donald graves - Academia.edu (original) (raw)
Papers by donald graves
Pharmacology Therapeutics, 1999
Although much can be learned about the specificity of protein kinases from studies with peptide s... more Although much can be learned about the specificity of protein kinases from studies with peptide substrates, the question remains, how do kinases recognize their three-dimensional protein substrates? Information derived from such studies provides further understanding of substrate recognition and can facilitate the design of specific protein kinase inhibitors. Phosphorylase kinase (PhK) catalyzes the phosphorylation of phosphorylase b (phos. b) to form the active phosphorylase a. No other protein kinase can duplicate this reaction. Why? To probe this question and establish what features in the protein are important for substrate binding and product release, mutants of phos. b have been studied. This report shows how mutations change the properties of the protein substrate and the ability of these mutants to be phosphorylated by PhK and other kinases. Action of protein kinases on their substrates is often regulated by autoinhibitory segments. The C-terminus of the catalytic ␥-subunit of PhK contains two inhibitory sites overlapping two calmodulin-binding regions. These two peptide segments resemble sequences in phos. b and may explain why peptides of these regions are potent inhibitors of PhK. We will show results with peptide inhibitors, using various expressed forms of the catalytic subunit, which describe their modes of interaction and mechanisms of inhibition. Metal ions can change molecular interactions. With PhK, Mn 2 ϩ facilitates the use of GTP as a phosphoryl group donor and greatly increases phosphorylation of a tyrosine residue in angiotensin II. This implies that the spatial arrangement of specificity determinants can be manipulated so that PhK can utilize other substrates. pharmacol. ther. 82(2-3):143-155, 1999.
Formation of a cyclic imide in aspartyl or asparaginyl glycyl peptides induced by heating in the dry state*
International Journal of Peptide and Protein Research, 2009
A common product was identified by HPLC from the heating of, alpha and beta isomers of, aspartyl ... more A common product was identified by HPLC from the heating of, alpha and beta isomers of, aspartyl glycine in the dry state. Fast atom-bombardment (FAB) mass spectrometry and Fourier transform infrared photoacoustic spectroscopy (FTIR-PAS) showed that the product is a cyclic imide. Cyclic imide formation also was shown to occur with the tripeptides Gly-Asp-Gly and Gly-Asn-Gly. The cyclization of aspartyl peptides depends upon protonation of the beta-carboxyl group and can be promoted by acidification or by heating the NH4+ salt in the dry state.
Archives of Biochemistry and Biophysics
Phosphorylase b and two peptides with sequences homologous to phosphorylation site 2 (syntide 2) ... more Phosphorylase b and two peptides with sequences homologous to phosphorylation site 2 (syntide 2) and site 3 (syntide 3) of glycogen synthase were compared as substrates for purified muscle phosphorylase kinase. The substrate specificity of phosphorylase kinase varied according to whether heparin (at pH 6.5) or Ca2+ (at pH 8.2) was used as a stimulator of its activity. Phosphorylase b was preferentially phosphorylated in the presence of Ca2'; the rate of syn tide 2 phosphorylation was the same for both stimulators; and the phosphorylation of syntide 3 was completely dependent on the presence of heparin. A kinetic analysis confirmed this stimulator-dependent substrate specificity since both the V
Journal of Biological Chemistry
Calcineurin, a calmodulin-activated protein phosphatase, is known to dephosphorylate certain low ... more Calcineurin, a calmodulin-activated protein phosphatase, is known to dephosphorylate certain low molecular weight phosphate esters. The low molecular weight phosphatase activity of calcineurin has been studied by utilizing tyrosine phosphate derivatives. Kinetic studies suggest that the substrate specificity is dependent upon the electronic nature of the substrate in contrast to results obtained with alkaline phosphatase from Escherichia coli. Comparison of calcineurinand acid-catalyzed hydrolyses indicates a 1:l correlation between the rate constants for the two processes. This correlation and other model studies have been utilized to provide insight into the chemical mechanism of calcineurin. Possible chemical mechanisms for calcineurin are discussed. 14932 This is an Open Access article under the CC BY license.
Journal of Biological Chemistry
The action of phosphorylase kinase on synthetic peptides is reported. These peptides are variants... more 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 6. The effects of size, the cluster of basic groups at the NH,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, K
Journal of Biological Chemistry
High-performance liquid chromatographic separation and renaturation of protein kinase subunits: application to catalytic subunit of phosphorylase kinase
Methods in Enzymology
Journal of Alzheimer's disease : JAD, 2009
An aqueous extract of Ceylon cinnamon (C. zeylanicum) is found to inhibit tau aggregation and fil... more An aqueous extract of Ceylon cinnamon (C. zeylanicum) is found to inhibit tau aggregation and filament formation, hallmarks of Alzheimer's disease (AD). The extract can also promote complete disassembly of recombinant tau filaments and cause substantial alteration of the morphology of paired-helical filaments isolated from AD brain. Cinnamon extract (CE) was not deleterious to the normal cellular function of tau, namely the assembly of free tubulin into microtubules. An A-linked proanthocyanidin trimer molecule was purified from the extract and shown to contain a significant proportion of the inhibitory activity. Treatment with polyvinylpyrolidone effectively depleted all proanthocyanidins from the extract solution and removed the majority, but not all, of the inhibitory activity. The remainder inhibitory activity could be attributed to cinnamaldehyde. This work shows that compounds endogenous to cinnamon may be beneficial to AD themselves or may guide the discovery of other pot...
