Disruption of the allosteric phosphorylase a regulation of the hepatic glycogen-targeted protein phosphatase 1 improves glucose tolerance in vivo

Type 2 diabetes is characterised by elevated blood glucose concentrations, which potentially could be normalised by stimulation of hepatic glycogen synthesis. Under glycogenolytic conditions, the interaction of hepatic glycogen-associated protein phosphatase-1 (PP1–G_L) with glycogen phosphorylase a is believed to inhibit the dephosphorylation and activation of glycogen synthase (GS) by the PP1–G_L complex, suppressing glycogen synthesis. Consequently, the interaction of G_L with phosphorylase a has emerged as an attractive anti-diabetic target, pharmacological disruption of which could provide a novel mechanism to lower blood glucose levels by increasing hepatic glycogen synthesis. Here we report for the first time the in vivo consequences of disrupting the G_L–phosphorylase a interaction, using a mouse model containing a Tyr284Phe substitution in the phosphorylase a-binding region of the G_L protein. The resulting G_L^Y284F/Y284F mice display hepatic PP1–G_L activity that is no longer sensitive to allosteric inhibition by phosphorylase a, resulting in increased GS activity under glycogenolytic conditions, demonstrating that regulation of G_L by phosphorylase a operates in vivo. G_L^Y284F/Y284F and G_L^Y284F/+ mice display improved glucose tolerance compared with G_L^+/+ littermates, without significant accumulation of hepatic glycogen. The data provide the first in vivo evidence in support of targeting the G_L–phosphorylase a interaction for treatment of hyperglycaemia. During prolonged fasting the G_L^Y284F/Y284F mice lose more body weight and display decreased blood glucose levels in comparison with their G_L^+/+ littermates. These results suggest that, during periods of food deprivation, the phosphorylase a regulation of G_L may prevent futile glucose–glycogen cycling, preserving energy and thus providing a selective biological advantage that may explain the observed conservation of the allosteric regulation of PP1–G_L by phosphorylase a in mammals.     

Reaction Kinetics of Substrate Transglycosylation Catalyzed by TreX of Sulfolobus solfataricus and Effects on Glycogen Breakdown

We studied the activity of a debranching enzyme (TreX) from Sulfolobus solfataricus on glycogen-mimic substrates, branched maltotetraosyl-β-cyclodextrin (Glc₄-β-CD), and natural glycogen to better understand substrate transglycosylation and the effect thereof on glycogen debranching in microorganisms. The validation test of Glc₄-β-CD as a glycogen mimic substrate showed that it followed the breakdown process of the well-known yeast and rat liver extract. TreX catalyzed both hydrolysis of α-1,6-glycosidic linkages and transglycosylation at relatively high (>0.5 mM) substrate concentrations. TreX transferred maltotetraosyl moieties from the donor substrate to acceptor molecules, resulting in the formation of two positional isomers of dimaltotetraosyl-α-1,6-β-cyclodextrin [(Glc₄)₂-β-CD]; these were 6¹,6³- and 6¹,6⁴-dimaltotetraosyl-α-1,6-β-CD. Use of a modified Michaelis-Menten equation to study substrate transglycosylation revealed that the k_cat and K_m values for transglycosylation were 1.78 × 10³ s⁻¹ and 3.30 mM, respectively, whereas the values for hydrolysis were 2.57 × 10³ s⁻¹ and 0.206 mM, respectively. Also, enzyme catalytic efficiency (the k_cat/K_m ratio) increased as the degree of polymerization of branch chains rose. In the model reaction system of Escherichia coli, glucose-1-phosphate production from glycogen by the glycogen phosphorylase was elevated ∼1.45-fold in the presence of TreX compared to that produced in the absence of TreX. The results suggest that outward shifting of glycogen branch chains via transglycosylation increases the number of exposed chains susceptible to phosphorylase action. We developed a model of the glycogen breakdown process featuring both hydrolysis and transglycosylation catalyzed by the debranching enzyme.     

