Integrated effects of multiple modulators on human liver glycogen phosphorylase a

Hepatic glucose production is increased in people with type 2 diabetes. Glucose released from storage in liver glycogen by phosphorylase accounts for approximately 50% of the glucose produced after an overnight fast. Therefore, understanding how glycogenolysis in the liver is regulated is of great importance. Toward this goal, we have determined the kinetic characteristics of recombinant human liver glycogen phosphorylase a (HLGPa) (active form) and compared them with those of the purified rat enzyme (RLGPa). The Michaelis-Menten constant (K(m)) of HLGPa for P(i), 5 mM, was about fivefold greater than the K(m) of RLGPa. Two P(i) (substrate) concentrations were used (1 and 5 mM) to cover the physiological range for P(i). Other effectors were added at estimated intracellular concentrations. When added individually, AMP stimulated, whereas ADP, ATP and glucose inhibited, activity. These results were similar to those of the RLGPa. However, glucose inhibition was about twofold more potent with the human enzyme. UDP-glucose, glucose 6-phosphate, and fructose 1-phosphate were only minor inhibitors of both enzymes. We reported previously that when all known effectors were present in combination at physiological concentrations, the net effect was no change in RLGPa activity. However, the same combination reduced HLGPa activity, and the inhibition was glucose dependent. We conclude that a combination of the known effectors of phosphorylase a activity, when present at estimated intracellular concentrations, is inhibitory. Of these effectors, only glucose changes greatly in vivo. Thus it may be the major regulator of HLGPa activity.     

Endogenous effectors of human liver glycogen phosphorylase modulate effects of indole-site inhibitors

Phosphorylase is regulated by a number of small-molecular-weight effectors that bind to three sites on the enzyme. Recently, a fourth site referred to as the indole-inhibitor site has been identified. Synthetic compounds bind to the site and inhibit activity. However, the effects of these compounds in the presence of other endogenous effectors are unknown. We have determined the effects of four indole derivative glycogen phosphorylase inhibitors (GPI) on recombinant human liver glycogen phosphorylase a activity. The GPIs tested were all potent inhibitors. However, the endogenous inhibitors (glucose, ADP, ATP, fructose 1-phosphate, glucose 6-phosphate, UDP-glucose) and the activator (AMP) markedly reduced the inhibitory effect of GPIs. Consistent with these in vitro findings, the IC50 for the inhibition of glycogenolysis in cells and the liver drug concentration associated with glucose-lowering activity in diabetic ob/ob mice in vivo were also significantly higher than those determined in in vitro enzyme assays. The inhibitory effect of indole-site effectors is modulated by endogenous small-molecular-weight effectors of phosphorylase a activity. However, at higher concentrations (10-30 microM), the GPI effect was dominant and resulted in inhibition of phosphorylase a activity irrespective of the presence or absence of the other modulators of the enzyme.     

Distinction between substrate- and enzyme-directed effects of modifiers of rabbit liver phosphorylase a phosphatases

The natural substrate (phosphorylase a) and two alternative ones (phosphorylated histone and a tetradecapeptide consisting of residues 5-18 of rabbit skeletal muscle phosphorylase a) were used to distinguish the modes of action of some physiologically important effectors of four different molecular forms of rabbit liver phosphorlase a phosphatases. In general, glucose, caffeine, AMP, ADP, Pi, and glucose-1-P showed substrate-directed effects for the holophosphatase forms, since they usually did not affect the activity on histone phosphate and, with one slight exception (Pi), never affected the activity on the tetradecapeptide phosphate. ADP, Pi, and glucose-1-P did affect directly the relative mass (Mr) 35,000 phosphatase, in addition to an inhibition mediated via phosphorylase a. ATP exerted both substrate- and enzyme-directed effects for the Mr 35,000 phosphatase and phosphatases 1 and 2A2, but only a substrate-directed effect for phosphatase 2A1, suggesting that the gamma-subunit of the type 2 phosphatases may prevent ATP binding to the phosphatase. Mg2+ showed substrate-directed effects for phosphatases 1, 2A1, and 2A2, and an additional enzyme-directed effect for the Mr 35,000 phosphatase form. Furthermore, Mg2+ could not abolish ATP inhibition of the tetradecapeptide phosphatase activity, but significantly overcame ATP inhibition of the phosphorylase a phosphatase activity, thus suggesting that its ability to reverse the ATP effect is by a substrate-directed mechanism. The substrate-directed effects seen for the different ligands on the different phosphatase forms strongly indicate the significance of this form of control in the regulation of phosphorylase a phosphatase activities and may serve to narrow the otherwise broad substrate specificities of the major phosphorylase a phosphatase activities in mammalian tissues: phosphatases 1 and 2A.     

