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D B Glass

Publications and source records attributed to D B Glass.

At least 37 records · Page 2Linked to original sources
PubMed1987

Modification of synthetic peptides related to lactate dehydrogenase (231-242) by protein carboxyl methyltransferase and tyrosine protein kinase: effects of introducing an isopeptide bond between aspartic acid-235 and serine-236.

The possibility that isoaspartyl residues contribute to the substrate specificity of eucaryotic protein carboxyl methyltransferases and/or tyrosine protein kinases has been investigated with two synthetic oligopeptides, Lys-Gln-Val-Val-Asp/isoAsp-Ser-Ala-Tyr-Glu-Val-Ile-Lys, which correspond to amino acids 231-242 of lactate dehydrogenase. One version of the peptide contains the normal amino acid sequence of the chicken muscle M4 isozyme. The other version contains an isoaspartyl residue in position 235 in place of the normal aspartyl residue; i.e., Asp-235 is linked to Ser-236 via its side-chain beta-carboxyl group, rather than via the usual alpha-carboxyl linkage. The normal peptide corresponds to the sequence around Tyr-238 that is phosphorylated in Rous sarcoma virus infected chick embryo fibroblasts [Cooper, J. A., Esch, F. S., Taylor, S. S., & Hunter, T. (1984) J. Biol Chem. 259, 7835]. Using protein carboxyl methyltransferase purified from bovine brain, we found that the normal peptide did not serve as a methyl-accepting substrate but that the isopeptide served as an excellent substrate, exhibiting a stoichiometry of one methyl group per peptide and Km of 0.54 microM. With tyrosine protein kinase partially purified from normal rat spleen both peptides were found to serve as phosphate acceptors at Tyr-238, exhibiting Km values of 4.7 and 8.9 mM for the normal and isopeptide versions, respectively. These results support the idea that protein carboxyl methyltransferase selectively methylates the alpha-carboxyl group of atypical isoaspartyl residues. In contrast, the presence of isoaspartate had a modest negative effect on substrate activity for a tyrosine protein kinase from rat spleen.

Amino Acid Sequence↗
PubMed1987

Phosphorylation of tyrosine hydroxylase by cyclic GMP-dependent protein kinase.

Tyrosine hydroxylase purified from rat pheochromocytoma was phosphorylated and activated by purified cyclic GMP-dependent protein kinase as well as by cyclic AMP-dependent protein kinase catalytic subunit. The extent of activation was correlated with the degree of phosphate incorporated into the enzyme. Comparable stoichiometric ratios (0.6 mol phosphate/mol tyrosine hydroxylase subunit) were obtained at maximal concentrations of either cyclic AMP-dependent or cyclic GMP-dependent protein kinases. The enzymes appeared to mediate the phosphorylation of the same residue based on the observation that incorporation was not increased when both enzymes were present. The major tryptic phosphopeptide obtained from tyrosine hydroxylase phosphorylated by each protein kinase exhibited an identical retention time following HPLC. The purified phosphopeptides also exhibited identical isoelectric points. These data provide support for the notion that the protein kinases are phosphorylating the same residue of tyrosine hydroxylase.

Animals↗
PubMed1986

Differential and common recognition of the catalytic sites of the cGMP-dependent and cAMP-dependent protein kinases by inhibitory peptides derived from the heat-stable inhibitor protein.

