibitor protein can thus be more properly designated as a "coenzyme A 3'-phosphohydrolase." In retrospect, it is now clear that this enzyme could have afforded no knowledge concerning the enzymatic mechanism of acetoacetate formation. The enzyme exhibits maximal activity at pH 3.6, with half-maximal activity at a "plateau" between pH 5.0 and pH 6.0, and at some point below pH 2.5. The rate of inactivation of coenzyme A by the phosphohydrolase is neither enhanced nor depressed by divalent cation or metal-binding agents. Although the most highly purified enzyme fraction obtained exhibits phosphohydrolase activity against a wide variety of phosphate esters, evidence is presented to indicate that the coenzyme A 3'-phosphohydrolase activity may well be a specific and unique enzyme. / Medicine, Faculty of / Anesthesiology, Pharmacology and Therapeutics, Department of / Graduate

Thesis (Ph. D.)--University of Wisconsin--Madison, 1959. / Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references (leaves 103-107).

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

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.)--School of Biological Sciences. University of Missouri--Kansas City, 2006. / "A dissertation in molecular biology and biochemistry and cell biology and biophysics." Advisor: Gerald M. Carlson. Typescript. Vita. Title from "catalog record" of the print edition Description based on contents viewed Nov. 9, 2007. Includes bibliographical references (leaves 103-113). Online version of the print edition.

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

München, Techn. Universiẗat, Diss., 1999.

Universiẗat, Diss., 2002--Bayreuth.

The initial goal was the conversion of Bifidobacterium adolescentis Sucrose Phosphorylase (BaSP) into a polyphenol glucosidase by structure based enzyme engineering. BaSP was chosen because of its ability to utilize sucrose, an economically viable and sustainable donor substrate, and transfer the glucosyl moiety to various acceptor substrates. The introduction of aromatic residues into the active site was considered a viable way to render it more suitable for aromatic acceptor compounds by reducing its polarity and potentially introducing π-π-interactions with the polyphenols. An investigation of the active site revealed Gln345 as a suitable mutagenesis target. As a proof of concept BaSP Q345F was employed in the glycosylation of (+)-catechin, (-)-epicatechin and resveratrol. The variant was selective for the aromatic acceptor substrates and the glucose disaccharide side reaction was only observed after almost quantitative conversion of the aromatic substrates. A crystal structure of BaSP Q345F in complex with glucose was obtained and it displayed an unexpected shift of an entire domain by 3.3 Å. A crystal structure of BaSP D192N-Q345F, an inactive variant in complex with resveratrol-3-α-D-glucosid, the glucosylation product of resveratrol, synthesized by BaSP Q345F was solved. It proved that the domain shift is in fact responsible for the ability of the variant to glycosylate aromatic compounds. Simultaneously a ligand free crystal structure of BaSP Q345F disproved an induced fit effect as the cause of the domain shift. The missing link, a crystal structure of BaSP Q345F in the F-conformation is obtained. This does not feature the domain shift, but is in outstanding agreement with the wildtype structure. The domain shift is therefore not static but rather a step in a dynamic process. It is further conceivable that the domain shifted conformation of BaSP Q345F resembles the open conformation of the wild type and that an adjustment of a conformational equilibrium as a result of the Q345F point mutation is observed. An investigation into the background reaction, the formation of glucose-glucose disaccharides of BaSP Q345F and three further variants that addressed the same region (L341C, D316C-L341C and D316C-N340C) revealed the formation of nigerose by BaSP Q345F. / Saccharose Phosphorylase aus Bifidobacterium adolescentis (BaSP) sollte durch strukturbasiertes Enzym-Engineering in die Lage versetzt werden Polyphenole zu glukosylieren. In die katalytische Tasche sollten aromatische Seitenketten eingeführt werden um die Polarität an jene der gewünschten Akzeptorsubstrate anzupassen und eine weitere Stabilisierung durch π-π-Wechselwirkungen zwischen Enzym und Substrat zu erlauben. BaSP Q345F war in der Lage die Zielsubstrate zu glukosylieren und behielt gleichzeitig ausreichen Aktivität bei. Die weitere Untersuchung dieses Enzyms ist in vier Studien beschrieben. Die Kristallstruktur einer inaktiven Variante, BaSP D192Q-Q345F in komplex mit dem Glukosylierungsprodukt Resveratrol-3-α-D-Glukosid wurde gelöst. Dadurch konnte gezeigt werden, dass einer Verschiebung einer Domäne für die Fähigkeit der Variante Glukose auf aromatische Substrate zu übertragen, verantwortlich ist. Die Orientierung des π-Systems von Resveratrol erlaubt weiterhin T-förmige π-π-Wechselwirkungen mit Phe156 und Phe345.Die detaillierte kinetische Untersuchung von BaSP Q345F mit acht Akzeptorsubstraten ergab eine starke Affinität der Variante zu den aromatischen Substraten (KM 0.08 bis 1.55 mM). Weitere Strukturdaten zeigen, dass die Verschiebung der Domäne Teil eines dynamischen Prozesses ist. Des weiteren ist die Q345F Variante in der Lage, den seltenen Zucker Nigerose zu synthetisisern.

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