Biomedical subjects

D J Graves

Publications and source records attributed to D J Graves.

At least 19 recordsLinked to original sources

PubMed2002

Four-laser scanning confocal system for microarray analysis.

We have constructed a confocal scanner suitable for routine microarray analysis from commercially available parts. We have outlined the details that should be considered when designing such an instrument and listed some of the specific components comprising the system [the full list of system components is available on CD from the corresponding author (D.J.G.) at no charge]. Here, we describe the methods used to test the linearity and sensitivity of the instrument. Performance was evaluated with two commonly used dyes, fluorescein and Cy5. While the instrument had a linear correlation between the dye concentration and fluorescence intensity, the observed deviation from a slope of 1.0 underscores the importance of running multipoint calibration experiments to obtain accurate dye quantitation over the full dynamic range of the scanner. This method has utility in testing commercial instruments in addition to the scanner described here. An array with over 300 spots dyed with Cy3 was scanned with our instrument and a high-end commercial instrument. The agreement between the two instruments was very good over a 1000-fold intensity range. Our scanner is a cost-effective alternative to more costly commercial scanners with similar capabilities.

Fluoresceins↗

PubMed2001

An inhibitory segment of the catalytic subunit of phosphorylase kinase does not act as a pseudosubstrate.

The C terminus of the catalytic gamma subunit of phosphorylase kinase contains two autoinhibitory calmodulin binding domains designated PhK13 and PhK5. These peptides inhibit truncated gamma(1-300). Previous data show that PhK13 (residues 302-326) is a competitive inhibitor with respect to phosphorylase b, with a K(i) of 1.8 microm. This result suggests that PhK13 may bind to the active site of truncated gamma(1-300). Variants of PhK13 were prepared to localize the determinants for interaction with the catalytic fragment gamma(1-300). PhK13-1, containing residues 302-312, was found to be a competitive inhibitor with respect to phosphorylase b with a K(i) of 6.0 microm. PhK13 has been proposed to function as a pseudosubstrate inhibitor with Cys-308 occupying the site that normally accommodates the phosphorylatable serine in phosphorylase b. A PhK13-1 variant, C308S, was synthesized. Kinetic characterization of this peptide reveals that it does not serve as a substrate but is a competitive inhibitor. Additional variants were designed based on previous knowledge of phosphorylase kinase substrate determinants. Variants were analyzed as substrates and as inhibitors for truncated gamma(1-300). Although PhK13-1 does not appear to function as a pseudosubstrate, several specificity determinants employed in the recognition of phosphorylase b as substrate are utilized in the recognition of PhK13-1 as an inhibitor.

Adenosine Triphosphatases↗

PubMed2001

The amino-terminal tail of glycogen phosphorylase is a switch for controlling phosphorylase conformation, activation, and response to ligands.

Glycogen phosphorylase is a muscle enzyme which metabolizes glycogen, producing glucose-1-phosphate, which can be used for the production of ATP. Phosphorylase activity is regulated by phosphorylation/dephosphorylation, and by the allosteric binding of numerous effectors. In this work, we have studied 10 site-directed mutants of glycogen phosphorylase (GP) in its amino-terminal regulatory region to characterize any changes that the mutations may have made on its structure or function. All of the GP mutants had normal levels of activity in the presence of the allosteric activator AMP. Some of the mutants were observed to have altered AMP-binding characteristics, however. R16A and R16E were activated at very low AMP concentration and crystallized at low temperature, like the phosphorylated form of GP, phosphorylase a, and unlike the dephospho-form, phosphorylase b. This indicates that even without phosphorylation, the structures of these mutants are more like phosphorylase a than phosphorylase b. These mutants were also very poorly phosphorylated in the presence of the inhibitor glucose, while phosphorylase b was phosphorylated normally with this inhibitor present. In contrast to R16A and R16E, four other mutants behaved like phosphorylase b after phosphorylation. R69E was only partially activated by phosphorylation, and I13G, R43E, and R43E/R69E were completely inactive after phosphorylation. We propose a model for the many functions of the amino terminus to explain the many varied effects of these mutations.