Assay for enzyme activity by following the absorbance change of pH-indicators

Based on the absorbance change of indicators with the concentration of hydrogen ion released from an enzyme-catalyzed reaction, a convenient colorimetric method was established for the assay of acidic phospholipase A₂ and glycogen phosphorylase b. Brilliant yellow and bromothymol blue were chosen as indicators for assays of acidic phospholipase A₂ and glycogen phosphorylase b by following the absorbance changes at 495 and 615 nm, respectively. The method is simple, sample-saving, sensitive and valid for a wide range of enzyme concentrations. It can be extended for assaying other enzymes catalyzing reactions with hydrogen ion concentration changes.     

Control of platelet glycogenolysis activation of phosporylase kinase by calcium

An apparent enigma during platelet aggregation is that increased glycogenolysis occurs despite a fall in cyclic AMP levels. Activation by a classical cascade is therefore unlikely, and an alternative stimulus for phosphorylase a formation was sought. It was found that low levels of Ca²⁺ markedly activate phosphorylase b kinase from human platelets, with a K_a of 0.89 μM Ca²⁺, which is similar to that for the skeletal muscle enzyme. The kinase activity is unstable, and on enzyme ageing there is a 50% loss in activity with the K_a decreasing to 0.33 μM Ca²⁺.In unstimulated platelets, phosphorylase a was 13.3% of total measured activity, and glycogen synthetase I was 32.3%. Aggregation induced by ADP did not change the percentage of I synthetase, while increasing that for phosphorylase a. Dibutyryl cyclic AMP did, as expected, increase the percentage of both phosphorylated enzymes.These findings suggest that the natural activator of platelet glycogenolysis during aggregation is Ca²⁺, which directly stimulates phosphorylase b kinase without altering glycogen synthetase activity. The cyclic AMP-dependent protein kinase does not appear to be involved.     

Studies on phosphorylase isozymes in lower vertebrates. Purification and properties of lamprey phosphorylase.

Skeletal muscle phosphorylase b has been purified from lamprey, Entosphenus japonicus, to a state of homogeneity as judged by the criterion of sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. The enzyme was completely dependent on AMP for activity and converted into the a form by rabbit muscle phosphorylase kinase in the presence of ATP and Mg²⁺. The subunit molecular weight determined by SDS-gel electrophoresis was 94,000 ± 1,600 (SE). The enzyme activity was stimulated by Na₂SO₄, but was not affected by mercaptoethanol. The K_m values of the a form for glucose 1-phosphate and glycogen were 3.5 mm and 0.13%, respectively, and those of the b form for glucose 1-phosphate, glycogen, and AMP were 15 mm, 0.4%, and 0.1 mm, respectively. These values were smaller than those reported with lobster phosphorylase and greater than those reported with mammalian skeletal muscle phosphorylases. Electrophoretic and immunological studies have indicated that lamprey phosphorylase b exists as a single molecular form in skeletal muscle, heart, brain, and kidney. Rabbit antibody against lamprey phosphorylase cross-reacted with phosphorylases from skate and shark livers more intensely than with those from skeletal muscles.     

Characterization of the spinach leaf phosphorylases

The chloroplastic and the cytoplasmic phosphorylases were purified and their kinetic properties characterized. The cytoplasmic enzyme was purified to homogeneity via affinity chromatography on a glycogen-Sepharose column. Subunit molecular weight studies indicated a value of 92,000, whereas a native molecular weight value of 194,000 was obtained by sucrose density gradient centrifugation. The chloroplast enzyme's native molecular weight was determined to be 203,800. The cytoplasmic enzyme shows the same V_max for maltopentaose, glycogen, amylopectin, amylose, and debranched amylopectin but is only slightly active toward maltotetraose. The K_m for phosphate at pH 7.0 is 0.9 millimolar and for glucose-1-phosphate, 0.64 millimolar. The K_m values for phosphorolysis of amylopectin, amylose, glycogen, and debranched amylopectin are 26, 165, 64, and 98 micrograms per milliliter, respectively. In contrast, the relative V_max values for the chloroplast enzyme at pH 7.0 are debranched amylopectin, 100, amylopectin, 63.7, amylose, 53, glycogen, 42, and maltopentaose, 41. K_m values for the above high molecular weight polymers are, respectively, 82, 168, 122 micrograms per milliliter, and 1.2 milligrams per milliliter. The K_m value for inorganic phosphate is 1.2 millimolar. The chloroplastic phosphorylase appears to have a lower apparent affinity for glycogen than the cytoplasmic enzyme. The results are discussed with respect to previous findings of multiple phosphorylase forms found in plant tissues and to possible regulatory mechanisms for controlling phosphorylase activity.     

