Biomedical subjects

D J Graves

Publications and source records attributed to D J Graves.

At least 37 records · Page 2Linked to original sources

PubMed1996

A structural basis for substrate specificities of protein Ser/Thr kinases: primary sequence preference of casein kinases I and II, NIMA, phosphorylase kinase, calmodulin-dependent kinase II, CDK5, and Erk1.

We have developed a method to study the primary sequence specificities of protein kinases by using an oriented degenerate peptide library. We report here the substrate specificities of eight protein Ser/Thr kinases. All of the kinases studied selected distinct optimal substrates. The identified substrate specificities of these kinases, together with known crystal structures of protein kinase A, CDK2, Erk2, twitchin, and casein kinase I, provide a structural basis for the substrate recognition of protein Ser/Thr kinases. In particular, the specific selection of amino acids at the +1 and -3 positions to the substrate serine/threonine can be rationalized on the basis of sequences of protein kinases. The identification of optimal peptide substrates of CDK5, casein kinases I and II, NIMA, calmodulin-dependent kinases, Erk1, and phosphorylase kinase makes it possible to predict the potential in vivo targets of these kinases.

Amino Acid Sequence↗

PubMed1995

Enzymatic deimination of glycogen phosphorylase and a peptide of the phosphorylation site: identification of modification and roles in phosphorylation and activity.

The functional role of arginine residues in glycogen phosphorylase b was probed by enzymatic modification by using peptidylarginine deiminase, which converts arginine residues to citrulline. A peptide with sequence LysArgLysGlnIleSerValArgGlyLeu, corresponding to the phosphorylation site of serine-14 in phosphorylase, was a substrate for the deiminase. Although both arginine residues could be converted to citrulline, modification of arginine-16 occurred more rapidly than modification of arginine-10. Previous studies have implicated a role for arginine, notably arginine-16, in determining phosphorylase kinase activity with the peptide. Deimination altered the phosphorylation of the peptide. Monodeimination of the peptide at arginine-16 slowed down the phosphorylation reaction, but did not diminish the total amount of phosphorylation that could be obtained. Deimination of both arginines produced a peptide that could not be phosphorylated. Modification of phosphorylase b resulted in activation or inactivation of enzyme activity depending on the extent of reaction with peptidylarginine deiminase. A low level of deiminase causes inactivation initially, but after prolonged incubation activation occurs. With high level of deiminase only activation can be observed. Because changes in activity are seen only at subsaturating AMP concentrations (50-100 microM), inactivation and activation are likely due to changes in affinity of the enzyme for AMP. The protein modified with the high level of deiminase has multiple sites of deimination. Arginine 16 was established as a major site of modification. Only protein modified with the high level of deiminase showed modification of arginine-16 and effects on the phosphorylation of phosphorylation b. As with the modified peptide substrate, the reaction was slower with modified phosphorylase in comparison with native phosphorylase b. The results show the importance of the guanidino group of arginine-16 of the protein substrate in modulating the phosphorylase kinase reaction.

Amino Acid Sequence↗

PubMed1995

Studies of calcineurin-calmodulin interaction: probing the role of arginine residues using peptidylarginine deiminase.

We have used an enzyme, peptidylarginine deiminase, to convert certain arginyl groups in calcineurin to citrulline. Amino acid analysis shows that only 3 of 34 arginines in calcineurin were deiminated; citrulline seems to be localized only in the calcineurin A (CaN A) subunit. Upon incubation with deiminase, the Mn2+/calmodulin-stimulated phosphatase activity decreases to 20-40% of the original activity within 1 h. However, the reduction in enzyme activity is fully protected by addition of calmodulin to the deimination reaction, and only 1.5 mol citrulline/mol calcineurin is found in this case. Removal of the calmodulin binding domain of the deiminated CaN A by limited proteolysis results in the reactivation of the phosphatase to the same level as digested native calcineurin and also results in the loss of all citrulline residues. The calmodulin activation curve of the deiminated enzyme is significantly shifted; the calculated apparent Kact using native calmodulin is 15-fold higher than that of native calcineurin while the apparent Kact using a fluorescent derivative of calmodulin, dansyl-calmodulin, is 10-fold higher. However, the Vm of deiminated calcineurin is similar to that of native if highly elevated levels of calmodulin are used to activate the modified calcineurin. To determine directly if the binding of calmodulin to calcineurin is affected upon deimination, fluorescence titrations using dansyl-calmodulin were performed. The Kd of deiminated calcineurin determined from these titrations is 10-fold higher than that of unmodified calcineurin, indicating that calmodulin binding is indeed affected. These data indicate that at least one arginine is important for calmodulin binding and is likely located at the calmodulin binding site of the CaN A subunit.