Formation of a cyclic imide in aspartyl or asparaginyl glycyl peptides induced by heating in the dry state
International journal of peptide and protein research, 1987
A common product was identified by HPLC from the heating of, alpha and beta isomers of, aspartyl ... more A common product was identified by HPLC from the heating of, alpha and beta isomers of, aspartyl glycine in the dry state. Fast atom-bombardment (FAB) mass spectrometry and Fourier transform infrared photoacoustic spectroscopy (FTIR-PAS) showed that the product is a cyclic imide. Cyclic imide formation also was shown to occur with the tripeptides Gly-Asp-Gly and Gly-Asn-Gly. The cyclization of aspartyl peptides depends upon protonation of the beta-carboxyl group and can be promoted by acidification or by heating the NH4+ salt in the dry state.
Production and Characterization of Monoclonal Antibodies to Purified Deglycosylated Cystic Fibrosis Respiratory Mucin: Evidence for the Presence of Four Immunologically Distinct Epitopes
Hybridoma, 1991
Respiratory mucus glycoproteins (mucins) were purified from the tracheobronchial secretions of th... more Respiratory mucus glycoproteins (mucins) were purified from the tracheobronchial secretions of three Cystic Fibrosis (CF) patients. The mucins were completely deglycosylated by treatment with trifluoromethanesulfonic acid and subsequent treatment with alpha-N-acetylgalactosaminidase. Over thirty hybrid clones secreting antibodies against the deglycosylated mucin (DGM) were obtained using standard hybridoma techniques. Hybrids with positive identification for CF-DGM were cloned twice using limiting dilution method to ensure the monoclonal nature of the antibodies. Eight stable clones (1a, 1b, 10a, 10c, 10d, 10e, 29d, and 30e) secreting monoclonal antibodies (MAbs) showing specificity of reaction to CF-DGM were obtained. Two clones, 29d and 30e, secreted antibodies of the IgM class while the other six clones secreted antibodies of the IgG1 subclass. Denaturation and reduction experiments suggested that MAbs 1b, 10e, 29d and 30e were directed against a given sequence of amino acids in the DGM while the other four MAbs, in addition to being sequence specific, were also conformation dependent. Further, competitive binding radioimmunoassays suggested that MAbs 1b, 10e, 29d and 30e recognize four distinct epitopes in the peptidic core of CF respiratory mucin. In summary, the MAbs may provide a promising approach to elucidate the structure of the polypeptide backbone of human respiratory mucins as well as for the screening of cDNA libraries for clones secreting mucin(s).
Pharmacology & Therapeutics, 1999
Although much can be learned about the specificity of protein kinases from studies with peptide s... more Although much can be learned about the specificity of protein kinases from studies with peptide substrates, the question remains, how do kinases recognize their three-dimensional protein substrates? Information derived from such studies provides further understanding of substrate recognition and can facilitate the design of specific protein kinase inhibitors. Phosphorylase kinase (PhK) catalyzes the phosphorylation of phosphorylase b (phos. b) to form the active phosphorylase a. No other protein kinase can duplicate this reaction. Why? To probe this question and establish what features in the protein are important for substrate binding and product release, mutants of phos. b have been studied. This report shows how mutations change the properties of the protein substrate and the ability of these mutants to be phosphorylated by PhK and other kinases. Action of protein kinases on their substrates is often regulated by autoinhibitory segments. The C-terminus of the catalytic ␥-subunit of PhK contains two inhibitory sites overlapping two calmodulin-binding regions. These two peptide segments resemble sequences in phos. b and may explain why peptides of these regions are potent inhibitors of PhK. We will show results with peptide inhibitors, using various expressed forms of the catalytic subunit, which describe their modes of interaction and mechanisms of inhibition. Metal ions can change molecular interactions. With PhK, Mn 2 ϩ facilitates the use of GTP as a phosphoryl group donor and greatly increases phosphorylation of a tyrosine residue in angiotensin II. This implies that the spatial arrangement of specificity determinants can be manipulated so that PhK can utilize other substrates. pharmacol. ther. 82(2-3):143-155, 1999.
Cell-growth quantitation methods for the evaluation of antiestrogens in human breast cancer cells in culture
Journal of Pharmacological and Toxicological Methods, 1992
The antiproliferative activity of the antiestrogen, tamoxifen, on the growth of MCF-7 human breas... more The antiproliferative activity of the antiestrogen, tamoxifen, on the growth of MCF-7 human breast cancer cells was evaluated using the hemocytometric trypan blue exclusion method, [3H]-thymidine incorporation, and a total protein determination. Tamoxifen was evaluated over a concentration range from 10(-9) to 10(-6) M. The hemocytometric trypan blue exclusion method and [3H]-thymidine incorporation were sensitive enough to demonstrate the inhibitory influence of tamoxifen on the proliferation of MCF-7 cells at a concentration as low as 10(-9) M. A very good correlation of these two methods was observed in the submicromolar concentration range of tamoxifen. The total protein determination method was only sensitive enough to detect the antiproliferative influence of tamoxifen at concentrations above 10(-6) M. In conclusion, the [3H]-thymidine incorporation method was found to be effective and much less time consuming than the hemocytometric trypan blue exclusion method for evaluating the antiproliferative effects of antiestrogens in cultured MCF-7 cells. However, when evaluating antiestrogens, which are cell-cycle specific, the results of the [3H]-thymidine incorporation method should be interpreted with caution.