A novel inhibitor of glycogen phosphorylase from crayfish hepatopancreas

• 1.1. A novel glycogen phosphorylase inhibitor was partially purified from crayfish hepatopancreas.
• 2.2. The inhibitor was found only in two species of crayfish examined, and not in lobster, fresh and salt water clams, mussels or cockroaches.
• 3.3. The inhibitor is a small protein (M_r = 23,000) which did not show proteolytic activity.
• 4.4. Preliminary kinetic analysis of the inhibitory mechanism indicated that it bound to both glycogen and the glycogen phosphorylase protein.
• 5.5. Inhibitor binding to glycogen resulted in a competitive inhibition pattern with respect to glycogen phosphorylase (inhibition constant of ca 10 μg/ml).
• 6.6. The inhibitor also bound glycogen phosphorylase directly with a binding coefficient of 100 μg/ml resulting in a partially non-competitive inhibition pattern with respect to phosphate.

     

Control of glycogen synthase and phosphorylase by insulin in rat adipocytes which is unrelated to the concentration of cyclic AMP

Incubation of adipocytes in glucose-free medium with adrenocorticotrophic hormone, epinephrine, isoproterenol, or norepinephrine increased the concentration of cyclic AMP and the percentage of phosphorylase a activity, and decreased the percentage of glycogen synthase I activity. Glucose was essentially without effect on glycogen synthase or phosphorylase in either the presence or absence of epinephrine. Although glucose potentiated the action of insulin to activate glycogen synthase, the hexose did not enhance the effectiveness of insulin in the presence of epinephrine. Likewise, glucose did not increase the ability of insulin to oppose the activation of phosphorylase by epinephrine.The activation of glycogen synthase by insulin was not associated with a decrease in the concentration of cyclic AMP. Insulin partially blocked the rise in cyclic AMP due to isoproterenol, adrenocorticotrophic hormone, and norepinephrine. The maximum effects of isoproterenol on glycogen synthase and phosphorylase were observed when the concentration of cyclic AMP was increased twofold. However, insulin clearly opposed the changes in enzyme activity produced by isoproterenol (and also adrenocorticotrophic hormone, epinephrine and norepinephrine) even though concentrations of cyclic AMP were still increased three- to fourfold. Nicotinic acid opposed the increases in cyclic AMP due to adrenocorticotrophic hormone, isoproterenol and norepinephrine to the same extent as insulin; however, nicotinic acid was ineffective in opposing the activation of phosphorylase and inactivation of glycogen synthase produced by these agents. Thus, it is unlikely that the effects of insulin on glycogen synthase and phosphorylase result from an action of the hormone to decrease the concentration of cyclic AMP.     

Calcium-dependent regulation of glycogen synthase activity in a muscle glycogen particle

The calcium-dependent inactivation of glycogen synthase in an isolated glycogen-protein complex (glycogen pellet) from rabbit skeletal muscle has been investigated. Addition of 1 mm Ca²⁺, 10 mm Mg²⁺, and 1 mm ATP-γ-S to a concentrated suspension of glycogen pellet resulted in a rapid activation of glycogen phosphorylase concomitant with an inactivation of glycogen synthase. These conversion reactions were blocked by ethylene glycol bis(β-aminoethyl ether) N, N′-tetraacetic acid or by pretreatment of the complex with an antiserum to purified phosphorylase kinase. These data suggest that in the glycogen-protein complex, which may be a functional unit of glycogen metabolism in vivo, phosphorylase kinase can catalyze a Ca²⁺-dependent activation of glycogen phosphorylase synchronized with an inactivation of glycogen synthase. If under similar conditions phosphoprotein phosphatase activity was assayed using exogenous [³²P]phosphorylase, there was an apparent inactivation of the phosphatase. Evidence is presented that this apparent inactivation of phosphatase was due to an accumulation of endogenous phosphorylase a which acted as an inhibitor to the exogenous [³²P]-phosphorylase.     

