Pyrophosphate : fructose-6-phosphate phosphotransferase (PPi-PFK) has been purified 150-fold from potato tubers and the kinetic properties of the purified enzyme have been investigated both in the forward and the reverse direction. Saturation curves for fructose 6-phosphate and also for fructose 1,6-bisphosphate were sigmoidal whereas those for PPi and Pi were hyperbolic. In the presence of fructose 2,6-bisphosphate, the affinity for fructose 6-phosphate and for fructose 1,6-bisphosphate were greatly increased and the kinetics became Michaelian. The effect of fructose 2,6-bisphosphate was increased by the presence of fructose 6-phosphate and decreased by the presence of Pi. Consequently, the K, for fructose 2,6-bisphosphate was as low as 5 nM for the forward reaction and reached 150 n M for the reverse reaction. On the basis of these properties, a procedure allowing one to measure fructose 2,6-bisphosphate in amounts lower than a picomole, is described.

1. The properties of phosphorylase a, phosphorylase b, phosphorylase kinase and phosphorylase phosphatase present in a human haemolysate were investigated. The two forms of phosphorylase have the same affinity for glucose 1-phosphate but greatly differ in Vmax. Phosphorylase b is only partially stimulated by AMP, since, in the presence of the nucleotide, it is about tenfold less active than phosphorylase a. In a fresh human haemolysate phosphorylase is mostly in the b form; it is converted into phosphorylase a by incubation at 20degreesC, and this reaction is stimulated by glycogen and cyclic AMP. Once activated, the enzyme can be inactivated after filtration of the haemolysate on Sephadex G-25. This inactivation is stimulated by caffeine and glucose and inhibited by AMP and fluoride. The phosphorylase kinase present in the haemolysate can also be measured by the rate of activation of added muscle phosphorylase b, on addition of ATP and Mg2+. 2. The activity of phosphorylase kinase was measured in haemolysates obtained from a series of patients who had been classified as suffering from type VI glycogenosis. In nine patients, all boys, an almost complete deficiency of phosphorylase kinase was observed in the haemolysate and, when it could be assayed, in the liver. A residual activity, about 20% of normal, was found in the leucocyte fraction, whereas the enzyme activity was normal in the muscle. These patients suffer from the sex-linked phosphorylase kinase deficiency previously described by others. Two pairs of siblings, each time brother and sister, displayed a partial deficiency of phosphorylase kinase in the haemolysate and leucocytes and an almost complete deficiency in the liver. This is considered as being the autosomal form of phosphorylase kinase deficiency. Other patients were characterized by a low activity of total (a+b) phosphorylase and a normal or high activity of phosphorylase kinase in their haemolysate.

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The addition of glucose to a liver Sephadex filtrate causes a several fold increase in the rate of inactivation of phosphorylase by phosphorylase phosphatase ; this enzyme is also several times more active in liver preparations obtained from mice treated with glucocorticoids several hours before sacrifice. The glucose effect is less apparent in an d t e r e d liver extract because of the high concentration of glucose and the inhibitory effect of AMP.In the preceding paper [l], it was shown that the addition of glucose to a liver Sephadex filtrate greatly accelerated the activation of glycogen synthetase, presumably through the stimulation of an enzyme that converts an inactive or b form of synthetase phosphatase into an active or a form. Activation of the phosphatase is also markedly faster in a liver preparation from mice that have been treated with glucocorticoids.Considering the many similarities known to exist between the enzymatic systems that catalyze the interconversion of the two forms of glycogen phosphorylase and of glycogen synthetase in both liver and muscle, we have investigated the possibility that the activity of liver phosphorylase phosphatase might also be under the control of glucose and glucocorticoids. It is of interest to recall that stimulation of muscle phosphorylase phosphatase by glucose has been described by Holmes and Mansour [2].Part of this work has been presented in a symposium [3] and published in two preliminary notes [4,51. MATERIAL AND METHODSAll experiments, except that reported in Fig.4, were performed on mice liver extracts that had been passed through a Sephadex 6-25 column; the preparation of these filtrates, treatment of the animals and the source of chemicals were described in the preceding paper [I].Phosphorylase was measured [6] a t 20" in the absence ofAMP but in the presence of 0.25°/0 glucose. A partially purified preparation of phosphorylase and phosphorylase phosphatase was obtained by isolating particulate glycogen according to Luck [9]. The following procedure was used : rats that had been fasted for 2 days and subsequently fed for 16 h received an intraperitoneal injection of 0.1 mg of glucagon and were killed 15 min later. A 25Ol0 liver homogenate was prepared in a 0.1 M NaF-0.1 M glycylglycine solution, pH 7.4, and centrifuged a t 1000 x g for 10 min. Six ml of supernatant layered on top of 5 ml of a 2.1 M sucrose solution, containing 0.1 M NaF and 0.05 M glycylglycine buffer, pH 7.4, were spun for 90 min at 105000 x g in a no 40 rotor of a Spinco preparative ultracentrifuge ; the glycogen packed a t the bottom of the tube was used as a source of enzyme. The specific activity of phosphorylase was 35 times greater than in the homogenate. RESULTS Stimulation of Phosphorylme Phosphatme by GlucoseThe rate of inactivation of phosphorylase was conveniently studied in liver extracts that had been filtered through a Sephadex column. When these preparations were incubated at 20", the addition of 0.5O/, glucose consistently caused a 5-to 10-fold increase in the rate of ...