Evidence for secretion of high molecular weight mucins by canine tracheal epithelial cells in primary culture: Effects of select secretagogues on mucin secretion
In Vitro Cellular & Developmental Biology - Animal, 1992
The purpose of this investigation was to provide evidence for the secretion of high molecular wei... more The purpose of this investigation was to provide evidence for the secretion of high molecular weight mucins, CTM-A and CTM-B, in primary culture of canine tracheal epithelial (CTE) cells. The cells were isolated from tracheas of mongrel dogs by pronase treatment. Primary cultures of the epithelial cells were established using ICN collagen inserts in Dulbecco's modified Eagle's/F12 medium supplemented with growth factors and could be maintained for up to 23 days. The evidence for the mucin secretion in culture medium and their localization in the cells was established by a) positive immunocytochemical staining using specific antibodies developed against purified native as well as deglycosylated CTM-A and CTM-B; b) incorporation of labeled amino acids, followed by electrophoresis and autoradiography detection of glycoconjugates purified from the culture medium; c) comparison of the amino acid compositions of mucin purified from canine tracheal pouch secretions and that purified from the culture medium; and d) Western blot analyses using specific polyclonal antibodies directed against deglycosylated CTM-A and CTM-B. Immunoaffinity purified secreted labeled glycoconjugates were resistant to hyaluronidase treatment. The effects of cyclic AMP (1 x 10(-5) M), dibutyryl cyclic AMP (1 x 10(-5) M), 8-bromocyclic AMP (1 x 10(-5) M), and prostaglandin E1 (1 x 10(-6) M) on mucin secretion by CTE cells were also investigated. Secretion of mucins by CTE cells in culture was considerably more enhanced by 8-bromocyclic AMP than that observed for other secretagogues used in this study.
TMAO Promotes Fibrillization and Microtubule Assembly Activity in the C-Terminal Repeat Region of Tau
Biochemistry, 2006
Alzheimer&amp... more Alzheimer's disease most closely correlates with the appearance of the neurofibrillary tangles (NFTs), intracellular fibrous aggregates of the microtubule-associated protein, tau. Under native conditions, tau is an unstructured protein, and its physical characterization has revealed no clues about the three-dimensional structural determinants essential for aggregation or microtubule binding. We have found that the natural osmolyte trimethylamine N-oxide (TMAO) induces secondary structure in a C-terminal fragment of tau (tau(187)) and greatly promotes both self-aggregation and microtubule (MT) assembly activity. These processes could be distinguished, however, by a single-amino acid substitution (Tyr(310) --> Ala), which severely inhibited aggregation but had no effect on MT assembly activity. The inability of this mutant to aggregate could be completely reversed by TMAO. We propose a model in which TMAO induces partial order in tau(187), resulting in conformers that may correspond to on-pathway intermediates of either aggregation or tau-dependent MT assembly or both. These studies set the stage for future high-resolution structural characterization of these intermediates and the basis by which Tyr(310) may direct pathologic versus normal tau function.
Biochemistry, 1970
Light-scattering measurements of molecular weight at different protein concentrations at 25 and 3... more Light-scattering measurements of molecular weight at different protein concentrations at 25 and 30" provided direct evidence for the concentration-dependent dissociation of phosphorylase a. The molecular weight of tetrameric phosphorylase a was estimated to be 380,000. Methods for evaluating the equilibrium constant and specific activities of the dimeric and tetrameric species from initial rate measurements were developed. It was found that the increase in specific activity upon dilution can be quantitatively accounted for by a decrease in molecular weight. Standard enthalpy and entropy changes of the dissociation reaction at
Biochemical and Biophysical Research Communications, 1984
The sarcoplasmic reticulum and glycogen pellet derived from rabbit skeletal muscle and the sarcol... more The sarcoplasmic reticulum and glycogen pellet derived from rabbit skeletal muscle and the sarcolemma and sarcoplasmic reticulum from pig skeletal muscle contains NAD:dependent mono ADP-ribosyltransferase activity toward the guanidine analog, P-nitrobenzylidine aminoguanidine. No or little activity could be found in the sarcolemma or sarcoplasmic reticulum derived from canine cardiac muscle. Seventy percent of activity extracted from rabbit skeletal muscle is localized in the sarcoplasmic reticulum. The enzyme has a pH optimum of 7.4, and K M of 0.5 mM and 0.35 mM for NAD and 9_-nitrobenzylidine aminoguanidine, respectively. Inorganic phosphate, KCI, and guanidine derivatives inhibit the reaction. Incubation of the sarcoplasmic reticulum or glycogen pellet with (adenylate-32p) NAD or [adenosine-14C(U)]labeled NAD results in the incorporation of radioactivity into proteins. A large number of proteins are labeled in the sarcoplasmic reticulum fraction. The major labeled band in the glycogen pellet corresponds to a protein of molecular weight of 83 K. ADP-ribosyltransferase that catalyze the transfer of ADP-ribosyl moiety from NAD to acceptors such as arginine, other guanidino compounds, and proteins are a part of a variety of bacterial toxins(l-6) and are present in a number of animal tissues(7-12). ADP-ribosylation of cellular proteins is described in different tissues(l-12), but the natural substrates for the ADP-ribosyltransferases and the physiological implications of the modifications are not well defined in many instances. A number of biological activities such as cyclic AMP formation(13), phosphate transport by renal brush border membranes(14), Ca 2+ release by mitrochondria(11), and phospho