Animals↗

PubMed1995

Identification of the substrate and pseudosubstrate binding sites of phosphorylase kinase gamma-subunit.

Using site-directed mutagenesis, we proposed that an autoinhibitory domain(s) is located at the C-terminal region (301-386) of the phosphorylase kinase gamma-subunit (Huang, C.-Y.F., Yuan C.-J., Livanova, N.B., and Graves, D.J. (1993) Mol. Cell. Biochem. 127/128, 7-18). Removal of the putative inhibitory domain(s) by truncation results in the generation of a constitutively active and calmodulin-independent form, gamma 1-300. To probe the structural basis of autoinhibition of gamma-subunit activity, two synthetic peptides, PhK13 (gamma 303-327) and PhK5 (gamma 343-367), corresponding to the two calmodulin-binding regions, were assayed for their ability to inhibit gamma 1-300. Competitive inhibition of gamma 1-300 by PhK13 was found versus phosphorylase b (Ki = 1.8 microM) and noncompetitive inhibition versus ATP. PhK5 showed noncompetitive inhibition with respect to both phosphorylase b and ATP. Calmodulin released the inhibition caused by both peptides. These results indicate that there are two distinct auto-inhibitory domains within the C terminus of the gamma-subunit and that these two domains overlap with the calmodulin-binding regions. Two mutant forms of gamma 1-300, E111K and E154R, were used to probe the enzyme-substrate-binding region using peptide substrate analogs corresponding to residues 9-18 of phosphorylase b (KRK11Q12ISVRGL). The data suggest that Glu111 interacts with the P-3 position of the substrate (Lys11) and Glu154 interacts with the P-2 site (Gln12). Both E111K and E154R were competitively inhibited with respect to phosphorylase b by PhK13, with 14- and 8-fold higher Ki values, respectively, than that observed with the wild-type enzyme. These data are consistent with a model for the regulation of the gamma-subunit of phosphorylase kinase in which PhK13 acts as a competitive pseudosubstrate that directly binds the substrate binding site of the gamma-subunit (Glu111 and Glu154).

Amino Acid Sequence↗

PubMed1995

The biophysics of DNA hybridization with immobilized oligonucleotide probes.

A mathematical model based on receptor-ligand interactions at a cell surface has been modified and further developed to represent heterogeneous DNA-DNA hybridization on a solid surface. The immobilized DNA molecules with known sequences are called probes, and the DNA molecules in solution with unknown sequences are called targets in this model. Capture of the perfectly complementary target is modeled as a combined reaction-diffusion limited irreversible reaction. In the model, there are two different mechanisms by which targets can hybridize with the complementary probes: direct hybridization from the solution and hybridization by molecules that adsorb nonspecifically and then surface diffuse to the probe. The results indicate that nonspecific adsorption of single-stranded DNA on the surface and subsequent two-dimensional diffusion can significantly enhance the overall reaction rate. Heterogeneous hybridization depends strongly on the rate constants for DNA adsorption/desorption in the non-probe-covered regions of the surface, the two-dimensional (2D) diffusion coefficient, and the size of probes and targets. The model shows that the overall kinetics of DNA hybridization to DNA on a solid support may be an extremely efficient process for physically realistic 2D diffusion coefficients, target concentrations, and surface probe densities. The implication for design and operation of a DNA hybridization surface is that there is an optimal surface probe density when 2D diffusion occurs; values above that optimum do not increase the capture rate. Our model predicts capture rates in agreement with those from recent experimental literature. The results of our analysis predict that several things can be done to improve heterogeneous hybridization: 1) the solution phase target molecules should be about 100 bases or less in size to speed solution-phase and surface diffusion; 2) conditions should be created such that reversible adsorption and two-dimensional diffusion occur in the surface regions between DNA probe molecules; 3) provided that 2) is satisfied, one can achieve results with a sparse probe coverage that are equal to or better than those obtained with a surface totally covered with DNA probes.