Amino Acid Substitution↗

PubMed2001

A hydroxychalcone derived from cinnamon functions as a mimetic for insulin in 3T3-L1 adipocytes.

OBJECTIVES: These studies investigated the ability of a hydroxychalcone from cinnamon to function as an insulin mimetic in 3T3-LI adipocytes. METHODS: Comparative experiments were performed with the cinnamon methylhydroxychalcone polymer and insulin with regard to glucose uptake, glycogen synthesis. phosphatidylinositol-3-kinase dependency, glycogen synthase activation and glycogen synthase kinase-3beta activity. The phosphorylation state of the insulin receptor was also investigated. RESULTS: MHCP treatment stimulated glucose uptake and glycogen synthesis to a similar level as insulin. Glycogen synthesis was inhibited by both wortmannin and LY294002, inhibitors directed against the PI-3-kinase. In addition, MHCP treatment activated glycogen synthase and inhibited glycogen synthase kinase-3beta activities, known effects of insulin treatment. Analysis of the insulin receptor demonstrated that the receptor was phosphorylated upon exposure to the MHCP. This supports that the insulin cascade was triggered by MHCP. Along with comparing MHCP to insulin, experiments were done with MHCP and insulin combined. The responses observed using the dual treatment were greater than additive, indicating synergism between the two compounds. CONCLUSION: Together, these results demonstrate that the MHCP is an effective mimetic of insulin. MHCP may be useful in the treatment of insulin resistance and in the study of the pathways leading to glucose utilization in cells.

3T3 Cells↗

PubMed2000

Site-directed mutants of glycogen phosphorylase are altered in their interaction with phosphorylase kinase.

Glycogen phosphorylase is found in resting muscle as phosphorylase b, which is inactive without AMP. Phosphorylation by phosphorylase kinase (PhK) produces phosphorylase a, which is active in the absence of AMP. PhK is the only kinase that can phosphorylate phosphorylase b, which in turn is the only physiological substrate for PhK. We have explored the reasons for this specificity and how these two enzymes recognize each other by studying site-directed mutants of glycogen phosphorylase. All mutants were assayed for changes in their interaction with a truncated form of the catalytic subunit of phosphorylase kinase, gamma(1-300). Five mutations (R69K, R69E, R43E, R43E/R69E, and E501A), made at sites that interact with the amino terminus in either phosphorylase b or a, showed little difference in phosphorylation by gamma(1-300) compared to wild-type phosphorylase b. Five mutations, made at three sites in the amino-terminal tail of phosphorylase (K11A, K11E, I13G, R16A, and R16E), however, produced decreases in catalytic efficiency for gamma(1-300), compared to that for phosphorylase b. R16E was the poorest substrate for gamma(1-300), giving a 47-fold decrease in catalytic efficiency. The amino terminus, and especially Arg 16, are very important factors for recognition of phosphorylase by gamma(1-300). A specific interaction between Lys 11 of phosphorylase and Glu 110 of gamma(1-300) was also confirmed. In addition, I13G and R16A were able to be phosphorylated by protein kinase A, which does not recognize native phosphorylase.

Amino Acid Substitution↗

PubMed2000

Arginine to citrulline replacement in substrates of phosphorylase kinase.

