Regulation of skeletal muscle phosphorylase phosphatase activity. I. Kinetic properties of the active and inactive forms.

Abstract 1. 1. The inactivation of phosphorylase a phosphatase decreased the maximum velocity of the phosphorylase a to phosphorylase b conversion reaction when it was assayed at different phosphorylase a concectrations. 2. 2. Maximal phosphorylase a phosphatase activities were found between pH 8 and 8.3. Inactivation of the phosphorylase a phosphatase led to a decrease in the activity in all the pH ranges tested. 3. 3. Theophylline and caffeine stimulated the phosphorylase a phosphatase. The effect of these substances was exerted in the reaction assay of the enzyme. 4. 4. ATP, ADP, AMP, GTP, UTP, CTP and pyrophosphate were found to decrease the rate of the reaction catalyzed by phosphorylase a phosphatase. This effect showed a striking parallelism with the capacity of these compounds to stimulate the phosphatase inactivation.

In this reaction the phosphate, bound to a seryl residue in each phosphorylase subunit, is hydrolyzed 1-3.
Evidence obtained in experiments carried out in vivo and in vitro indicates that the product of the phosphorylase a phosphatase reaction, i.e. phosphorylase b, is almost inactive at the metabolite concentrations measured in the muscle cells4, 5. Under these conditions, it could be expected that a stimulation in the rate of phosphorylase a to b conversion through an increase in the phosphatase activity could reduce the rate of phosphorolysis of glycogen.
The regulation of glycogen metabolism in mammalian cells has been studied in some detail. It seems that phosphorylase b kinase and glycogen synthetase are the two enzymes of the glycogen-metabolizing system subject to hormonal control. This supposition is supported by evidence from this and other laboratories 6-1°. Another kind of regulation was, however, demonstrated by RILEY and co-workersn, 1~ in adrenal cortex. In this tissue, they found a control step at the level of phosphorylase a phosphatase activation.
In a preliminary report, evidence was given indicating that muscle phosphorylase phosphatase exists in two interconvertible forms 13. In the present paper, experiments are reported which were designed to find the kinetic properties that differentiate each form of the phosphatase.

EXPERIMENTAL PROCEDURES
Phosphorylase phosphatase activity was assayed using 32P-labeled rabbitmuscle phosphorylase a as substrate, following the liberation of trichloroacetic acidsoluble radioactive phosphate. The labeled phosphorylase was prepared by enzymatic phosphorylation. A mixture containing I ml of crystalline rabbit-muscle phosphorylase b (60 ooo Cori units), o.o15 ml of I M mercaptoethanol, o.2 ml of 1 M Tris-HC1 buffer (pH 8.6), o.I ml of I M magnesium acetate, 1.2 ml of a2P-labeled ATP (5 "lO9 counts/min, specific activity I mC/mmole), and o.2 ml of rabbit-muscle phosphorylase b kinase (11. 4 mg protein per ml) was incubated for 2o min at 3 o°. The reaction was stopped by the addition of 3 ml of a solution containing IOO mM NaF, 8o mM glycerophosphate buffer (pH 6.8), 4 ° mM mercaptoethanol, and 2o mM EDTA. After the addition ofo.o 5 ml of IOO mM ATP and o.o5 ml of i M phosphate buffer (pH 7.o), the mixture was cooled in ice for 6o min. The crystalline labeled phosphorylase a was collected by centrifugation at IO ooo × g for 15 min. The supernatant was discarded and the precipitate was resuspended in IO ml of a solution containing 5o mM NaF, 4 ° mM glycerophosphate buffer (pH 6.8), IO mM EDTA, and 20 mM mercaptoethanol plus o.o5 ml of IOO mM ATP and o.o5 ml of I M phosphate buffer (pH 7.o). The mixture was shaken at 37 ° until the phosphorylase was dissolved and then it was left to crystallize at o ° for 6o min. After 5-8 crystallizations the precipitate of crystalline labeled phosphorylase a was resuspended in 5 ml of a solution containing 5 mM glycerophosphate buffer (pH 6.5), IO mM mercaptoethanol, and i nlM EDTA plus ATP and phosphate buffer as indicated above, and dialyzed against the same solution except that ATP and phosphate buffer were omitted. After 6-16 h more ATP and phosphate buffer were added into the dialysis hag and the dialysis medium was renewed. The addition of ATP and the phosphate buffer and the change of the dialysis medium were repeated 3-5 times. Finally, the enzyme was dialyzed against the same solution, omitting the addition of ATP and phosphate buffer to the dialysis bag. In a typical preparation, 3 ml of labeled phosphorylase a were obtained with the following characteristics: enzymatic activity, 14 ooo Cori units/ml; specific activity, 5000 counts/min per Cori unit; ratio of bound to unbound radioactive phosphate (5% Biochim. Biophys. Acta, 198 (I97 o) 495-503 trichloroacetic acid-insoluble radioactivity/5% trichloroacetic acid-soluble radioactivity), 138.
3~P-Labeled ATP was prepared according to LOWENSTEIN 14 with some modifications. Ten/,moles of ADP and IO mC of carrier-free radioactive phosphate were converted into the pyridinium salt by passing through a 0.8 × Io-cm Dowex 5o column (X4, 20-50 mesh, H + form) and adding pyridine in excess to the percolate. The mixture was dried in a rotatory evaporator and mixed with o.45 ml of pyridine, 0.05 ml of water and ioo mg of dicyclohexylcarbodiimide in a heavy-wail glass centrifuge tube. After addition of a glass bead (3-mm diameter), the tube was plugged with a rubber stopper covered with aluminum foil and was shaken for 16 h at room temperature in a Griffin flask shaker. The reaction was stopped by the addition of IO ml of water, and the mixture was filtered through a funnel plugged with cotton. The filtrate was concentrated on a rotatory evaporator and was then spread in a Io-cm band on washed Whatman 3 MM paper. Chromatography was carried out on ethanol-ammonium acetate at pH 7.2 (ref. 15) for 20 h. The ammonium acetate was washed out with absolute ethanol and the radioactive band which had a mobility like that of ATP was eluted with water. Usually, 60% of the radioactive phosphate was recovered as ATP.
The rabbit-muscle acid precipitate used as the source of phosphorylase b kinase was prepared as previously indicated TM. Crystalline rabbit-muscle phosphorylase b was prepared as described by FISCHER AND KREBSlL The mixture for the assay of phosphorylase phosphatase contained 0.02 ml of enzyme sample (diluted in IO mM mercaptoethanol, 5 mM EDTA, and 4 ° mM glycerophosphate buffer of pH 6.8) and o.oi ml of 32P-labeled phosphorylase a (48 Cori units ; lOOO-3OOO counts/min per Cori unit). Incubations were carried out at 30 ° for 5 rain. Reactions were stopped by the addition of I ml of 5% trichloroacetic acid. After centrifugation at 3000 rev./min in a clinical centrifuge for 30 min, the supernatant was transferred to a glass vial containing 0.04 ml of 60% KOH. After the addition of 5 ml of BRAY solution TM the radioactivity was measured in a liquid scintillation spectrometer. The radioactive phosphate liberated during the incubation was expressed in terms of consumed phosphorylase a according to the following equation: liberated counts/rain Pa pmoles of phosphorylase a consumed per unit of time = × -initial counts/min t. k t, incubation time (5 min); P a, initial phosphorylase a activity in the assay mixture (48 units) ; k, Cori units per pmole of phosphorylase a (0.93) ; this value was estimated assuming a specific activity of the phosphorylase a of 2515 Cori units/mg (ref. 19) and a molecular weight of 37 ° ooo (ref. 20). Pigeon breast muscle phosphorylase phosphatase was prepared as described in the following paper 21.

