Thesis (Ph. D.)--University of Michigan, 1932. / "By Mabel M. Miller and Howard B. Lewis." "Reprinted from the journal of biological chemistry, vol. XCVIII, no. 1 ... October, 1932." Bibliography: p. 140, 149-150.

Thesis (M.S.)--University of Wisconsin--Madison, 1960. / Typescript. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references (leaves [38]-39).

Thesis (M.S.)--University of Wisconsin--La Crosse, 2002. / Includes bibliographical references.

Although the quantity of glycogen in nervous tissue is small, in recent years it has been found that there is a significantly high turnover rate of this polysaccharide. Glycogen is present in sympathetic ganglia, midbrain structures, white matter, and synaptic regions. The enzymes of glycogen synthesis and breakdown exist in brain in high concentrations, which suggests that glycogenolysis may be coupled in some way to the energy-requiring processes concerned with electrical activity. In this thesis, the occurrence and activity of phosphorylase a, phosphorylase b kinase and phosphofructokinase were determined in peripheral and central nerve tissue, an attempt to purify phosphorylase b kinase was made, and the mechanisms controlling the activity of the brain kinase were studied. These experiments were carried out in an effort to learn more about the importance of glycogenolysis in the nervous system and the factors which control it. The results of a survey of dog nerve tissue and some experiments on rabbit tissue indicated that the enzyme levels in brain, superior cervical and stellate ganglia were comparable to those in heart. Other peripheral nerve tissue has approximately one-tenth the level found in brain. Heavily myelinated axonal tissue contained higher phosphorylase activity and much higher kinase activity than axons with relatively little myelination. It is suggested that glycogenolysis is likely to be concerned with synaptic transmission and Schwann cell metabolism. Attempts to purify one of the controlling enzymes in glycogenolysis, phosphorylase b kinase, from brain tissue were hampered by the instability of the enzyme and the consequent loss of activity that occurred at each purification step. A modest 3.0 to 3.5-fold purification was achieved by differential ultracentrifugation, freezing and thawing of the 100,000 x g precipitate, and a 0-35 per cent ammonium sulfate fractionation at pH 5.7. The enzyme exhibited increased stability when stored in 50 per cent glycerol at -18°, especially when it was present in a more purified state, but other attempts to increase the stability of the kinase met with little success. The maximum activity of brain phosphorylase b kinase occurred between pH 8.75 and 8.9, and a significant activity was noted at pH 7.0, which is somewhat different from that found in other tissues. However, the brain kinase is activated by mechanisms similar to those found for skeletal and cardiac muscle. The activity at pH 6.8 is increased by preincubation with Ca⁺⁺, and further increased by the addition of rabbit skeletal muscle calcium-activating factor. This is partially prevented by beef heart calcium inhibitory factor. ATP-Mg⁺⁺ and cyclic AMP also cause activation of the brain kinase. The total activation of the brain enzyme appeared much lower than that found in skeletal or cardiac muscle due to the already high activity at pH 7.0. Experiments designed to produce an accurate value for the pH 6.8/8.2 ratios of kinase activity utilized freezing of mouse brain in vivo in liquid Freon cooled to its freezing point. The value of 0.44 obtained from these procedures was similar to, or slightly higher than, the ratios in rabbit and beef brain. The kinase was not inactivated by room temperature incubation for two hours, although similar treatment would convert phosphorylase to its inactive form. The possibility remains that the pH 7.0 activity is artificially high, assuming the freezing techniques used are not fast enough. Otherwise, the high enzymatic activity may be due to the continuous electrical activity present in the brain. The possibility that glycogen catabolism is a more important source of energy in the nervous system than previously thought is supported by the high levels of enzymatic activity and similar phosphorylase b kinase control mechanisms to those in other tissues. / Medicine, Faculty of / Anesthesiology, Pharmacology and Therapeutics, Department of / Graduate

The concentration of glycolytic intermediates and co-factors in and pH values of P. superficlalis muscles from each of five 15 week-old and five 25 week-old White Cannon torn turkeys was measured at varying times between 0 and 180 min. post-mortem. Different rates of post-mortem glycolysis were evident among birds, independent of age. On the basis of ATP catabolism, pH and lactic acid accumulation, two groups categorized as "fast-" and "slow-glycolyzing" were evident. The different rates of glycolysis could not be explained by qualitative or quantitative differences in control of the glycolytic flux. The patterns of change in concentration of intermediates and co-factors expressed as mass action ratios suggested that regardless of glycolytic rate, post-mortem glycolysis in turkey breast muscle is susceptible to control at the reactions catalyzed by hexokinase, phosphofructo kinase, aldolase and triosephosphate isomerase, glyceraldehyde phosphate dehydrogenase and phosphoglycerokinase and pyruvate kinase. P. superficialis muscles from six 19 and six 27 week-old White Cannon torn turkeys were analyzed for ATP concentration at "0" and "60" min. post-mortem and muscle pH was determined during a three hour post-mortem period. The time required for excised muscle strips to achieve maximum contraction was determined by periodic measurement of strip length. Analysis of the data indicated a relationship between rate of ATP catabolism and time to maximum contraction. "Slow-" and "fast-glycolyzing" groups were evident and were independent of age. The "slow-glycolyzing" group had a higher initial ATP concentration, a larger proportion of initial ATP remaining at 60 min. and required a longer time for the muscle strips to achieve maximum contraction. / Land and Food Systems, Faculty of / Graduate

