Modification Of Arginine Residues Research Articles

Increased Citrullination of Histone H3 in Multiple Sclerosis Brain and Animal Models of Demyelination: A Role for Tumor Necrosis Factor-Induced Peptidylarginine Deiminase 4 Translocation

Modification of arginine residues by citrullination is catalyzed by peptidylarginine deiminases (PADs), of which five are known, generating irreversible protein structural modifications. We have shown previously that enhanced citrullination of myelin basic protein contributed to destabilization of the myelin membrane in the CNS of multiple sclerosis (MS) patients. We now report increased citrullination of nucleosomal histones by PAD4 in normal-appearing white matter (NAWM) of MS patients and in animal models of demyelination. Histone citrullination was attributable to increased levels and activity of nuclear PAD4. PAD4 translocation into the nucleus was attributable to elevated tumor necrosis factor-alpha (TNF-alpha) protein. The elevated TNF-alpha in MS NAWM was not associated with CD3+ or CD8+ lymphocytes, nor was it associated with CD68+ microglia/macrophages. GFAP, a measure of astrocytosis, was the only cytological marker that was consistently elevated in the MS NAWM, suggesting that TNF-alpha may have been derived from astrocytes. In cell cultures of mouse and human oligodendroglial cell lines, PAD4 was predominantly cytosolic but TNF-alpha treatment induced its nuclear translocation. To address the involvement of TNF-alpha in targeting PAD4 to the nucleus, we found that transgenic mice overexpressing TNF-alpha also had increased levels of citrullinated histones and elevated nuclear PAD4 before demyelination. In conclusion, high citrullination of histones consequent to PAD4 nuclear translocation is part of the process that leads to irreversible changes in oligodendrocytes and may contribute to apoptosis of oligodendrocytes in MS.

Peptide Mapping of Human Serum Albumin Modified Minimally by Methylglyoxal in Vitro and in Vivo

Methylglyoxal is a potent glycating agent and important precursor of advanced glycation end products (AGEs) in physiological systems. Unlike glucose, methylglyoxal is predominantly an arginine-directed glycating agent. Methylglyoxal reacts with proteins to form mainly the arginine-derived hydroimidazolone AGE, Ndelta-(5-hydro-5-methyl-4-imidazolon-2-yl)-ornithine (MG-H1), argpyrimidine, the lysine-derived AGEs, N(epsilon)-(1-carboxyethyl)lysine (CEL), and methylglyoxal-derived lysine dimer (MOLD). Sites within proteins susceptible to modification by methylglyoxal have not been identified. Here we show that modification of human serum albumin by methylglyoxal forms mainly hydroimidazolone MG-H1 residues. The location of MG-H1 residues was identified by mass spectrometric peptide mapping. This method identified a hot spot of hydroimidazolone formation at Arg-410, with other minor MG-H1 modifications at Arg-114, Arg-186, Arg-218, and Arg-428. Other extracellular and intracellular proteins are modified by methylglyoxal in physiological systems. Modification of arginine residues by methylglyoxal may be particularly damaging because arginine residues have a high frequency of occurrence in ligand and substrate recognition sites in receptor and enzyme active sites.

Dicarbonyl Intermediates in the Maillard Reaction

The complexity of the Maillard reaction arises partly from multiple fragmentation reactions of the sugar moiety, constituting branch points in the reaction progress and establishing many parallel reaction pathways. Reactive intermediates produced by these processes are often alpha-oxoaldehydes. The formation of alpha-oxoaldehydes enhances and redirects glycating activity in the Maillard reaction since alpha-oxoaldehydes are up to 20,000-fold more reactive than glucose in glycation processes and are predominantly arginine-directed glycating agents. alpha-Oxoaldehydes bypass a requirement for a fructosamine precursor in the formation of advanced glycation end products (AGEs) since alpha-oxoaldehydes react with proteins (also nucleotides and basic phospholipids) to form AGEs directly. The major AGE formed from alpha-oxoaldehydes is generally a hydroimidazolone with other products-although for glyoxal, N(omega)-carboxymethylarginine is a major product. alpha-Oxoaldehyde formation also occurs in the absence of an amine substrate, particularly during heat processing of sugar solutions and lipid peroxidation processes-in the latter case, the glycation adducts are advanced lipoxidation products (ALEs). Hydroimidazolones are quantitatively important AGEs in cellular and extracellular proteins in physiological systems. Hydroimidazolone free adducts are liberated by cellular proteolysis and digestion. They are released into blood plasma for urinary excretion. Modification of arginine residues by alpha-oxoaldehydes may be particularly damaging since arginine residues have high-frequency occurrence in ligand and substrate recognition sites in receptor and enzyme active sites. Along with fructosamine formation, alpha-oxoaldehyde intermediates of the Maillard reaction represent a major source of damage to the proteome and genome.

