Fractionation of particulate glycogen and bound enzymes using high-performance liquid chromatography

During a study aimed at the specificity of binding of phosphorylase and glycogen synthase to glycogen particles we investigated the separation of these particles with the technique of high-performance liquid chromatography using several media. The technique allowed relatively rapid fractionations with little, if any, loss of enzyme activity. The basis of some separations was molecular weight of the particle. Of the molecular exclusion media used, Superose 6 had an exclusion limit large enough to accommodate most glycogens but excluded mouse liver and bovine liver glycogen particles. Toyopearl HW-75 F was a medium that could accommodate liver glycogen and gave us reasonably well-defined separation of the particles that carried phosphorylase and glycogen synthase. Separations on ion-exchange columns, that were obviously based on the overall charge of proteins associated with the glycogen particles, were not very effective in terms of allowing the separation of different types of particle. Of all the media used the hydrophobic interaction column was best in terms of separating what appeared to be distinct populations of particles that had definite preferences in terms of binding phosphorylase and glycogen synthase. As expected, these separations were based on the hydrophobic interaction of the proteins associated with the glycogen particles, since particles that had been deproteinized by a cold water extraction procedure did not bind to the column.     

Glucose 6-phosphate plays a central role in the regulation of glycogen synthesis in a glycogen-storing liver cell line

Two substrains of the epithelial liver cell line C1I, one storing large amounts of glycogen, the other one being very poor in glycogen were used as a model for studying glycogen synthesis. The glycogen content of glycogen-rich cells doubled during the proliferative phase and remained high in plateau phase although glycogen synthase I activity was not significantly altered during growth cycle and was too low to account for the increase in glycogen. However, the activity of the glucose 6-phosphate (Glc6-P)-dependent synthase rose continuously during growth cycle, and intracellular Glc6-P-concentration increased about 10-fold in log phase cells to 0.72 mumol g-1 wet weight. A0.5 of synthase for Glc6-P was 0.79 mM. It was also found that in contrast to the enzyme from normal liver, glycogen phosphorylase a from C1I cells was inhibited by Glc6-P, the apparent Ki being 0.45 mM. It was concluded that glycogen accumulation in C1I cells was due to stimulation of synthase and inhibition of phosphorylase by Glc6-P. Findings from the glycogen-poor cell line which revealed similar specific activities of synthase and phosphorylase but only low Glc6-P (0.056 mumol g-1 wet weight) supported this conclusion. Addition of glucose to starved cells resulted in a transient activation of synthase in both cell lines. Net glycogen synthesis, was, however, only observed in the cells with a high Glc6-P-content. Thus, modulation of synthase and phosphorylase by Glc6-P and not activation/inactivation of the enzymes seems to play a predominant role in glycogen accumulation in this cell line.     

Synthesis of glycogen from fructose in the presence of elevated levels of glycogen phosphorylase a in rat hepatocytes

Incubation of hepatocytes with glucose promoted the increase in the glycogen synthase (-glucose 6-phosphate/+glucose 6-phosphate) activity ratio, the decrease in the levels of phosphorylase a and a marked increase in the intracellular glycogen level. Incubation with fructose alone promoted the simultaneous activation of glycogen synthase and increase in the levels of phosphorylase a. Strikingly, glycogen deposition occurred in spite of the elevated levels of phosphorylase a. When glucose and fructose were added to the media the activation of glycogen synthase was always higher than when the hexoses were added separately. On the other hand the effects on glycogen phosphorylase were a function of the relative concentrations of both sugars. Inactivation of glycogen phosphorylase occurred when the fructose to glucose ratio was low while activation took place when the ratio was high. The simultaneous presence of glucose and fructose resulted, in all cases, in an enhancement in the deposition of glycogen. The effects described were not limited to fructose as D-glyceraldehyde, dihydroxyacetone, L-sorbose, D-tagatose and sorbitol, compounds metabolically related to fructose, provoked the same behaviour.     