Interaction of calmodulin with muscle phosphofructokinase. Interplay with metabolic effectors of the enzyme under physiological conditions

The hysteretic calmodulin-induced inactivation of muscle phosphofructokinase and the calmodulin-mediated reactivation are essentially dependent on environmental conditions. The interplay of calmodulin during these reactions and at allosteric conditions with Mg . ATP, fructose 6-phosphate, adenosine 5'-[beta, gamma-imido]triphosphate and with the allosteric effectors AMP, ADP, fructose 1,6-bisphosphate, fructose 2,6-bisphosphate and glucose 1,6-bisphosphate was studied by two techniques. (a) A two-step technique with a preincubation of enzyme, calmodulin and effectors in close to physiological concentrations before dilution into an optimal activity assay. It reveals aggregation and slowly reversible conformation changes. (b) A direct assay of dilute enzyme at allosteric conditions. Dominating in the interplay of calmodulin with metabolic effectors is the competitive-like action of calmodulin on Mg . ATP binding to the regulatory sites of the enzyme. At high enzyme concentrations in the absence of hexose phosphates, i.e. at noncatalytic conditions calmodulin counteracts the stabilization of the highly active tetrameric form caused by Mg . ATP. In the allosteric assay it counteracts the ATP-induced allosteric inhibition. In both cases calmodulin acts synergistic with AMP and ADP. To a minor degree calmodulin also counteracts the stabilization of the tetrameric form caused by fructose 6-phosphate and hexose bisphosphate, now however antagonistically to AMP and ADP. By the demonstrated interactions the enzyme can be slowly and hysteretically shifted between an active tetrameric and an inactive dimeric state under control metabolic conditions and of Ca2+ and calmodulin. Resting conditions will inactivate and high contractile activity reactivate available enzyme.     

Properties of phsophofructokinase from the mucosa of rat jejunum and the relation to the lack of Pasteur effect

1. The properties of phosphofructokinase after its slight purification from the mucosa of rat jejunum were studied. 2. The enzyme is inhibited by almost 100% by an excess of ATP (1.6mm), with 0.2mm-fructose 6-phosphate. AMP, ADP, P(i) and NH(4) (+) at 0.2, 0.76, 1.0 and 2mm respectively do not individually prevent the inhibition of phosphofructokinase activity by 1.6mm-ATP with 0.2mm-fructose 6-phosphate to any great extent, but all of them together completely prevent the inhibition of phosphofructokinase by ATP. 3. One of the effects of high concentrations of ATP on the enzyme was to increase enormously the apparent K(m) value for the other substrate fructose 6-phosphate, and this increase is largely counteracted by the presence of AMP, ADP, P(i) and NH(4) (+). At low concentrations of ATP the above effectors individually decrease the concentration of fructose 6-phosphate required for half-maximum velocity and when present together they decrease it further, in a more than additive way. 4. When fructose 6-phosphate is present at a saturating concentration (5mm), 0.3mm-NH(4) (+) increases the maximum velocity of the reaction 3.3-fold; with 0.5mm-fructose 6-phosphate, 4.5mm-NH(4) (+) is required for maximum effect. The other effectors do not change the maximum reaction velocity. 5. The results presented here suggest that NH(4) (+), AMP, ADP and P(i) synergistically decrease the inhibition of phosphofructokinase activity at high concentrations of ATP by decreasing the concentration of fructose 6-phosphate required for half-maximum velocity. Such synergism among the effectors and an observed, low ;energy charge' [(ATP+(1/2)ADP)/(AMP+ADP+ATP)] in conjunction with the possibility of a relatively high NH(4) (+) and fructose 6-phosphate concentration in this tissue, may keep the mucosal phosphofructokinase active and uninhibited by ATP under aerobic conditions, thus explaining the high rate of aerobic glycolysis and the lack of Pasteur effect in this tissue.     