Effect of Amino Acid Residue and Oligosaccharide Chain Chemical Modifications on Spectral and Hemagglutinating Activity of <italic>Millettia dielsiana</italic> Harms. ex Diels. Lectin

The effects of modifying the carbohydrate chain and amino acids on the conformation and activity of Millettia dielsiana Harms. ex Diels. lectin (MDL) were studied by hemagglutination, fluorescence and circular dichroism analysis. The modification of tryptophan residues led to a compete loss of hemagglutinating activity; however, the addition of mannose was able to prevent this loss of activity. The results indicate that two tryptophan residues are involved in the carbohydrate-binding site. Modifications of the carboxyl group residues produced an 80% loss of activity, but the presence of mannose protected against the modification. The results suggest that the carboxyl groups of aspartic and glutamic acids are involved in the carbohydrate-binding site of the lectin. However, oxidation of the carbohydrate chain and modification of the histidine and arginine residues did not affect the hemagglutinating activity of MDL. Fluorescence studies of MDL indicate that tryptophan residues are present in a relatively hydrophobic region, and the binding of mannose to MDL could quench tryptophan fluorescence without any change in lambda(max). The circular dichroism spectrum showed that all of these modifications affected the conformation of the MDL molecule to different extents, except the modification of arginine residues. Fluorescence quenching showed that acrylamide and iodoacetic acids are able to quench 77% and 98% of the fluorescence of tryptophan in MDL, respectively. However, KI produced a barely perceptible effect on the fluorescence of MDL, even when the concentration of I(-) was 0.15 M. This demonstrates that most of tryptophan residues are located in relatively hydrophobic or negatively charged areas near the surface of the MDL molecule.

Diagnostic Performance and Predictive Value of Anti-citrullinated Peptide Antibodies for Diagnosis of Rheumatoid Arthritis: Toward More Accurate Detection?

During the past few years, much enthusiasm has greeted a new laboratory test that measures autoantibodies reactive with synthetic peptides containing the unusual amino acid citrulline, a posttranslationally modified arginine residue (1). Data from several investigators indicated that anti-citrullinated peptide autoantibodies (ACPAs), including anti-cyclic citrullinated peptide (anti-CCP) antibodies, were specifically present in the sera of patients with rheumatoid arthritis (RA); increased specificity is the major advantage of this test (1)(2). Now, in this issue of the Journal, Hoffman et al. (3) prospectively evaluated, in a setting reflecting everyday rheumatologic practice for prevalence of RA, the diagnostic performance of two classic assays for rheumatoid factor (RF) and methods for ACPA detection. Using ROC curve analysis, their study showed that RF has a better overall diagnostic performance than ACPAs. However, when high-specificity cutoffs were selected, one of the ACPA assays (anti-pepA) performed better than RF assays. Importantly, combining one RF assay with one of the ACPA assays increased positive predictive value. Combining one serologic marker with the finding of swollen joints also reveals high positive predictive value. Two of the strengths of their study were that its design reflected everyday rheumatology practice and that the composition of the control group reflected the natural prevalence of diseases in cases for which serologic markers for RA are requested. Their data for specificity, sensitivity, and predictive values are thus easier to interpret. The study included 1003 consecutive patients in situations where the rheumatologist would typically request RF determination. Importantly, diagnoses were established after …

Proteomic Analysis of Arginine Adducts on Glyoxal-modified Ribonuclease

Accumulation of advanced glycation end-products (AGEs) on proteins is associated with the development of diabetic complications. Although the overall extent of modification of protein by AGEs is limited, localization of these modifications at a few critical sites might have a significant effect on protein structure and function. In the present study, we describe the sites of modification of RNase by glyoxal under physiological conditions. Arg39 and Arg85, which are closest to the active site of the enzyme, were identified as the primary sites of formation of the glyoxal-derived dihydroxyimidazolidine and hydroimidazolone adducts. Lower amounts of modification were detected at Arg10, while Arg33 appeared to be unmodified. We conclude that dihydroxyimidazolidine adducts are the primary products of modification of protein by glyoxal, that Arg39 and Arg85 are the primary sites of modification of RNase by glyoxal, and that modification of arginine residues during Maillard reactions of proteins is a highly selective process.