Synthetic peptides corresponding to the active domain of the heat-stable inhibitor protein of cAMP-dependent protein kinase (Cheng, H.-C., Kemp, B. E., Pearson, R. B., Smith, A. J., Misconi, L., Van Patten, S. M., and Walsh, D. A. (1986) J. Biol. Chem. 261, 989-992) were tested as inhibitors of cGMP-dependent protein kinase. The peptides themselves were not substrates. cGMP-dependent protein kinase activity was assayed using histone H2B and two synthetic peptide substrates. Consistent with previous observations of other peptide inhibitors of this enzyme (Glass, D. B. (1983) Biochem. J. 213, 159-164), the inhibitory peptides had no effect on the phosphorylation of histone H2B, but they competitively inhibited cGMP-dependent phosphorylation of the two peptide substrates. The parent inhibitor peptide, PKI(5-24)amide, and a series of analogs had Ki (or IC50) values for cGMP-dependent protein kinase in the range of 15-190 microM. In contrast to their effects on the cAMP-dependent protein kinase, the inhibitory peptides were substantially less potent with cGMP-dependent protein kinase, and potency was reduced by the presence of the NH2-terminal residues (residues 5-13). We conclude that the two protein kinases share a recognition of the basic amino acid cluster within the pseudosubstrate region of the peptide, but that the cGMP-dependent protein kinase does not recognize additional NH2-terminal determinants that make the inhibitor protein extremely potent toward the cAMP-dependent enzyme. Even- when tested at high concentrations and with peptide substrates, the native inhibitor protein did not inhibit cGMP-dependent protein kinase under assay conditions in which the peptides derived from it were inhibitory. Thus, the native inhibitor protein appears to have structural features which block interaction with the cGMP-dependent enzyme and enhance its selectivity for cAMP-dependent protein kinase.

Amino Acid Sequence↗
PubMed1986

In vitro phosphorylation of type I collagen by cyclic AMP-dependent protein kinase.

Both the triple-helical and denatured forms of nonfibrillar bovine dermal type I collagen were tested as substrates for the catalytic subunit of cAMP-dependent protein kinase in an in vitro reaction. Native, triple-helical collagen was not phosphorylated, but collagen that had been thermally denatured into individual alpha chains was a substrate for the protein kinase. Catalytic subunit of cAMP-dependent protein kinase phosphorylated denatured collagen to between 3 to 4 mol of phosphate/mol of (alpha 1(I)2 alpha 2(I). Pepsin-solubilized and intact collagens were phosphorylated similarly, as long as each was in a nonhelical conformation. The first 2 mol of phosphate incorporated into type I collagen by the protein kinase were present in the alpha 2(I) chain. The alpha 1(I) chain was only phosphorylated during long incubations in which the stoichiometry exceeded 2 mol of phosphate/mol of (alpha 1(I)2 alpha 2(I). Phosphoserine was the only phosphoamino acid identified in collagen that had been phosphorylated to any degree by the protein kinase. The 2 mol of phosphate incorporated into the alpha 2(I) chain were localized to the alpha 2(I)CB4 cyanogen bromide fragment. The catalytic subunit of cAMP-dependent protein kinase phosphorylated denatured pepsin-solubilized collagen with a Km of 8 microM and a Vmax of approximately 0.1 mumol/min/mg of enzyme. Denatured, but not triple-helical, type I collagen was also phosphorylated by cGMP-dependent protein kinase, although it was a poorer substrate for this enzyme than for the cAMP-dependent protein kinase. Collagen was not a substrate for phospholipid-sensitive Ca2+-dependent protein kinase. These results suggest the potential for nascent alpha chains of type I collagen to be susceptible to phosphorylation by cAMP-dependent protein kinase in vivo prior to triple-helix formation. Such a phosphorylation of collagen could be relevant to the action of cAMP to increase the intracellular degradation of newly synthesized collagen.

Animals↗
PubMed1986

Synthetic peptides corresponding to the site phosphorylated in 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase as substrates of cyclic nucleotide-dependent protein kinases.

