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N B Madsen

Publications and source records attributed to N B Madsen.

At least 37 records · Page 2Linked to original sources
PubMed1983

Site-site interactions in glycogen phosphorylase b probed by ligands specific for each site.

Three ligand binding sites on glycogen phosphorylase b which were originally described by kinetic and physicochemical means, and more recently located and defined in molecular terms by X-ray crystallography, have been probed by ligands specific for each site. Kinetic analyses, supplemented by X-ray crystallographic binding studies, permit assignment of each ligand to a primary binding site, as well as determination of its dissociation constant and interaction with ligands binding to the other sites. 8-Anilino-1-naphthalenesulfonate binds most strongly to the activator site, in competition with adenosine 5'-phosphate, presumably because its sulfonate group interacts with several arginine residues, and binds only weakly to the hydrophobic inhibitor site, possibly because of charge repulsion. It is itself a weak activator and decreases binding affinities for compounds specific for the inhibitor site. Our results with 8-anilino-1-naphthalenesulfonate are not consistent with predictions of its expected behavior and suggest caution in the use of this reagent as an indicator of hydrophobicity. Our second major probe, caffeine, binds primarily to the inhibitor site, shows competitive inhibition with substrate binding to the catalytic site, and decreases the affinity for the activator at the activator site. The catalytic site was probed with two different types of ligand. Glucose, known to stabilize the inactive T conformation of the enzyme, competes with the substrate alpha-D-glucose 1-phosphate for the catalytic site and decreases the affinity of adenosine 5'-phosphate for the activator site. Glucose also improves the binding affinity of caffeine for the inhibitor site by 3-5-fold, both compounds synergistically stabilizing the inactive T conformation.(ABSTRACT TRUNCATED AT 250 WORDS)

Anilino Naphthalenesulfonates↗
PubMed1982

Covalently activated glycogen phosphorylase: a phosphorus-31 nuclear magnetic resonance and ultracentrifugation analysis.

Glycogen phosphorylase b reconstituted with pyridoxal pyrophosphate in place of the natural coenzyme, pyridoxal phosphate, is shown to exist in a more activated (R) conformation than does native phosphorylase. Addition of nucleotide activator to the reconstituted enzyme traps it totally in this activated conformation. These conclusions were arrived at on the basis of tertiary structural information obtained from 31P nuclear magnetic resonance studies, which allowed measurement of the nucleotide binding constant, and on the basis of quaternary structural information obtained via ultracentrifugal analysis of the enzyme in the presence of various effectors. Control experiments were performed with another modified form of the enzyme, pyridoxal phosphorylase. It is suggested that the transition-state analogue pyridoxal pyrophosphate, bound at the active site, mimics the actual configuration of enzyme plus substrate achieved during the normal catalytic reaction and therefore traps the enzyme in an activated conformation. These findings agree well with recent results obtained with the alternate transition-state analogue pyridoxal pyrophosphate glucose [Withers, S. G., Madsen, N. B., Sykes, B. D., Takagi, M., Shimomura, S., & Fukui, T. (1981) J. Biol. Chem. 256, 10759] and therefore provide further evidence for the "interacting phosphates" hypothesis presented in the latter paper.

Enzyme Activation↗
PubMed1981

Active form of pyridoxal phosphate in glycogen phosphorylase. Phosphorus-31 nuclear magentic resonance investigation.

The substrate analogue alpha-D-glucopyranosyl cyclic 1,2-phosphate has been confirmed to be a good competitive inhibitor of glycogen phosphorylases a and b isolated from rabbit muscle. Effects of tertiary and quaternary structure of the enzyme have been shown to be similar to those induced by the substrate glucose 1-phosphate and different from those of the coincidently binding inhibitor glucose. This information was obtained from study of the ultracentrifugation patterns of the enzyme-inhibitor complex and by determination of its effect on the binding constant for the activator AMP. 31P NMR investigation of the binding of this inhibitor to the enzyme has demonstrated that it both tightens the binding of the nucleotide activator and shifts the resonance of the phosphate group of the pyridoxal phosphate residue to a broad signal around 0 ppm. This situation is further reinforced in the presence of the second substrate, maltopentaose, giving a fully potentiated, but inactive, enzyme-substrate complex. This has not been studied previously by 31 P NMR. The active form of the pyridoxal phosphate (PLP), in the presence of substrates or their analogues, is not therefore a mobile dianionic phosphate as has been previously proposed. It may represent a tightly bound and constrained dianionic phosphate or possible a protonated phosphate in intermediate exchange. The implications of this finding are discussed.