Skeletal muscle glycogen content, structure, and metabolism are normal in rats with hepatic glycogen phosphorylase kinase deficiency

Skeletal muscle glycogen content and structure, and the activities of several enzymes of glycogen metabolism are reported for the hepatic glycogen phosphorylase b kinase deficient (gsd/gsd) rat. The skeletal muscle glycogen content of the fed gsd/gsd rat is 0.50 +/- 0.11% tissue wet weight, and after 40 hours of starvation this value is lowered 40% to 0.30 +/- 0.05% tissue wet weight. In contrast the gsd/gsd rat liver has an elevated glycogen content which remains high after starvation. The skeletal muscle phosphorylase b kinase, glycogen phosphorylase, glycogen synthase and acid alpha-glucosidase activities are 17.2 +/- 2.9 units/g tissue, 119.9 +/- 6.4 units/g tissue, 12.2 +/- 0.4 units/g tissue and 1.4 +/- 0.4 milliunits/g tissue, respectively, with approx. 20% of phosphorylase and approx. 24% of synthase in the active form (at rest). These enzyme activities resemble those of Wistar skeletal muscle, and again this contrasts with the situation in the liver where there are marked differences between the Wistar and the gsd/gsd rat. Fine structural analysis of the purified glycogen showed resemblance to other glycogens in branching pattern. Analysis of the molecular weight distribution of the purified glycogen indicated polydispersity with approx. 66% of the glycogen having a molecular weight of less than 250 X 10(6) daltons and approx. 25% greater than 500 X 10(6) daltons. This molecular weight distribution resembles those of purified Wistar liver and skeletal muscle glycogens and differs from that of the gsd/gsd liver glycogen which has an increased proportion of the low molecular weight material.(ABSTRACT TRUNCATED AT 250 WORDS)     

Glycogen synthesis by rat hepatocytes.

1. Hepatocytes from starved rats or fed rats whose glycogen content was previously depleted by phlorrhizin or by glucagon injections, form glycogen at rapid rates when incubated with 10mM-glucose, gluconeogenic precursors (lactate, glycerol, fructose etc.) and glutamine. There is a net synthesis of glucose and glycogen. 14C from all three types of substrate is incorporated into glycogen, but the incorporation from glucose represents exchange of carbon atoms, rather than net incorporation. 14C incorporation does not serve to measure net glycogen synthesis from any one substrate. 2. With glucose as sole substrate net glucose uptake and glycogen deposition commences at concentrations of about 12--15mM. Glycogen synthesis increases with glucose concentrations attaining maximal values at 50--60mM, when it is similar to that obtained in the presence of 10mM glucose and lactate plus glutamine. 3. The activities of the active (a) and total (a+b) forms of glycogen synthase and phosphorylase were monitored concomitant with glycogen synthesis. Total synthase was not constant during a 1 h incubation period. Total and active synthase activity increased in parallel with glycogen synthesis. 4. Glycogen phosphorylase was assayed in two directions, by conversion of glycose 1-phosphate into glycogen and by the phosphorylation of glycogen. Total phosphorylase was assyed in the presence of AMP or after conversion into the phosphorylated form by phosphorylase kinase. Results obtained by the various methods were compared. Although the rates measured by the procedures differ, the pattern of change during incubation was much the same. Total phosphorylase was not constant. 5. The amounts of active and total phosphorylase were highest in the washed cell pellet. Incubation in an oxygenated medium, with or without substrates, caused a prompt and pronounced decline in the assayed amounts of active and total enzyme. There was no correlation between phosphorylase activity and glycogen synthesis from gluconeogenic substrates. With fructose, active and total phosphorylase activities increased during glycogen syntheses. 6. In glycogen synthesis from glucose as sole substrate there was a decline in phosphorylase activities with increased glucose concentration and increased rates of glycogen deposition. The decrease was marked in cells from fed rats. 7. To determine whether phosphorolysis and glycogen synthesis occur concurrently, glycogen was prelabelled with [2-3H,1-14C]-galactose. During subsequent glycogen deposition there was no loss of activity from glycogen in spite of high amounts of assayable active phosphorylase.     

Identification of a glycogen synthase phosphatase from yeast Saccharomyces cerevisiae as protein phosphatase 2A.