Chemical modification of arginine residues of Notechis scutatus scutatus notexin

Notexin, a presynaptic phospholipase A 2 (PLA 2) neurotoxin isolated from Notechis scutatus scutatus venom, was inactivated by arginine-specific reagents, phenylglyoxal and 1,2-cyclohexanedione. Kinetic analyses of the modification reaction revealed that the inactivation of notexin followed pseudo-first order kinetics and the loss of PLA 2 activity was correlated with the incorporation of one molecule of modification reagent per toxin molecule. However, the results of amino acid analysis and sequence determination revealed that two arginine residues at positions 43 and 79 of notexin were modified simultaneously. Modification of the arginine residues was accompanied with a decrease in the ability to inhibit the indirectly evoked contraction of chick biventer cervicis muscle and bind with synaptic membranes. The secondary structure of the toxin molecule did not significantly change after modification with phenylglyoxal as revealed by the CD spectra. The modified derivative retained its affinity for Ca 2+, indicating that the modified arginine residues did not participate in Ca 2+-binding. Together with the notion that Arg-43 and Arg-79 of notexin are located in the proximity of its catalytic site and toxic site, respectively, our results suggest that modification of Arg-43 and Arg-79 should differently contribute to the observed decrease in the PLA 2 activity and neurotoxic effect of notexin.

Defects in the regulation of B cell apoptosis are required for the production of citrullinated peptide autoantibodies in mice.

Protein deimination, a process to modify arginine residues to citrulline by the addition of a neutral oxygen group, is associated with apoptosis. The presence of autoantibodies recognizing citrullinated peptides is highly specific to rheumatoid arthritis (RA) and is therefore a useful marker for the early diagnosis of RA. In this study, we explored whether anti-cyclic citrullinated peptide (anti-CCP) autoantibodies are produced in several experimental models of autoimmune diseases in mice. The levels of anti-CCP autoantibodies were analyzed by enzyme-linked immunosorbent assay in several lupus-prone strains of mice, in animals with type II collagen (CII)-induced arthritis, and after induction of neonatal tolerance to alloantigens. We observed the production of these autoantibodies in 2 different lupus-prone mice, MRL-lpr/lpr and (NZW x B6)F(1)-hbcl-2 transgenic mice, characterized by the presence of abnormalities in the regulation of B cell apoptosis. Other genetic defects, determining autoimmune susceptibility, present in MRL and NZW mice were additionally required for anti-CCP autoantibody production. The induction of autoantibodies in normal BALB/c mice injected at birth with semiallogeneic spleen cells from (BALB/c x B6)F(1)-hbcl-2 transgenic mice suggested that these additional autoimmune defects may be related, at least in part, to the establishment of abnormal interactions between T cells and B cells. In addition, anti-CCP autoantibodies were not produced in the course of CII-induced arthritis, an experimental model of RA in mice. Our study provides evidence for the association between defects in the regulatory cell death machinery of B lymphocytes and the production of certain autoantibody specificities.

Identification of the Binding Site of Methylglyoxal on Glutathione Peroxidase: Methylglyoxal Inhibits Glutathione Peroxidase Activity via Binding to Glutathione Binding Sites Arg 184 and 185