Conversion of 32P-labeled phosphorylase a to phosphorylase b
The conversion of phosphorylase a to phosphorylase b may be followed either by the disappearance of phosphorylase a activity or by the release of radioactive inorganic phosphate. As can be seen in  radioactive phosphate liberated and the decrease of phosphorylase a activity in the standard assay. Fig. 2 shows that the release of radioactivity from the 32P-labeled phosphorylase a is proportional to the incubation time and to the concentration of the enzyme.
Properties of the 32P-labeled phosphorylase a to phosphorylase b conversion Substrate dependence Fig. 3 shows the reciprocal plots of the initial velocities of the reaction catalyzed by muscle phosphorylase phosphatase, against 32p-labeled phosphorylase a concentration. The inactivation of phosphorylase phosphatase decreased the maximum velocity; no appreciable change in the apparent Km for 32P-labeled phosphorylase a was observed (Curves a and b). At pH 6.8, in the presence of 0.45 mM theophylline, the value for the apparent Michaelis constant was 0.25 • lO -6 M of n~P-labeled phosphorylase a (230 Cori units/ml). When the inactivated phosphatase was reactivated, an enzymatic preparation was obtained which behaved as the initial partially active phosphatase (Curve c).

pH curves
The pH-dependence of the phosphatase reaction is shown in Fig. 4. In the presence of 0.45 mM theophylline, no major differences were evident between an active phosphatase preparation and an inactivated-reactivated enzyme. Maximum activities were found between pH 8.0 and 8.3 (Curves a and b) Fig. 3. Effect of phosphorylase a concentration on the activity of muscle phosphorylase phosphatase.
In the experiments corresponding to Curves a, d and e, the enzyme was a crude preparation obtained from a homogenate that was not incubated at 37 ° (active enzyme). In Curve b the enzyme was a crude preparation obtained from a homogenate treated for 4 ° min at 37 ° (inactive enzyme). This enzyme preparation (0.25 ml) was further incubated with Io mM mercaptoethanol, 6. 7 mM theophylline,-2. 5 mM ATP-MgCI2, and 5 mM phosphocreatine-MgC12 for 5 min at 37 ° in a total volume of 0. 3 ml. The reaction was stopped by the addition of 2. 7 ml of a solution containing 4 ° mM glycerophosphate buffer (pH 6.8), 5 mM EDTA and io mM mercaptoethano] (reactivated enzyme). Curve c represents the experiment carried out with that enzyme. Before the assay all the enzymatic preparations were diluted in the glycerophosphate-EDTA-mercaptoethanol solution, and aliquots of these dilutions were again passed through Sephadex G-25 columns equilibrated with io mM NaC1 containing 5 mM EDTA and IO mM mercaptoethanol. After that, the pH of the samples was adjusted to 6.8. Incubations for phosphatase activity were carried out as indicated under EXPERIMENTAL PROCEDURES except that the total volume was 0.  phosphatase led to a decrease in the activity at all the pH ranges tested (Curve c).

