Effect of fructose 1-phosphate on the activation of liver glycogen synthase.
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Animals↗
Biomedical subjects
G Bot
Publications and source records attributed to G Bot.
Hormonal regulation of phosphorylase phosphatase activity in rat liver.
The effect of glucagon and insulin on rat liver phosphorylase phosphatase activity in vivo was investigated. The activity of phosphatase was found to decrease following the administration of glucagon and increase with insulin in a reversible manner. No change was detected in the activity of heat-stable phosphatase inhibitors in the hormone-treated samples. Liver protein kinases (regulatory subunit of cAMP-dependent protein kinase and/or Ca2+-dependent phosphorylase kinase) are suggested to regulate the activity of hepatic phosphorylase phosphatase (type 1 and 2A).
Animals↗
Regulatory subunit of type II cAMP-dependent protein kinase as substrate and inhibitor of protein phosphatase-1 and -2A.
The dissociated regulatory subunit (RII) of autophosphorylated cAMP-dependent protein kinase II was dephosphorylated by the catalytic subunits of protein phosphatase-1 and -2A (phosphatase-1c and -2Ac) and by a high-Mr polycation-dependent form of phosphatase-2A (2Ao) with Km values of 5, 0.3 and 1 microM, respectively. Dissociation of protein kinase by cAMP preferentially increased the dephosphorylation of RII by phosphatase-1c, whereas polycations (histone Hl or polybrene) markedly stimulated phosphatase-2Ac and -2Ao even in the absence of cAMP. Thiophosphorylated RII inhibited the dephosphorylation of phosphorylase a by these phosphatases with half-maximum inhibitory concentrations of 0.1-0.36 microM.
Animals↗
Formation of partially phosphorylated phosphorylase in isoproterenol stimulated rat hearts.
Phosphorylase ab hybrid was demonstrated in perfused rat hearts and during the in vitro conversion of purified rat heart phosphorylase b. Phosphorylase ab hybrid was determined in rat heart extracts by the activating effect of AMP in the presence of caffeine. These results were confirmed by the quantitative determination of incorporated 32P in vitro and through the characteristic inhibition of ab hybrid by glucose-6-phosphate. As shown by our results, in aerobically perfused control hearts only the ab hybrid represents the active form of phosphorylase, its activity reaching about 20% of the total. In response to isoproterenol (5-1000 ng), the amount of ab hybrid rose to about 30-40%, preceding the rise of the a form, which increased in a dose-dependent manner up to 45% of the total. The great sensitivity of the ab form to AMP activation and glucose-6-phosphate inhibition supports its physiological significance in heart under in vivo conditions as well. Our results strongly suggest that the activity ratio -AMP/ + AMP reflects rather the percentage ratio of phosphorylated subunits than that of the activated (partially or totally phosphorylated) phosphorylase molecules.
Adenosine Monophosphate↗
Structural and functional properties of Drosophila melanogaster phosphorylase: comparison with the rabbit skeletal muscle enzyme.
Glycogen phosphorylase isolated from Drosophila melanogaster contains one pyridoxal 5'-phosphate per subunit; the coenzyme is in a hydrophobic environment. Fruit-fly phosphorylase a has lower KM for glucose-1-phosphate and is less sensitive to allosteric inhibitors than the b form of the enzyme. The amino acid composition of Drosophila phosphorylase differs from that of rabbit skeletal muscle phosphorylase. These two enzymes give distinct one dimensional peptide maps. The distribution of reactive SH-groups is markedly different in the insect and vertebrate phosphorylase. Fruit-fly phosphorylase a is dephosphorylated by either rabbit or Drosophila protein phosphatase-1 at a slower rate than rabbit muscle phosphorylase a.
Amino Acids↗
Effect of fructose 1-phosphate on the activation of liver glycogen synthase.
The activation (dephosphorylation) of glycogen synthase and the inactivation (dephosphorylation) of phosphorylase in rat liver extracts on the administration of fructose were examined. The lag in the conversion of synthase b into a was cancelled, owing to the accumulation of fructose 1-phosphate. A decrease in the rate of dephosphorylation of phosphorylase a was also observed. The latency re-appeared in gel-filtered liver extracts. Similar latency was demonstrated in extracts from glucagon-treated rats. Addition of fructose 1-phosphate to the extract was able to abolish the latency, and the activation of glycogen synthase and the inactivation of phosphorylase occurred simultaneously. Fructose 1-phosphate increased the activity of glycogen synthase b measured in the presence of 0.2-0.4 mM-glucose 6-phosphate. According to kinetic investigations, fructose 1-phosphate increased the affinity of synthase b for its substrate, UDP-glucose. The accumulation of fructose 1-phosphate resulted in glycogen synthesis in the liver by inducing the enzymic activity of glycogen synthase b in the presence of glucose 6-phosphate in vivo and by promoting the activation of glycogen synthase.
Animals↗
Separation of rabbit liver latent and spontaneously active phosphorylase phosphatases by chromatography on heparin-sepharose.
