Daniel P. Gilboe

Radioactive method for the assay of glycogen phosphorylases

Abstract A new assay for glycogen phosphorylase (EC 2.4.1.1) is presented. This assay employs a filter paper technique in which newly formed glycogen-14C is precipitated on paper squares using 66% ethanol while other radioactivity is removed by washing. It is rapid and reproducible and may be employed with crude as well as purified enzyme preparations. An additional advantage of the method is the potential for increased sensitivity. It should find utility for assay of extremely small tissue samples and should be useful in kinetic studies.

Stimulation of liver glycogen particle synthase D phosphatase activity by caffeine, AMP, and glucose 6-phosphate

Abstract Caffeine stimulates synthase and phosphorylase phosphatase activities by independent mechanisms. Both effects of caffeine are concentration-dependent with different apparent A 0.5 for each reaction. Stimulation of the synthase phosphatase reaction was independent of the initial phosphorylase a concentration, was immediate, and did not follow in sequence the depletion of phosphorylase a . Glucose 6-phosphate also was stimulatory to the synthase phosphatase reaction ( A 0.5 = 0.14 mM) with little effect on phosphorylase phosphatase. In combination glucose 6-phosphate and caffeine effects were additive suggesting the existence of separate binding sites. The synthase phosphatase reaction also was stimulated by AMP (binding affinity 2.3 m m ) but with no effect on phosphorylase phosphatase activity. Together caffeine and AMP effects were not additive suggesting that they share a common binding site or closely interrelated sites. The location of the AMP and caffeine site(s) has not yet been determined.

The role of ATP and glucose 6-phosphate in the regulation of glycogen synthetase D phosphatase

Abstract Glycogen synthetase D phosphatase catalyzes the conversion of synthetase D to synthetase I. The phosphatase reaction has been found to be inhibited by ATP, ADP and UDP; however, only ATP inhibited at a physiological concentration. ATP inhibition was enhanced by glycogen. Glucose 6-phosphate (G 6-P) stimulated the phosphatase reaction and at least partially relieved the ATP inhibition. A possible physiological role for ATP, G 6-P and glycogen in the regulation of the synthetase D phosphatase reaction has been proposed.

The regulation of liver glycogen synthetase D phosphatase by ATP and glucose.

Abstract Synthetase D phosphatase activity in a liver glycogen pellet preparation is inhibited by ATP (physiological concentration). The inhibition can be reversed by glucose concentrations within the usual physiological range. Phosphophosphorylase activity decreases concomitantly with increasing synthetase I activity during the phosphatase incubation but the decrease is modest even in the presence of glucose. The glucose reversal of ATP inhibition is not the result of ATPase or glucokinase activities, which would reduce the ATP concentration. ATP, glucose and glucose 6-phosphate concentrations remain stable during the phosphatase assay.

Direct glucose stimulation of glycogen synthase phosphatase activity in a liver glycogen particle preparation

In glycogen particle suspensions prepared from fed rats given either glucagon or glucose in order to increase or decrease the phosphorylase a concentration, respectively, glucose stimulation of synthase phosphatase activity was observed. In preparations from glucagon-treated rats, addition of glucose stimulated synthase and phosphorylase phosphatase simultaneously and not sequentially. Synthase phosphatase stimulation was glucose concentration dependent even when phosphorylase a had been rapidly reduced to a low level. The estimated A0.5 for glucose stimulation of synthase phosphatase activity was 27 mM. An A0.5 for glucose stimulation of phosphorylase phosphatase activity could not be estimated since activity was still increasing with concentrations of glucose as high as 200 mM. In preparations from glucose-treated rats which contain virtually no phosphorylase a, glucose stimulation was still apparent but the A0.5 was increased modestly (36 mM). Stimulation of synthase phosphatase activity was specific for glucose. Several other monosaccharides and the polyhydric alcohol sorbitol were ineffective.

Influence of fructose on the glycogen synthase and phosphorylase systems in rat liver

Fructose and glucose, when administered as a single, large intravenous dose (500 mg/kg) produced opposite effects on key regulatory enzymes of glycogen metabolism in intact normal fed animals. Glucose rapidly stimulated glycogen synthase phosphatase activity and increased the proportion of glycogen synthase in the active (I) form as expected; fructose reduced synthase phosphatase activity and the proportion of synthase in the I form. Glucose also stimulated a reduction in the proportion of phosphorylase in the active (a) form, whereas fructose stimulated an increase in the proportion of phosphorylase in thea form. The effect of fructose was not mediated by an increase in cyclic adenylate (cAMP) concentration nor by a conversion of phosphorylase kinase b to phosphorylase kinase a. As expected, the concentration of ATP decreased significantly. The increase in proportion of phosphorylase in the a form may be due to stimulation of phosphorylase kinase b activity by a decrease in the intracellular ATP:Mg++ ratio or by increase in intracellular Ca++ concentration. The mechanism of the fructose-induced change in synthase phosphatase activity and in synthase I activity is unknown.