Base Composition↗

PubMed1994

Use of photoacoustic Fourier-transform infrared spectroscopy to study phosphates in proteins.

Photoacoustic Fourier-transform infrared spectroscopy was used to study phosphoamino acids and phosphoproteins. Using this method, we have found that the spectral properties of phosphate esters depend on the nature of the linkage, pH, and binding of metal ion. At high pH values, dianionic symmetric stretching of phosphotyrosine and phosphoserine occurs at 984 and 974 reciprocal centimeters, respectively. Analysis of the IR bands of bound phosphate in phosvitin at different pH values gives the pKa value for the esterified phosphates. Addition of aluminum ions to phosvitin at different pH values causes a shift in the phosphate band consistent with a direct binding of aluminum ions to the esterified phosphate. The phosphate signal for 40 micrograms of Pepsin (1 P/mole) is detectable by this method.

Acoustics↗

PubMed1994

Oxidation and site-directed mutagenesis of the sulfhydryl groups of a truncated gamma catalytic subunit of phosphorylase kinase. Functional and structural effects.

A truncated form of the gamma subunit of phosphorylase kinase is inactivated by Cu2+ with the formation of two intra-molecular disulfide bonds. The formation of a disulfide bond between Cys-36 and Cys-172 (semioxidized form) results in approximately 50% loss of specific activity because the Km for MgATP is about 10-fold higher. The second disulfide bond is between Cys-184 and Cys-197 and causes further loss of activity. Eight Cys mutants, i.e. C36S, C36A, C42S, C138S, C172S, C184S, C184A, and C197S, were expressed and purified. Kinetic studies suggest that Cys-36 is important for interaction at the nucleotide site because of its hydrophobicity. With Cys-184 mutants, C184S and C184A, tyrosyl phosphorylation of angiotensin II is affected much more than serine kinase activity. The loss of tyrosine kinase activity is related to a lowered activity with Mn2+. With Mn2+, angiotensin II is a competitive inhibitor with respect to seryl kinase activity of C184S. With Mg2+, however, angiotensin II is a noncompetitive inhibitor. We suggest that metal ions influence the conformation of truncated gamma and that the protein substrate binding region containing Cys-184 is important for the dual specificity of this kinase.

Amino Acid Sequence↗

PubMed1994

Isotope effects on the mechanism of calcineurin catalysis: kinetic solvent isotope and isotope exchange studies.

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.

Animals↗

PubMed1994

Mutational analyses of the metal ion and substrate binding sites of phosphorylase kinase gamma subunit.

Phosphorylase kinase (PhK) and truncated gamma subunit, denoted gamma 1-300, can phosphorylate seryl and tyrosyl residues dependent on the metal ion [Yuan, C.-J., Huang, C. F., & Graves, D. J. (1993) J. Biol. Chem. 268, 17683-17686]. Recombinant gamma 1-300 was used to explore its dual specificity and the location of the metal ion binding sites by using site-directed mutagenesis. Two approaches were taken to generate 26 mutants. First, on the basis of the crystal structure of cAMP-dependent protein kinase (cAPK), the invariant Asn155 and highly conserved Asp168-Phe169-Gly170 residues were mutated. Changes included production of N155H, D168E, D168N, F169R, G170V, G170I, G170L (less than 1% of enzymatic activities were found in these mutants), F169W, and G170A mutants. Second, charge to alanine and charge reversal scanning mutations were used to probe the metal ion binding sites. Two mutants, E111K and E154R, showed very different metal ion response compared to wild-type gamma and were further characterized. The mutants F169W, G170A, E111K, and E154R had 15%, 5%, 8%, and 25% specific activity relative to wild-type gamma, respectively. The folding pattern of wild-type and mutated enzyme forms of gamma was determined by photoacoustic infrared spectroscopy. Conformational disruptions were found in G170V, G170I, and G170L mutants, but the conformation of the rest of the mutants was similar to that of wild-type gamma, suggesting that the loss of enzymatic activities of these mutants was not because of incorrect refolding.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1994

A study of protein secondary structure by Fourier transform infrared/photoacoustic spectroscopy and its application for recombinant proteins.

