Search

Perfusion of dog liver in situ with NaF or incubation of liver extracts with NaF resulted in inactivation of phosphorylase phosphatase (phosphorylase phosphohydrolase, EC 3.1.3.17). In vitro this process was found to be dependent on time, temperature and concentration of fluoride. Reactivation could be obtained with ATP in the presence of Mg 2+ . These interconversions were of a stable character. This inactivation of phosphorylase phosphatase is unrelated to the well known inhibition of the enzyme by NaF. The inactive forms isolated from livers perfused with either NaF or NaCl displayed identical properties, as judged from their activation by ATP and Mg 2+ .

Purification of the phospho-protein phosphatase of skeletal muscle which promotes the conversion of the phospho- (D or b) form to the dephospho- (I or a) form of glycogen synthetase (UDPG:glycogen α-4-glucosyltransferase, EC 2.4.I.II) was aided by the use of buffers which include manganese chloride. The Mn2+ (5 mm) appeared to stabilize the enzyme and to insure greater recovery from DEAE-cellulose chromatography than reported previously. The enzyme was purified more than 1,000-fold from the 78,000 x g supernatant of rabbit muscle homogenate. It was essentially free of phosphorylase and synthetase. Phosphatase activity was measured by two methods: (a) as the rate of conversion of glycogen synthetase-D to the I form (D to I conversion) and (b) as the rate of orthophosphate release from 32P-labeled synthetase-D (32Pi release). Inhibition of the phosphatase reaction (more than 50%) was found in the presence of F- (10 mm), Na2SO3 (1 mm), and Pi or PPi (0.2 mm). More than a 2-fold increase in phosphatase activity was found in the presence of divalent metal cations: Mn2+ g Ca2+ g Mg2+ (half-maximal concentration was 0.6 to 1.2 mm). Glucose-6-P (up to 1 mm) had no effect on the rate of 32Pi release, but increased about 2-fold the rate of D to I conversion (at 0.1 mm), as did galactose-6-P and glucosamine-6-P to a lesser extent. A glycogen concentration of 0.5 to 1.5 mg per ml was found to be optimal compared to the slower reaction rates found with greater or lesser concentrations. A histone phosphatase activity found in this preparation, measured using 32P-labeled histone phosphate as substrate, had properties similar to and was copurified with glycogen synthetase-D phosphatase. However, the above carbohydrate-type effectors of synthetase and synthetase-D phosphatase reactions were without effect on the rate of histone dephosphorylation. These findings suggest that these two phosphatase activities may reside in the same enzyme protein.

Niridazole was administered to 57 rhesus monkeys at a dosage schedule approximating the minimally curative dose for schistosomiasis mansoni of this species. This resulted in a marked depletion of the muscle glycogen stores, which was attributed in part to a reduced food intake due to anorexia produced by the drug and in part to an inhibition of phosphorylase phosphatase, activity bringing about an accumulation of active glycogen phosphorylase. Restoration of muscle glycogen levels to control values was observed about 8 days after discontinuation of drug administration.

Brain glycogen phosphorylase has been shown to exist in an inactive and an active form. The inactive enzyme shows no activity in the absence of AMP but is maximally active in the presence of this nucleotide. The inactive enzyme has been purified 100-fold by adsorption on starch, followed by elution of other protein and finally elution of the enzyme with glycogen solution. The presence of phosphorylase phosphatase and phosphorylase kinase in brain is also established.

The administration of glucose to anesthetized fed rats causes within 1 to 2 minutes the inactivation of phosphorylase; glycogen synthetase is activated only when and if the level of phosphorylase α has been taken down below a threshold value equal to approximately 10% of the total phosphorylase. The same conclusion has been reached in a study with anesthetized fed mice. These observations are adequately explained by the previously described stimulation of the phosphorylase phosphatase reaction by glucose and inhibition of synthetase phosphatase by phosphorylase α. In some experiments, insulin caused a partial inactivation of liver phosphorylase in the liver of normal rats, but more often was without effect. When effective in the diabetic animal, insulin produced the inactivation of glycogen phosphorylase and the activation of glycogen synthetase. Glucocorticoids displayed two effects: a greater activity of phosphorylase phosphatase and a decreased inhibition of synthetase phosphatase by phosphorylase α.

The gamma1-isoform of protein phosphatase-1 expressed in Escherichia coli (PP1gamma) and the native PP1 catalytic subunit (PP1C) isolated from skeletal muscle dephosphorylated Ser-14 of glycogen phosphorylase at comparable rates. In contrast, PP1gamma dephosphorylated several tyrosine-phosphorylated proteins at similar rates to authentic protein tyrosine phosphatases (PTPases), but native PP1C was almost inactive towards these substrates. The phosphorylase phosphatase (PhP) and PTPase activities of PP1gamma were inhibited by vanadate with IC50 values (30-100 microM) comparable to authentic PTPases, whereas the PhP activity of native PP1C was insensitive to vanadate. PP1gamma lost its PTPase activity, and its PhP activity became insensitive to vanadate, after interaction with inhibitor-2, followed by the reversible phosphorylation of inhibitor-2 at Thr-72. These findings support and extend the hypothesis that inhibitor-2 functions like a chaperone to fold PP1 into its native conformation, and suggest that the correct folding of PP1 may be critical to prevent the uncontrolled dephosphorylation of cellular phosphotyrosine residues.