Isotope effects on the mechanism of calcineurin catalysis: kinetic solvent isotope and isotope exchange studies
Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology, 1994
The reaction scheme of calcineurin was examined with kinetic and physical approaches. Proton inve... more The reaction scheme of calcineurin was examined with kinetic and physical approaches. Proton inventory studies of the calcineurin-catalyzed hydrolysis of para-nitrophenyl phosphate were done to probe the role of proton transfer in the mechanism. Control experiments determined that the solvent did not cause the irreversible inactivation of the enzyme and had no effect on the dependence on metal ion or calmodulin. A solvent isotope effect was observed on the Vmax/Km term, but not the Vmax term. The isotope effect was modest with a value of 1.35. Proton inventory data could be fit by multiple parameter sets. The parameter sets yielded fractionation factors of 0.73 for a one-proton transfer or 0.85 for a two-proton transfer. These values compare to the value of 0.69 for reactions involving a water molecule or hydroxide coordinated to metal ion. A chemical mechanism consistent with the proton inventory data and other information about calcineurin catalysis is presented. The simplest model for catalysis involves a single proton transfer from water coordinated to metal that is reasoned to occur during association of the substrate with calcineurin. Questions about the reaction intermediate were also addressed. Attempts to monitor a phosphate-water exchange reaction with 31P nuclear magnetic resonance spectroscopy were unsuccessful. Failure to observe an exchange reaction suggests that no phosphoryl enzyme is formed during the progress of the reaction. Together these data are explained by a model in which cleavage of the phosphate ester bond is catalyzed by a water (hydroxide) molecule coordinated to a divalent metal ion without the formation of a covalent intermediate.
Biochimica et Biophysica Acta (BBA) - General Subjects, 2001
Akt is a serine/threonine kinase that plays a critical role in cell survival signaling and its ac... more Akt is a serine/threonine kinase that plays a critical role in cell survival signaling and its activation has been linked to tumorigenesis. Upregulation of Akt as well as its upstream regulator phosphatidylinositol-3 kinase (PI3K) has been found in many tumors and the negative regulator of this pathway PTEN/MMAC is a tumor suppressor. As a target for drug discovery, we have expressed and purified an active Akt1 enzyme from a recombinant baculovirus-infected Sf9 cell culture. Coexpression of Akt1 with the catalytic subunit of PI3K or treatment with okadaic acid during expression was found to generate an active enzyme in the insect cell culture system. We have optimized the kinase activity and developed a simple quantitative kinase assay using biotinylated peptide substrates. Using the purified active enzyme, we have characterized its physical, catalytic and kinetic properties. Since Akt is closely related to protein kinase C (PKC) and protein kinase A, the issue of obtaining selective inhibitors of this enzyme was addressed by comparison of the structures of catalytic domains of Akt and PKC, derived by homology modeling methods. A number of amino acid differences in the ATP binding regions of these kinases were identified, suggesting that selective inhibitors of Akt can be discovered. However, the ATP binding regions are highly conserved in the three isoforms of Akt implying that the discovery of isoform-selective inhibitors would be very challenging.
Archives of Biochemistry and Biophysics, 1998
Phosphorylated and nonphosphorylated forms of a decapeptide corresponding to residues 9 to 18 of ... more Phosphorylated and nonphosphorylated forms of a decapeptide corresponding to residues 9 to 18 of glycogen phosphorylase were compared using twodimensional nuclear magnetic resonance with assignment of both peptides done by the sequential method. Both forms had little secondary structure, but there was evidence for an interaction between arginine-16 and phosphorylated serine at position 14. A change in the chemical shift for the ⑀-nitrogen hydrogen of arginine in position 16 was observed in the spectrum of the phosphorylated peptide and was not evident in a phosphopeptide having citrulline in place of arginine-16. Hydrolysis catalyzed by protein phosphatase-1 was decreased with the citrulline-containing phosphopeptide compared to the arginine-containing phosphopeptide with effects observed on both k cat and K m of the phosphatase reaction. Alkaline phosphatase hydrolyzed these peptides and a di-citrulline peptide equally well. These results are consistent with arginine being favorable in the recognition of substrates by phosphatase-1, possibly recognition as an argininephosphoserine complex. As a model study, arginine and two analogs, citrulline and canavanine, were examined for association with inorganic phosphate by nuclear magnetic resonance spectrometry. 31 P-NMR measurements showed that arginine and canavanine caused a shift in the phosphate resonance at 20°C. Citrulline caused no change. Changes in chemical shift were measured over the pH range 5-9 with arginine and canavanine both causing a slight decrease in the apparent pK a of inorganic phosphate (⌬pK a Ϸ 0.15). NaCl, NH 4 Cl, and guanidine hydrochloride showed little effect on the resonance signal position of inorganic phosphate at pH 6.5, consistent with selectivity for the guanidino group. Temperature (6°, 20°, and 37°C) caused little change in the effect of arginine, but there was some dependency with canavanine, decreasing with temperature. Citrulline caused no change in the chemical shift of phosphate at any temperature. It was concluded that hydrogen bonded complexes were formed between the dianion of phosphate and the protonated form of arginine or canavanine with a bifurcated structure having preference for the-hydrogens.