FT-IR/PAS (Fourier transform infrared/photoacoustic spectroscopy) was used to evaluate the secondary structure of proteins. Four well-studied proteins, concanavalin A, hemoglobin, lysozyme, and trypsin, which have different distributions of secondary structures, were used for assignments of the infrared bands and evaluating the accuracy of FT-IR/PAS methods. Secondary structure contents estimated from FT-IR/PAS and other physical methods (e.g., X-ray diffraction, CD, and traditional FT-IR) show good agreement. In addition, the secondary structure can be evaluated with as little as 0.5 micrograms of protein (concanavalin A), suggesting that FT-IR/PAS is a sensitive and useful technique that could be applied to studies of the folding of recombinant and mutant proteins where only small amounts of material are available. Recombinant phosphorylase kinase gamma 1-300 subunit expressed in Escherichia coli was found in the inclusion bodies. We found that renatured phosphorylase kinase gamma 1-300 subunit has two kinase forms: one has a 10-fold higher activity than the other one. Both fractions, however, are the same as judged from sodium dodecylsulfate-polyacrylamide gel electrophoresis. Differences in conformation were demonstrated by using the FT-IR/PAS method, which showed that the low-activity form has more beta-sheet structure than the form with high activity. Analysis of these kinase forms by CD confirms the interpretation made by the FT-IR/PAS method.

Protein Structure, Secondary↗

PubMed1993

ADP-ribosylation of the intermediate filament protein desmin and inhibition of desmin assembly in vitro by muscle ADP-ribosyltransferase.

Arginine-specific mono(ADP-ribosyl)transferase purified from rabbit skeletal muscle catalyzes stoichiometric ADP-ribosylation of the intermediate filament protein, desmin. In contrast, cholera toxin catalyzes a much lower level of ADP-ribosylation of desmin. Modification results in potent inhibition of desmin's ability to assemble into filaments. Phosphorylation of desmin by the catalytic subunit of cAMP dependent protein kinase is also inhibited by ADP-ribosylation. ADP-ribosylation site(s) are located within the N-terminal head domain of desmin.

ADP Ribose Transferases↗

PubMed1993

Phosphorylase kinase, a metal ion-dependent dual specificity kinase.

Phosphorylase kinase is shown to be a dual specificity kinase. The specificity of phosphorylation is determined by divalent cation. Mg2+ causes seryl phosphorylation of phosphorylase b, but Mn2+ activates tyrosine phosphorylation of angiotensin II. In contrast to seryl phosphorylation, the tyrosine kinase activity of holoenzyme is not regulated by Ca2+. Preincubation of the holoenzyme with Ca2+, Mg2+ and ATP that causes autophosphorylation activates tyrosine kinase activity. The tyrosyl kinase activity is a property of the gamma subunit. Addition of varying amounts of Mn2+ to a truncated form of the gamma subunit of phosphorylase kinase containing MgATP inhibits serine kinase but activates tyrosine kinase activity. This result along with an oxidative reaction caused by Cu2+ and site-directed mutagenesis of the putative catalytic base inhibiting both serine and tyrosine kinase activity suggest that one active site is involved in both activities. Kinetic studies with Mn2+ and ATP show that Km for nucleotide is not changed with a seryl or tyrosyl substrate. The Vm values are different, and the value for tyrosyl phosphorylation is similar to other tyrosyl kinases. We propose two conformations for the active site; one favors seryl phosphorylation, and the second tyrosyl phosphorylation is caused by the binding of divalent cation at a second metal ion binding site.

Adenosine Triphosphate↗

PubMed1993

Hydrolysis of trifluoroethyl phosphate as evidence that the serine and tyrosine phosphatase activities of calcineurin share the same specificity determinant.

Calcineurin is shown to hydrolyze 2,2,2-trifluoroethyl phosphate. The time course of hydrolysis showed a lag period. The lag was present at all substrate concentrations used and was dependent on substrate concentration. Evaluation of the kinetic constants from the steady state portion of the enzymatic reaction indicated that hydrolysis of this compound fit the Bronsted relationship determined previously for Vmax/Km term for the hydrolysis of aromatic phosphate esters. Moreover, a Bronsted relationship for the Vmax was developed for the substrates containing leaving groups that are more basic than 2,3,4,5-tetrafluorotyrosine. The hydrolysis of 2,3,4,5-tetrafluorotyrosine phosphate did not fit this relationship implying a change in the rate limiting step with this substrate. The significance of these data to the serine and tyrosine phosphatase activities of calcineurin is discussed. A rationale for understanding the determinant of both activities of calcineurin is presented.

