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D J Graves

Publications and source records attributed to D J Graves.

At least 109 records · Page 6Linked to original sources
PubMed1979

The effect of solvents on nucleotide regulation of glycogen phosphorylase.

The activity of glycogen phosphorylase is controlled by two nucleotide sites. We have found that organic solvents affect the regulatory properties of phosphorylase by altering the binding at these two sites. At the activator site, the Ka for AMP is lowered 10-fold in the presence of 10% 1,2-dimethoxyethane while, at the inhibitor site, the Ki for caffeine is increased 6-fold. The stimulation of activity by organic solvents is highly dependent on the enzyme's activity state. Phosphorylase b, which has a requirement for a nucleotide activator, loses this requirement in the presence of organic solvents, while the active form of the enzyme, phosphorylase a, is only slightly stimulated by organic solvents. The activation profile obtained with rabbit liver phosphorylase suggests that differences in the properties of this enzyme from rabbit muscle phosphorylase might be explained by a change in the relative affinity for AMP at the two nucleotide sites. The results also suggest that 1,2-dimethoxyethane may be useful to determine accurately the activities of different forms of liver phosphorylase.

Adenosine Monophosphate↗
PubMed1978

Use of a double-headed peptide substrate to study the specificity of cAMP-dependent protein kinase and posphorylase kinase.

A peptide containing 2 seryl residues, (1)Leu(2)Ser(3)Tyr(4)Arg(5)Aly(6)Tyr(7)Ser(8)Leu, was chemically synthesized and used as a substrate for phosphorylase kinase and cyclic AMP-dependent protein kinase. The sequence, TryArgGlyTyr, makes up a beta turn in the native protein. Phosphorylase kinase was found to phosphorylate specifically seryl residue2 and protein kinase seryl residue7. Km and Vmax values were obtained and compared with natural substrates. The differences in the specificity of the two enzymes might be explained by a different requirement for organized structure. As a working hypothesis, it is suggested the results could be explained if the two enzymes interacted with seryl residues at different sides of a beta turn.

Animals↗
PubMed1977

Studies on the specificity of phosphorylase kinase using peptide substrates.

The action of phosphorylase kinase on synthetic peptides is reported. These peptides are variants of the amino acid sequence. Ser-Asp-Gln-Glu-Lys-Arg-Lys-Gln-Ile-Ser-Val-Arg-Gly-Leu, found in the natural substrate, phosphorylase b. The effects of size, the cluster of basic groups at the NH2-terminal side, the phosphorylatable seryl residue, the hydrophobic groups surrounding serine, and the arginyl function at the COOH-terminal side were tested and analyzed by evaluation of the kinetic parameters, Km and Vmax. The first 6 residues were found to be nonessential, but substitution of residues in the sequence. Lys-Gln-Ile-Ser-Val-Arg, had a large effect on phosphorylation. A comparison was made between the action of nonactivated and activated phosphorylase kinase on selected peptides and phosphorylase b. Various forms of phosphorylase b were tested as substrates for cyclic AMP-dependent protein kinase in the presence of effectors and salts. Although phosphorylase would not serve as a substrate for protein kinase, the aforementioned synthetic peptide of the phosphorylase b sequence would do so, indicating that the primary sequence surrounding the phosphorylatable serine did not block phosphorylation, which suggests that higher order structural features prohibit the phosphorylation.

Amino Acids↗
PubMed1976

Stimulation of phosphorylase kinase autophosphorylation by peptide analogs of phosphorylase.

Autoactivation of phosphorylase kinase in the presence of substrates has been studied to determine the cause of the hysteresis, or lag, in the phosphorylase kinase reaction. Peptide analogs corresponding to the convertible serine region of phosphorylase have been used as low molecular weight alternative substrates. Autophosphorylation of the kinase molecule was measured under conditions that favored autoactivation. Phosphorylase b and a tetradecapeptide, which was found to be a good model of phosphorylase, stimulated autoactivation by 86- and 37-fold, respectively. The tetradecapeptide also stimulated autophosphorylation of subunits A and B of the kinase molecule. This increased autophosphorylation coincided with an increased ability to convert phosphorylase. This finding supports the hypothesis that autophosphorylation is responsible for the lag in the phosphorylase kinase reaction. No evidence was obtained to suggest that the lag could be due to dissociation of the kinase. The stoichiometry of phosphate incorporation into phosphorylase kinase subunits by autophosphorylation was much greater than that reported to occur by protein kinase phosphorylation. Multiple phosphorylation sites in subunit A accounted for most of the phosphate incorporation during autophosphorylation. Saturating levels of hexa- and octapeptide analogs also caused stimulation of autophosphorylation. Possible mechanisms and experimental implications of substrate-stimulated autophosphorylation are discussed. Consideration also is given to the possible role of effectors in autophosphorylation in vivo.

Animals↗
PubMed1976

Site of action and biphasic effect of neutral salts in the phosphorylase kinase reaction.

The inhibition of phosphorylase kinase catalytic activity by 0.1 M neutral salts was predicted by the Hofmeister series of anions. The site of action of the salts was determined by the following evidence to be on the phosphorylase kinase molecule directly, rather than on its protein substrate. (1) Nonactivated kinase was more sensitive to salt inhibition than the activated form. (2) Ca2+ partially overcame the inhibition of nonactivated kinase. (3) Inhibition by Cl- occurred with either phosphorylase or a tetradecapeptide containing the convertible seryl residue as substrate. (4) Phosphorylation of nonactivated phosphorylase kinase by protein kinase was markedly inhibited by NaNO3, but this salt had little effect on the phosphorylation of histone by protein kinase. The influence of neutral salts on phosphorylase kinase activity was biphasic. Although activity was inhibited at low salt concentrations, it actually was stimulated as the salt concentration was increased. A similar biphasic response to various salt concentrations was observed in the velocities of autophosphorylation of phosphorylase kinase. The lag in the rate of product formation seen during the activity assay was less pronounced at inhibitory salt concentrations and was abolished at stimulatory salt concentrations. How the influence of salts relates to autophosphorylation and the lag is considered.

Animals↗
PubMed1975

Regulation of glycogen phosphorylase. Role of the peptide region surrounding the phosphoserine residue in determining enzyme properties.

A phosphopeptide which contains 14 residues including phosphoserine and which is derived from the NH2-terminal region of skeletal muscle glycogen phosphorylase (Nolan, C., Novoa, W. B., Krebs, E. G., and Fischer, E. H. (1964) Biochemistry 3, 542-551) has been shown to induce the enzymic properties of phosphorylase a in phosphorylase b and b'. When phosphorylase b is incubated with the phosphorylated tetradecapeptide, the following changes occur: (1) the enzyme becomes partially catalytically active in the absence of AMP; (2) the allosteric interactions of the enzyme are altered, as evidenced by the fact that phosphorylase b does not bind AMP cooperatively, and is no longer inhibited by glucose-6-P; and (3) the enzyme, normally present as a dimer, associates to a tetramer. Phosphorylase b' is a modified form of phosphorylase in which the phosphorylation site has been removed by limited tryptic attack. In the presence of phosphopeptide, 86% of the total enzyme activity can be induced in the absence of AMP. The properties of phosphorylases b and b' with phosphopeptide, cited above, are all characteristics of the phosphonenzyme, phosphorylase a. In addition, evidence is presented that these effects are specific. They are not the result of the polycationic nature of the peptide since they cannot be duplicated by spermine, and the phosphate group must also be present for the peptide to effect changes on the enzyme.

Adenosine Monophosphate↗
PubMed1975

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.

Apoenzymes↗