Pharmacology Therapeutics, 1999
Although much can be learned about the specificity of protein kinases from studies with peptide s... more Although much can be learned about the specificity of protein kinases from studies with peptide substrates, the question remains, how do kinases recognize their three-dimensional protein substrates? Information derived from such studies provides further understanding of substrate recognition and can facilitate the design of specific protein kinase inhibitors. Phosphorylase kinase (PhK) catalyzes the phosphorylation of phosphorylase b (phos. b) to form the active phosphorylase a. No other protein kinase can duplicate this reaction. Why? To probe this question and establish what features in the protein are important for substrate binding and product release, mutants of phos. b have been studied. This report shows how mutations change the properties of the protein substrate and the ability of these mutants to be phosphorylated by PhK and other kinases. Action of protein kinases on their substrates is often regulated by autoinhibitory segments. The C-terminus of the catalytic ␥-subunit of PhK contains two inhibitory sites overlapping two calmodulin-binding regions. These two peptide segments resemble sequences in phos. b and may explain why peptides of these regions are potent inhibitors of PhK. We will show results with peptide inhibitors, using various expressed forms of the catalytic subunit, which describe their modes of interaction and mechanisms of inhibition. Metal ions can change molecular interactions. With PhK, Mn 2 ϩ facilitates the use of GTP as a phosphoryl group donor and greatly increases phosphorylation of a tyrosine residue in angiotensin II. This implies that the spatial arrangement of specificity determinants can be manipulated so that PhK can utilize other substrates. pharmacol. ther. 82(2-3):143-155, 1999.
Formation of a cyclic imide in aspartyl or asparaginyl glycyl peptides induced by heating in the dry state*
International Journal of Peptide and Protein Research, 2009
A common product was identified by HPLC from the heating of, alpha and beta isomers of, aspartyl ... more A common product was identified by HPLC from the heating of, alpha and beta isomers of, aspartyl glycine in the dry state. Fast atom-bombardment (FAB) mass spectrometry and Fourier transform infrared photoacoustic spectroscopy (FTIR-PAS) showed that the product is a cyclic imide. Cyclic imide formation also was shown to occur with the tripeptides Gly-Asp-Gly and Gly-Asn-Gly. The cyclization of aspartyl peptides depends upon protonation of the beta-carboxyl group and can be promoted by acidification or by heating the NH4+ salt in the dry state.
Archives of Biochemistry and Biophysics
Phosphorylase b and two peptides with sequences homologous to phosphorylation site 2 (syntide 2) ... more Phosphorylase b and two peptides with sequences homologous to phosphorylation site 2 (syntide 2) and site 3 (syntide 3) of glycogen synthase were compared as substrates for purified muscle phosphorylase kinase. The substrate specificity of phosphorylase kinase varied according to whether heparin (at pH 6.5) or Ca2+ (at pH 8.2) was used as a stimulator of its activity. Phosphorylase b was preferentially phosphorylated in the presence of Ca2'; the rate of syn tide 2 phosphorylation was the same for both stimulators; and the phosphorylation of syntide 3 was completely dependent on the presence of heparin. A kinetic analysis confirmed this stimulator-dependent substrate specificity since both the V
Journal of Biological Chemistry
Calcineurin, a calmodulin-activated protein phosphatase, is known to dephosphorylate certain low ... more Calcineurin, a calmodulin-activated protein phosphatase, is known to dephosphorylate certain low molecular weight phosphate esters. The low molecular weight phosphatase activity of calcineurin has been studied by utilizing tyrosine phosphate derivatives. Kinetic studies suggest that the substrate specificity is dependent upon the electronic nature of the substrate in contrast to results obtained with alkaline phosphatase from Escherichia coli. Comparison of calcineurinand acid-catalyzed hydrolyses indicates a 1:l correlation between the rate constants for the two processes. This correlation and other model studies have been utilized to provide insight into the chemical mechanism of calcineurin. Possible chemical mechanisms for calcineurin are discussed. 14932 This is an Open Access article under the CC BY license.
Journal of Biological Chemistry
The action of phosphorylase kinase on synthetic peptides is reported. These peptides are variants... more 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 6. The effects of size, the cluster of basic groups at the NH,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, K
Journal of Biological Chemistry
High-performance liquid chromatographic separation and renaturation of protein kinase subunits: application to catalytic subunit of phosphorylase kinase
Methods in Enzymology
Journal of Alzheimer's disease : JAD, 2009
An aqueous extract of Ceylon cinnamon (C. zeylanicum) is found to inhibit tau aggregation and fil... more An aqueous extract of Ceylon cinnamon (C. zeylanicum) is found to inhibit tau aggregation and filament formation, hallmarks of Alzheimer's disease (AD). The extract can also promote complete disassembly of recombinant tau filaments and cause substantial alteration of the morphology of paired-helical filaments isolated from AD brain. Cinnamon extract (CE) was not deleterious to the normal cellular function of tau, namely the assembly of free tubulin into microtubules. An A-linked proanthocyanidin trimer molecule was purified from the extract and shown to contain a significant proportion of the inhibitory activity. Treatment with polyvinylpyrolidone effectively depleted all proanthocyanidins from the extract solution and removed the majority, but not all, of the inhibitory activity. The remainder inhibitory activity could be attributed to cinnamaldehyde. This work shows that compounds endogenous to cinnamon may be beneficial to AD themselves or may guide the discovery of other pot...