Proteins of 60–70 kDa copurify with some preparations of type-1 or type-2 phosphatases. In our system chromatography on polylysine-Affi-Gel 10 separates a 68 kDa protein from rabbit muscle glycogen particle phosphorylase phosphatase. The separation affects neither the activity nor the size of the phosphatase. The 68 kDa protein, although pure by SDS gel electrophoresis criteria, still displays phosphatase activity of approx. 6–8 Umg. However, rechromatography either on Bio-Gel A-0.5 m or on Blue Sepharose CL-6B followed by gel filtration shows that the activity is due to a contamination with phosphatases of type 1 and type 2, displaying a molecular mass of 35 kDa, which can be totally removed from the 68 kDa protein. The amino acid composition of the 68 kDa protein is identical to that of rabbit serum albumin, within the limits of variation of the method. Furthermore, the sequence of the 38 N-terminal amino acids is the same in the isolated 68 kDa protein and in rabbit serum albumin.

Different enzymes, related to the glycogen metabolism have been reported to be associated in the rabbit skeletal muscle within a ‘glycogen particle’ [l-5]. In this purified particle one can detect the presence of glycogen phosphorylase [ 1,351, phosphorylase kinase [ 1,5] , phosphorylase phosphatase [l] , amylase [l] , myokinase [l] , adenylic deaminase [ 11, the enzymes of the glycolytic pathway [l] , g$xgen synthetase [3,5] and amylo I-6-glucosidase

The dephosphorylation of the modulator subunit is an essential step in the kinase FA-mediated activation of the ATP,Mg-dependent protein phosphatase. Mg2+ is implicated in this autocatalytic dephosphorylation which is not effected by the addition of phosphoinhibitor-1. Dephosphorylation of free modulator by the catalytic subunit is also largely Mg2+-dependent but can be abolished by phosphoinhibitor-1 in concentrations comparable to the amount of modulator used as substrate (micromolar). The phosphorylase phosphatase activity of the catalytic subunit is inhibited by nanomolar concentrations of phosphoinhibitor-1 and is completely independent of divalent cations.

Phosphorylase phosphatase from rabbit skeletal muscle, when purified 700-fold, dephosphorylated the 22,000 molecular weight subunit of troponin (TNI). The endogenous phosphate in TNI and that incorporated by phosphorylase kinase were both totally removed at identical rates. Five criteria were established to show that dephosphorylation of TNI was catalyzed by phosphorylase phosphatase and not a contaminant in the preparation: (a) co-purification of TNIand phosphorylase-phosphatase activities; (b) identical patterns of the activities on polyacrylamide gel electrophoresis; (c) parallel heat denaturation and (d) fluoride inhibition of the activities; (e) competitive effects when both TNI and phosphorylase a were present. The Km for phosphorylase a was 6.8 µm, and 21.2 µm for TNI. The Vmax with TNI was approximately 4-fold greater than with phosphorylase a.

The distribution df phosphorylase, phosphatase, and β-amylase in fruits, vegetables (except root vegetables), and some leaves was examined. Fruits contained little or no enzyme activities. But chestnut and gingko, which contained large amounts of strch were rich in phosphorylase activities. Almost all vegetables and leaves, except onion, showed more or less phosphorylase activities and little or no β-amylase activities, which suggested that phosphorylase might take part in the formation of assimilation starch as well as reserve starch in plants.

Phosphorylase phosphatase in crude tissue extracts of rat liver, skeletal and heart muscle can be markedly activated by a treatment involving precipitation with ammonium sulfate and ethanol. The activation of the enzyme in rat liver extracts is shown to occur with the concomitant conversion of the enzyme from multiple molecular weight forms to a single form of lower molecular weight (M.W.∼30,000). After activation by ethanol treatment, phosphorylase phosphatase activities of rat liver, skeletal and heart muscle were shown by sucrose density ultracentrifugation to sediment in a similar manner with a sedimentation coefficient of 2.8S and M.W. 32,000.

Neural and humoral induction of hepatic glycogenolysis occurs via different pathways. Sympathetic nerve stimulation activates alpha adrenergic receptors to produce what is probably a cyclic AMP independent, Ca++ dependent activation of glycogen phosphorylase. Evidence suggests that phosphorylase 'a' is activated by inhibition of phosphorylase phosphatase and perhaps by activation of phosphorylase kinase. Blood borne catecholamines act via beta adrenergic receptors to produce a cyclic AMP dependent activation of phosphorylase kinase. Additionally, hepatic parasympathetic nerves can produce a dramatic shut-down of hepatic glucose output by activation of glycogen synthetase and inhibition of glycogen phosphorylase. The observed minor effect of insulin or intravenous glucose on hepatic glucose uptake compared to the effect of oral doses of glucose suggests that an additional factor other than insulin can produce hepatic glucose uptake: this factor could be, and probably is, the hepatic parasympathetic nerves. The present hypotheses result from a combination and extrapolation of whole animal physiological and in vitro biochemical data.