Methylglyoxal (MG), a physiological f -dicarbonyl compound is derived from glycolytic intermediates and produced during the Maillard reaction. The Maillard reaction, a non-enzymatic reaction of ketones and aldehydes with amino group of proteins, contributes to the aging of proteins and to complications associated with diabetes. In our previous studies (Che, et al. (1997) "Selective induction of heparin-binding epidermal growth factor-like growth factor by MG and 3-deoxyglucosone in rat aortic smooth muscle cells. The involvement of reactive oxygen species formation and a possible implication for atherogenesis in diabetes". J. Biol. Chem., 272 , 18453-18459), we reported that MG elevates intracellular peroxide levels, but the mechanisms for this remain unclear. Here, we report that MG inactivates bovine glutathione peroxidase (GPx), a major antioxidant enzyme, in a dose- and time-dependent manner. The use of BIAM labeling, it was showed that the selenocysteine residue in the active site was intact when GPx was incubated with MG. MALDI-TOF-MS (matrix-assisted laser desorption/ionization time-of-flight mass spectrometry) and protein sequencing examined the possibility that MG modifies arginine residues in GPx. The results show that Arg 184 and Arg 185, located in the glutathione binding site of GPx was irreversively modified by treatment with MG. Reactive dicarbonyl compounds such as 3-deoxyglucosone, glyoxal and phenylglyoxal also inactivated GPx, although the rates for this inactivation varied widely. These data suggest that dicarbonyl compounds are able to directly inactivate GPx, resulting in an increase in intracellular peroxides which are responsible for oxidative cellular damage.

3-Deoxyosone-related advanced glycation end products in foods and biological systems

In an intermediate Maillard reaction of proteins with glucose, 3-deoxyglucosone (3DG) was generated from Amadori compounds in an early stage, leading to generation of advanced glycation end products (AGEs). 3DG modified arginine residues to form imidazolone compounds, and lysine residues to form pyrrole aldehydes (lysyl-pyrraline) and lysyl-pyrropyridine. S16 and S17, imidazolone compounds, are formed via S12, imidazoline-4-one compound, by dehydration and oxidation from the Nα-benzoylarginine amide (BzArgNH2)–3DG reaction system to be the major pathway. A novel spiro compound, S17, was formed by intramolecular cyclization of dehydrogenated S12. S12 was also identified in a 3DG–insulin or angiotensin II reaction system under physiologic conditions at 37 °C. These 3DG-related AGEs, imidazolone, pyrraline, lysyl-pyrropiridine, and pentosidine were detected in browned foods, such as soy sauce, as well as in vivo. 3DG, which has weaker mutagenicity, reacted readily with 2′-deoxyguanosine in DNA. Two major products (G–A and G–B) were isolated, and identified as diastereomers of N-(1-oxo-2,4,5,6-hydroxyhexyl)-2′-deoxyguanosine. Blue pigment was isolated from the reaction between d-xylose and glycine. Blue pigment which was designated Blue-M1 (blue Maillard reaction intermediate-1) was identified as novel pyrrolopyrrolylium compound and is assumed to be a dimer of yellow pyrrolopyrrole-2-carboxaldehyde compounds. Blue-M1 that reacts readily to yellow compounds has a polymerizing activity, suggesting that it is an important Maillard reaction intermediate through the formation of melanoidins. Blue-M1 had strong antioxidative effects as well as melanoidins.

Effects of modifications of alpha-crystallin on its chaperone and other properties.

The role of alpha-crystallin, a small heat-shock protein and chaperone, may explain how the lens stays transparent for so long. alpha-Crystallin prevents the aggregation of other lens crystallins and proteins that have become unfolded by 'trapping' the protein in a high-molecular-mass complex. However, during aging, the chaperone function of alpha-crystallin becomes compromised, allowing the formation of light-scattering aggregates that can proceed to form cataracts. Within the central part of the lens there is no turnover of damaged protein, and therefore post-translational modifications of alpha-crystallin accumulate that can reduce chaperone function; this is compounded in cataract lenses. Extensive in vitro glycation, carbamylation and oxidation all decrease chaperone ability. In the present study, we report the effect of the modifiers malondialdehyde, acetaldehyde and methylglyoxal, all of which are pertinent to cataract. Also modification by aspirin, which is known to delay cataract and other diseases, has been investigated. Recently, two point mutations of arginine residues were shown to cause congenital cataract. 1,2-Cyclohexanedione modifies arginine residues, and the extent of modification needed for a change in chaperone function was investigated. Only methylglyoxal and extensive modification by 1,2-cyclohexanedione caused a decrease in chaperone function. This highlights the robust nature of alpha-crystallin.

HlyC, the internal protein acyltransferase that activates hemolysin toxin: the role of conserved tyrosine and arginine residues in enzymatic activity as probed by chemical modification and site-directed mutagenesis.