Adenylate deaminase from rat muscle. Regulation by purine nucleotides and orthophosphate in the presence of 150 mM KCl.

Adenylate deaminase from rat skeletal muscle has been studied with the objective of understanding how the activity of the enzyme is regulated in vivo. ATP and GTP inhibit the enzyme at low concentrations in the presence of 150 mM KCl. The ATP inhibition is reversed as the ATP concentration is raised to physiological levels. The GTP inhibition is reversed as the GTP concentration is raised to unphysiologically high levels. In the presence of physiological concentrations of ATP, the GTP inhibition is also greatly diminished, but inhibition by orthophosphate remains strong. The apparent affinities of the enzyme for GTP, ATP, and orthophosphate are reduced as the pH is decreased from 7.0 to 6.2. ADP also reduces the apparent affinities of the enzyme for the inhibitors. The regulatory effects of GTP, ATP, and ADP are produced primarily by their unchelated forms. Comparison of the kinetic behavior of the enzyme in vitro with metabolite concentrations in vivo indicates that the major variables that regulate the activity of adenylate deaminase of muscle in vivo are the concentrations of AMP, ADP, orthophosphate, and H+.     

Intracellular mechanism of action of sympathetic hepatic nerves on glucose and lactate balance in perfused rat liver

In rat liver perfused in situ stimulation of the nerve plexus around the hepatic artery and the portal vein caused an increase in glucose output and a shift from lactate uptake to output. The effects of nerve stimulation on some key enzymes, metabolites and effectors of carbohydrate metabolism were determined and compared to the actions of glucagon, which led to an increase not only of glucose output but also of lactate uptake. 1. Nerve stimulation caused an enhancement of the activity of glycogen phosphorylase a to 300% and a decrease of the activity of glycogen synthase I to 40%, while it left the activity of pyruvate kinase unaltered. Glucagon, similarly to nerve action, led to a strong increase of glycogen phosphorylase and to a decrease of glycogen synthase; yet in contrast to the nerve effect it lowered pyruvate kinase activity clearly. 2. Nerve stimulation increased the levels of glucose 6-phosphate and of fructose 6-phosphate to 200% and 170%, respectively; glucagon enhanced the levels to about 400% and 230%, respectively. The levels of ATP and ADP were not altered, those of AMP were increased slightly by nerve stimulation. 3. Nerve stimulation enhanced the levels of the effectors fructose 2,6-bisphosphate and cyclic AMP only slightly to 140% and 125%, respectively; glucagon lowered the level of fructose 2,6-bisphosphate to 15% and increased the level of cyclic AMP to 300%. 4. In calcium-free perfusions the metabolic responses to nerve stimulation showed normal kinetics, if calcium was re-added 3 min before, but delayed kinetics, if it was re-added 2 min after the onset of the stimulus. The delay may be due to the time required to refill intracellular calcium stores. The hemodynamic alterations dependent on extracellular calcium were normal in both cases. The activation of glycogen phosphorylase, the inhibition of glycogen synthase and the increase of glucose 6-phosphate can well explain the enhancement of glucose output following nerve stimulation. The unaltered activity of pyruvate kinase and the marginal increase of fructose 2,6-bisphosphate cannot be the cause of the nerve-stimulation-dependent shift from lactate uptake to output. The very slight increase of the level of cyclic AMP after nerve stimulation cannot elicit the observed activation of glycogen phosphorylase.(ABSTRACT TRUNCATED AT 400 WORDS)     

Heterotropic interactions of ligands with phosphorylase b.