Phosphorylase phosphatase purified from the protein-glycogen complex of rabbit muscle has a M r of 34,000 by gel filtration and migrates as a single band of M r 38,000 on sodium dodecyl sulfate gel electrophoresis, i.e., of the same size as the catalytic subunit of the sarcoplasmic complex of type 1 phosphatase (L. M. Ballou, D. L. Brautigan, and E. H. Fischer (1983) Biochemistry 22 , 3393–3399) . The enzyme, called PG-E a , has a specific activity of 12,000 units/mg of protein and is essentially fully active, displaying at most a 20% further increase in activity on treatment with trypsin. As in the case of the catalytic subunit of the sarcoplasmic enzyme, tryptic attack decreases the size of PG-E a to 33,000. PG-E a is completely inhibited by the modulator protein (inhibitor 2) after formation of a complex of M r 70,000. On incubation of this complex at 30 °C, the catalytic subunit is converted ( t 1 2 = 30 min ) to an inactive form (E i ) that can be reactivated by the protein kinase F A (J. R. Vandenheede, S.-D. Yang, J. Goris, and W. Merlevede (1980) J. Biol. Chem. 255 , 11,768–11,774) or to a lower extent by trypsin-Mn 2+ . Also the trypsinized PG-E a is inhibited by inhibitor 2, it forms with this a M r 70,000 complex and undergoes an even faster ( t 1 2 = 10 min ) conversion to an E i form that can be reactivated by the kinase F A or to a lower extent by trypsin-Mn 2+ . Enzymological comparison of PG-E a and trypsinized PG-E a with the F A -activated, isolated catalytic subunit of the sarcoplasmic phosphatase (called E a F A , E. Villa-Moruzzi, L. M. Ballou, and E. H. Fischer (1984) J. Biol. Chem. 259 , 5857–5863) and with its trypsinized form shows several similarities. The most relevant of these is the very specific interaction with inhibitor 2, that takes to inactivation and allows the following reactivation by F A or by trypsin-Mn 2+ . However, the inactivation-reactivation patterns show also some differences, namely (i) the t 1 2 of the conversion to E i is longer for PG-E a than for E a F A , and (ii) the reactivation of PG-E a and trypsinized PG-E a with trypsin-Mn 2+ is only partial. Altogether the similarity but not identity of PG-E a and E a F A would suggest that they are either two different conformations of the same molecule or two isozymes.

An assay method was devised for accurately measuring the activity of phosphorylase phosphatase in the presence of glycogen. Both oyster glycogen and rabbit liver glycogen inhibited the activity of purified phosphorylase phosphatase catalytic subunit in a concentration-dependent manner. In the presence of 120 mM KCl, micromolar AMP increased the sensitivity of the phosphatase to glycogen inhibition. The present studies suggest that glycogen feedback inhibition may be a coordinated mechanism producing a decrease in phosphorylase phosphatase activity as well as glycogen synthase phosphatase activity.

Rabbit muscle nonactivated phosphorylase kinase (EC 2.7.1.38) is converted to thiophosphate-activated phosphorylase kinase by cyclic AMP dependent protein kinase, Mg2+ and ATP-gamma-S/adenosine-5'-O-(s-thiotriphosphate)/. The formation of thiophosphate-activated phosphorylase kinase wal also observed in the protein-glycogen complex from skeletal muscle. This new form of kinase is resistant to the action of phosphatase and behaves as a competitive inhibitor in the dephosphorylation of phosphorylase alpha by phosphorylase phosphatase (Ki = 0.04 mg per ml). The fact that the inhibitory effect of thiophosphate-activated phosphorylase kinase is 3 times higher than in the case of nonactivated kinase, may explain the transient inhibition of phosphorylase phosphatase in the protein-glycogen complex. The use of activated (phosphorylated) phosphorylase kinase supports this assumption since it causes a delay in the dephosphorylation of phosphorylase alpha, i.e. the conversion of phosphorylase alpha into beta could start only after the dephosphorylation of activated phosphorylase kinase.

Acetyl-CoA carboxylase from rat epididymal fat tissue is activated by phosphorylase phosphatase, a reaction which is inhibited by phosphatase inhibitor-1. This activation is accompanied by a corresponding loss of 32P from the labelled enzyme. These results establish that dephosphorylation of the enzyme causes its activation.

Hamster adrenal HMG-CoA reductase activity was enhanced with rat liver cytosolic phosphorylase phosphatase as well as with similarly isolated beef and hamster adrenal cytosolic preparations. HMG-CoA reductase was inactivated when microsomes were incubated in an EDTA-free medium but containing MgCl 2 and ATP. The reductase activity of microsomes isolated from adrenals of hamsters sacrificed at 1100 h and 1900 h were (mean ± SEM, pmo1/mg protein/min.) 299.6±62.3 and 588.3 ± 96.6 respectively and could be enhanced by a factor of four when preincubated in the presence of liver phosphatase.