Animals↗

PubMed1993

Expression, purification, characterization, and deletion mutations of phosphorylase kinase gamma subunit: identification of an inhibitory domain in the gamma subunit.

A catalytic fragment, gamma 1-298, derived from limited chymotryptic digestion of phosphorylase b kinase (Harris, W.R. et al., J. Biol. Chem., 265: 11740-11745, 1990), is reported to have about six-fold greater specific activity than does the gamma subunit-calmodulin complex. To test whether there is an inhibitory domain located outside the catalytic core of the gamma subunit, full-length wild-type and seven truncated forms of gamma were expressed in E. coli. Recombinant proteins accumulate in the inclusion bodies and can be isolated, solubilized, renatured, and purified further by ammonium sulfate precipitation and Q-Sepharose column. Four out of seven truncated mutants show similar (gamma 1-353 and gamma 1-341) or less (gamma 1-331 and gamma 1-276) specific activity than does the full-length wild-type gamma, gamma 1-386. Three truncated forms, gamma 1-316, gamma 1-300, and gamma 1-290 have molar specific activities approximately twice as great as those of the full-length wild-type gamma and the nonactivated holoenzyme. All recombinant gamma s exhibit similar Km values for both substrates, i.e., about 18 microM for phosphorylase b and about 75 microM for MgATP. Three truncated gamma s, gamma 1-316, gamma 1-300, and gamma 1-290, have a 1.9- to 2.5-fold greater catalytic efficiency (Vmax/Km) than that of the full-length wild-type gamma and a 3.5- to 4.5-fold greater efficiency than that of the truncated gamma 1-331. This evidence suggests that there is at least one inhibitory domain in the C-terminal region of gamma, which is located at gamma 301-331. gamma 1-290, but not gamma 1-276, which contains the highly conserved kinase domain, is the minimum sequence required for the gamma subunit to exhibit phosphotransferase activity. Both gamma 1-290 and gamma 1-300 have several properties similar to full-length wild-type gamma, including metal ion responses (activation by free Mg2+ and inhibition by free Mn2+), pH dependency, and substrate specificities.

Amino Acid Sequence↗

PubMed1992

Effect of an arginine-specific ADP-ribosyltransferase inhibitor on differentiation of embryonic chick skeletal muscle cells in culture.

Primary cultures of embryonic chick skeletal myogenic cells were used as an experimental model to examine the possible role of mono(ADP-ribosyl)ation reactions in myogenic differentiation. Initial studies demonstrated arginine-specific mono(ADP-ribosyl)transferase activity in the myogenic cell cultures. We then examined the effect of a novel inhibitor of cellular arginine-specific mono(ADP-ribosyl)transferases, meta-iodobenzylguanidine (MIBG), on differentiation of cultured embryonic chick skeletal myoblasts. MIBG reversibly inhibited both proliferation and differentiation of embryonic chick myoblasts grown in culture. Micromolar (15-60 microM) concentrations of MIBG blocked myoblast fusion, the differentiation-specific increase in creatine phosphokinase activity, and both DNA and protein accumulation in myogenic cell cultures. Meta-iodobenzylamine, an analog of MIBG missing the guanidine group, had no effect. Low concentrations of methylglyoxal bis-guanylhydrazone, a substrate for cholera toxin with a higher Km than MIBG, also had no effect, but higher concentrations reversibly inhibited fusion. These findings suggest a possible role for mono(ADP-ribosyl)ation reactions in myogenesis. In addition, the total arginine-specific mono(ADP-ribosyl)transferase activity increased with differentiation in the myogenic cell cultures, and this increase was also blocked by MIBG treatment. Because high levels of activity were found in the membrane fraction derived from later, myotube cultures, the membrane fraction from 96-h cultures was incubated with [32P]NAD+ and subjected to electrophoresis and autoradiography. Three proteins, migrating at 21, 20, and 17 kDa, that were ADP-ribosylated in the absence, but not the presence, of MIBG were identified. These proteins may be endogenous substrates for this enzyme.