Latent and spontaneously active forms of phosphorylase phosphatase were separated by heparin-Sepharose chromatography of rabbit liver extract. The latent enzyme had an absolute polycation (histone H1, polybrene) requirement for the activity assayed with phosphorylase a and phosphorylase kinase substrates. Ethanol treatment resulted in the activation of both phosphatases by dissociating of 150-180 kDa holoenzymes to 33-38 kDa catalytic subunits as judged by gel filtration. The latent and spontaneously active phosphatases were differentiated according to their abilities to dephosphorylate the alpha and the beta subunits of phosphorylase kinase and sensitivities to inhibition by inhibitor-2 or heparin, and were classified as type-2A and type-1 phosphatases, respectively.
Animals↗
Effects of acidic and basic macromolecules on the activity of protein phosphatase-1.
The dephosphorylation of phosphorylase a by the catalytic subunit of protein phosphatase-1 obtained from rabbit skeletal muscle is inhibited by heparin in a noncompetitive manner with respect to phosphorylase a (Ki = 8 micrograms/ml). The inhibitory effect of heparin is also observed in the presence of effectors (e.g., glucose and AMP) modifying the dephosphorylation of phosphorylase a. Heat-stable protein inhibitors of protein phosphatase-1 can develop their inhibitory effect of the activity of protein phosphatase-1 even in the presence of heparin. The inhibitory effect of heparin and the heat-stable inhibitor-2 of phosphatase is additive. Polybrene, a heparin antagonist, prevented phosphatase-1 from the inhibition caused by heparin or the inhibitors. Proteins with basic character, histone fractions (H1, H3) and protamine sulfate, can counteract with the inhibitory effect of heparin, but they cannot intercept the actions of inhibitor-1 or -2.
Animals↗
Heparin inhibits the activity of protein phosphatase-1.
Heparin inhibited the dephosphorylation of rabbit skeletal muscle or liver phosphorylase a by protein phosphatase-1. Other glycosaminoglycans (chondroitin sulfates) and their constituents were found to be without effect. The chromatography of a partially purified phosphatase preparation on heparin-Sepharose CL-6B resulted in a fraction that did not bind to the matrix and its activity was not inhibited by heparin or inhibitor-1. The phosphatase bound to heparin-Sepharose was eluted by 0.2 M NaCl and was inhibited by heparin or inhibitor-1.
Animals↗
Ca2+-dependent stimulation of muscle and liver phosphorylase kinase by heparin.
Heparin stimulates the activity of nonactivated and activated skeletal muscle phosphorylase kinase in a Ca2+-dependent manner. The stimulatory effect of heparin on the activity of nonactivated phosphorylase kinase is also expressed in the presence of calmodulin and glycogen. Heparin acted in synergism with glycogen. Heparin increases the affinity of phosphorylase kinase to Ca2+ 5-12 fold depending upon the activation conditions. Ca2+ influences the stimulation of liver phosphorylase kinase by heparin in a similar way.
Animals↗
Effect of immobilization on some glycolytic enzymes of skeletal muscle.
The effect of immobilization by plaster cast was studied on the activities of phosphoglucomutase (PGM), pyruvate kinase ( PyK ) and cAMP-dependent protein kinase in fast (extensor digitorum longus [EDL]), and slow (soleus) muscles of rats. In control untreated animals, PGM and PyK activities are 3 and 5 fold higher respectively in EDL than in soleus in correlation with the high glycolytic activity of fast muscles. During the four weeks period of immobilization a 20% decrease occurred in PGM activity, to which no limiting role in glycolysis is attributed, while PyK which has a regulatory function, showed a 35% decrease in EDL; at the same time in the soleus the activity of these enzymes did not change. The decrease of PGM and PyK activity in EDL diminished the difference between the slow and fast muscles, and it was evaluated as a tendency to dedifferentiation (transformation). The activity of cAMP-dependent protein kinase, in contrast to the glycolytic enzymes, was higher in the soleus than in the EDL and during immobilization it did not change significantly in either the muscles.
Animals↗
Structural changes in phosphorylase b as revealed by proteolysis with subtilisin BPN'.
The proteolysis of rabbit skeletal muscle phosphorylase b was studied with Sepharose 4B bound subtilisin BPN' in the absence and presence of various ligands. The proteolysis was carried out at pH 7.0 and pH 8.5 and was followed by measuring phosphorylase b activity and by SDS gel electrophoresis. The effect of ligands proved to be qualitatively the same at both pH values. It was found that AMP and alpha-D-glucose-1-phosphate accelerated the inactivation of phosphorylase b by subtilisin, two main proteolytic products (Mr 70 000 and 30 000) were formed in the presence of these ligands. IMP and glycogen protected phosphorylase b against proteolytic attack. Subtilisin treatment in the presence of D-glucose, caffeine and D-glucose-6-phosphate produced a reproducible increase (about 20%) of phosphorylase b activity. This "activation" resulted in an increased Vmax of phosphorylase b though did not alter the subunit size, the aggregation state and the ligand binding capacity of the enzyme.
Animals↗
Heterotropic interactions of AMP and glucose binding sites in phosphorylase a are destroyed by limited proteolysis.