1. Phosphoprotein phosphatases with activity towards the inhibitory subunit of troponin (troponin I), phosphorylase a and lysine-rich histone (fraction F1) have been fractionated from rat skeletal muscle by chromatography on Sephadex G-200 and polylysine-Sepharose. Six separate fractions were identified on the basis of substrate specificity and behaviour during chromatography. 2. All fractions showed similar Km values for any given protein substrate. The Km for troponin I (5 muM) was significantly lower than that previously reported. 3. Phosphatase activities towards troponin I and hosphorylase a did not show a requirement for bivalent-metal ions. Two of the fractions with only minor activity towards histone were activated by Mn2+. 4. Discontinuous polyacrylamide-gel-electrophoresis studies indicated that several of the fractions contained more than one phosphatase activity, and additionally showed that several of the activities could exist in different aggregation states. On the basis of these studies at least two phosphatases with activity only towards troponin I were identified. In addition, phosphorylase phosphatase (which has considerable activity towards troponin I) and a general phosphatase with activity towards all three substrates were found. 5. A fraction with mol.wt. of 150000 could be activated by freezing with 2-mercaptoethanol or by heating to 55 degrees C. This activation was accompanied by a decrease in mol.wt. to 25000. 6. The total amount of phosphatase with activity towards troponin I which was extracted would be sufficient to dephosphorylate all the troponin I present in skeletal muscle in approximately 10s.

Analogs of the 5′-phosphoryl group of pyridoxal-5′-phosphate were used to test the function of this position of the coenzyme in glycogen phosphorylase. Trans-5′-phosphonomethylene-5′-deoxypyridoxal and pyridoxal in the presence of noncovalently bound phosphate analogs produce active enzyme forms. With the pyridoxal enzyme 20% of the activity of the native enzyme is seen in the presence of inorganic phosphite. Fluorophosphate with a pKa of 4.8 also activates suggesting that the dianionic form is involved. Pyrophosphate was found to be a potent competitive inhibitor of phosphite activation and glucose-1-P. One mole of pyrophosphate bound per mole of phosphorylase b monomer and suggests that the phosphoryl group of the coenzyme and substrate are in close proximity. 3-amino pyridoxal phosphate and 6-fluoropyridoxal in the presence of inorganic phosphite activate apophosphorylase b . These experiments suggest that it is unlikely that the pyridine nitrogen of the coenzyme is directly involved in the catalytic reaction. Studies with the enzymic dephosphorylation of pyridoxal phosphorylase a suggest that the 5′-position of the coenzyme is necessary for the glucose activation of the phosphorylase phosphatase reaction.

At the 1973 meeting on interconvertible enzymes, we summarized the data which establish that the activity of phosphorylase phosphatase in the liver is regulated by the concentration of glucose and that the activity of synthetase phosphatase is controlled by the concentration of phosphorylase a (Hers et al., 1974). The two phosphatases oppose the kinase system which is controlled by the concentration of cyclic AMP, according to the general scheme shown in Figure 1.

Two heat-stable and trypsin-labile inhibitors of phosphorylase phosphatase, designated inhibitor-1 and inhibitor-2, were partially purified from extracts of rabbit skeletal muscle by heating and coloumn chromatography using DEAE-dellulose and Bio-gel P-60. Inhibitor-1 exists in an active phosphorylated form and an inactive dephosphorylated form. The interconversion of phosphorylated inhibitor-1 and dephosphorylated inhibitor-1 is mediated by protein kinase dependent on adenosine 3':5'-monophosphate (cyclic AMP) and a Mn2+-stimulated phosphoprotein phosphatase. Inhibitory activity of inhibitor-2 is not influenced by treatment with either the kinase or the Mn2+-stimulated phosphatase. The molecular weights of inhibitor-1 and inhibitor-2 estimated by sodium dodecylsulfate-polyacrylamide gel electrophoresis are 26000 and 33000 respectively. Both inhibitor-1 and inhibitor-2 inhibit phosphorylase phosphatase by a mechanism which appears to be non-competitive with respect to the substrate phosphorylase a. Inhibitor fractions at early stages of purification also inhibit cyclic-AMP-dependent histone phosphorylation, but this kinase inhibitory activity resides with a protein moiety which is separable from inhibitor-1 and inhibitor-2.

Publisher Summary The low-molecular-weight form of phosphorylase phosphatase proved readily amenable to purification by a combination of ion-exchange chromatography and gel filtration, and was first purified from rabbit liver. The availability of the purified enzyme allowed the initial characterization of phosphorylase phosphatase as an enzyme of broad specificity. In addition, two such enzymes were isolated from rabbit liver, one with a high specific activity toward phosphorylase, and one with a lower specific activity to this substrate. The examination of the 35K phosphatases from rabbit muscle also led to the identification of two such enzymes, which are designated phosphatase C-I and C-II for convenience. It was proposed that the ∼35K low-molecular-weight phosphatases were the catalytic subunits of larger holoenzymes, based on the dissociation of phosphorylase phosphatase from higher molecular weight forms, and the existence of heat-stable protein inhibitors which formed larger complexes in association with the 35K enzyme. This hypothesis is now generally accepted, and is applicable to both the type-1 and type-2A phosphatases.

The chronically hyperinsulinemic Zucker fatty rat, with peripheral insulin resistance and glucose intolerance, represents a model of noninsulin dependent diabetes mellitus (NIDDM). These animals have elevated hepatic glycogen levels. Hepatic levels of synthase phosphatase and phosphorylase phosphatase, which are diminished in the IDDM rat, were markedly increased in the obese rats. Glyburide, a sulfonylurea used in treatment of NIDDM, resulted in reduced levels of glycemia and increased insulin levels in Zucker rats. Hepatic glycogen levels were increased, as was the activation of glycogen synthase, although there were no effects of drug administration on synthase phosphatase or phosphorylase phosphatase activities. G6P levels were increased by glyburide in lean rats but not in obese animals. These effects of glyburide on liver glycogen metabolism are accounted for via potentiation of the glycogenic effects of insulin.