The specificities of cAMP-dependent and cGMP-dependent protein kinases were studied using synthetic peptides corresponding to the phosphorylation site in 6-phosphofructo-2-kinase/Fru-2,6-P2ase (Murray, K.J., El-Maghrabi, M.R., Kountz, P.D., Lukas, T.J., Soderling, T.R., and Pilkis, S.J. (1984) J. Biol. Chem. 259, 7673-7681) as substrates. The peptide Val-Leu-Gln-Arg-Arg-Arg-Gly-Ser-Ser-Ile-Pro-Gln was phosphorylated by the catalytic subunit of cAMP-dependent protein kinase on predominantly the first of its 2 seryl residues. The Km (4 microM) and Vmax (14 mumol/min/mg) values were comparable to those for the phosphorylation of this site within native 6-phosphofructo-2-kinase/Fru-2,6-P2ase. An analog peptide containing only two arginines was phosphorylated with poorer kinetic constants than was the parent peptide. These results suggest that the amino acid sequence at its site of phosphorylation is a major determinant that makes 6-phosphofructo-2-kinase/Fru-2,6-P2ase an excellent substrate for cAMP-dependent protein kinase. Although 6-phosphofructo-2-kinase/Fru-2,6-P2ase was not phosphorylated by cGMP-dependent protein kinase, the synthetic peptide corresponding to the cAMP-dependent phosphorylation site was a relatively good substrate (Km = 33 microM, Vmax = 1 mumol/min/mg). Thus, structures other than the primary sequence at the phosphorylation site must be responsible for the inability of cGMP-dependent protein kinase to phosphorylate native 6-phosphofructo-2-kinase/Fru-2,6-P2ase. Peptides containing either a -Ser-Ser- or -Thr-Ser- moiety were all phosphorylated by cGMP-dependent kinase to 1.0 mol of phosphate/mol of peptide, but the phosphate was distributed between the two hydroxyamino acids. Substitution of a proline in place of the glycine between the three arginines and these phosphorylatable amino acids caused the protein kinase selectively to phosphorylate the threonyl or first seryl residue and also enhanced the Vmax values by 4-6-fold. These results are consistent with a role for proline in allowing an adjacent threonyl residue to be readily phosphorylated by cGMP-dependent protein kinase.

Amino Acid Sequence↗
PubMed1985

Inactivation of guanosine cyclic 3',5'-monophosphate dependent protein kinase from bovine lung by o-phthalaldehyde.

Guanosine cyclic 3',5'-monophosphate (cGMP) dependent protein kinase is inactivated by o-phthalaldehyde. The loss of phosphotransferase activity following treatment with o-phthalaldehyde was rapid, and the second-order rate constant at 25 degrees C and pH 7.3 was 35 M-1 s-1. The inactivation reaction did not follow saturation kinetics. The cGMP-dependent protein kinase was protected from inactivation by its substrates, MgATP and Ser-peptide. Fluorescence excitation and emission spectroscopic data showed that an isoindole derivative was formed following the reaction between cGMP-dependent protein kinase and o-phthalaldehyde. Four moles of isoindole per mole of the cGMP-dependent protein kinase dimer was formed following complete inactivation by o-phthalaldehyde. In the absence of cGMP, the protein kinase lost only 50% of its cGMP binding activity while there was almost a complete loss of its phosphotransferase activity. Studies in the presence of 20 microM cGMP, however, showed that about 2 mol of isoindole groups per mole of the protein kinase dimer was formed following complete inactivation by o-phthalaldehyde. The second-order rate constant for inactivation of cGMP-dependent protein kinase by o-phthalaldehyde in the presence of 20 microM cGMP was 40 M-1 s-1. Fluorescence measurements of samples containing inactivated, iodoacetamide-modified, or 5'-[p-(fluorosulfonyl)benzoyl]adenosine-modified, cGMP-dependent protein kinase and o-phthalaldehyde showed that the intensity of fluorescence in each case was about 50% of that obtained from unmodified, active cGMP-dependent protein kinase and o-phthalaldehyde.(ABSTRACT TRUNCATED AT 250 WORDS)

Aldehydes↗
PubMed1985

A [3H]lysine-containing synthetic peptide substrate for human protocollagen lysyl hydroxylase.

A tridecapeptide containing tritium-labelled lysine and corresponding closely to residues 98 to 110 of the alpha chain of type I collagen was synthesized by the solid-phase method. Gly-Leu-Hyp-Gly-Nle-[4,5-3H]Lys-Gly-His-Arg-Gly-Phe-Ser-Gly was used as a substrate of human protocollagen lysyl hydroxylase (peptidyllysine, 2-oxoglutarate: oxygen 5-oxidoreductase, EC 1.14.11.4) obtained from dermal fibroblasts. L-[4,5-3H]Lysine was converted to N alpha-t-butyloxycarbonyl-N epsilon-o-chlorobenzyloxycarbonyl [3H]lysine which was incorporated during stepwise synthesis of the peptide. The chemical and radiochemical purities and specific activity of the completed peptide were characterized. A non-radiolabelled analogue of the peptide inhibited the hydroxylation of [3H]lysine-containing protocollagen by human lysyl hydroxylase, indicating that the synthetic peptide interacted with the enzyme. The peptide containing [3H]lysine was a substrate for lysyl hydroxylase and permitted direct measurement of enzyme activity in relatively crude cell extracts by a tritium-release assay. Extracts of cultured fibroblasts from a patient with an autosomal recessive pattern of inheritance for Ehlers-Danlos syndrome type VI had activities for tritium release from either the radiolabelled synthetic peptide or from [3H]lysine-containing protocollagen that were only 30% of those from control cells. These data indicate that a stable, well-defined synthetic peptide containing [3H]lysine is a useful substrate for studies of genetically variant lysyl hydroxylase from cultured human cells.