3-Iodobenzylguanidine↗

PubMed1991

Solution conformations of the N-terminal CNBr fragment of glycogen phosphorylase and its interaction with calmodulin.

The CNBr peptides, CBPa and CBPb, corresponding to the N-terminal 1-91 amino acid residues of glycogen-phosphorylase a and b, respectively, were purified and characterized. CD, 31P-NMR and fluorescence spectroscopy were used to assess the structural organization of the cyanogen bromide peptides in solution. The cyanogen bromide peptides yielded 21% of alpha-helical structures by CD compared to a calculated value of 36.3%. These peptides interact with calmodulin which induces measurable alpha-helices in the cyanogen bromide peptides. The helix stabilizing reagent, trifluoroethanol, induces high numbers of alpha-helices in CBP, thereby demonstrating the conformation fluidity of this peptide. The dissociation constants for calmodulin and CBP estimated by fluorescence titrations were 36.0 and 29.9 nM for CBPb in the presence of Ca2+ and EGTA, respectively. The phosphorylated residue in CBPa causes a decrease in binding interactions with calmodulin and corresponding values obtained for CBPa by fluorescence titration are 56.0 and 141.0 nM, respectively. The Ser-P-14 of CBPa is titratable, yielding a pKa = 5.45 and a Hill coefficient of 1.5. A helical wheel analysis using a computer program in PC/GENE of the CBP shows that peptide stretches in the alpha-1 and alpha-2 helices are most basic and fairly amphiphilic and therefore represent the most probable segment for CaM binding. It is this structural character of these segments which presumably confer the ability to bind CaM and facilitate some of the allosteric transitions of glycogen phosphorylase.

Calcium↗

PubMed1991

Calcineurin-catalyzed reaction with phosphite and phosphate esters of tyrosine.

A convenient synthesis is reported for the preparation of the phosphite ester of tyrosine methyl ester. By use of calcineurin, at 30 degrees C, a phosphite ester was hydrolyzed with a VM value [119 nmol/(min.micrograms of E)] approximately 500 times greater than that obtained with tyrosine phosphate [0.23 nmol/(min.microgram of E)] as substrate, but with similar KM values (12 mM for Tyr-PH ME, 11 mM for Tyr-P). Acid phosphatase, on the other hand, hydrolyzed the phosphite ester with a VM and KM value lower than those obtained with tyrosyl phosphate. The temperature dependence of the kinetic parameters (KM and VM) was evaluated, and the activation parameters were obtained with both substrates. The entropy of activation associated with the enzymatic hydrolysis of tyrosine phosphate agrees with the entrophy change for the hydrolysis of the monoanion of phosphate monoesters. The energy of activation for both substrates was in agreement with the energy change for hydrolysis of the oxygen-phosphorous linkage of phosphate monoester monoanions and phosphite esters. These results are consistent with a scheme of general acid catalysis in the action of calcineurin.

Calcineurin↗

PubMed1991

Determination of the kinetic mechanism of arginine-specific ADP-ribosyltransferases using a high performance liquid chromatographic assay.

A high performance liquid chromatographic method has been developed for the assay of arginine-specific ADP-ribosyl transferases. The assay utilizes L-arginine methyl ester (LAME) as the acceptor substrate. ADP-ribosylated-LAME is separated from the reaction mixture using a C-8 reversed-phase column. Before injection, the assay mixture is derivatized with an orthophthaldialdehyde/2-mercaptoethanol reagent. Fluorescence detection of the orthophthaldialdehyde-derivatized product provides excellent sensitivity and a limit of detection of less than 100 fmol. The kinetic mechanism of two arginine-specific ADP-ribosyltransferases, cholera toxin A subunit and an endogenous transferase from rabbit skeletal muscle, were both determined to be random sequential. The kinetic studies utilized 3-aminobenzamide and NG-monomethylarginine as competitive inhibitors for NAD and LAME, respectively. Cholera toxin was reported to have Km values of 5.6 and 39 mM for NAD and LAME, respectively. Km values of 0.56 and 1.2 mM were determined for NAD and LAME, respectively, using the transferase from rabbit skeletal muscle.

ADP Ribose Transferases↗