Synthetic peptides based on residues 9 to 18 of glycogen phosphorylase were prepared containing citrulline at position 10 or 16, or at both positions 10 and 16. The peptides were compared as substrates for a recombinant, truncated form of the catalytic subunit of phosphorylase kinase (residues 1-300). The peptide having citrulline at position 10 was phosphorylated the same as the parent peptide. Both the peptides with a single citrulline at position 16 and with two citrullines were phosphorylated less effectively than the parent peptide; k(cat)/K(m) values were approximately 20% the value with the parent peptide. Incorporation of the second citrulline had little change in the effectiveness of the peptide as a substrate although the kinetic parameters with the citrulline peptides did show differences. The change in peptide phosphorylation did not seem to result from a change in peptide structure. Two-dimensional nuclear magnetic resonance studies of di-citrulline peptide are reported and showed no change in the solution structure of the peptide compared to the parent peptide. Thus, the change in kinetic parameters with the modified peptides seemed an effect of arginine replacement and was likely a consequence of the loss of charge inasmuch as the size of arginine and citrulline are similar. Arginine-16 was concluded to be more important for phosphorylase kinase recognition than arginine-10. These findings were consistent with the earlier studies using alanine replacement of arginine in synthetic peptides as substrates for the holoenzyme form of phosphorylase kinase.

Amino Acid Sequence↗

PubMed1999

Phosphorylated tau can promote tubulin assembly.

Phosphorylation can affect the function of microtubule-associated protein tau. Here, the human brain tau with 441 amino acids was phosphorylated by cyclic-AMP-dependent protein kinase (PKA) or glycogen synthase kinase-3beta. PKA-phosphorylated tau (2.7 mol phosphates/mol) does not promote tubulin assembly as judged by spectrophotometric and atomic force microscopy measurements, unless trimethylamine N-oxide (TMAO), a natural occurring osmolyte, is included in these assays. TMAO is also found to promote tubulin assembly of glycogen synthase kinase-3beta-phosphorylated tau (1.6 mol phosphates/mol). TMAO does not act by causing a chemical dephosphorylation of phosphorylated tau, but it acts to overcome the functional deficit caused by phosphorylation. PKA-phosphorylated tau binds to tubulin in the presence of TMAO and lowers the critical concentration of tubulin needed for assembly. From these data, we conclude that PKA-phosphorylated tau retains the ability to bind tubulin and promote tubulin assembly. TMAO is required, however, to sensitize the reaction. Possible uses of TMAO in relation to studies of tubulin assembly in vitro, in intact cells, and in relation to Alzheimer's disease are presented in this report.

Brain Chemistry↗

PubMed1999

A recombinant form of the catalytic subunit of phosphorylase kinase that is soluble, monomeric, and includes key C-terminal residues.

Residues 302-326 of the catalytic (gamma) subunit of phosphorylase kinase (PhK) may comprise an autoinhibitory, pseudosubstrate domain that binds calmodulin. To study this, the cDNA corresponding to rabbit muscle PhKgamma was expressed using Escherichia coli. This yielded two stable, high-activity PhKgamma forms (35 and 42 kDa by SDS-PAGE) that were smaller than an authentic sample of rabbit muscle PhKgamma (45 kDa by SDS-PAGE). Each recombinant form was purified to homogeneity. The N-terminal sequence of the larger, 42-kDa form (pk42) matched that of the rabbit muscle enzyme. This suggested that pk42 consisted of PhKgamma residues 1-362, including the putative calmodulin-binding, autoinhibitory domain. Kinetic parameters obtained for pk42 were like those previously reported for the intact gamma subunit. This implied that the lack of 25 PhKgamma C-terminal residues did not affect phosphorylase kinase activity, but greatly improved enzyme stability. An additional 60 residues were removed from the C-terminus of pk42 using the protease m-calpain. This increased the kinase activity 1.5-fold. Consistent with this, the activity of a mutant PhKgamma that consisted of residues 1-300, denoted gamma1-300, was like that of the m-calpain-treated enzyme. Therefore, although the effect was small, some influence by the C-terminus of pk42 was noted. Moreover, when pk42 was incubated with ATP alone, a C-terminal threonine residue became phosphorylated. Although the influence of this autophosphorylation cannot be inferred from this data, it was evidence that the C-terminus accessed the enzyme's active site. Taken together, these data imply that pk42 will be useful to study phosphorylase kinase structure/activity relationships.

