Phosphate In Phosphorylase Research Articles

Insulin Stimulates Dephosphorylation of Phosphorylase in Rat Epitrochlearis Muscles

We have investigated the effects of insulin on the phosphorylation of glycogen phosphorylase in skeletal muscle. Rat epitrochlearis muscles were incubated in vitro with 32Pi to label cellular phosphoproteins, before being treated with hormones. Phosphorylase, phosphorylase kinase, and glycogen synthase were immunoprecipitated under conditions that prevented changes in their phosphorylation states. Based on measurements of the activity ratio (-AMP/+AMP) and the 32P content of phosphorylase, 4-8% of the phosphorylase in untreated muscles appeared to be phosphorylated. Epinephrine promoted increases of approximately 4-fold in the 32P content and activity ratio. Neither these effects nor the epinephrine-stimulated increases in phosphorylation of glycogen synthase and phosphorylase kinase were attenuated by insulin. However, insulin at physiological concentrations rapidly decreased the 32P content of phosphorylase in muscles incubated without epinephrine. Results from peptide mapping experiments indicate that phosphorylase was phosphorylated at a single site in both control and insulin on phosphorylase represented a decrease in 32P of approximately 50%. By comparison, the 32P content of glycogen synthase and the beta subunit of phosphorylase kinase were decreased by only 20 and 16%, respectively; the 32P content of the kinase alpha subunit was not affected by insulin. The results provide direct evidence that insulin decreases the amount of phosphate in phosphorylase and phosphorylase kinase. These findings have important implications with respect to both the regulation of glycogen metabolism in skeletal muscle and the mechanism of insulin action.

Functions of the 5'-phosphoryl group of pyridoxal 5'-phosphate in phosphorylase: a study using pyridoxal-reconstituted enzyme as a model system.

Pyridoxal-reconstituted phosphorylase was used as a model system to study the possible functions of the 5'-phosphoryl group of pyridoxal 5'-phosphate (PLP) in rabbit muscle glycogen phosphorylase. Kinetic study was conducted by using competitive inhibitors of phosphite, an activator, and alpha-D-glucopyranose 1-phosphate (glucose-1-P) to study the relationship between the PLP phosphate and the binding of glucose-1-P to phosphorylase. Fluorine-19 nuclear magnetic resonance (19F NMR) spectroscopy of fluorophosphate bound to pyridoxal phosphorylase showed that its ionization state did not change during enzymatic catalysis. Evaluation of the apparent kinetic parameters for the activation of pyridoxal phosphorylase with different analogues having varied pKa2 values demonstrated a dependency of KM on pKa2. Molybdate, capable of binding as chelates in a trigonal-bipyramidal configuration, was tested for its inhibitory property with pyridoxal phosphorylase. On the basis of the results in this study, several conclusions may be drawn: (1) The bound phosphite in pyridoxal phosphorylase and, possibly, the 5'-phosphoryl group of PLP in native phosphorylase do not effect the glucose-1-P binding. (2) One likely function of the 5'-phosphoryl group of PLP in native phosphorylase is acting as an anchoring point to hold the PLP molecule and/or various amino acid side chains in a proper orientation for effective catalysis. (3) The force between the PLP phosphate and its binding site in phosphorylase is mainly electrostatic; a change of ionization state during catalysis is unlikely. (4) Properties of the central atoms of different anions are important for their effects as either activators or inhibitors of pyridoxal phosphorylase.(ABSTRACT TRUNCATED AT 250 WORDS)

Inactivation of glucocorticoid-binding capacity by protein phosphatases in the presence of molybdate and complete reactivation of dithiothreitol.

Glucocorticoid receptor in rat liver cytosol is inactivated (rendered unable to bind steroid) by incubation with calf intestine alkaline phosphatase or highly purified rabbit muscle phosphoprotein phosphatase (phosphorylase phosphate, protein phosphatase C). The receptor is inactivated by both enzymes even when 10 mM sodium molybdate is present. Receptors that are inactivated by phosphatases in the presence of molybdate can be reactivated to the steroid-binding state by addition of dithiothreitol, but receptors that are inactivated in the absence of molybdate cannot be reactivated. These observations suggest that dephosphorylation leads to oxidation of a moiety (-SH) on the receptor that is required for steroid binding. Molybdate apparently preserves the receptor in a form such that reduction returns the receptor to the steroid binding state. We would propose that molybdate may act by complexing with sulfur groups on the receptor.

Determination of sites in rabbit muscle glycogen phosphorylase b modified by an adenosine 5'-monophosphate analog, N6-p-bromoacetaminobenzyladenosine-5'-phosphate.

To identify the residues modified by radioactive N6-p-bromoacetaminobenzyladenosine-5'-phosphate in phosphorylase b, the peptic peptides of the modified enzyme were purified. Two radioactive peptides were isolated, each of which had a cysteine residue modified. The cysteine residue involved in the AMP site was determined to be cystein-317 and the other eactive cysteine residue was cysteine-171.

Pyridoxal phosphate-dependent conformational states of glycogen phosphorylase as probed by interconverting enzymes.