It has long been observed that some organo-fluorine compounds exhibit enhanced biological activity over their non-fluorinated precursors, however reasons for these unusual properties still remain poorly understood. An explanation which has been widely used relates to the ability of the C-F fragment of the analog to participate in hydrogen-bonding interactions with its protein receptor. For this reason, fluorinated carbohydrates have been used as hydrogen-bonding probes with a number of proteins. Thus there exists a need for a systematic investigation into the hydrogen-bonding ability of the C-F fragment, and the enzyme glycogen phosphorylase provides an excellent subject for such a study. The glucopyranose binding site in the inactive (T-state) conformation of the enzyme has been well characterised and high resolution crystallographic data is available. Thus by comparison of kinetic and crystallographic data for the natural effectors and the fluorinated substrate analogs considerable insight into the hydrogen bonding ability of the C-F fragment and the nature of carbohydrate-protein interactions should be gained. Little is known about the active (R-state) conformation of the enzyme and about the T-state to R-state transition. Use of fluorinated analogs of the enzymes natural substrate, glucose-l-phosphate, could also shed light on these questions. With these aims in mind, all of the isomeric mono-fluorinated derivatives of glucose and glucose-l-phosphate have been synthesised. Some deoxy and difluorinated analogs of glucose and mannose have also been prepared. Kinetic results obtained using the analogs of glucose indicate that the 3 and 6 positions of the sugar participate in strong hydrogen-bonding interactions with the protein while the other positions are only involved in relatively weak interactions. These results agree well with recent X-ray crystallographic data. None of the analogs of glucose-l-phosphate exhibited any substrate activity. The 2-deoxyfluoro analog had a similar affinity to glucose-1-phosphate and therefore probably binds in the same mode. The lack of substrate activity in this case can be explained by the destabi1isation of the putative oxo-carbonium ion intermediate at C(l), by the adjacent fluorine substituent. The other analogs of glucose-l-phosphate showed lower affinity for the enzyme. The similar inhibition constants obtained for these compounds suggested a binding mode in which the glucopyranose ring contributes little to the overall binding energy. This has led to the proposal of a molecular mechanism for the T-state to R-state transition. / Science, Faculty of / Chemistry, Department of / Graduate

This study examined the effects of a sports drink on the performance of young soccer players. Ten competitive young male soccer players, ages 10 and 11, performed two experimental trials while consuming 32 ounces of either a sports drink (G)- Gatorade or a placebo (P)- Crystal Light in a double-blind, crossover design. Both trials consisted of a 15-minute warm-up period, a pre and post exercise test protocol and a 40-minute indoor scrimmage with a five-minute half time. The assigned fluid was consumed just prior to the warm-up, pre-test protocol, scrimmage and post-test protocol as well as during the half time of the scrimmage. The exercise tests included six activities such as shooting velocity, dribbling, passing, jumping, backward running, and sprinting in order to measure skill, agility, power, and speed. The results showed that due to the interaction of the 40-minute scrimmage and the consumption of Gatorade, the post-test shot velocity measurement was significantly (p<0.01) lower for P while G remained similar to the pre-test measurement. Also, there was a significant (p<0.05) decrease in the number of jumps completed for both P and G during the post-test jumping exercise when compared to the pre-test measurement. However, there were no significant difference of treatment, time and/or their interaction for the dribbling, passing, backward running, and sprinting. Many possible reasons may account for this lack of effect. 1) Muscle glycogen may not have been substantially depleted, possibly because the prescribed exercise during the trial was not intense or long enough. 2) Prior to the experimental trials, muscle glycogen stores were sufficient where no additional CHO was necessary (due to the subject's diet on the day of the trial or the short fast prior to the experimental trials). 3) Alternative mechanism, such as increased lactate production or dehydration and not muscle glycogen depletion, may be the cause of impaired skills. 4) A child's increased fat utilization allows for less of a need for manipulation of glycogen stores. / Master of Science

The existence of a glycogen-sarcoplasmic reticulum has been demonstrated by a number of researchers. This complex is suspected to participate in the calcium uptake activities of the sarcoplasmic reticulum (SR). Removal of glycogen particles associated with this complex may alter the calcium handling abilities of the SR. This experiment sought to determine what effect exercise and treatment with a-amylase had on the abilities of the SR to regulate intracellular calcium ([Ca2+]i). Rats were either run on a treadmill for 60 min at a speed of 21 m/min and a 10% grade or were not exercised. Animals were then killed by decapitation after inhalation of CO2. Left and right gastrocnemius muscles were excised from both groups and underwent SR vesicle preparation to separate the heavy and light SR fractions (HSR and LSR respectively). Left hindlimb muscle homogenate also underwent 60 min incubation with a-amylase to digest glycogen before differential centrifugation. Treatment with a-amylase significantly depressed rate of calcium uptake by LSR and HSR fractions by 22.89% and 25.22% respectively (p<0.05). alpha-Amylase had no effect on SR's rate of calcium release. There was no effect of exercise on calcium uptake or release rates. Glycogen concentration associated with the SR was unaffected by either alpha-amylase treatment or exercise. These results indicate that treatment with alpha-amylase decreases the ability of the SR to sequester calcium ions. / Master of Science

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