Dephosphorylation of skeletal muscle phosphorylase,glycogen synthase,and phosphorylase kinase β-subunit by a Mn2+-activated protein phosphatase

An Mn²⁺-activated phosphoprotein phosphatase of M_r = 80,000 from rabbit muscle catalyzes the dephosphorylation of skeletal muscle proteins that are phosphorylated by either phosphorylase kinase or cAMP-dependent protein kinase. Phosphorylase or glycogen synthase labeled by phosphorylase kinase at seryl residues 14 or 7, respectively, are both dephosphorylated by the phosphatase. Phosphorylase a and glycogen synthase compete with one another for the phosphatase. The phosphatase discriminates between different sites labeled by the cAMP-dependent protein kinase: glycogen synthase phosphorylated either to 1.0 or 1.8 mol phosphate/mol, or phosphorylase kinase phosphorylated on its β-subunit serve as substrates for the phosphatase, but the phosphorylase kinase α-subunit, the phosphorylated phosphatase inhibitor 1, or casein do not. Histone fraction IIA, phosphorylated by the catalytic subunit, was a poor substrate even at a concentration of 100 μm. Phosphorylation of the α-subunit of phosphorylase kinase had no influence on the kinetics of dephosphorylation of the β-subunit. Thus, the M_r = 80,000 phosphatase meets the functional definition of a protein phosphatase 1 [Cohen, P. (1978) Curr. Top. Cell. Regul.14, 117–196]. Furthermore, from a comparison of the known phosphorylated sites of these proteins, it appears that the phosphatase discriminates between different sites present in the phosphoproteins tested on the basis of the K_m values for the reactions. It displays a preferential activity toward proteins with a primary structure wherein basic residues are two positions amino-terminal from the phosphoserine, AgrLysX-YSer(P) or LysArgX-YSer(P), rather and one residue away, ArgArgX-Ser(P).     

Purification and characterization of the Novikoff hepatoma glycogen phosphorylase and its relations to a fetal form

The Novikoff hepatoma glycogen phosphorylase b has been purified over 300-fold, free of glycogen synthetase, some of its properties have been studied, and its relationship to fetal forms of rat muscle and liver phosphorylase has been established immunochemically. Its molecular weight is approximately 200,000, and, like the liver but unlike the muscle isozyme, it does not dimerize on conversion to the a form. However, it differs from the liver isozyme in being activated by AMP (K_a = 0.2 mM) and in not being activated by sulfate ion. Antibody to the adult rat muscle phosphorylase did not inhibit the activity of the tumor or liver isozyme. Although antibody to liver or hepatoma phosphorylase had no effect on adult muscle phosphorylase, each of these antibodies partially inhibited the other enzyme. These findings indicate the presence of some liver isozyme in the tumor, and this was confirmed by isoelectric focusing. Rat liver and muscle phosphorylase (and synthetase) were low during embryonal development but rose rapidly at or shortly after birth. Immunochemical studies revealed that both fetal liver and fetal muscle phosphorylases are immunologically identifiable with the tumor enzyme; and the fetal form is also present as a major form in rat kidney and brain.     

A kinetic study of glycogen phosphorylase b from the mantle tissue of Mytilus galloprovincialis,Lmk

• 1.1. In order to assign a meaningful role to the phosphorolytic pathway in Mytilus glycogen metabolism the kinetic mechanism of phosphorylase b, and its allosteric control, were studied.
• 2.2. The kinetic parameters of phosphorylase b from the mussel Mytilus galloprovincialis were determined. Michaelis constants (K_m or S_0.5) were in the range of 0.32–2.49 mg/ml for glycogen, 7–16 mM for P_i and 114–423 μM for AMP. In the direction of glycogen synthesis, the K_m value for glucose-1-P was approximately 180 mM.
• 3.3. The enzyme displayed homotropic co-operativity towards the binding of co-substrate and AMP (Hill coefficients of 2 and 1.4, respectively) and heterotropic co-operativity between substrates and AMP.
• 4.4. The concentration of glycogen in the Mytilus mantle is between 38- and 125-fold higher than the apparent K_m of phosphorylase b; the concentration of AMP varies throughout the year from 10 to 175 μM, up to a value close to the apparent K_m for the effector.
• 5.5. The apparent K_m for P_i is close to the concentration found in the mantle. This ligand showed more important regulatory effects than the effector AMP.