1. The interaction of rabbit muscle glycogen phosphorylase b with pairs of ligands has been examined. 2. The electron spin resonance spectrum of a spin label, covalently attached to the protein, provided information about dissociation constants, formation of ternary complexes and both negative and positive interactions between different ligand pairs. 3. AMP competes with a series of nucleotides (ADP, ATP, CMP aand cytosine) but with adenosine a ternary enzyme - AMP - adenosine complex can be formed. 4. ADP binding is tight and ADP inhibits the AMP activation of phosphorylase b in a physiologically important concentration range. 5. The substrates glucose 1-phosphate and glycogen tighten AMP binding in the ternary complex as does the competitive inhibitor UDPG. Inorganic phosphate is different in this respect. Gluconolactone, a transition state analogue, competes with glucose 1-phosphate (but not with glycogen) but does not prevent completely the binding of the sugar phosphate. 6. The effect of glucose b-phosphate on phosphorylase is rather complex as it 'formally competes' with both AMP and UDPG probably mediated by a conformational changes and not by 'direct' interactions with these two ligands. Glycerol 2-phosphate, a commonly used buffer for phosphorylase, also shows complex interactions.     

Enzymes regulating glycogen metabolism in swine subcutaneous adipose tissue. I. Phosphorylase and phosphorylase phosphatase.

Glycogen phosphorylase from swine adipose tissue was purified nearly 700-fold using ethanol precipitation, DEAE-cellulose adsorption, AMP-agarose affinity chromatography, and agarose gel filtration. The purified enzyme migrated as one major and several minor components during polyacrylamide gel electrophoresis. Activity was associated with the major component and at least one of the minor components. The molecular weight of the disaggregated, reduced, and alkylated enzyme, estimated by polyacrylamide gel electrophoresis performed in the presence of sodium dodecyl sulfate, was 90,000. Stability of the purified enzyme was considerably increased in the presence of AMP. The isoelectric pH of the enzyme in crude homogenates was 6.3. The sedimentation coefficient of the purified enzyme (7.9 S) and that in crude homogenates (7.3 S) was determined by sucrose density gradient sedimentation. Optimal pH for activity was between pH 6.5 and 7.1. Apparent Km values for glycogen and inorganic phosphate were 0.9 mg/ml and 6.6 mM, respectively. The Ka for AMP was 0.21 mM. Enzyme activity was increased by K2SO4, KF, KCl, and MgCl2 and decreased by NaCl, Na2SO4, D-glucose, and ATP. Inhibition by glucose was noncompetitive with the activator AMP; inhibition by ATP was partially competitive with AMP. The purified enzyme was activated by incubation with skeletal muscle phosphorylase kinase. Enzyme in crude homogenates was activated by the addition of MgCl2 and ATP; activation was not blocked by addition of protein kinase inhibitor, suggesting that phosphorylase kinase in homogenates of swine adipose tissue is present largely in an activated form. Deactivation of phosphorylase a by phosphorylase phosphatase was studied using enzyme purified approximately 200-fold from swine adipose tissue by ethanol precipitation, DEAE-cellulose chromatography, and gel filtration. The Km of the adipose tissue phosphatase for skeletal muscle phosphorylase a was 6 muM. The purified swine adipose tissue phosphorylase, labeled with 32-P, was inactivated and dephosphorylated by the adipose tissue phosphatase. Dephosphorylation of both skeletal muscle and adipose tissue substrates was inhibited by AMP and glucose reversed this inhibition. Several lines of evidence suggest that AMP inhibition was due to an action on the substrate rather than on the enzyme. We have previously reported that the system for phosphorylase activation in rat fat cells differs in some important characteristics from that in skeletal muscle. However, both swine fat phosphorylase and phosphorylase phosphatase have major properties very similar to those described for the enzymes from skeletal muscle.     