Adenosine Triphosphate↗

PubMed1999

The effects of mono-ADP-ribosylation on desmin assembly-disassembly.

Previous studies have shown that desmin, the muscle-specific intermediate filament protein, is a substrate for the endogenous muscle arginine-specific mono-ADP-ribosyltransferase and that ADP-ribosylation inhibits assembly of desmin into intermediate filaments (Huang et al., Exp. Cell Res. 226, 147-153, 1996). In this paper, the effects of mono-ADP-ribosylation on assembly and disassembly of desmin intermediate filaments were further characterized. First, it was found that ADP-ribosylated desmin does not coassemble with unmodified desmin and has no effect on assembly of unmodified desmin. Second, incubation of assembled desmin filaments with mono-ADP-ribosyltransferase and NAD+ results in disassembly of the filaments. Finally, the structural components of the attached ADP-ribose moiety responsible for altering the assembly of desmin into filaments were investigated by a stepwise cleavage of ADP-ribose with snake venom phosphodiesterase and alkaline phophatase, followed by analysis of assembly. The reactions catalyzed by these two enzymes were established using a desmin peptide as a substrate. Our results show that ribosylated desmin, but not phosphoribosylated desmin, was able to self-assemble into intermediate filaments, suggesting that the presence of a phosphate group is needed to alter desmin's assembly ability.

ADP Ribose Transferases↗

PubMed1999

Powerful tools for genetic analysis come of age.

Microscopic arrays of oligonucleotides or cDNA containing up to several hundred thousand different sequences are starting to influence methodologies and paths to discovery in genomics. Gene polymorphisms and mutations can be found and gene expression measured with unprecedented speed and parallelism. The principles of this modern technology and some of the problems awaiting further study are discussed.

Biotechnology↗

PubMed1998

System for preparing microhybridization arrays on glass slides.

We describe the construction and operation of an arrayer system to produce patterns of DNA sequences for analytical uses such as microarrays of oligonucleotide on microchips. Detailed documentation on construction is provided, as well as added electronic circuitry and the software for the instrument, including programs to machine its own working surface as well as those to operate it as an arrayer. Its cost is modest, and with a single droplet tip it can deposit 96 spots per slide on 32 slides in about 200 min (readily upgraded to higher speeds). As currently operated, it can place 400 spots in 1 cm2, and this density, too, can be increased easily. We discuss design features and performance to demonstrate utility and flexibility.

Microchemistry↗

PubMed1998

Effect of citrulline for arginine replacement on the structure and turnover of phosphopeptide substrates of protein phosphatase-1.

Phosphorylated and nonphosphorylated forms of a decapeptide corresponding to residues 9 to 18 of glycogen phosphorylase were compared using two-dimensional 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 epsilon-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 kcat and Km 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 arginine-phosphoserine complex. As a model study, arginine and two analogs, citrulline and canavanine, were examined for association with inorganic phosphate by nuclear magnetic resonance spectrometry. 31P-NMR measurements showed that arginine and canavanine caused a shift in the phosphate resonance at 20 degreesC. 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 pKa of inorganic phosphate (DeltapKa approximately 0.15). NaCl, NH4Cl, 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 degrees, 20 degrees, and 37 degreesC) 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 omega-hydrogens.

Alkaline Phosphatase↗

PubMed1998

Natural methylamine osmolytes, trimethylamine N-oxide and betaine, increase tau-induced polymerization of microtubules.

The natural osmolyte trimethylamine N-oxide (TMAO) at 200 mM increases the extent and the rate of formation of polymerized microtubule (MT) complex caused by tau. TMAO at this concentration has no effect on tubulin alone. Urea at 200 mM blocks tubulin assembly caused by tau, but this inhibition can be reversed by an equal amount of TMAO. Besides TMAO, betaine, another natural osmolyte, was found to have the same effects on MT as TMAO. On the contrary, glycerol (a carbohydrate osmolyte) and glycine (an amino acid osmolyte) do not increase tau-induced MT assembly. The mechanism by which TMAO and betaine enhance tau's effectiveness is not known, but physical studies suggest that the secondary structure of tau is not appreciably changed by 200 mM TMAO. This is the first report showing that natural osmolytes, TMAO and betaine, at a near physiological concentration are able to stimulate tau-induced tubulin assembly.