     

Kinetics of glycogen phosphorylase a from the muscle of the blue crab, Callinectes danae

The kinetics of purified glycogen phosphorylase a from the muscle of the blue crab (Callinectes danae) were studied in the direction of glycogen synthesis, and in the direction of glycogen degradation with P_i or arsenate as substrates. The effects of AMP, UDPG, G-6-P, glucose, and arsenate on the appropriate systems were studied. AMP is an activator of the enzyme. Inhibition by UDPG with respect to P_i changes from noncompetitive to competitive when AMP is added; it changes from noncompetitive to mixed with respect to glycogen when AMP is added. G-6-P is a competitive inhibitor of G-1-P and arsenate. Inhibition by glucose with respect to glycogen changes from noncompetitive to competitive when AMP is added in the direction of glycogen breakdown; it is noncompetitive with respect to P_i. Arsenate is a competitive inhibitor with respect to P_i. The K_m for AMP increases in the presence of UDPG, and decreases with increasing concentrations of P_i or glycogen. We propose a model in which the enzyme bears three interacting sites: an active site, an activator (AMP) site, and an inhibitor (glucose) site. The active site has three subsites: one for P_i, one for glycogen, and one for a glucose moiety which may be part of the substrates or inhibitors.     

Liver glycogen synthase phosphatase and phosphorylase phosphatase activities in vitro following glucose and glucagon administration

Correlation of the changes in phosphorylase a concentration with the synthase phosphatase velocity in a glycogen particle preparation in the presence of EDTA revealed that the initial synthase phosphatase rate was greatest in extracts from glucose-treated rats and least in extracts from glucagon-treated rats. In all cases the velocity increased with time and with a decrease in phosphorylase a. However, a threshold release of phosphatase activity when phosphorylase a reached a critical level was not observed. The data are compatible with either an independent regulation of synthase phosphatase by glucose and glucagon or regulation of the activity by phosphorylase over a range of phosphorylase a concentrations.     

Effect of 1,2-dimethoxyethane on potato phosphorylase for polysaccharide specificity

Potato phosphorylase normally utilizes starch as a substrate but will not use glycogen effectively. In the presence of 20% 1,2-dimethoxyethane, this specificity is lost because of marked activation with glycogen and some inhibition with starch. The effect of dimethoxyethane on β-amylase was the same for both starch and glycogen. These was no contaminating enzyme that would make glycogen more “starch-like.” The 20-fold decrease in K_m for glycogen, a change in pH profile, and elimination of inhibition by cyclodextrin suggest that dimethoxyethane causes a change in phosphorylase structure.     

Interaction between glycogen and glycogen phosphorylase of Tetrahymena

The glycogen phosphorylase of Tetrahymena pyriformis complexes with glycogen as judged by its elution pattern from columns of Sepharose 6B. Complex formation does not occur with starch, amylose, or amylopectin, and neither do these polyglucans serve as primers for the enzyme. To study the association between the phosphorylase and glycogen particles in situ, Tetrahymena were grown under differing physiological conditions, phosphorylase was isolated and chromatographed on a Sepharose 6B column. Phosphorylase activity isolated from cells grown in the absence of glucose was only partially associated with glycogen, while in cells exposed to glucose for 30 min or more all the phosphorylase activity was associated with glycogen. The effects of culture age and anaerobiosis on the relative amounts of free and glycogen-bound enzyme in the cells were also studied. It was concluded from the in vivo experiments that there was no simple relation between the fraction of enzyme bound to glycogen and between cell glycogen content.     