Studies with a reconstituted muscle glycolytic system. The rate and extent of creatine phosphorylation by anaerobic glycolysis

A mixture of purified muscle glycolytic enzymes was reconstituted and the mixture shown to behave in a fashion analogous to that occurring in vivo. Glycolysis leads to ATP production in muscle and results in the phosphorylation of creatine. The extent of this phosphorylation by anaerobic glycolysis was shown to depend to a small extent on the relative proportions of available P(i) and creatine initially, but more importantly on the first step in glycolysis, in this case the enzyme phosphorylase. With less than 0.1% of the phosphorylase in the a form, only about one-third of the creatine was phosphorylated in 30min, whereas with 4% or more of phosphorylase a, 90% of the creatine was phosphorylated within this time. Inclusion of an adenosine triphosphatase decreased the steady-state concentration of phosphocreatine in the system. Calculations of the theoretical concentrations of ADP and AMP showed that phosphorylase b was almost inactive even in the presence of 9mum-AMP, because of ATP inhibition. With phosphorylase a present, glycolysis was able to continue at least until the calculated concentration of MgADP(-) was only 7mum, and AMP in the sub-mumolar range. The relation of these values to measured concentrations of nucleotides and to phosphorylase a percentages in intact muscle is discussed.     

Glucose stimulation of heart phosphorylase phosphatase activity in vitro and in vivo

Summary To determine the mechanism of the glucose stimulation, glucose or glucose-6-phospate was added to dilute heart extracts in the presence or absence of AMP. The intracellular glucose, tissue glucose-6-phosphate, and tissue AMP concentrations were also determined in 24-h starved animals given glucose; 24-h starved animals given insulin as well as diabetic starved and diabetic starved insulin-treated animals were also studied.The A_0.5 for glucose stimulation of cardiac phosphorylase phosphatase activity was approximately 1 .2 mM. The A_0.5 for glucose-6-phosphate was approximately 0.02 mM. The glucose-6-phosphate concentration in all animals exceeded the Ao.5 by 10-fold. However, the intracellular glucose concentration in the glucose-treated, insulin-treated, diabetic, and diabetic insulin-treated rats was in the range of the A_0.5 for stimulation of phosphorylase phosphatase activity. AMP completely inhibited phosphorylase phosphatase activity at a concentration of 0.2 mM. Physiological concentrations of glucose and glucose-6-phosphate partially reversed this inhibition. Administration of glucose or insulin resulted in an increase in intracellular glucose concentration, an increase in tissue glucose-6-phosphate and a decrease in tissue AMP concentrations. These data suggest that glucose may be a physiological regulator of phosphorylase phosphatase in heart muscle as it is in liver.Recipient ofaMedical InvestigatorshipAward from theVeterans Administration.     

The catalytic activity of phosphorylase b in the liver. With a note on the assay in the glycogenolytic direction.

1. The activity and the kinetic properties of purified hepatic phosphorylases a and b from rabbit and rat have been investigated in the glycogenolytic direction with a radiochemical assay. 2. In contrast with the a form, phosphorylase b has an absolute requirement for both AMP and a lyotropic salt. When the latter effectors are included, the b/a-form activity ratio remains low (0.03-0.15) at the hepatic concentration of Pi, because the b form has an exceedingly low affinity for this substrate. 3. Only phosphorylase b is significantly inhibited by glucose, glucose 6-phosphate and MgATP2-. Assays in the presence of substrastes, stimulators and inhibitors in the physiological concentration range indicate that glycogenolysis in the liver depends strictly on the conversion of phosphorylase b into a. Even at 1 mM-AMP the b/a-form activity ratio does not exceed 0.01. 4. Current spectrophotometric procedures for the glycogenolytic assay of phosphorylase in crude liver preparations are highly specific for the a form; the measurement of total phosphorylase (a + b) would require impractical modifications, and is better performed in the direction of glycogen synthesis.     

