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Some studies on enzymes involved in glycogen catabolism in nervous tissue Klier, Gail Dianne Bellward

Abstract

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.

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