Formation of a cyclic imide in aspartyl or asparaginyl glycyl peptides induced by heating in the dry state
International journal of peptide and protein research, 1987
A common product was identified by HPLC from the heating of, alpha and beta isomers of, aspartyl ... more A common product was identified by HPLC from the heating of, alpha and beta isomers of, aspartyl glycine in the dry state. Fast atom-bombardment (FAB) mass spectrometry and Fourier transform infrared photoacoustic spectroscopy (FTIR-PAS) showed that the product is a cyclic imide. Cyclic imide formation also was shown to occur with the tripeptides Gly-Asp-Gly and Gly-Asn-Gly. The cyclization of aspartyl peptides depends upon protonation of the beta-carboxyl group and can be promoted by acidification or by heating the NH4+ salt in the dry state.
Production and Characterization of Monoclonal Antibodies to Purified Deglycosylated Cystic Fibrosis Respiratory Mucin: Evidence for the Presence of Four Immunologically Distinct Epitopes
Hybridoma, 1991
Respiratory mucus glycoproteins (mucins) were purified from the tracheobronchial secretions of th... more Respiratory mucus glycoproteins (mucins) were purified from the tracheobronchial secretions of three Cystic Fibrosis (CF) patients. The mucins were completely deglycosylated by treatment with trifluoromethanesulfonic acid and subsequent treatment with alpha-N-acetylgalactosaminidase. Over thirty hybrid clones secreting antibodies against the deglycosylated mucin (DGM) were obtained using standard hybridoma techniques. Hybrids with positive identification for CF-DGM were cloned twice using limiting dilution method to ensure the monoclonal nature of the antibodies. Eight stable clones (1a, 1b, 10a, 10c, 10d, 10e, 29d, and 30e) secreting monoclonal antibodies (MAbs) showing specificity of reaction to CF-DGM were obtained. Two clones, 29d and 30e, secreted antibodies of the IgM class while the other six clones secreted antibodies of the IgG1 subclass. Denaturation and reduction experiments suggested that MAbs 1b, 10e, 29d and 30e were directed against a given sequence of amino acids in the DGM while the other four MAbs, in addition to being sequence specific, were also conformation dependent. Further, competitive binding radioimmunoassays suggested that MAbs 1b, 10e, 29d and 30e recognize four distinct epitopes in the peptidic core of CF respiratory mucin. In summary, the MAbs may provide a promising approach to elucidate the structure of the polypeptide backbone of human respiratory mucins as well as for the screening of cDNA libraries for clones secreting mucin(s).
Pharmacology & Therapeutics, 1999
Although much can be learned about the specificity of protein kinases from studies with peptide s... more Although much can be learned about the specificity of protein kinases from studies with peptide substrates, the question remains, how do kinases recognize their three-dimensional protein substrates? Information derived from such studies provides further understanding of substrate recognition and can facilitate the design of specific protein kinase inhibitors. Phosphorylase kinase (PhK) catalyzes the phosphorylation of phosphorylase b (phos. b) to form the active phosphorylase a. No other protein kinase can duplicate this reaction. Why? To probe this question and establish what features in the protein are important for substrate binding and product release, mutants of phos. b have been studied. This report shows how mutations change the properties of the protein substrate and the ability of these mutants to be phosphorylated by PhK and other kinases. Action of protein kinases on their substrates is often regulated by autoinhibitory segments. The C-terminus of the catalytic ␥-subunit of PhK contains two inhibitory sites overlapping two calmodulin-binding regions. These two peptide segments resemble sequences in phos. b and may explain why peptides of these regions are potent inhibitors of PhK. We will show results with peptide inhibitors, using various expressed forms of the catalytic subunit, which describe their modes of interaction and mechanisms of inhibition. Metal ions can change molecular interactions. With PhK, Mn 2 ϩ facilitates the use of GTP as a phosphoryl group donor and greatly increases phosphorylation of a tyrosine residue in angiotensin II. This implies that the spatial arrangement of specificity determinants can be manipulated so that PhK can utilize other substrates. pharmacol. ther. 82(2-3):143-155, 1999.
Cell-growth quantitation methods for the evaluation of antiestrogens in human breast cancer cells in culture
Journal of Pharmacological and Toxicological Methods, 1992
The antiproliferative activity of the antiestrogen, tamoxifen, on the growth of MCF-7 human breas... more The antiproliferative activity of the antiestrogen, tamoxifen, on the growth of MCF-7 human breast cancer cells was evaluated using the hemocytometric trypan blue exclusion method, [3H]-thymidine incorporation, and a total protein determination. Tamoxifen was evaluated over a concentration range from 10(-9) to 10(-6) M. The hemocytometric trypan blue exclusion method and [3H]-thymidine incorporation were sensitive enough to demonstrate the inhibitory influence of tamoxifen on the proliferation of MCF-7 cells at a concentration as low as 10(-9) M. A very good correlation of these two methods was observed in the submicromolar concentration range of tamoxifen. The total protein determination method was only sensitive enough to detect the antiproliferative influence of tamoxifen at concentrations above 10(-6) M. In conclusion, the [3H]-thymidine incorporation method was found to be effective and much less time consuming than the hemocytometric trypan blue exclusion method for evaluating the antiproliferative effects of antiestrogens in cultured MCF-7 cells. However, when evaluating antiestrogens, which are cell-cycle specific, the results of the [3H]-thymidine incorporation method should be interpreted with caution.