Subtilisin BPN' hydrolyses a single peptide bond in phosphorylase a. The two proteolytic fragments are attached to each other by noncovalent bonds in solution as shown by gel filtration and ultracentrifugation studies. The subtilisin nicked phosphorylase a is inactive, however, still binds AMP and glucose as judged by equilibrium dialysis and fluorescence experiments. The modified enzyme can be dephosphorylated by protein phosphatase and AMP is an effective inhibitor of the dephosphorylation reaction. Glucose cannot cancel the AMP inhibition as well as cannot expel AMP from the nucleotide binding site. Thus a single nick in the polypeptide chain breaks the "communication" between the two ligand binding domains.
Adenosine Monophosphate↗
Limited proteolysis of glycogen phosphorylase a by subtilisin BPN'.
The limited proteolysis of rabbit skeletal muscle phosphorylase a was undertaken with subtilisin BPN' immobilized to Sepharose 4B. The effect of substrates, activators and inhibitors of phosphorylase a was investigated by monitoring the changes in phosphorylase activity in the SDS gel electrophoretic pattern and in the 32P-content of 32P-labeled phosphorylase a. Phosphorylase a loses its activity upon subtilisin treatment. All ligands tested protect phosphorylase a activity against subtilisin action, probably by inducing structural changes in the tower loop of the enzyme. Glucose-6-P significantly accelerates [32P]peptide release from phosphorylase a through altering the structure of the N-terminal tail segment. The two subunits of dimeric phosphorylase a are held together by strong interactions--deduced from the correlation of the rate of proteolysis and the disappearance of catalytic activity.
Adenosine Monophosphate↗
Limited proteolysis by subtilisin reveals structural differences between phosphorylase a and b.
The limited proteolysis of rabbit skeletal muscle phosphorylase a and b was studied with subtilisin BPN' immoblized to Sepharose 4B. The activity of phosphorylase b is nearly resistant to subtilisin under the conditions (pH 7.0, 30 degrees C) where phosphorylase a rapidly loses its activity. The pH profile of phosphorylase a and b digestion is different. Proteolytic fragments of mol. wt 70,000 and 30,000 generated from phosphorylase a, while mol. wt 80,000 and 70,000 generated from phosphorylase b can be detected by SDS gel electrophoresis. Addition of AMP to phosphorylase b favours a conformation similar to--but not identical with--phosphorylase a as recognised by subtilisin action.
Adenosine Monophosphate↗
Ligand effects on the dephosphorylation of heart and skeletal muscle specific phosphorylases.
Pseudo first order rate constants were determined for the dephosphorylation of heart and skeletal muscle specific phosphorylase a isoenzymes isolated from rabbit and pig using rabbit muscle phosphorylase phosphatase (mol. wt 34,000). The rate constants determined in the absence of ligands, were 4-5 fold lower for heart specific phosphorylases than for skeletal muscle specific ones. Glucose 6-phosphate (0.5-1 mM) enhances the rate of dephosphorylation of heart specific isophosphorylases 3-fold and suspends inhibition by 10(-5) M AMP, however, it has no significant effect on the dephosphorylation of skeletal muscle specific enzymes under the same conditions. Our data support characteristic functional differences between heart and skeletal muscle specific phosphorylases both in rabbit and pig.
Adenosine Monophosphate↗
Effect of immobilization on the enzymes of glycogen metabolism.
Activity of the enzymes regulating glycogen metabolism was determined for glycogen phosphorylase (Ph), phosphorylase kinase (PhK) and glycogen synthetase (GS) in fast extensor digitorum longus (EDL) and slow (soleus) muscles of rats following immobilization by plastering. It was shown that the activity not only of Ph but also of PhK was about 8 times higher in the EDL than in the soleus muscle in correlation with the higher glycogenolytic activity of fast muscles. The activity of GS was approximately similar in the two muscles. PhK activity decreased significantly in the immobilized EDL muscle while in the soleus an early reduction of GS activity ensued. Reduction of the activity of the glycogenolytic enzymes caused dedifferentiation in the muscles with anaerobic metabolism and containing large amounts of glycogenolytic enzymes. The similar rate of Ph and PhK in fast and slow muscles, as well as the similar rate of their reduced activity, during immobilization indicated a close correlation between the two enzymes and raised the possibility of a combined regulation of their turnover.
Animals↗
Ligand effects on the activity of the a form of various mammalian heart specific isophosphorylases.
1. The allosteric sensitivity of heart specific and skeletal muscle specific phosphorylase a-s from rabbit, pig, bovine and dog was compared. 2. 0.2-0.3 mM glucose 6-phosphate exerts 50% inhibition on the heart specific phosphorylase a-s without any inhibitory effect on the skeletal muscle specific ones. 3. AMP decreases the Km for substrates (glucose 1-phosphate, or Pi) of all heart specific phosphorylase a-s. It also enhances the Vmax of pig and bovine phosphorylase a-s 2-fold, but has no effect on the Vmax of rabbit and dog enzymes.
Adenosine Monophosphate↗