PhD

dissertationPhosphorylase and the enzymes which regulate its activity occur as complexes with glycogen in liver and can therefore be considered as compartmentalized enzymes. A study of the metabolism of glycogen phosphorylase was undertaken to determine whether or not this association with glycogen was an important factor in establishing the phosphorylase concentration at equilibrium. We also wanted to investigate if regulating phosphorylase levels supplemented the hormonal control of the enzyme's activity. Homogeneous glycogen phosphorylase b, free of phosphorylase a contamination, was isolated from rabbit liver by differential sedimentation of the glycogen-phosphorylase complex, digestion of the glycogen with a-amylase, and fractionation of the resulting sample by consecutive n-butyl Sepharose and DEAE cellulose chromatographies, followed by isoelectric focusing. Yields approaching 50 percent and 1600-fold purification were obtained, with resulting specific activities that were significantly higher than those expressed by the enzyme isolated by previous methods. Isolation of the enzyme in the absence of glycogen was accomplished by ethanol fractionation, adsorption of contaminating protein on calcium phosphate gel, ammonium sulfate precipitation, and slight modifications of the Sepharose, DEAE cellulose, and isoelectric focusing purification steps. After demonstrating that the isolated phosphorylase was representative of the total cellular content, its synthesis and degradation in fed and fasted animals was studied using both radioactive tracers and direct enzyme assay to monitor the turnover. A pulse of labeled leucine or lysine was injected into each of three rabbits. Incorporation of the label into phosphorylase and liver aldolase was simultaneously monitored by isolating the enzymes in homogeneous form at various times after injection and calculating their normalized specific radioactivilies. Results showed that phosphorylase was a more stable molecule, turning over at one-half to one-third the rate of aldolase. Approximations of half lives yielded values of 2-3 days for phosphorylase and slightly less than one day for aldolase. An independent approach to phosphorylase turnover was made possible through changes observed in the total content of the enzyme during adjustments to differing dietary conditions. Phosphorylase levels were determined directly in whole-liver homogenates after conversion to the b form at intervals during fasting and subsequent refeeding. On fasting, the total phosphorylase content decreased 63 percent within about four days to a new steady-state level and, upon refeeding, returned at a significantly slower rate to its normal concentration. It was established by isotope dilution analysis that the changes in phosphorylase activity were due to changes in the amount of enzyme present and not to alterations in its enzymatic activity. Therefore, significant changes in the rate constants for phosphorylase metabolism had occurred as a result of the fast. Analysis of the time approaches to end from the two steady states established that phosphorylcse exhibited a 66--hour half life under normal dietary conditions and a 21-hour half life under fasting conditions. Ratios of normalized specific radioactivities shortly after a labeled pulse confirmed the half lives, yielding a 2.6 ratio between the degradative rate constants for phosphorylase under fasting and normal! dietary conditions. Furthermore, the synthetic rate constant for phosphorylase was shown to be equivalent in each of the two steady states. The observed changes in the concentration of phosphorylase during fasting and refeeding appear, therefore, to be due only to variations in the rate of degradation. This decreased stability of phosphorylase from fasted rabbits was also demonstrated in liver extracts at 37 ° C. Since glycogen is depleted within one day after food withdrawal, it seems probable that stabilization by glycogen is mainly responsible for the lower rate of phosphorylase degradation under normal dietary conditions. A lag period equal to the time needed for glycogen depletion, occurring between the start of the fasting interval and the beginning of the phosphorylase decline, strongly implicates glycogen as an important factor in the observed phenomenon