Cells, Cultured↗
PubMed1985

Interaction of guanosine cyclic 3',5'-phosphate dependent protein kinase with lin-benzoadenine nucleotides.

Using the activated cGMP-dependent protein kinase in the presence of the phosphorylatable peptide [[Ala34]histone H2B-(29-35)], we found that lin-benzoadenosine 5'-diphosphate (lin-benzo-ADP) was a competitive inhibitor of the enzyme with respect to ATP with a Ki (22 microM) similar to the Kd (20 microM) determined by fluorescence polarization titrations. The Kd for lin-benzo-ADP determined in the absence of the phosphorylatable peptide, however, was only 12 microM. ADP bound with lower affinity (Ki = 169 microM; Kd = 114 microM). With [Ala34]histone H2B-(29-35) as phosphoryl acceptor, the Km for lin-benzo-ATP was 29 microM, and that for ATP was 32 microM. The Vmax with lin-benzo-ATP, however, was only 0.06% of that with ATP as substrate [0.00623 +/- 0.00035 vs. 11.1 +/- 0.17 mumol (min.mg)-1]. Binding of lin-benzo-ADP to the kinase was dependent upon a divalent cation. Fluorescence polarization revealed that Mg2+, Mn2+, Co2+, Ni2+, Ca2+, Sr2+, and Ba2+ supported nucleotide binding to the enzyme; Ca2+, Sr2+, and Ba2+, however, did not support any measurable phosphotransferase activity. The rank order of metal ion effectiveness in mediating phosphotransferase activity was Mg2+ greater than Ni2+ greater than Co2+ greater than Mn2+. Although these results were similar to those observed with the cAMP-dependent protein kinase [Hartl, F. T., Roskoski, R., Jr., Rosendahl, M. S., & Leonard, N. J. (1983) Biochemistry 22, 2347], major differences in the Vmax with lin-benzo-ATP as substrate and the effect of peptide substrates on nucleotide (both lin-benzo-ADP and ADP) binding were observed.

Animals↗
PubMed1984

Phosphorylation of purified cardiac muscle C-protein by purified cAMP-dependent and endogenous Ca2+-calmodulin-dependent protein kinases.

C-protein, a component of the thick filament of striated muscles, becomes phosphorylated in response to beta-adrenergic receptor stimulation and dephosphorylated in response to cholinergic receptor stimulation in heart. We have purified C-protein in high yield from cardiac muscle (approximately 50% yield: 0.3 mg of C-protein/g of frozen chicken heart). C-protein has a molecular weight on sodium dodecyl sulfate polyacrylamide gels of 155,000 but the native protein migrates as a globular protein of 209,000 daltons in gel filtration on Sephacryl S-300, suggesting that it is an asymmetric molecule composed of a single 155,000-dalton polypeptide. C-protein from chicken cardiac muscle has an amino acid composition similar to that of C-proteins from other muscles. The purified protein contains approximately 0.2 mol of phosphate/mol of C-protein. The purified C-protein has no endogenous protein phosphatase activity but does exhibit protein kinase activity in the presence of calcium and calmodulin (approximately 160 pmol of phosphate incorporated/min/mg of C-protein). This endogenous kinase catalyzes the incorporation of approximately 1 mol of phosphate/mol of C-protein. C-protein is an excellent substrate for catalytic subunit of cAMP-dependent protein kinase (Km = 4 microM, Vmax = 18.6 mumol/min/mg). Phosphorylation by catalytic subunit of cAMP-dependent protein kinase exhibits a broad pH optimum between pH 8 and 9 and results in the incorporation of up to 3 mol of phosphate/mol of C-protein. Phosphate is incorporated into 3-5 different sites at both phosphothreonine and phosphoserine residues. The phosphorylated C-protein does not differ from unphosphorylated C-protein with regard to Stokes radius, migration on sodium dodecyl sulfate-polyacrylamide gels, or UV spectrum.