A glycogen synthase phosphatase was purified from the yeast Saccharomyces cerevisiae. The purified yeast phosphatase displayed one major protein band which coincided with phosphatase activity on nondenaturing polyacrylamide gel electrophoresis. This phosphatase had a molecular mass of about 160,000 Da determined by gel filtration and was comprised of three subunits, termed A, B, and C. The subunit molecular weights estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis were 60,000 (A), 53,000 (B), and 37,000 (C), indicating that this yeast glycogen synthase phosphatase is a heterotrimer. On ethanol treatment, the enzyme was dissociated to an active species with a molecular weight of 37,000 estimated by gel filtration. The yeast phosphatase dephosphorylated yeast glycogen synthase, rabbit muscle glycogen phosphorylase, casein, and the alpha subunit of rabbit muscle phosphorylase kinase, was not sensitive to heat-stable protein phosphatase inhibitor 2, and was inhibited 90% by 1 nM okadaic acid. Dephosphorylation of glycogen synthase, phosphorylase, and phosphorylase kinase by this yeast enzyme could be stimulated by histone H1 and polylysines. Divalent cations (Mg2+ and Ca2+) and chelators (EDTA and EGTA) had no effect on dephosphorylation of glycogen synthase or phosphorylase while Mn2+ stimulated enzyme activity by approximately 50%. The specific activity and kinetics for phosphorylase resembled those of mammalian phosphatase 2A. An antibody against a synthetic peptide corresponding to the carboxyl terminus of the catalytic subunit of rabbit skeletal muscle protein phosphatase 2A reacted with subunit C of purified yeast phosphatase on immunoblots, whereas the analogous peptide antibody against phosphatase 1 did not. These data show that this yeast glycogen synthase phosphatase has structural and catalytic similarity to protein phosphatase 2A found in mammalian tissues.     

High-affinity binding of glycogen-synthase phosphatase to glycogen particles in the liver. Role of glycogen in the inhibition of synthase phosphatase by phosphorylase a.

1. Post-mitochondrial supernatants were prepared from the livers of 24 h-fasted rats. Upon centrifugation at high speed, the major part of the glycogen-synthase phosphatase activity sedimented with the microsomal fraction. However, two approaches showed that the enzyme was associated with residual glycogen rather than with vesicles of the endoplasmic reticulum. Indeed, the activity was entirely solubilized when the remaining glycogen was degraded either by glucagon treatment in vivo or by alpha-amylolysis in vitro. No evidence could be found for an association of glycogen-synthase phosphatase with the smooth endoplasmic reticulum, as isolated with the use of discontinuous sucrose gradients. 2. After solubilization by glucagon treatment in vivo, synthase phosphatase could be transferred to glycogen particles with very high affinity. Half-maximal binding occurred at a glycogen concentration of about 0.25 mg/ml, whereas glycogen synthase and phosphorylase required 1.5-2 mg/ml. 3. In gel-filtered extracts prepared from glycogen-depleted livers, the activation of glycogen synthase was not inhibited at all by phosphorylase alpha. The inhibition was restored when the liver homogenates were prepared in a glycogen-containing buffer. The effect was half-maximal at a glycogen concentration of about 0.25 mg/ml, and virtually complete at 1 mg/ml. These findings explain long-standing observations that in fasted animals the liver contains appreciable amounts of both synthase and phosphorylase in the active form.     

Methadone inhibition of glycogen synthase and phosphorylase in rat skeletal muscle

The activities of glycogen synthase (I and total) and phosphorylase ($\text{a}$ and total) in crude extracts of isolated extensor digitorum longus and soleus muscles of the rat incubated $\text{in vitro}$ in the absence or presence of methadone were very low. Addition of glycogen during homogenization increased the activities of both enzymes in control muscles. Even at optimal concentrations of glycogen, however, the activities of both enzymes from methadone-treated muscles were significantly lower than their activities in control muscles. The activity of phosphoglucomutase was not altered by incubation with methadone or by homogenization with glycogen. It is suggested that the addition of optimal amounts of glycogen during extraction of the enzymes enhances the extractability of glycogen synthase and increases the activity of phosphorylase by some other mechanism and that these processes are interfered with when the muscles are pretreated with methadone.     

The effects of fasting and refeeding on liver glycogen synthase and phosphorylase in obese and lean mice.