Evidence for secretion of high molecular weight mucins by canine tracheal epithelial cells in primary culture: Effects of select secretagogues on mucin secretion
In Vitro Cellular & Developmental Biology - Animal, 1992
The purpose of this investigation was to provide evidence for the secretion of high molecular wei... more The purpose of this investigation was to provide evidence for the secretion of high molecular weight mucins, CTM-A and CTM-B, in primary culture of canine tracheal epithelial (CTE) cells. The cells were isolated from tracheas of mongrel dogs by pronase treatment. Primary cultures of the epithelial cells were established using ICN collagen inserts in Dulbecco's modified Eagle's/F12 medium supplemented with growth factors and could be maintained for up to 23 days. The evidence for the mucin secretion in culture medium and their localization in the cells was established by a) positive immunocytochemical staining using specific antibodies developed against purified native as well as deglycosylated CTM-A and CTM-B; b) incorporation of labeled amino acids, followed by electrophoresis and autoradiography detection of glycoconjugates purified from the culture medium; c) comparison of the amino acid compositions of mucin purified from canine tracheal pouch secretions and that purified from the culture medium; and d) Western blot analyses using specific polyclonal antibodies directed against deglycosylated CTM-A and CTM-B. Immunoaffinity purified secreted labeled glycoconjugates were resistant to hyaluronidase treatment. The effects of cyclic AMP (1 x 10(-5) M), dibutyryl cyclic AMP (1 x 10(-5) M), 8-bromocyclic AMP (1 x 10(-5) M), and prostaglandin E1 (1 x 10(-6) M) on mucin secretion by CTE cells were also investigated. Secretion of mucins by CTE cells in culture was considerably more enhanced by 8-bromocyclic AMP than that observed for other secretagogues used in this study.
TMAO Promotes Fibrillization and Microtubule Assembly Activity in the C-Terminal Repeat Region of Tau
Biochemistry, 2006
Alzheimer&amp... more Alzheimer's disease most closely correlates with the appearance of the neurofibrillary tangles (NFTs), intracellular fibrous aggregates of the microtubule-associated protein, tau. Under native conditions, tau is an unstructured protein, and its physical characterization has revealed no clues about the three-dimensional structural determinants essential for aggregation or microtubule binding. We have found that the natural osmolyte trimethylamine N-oxide (TMAO) induces secondary structure in a C-terminal fragment of tau (tau(187)) and greatly promotes both self-aggregation and microtubule (MT) assembly activity. These processes could be distinguished, however, by a single-amino acid substitution (Tyr(310) --> Ala), which severely inhibited aggregation but had no effect on MT assembly activity. The inability of this mutant to aggregate could be completely reversed by TMAO. We propose a model in which TMAO induces partial order in tau(187), resulting in conformers that may correspond to on-pathway intermediates of either aggregation or tau-dependent MT assembly or both. These studies set the stage for future high-resolution structural characterization of these intermediates and the basis by which Tyr(310) may direct pathologic versus normal tau function.
Biochemistry, 1970
Light-scattering measurements of molecular weight at different protein concentrations at 25 and 3... more Light-scattering measurements of molecular weight at different protein concentrations at 25 and 30" provided direct evidence for the concentration-dependent dissociation of phosphorylase a. The molecular weight of tetrameric phosphorylase a was estimated to be 380,000. Methods for evaluating the equilibrium constant and specific activities of the dimeric and tetrameric species from initial rate measurements were developed. It was found that the increase in specific activity upon dilution can be quantitatively accounted for by a decrease in molecular weight. Standard enthalpy and entropy changes of the dissociation reaction at
Biochemical and Biophysical Research Communications, 1984
The sarcoplasmic reticulum and glycogen pellet derived from rabbit skeletal muscle and the sarcol... more The sarcoplasmic reticulum and glycogen pellet derived from rabbit skeletal muscle and the sarcolemma and sarcoplasmic reticulum from pig skeletal muscle contains NAD:dependent mono ADP-ribosyltransferase activity toward the guanidine analog, P-nitrobenzylidine aminoguanidine. No or little activity could be found in the sarcolemma or sarcoplasmic reticulum derived from canine cardiac muscle. Seventy percent of activity extracted from rabbit skeletal muscle is localized in the sarcoplasmic reticulum. The enzyme has a pH optimum of 7.4, and K M of 0.5 mM and 0.35 mM for NAD and 9_-nitrobenzylidine aminoguanidine, respectively. Inorganic phosphate, KCI, and guanidine derivatives inhibit the reaction. Incubation of the sarcoplasmic reticulum or glycogen pellet with (adenylate-32p) NAD or [adenosine-14C(U)]labeled NAD results in the incorporation of radioactivity into proteins. A large number of proteins are labeled in the sarcoplasmic reticulum fraction. The major labeled band in the glycogen pellet corresponds to a protein of molecular weight of 83 K. ADP-ribosyltransferase that catalyze the transfer of ADP-ribosyl moiety from NAD to acceptors such as arginine, other guanidino compounds, and proteins are a part of a variety of bacterial toxins(l-6) and are present in a number of animal tissues(7-12). ADP-ribosylation of cellular proteins is described in different tissues(l-12), but the natural substrates for the ADP-ribosyltransferases and the physiological implications of the modifications are not well defined in many instances. A number of biological activities such as cyclic AMP formation(13), phosphate transport by renal brush border membranes(14), Ca 2+ release by mitrochondria(11), and phospho