Purification and characterization of glycogen phosphorylase A and B from the freeze-avoiding gall moth larvae Epiblema scudderiana

The active a and inactive b forms of glycogen phosphorylase from cold-hardy larvae of the gall moth, Epiblema scudderiana, were purified using DEAE⁺ ion exchange and 3-5-AMP-agarose affinity chromatography. Maximum activities for glycogen phosphorylases a and b were 6.3±0.74 and 2.7±0.87 mol glucose-1-P·min^-1·g wet weight^-1, respectively, in -4°C-acclimated larvae. Final specific activities of the purified enzymes were 396 and 82 units·mg protein^-1, respectively. Both enzymes were dimers with native molecular weights of 215000±18000 for glycogen phosphorylase a and 209000±15000 for glycogen phosphorylase b; the subunit molecular weight of both forms was 87000±2000. Both enzymes showed pH optima of 7.5 at 22°C and a break in the Arrhenius relationship with a two- to four-fold increase in activation energy below 10°C. Michaelis constant values for glycogen at 22°C were 0.12±0.004 mg·ml^-1 for glycogen phosphorylase a and 0.87±0.034 mg·ml^-1 for glycogen phosphorylase b; the Michaelis constant for inorganic phosphate was 6.5±0.07 mmol·l^-1 for glycogen phosphorylase a and 23.6 mmol·l^-1 for glycogen phosphorylase b. Glycogen phosphorylase b was activated by adenosine monophosphate with a K _a of 0.176±0.004 mmol·l^-1. Michaelis constant and K _a values decreased by two- to fivefold at 5°C compared with 22°C. Glycerol had a positive effect on the Michaelis constant for glycogen for glycogen phosphorylase a at intermediate concentrations (0.5 mol·l^-1) but was inhibitory to both enzyme forms at high concentrations (2 mol·l^-1). Glycerol production as a cryoprotectant in E. scudderiana larvae is facilitated by the low temperature-simulated glycogen phosphorylase b to glycogen phosphorylase a conversion and by positive effects of low temperature on the kinetic properties of glycogen phosphorylase a. Enzyme shut-down when polyol synthesis is complete appears to be aided by strong inhibitory effects of glycerol and KCl on glycogen phosphorylase b.Abbreviations E _a activation energy - GPa glycogen phosphorylase a - GPb glycogen phosphorylase b - h Hill coefficient - I ₅₀ concentration of inhibitor that reduces enzymes velocity by 50% - K _a concentration of activator that produces half-maximal activation of enzyme activity - K _m Michaelis-Menten substrate affinity constant - MW molecular weight - PEG polyethylene glycol - P_i morganic phosphate - SDS PAGE sodium dodecyl sulphate polyacrylamide gel electrophoresis - V _max enzyme maximal velocity     

Purification and properties of glycogen phosphorylase from Streptococcus salivarius

Glucose-grown cells of Streptococcus salivarius have been shown to contain a polyglucose phosphorylase which had maximum activity in the stationary phase of growth. Despite the fact that activity in crude cell-free extracts was two- to threefold greater in the presence of corn dextrin than with oyster glycogen, subsequent purification (200-fold) of the enzyme from the soluble fraction of the organism by protamine sulfate treatment, ammonium sulfate fractionation (30–50%), ion exchange chromatography on DEAE-cellulose and gel filtration on Sephadex G-200 demonstrated that this dextrin/glycogen activity was associated with a single enzyme. Since glucose-grown cells of S. salivarius are known to synthesize a typical glycogen polymer, the enzyme was named: glycogen phosphorylase. The purified enzyme preparation was devoid of phosphoglucomutase and ADP-glucose pyrophosphorylase, but contained a small amount of ADP-glucose: α-1,4 glucan transferase activity. The enzyme was stable at ?10 °C in the presence of 0.2 m NaF, while the pH optimum for the enzyme was 6.0 both with glycogen and with dextrin. With the purified enzyme, corn dextrin was the best primer, both in the direction of synthesis and in the direction of phosphorolysis, being 1.8–1.9 times more effective than purified S. salivarius glycogen. When the enzyme was assayed in the direction of glycogen synthesis, a K_m value of 3.4 mm was obtained for glucose-1-P, while the values for S. salivarius glycogen, oyster glycogen and corn dextrin were 25, 42, and 40 mg/ml, respectively. In the direction of phosphorolysis, K_m values were 20 mm for P_i obtained with oyster glycogen, 25 mm for P_i with corn dextrin, and 20 mg/ml and 26 mg/ml for oyster glycogen and corn dextrin, respectively. Present data suggests no involvement of -SH groups in enzyme catalysis, while the enzyme was inhibited by divalent ions with the severest inhibition being observed with Ca²⁺, Zn²⁺ and Fe²⁺. The two ion chelators, EDTA and EGTA, had no effect on enzyme activity.     