Modification of yeast phosphofructokinase by trypsin and subtilisin in the presence of effectors.

Yeast phosphofructokinase was subjected to limited proteolysis by trypsin in the presence of different effectors. It could be demonstrated that the substrates MgATP and fructose-6-phosphate are able to protect the enzyme from inactivation by trypsin. Other effectors like AMP, ADP, phosphoenolpyruvate, citrate and ammonium ions exhibit only negligible effects. During the first step of degradation consisting in the conversion of the subunits from Mr 120,000 to 90,000 no significant effects of the substrates and effectors on the proteolytic inactivation of yeast phosphofructokinase can be observed. In the presence of ATP as well as of ADP the sensitivity of the enzyme against ATP inhibition is either not or only slightly influenced by proteolytic modification. The modified enzyme retains its sensitivity against activation by AMP, independently of whether effectors are present or absent during proteolysis. The kinetic parameters of the enzyme modified by subtilisin in the presence of ATP or of fructose-6-phosphate have been determined.     

Skeletal muscle glycogen phosphorylase a kinetics: effects of adenine nucleotides and caffeine.

This study aimed to determine physiologically relevant kinetic and allosteric effects of P(i), AMP, ADP, and caffeine on isolated skeletal muscle glycogen phosphorylase a (Phos a). In the absence of effectors, Phos a had Vmax = 221 +/- 2 U/mg and Km = 5.6 +/- 0.3 mM P(i) at 30 degrees C. AMP and ADP each increased Phos a Vmax and decreased Km in a dose-dependent manner. AMP was more effective than ADP (e.g., 1 microM AMP vs. ADP: Vmax = 354 +/- 2 vs. 209 +/- 8 U/mg, and Km = 2.3 +/- 0.1 vs. 4.1 +/- 0.3 mM). Both nucleotides were relatively more effective at lower P(i) levels. Experiments simulating a range of contraction (exercise) conditions in which P(i), AMP, and ADP were used at appropriate physiological concentrations demonstrated that each agent singly and in combination influences Phos a activity. Caffeine (50-100 microM) inhibited Phos a (Km approximately 8-14 mM, approximately 40-50% reduction in activity at 2-10 mM P(i)). The present in vitro data support a possible contribution of substrate (P(i)) and allosteric effects to Phos a regulation in many physiological states, independent of covalent modulation of the percentage of total Phos in the Phos a form and suggest that caffeine inhibition of Phos a activity may contribute to the glycogen-sparing effect of caffeine.     