The responses of hepatic glycogen synthase and phosphorylase to fasting and refeeding were assessed as part of an investigation into possible sites of insulin resistance in gold thioglucose (GTG) obese mice. The active forms glycogen synthase and phosphorylase (synthase I and phosphorylase a) and the total activity of these enzymes were estimated in lean and GTG mice over 48 h of food deprivation, and for 120 min after glucose gavage (1 g/kg wt). In lean mice there was a maximal reduction in hepatic glycogen content after 12 h of starvation and the activity of phosphorylase a decreased from 23.8 +/- 1.9 to 6.8 +/- 0.7 mumol/g protein/min. These changes were accompanied by an increase in the activity of synthase I (from 0.14 +/- 0.01 to 0.46 +/- 0.04 mumol/g protein/min). In obese mice, similar changes in enzyme activity occurred after 48 h of starvation. These changes were accompanied by a significant reduction in the hyperinsulinemia and hyperglycemia of the GTG mice. After glucose gavage in both lean and obese mice, the activity of synthase I further increased over the first 30 min and declined thereafter. The activity of phosphorylase a increased progressively after refeeding. Results from this study suggest that despite increased hepatic glycogen deposition, the responses of glycogen synthase and phosphorylase, in livers of obese mice, to fasting and refeeding are similar to those of control mice even in the presence of insulin resistance.     

Isolation and characterization of cyclic AMP-independent glycogen synthase kinase from rat skeletal muscle

Glycogen synthase kinase was isolated from rat skeletal muscle. This kinase, which is cyclic nucleotide-independent and calcium-independent, was separated from phosphorylase kinase, cyclic AMP-dependent protein kinase and phosvitin kinase by phosphocellulose chromatography. Gel filtration on Sephadex G-100 resolved the glycogen synthase kinase into two fractions with apparent molecular weights of 68 000 (peak I) and 52 000 (peak II). This step also separated glycogen synthase kinase from the catalytic subunit of the cyclic AMP-dependent protein kinase, which had an apparent molecular weight of 39 000. Peak II glycogen synthase kinase activity was not affected by the addition of calcium, EGTA or a number of cyclic nucleotides. In addition to ATP, dATP would serve as the phosphate donor. Other trinucleotides tested were either poor or ineffective substrates. Activity was about 5-fold greater with Mg2+ than with Mn2+. Glycogen stimulated activity about 25%. Modifications of the methods of Soderling et al. ((1970) J. Biol. Chem. 245, 6317--6328) and Nimmo et al. ((1976) Eur. J. Biochem. 68, 21--30) were developed for purification of glycogen synthease (UDPglucose:glycogen 4-alpha D-glucosyltransferase, EC 2.4.1.11) to specific activity of 35 units/mg of protein. Using this preparation of glycogen synthase as substrate, the phosphorylation and inactivation catalyzed by glycogen synthase kinase was compared to that catalyzed by cyclic AMP-dependent protein kinase or phosphorylase kinase. Each of the kinases had different specificities for phosphorylation sites on glycogen synthase.     

On the activities of glycogen phosphorylase and glycogen synthase in the liver of the rat

A procedure was developed for determination of glycogen synthase and phosphorylase activities in liver after various in vivo physiological treatments. Liver samples were obtained from anaesthetised rats by freeze-clamping in situ. Other procedures were shown to stimulate the activity of phosphorylase and depress the activity of glycogen in the liver. The direction of glycogen metabolism appears to be regulated by the relative proportions of the two enzymes, as shown by a strong positive correlation between total activities and active forms of phosphorylase and synthase. The enzyme activities responded as expected to stimuli such as insulin and glucose, which depressed phosphorylase and increased synthase activity, and glucagon, which increased phosphorylase and decreased synthase activity. In fasted animals approximately 50% of each enzyme was in the active form, which suggests the existence of a potential futile cycle for glycogen metabolism. The role for such a cycle in the regulation of glycogen synthesis and degradation is discussed.     

On the activities of glycogen phosphorylase and glycogen synthase in the liver of the rat.

A procedure was developed for determination of glycogen synthase and phosphorylase activities in liver after various in vivo physiological treatments. Liver samples were obtained from anaesthetised rats by freeze-clamping in situ. Other procedures were shown to stimulate the activity of phosphorylase and depress the activity of glycogen in the liver. The direction of glycogen metabolism appears to be regulated by the relative proportions of the two enzymes, as shown by a strong positive correlation between total activities and active forms of phosphorylase and synthase. The enzyme activities responded as expected to stimuli such as insulin and glucose, which depressed phosphorylase and increased synthase activity, and glucagon, which increased phosphorylase and decreased synthase activity. In fasted animals approximately 50% of each enzyme was in the active form, which suggests the existence of a potential futile cycle for glycogen metabolism. The role for such a cycle in the regulation of glycogen synthesis and degradation is discussed.     