The interaction between pyridoxal 5'-phosphate and the convertible serine of glycogen phosphorylase has been investigated by using: specific interconverting enzymes, phosphorylase kinase and phosphorylase phosphatase; effectors, glucose and glucose 6-phosphate; and a protein kinase and trypsin. Both phosphorylase kinase and phosphorylase phosphatase utilized the native protein while having little influence on the apoprotein. Removal of a peptide containing the critical serine residue gave phosphorylase b' from which the pyridoxal 5'-phosphate in phosphorylase has an important effect on enzymic interconversion.

Factors Contributing to the Absorption and Fluorescence Characteristics of Pyridoxal Phosphate in Glycogen Phosphorylase b

AbstractThe Schiff base formed between salicylaldehyde and n‐hexylamine has absorption bands peaked at 255 and 317 nm in non‐polar solvents. In a polar solvent (ethanol) an additional band appears at 405 nm and a shoulder is resolved at 275 nm. The fluorescence band has a peak at 535 nm in non‐polar solvents (with the exception of CCl4) which is blue shifted to 505 nm in ethanol. These spectral data are similar to those of Schiff bases formed between pyridoxal phosphate and aliphatic amines. On the other hand, the spectral properties of the Schiff base of o‐methoxybenzaldehyde with n‐hexylamine are very different. It may thus be concluded that the nitrogen atom of the aromatic ring does not have to be invoked in explaining the spectral properties of pyridoxal phosphate Schiff bases and of pyridoxal phosphate in glycogen phosphorylase. The hydroxyl group seems however to play an important role in determining the spectral properties of Schiff bases of o‐hydroxyaldehydes. The optical rotatory power of the pyridoxal phosphate in phosphorylase is of comparable magnitude for the chromophore in the ground state or the electronically excited state, indicating similarity of mode of binding of the chromophore to the protein in the two electronic states. Measurements of circular dichroism indicate that the “deformer” imidazole citrate affects the interaction of the pyridoxal phosphate with the protein when the cofactor Schiff base is in the ketoenamine form but not when in the enolimine form.

Glucose as a regulator of glycogen phosphorylase in rat diaphragm: II. Effect of glucose and related compounds on phosphorylase phosphate

1. 1. The mechanism by which glucose causes a decrease in phosphorylase a levels in isolated rat diaphragm was investigated. Although glucose is known to be an inhibitor of both phosphorylases a and b, the effect of glucose could not be explained by a direct inhibitory action of the hexose on these enzymes. In contrast, it has been found that glucose markedly activates phosphorylase phosphatase, thus decreasing the level of phosphorylase a. 2. 2. In addition to glucose, glycogen and Glc-6- P also enhanced phosphatase activity, while Fru-1,6- P 2 and P i were inhibitory. 3. 3. Possible mechanismsof glucose activation of diaphragm phosphorylase phosphatase were investigated. Results indicated that the observed effect cannot be explained by a change in the molecular form of the substrate, phosphprylase a. Glucose may alter the susceptibility of the substrate and/or its partially phosphorylated intermediates to phosphatase action. Alternatively, glucose may directly activate the phosphatase itself.

On the role of pyridoxal 5'-phosphate in phosphorylase. 3. Physicochemical properties and reconstitution of apophosphorylase b.

On the role of pyridoxal 5'-phosphate in phosphorylase. 3. Physicochemical properties and reconstitution of apophosphorylase b.

On the role of pyridoxal 5'-phosphate in phosphorylase. II. Resolution of rabbit muscle phosphorylase b.

On the role of pyridoxal 5'-phosphate in phosphorylase. II. Resolution of rabbit muscle phosphorylase b.

On the role of pyridoxal 5'-phosphate in phosphorylase. I. Absence of classical vitamin B6--dependent enzymatic activities in muscle glycogen phosphorylase.

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTOn the Role of Pyridoxal 5'-Phosphate in Phosphorylase. I. Absence of Classical Vitamin B6—dependent Enzymatic Activities in Muscle Glycogen Phosphorylase*Jerry L. Hedrick and Edmond H. FischerCite this: Biochemistry 1965, 4, 7, 1337–1343Publication Date (Print):July 1, 1965Publication History Published online1 May 2002Published inissue 1 July 1965https://doi.org/10.1021/bi00883a018RIGHTS & PERMISSIONSArticle Views144Altmetric-Citations174LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (717 KB) Get e-Alerts Get e-Alerts

Phosphorylase and Related Enzymes of Glycogen Metabolism

Publisher Summary The finding that pyridoxal phosphate is an essential constituent or coenzyme of glycogen phosphorylase created a new dimension for thought concerning the functions of vitamin B6. This chapter explores questions, such as “Did the discovery of vitamin B6 in phosphorylase stimulate avenues of approach that have led to new disclosures concerning functions of vitamin B6? Have any of the classical findings of vitamin B6 deficiency been explained as a consequence of impaired phosphorylase activity?” The chapter reviews the progress that has been made in several laboratories as to the mechanism of action of pyridoxal phosphate in phosphorylase. In view of the complex role of phosphorylase in the regulation of glycogen metabolism, the chapter focuses on the phosphorylase system. The interconversion reactions of the two forms of phosphorylase catalyzed by phosphorylase b kinase and phosphorylase phosphatase are briefly reviewed. The chapter evaluates the role of vitamin B6 in carbohydrate metabolism in general.