Animals↗
PubMed1984

In vitro phosphorylation of a synthetic collagen peptide by cyclic AMP-dependent protein kinase.

A synthetic tridecapeptide that corresponds closely to amino acid residues 98 to 110 in chick collagen alpha 1(I) contains several determinants of specificity required for recognition and phosphorylation by the cyclic AMP-dependent protein kinase. The peptide Gly-Leu-Hyp-Gly-Nle-Lys-Gly-His-Arg-Gly-Phe-Ser-Gly was predicted to be a substrate for the cyclic AMP-dependent protein kinase because it contained multiple basic amino acids NH2-terminal to a potentially phosphorylatable seryl residue. When tested as a substrate for the enzyme, the peptide was stoichiometrically phosphorylated. Phosphoserine was identified as the only phosphoamino acid in a partial hydrolysate of the phosphorylated peptide. The peptide and several of its analogs were phosphorylated by the cyclic AMP-dependent protein kinase with Km values from 1 to 6 mM and Vmax values from 1 to 3 mumol of phosphate/min/mg of enzyme. Although the Km of the kinase for the collagen peptide was high, these results confirmed the prediction made from knowledge of the substrate specificity of cyclic AMP-dependent protein kinase. The potential for such a phosphorylation reaction to occur in vivo is discussed.

Amino Acid Sequence↗
PubMed1983

Phosphorylation by cyclic GMP-dependent protein kinase of a synthetic peptide corresponding to the autophosphorylation site in the enzyme.

The substrate specificity of cGMP-dependent protein kinase has been investigated by examining the ability of the enzyme to phosphorylate a series of synthetic peptides that correspond to the amino acid sequence at its site of autophosphorylation. The undecapeptide Ile53-Gly-Pro-Arg-Thr-Thr58-Arg-Ala-Gln-Gly-Ile63 which corresponds to the sequence around threonine-58 in cGMP-dependent protein kinase (Takio, K., Smith, S.B., Walsh, K.A., Krebs, E.G., and Titani, K. (1983) J. Biol. Chem. 258, 5531-5536) was synthesized and tested as a substrate for that enzyme. It was phosphorylated to the extent of 1.0 mol of phosphate/mol of peptide. Analysis of the products of Edman degradation of the phosphopeptide indicated that only threonine-58 was phosphorylated, as is the case for the autophosphorylation reaction in the native enzyme. The peptide was phosphorylated by cGMP-dependent protein kinase with a Km value of 578 +/- 25 microM and a Vmax of 0.069 +/- 0.003 mumol/min/mg of enzyme. This low Vmax value is consistent with the relatively slow rate of the autophosphorylation reaction. An analog peptide that contained serine in place of threonine-58 was also phosphorylated to 1.0 mol of phosphate/mol of peptide. That phosphopeptide contained only phosphoserine. The serine-containing analog peptide had a Km value similar to that of the parent peptide but was phosphorylated with a 70-fold higher Vmax value. Substitution of arginine-56 in the parent peptide by an alanine residue resulted in a peptide that was essentially not a substrate. Substitution of arginine-59, COOH-terminal to the phosphorylatable threonine, yielded a peptide with a Vmax similar to that of the parent peptide but a Km value of almost 22,000 microM. These results indicate that serine is a better phosphate-accepting residue than is threonine and that both arginine residues around the site of autophosphorylation are important specificity determinants for the cGMP-dependent protein kinase.

Amino Acid Sequence↗
PubMed1983

Differential responses of cyclic GMP-dependent and cyclic AMP-dependent protein kinases to synthetic peptide inhibitors.