Isotope effects on the mechanism of calcineurin catalysis: kinetic solvent isotope and isotope exchange studies
Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology, 1994
The reaction scheme of calcineurin was examined with kinetic and physical approaches. Proton inve... more The reaction scheme of calcineurin was examined with kinetic and physical approaches. Proton inventory studies of the calcineurin-catalyzed hydrolysis of para-nitrophenyl phosphate were done to probe the role of proton transfer in the mechanism. Control experiments determined that the solvent did not cause the irreversible inactivation of the enzyme and had no effect on the dependence on metal ion or calmodulin. A solvent isotope effect was observed on the Vmax/Km term, but not the Vmax term. The isotope effect was modest with a value of 1.35. Proton inventory data could be fit by multiple parameter sets. The parameter sets yielded fractionation factors of 0.73 for a one-proton transfer or 0.85 for a two-proton transfer. These values compare to the value of 0.69 for reactions involving a water molecule or hydroxide coordinated to metal ion. A chemical mechanism consistent with the proton inventory data and other information about calcineurin catalysis is presented. The simplest model for catalysis involves a single proton transfer from water coordinated to metal that is reasoned to occur during association of the substrate with calcineurin. Questions about the reaction intermediate were also addressed. Attempts to monitor a phosphate-water exchange reaction with 31P nuclear magnetic resonance spectroscopy were unsuccessful. Failure to observe an exchange reaction suggests that no phosphoryl enzyme is formed during the progress of the reaction. Together these data are explained by a model in which cleavage of the phosphate ester bond is catalyzed by a water (hydroxide) molecule coordinated to a divalent metal ion without the formation of a covalent intermediate.
Biochimica et Biophysica Acta (BBA) - General Subjects, 2001
Akt is a serine/threonine kinase that plays a critical role in cell survival signaling and its ac... more Akt is a serine/threonine kinase that plays a critical role in cell survival signaling and its activation has been linked to tumorigenesis. Upregulation of Akt as well as its upstream regulator phosphatidylinositol-3 kinase (PI3K) has been found in many tumors and the negative regulator of this pathway PTEN/MMAC is a tumor suppressor. As a target for drug discovery, we have expressed and purified an active Akt1 enzyme from a recombinant baculovirus-infected Sf9 cell culture. Coexpression of Akt1 with the catalytic subunit of PI3K or treatment with okadaic acid during expression was found to generate an active enzyme in the insect cell culture system. We have optimized the kinase activity and developed a simple quantitative kinase assay using biotinylated peptide substrates. Using the purified active enzyme, we have characterized its physical, catalytic and kinetic properties. Since Akt is closely related to protein kinase C (PKC) and protein kinase A, the issue of obtaining selective inhibitors of this enzyme was addressed by comparison of the structures of catalytic domains of Akt and PKC, derived by homology modeling methods. A number of amino acid differences in the ATP binding regions of these kinases were identified, suggesting that selective inhibitors of Akt can be discovered. However, the ATP binding regions are highly conserved in the three isoforms of Akt implying that the discovery of isoform-selective inhibitors would be very challenging.
Archives of Biochemistry and Biophysics, 1998
Phosphorylated and nonphosphorylated forms of a decapeptide corresponding to residues 9 to 18 of ... more Phosphorylated and nonphosphorylated forms of a decapeptide corresponding to residues 9 to 18 of glycogen phosphorylase were compared using twodimensional nuclear magnetic resonance with assignment of both peptides done by the sequential method. Both forms had little secondary structure, but there was evidence for an interaction between arginine-16 and phosphorylated serine at position 14. A change in the chemical shift for the ⑀-nitrogen hydrogen of arginine in position 16 was observed in the spectrum of the phosphorylated peptide and was not evident in a phosphopeptide having citrulline in place of arginine-16. Hydrolysis catalyzed by protein phosphatase-1 was decreased with the citrulline-containing phosphopeptide compared to the arginine-containing phosphopeptide with effects observed on both k cat and K m of the phosphatase reaction. Alkaline phosphatase hydrolyzed these peptides and a di-citrulline peptide equally well. These results are consistent with arginine being favorable in the recognition of substrates by phosphatase-1, possibly recognition as an argininephosphoserine complex. As a model study, arginine and two analogs, citrulline and canavanine, were examined for association with inorganic phosphate by nuclear magnetic resonance spectrometry. 31 P-NMR measurements showed that arginine and canavanine caused a shift in the phosphate resonance at 20°C. Citrulline caused no change. Changes in chemical shift were measured over the pH range 5-9 with arginine and canavanine both causing a slight decrease in the apparent pK a of inorganic phosphate (⌬pK a Ϸ 0.15). NaCl, NH 4 Cl, and guanidine hydrochloride showed little effect on the resonance signal position of inorganic phosphate at pH 6.5, consistent with selectivity for the guanidino group. Temperature (6°, 20°, and 37°C) caused little change in the effect of arginine, but there was some dependency with canavanine, decreasing with temperature. Citrulline caused no change in the chemical shift of phosphate at any temperature. It was concluded that hydrogen bonded complexes were formed between the dianion of phosphate and the protonated form of arginine or canavanine with a bifurcated structure having preference for the-hydrogens.