Effect of theophylline
In order to inhibit cyclic phosphodiesterase activity in the phosphorylase phosphatase preparation, theophylline was added as a standard component of the activation and inactivation reaction mixtures. It can be seen in Fig. 5 of this paper and Table I (Expts. I and II) of the following one 21, that theophylline stimulated the phosphatase and that the effect of this substance was exerted in the reaction assay of the enzyme.
Some insight into the nature of the phosphatase activation by theophylline was obtained by comparing the activity of the enzyme measured at different concentrations of s2P-labeled phosphorylase a in the presence and absence of theophylline. Fig. 3 (Curves a and d) shows that theophylline increases both the maximum velocity and the apparent Michaelis constant of the enzyme for 32p-labeled phosphorylase a in such a manner that the reciprocal plots of the experiments carried out without the modifier were roughly parallel. On the other hand, activation by theophylline was evident at all the pH's tested in the range between 6.1 and 8.6 (Fig. 4, Curves a and d). Activation of the phosphatase was also observed with caffeine. As can be seen in Fig. 6 both methylxanthines stimulated the enzyme to a similar extent. The optimum effect was found at IO raM.

Effect of A TP and other modifiers
Several substances were found to decrease the rate of the reaction catalyzed by phosphorylase phosphatase. At 5 mM ATP, ADP, AMP, GTP, UTP, CTP and pyrophosphate were all effective. Pi, carbamyl phosphate and creatine phosphate also decreased the rate of the reaction but only slightly (Table I).
The effect of ATP was studied in more detail. As can be seen in Fig. 3 (Curve e), in the presence of 0.45 mM theophylline, the nucleotide decreases the maximum velocity of the reaction, when the enzyme was assayed at high concentrations of 32P-labeled phosphorylase a. No appreciable change in the apparent Michaelis constant for 32p-labeled phosphorylase a was observed. The ATP effect seemed to be independent of the degree of inactivation of the phosphatase preparation (Fig. 7 a) and increased with the concentration of this metabolite (Fig. 7b).
Some facts indicate the complex nature of the ATP effect. Indeed, the rate of the phosphatase reaction carried out in the presence of the metabolite declines sharply with the incubation time (Fig. 5). This suggests that in the presence of ATP and in the conditions of the assay, dilute preparations of phosphorylase phosphatase can be converted to the inactive form in a time-dependent reaction.
On the other hand the experiments shown in Table I and Fig. 7 of this paper and Table IV and Fig. 7 of the following one °~, clearly evidence a striking parallelism between the capacity of a given compound to stimulate the phosphatase inactivation and the corresponding capacity of the same compound to decrease the rate of the phosphatase reaction. A comparison between these two effects is shown in Fig. 8.   Table I  of this paper and Table IV of the following one ~1 were normalized and used in the construction of the curve. Both experiments were carried out with the same enzymatic preparation.

DISCUSSION
The main difference between the active and inactive forms of pigeon breast muscle phosphorylase phosphatase appears to be the maximal velocity measured at saturating levels of its substrate, phosphorylase a. No changes were found in the affinity for phosphorylase a after the activation or the inactivation of the phosphatase. If these conversions have some physiological significance in vivo, it seems evident that phosphatase regulation might operate through the change of the maximum velocity, since physiologically the enzyme appears to work at a concentration of phosphorylase a above the half saturation point. Indeed, pigeon breast muscle contains about 3000 Cori units per g of wet tissue, calculated as total phosphorylase activity, and the value for the apparent Michaelis constant for phosphorylase a (as substrate of the phosphatase) was about 230-380 Cori units/ml (0.25" lO-6-o.4 °. lO -6 M). This value is about io times lower than that reported by HuRD et al. 24 for a 2ooo-fold purified phosphorylase phosphatase preparation from rabbit muscle.
Several substances have been found to decrease the phosphatase-catalyzed reaction. An inhibitory effect of ATP and AMP on phosphatase activity was previously reported by HURD et al. ~4. The evidence obtained in this paper and in the following one indicates that the effect of these metabolites could be due to a timedependent inactivation of the enzyme during the assay of the phosphatase.
Another interesting property of skeletal muscle phosphorylase phosphatase is the activation by methylxanthines. The results obtained on this point corroborate the observation of WOSILAIT AND SUTHERLAND 2~ concerning the liver enzyme and these results expose the possibility that a metabolite modulation of phosphatase activity may play some role in the control in vivo of the phosphatase activity. However, efforts to demonstrate a metabolite activation of the phosphatase in the standard assay were unsuccessful. Under these conditions, the reported activation by glucose 23 and glucose 6-phosphate 24 could not be reproduced.