The peptide Arg-Lys-Arg-Ala-Arg-Lys-Glu was synthesized and tested as an inhibitor of cyclic GMP-dependent protein kinase. This synthetic peptide is a non-phosphorylatable analogue of a substrate peptide corresponding to a phosphorylation site (serine-32) in histone H2B. The peptide was a competitive inhibitor of cyclic GMP-dependent protein kinase with respect to synthetic peptide substrates, with a Ki value of 86 microM. However, it did not inhibit phosphorylation of intact histones by cyclic GMP-dependent protein kinase under any conditions tested. Arg-Lys-Arg-Ala-Arg-Lys-Glu competitively inhibited the phosphorylation of either peptides or histones by the catalytic subunit of cyclic AMP-dependent protein kinase, with similar Ki values (550 microM) for both of these substrates. The peptide Leu-Arg-Arg-Ala-Ala-Leu-Gly, which was previously reported to be a selective inhibitor of both peptide and histone phosphorylation by cyclic AMP-dependent protein kinase, was a poor inhibitor of cyclic GMP-dependent protein kinase acting on peptide substrates (Ki = 800 microM), but did not inhibit phosphorylation of histones by cyclic GMP-dependent protein kinase. The selectivity of these synthetic peptide inhibitors toward either cyclic GMP-dependent or cyclic AMP-dependent protein kinases is probably based on differences in the determinants of substrate specificity recognized by these two enzymes. It is concluded that histones interact differently with cyclic GMP-dependent protein kinase from the way they do with the catalytic subunit of cyclic AMP-dependent protein kinase.

Carrier Proteins↗
PubMed1982

Phosphorylation by guanosine 3':5'-monophosphate-dependent protein kinase of synthetic peptide analogs of a site phosphorylated in histone H2B.

Analogs of a synthetic heptapeptide substrate corresponding to the sequence around a phosphorylation site in histone H2B were used to assess the substrate specificity of cGMP-dependent protein kinase. cGMP-dependent kinase phosphorylated the oligopeptide Arg-Lys-Arg-Ser32-Arg-Lys-Glu with favorable kinetic parameters as compared to those for cAMP-dependent kinase (Glass, D. B., and Krebs, E. G. (1979) J. Biol. Chem. 254, 9728-9738). The contribution of each amino acid to the ability of the peptide to be phosphorylated by cGMP-dependent or cAMP-dependent kinase was studied by replacement of individual residues and evaluation of the kinetic constants of the substituted peptides. Peptides containing acetylated lysine residues or nitroarginine residues were poor substrates for both kinases. Substitution of either arginine 29 or lysine 30 with alanine increased the Km values and decreased the Vmax values for both kinases. Substitution of lysine 34 with alanine increased the Vmax values for both kinases but did not affect the Km values for either enzyme. Substitution of the phosphorylatable serine with a threonine residue greatly depressed the Vmax for both kinases. Peptides in which arginine 31 or arginine 33 were replaced by an alanine residue revealed several apparent differences in the specificity requirements between cGMP-dependent and cAMP-dependent kinases.

Amino Acid Sequence↗
PubMed1981

Glucocorticoid-stimulated increase in chemotactic peptide receptors on differentiating human myeloid leukemia (HL-60) cells.

The effect of dexamethasone on the HL-60 human promyelocytic leukemia cell line was studied using a radioligand-binding assay for a chemotactic peptide receptor that is found on the surface of mature polymorphonuclear leukocytes. When dimethyl sulfoxide (DMSO) is added to cultures, HL-60 cells undergo morphological and functional differentiation over a period of 6 to 7 days. Differentiation is accompanied by increased binding of the synthetic N-formylated chemotactic peptide N-formylmethionylleucyl[3H]phenylalanine. Binding of N-formylmethionylleucyl[3H]phenylalanine to cells induced to differentiate with DMSO was temperature dependent, specific, saturable, of high affinity, reversible, and proportional to cell number. Dexamethasone increased the number of N-formylated chemotactic peptide receptors in cultures of differentiating HL-60 cells without affecting the affinity of the receptors for the tritiated peptide. This steroid response was dose dependent, proportional in magnitude to glucocorticoid activity, and abolished by cycloheximide. Dexamethasone had little effect on N-formylmethionylleucyl[3H]phenylalanine binding unless DMSO was also present in culture or the cells were first induced by DMSO to differentiate. These results suggest that the glucocorticoid stimulated increase in number of N-formylated chemotactic peptide receptors was mediated by the high-affinity glucocorticoid receptor, involved protein synthesis, and was apparently dependent on differentiation.

Cell Differentiation↗