Effects of added adenine nucleotides on renal carbohydrate metabolism

1. The regulatory effects that adenine nucleotides are known to exert on enzymes of glycolysis and gluconeogenesis were demonstrated to operate in kidney-cortex slices and in the isolated perfused rat kidney by the addition of exogenous ATP, ADP and AMP to the incubation or perfusion media. 2. Both preparations rapidly converted added ATP into ADP and AMP, and ADP into AMP; added AMP was rapidly dephosphorylated. AMP formed from ATP was dephosphorylated at a lower rate than was added AMP, especially when the initial ATP concentration was high (10mm). Deamination of added AMP occurred more slowly than dephosphorylation of AMP. 3. Gluconeogenesis from lactate or propionate by rat kidney-cortex slices, and from lactate by the isolated perfused rat kidney, was inhibited by the addition of adenine nucleotides to the incubation or perfusion media. In contrast, oxygen consumption and the utilization of propionate or lactate by slices were not significantly affected by added ATP or AMP. 4. The extent and rapidity of onset of the inhibition of renal gluconeogenesis were proportional to the AMP concentration in the medium and the tissue, and were not due to the production of acid or P(i) or the formation of complexes with Mg(2+) ions. 5. Glucose uptake by kidney-cortex slices was stimulated 30-50% by added ATP, but the extra glucose removed was not oxidized to carbon dioxide and did not all appear as lactate. Glucose uptake, but not lactate production, by the isolated perfused kidney was also stimulated by the addition of ATP or AMP. 6. In the presence of either glucose or lactate, ATP and AMP greatly increased the concentrations of C(3) phosphorylated intermediates and fructose 1,6-diphosphate in the kidney. There was a simultaneous rise in the concentration of malate and fall in the concentration of alpha-oxoglutarate. 7. The effects of added adenine nucleotides on renal carbohydrate metabolism seem to be mainly due to an increased concentration of intracellular AMP, which inhibits fructose diphosphatase and deinhibits phosphofructokinase. This conclusion is supported by the accumulation of intermediates of the glycolytic pathway between fructose diphosphate and pyruvate. 8. ATP or ADP (10mm) added to the medium perfusing an isolated rat kidney temporarily increased the renal vascular resistance, greatly diminishing the flow rate of perfusion medium for a period of several minutes.     

Studies on alpha-adrenergic activation of hepatic glucose output. Studies on role of calcium in alpha-adrenergic activation of phosphorylase.

The role of Ca2+ ions in alpha-adrenergic activation of hepatic phosphorylase was studied using isolated rat liver parenchymal cells. The activation of glucose release and phosphorylase by the alpha-adrenergic agonist phenylephrine was impaired in cells in which calcium was depleted by ethylene glycol bis(beta-aminoethyl ether)N,N'-tetraacetic acid (EGTA) treatment and restored by calcium addition, whereas the effects of a glycogenolytically equivalent concentration of glucagon on these processes were unaffected. EGTA treatment also reduced basal glucose release and phosphorylase alpha activity, but did not alter the level of cAMP or the protein kinase activity ratio (-cAMP/+cAMP) or impair viability as determined by trypan blue exclusion, ATP levels, or gluconeogenic rates. The effect of EGTA on basal phosphorylase and glucose output was also rapidly reversed by Ca2+, but not by other ions. Phenylephrine potentiated the ability of low concentrations of calcium to reactivate phosphorylase in EGTA-treated cells. The divalent cation inophore A23187 rapidly increased phosphorylase alpha and glucose output without altering the cAMP level, the protein kinase activity ratio, and the levels of ATP, ADP, or AMP, The effects of the ionophore were abolished in EGTA-treated cells and restored by calcium addition. Phenylephrine rapidly stimulated 45Ca uptake and exchange in hepatocytes, but did not affect the cell content of 45Ca at late time points. A glycogenolytically equivalent concentration of glucagon did not affect these processes, whereas higher concentrations were as effective as phenylephrine. The effect of phenylephrine on 45Ca uptake was blocked by the alpha-adrenergic antagonist phenoxybenzamine, was unaffected by the beta blocker propranolol, and was not mimicked by isoproterenol. The following conclusions are drawn: (a) alpha-adrenergic activation of phosphorylase and glucose release in hepatocytes is more dependent on calcium than is glucagon activation of these processes; (b) variations in liver cell calcium can regulate phosphorylase alpha levels and glycogenolysis; (c) calcium fluxes across the plasma membrane are stimulated more by phenylephrine than by a glycogenolytically equivalent concentration of glucagon. It is proposed that alpha-adrenergic agonists activate phosphorylase by increasing the cytosolic concentration of Ca2+ ions, thus stimulating phosphorylase kinase.     

4-pyridylmethyl, a thiol-protecting group suitable for the partial synthesis of proteins.