Publisher Summary This chapter focuses on the subunit structure and regulation of phosphorylase phosphatase. Phosphorylation–dephosphorylation reactions catalyzed by protein kinases and phosphatases are known to play a major role in controlling cellular processes. Phosphorylase phosphatase is found in skeletal muscle extracts in a high-molecular weight form. When subjected to a purification procedure that includes acetone precipitation followed by gel filtration chromatography, three enzyme species can be recovered. The first and major form is completely inactive until subjected to treatment with trypsin in the presence of Mn 2+ . This mode of activation, although it is not physiological, has proved to be useful in measuring the activity of the enzyme. The phosphatase complex is unlike the cAMP-dependent protein kinase, in which dissociation of the enzyme releases active catalytic subunits. In the case of the phosphatase, any dissociation of the complex would only yield inactive enzymes. There are several observations that do not readily fit within this scheme and that require further explanation. One major difficulty is that while there is a correlation between phosphorylation of inhibitor 2 and activation of the enzyme, the total amount of 32 P incorporated is substoichiometric, usually between 0.2 and 0.3 mol per mole of regulatory subunit. Glycogen degradation is triggered when a number of enzymes become phosphorylated following hormonal stimulation of adenylate clase. To bring about glycogen synthesis and an arrest of glycogenolysis, as observed under the influence of insulin, either the phosphorylation reactions must be blocked or the protein phosphatases must be activated. This phosphorylation, however, is silent in that it does not cause an activation of the enzyme. Understanding the basic structural and regulatory properties of the enzyme should be of help in defining the hormonal control of the system.

The effects of glucose and of AMP on several properties of purified dog liver phosphorylase a have been investigated. Glucose stimulated several-fold the conversion of phosphorylase a into b, catalyzed by purified phosphorylase phosphatase. The reaction was strongly inhibited by AMP in the absence of glucose, and much less in the presence of glucose. Conversely, the concentration of glucose that gave half-maximal stimulation was increased several-fold by the nucleotide. Similar effects were observed when the phosphorylase phosphatase was studied in crude liver preparations. 1,5-Anhydroglucitol, α-methylglucoside and 2-deoxyglucose, quoted in order of decreasing efficiency, also stimulated the phosphorylase phosphatase reaction. Similar results were obtained when the conversion of phosphorylase a into b′ by trypsin was studied. The effect of glucose on the kinetics of purified liver phosphorylase a was mostly to decrease affinity for glucose 1-phosphate and to increase cooperativity between glucose 1-phosphate binding sites. AMP had the opposite effect and each of the two ligands antagonized the binding of the other. Glucose derivatives, in the order of efficiency given above, mimicked the effects of glucose. Glucose and the same glucose analogues, as well as AMP, protected phosphorylase a against denaturation at 55°C. These results indicate that the stimulation of the phosphorylase phosphatase reaction by glucose is secondary to the binding of the hexose to phosphorylase a, and that this binding is modulated by the concentration of AMP. The physiological implication of these findings is discussed.

The effect of three naturally occurring polyamines (putrescine, spermidine, and spermine) on the activity of rabbit skeletal muscle phosphorylase phosphatase was investigated. Only spermine significantly inhibited the enzyme. The mode of inhibition (ki value of 0.3 mM) of the phosphatase by spermine appears to be different from that caused by divalent metal ions or by other organic cations, such as arginine and lysine esters, since it is noncompetitive with respect to the substrate, phosphorylase a.

Mixed skeletal muscles of ICR/IAn mice contain 0.2% of the phosphorylase kinase activity found in C3H/He-mg mice at pH 8.2 in the presence of Ca2+. This lack of activity is caused by the absence of the normal phosphorylase kinase protein (Cohen, P. T. W. et al. (1976) Eur. J. Biochem. 66, 347–356). The trace residual activity in ICR/IAn mice has quite different properties from the normal enzyme. It has a much higher activity ratio (pH 6.8/8.2), its activity is less dependent on Ca2+ and it is not activated by cyclicAMP-dependent protein kinase or by trypsin. Its elution behaviour on Sepharose 413 also differs from the normal enzyme. A consequence of the higher activity ratio (pH 6.8/8.2) and decreased effect of Ca2+, is that mixed skeletal muscles of ICR/IAn mice contain 7% of normal activity at pH 6.8 in the absence of Ca2+. The activity of phosphorylase kinase in C3H/He-mg control mice is sixfold lower in red oxidative (soleus) muscle and 15-fold lower in cardiac muscle than in mixed skeletal muscle. In contrast, the trace residual activity in ICR/IAn mice is present at the same level in soleus muscle, cardiac muscle and mixed skeletal muscle. Soleus muscles of ICR/IAn mice therefore contain 1.5% of normal activity at pH 8.2 in the presence of Ca2+ and 30% of normal activity at pH 6.8 in the absence of Ca2+. Cardiac muscle contains 6% of normal activity at pH 8.2 in the presence of Ca2+ and 60% of normal activity at pH 6.8 in the absence of Ca2+. When isolated soleus muscles were incubated with adrenalin, the level of phosphorylase a in ICR/IAn mice rose from 2.8% in the absence to 5.7% in the presence of the hormone. In contrast, the level of phosphorylase a in normal mice varied from 2.4% to 11.1% in the absence of adrenalin, and increased to 17.7–23.9% in the presence of the hormone. It is concluded that the trace phosphorylase kinase activity in ICR/IAn mice is capable of phosphorylating phosphorylase b in vivo and that this enzyme is responsible for the very low level of phosphorylase a in resting soleus muscle of ICR/IAn mice, and perhaps even in normal mice. Since the residual enzyme cannot be activated by cyclic-AMP-dependent protein kinase, the elevation of phosphorylase a by adrenalin may be caused by an inhibition of phosphorylase phosphatase. The possibility that this occurs through the activation of protein phosphatase inhibitor 1 by cyclic-AMP-dependent protein kinase is discussed. Incubation of soleus muscles with adrenalin decreased the activity ratio (+ glucose 6-phosphate) of glycogen synthase from 0.24 to 0.08 in ICR/IAn mice and 0.31 to 0.14 in C3H/He-mg or Swiss albino mice. These results demonstrate that the inactivation of glycogen synthase by adrenalin in resting soleus muscle does not result from the activation of phosphorylase kinase by cyclic-AMP-dependent protein kinase. It is most likely mediated by a direct phosphorylation catalysed by cyclic-AMP-dependent protein kinase, although the activation of protein phosphatase inhibitor 1 may also contribute to the effect.