Stimulation of autophosphorylation of rabbit skeletal muscle phosphorylase kinase by glycogen synthase on glycogen particles

Glycogen synthase stimulated the autophosphorylation and autoactivation of phosphorylase kinase from rabbit skeletal muscle. This stimulation was additive to that by glycogen and the reaction was dependent on Ca2+. The effect by glycogen synthase was maximum within the activity ratio (the activity of enzyme without glucose-6-P divided by the activity with 10 mM glucose-6-P) of 0.3 and over 0.3 it was rather inhibitory. The results suggest that autophosphorylation of phosphorylase kinase in the presence of glycogen synthase on glycogen particles may be an important regulatory mechanism of glycogen metabolism in skeletal muscle.     

Effect of weaning on the activities of glycogen synthase and phosphorylase in rat liver

The effects of weaning on the level of glycogen and the activities of glycogen synthase and phosphorylase were determined in rat liver. Glycogen levels in rat liver increased at the start of the weaning period and reached a plateau on postnatal day 20. The active form of glycogen synthase increased until postnatal day 19 and then declined. Total glycogen synthase (active + inactive) remained high during the suckling period and declined to a new low level during the weaning period. The activity ratio (active/total) increased from day 16 to days 18-22 and then decreased to the same level as found during the suckling period. At the onset of weaning the active form of phosphorylase decreased, whereas total phosphorylase initially increased and then decreased after postnatal day 20. Both forms of phosphorylase increased again at the end of the weaning period. The activity ratio decreased at the start of weaning and remained low throughout the rest of the weaning period. The effects of premature weaning were similar to those observed in normally weaned animals, but the changes occurred sooner and were more pronounced.     

The glycogenic action of protein targeting to glycogen in hepatocytes involves multiple mechanisms including phosphorylase inactivation and glycogen synthase translocation

Expression of the glycogen-targeting protein PTG promotes glycogen synthase activation and glycogen storage in various cell types. In this study, we tested the contribution of phosphorylase inactivation to the glycogenic action of PTG in hepatocytes by using a selective inhibitor of phosphorylase (CP-91149) that causes dephosphorylation of phosphorylase a and sequential activation of glycogen synthase. Similar to CP-91194, graded expression of PTG caused a concentration-dependent inactivation of phosphorylase and activation of glycogen synthase. The latter was partially counter-acted by the expression of muscle phosphorylase and was not additive with the activation by CP-91149, indicating that it is in part secondary to the inactivation of phosphorylase. PTG expression caused greater stimulation of glycogen synthesis and translocation of glycogen synthase than CP-91149, and the translocation of synthase could not be explained by accumulation of glycogen, supporting an additional role for glycogen synthase translocation in the glycogenic action of PTG. The effects of PTG expression on glycogen synthase and glycogen synthesis were additive with the effects of glucokinase expression, confirming the complementary roles of depletion of phosphorylase a (a negative modulator) and elevated glucose 6-phosphate (a positive modulator) in potentiating the activation of glycogen synthase. PTG expression mimicked the inactivation of phosphorylase caused by high glucose and counteracted the activation caused by glucagon. The latter suggests a possible additional role for PTG on phosphorylase kinase inactivation.     

Effects of diabetes and glycogen on the distribution between soluble and particulate fractions of activities of enzymes involved in hepatic glycogen metabolism.

The large decreases in hepatic glycogen associated with alloxan diabetes in fed rats were accompanied by apparent decreases in total activities of glycogen synthase, phosphorylase, protein kinase and synthase phosphatase determined on 8000 × g supernatants of liver homogenates. Inclusion of 4% glycogen in the extraction buffer normalized total soluble activities of synthase in the diabetic. Whereas inclusion of 4% glycogen in the extraction buffer doubled total soluble phosphorylase, total activity remained lower in the diabetic than in the normal. Extraction and assay of soluble protein kinase were unaffected by added glycogen. When activities were determined on whole homogenates, total glycogen synthase activities were the same in normal and diabetic liver. Although the decreases in total activities of phosphorylase, kinase and phosphatase were less when determined on whole homogenates of livers from diabetic rats, the diabetes-related decreases in total activities remained significant. Therefore, it appears that while alloxan diabetes results in absolute decreases in total hepatic activities of phosphorylase, kinase and phosphatase, it may also result in redistribution of hepatic synthase and phosphorylase between soluble and particulate fractions, a phenomenon possibly related to tissue glycogen concentrations. Such a redistribution might be involved in the lack of control of hepatic glycogenesis observed in alloxan diabetic rats.     