1. A dose-dependent activation of phosphorylase and consumption of ATP was observed in isolated hepatocytes incubated in the presence of fructose; histone kinase and phosphorylase kinase activities were unchanged at doses of this sugar that were fully effective on phosphorylase. The activation of phosphorylase by fructose was also observed in cells incubated in a Ca2+-free medium as well as in the livers of rats in vivo. 2. In a liver high-speed supernatant, fructose, tagatose and sorbose stimulated the activity of phosphorylase kinase; this effect was dependent on the presence of K+ ions, which are required for the activity of fructokinase; it was accompanied by the transformation of ATP into ADP. In the presence of hexokinase, glucose also stimulated phosphorylase kinase, both in an Na+ or a K+ medium. 3. The activities of partially purified muscle or liver phosphorylase kinase were unchanged in the presence of fructose. 4. Some properties of liver phosphorylase kinase are described, including a high molecular weight and an inhibition at ATP/Mg ratios above 0.5, as well as an effect of ATP concentration on the hysteretic behaviour of this enzyme. 5. The effect of fructose on the activation of phosphorylase is discussed in relation to the comsumption of ATP.     

Effects of adenosine phosphates and nicotinamide nucleotides on pyruvate carboxylase from baker''s yeast

1. Pyruvate carboxylase from baker's yeast is inhibited by ADP, AMP and adenosine at pH8.0 in the presence of magnesium chloride concentrations equal to or higher than the ATP concentration. The adenine moiety is essential for the inhibitory effect. 2. In the absence of acetyl-CoA (an allosteric activator) ADP, AMP and adenosine are competitive inhibitors with respect to ATP. In the presence of acetyl-CoA, besides the effect with respect to ATP, AMP competes with acetyl-CoA, whereas ADP and adenosine are non-competitive inhibitors with respect to the activator. 3. Pyruvate carboxylase is inhibited by NADH. The inhibition is competitive with respect to acetyl-CoA and specific with respect to NADH, since NAD(+), NADP(+) and NADPH do not affect the enzyme activity. In the absence of acetyl-CoA, NAD(+), NADH, NADP(+) and NADPH do not inhibit pyruvate carboxylase. 4. Pyruvate carboxylase is inhibited by ADP, AMP and NADH at pH6.5, in the presence of 12mm-Mg(2+), 0.75mm-Mn(2+) and 0.5mm-ATP, medium conditions similar to those existing inside the yeast cell. The ADP and NADH effects are consistent with a regulation of enzyme activity by the intracellular [ATP]/[ADP] ratio and secondarily by NADH concentration. These mechanisms would supplement the already known control of yeast pyruvate carboxylase by acetyl-CoA and l-aspartate. Inhibition by AMP is less marked and its physiological role is perhaps limited.     

H Efflux and Hexose Transport under Imposed Energy Status in Maize Root Tips

The relationship between changes in H⁺ flux and sugar transport in maize Zea mays L. DEA root tips have been investigated using two methods for controlling the cellular nucleotide level: (a) incubation in the presence of a glucose analog, the 2-deoxyglucose, which decreased the ATP level to less than 15% of its initial value within 60 minutes without changing the ADP and AMP levels; (b) an hypoxic treatment which also decreased the ATP level but with a concomitant rise in ADP and AMP. In both cases the rate of hexose transport was not modified until ATP had dropped to 70% of its initial value; then it decreased with the cellular ATP level. The residual uptake rate at very low ATP concentrations still represented 50% of the maximum rate with the dGlc treatment but only the diffusion rate in anoxia. H⁺ efflux was abolished in anoxia but not by the 2-deoxyglucose treatment, in spite of a lower cellular ATP concentration. Our results are consistent with an inhibition of H⁺-ATPase activity in anoxia by the high levels of cellular ADP and AMP, and provide in vivo evidence that sugar uptake is dependent upon the proton motive force rather than cellular ATP concentration. The absence of stimulation of H⁺ extrusion by ferricyanide in either normoxic or hypoxic conditions suggests that a redox system does not appear to contribute to H⁺ secretion under the conditions of this investigation.