Adrenalectomized 24-h fasted rats lack the ability to synthesize and accumulate hepatic glycogen. In addition, the ability of liver glycogen synthase to respond to acute glucose administration is muted. The defect has been localized to the level of smooth endoplasmic reticulum-associated synthase phosphatase activity which is greatly reduced in adrenalectomized fasted rat liver. In vivo exposure to dexamethasone increases hepatic glucose output and hepatic glycogen synthesis and accumulation by 2-6 h after administration. Smooth endoplasmic reticulum synthase phosphatase activity is increased, and activation of glycogen synthase is restored. Ambient insulin concentrations are increased by steroid administration and appear to have a role in restoration of the ability to activate glycogen synthase, and consequently to restore the ability to synthesize and accumulate hepatic glycogen.

Publisher Summary This chapter describes the purification from rabbit skeletal muscle of an enzyme complex of Mr 70,000, which accounts for most of the total phosphorylase phosphatase activity in crude extracts. The enzyme is a heterodimer consisting of two subunits: an Mr 38,000 catalytic subunit and an Mr 31,000 regulatory subunit that is identical to inhibitor-2. The enzyme as isolated is totally inactive but can be activated in two ways. First, full activation can be obtained by limited proteolysis with trypsin in the presence of Mn2+. Second, the enzyme can be activated by an ATP. Mg2+-dependent reaction first described by Merlevede and Riley that also requires a factor known as Fa that appears to be identical to glycogen synthase kinase-3. The purification procedure is essentially that used by Ballou et al. and involves DEAE chromatography, acetone precipitation, gel filtration, and finally affinity chromatography using polylysine as the bound ligand. Throughout the procedure, the phosphatase is monitored by its activity toward phosphorylase a as substrate. Several other similar procedures for the isolation of the Mr 70,000 ATP Mg2+-dependent phosphatase are also described in the chapter.

1. 1. The effects of ATP, ADP, Mg 2+ and pH on three different preparations of hepatic phosphorylase phosphatase (phosphorylase α phosphohydrolase, EC 3.1.3.17) were investigated. 2. 2. The phosphorylase phosphatase activity in these preparations was decreased by ATP, ADP, adenylyl imidodiphosphate and by Mg 2+ . The effect produced by the adenine nucleotides was less pronounced at high pH than at low pH. 3. 3. Increasing concentrations of Mg 2+ , Mn 2+ and Ca 2+ in the presence of a constant concentration of ATP prevented the effect of ATP. At high concentrations the divalent cations decreased the reaction rate of the phosphatase in the presence of ATP as in its absence. At pH 7.5 and 8.5, ATP and Mg 2+ in equimolar concentrations decreased the phosphatase activity to a smaller extent than ATP alone. 4. 4. The activity of phosphorylase phosphatase was progressively less affected if ATPMg 2− and free Mg 2+ were increased at constant concentrations of uncomplexed ATP. In the concentration range expected to occur in the intact cell an increasing degree of complexation of ATP with Mg 2+ diminished the inhibitory effect of the nucleotide. Since addition of Mg 2+ after the preincubation with free ATP did not reverse the loss of phosphatase activity we concluded that the mechanism of action of ATP and ADP is not a simple inhibition as defined by an immediate and reversible loss of activity after the binding of a ligand.

The effects of both high voltage and low voltage electrical stimulation were studied in rabbit Longissimus dorsi muscles. The rate of fall of pH as well as the activities of phosphorylase a, phosphorylase kinase, and phosphorylase phosphatase were measured. The effects on the yield, ATPase activities, and calcium permeability of the sarcoplasmic reticulum (SR) were also measured. Although only high voltage stimulation increased the post-stimulation rate of pH fall, both types of electrical stimulation increased the phosphorylase a activity, apparently by increasing the activity of phosphorylase kinase and destroying a large part of the phosphorylase phosphatase activity. Electrical stimulation reduced the yield of SR and increased its basal ATPase activity. It also increased the ability of the SR to retain accumulated calcium. We conclude that the different rates of pH fall observed following the two types of stimulation are due to the differential effects of these treatments on one of the ATPase activities, probably the myofibrillar ATPase.