Binding of streptonigrin to DNA

Abstract 1. This is a report of investigations on the nature and the site of binding of streptonigrin to DNA and some of the factors which influence this interaction in vitro and in vivo . 2. There are at least two types of binding of streptonigrin to calf-thymus DNA in vitro : one, which is reversible by dialysis and the other, which is irreversible. The extent of stably bound streptonigrin to DNA was estimated to be 1 mole per 2000 moles deoxynucleotides. 3. More streptonigrin was bound to denatured DNA, apurinic acid and poly dC: poly dG than to native DNA. It was associated preferentially with the dCMP moiety of DNA. Reduction of streptonigrin was not found to be required for binding. 4. Alkaline sucrose gradient centrifugation showed evidence of single strand breaks in streptonigrin-treated DNA, and increased denatured state was indicated by hyperchromicity at 260 nm and increased buoyant density by isopycnic centrifugation in CsCl. 5. With synchronized tissue culture cells, streptonigrin was bound preferentially to DNA during the S (DNA synthesis) period of the cell cycle.

The mechanism of caffeine-enhanced glucose stimulation of liver glycogen synthase phosphatase activity

Our report that glucose within its physiological range stimulates glycogen synthase phosphatase activity, provided an appropriate second effector is present, has been expanded. The nature of the stimulatory process, particularly the roles of glucose, and of caffeine which represents the potential second effectors, has been studied. Glucose and caffeine stimulated synthase phosphatase activity in a synergistic manner. With 0.5 mM caffeine the A0.5 for glucose was 11 mM (from 27 mM), whereas in the presence of 30 mM glucose the A0.5 for caffeine was 0.06 mM (from 0.7 mM). At 10 mM glucose the A0.5 for caffeine was 0.1 mM. Glucose stimulation remained non-cooperative, unaffected by the presence of caffeine, whereas the cooperative stimulation of caffeine was unaffected by glucose. Some slight stimulation of synthase activity was observed with caffeine and with glucose over a wide concentration range. However, they did not act synergistically to influence the measurement of synthase activity. Glucose-6-phosphate, which also stimulates synthase phosphatase activity, acted independently, not synergistically with caffeine. All the methylxanthines were tested as potential second effectors in an effort to discover the essential structural elements of the agent. All dimethylxanthines, 3- and 7-methylxanthine and 1-methyl-3-isobutylxanthine enhanced glucose stimulation but none of them alone was stimulatory. Judged from the half-maximal concentrations, in the presence of 10 mM glucose, caffeine was the most potent second effector by a significant margin. The maximum velocity was also greatest with caffeine, whereas that with other methylxanthines was generally lower, and varied. 1-Methylxanthine with increased concentration was slightly inhibitory even in the presence of 10 mM glucose. Xanthine (0.5 mM), itself, strongly inhibited synthase phosphatase activity, an effect not influenced by glucose. Xanthine did not influence the measurement of synthase or phosphorylase phosphatase activity with or without glucose. In general, conditions of methylxanthine-enhanced, glucose stimulation of synthase phosphatase and phosphorylase phosphatase activities differed markedly, confirming that separate, distinct mechanisms are involved.

The effect of glucose on liver glycogen synthase phosphatase activity in the presence of ATP-Mg.

Glycogen particle synthase phosphatase activity is stimulated by glucose with an A0.5 of approximately 27 mM. The A0.5 is higher than the usual concentrations present in the liver. However, in vitro, certain methylxanthines such as caffeine or theophylline reduce the glucose A0.5 to approximately 10 mM, a concentration well within the normal range of liver glucose concentrations. Methylxanthines do not affect the maximum stimulation by glucose (2.3-fold greater than control rate). The phosphatase reaction also is inhibited by ATP-Mg (I0.5 = 0.1 mM). In the present studies, we have determined the interaction of these effectors. The presence of ATP-Mg at a concentration of 3 mM only slightly reduced the maximal stimulation by glucose. The A0.5 for glucose was unaffected (24 mM). The synergistic effect of caffeine with glucose also was not changed by the presence of ATP-Mg. The A0.5 for glucose was reduced to 11 mM, similar to that in the absence of ATP-Mg. In addition, maximum stimulation by glucose was unchanged. Similar results were obtained when theophylline replaced caffeine. We conclude that the ATP-Mg binding site on either the phosphatase or its substrate, synthase D, does not influence the glucose and methylxanthine binding sites. Effectively, ATP-Mg increased the range over which glucose stimulates the phosphatase activity. In the presence of ATP-Mg, the maximum stimulation by glucose is approximately 7-fold; whereas, in the absence of ATP-Mg it is approximately 2.3-fold. Thus, ATP-Mg may serve to increase the sensitivity of the synthase phosphatase reaction to glucose regulation under in vivo conditions.

Preparation of tissue extracts for glycogen phosphorylase assay

Abstract The conditions for preparing heart and skeletal muscle extracts for phosphorylase assay following homogenization and centrifugation have been investigated. It is concluded that extracts should be kept at room temperature to avoid cold inactivation of the phosphorylase b component of total phosphorylase activity. Prolonged delay in assaying total phosphorylase activity especially in dilute solution also results in the loss of phosphorylase b activity but not phosphorylase a activity. The accurate determination of phosphorylase a activity is dependent on an efficient means to reduce the AMP concentration. Although dilution of extracts several-fold has been effective in the past for this purpose, Dowex 1 treatment is recommended. Norit A, another agent used to remove AMP, is less satisfactory because it results in a loss of enzyme activity. Several buffers have been investigated and MES or maleate appear to give the best results.