Adenosine Monophosphate↗
PubMed1981

Regulation of the dephosphorylation of glycogen phosphorylase a and synthase b by glucose and caffeine in isolated hepatocytes.

Synergistic regulation of glycogen phosphorylase a by the competitive inhibitors glucose and caffeine in vitro indicates a possible physiological role for the negative effector site which binds caffeine (nucleoside site). In intact viable hepatocytes glucose promotes the phosphorylase a to be conversion by phosphorylase phosphatase. This conversion is considered to be a necessary prelude to the activation of glycogen synthase by phosphatase and of importance in hepatic regulation of glucose homeostasis. The effects of glucose and(or) caffeine on the conversion of phosphorylase a to b and synthase b to a were studied. Assays of phosphorylase a were used which limited synergistic inhibition (in the assay) by these ligands. Such an approach is necessary to achieve an accurate measure of phosphatase activity in the viable hepatocyte when the combination of ligands is used. The data indicate that in the presence of caffeine and glucose together, the rate of loss of phosphorylase a is significantly increased (1.7-fold) over that in the presence of glucose alone. Phosphorylase phosphatase is activated. The sequential activation of glycogen synthase was also accelerated in the presence of both ligands. The results are consistent with an in vivo function for the nucleoside site, similar to that of glucose. A controlling role or phosphorylase in the regulation of glycogen metabolism by glucose is supported. Although the existence and nature of an intracellular effector is as yet unknown, crystallographic analyses of phosphorylase a crystals soaked in perchloric acid extracts of liver demonstrate that the negative effector site binds a natural metabolite.

Animals↗
PubMed1977

Location of pyridoxal phosphate in glycogen phosphorylase a.

The pyridoxal 5'-phosphate cofactor of glycogen phosphorylase a (1,4-alpha-D-glucan:orthophosphate alpha-glucosyltransferase, EC2.4.1.1.) has been positioned on the protomer with x-ray diffraction data, chemical markers, and sequence information. The electron density was computed from 3.0-A resolution phases calculated from four heavy-atom derivatives. The cofactor is buried inside the protomer adjacent to the glucose-binding site. The phosphoryl substrates Pi and glucose-1-P each bind at two sites on the protomer. At low concentrations, Pi and glucose-1-P bind in the same location as does the allosteric effector AMP, near the monomer-monomer interface and some 30 A from the glucose site. At high concentrations glucose-1-P also binds strongly at the glucose site, with its phosphate only 7.2 A from that of the cofactor. Inorganic phosphate can also bind at this site. Implications for the participation of the pyridoxal phosphate in the catalytic mechanism are discussed in the light of these structural findings as well as the wealth of indirect evidence in the literature.

Binding Sites↗
PubMed1977

An essential arginine residue in the active-site pocket of glycogen phosporylase.

Phosphorylases a and b (EC 2.4.1.1) were inactivated by selective modification of arginyl residues on reaction with 2,3-butanedione in sodium borate buffer. The rate of inactivation was slightly greater for phosphorylase a than b. The course of inactivation followed pseudo-first-order kinetics with some deviations at low rates or at more than 60% inactivation. The rate of inactivation was first order with respect to butanedione concentration. The inactivation was partially reversible, and ultracentrifugal studies showed no change in subunit association or dissociation. Amino acid analyses indicated that several arginines were modified during inactivation and that no other amino acid was affected. Protection from inactivation was provided by the substrate glucose 1-phosphate (G1P), alone or together with the allosteric activator AMP, as well as by the competitive inhibitor UDP-glucose. The rate of inactivation of phosphorylase b was also retarded by the presence of AMP alone. Glycogen did not have any appreciable effect on inactivation. The Km of G1P for phosphorylase a remained constant over the course of inactivation, while the Km values of G1P and AMP for phosphorylase b increased. The modification of cross-linked tetragonal microcrystals of phosphorylase a followed the same trend as the enzyme in solution, although the rate of inactivation was slower. The X-ray crystallography studies at 6 A (1 A = 0.1 nm) resolution, of butanedione-treated cross-linked tetragonal crystals of phosphorylase a showed a large new peak of electron density at the end of a long side chain in the active-site pocket. The substrates G1P and arsenate, as well as UDP-glucose, had previously been shown to bind in that location. Other, small peaks of electron density were found in locations on the outside of the protein molecule. UDP-glucose failed to bind to the active site of crystals which had been treated with butanedione, while AMP, which also binds in the active-site pocket, showed a lower occupancy. This work indicates the presence of a functional arginine residue at the binding site for G1P in glycogen phosphorylases a and b.

Adenosine Monophosphate↗