Insulin alone at concentrations of less than 1 to 5 uU/ml increased the enzyme activities of glycogen synthase, synthase phosphatase, phosphorylase, and phosphorylase phosphatase in hepatoma H4 cells in culture in the presence and absence of serum. Increase in total and active forms of glycogen synthase and phosphorylase were observed. Cycloheximide blocked the action of insulin on glycogen synthase, glycogen synthase phosphatase and phosphorylase phosphatase. The enzymes with the exception of glycogen synthase phosphatase were expressed with greater hormonal sensitivity in the absence as compared to the presence of serum in terms of hormone concentration required and or time of onset. These results demonstrate that these glycogen metabolizing enzymes are under long term control by insulin, with glycogen synthase being the most sensitive of the enzymes studied to the action of the hormone.

In a protein-glycogen complex preparation isolated from rabbit skeletal muscle phosphorylase kinase and phosphorylase phosphatase activities are found in membrane fractions of the sarcoplasmic reticulum and proteins adsorbed to the carbohydrate (Meyer et al., 1970). This pattern led to the question where phosphorylase kinase is localized intracellularly. In addition the synthesis of this enzyme was studied, which is a large oligomer (M.W. 1.26 × 106) containing three types of subunits (Hayakawa et al., 1973a,b) of the stoichiometry α4β4γ4 (Cohen, 1973).

Inhibitor-1 from rabbit skeletal muscle was phosphorylated by protein kinase dependent on adenosine 3' :5'-monophosphate (cyclic AMP), but not by phosphorylase kinase or by glycogen synthetase kinase-2. Protein phosphatase-III, isolated and stored in the presence of manganese ions to keep it stable, was in a form which catalysed a rapid dephosphorylation and inactivation of inhibitor-1. The kinetic constants for the dephosphorylation of inhibitor-1 [Km = 0.7 micron, V(rel) = 40] were comparable to those for the dephosphorylation of phosphorylase kinase [Km =1.1 micron, V (rel) = 62] and phosphorylase [Km = 5.0 micron, V (rel) = 100]. The dephosphorylation of inhibitor -1 was inhibited by inhibitor-2, indicating that it was catalysed by protein phosphatase-III, and not by another enzyme that might be contaminating the preparation. When protein phosphatase-III was diluted into buffers containing excess EDTA, it lost activity initially, but after 90 min, the activity reached a plateau that remained stable for at least 20h. The initial loss in activity varied with the substrate that was tested; it was 20-30% with phosphorylase a, 50-60% with phosphorylase kinase and greater than or equal to 95% with inhibitor-1. This form of protein phosphatase-III was inhibited by inhibitor-1 in a noncompetitive manner, and the Ki for inhibitor-1 was 1.6 +/- 0.3 nM. The phosphorylase phosphatase, phosphorylase kinase phosphatase and glycogen synthetase phosphatase activities of protein phosphatase-III were inhibited in an identical manner by inhibitor-1. This result emphasizes the potential importance of inhibitor-1 in the regulation of glycogen metabolism, since it can influence the state of phosphorylation of three different enzymes. The formation of the inactive complex between inhibitor-1 and protein phosphatase-III was reversed by incubation with trypsin (which destroyed inhibitor-1, but not protein phosphatase-III) or by dilution of the inactive complex. Kinetic studies, using the form of protein phosphatase-III which dephosphorylated inhibitor-1 very rapidly, demonstrated three unusual features of the system: (a) inhibitor-1 was still as powerful and inhibitor of the dephosphorylation of phosphorylase a and phosphorylase kinase a even under conditions where it was being rapidly dephosphorylated; (b) inhibitor-1 was not an inhibitor of its own dephosphorylation; (c) phosphorylase a did not effect the rate of dephosphorylation of inhibitor-1 even when it was present in a 50-fold molar excess over inhibitor-1. The result of these three properties is that inhibitor-1 is preferentially dephosphorylated by protein phosphatase-III even in the presence of a large excess of other phosphoprotein substrates. Inhibitor-1 was also dephosphorylated by protein phosphatase-II. The kinetic constants for the dephosphorylation of inhibitor-1 [Km = 2.8 micron, V (rel) = 200] and the alpha-subunit of phosphorylase kinase [Km = 3.7 micron, V (rel) = 100]were comparable...

The possibility of a “futile cycling” of fructose-6-phosphate (F-6-P) through the phosphofructokinase (PFK) and fructose-1,6-bisphosphatase (FDPase) catalyzed reactions in liver has been discussed first by Gevers and Krebs (1966). Later on, several groups (Clark et al. 1974; Rognstadt and Katz 1976; Van Schaftingen et al. 1980a) could indeed demonstrate the occurence of such a cycling, which is increased in the presence of a high concentration of glucose but will decrease in the presence of glucagon.