Glycogen metabolism in the liver of the developing rat.

1. The total activity of glycogen synthease increased 20-fold from day 17 of gestation to birth at day 22, with a further increase of 18% in the 24h after birth. Active synthase (I) rose 45-fold to a maximum at day 21, fell 40% before birth, and then increased by a similar amount 24h after birth. The fraction of synthase in the active form correlated very well with the deposition of glycogen in the liver. 2. Total phosphorylase had a similar developmental pattern of total synthease with an 18-fold increase from day 17 to day 22. The appearance of active phosphorylase showed a lag-period compared with total phosphorylase and did not increase significantly until day 20. The fraction of phosphorylase in the active form did not correlate at all with glycogen deposition or mobilization. 3. There was a close relationshp between the ratio of phosphorylase a/synthase I and the glycogen content of the liver. An increase or decrease in this ratio would result in glycogenolysis of glycogenesis respectively. 4. It is postulated that a cycle between the two enzymes under basal conditions could exist which permits a continuous turnover of glycogen. Such a system would explain why active phosphorylase is always seen, even under conditions of net glycogen synthesis. The differences in hormone sensitivity of synthase and phosphorylase would also be accounted for as only one enzyme would have to respond acutely to hormonal influences.     

The effects of starvation and re-feeding on glycogen metabolism in mouse tail skin.

Although the glycogen content of mouse tail skin was decreased during starvation and was restored on re feeding, the proportion of glycogen synthase in the I form remained constant throughout at about 10% of the total. During the phase of net glycogen synthesis 1.5h after access to food was restored, the concentration of UDP glucose was markedly increased and the proportion of phosphorylase in the a form was significantly decreased.     

Role of glucose and insulin in regulating glycogen synthase and phosphorylase activities in rainbow trout hepatocytes

This study, using ¹³C nuclear magnetic resonance spectroscopy showed enrichment of glycogen carbon (C1) from ¹³C-labelled (C1) glucose indicating a direct pathway for glycogen synthesis from glucose in rainbow trout (Oncorhynchus mykiss) hepatocytes. There was a direct relationship between hepatocyte glycogen content and total glycogen synthase, total glycogen phosphorylase and glycogen phosphorylase a activities, whereas the relationship was inverse between glycogen content and % glycogen synthase a and glycogen synthase a/glycogen phosphorylase a ratio. Incubation of hepatocytes with glucose (3 or 10 mmol·1^-1) did not modify either glycogen synthase or glycogen phosphorylase activities. Insulin (porcine, 10^-8 mol·1^-1) in the medium significantly decreased total glycogen phosphorylase and glycogen phosphorylase a activities, but had no significant effect on glycogen synthase activities when compared to the controls (absence of insulin). In the presence of 10 mmol·1^-1 glucose, insulin increased % glycogen synthase a and decreased % glycogen phosphorylase a activities in trout hepatocytes. Also, the effect of insulin on the activities of % glycogen synthase a and glycogen synthase a/glycogen phosphorylase a ratio were more pronounced at low than at high hepatocyte glycogen content. The results indicate that in trout hepatocytes both the glycogen synthetic and breakdown pathways are active concurrently in vitro and any subtle alterations in the phosphorylase to synthase ratio may determine the hepatic glycogen content. Insulin plays an important role in the regulation of glycogen metabolism in rainbow trout hepatocytes. The effect of insulin on hepatocyte glycogen content may be under the control of several factors, including plasma glucose concentration and hepatocyte glycogen content.     

Interaction between glycogen and glycogen phosphorylase of Tetrahymena

The glycogen phosphorylase of Tetrahymena pyriformis complexes with glycogen as judged by its elution pattern from columns of Sepharose 6B. Complex formation does not occur with starch, amylose, or amylopectin, and neither do these polyglucans serve as primers for the enzyme. To study the association between the phosphorylase and glycogen particles in situ, Tetrahymena were grown under differing physiological conditions, phosphorylase was isolated and chromatographed on a Sepharose 6B column. Phosphorylase activity isolated from cells grown in the absence of glucose was only partially associated with glycogen, while in cells exposed to glucose for 30 min or more all the phosphorylase activity was associated with glycogen. The effects of culture age and anaerobiosis on the relative amounts of free and glycogen-bound enzyme in the cells were also studied. It was concluded from the in vivo experiments that there was no simple relation between the fraction of enzyme bound to glycogen and between cell glycogen content.