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The removal of glucose, AMP and other small molecules from mouse liver extract by filtration through Sephadex 6-25 has allowed us t o demonstrate that the activation in vitro of glycogen synthetase is much more rapidly attained in the presence of glucose and also when the animals have received prednholone 3 h before sacrifice. These effects are the result of a shortening of the lag period that precedes the activation of the synthetase; they are conveniently studied in filtrates that have been enriched with sulfate or phosphate ions. Like glucose, caffeine and nicotinamide shorten the lag period and are without action when this period is over; a-particulate glycogen has the opposite effect. No lag period is observed in the presence of AMP, particularly when associated with magnesium acetate. When a liver Sephadex filtrate is incubated in the absence of salts, glycogen synthetase b is converted into a partially active form that is rapidly transformed into synthetase a upon the addition of salt.These results support the hypothesis that glycogen synthetase phosphatase is initially present in the liver filtrate as a b enzyme, active only in the presence of AMP and magnesium; this b form is converted into its active, a, homologue through the action of a synthetase phosphatase activating enzyme, which is more active after a treatment with glucocorticoids, is stimulated by glucose, caffeine and nicotinamide and inhibited by glycogen, AMP and fluoride. I n the liver of an untreated animal, the enzyme i s predominantly in the b form [2,3]; it is activated within a few min after an intravenous load of glucose [4,6] or within 2 to 3 h after the administration of glucocorticoids [5-71. Once activated, the enzyme is rapidly reconverted into b after the injection of glucagon [5,8] epinephrine or cyclic AMP [5].The studies in Witro (9-111 support the hypothesis that the activation and the inactivation of the synthetase in liver occurs, as in muscle [12], by dephosphorylation and rephosphorylation, with a phosphatase and b a s e , respectively. The latter enzyme is stimulated by cyclic AMP and this effect adequately explains the inactivation of the synthetase under the action of glucagon or epinephrine. Up to now, it had not been possible to show unequivocally what part of the system was mod5ed by glucose and by glucocorticoids.Enzymes. Glycogen synthetase or UDPG: a-l,4-gluoan a-4-glucosyltransferase (EC 2.4.1.11) ; glycogen phosphorylaae or a-1,4-glucan: orthophosphate glucosyltransferese (EC 2.4.1.1) ; phosphorylase phosphatase or phosphorylase phosphohydrolaae (EC 3.1.3.17).

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1. The two forms of glycogen phosphorylase were purified from human liver, and some kinetic properties were examined in the direction of glycogen synthesis. The b form has a limited catalytic capacity, resembling that of the rabbit liver enzyme. It is characterized by a low affinity for glucose 1-phosphate, which is unaffected by AMP, and a low V, which becomes equal to that of the a form in the presence of the nucleotide. Lyotropic anions stimulate phosphorylase b and inhibit phosphorylase a by modifying the affinity for glucose 1-phosphate. Both enzyme forms are easily saturated with glycogen. 2. These kinetic properties have allowed us to design a simple assay method for total (a + b) phosphorylase in human liver. It requires only 0.5 mg of tissue, and its average efficiency is 90% when the enzyme is predominantly in the b form. 3. The assay of total phosphorylase allows the unequivocal diagnosis of hepatic glycogen-storage disease caused by phosphorylase deficiency. One patient with a complete deficiency is reported. 4. The assay of human liver phosphorylase a is based on the preferential inhibition of the b form by caffeine. The a form displays the same activity when measured by either of the two assays.