Internal fatty acylation of proteins is a recognized means of modifying biological behavior. Escherichia coli hemolysin A (HlyA), a toxic protein, is transcribed as a nontoxic protein and made toxic by internal acylation of two lysine residue epsilon-amino groups; HlyC catalyzes the acyl transfer from acyl-acyl carrier protein (ACP), the obligate acyl donor. Conserved residues among the respective homologous C proteins that activate 13 different RTX (repeats in toxin) toxins of which HlyA is the prototype likely include some residues that are important in catalysis. Possible roles of two conserved tyrosines and two conserved arginines were investigated by noting the effects of chemical modifiers and site-directed mutagenesis. TNM modification of HlyC at pH 8.0 led to extensive inhibition that was prevented by the presence of the substrate myristoyl-ACP but not by the product, ACPSH. NAI had no effect. Y70G and Y150G greatly diminished enzyme activity, whereas mutations Y70F and Y150F exhibited wild-type activity. Modification of arginine residues with PG markedly lowered acyltransferase activity with moderate protection by both myristoyl-ACP and ACPSH. Under optimum conditions, four separate mutations of the two conserved arginine residues (R24A, R24K, R87A, and R87K) had little effect on acyltransferase activity.

Active site topology of artificial peroxidase-like hemoproteins based on antibodies constructed from a specifically designed ortho-carboxy-substituted tetraarylporphyrin.

The topology of the binding site has been studied for two monoclonal antibodies 13G10 and 14H7, elicited against iron(III)-alpha,alpha,alpha,beta-meso-tetrakis(ortho-carboxyphenyl)porph yrin [alpha,alpha,alpha, beta-Fe[(o-COOHPh)4-porphyrin]], and which exhibit in the presence of this alpha,alpha,alpha, beta-Fe[(o-COOHPh)4-porphyrin] cofactor a peroxidase activity. A comparison of the dissociation constants of the complexes of 13G10 and 14H7 with various tetra-aryl-substituted porphyrin has shown that: (a) the central iron(III) atom of alpha,alpha,alpha,beta-Fe[(o-COOHPh)4-porphyrin] is not recognized by either of the two antibodies; and (b) the ortho-carboxylate substituents of the meso-phenyl rings of alpha,alpha,alpha, beta-Fe[(o-COOHPh)4-porphyrin] are essential for the recognition of the porphyrin by 13G10 and 14H7. Measurement of the dissociation constants for the complexes of 13G10 and 14H7 with the four atropoisomers of (o-COOHPh)4-porphyrinH2 as well as mono- and di-ortho-carboxyphenyl-substituted porphyrins suggests that the three carboxylates in the alpha, alpha, beta position are recognized by both 13G10 and 14H7 with the two in the alpha, beta positions more strongly bound to the antibody protein. Accordingly, the topology of the active site of 13G10 and 14H7 has roughly two-thirds of the alpha,alpha,alpha,beta-Fe[(o-COOHPh)4-porphyrin] cofactor inserted into the binding site of the antibodies, with one of the aryl ring remaining outside. Three of the carboxylates are bound to the protein but no amino acid residue acts as an axial ligand to the iron atom. Chemical modification of lysine, histidine, tryptophan and arginine residues has shown that only modification of arginine residues causes a decrease in both the binding of alpha,alpha,alpha, beta-Fe[(o-COOHPh)4-porphyrin] and the peroxidase activity of both antibodies. Consequently, at least one of the carboxylates of the hapten is bound to an arginine residue and no amino acids such as lysine, histidine or tryptophan participate in the catalysis of the heterolytic cleavage of the O-O bond of H2O2. In addition, the amino acid sequence of both antibodies not only reveals the presence of arginine residues, which could be those involved in the binding of the carboxylates of the hapten, but also the presence of several amino acids in the complementary determining regions which could bind other carboxylates through a network of H bonds.

The asymmetric synthesis of arginine mimetics: derivatives of (S)-2-, 3- and 4-amidinophenylalanine suitable for incorporation into enzyme inhibitors and/or peptides.