Animals↗

PubMed1998

Regulation of PTP-1 and insulin receptor kinase by fractions from cinnamon: implications for cinnamon regulation of insulin signalling.

Bioactive compound(s) extracted from cinnamon potentiate insulin activity, as measured by glucose oxidation in the rat epididymal fat cell assay. Wortmannin, a potent PI 3'-kinase inhibitor, decreases the biological response to insulin and bioactive compound(s) from cinnamon similarly, indicating that cinnamon is affecting an element(s) upstream of PI 3'-kinase. Enzyme studies done in vitro show that the bioactive compound(s) can stimulate autophosphorylation of a truncated form of the insulin receptor and can inhibit PTP-1, a rat homolog of a tyrosine phosphatase (PTP-1B) that inactivates the insulin receptor. No inhibition was found with alkaline phosphate or calcineurin suggesting that the active material is not a general phosphatase inhibitor. It is suggested, then, that a cinnamon compound(s), like insulin, affects protein phosphorylation-dephosphorylation reactions in the intact adipocyte. Bioactive cinnamon compounds may find further use in studies of insulin resistance in adult-onset diabetes.

Androstadienes↗

PubMed1997

Regulatory role of arginine-specific mono(ADP-ribosyl)transferase in muscle cells.

Earlier we demonstrated that meta-iodobenzylguanidine (MIBG), a specific inhibitor of arginine mono-ADP-ribosylation blocks proliferation and differentiation of chick skeletal myogenic cells in culture (Exp. Cell Res., 1992, 201:33-42). Membrane fractions from 4-day, myotube cultures of embryonic chick muscle cells were incubated with 32P-NAD+. Several proteins were labeled, but labeling of two hands of about 53 and 36 kDa appeared to be due to arginyl ADP-ribosylation. Immunoprecipitation with D3 monoclonal antibody to the intermediate filament protein desmin, SDS-PAGE and autoradiography demonstrated that the 53 kDa band contained desmin, and that this desmin is ADP-ribosylated by the endogenous arginine-specific mono(ADP-ribosyl)transferase (Exp. Cell Res., 1996, in press). Desmin is the muscle-specific intermediate filament protein, and it appears to be one of the first muscle-specific proteins expressed during terminal myogenic differentiation. We have examined whether desmin can be ADP-ribosylated in muscle cells by use of polyclonal antibodies for ADP-ribosylated arginyl residues. We have found that soluble desmin is present in 5-6 day myogenic cell cultures and that this desmin contains ADP-ribose, demonstrating that desmin is ADP-ribosylated in skeletal muscle cells. We also found that purified avian desmin contains antigenic material that reacts with these antibodies. In both cases, NaCl had no effect on the reactivity, but NH2OH did, which is consistent with an arginine-ADPR linkage. In summary, these results suggest that ADP-ribosylation is an important regulatory mechanism in differentiating muscle cells, and that the intermediate filament protein desmin is an important substrate for modification in muscle cells.

ADP Ribose Transferases↗

PubMed1996

Characterization of ADP-ribosylation sites on desmin and restoration of desmin intermediate filament assembly by de-ADP-ribosylation .