The Ca2+-independent activity of fast skeletal muscle phosphorylase kinase, A0, can be reversibly stimulated by heparin more than 20-fold; concomitantly the Ca2+-dependent A2 activity is abolished completely. Heparin also drastically changes the aggregation state of the enzyme; aggregated species contain significantly less delta and show an about fivefold higher A0 activity than the tetrameric form containing delta stoichiometrically. We interpret this to mean that delta has two functions in the phosphorylase kinase: an inhibitory one with respect to A0 and an activating one with respect to A2. The inhibition of A0 by Ca2+-free delta is released, i.e. A0 increases when this subunit dissociates from the holoenzyme. The maximally heparin-stimulated A0 activity, A0,hep, is enriched from a crude extract to the same degree and approximately with the same yield as the major activity, A2. The phosphorylase kinase is not eluted from DEAE-cellulose as a symmetrical bell-shaped protein peak. The peak fraction contains the activities A2 and A0,hep superimposed and yields a nearly homogeneous sedimentation boundary with an S20,w value of 25.5 S. The A0 yields a much broader eluation profile showing a distinct maximum from the A2 activity which contains slower sedimenting species of 12.1 S, some tetrameric enzyme of 22.7 S and higher aggregated material. Over the whole profile the activity ratio A2/A0 decreases about sevenfold whereas the ratio A2/A0,hep is constant on average. This shows that A0 is an intrinsic activity of phosphorylase kinase. The heparin-activated A0 activity or A0 itself in the presence of the phosphorylase phosphatase inhibitor, fluoride, can trigger a Ca2+-independent flash activation of phosphorylase in a protein-glycogen complex. Thus, A0 could be responsible for the conversion of phosphorylase b to a at 20 nM free Ca2+ in resting, hormone-stimulated, muscle.

1. Livers from gsd/gsd rats, which do not express phosphorylase kinase activity, also contain much less particulate type-1 protein phosphatases. In comparison with normal Wistar rats, the glycogen/microsomal fraction contained 75% less glycogen-synthase phosphatase and 60% less phosphorylase phosphatase activity. This was largely due to a lower amount of the type-1 catalytic subunit in the particulate fraction. In the cytosol, the synthase phosphatase activity was also 50% lower, but the phosphorylase phosphatase activity was equal. 2. Both Wistar rats and gsd/gsd rats responded to an intravenous injection of insulin plus glucose with an acute increase (by 30-40%) in the phosphorylase phosphatase activity in the liver cytosol. In contrast, administration of glucagon or vasopressin provoked a rapid fall (by about 25%) in the cytosolic phosphorylase phosphatase activity in Wistar rats, but no change occurred in gsd/gsd rats. 3. Phosphorylase kinase was partially purified from liver and subsequently activated. Addition of a physiological amount of the activated enzyme to a liver cytosol from Wistar rats decreased the V of the phosphorylase phosphatase reaction by half, whereas the non-activated kinase had no effect. The kinase preparations did not change the activity of glycogen-synthase phosphatase, which does not respond to glucagon or vasopressin. Furthermore, the phosphorylase phosphatase activity was not affected by addition of physiological concentrations of homogeneous phosphorylase kinase from skeletal muscle (activated or non-activated). 4. It appears therefore that phosphorylase kinase plays an essential role in the transduction of the effect of glucagon and vasopressin to phosphorylase phosphatase. However, this inhibitory effect either is specific for the hepatic phosphorylase kinase, or is mediated by an unidentified protein that is a specific substrate of phosphorylase kinase.

Fat body glycogen phosphorylase in some overwintering insects is known to be activated by cold and, therefore, this enzyme acts as a key enzyme that regulates the production of glycerol or trehalose from glycogen during winter. In this paper we report the mechanism of phosphorylase activation by cold: the major phosphorylase kinase (EC 2.7.1.38) of fat body is bound to glycogen and functions at 0 degrees C, whereas phosphorylase phosphatase does not; thus this may cause a slow but continuous accumulation of the active form of phosphorylase in the cold.

The chromatography of rat liver crude extract (10 000 × g supernatant) on a Biogel A-0.5 m column resulted in two peaks of phosphorylase phosphatase activity. The first peak, eluted in void volume, is of microsomal origin whereas the second peak, a cytoplasmic origin, was eluted with an apparent molecular weight of 260 000. In the presence of NaCl, the cytosolic enzyme was dissociated into intermediary ( M r = 135 000 and 68 000) and low molecular weight ( M r = 35 000) forms with an apparent activation of the enzyme activity. Similar dissociation was observed in the presence of KCl and (NH 4 ) 2 SO 4 . A partially purified high molecular weight phosphorylase phosphatase was also dissociated into the low molecular weight form by 0.2 M NaCl. However, a protein phosphatase showing activity towards 32 P-labelled histone was not dissociated by 0.2 M NaCl or 0.1 M (NH 4 ) 2 SO 4 .

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.

Acetyl-CoA carboxylase from rat epididymal fat tissue is activated by incubation at 30 C in the absence of citrate or metal ions. This activation is accompanied by a corresponding loss of 32P from the labeled enzyme, and it is not blocked by the heat-stable phosphorylase phosphatase inhibitor proteins from rabbit muscle. We have succeeded in separating an activity which activates and dephosphorylates acetyl-CoA carboxylase from the carboxylase using polyethylene glycol-6000. These results suggest that the temperature-dependent activation of acetyl-CoA carboxylase in crude or partially purified preparations results from dephosphorylation of the carboxylase by bound phosphatase.