The specific inhibition of trypsin-like serine proteases has become an important therapeutic target. These proteases have been implicated in several physiological and pathophysiological processes, including blood coagulation, digestion, and inflammation. Proteases of this class cleave polypeptide chains C-terminal to a basic residue (arginine or lysine). It has been shown that selectivity for a particular serine protease can be conferred based upon the structural moiety incorporated in the P1 position. In this regard, the three isomers (ortho, meta, and para) of amidinophenylalanine represent modified arginine residues and are important synthetic targets. Herein, a convenient asymmetric synthesis of (S)-Nalpha-(tert-butyloxycarbonyl)-2-, (S)-Nalpha-(tert-butyloxycarbonyl)-3-, and (S)-Nalpha-(tert-butyloxycarbonyl)-4-amidinophenyl-alanine N,O-dimethylamides (Weinreb amides) will be described. These derivatives represent key synthetic intermediates for the synthesis of enzyme inhibitors because the amidine can be readily orthogonally protected, while the Weinreb amide is easily converted to a variety of electrophilic carbonyls via reduction to the corresponding aldehyde or by reaction with various lithiated heterocycles. Likewise, the Weinreb amide can be reduced to the aldehyde and subsequently oxidized to the corresponding carboxylate, which is suitable for solid- or solution-phase peptide synthetic strategies.

Citrulline is an essential constituent of antigenic determinants recognized by rheumatoid arthritis-specific autoantibodies.

Only a few autoantibodies that are more or less specific for RA have been described so far. The rheumatoid factor most often tested for is not very specific for RA, while the more specific antiperinuclear factor for several reasons is not routinely used as a serological parameter. Here we show that autoantibodies reactive with synthetic peptides containing the unusual amino acid citrulline, a posttranslationally modified arginine residue, are specifically present in the sera of RA patients. Using several citrulline-containing peptide variants in ELISA, antibodies could be detected in 76% of RA sera with a specificity of 96%. Sera showed a remarkable variety in the reactivity pattern towards different citrulline-containing peptides. Affinity-purified antibodies were shown to be positive in the immunofluorescence-based antiperinuclear factor test, and in the so-called antikeratin antibody test, and were reactive towards filaggrin extracted from human epidermis. The specific nature of these antibodies and the presence of these antibodies early in disease, even before other disease manifestations occur, are indicative for a possible role of citrulline-containing epitopes in the pathogenesis of RA.

Improving mass spectrometric sequencing of arginine-containing peptides by derivatization with acetylacetone.

Modification of arginine residues in bradykinin, [1-5]-bradykinin, splenopentin and two synthetic pentapeptides with acetylacetone (pentane-2,4-dione) significantly increases the relative abundance of sequence-specific fragment ions produced by matrix-assisted laser desorption/ionization (MALDI). The fragmentation efficiency as measured by post-source decay in a reflectron time-of-flight mass spectrometer increases by a factor of 2-3.5. Peptide bonds adjacent to modified residues are more susceptible to cleavage than in the non-derivatized peptide ions. The increased lability of these bonds gives rise to more complete sequence information. In addition, the relative abundances of sequence-specific fragment ions are enhanced. This strategy makes it possible to obtain valuable structural information from arginine-containing peptides that otherwise do not fragment well.

The Mammalian Immediate-early TIS21 Protein and the Leukemia-associated BTG1 Protein Interact with a Protein-arginine N-Methyltransferase

The TIS21 immediate-early gene and leukemia-associated BTG1 gene encode proteins with similar sequences. Two-hybrid analysis identified a protein that interacts with TIS21 and BTG1. Sequence motifs associated with S-adenosyl-L-methionine binding suggested this protein might have methyltransferase activity. A glutathione S-transferase (GST) fusion of the putative methyltransferase modifies arginine residues, in appropriate protein substrates, to form NG-monomethyl and NG,NG-dimethylarginine (asymmetric). We term the protein- arginine N-methyltransferase (EC 2.1.1.23) gene "PRMT1, " for protein-arginine methyltransferase 1. GST-TIS21 and GST-BTG1 fusion proteins qualitatively and quantitatively modulate endogenous PRMT1 activity, using control and hypomethylated RAT1 cell extracts as methyl-accepting substrates. PRMT1 message appears ubiquitous, and is constitutive in mitogen-stimulated cells. Modulation of PRMT1 activity by transiently expressed regulatory subunits may be an additional mode of signal transduction following ligand stimulation.