Desmin is an intermediate filament protein that can be ADP-ribosylated by arginine-specific mono(ADP-ribosyl) transferase. Stoichiometric modification of desmin by the transferase causes inhibition of assembly of desmin into 10-nm intermediate filaments (Huang et al., 1993, Biochem. Biophys. Res. Commun. 197, 570-577). In this work, the sites of modification that can affect disassembly have been identified. ADP-ribosylated desmin (1.2 mol ADP-ribose/mol desmin) was digested with lysyl endopeptidase followed by trypsin. Two ADP-ribosylated peptides were obtained, sequenced by Edman degradation, and analyzed by the use of matrix-assisted laser desorption/ionization mass spectrometry. Arginines 48 and 68 of desmin's head domain were shown to be sites of modification, with arginine 48 the major ADP-ribosylation site. ADP-ribosylated desmin (4 mol ADP-ribose/mol desmin) was treated with ADP-ribosylarginine hydrolase. Removal of more than three ADP-ribose groups results in partial restoration of desmin's ability to form intermediate filaments. It is necessary to remove all ADP-ribose groups from desmin to restore its complete ability to form intermediate filaments. The fact that the effect of ADP-ribosylation on the filament-forming properties of desmin is fully reversible suggests that ADP-ribosylation alone is responsible for the changes noted in desmin.

ADP Ribose Transferases↗

PubMed1996

Target proteins for arginine-specific mono(ADP-ribosyl) transferase in membrane fractions from chick skeletal muscle cells.

In a previous study, we found that a specific inhibitor of cellular arginine-specific mono(ADP-ribosyl) transferase, meta-iodobenzylguanidine (MIBG), reversibly inhibited both proliferation and differentiation of cultured embryonic chick primary muscle myoblasts. In addition, we observed that arginine-specific ADP-ribosyltransferase activity increased with muscle-cell differentiation in cultures. Therefore, muscle-cell cultures, especially the 96-h myotube cultures that contain the highest levels of ADP-ribosyltransferase, were used as a working system to determine the cellular protein substrates for arginine-specific ADP-ribosyltransferase. When membrane fractions extracted from 96-h chick myotubes were incubated with [32P]NAD at 30 degrees C for 30 min, only a few proteins were labeled. The labeling of two proteins of 36 and 56 kDa was inhibited by the presence of an arginine-specific mono(ADP-ribosyl) transferase inhibitor, MIBG, and by novobiocin. To prove that these proteins are indeed the targets for arginine-specific mono(ADP-ribosyl)ation, active recombinant muscle ADP-ribosyltransferase was incubated with membrane proteins under the same conditions. ADP-ribosylation of these two membrane proteins, as seen in the endogenous reactions, was also catalyzed by the added muscle transferase and was also inhibited by MIBG and novobiocin. By using antibody specific for desmin for immunoprecipitation and immunoblot analysis, we found that a 56-kDa protein associated with the membrane of myotubes is desmin. Our results showed that incorporation of isotope into this protein band from [32P]NAD is due to ADP-ribosylation of desmin.

3-Iodobenzylguanidine↗

PubMed1996

Deimination of glycogen phosphorylase b by peptidylarginine deiminase. Influence on the kinetical characteristics and dimer-tetramer transition.

The kinetics of the native glycogen phosphorylase b from rabbit skeletal muscle and of the enzyme specifically deiminated by peptidylarginine deiminase have been studied. One arginine residue per phosphorylase b monomer is transformed into citrulline after 3 h of incubation with peptidylarginine deiminase. The maximal velocity of the enzymatic reaction for the modified phosphorylase b is 7-20% higher than that for the native enzyme. Deiminated phosphorylase b, like the native enzyme, shows a positive kinetic cooperativity with respect to glucose-1-phosphate. The affinity of the modified phosphorylase b for the allosteric activator AMP is one order of magnitude higher than that of the native enzyme. Deimination caused a pronounced reduction of the values of [I]0.5 for FMN and glucose, but the sensitivity of the deiminated enzyme to glucose-6-phosphate is much lower than that of the native phosphorylase b. Deiminated phosphorylase b, unlike the native enzyme, shows the positive cooperativity for FMN binding. Deiminated phosphorylase b, unlike the native enzyme, shows less capability to form tetramers in the presence of AMP as compared to the native enzyme.

Adenosine Monophosphate↗