Rat liver glycogen metabolism in the perinatal period

The correlation between blood glucose levels, the concentration of glycogen, the activities of glycogen sythase and phosphorylase and their respective kinases and phosphatases was examined in liver of rat fetuses between day 18 of gestation and one day after birth. Between day 18 and 21 there is a rapid increase in the concentration of glycogen and in the activity of synthase a and a much slower increase in the activity of phosphorylase a. The activity of the respective kinases increased rapidly during this period and reached maximun on day 21. The activity of synthase phosphatase and phosphorylase phosphatase increased after day 18, to reach a maximum on day 19 and 20, respectively, but decreased again towards day 21. The possibility that the changes in glycogen concentration and enzyme activities were related to an effect of glucose of AMP on the respective phosphatases was considered. It was found that the Km of phosphatase for glucose in the prenatal period was 5–7 mM, as in the adult. Since the level of blood glucose during this period was constant (2.8 mM), an effect of glucose on phosphatase activity seems unlikely. AMP concentration increased between day 18 and 21 from 6–15 nmol/g. In view of the low level of phosphorylase a activity during this period, the increase in AMP concentration is not considered to be important in the regulation of glycogen breakdown at this time.Immediately after birth blood glucose levels dropped to 5 mg/dl. This was accompanied by a rapid decrease in glycogen concentration and in the activity of glycogen synthase and a rise in phosphorylase activity. Blood glucose levels returned to the initial level within 1 h after birth, whereas the changes in glycogen concentration and enzyme activities continued for at least 3 h after birth. On day 22 all parameters examined had reached the level found in adult rat liver.It is suggested that the rapid changes observed immediately after birth are due to an effect of hypoglycemia mediated by hormones and cannot be ascribed to direct effects of metabolites on the enzyme systems involved.     

Purification and Properties of Mesophyll and Bundle Sheath Cell alpha-Glucan Phosphorylases from Zea mays L. : Equivalence of the Enzymes with the Cytosol and Plastid Phosphorylases from Spinach

Two major α-glucan phosphorylases (I and II) from leaves of the C₄ plant corn (Zea mays L.) were previously shown to be compartmented in mesophyll and bundle sheath cells, respectively (C Mateyka, C Schnarrenberger 1984 Plant Sci Lett 36: 119-123). The two enzymes were separated by chromatography on DEAE-cellulose and purified to homogeneity by affinity chromatography on immobilized starch, according to published procedures, as developed for the cytosol and chloroplast phosphorylase from the C₃ plant spinach. The two α-glucan phosphorylases have their pH optimum at pH 7. The specificity for polyglucans was similar for soluble starch and amylopectin, however, differed for glycogen (K_m = 16 micrograms per milliliter for the mesophyll cell and 250 micrograms per milliliter for the bundle sheath cell phosphorylase). Maltose, maltotriose, and maltotetraose were not cleaved by either phosphorylase. If maltopentaose was used as substrate, the rate was about twice as high with the bundle sheath cell phosphorylase, than with the mesophyll cell phosphorylase. The phosphorylase I showed a molecular mass of 174 kilodaltons and the phosphorylase II of 195 kilodaltons for the native enzyme and of 87 and of 53 kilodaltons for the SDS-treated proteins, respectively. Specific antisera raised against mesophyll cell phosphorylase from corn leaves and against chloroplast phosphorylase from spinach leaves implied high similarity for the cytosol phosphorylase of the C₃ plant spinach with mesophyll cell phosphorylase of the C₄ plant corn and of chloroplast phosphorylase of spinach with the bundle sheath cell phosphorylase of corn.