Effect of Modification of Arginine Residues on the Activity of Soybean Lipoxygenase-1

Abstract Arginine residues of soybean lipoxygenase-1 was modified with an arginine-directed chemical modifier, 2,3-butanedione. Although inactivation was not visible if the enzyme reaction was monitored under the standard assay condition (83.3 μᴍ linoleic acid dispersed in 200 mᴍ sodium borate, pH 9.0), rapid inactivation was observed with 5 mᴍ sodium borate, pH 8.0. The inactivation was protected by the addition of a substrate, linoleic acid, in the modification mixture. Kinetic analyses indicated that one arginine residue accounted for the inactivation. Enzymological analyses showed that the modification narrowed the pH-activity profile of L-1 and made L-1 sensitive to salt concentration of the assay solution. Strong inactivation by modification was found at low salt concentration and low pH. This was not due to a physical change of the linoleic acid. On the other hand, product specificity of L-1 was not altered after modification. Taken together, the modified arginine residue(s) was thought to be not essential to the catalysis but have an important role in supporting an ideal electrostatic interaction within L-1 and/or between L-1 and a substrate even in sub-optimal reaction conditions.

Ca(2+)-calmodulin-dependent phosphorylation of arginine in histone 3 by a nuclear kinase from mouse leukemia cells.

A Ca(2+)-calmodulin dependent histone 3 kinase was partially purified from a low salt (150 mM NaCl) nuclear extract of mouse leukemia cells by calmodulin-Sepharose affinity chromatography. In vitro, the kinase activity transferred gamma-phosphate from ATP to histone 3 to form an acid-labile and alkaline-stable linkage. Under the assay conditions 1.8 mol of phosphate are incorporated per mol of histone 3. Upon modification of arginine residues with phenylglyoxal prior to phosphorylation, a considerable decrease in the amount of phosphate transferred to histone 3 was observed. Amino acid analysis revealed that H3 was phosphorylated on arginine residues. To identify the phosphorylated peptide(s), histone 3 was cleaved with cyanogen bromide prior to phosphorylation. The phosphorylated mixture was then separated by gel filtration high-performance liquid chromatography under denaturing conditions. Fragments I (N-terminal 10.3-kDa peptide) and III (C-terminal 1.7-kDa peptide) were both phosphorylated. Amino acid sequencing further revealed that the molar yields of 3 of the 4 arginines present in the phosphorylated cyanogen bromide fragment III were reduced by a factor of about 10 compared with the corresponding arginines from the unphosphorylated fragment. In the case of fragment I, 25 cycles of Edman degradation revealed that the recovery of only arginine 2 was reduced by a factor of 20. The putative phosphorylation sites are arginines 2, 128, 129, and 131. The sequence information offered an indirect evidence that these arginines were the sites of phosphorylation. The kinase described in this report represents a first member of a potentially important new class of kinases which are Ca(2+)-calmodulin dependent and which phosphorylate arginine.

Studies on pig aldose reductase. Identification of an essential arginine in the primary and tertiary structure of the enzyme.

Reaction of pig muscle aldose reductase with phenylglyoxal resulted in the chemical modification of 2 arginine residues with accompanying loss of catalytic activity. The amino acid sequences of radioactive peptides resulting from the reaction of aldose reductase with [14C]phenylglyoxal followed by tryptic digestion and high performance liquid chromatography separation allowed identification of the modified arginine residues as R268 and R293. In the presence of the coenzyme NADP+, R268 is protected from modification by phenylglyoxal, while R293 becomes hyper-reactive. Phenylglyoxal modification of aldose reductase is slowed 3-fold by the presence of the coenzyme analog ADPRP; however, both arginines are still modified. These chemical modification results are in complete accord with the previously determined crystal structures of human and porcine aldose reductase complexed with NADPH, NADP+, and ADPRP. These structures indicate that R268 is located at the adenosine binding site, salt bridged to the 2'-phosphate group of NADP(H) and ADPRP. Arginine 293 is near the surface of the enzyme and is part of the C-terminal loop. In the apoenzyme or the ADPRP complex, R293 is partially protected by loop 7; upon binding NADP(H), loop 7 folds down over the coenzyme, thus exposing R293 to solvent. Our modification studies provide further evidence of the conformational change that occurs during the aldose reductase catalytic cycle.