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PubMed2006

Elevated level of methylglyoxal during diabetic ketoacidosis and its recovery phase.

Carbonyl stress is hypothesized to be an associated complication of diabetic ketoacidosis. The production of the glycolytic intermediate methylglyoxal (MG) was followed up in 7 diabetic patients treated for ketoacidosis during pretreatment and recovery phase. Blood samples for methylglyoxal analysis were collected upon patient arrival in emergency department (0 h), and during ketoacidosis treatment between 12-24 h and at 168 h. The study also included 10 normoglycaemic healthy volunteers and 31 type 1 diabetic patients (control diabetes group). The methylglyoxal assay, based on methylglyoxal derivation with 1,2-diamino-4,5-dimethoxybenzene (DDB), was performed by HPLC, only assessing the level of free methylglyoxal. The baseline level of methylglyoxal recorded in normoglycemic healthy controls was 338 +/- 62 nmol/l versus 374 +/- 89 nmol/l in control diabetes group (P = 0.0407). A consistent feature of diabetic ketoacidosis before and during treatment was striking elevation of methylglyoxal as compared with control diabetes group (median test chi(2) = 14.6, df = 3, P = 0.0021). Friedman's ANOVA indicated differences (P = 0.04) among the three sampling times with a peak value (601 +/- 95 nmol/l) at 12-24 h following therapy initiation. However, fasting treatment values at 168 h were still significantly higher than the mean fasting methylglyoxal level in control diabetes group (P = 0.008). The study indicated that diabetic ketoacidosis results in an increase in methylglyoxal level. Excessive production of toxic intermediates such are alpha-dicarbonyls may be a link connecting an acute metabolic event with accelerated tissue damage, a feature characteristic of long-term complications of diabetes.

Bicarbonates↗
PubMed2001

Morphofunctional mitochondrial response to methylglyoxal toxicity in Bufo bufo embryos.

Methylglyoxal (2-oxopropanal) is a reactive alpha-oxoaldehyde that can be formed endogenously mainly as a by-product of glycolytic pathway. It is a cytotoxic compound with significant antiproliferative properties as it can bind, under physiological conditions, to nucleic acids and proteins, forming stable adducts. We have recently shown that exogenous methylglyoxal (150-600 microM) is highly toxic for amphibian embryos where it produces, when added to the culture water, inhibition of cell proliferation in the early developmental stages, followed by severe malformations and strongly reduced embryonic viability. In this work we investigate the morphofunctional effect of methylglyoxal on the common toad B. bufo embryo mitochondria in order to verify if its dysmorphogenetic action might be also ascribed to impairment of mitochondrial functions. The mitochondria were isolated from embryos at the developmental stages of morula, neural plate and operculum complete and developing in the presence of 600 microM methylglyoxal. The results show that exogenous methylglyoxal is highly toxic at mitochondrial level, where it produces proliferation, swelling and membrane derangement. As a consequence, mitochondria from treated embryos show decreased oxidative phosphorylation efficiency, as indicated by the significant reduction both of the respiratory control index values and of the embryonic ATP content. On the basis of these data, it is possible that the methylglyoxal-induced embryonic malformations as well as the strongly reduced viability might be also ascribed to energy depletion.

Adenosine Diphosphate↗
PubMed1999

The effect of advanced glycation end-product formation upon cell-matrix interactions.

The formation of advanced glycation end-products plays a central role in the progressive deterioration of tissues with age, a process that is accelerated in diabetes. Collagen in addition to providing structure and tensile strength to tissues also provides a dynamic matrix for cells to interact with, and due to its long-lived nature is particularly susceptible to modification with age and disease. We have recently identified methylglyoxal as a key intermediate in this process, reacting predominantly with arginine residues to form imidazolone compounds. We therefore postulated that modification of RGD sequences in collagen with methylglyoxal would interfere with crucial cell-matrix interactions. To investigate this concept we studied the interaction of two cell lines, MG63 and HT1080, with collagen modified to varying degrees with respect to arginine. Adhesion and subsequent spreading of both cell lines was significantly decreased by minimal methylglyoxal modification leading to the conclusion that such modification of collagen severely inhibits cell matrix interactions, most likely via the loss of specific arginine residues involved in integrin mediated cell attachment. This is the first demonstration that methylglyoxal modification of collagen can affect cell-matrix interactions and introduces a possible mechanism by which some of the deleterious changes in tissues with age and disease are occurring.

Antibodies, Monoclonal↗
PubMed2000

Methylglyoxal induces G:C to C:G and G:C to T:A transversions in the supF gene on a shuttle vector plasmid replicated in mammalian cells.

We previously reported that the majority of base-pair substitutions induced by an endogenous mutagen, methylglyoxal, were G:C-->T:A transversions and G:C-->A:T transitions in wild-type and nucleotide excision repair (NER)-deficient (uvrA or uvrC) Escherichia coli strains. To investigate the mutation spectrum of methylglyoxal in mammalian cells and to compare the spectrum with those detected in other experimental systems, we analyzed mutations in a bacterial suppressor tRNA (supF) gene in the shuttle vector plasmid pMY189. We treated pMY189 with methylglyoxal and immediately transfected it into simian COS-7 cells. The cytotoxicity and the mutation frequency (MF) increased according to the dose of methylglyoxal. In the mutants induced by methylglyoxal, multi-base deletions were predominant (50%), followed by base-pair substitutions (35%), in which 89% of the substitutions occurred at G:C sites. Among them, G:C-->C:G and G:C-->T:A transversions were predominant. The overall distribution of methylglyoxal-induced mutations detected in the supF gene was different from that for the spontaneous mutations. These results suggest that methylglyoxal may take part in causing G:C-->C:G and G:C-->T:A transversions in vivo.

Animals↗
PubMed1997

Base-pair mutation caused by four nitro-group containing aromatic amines in Salmonella typhimurium TA100, TA104, TA4001 and TA4006.

Among p-phenylenediamine, benzidine and the analogues we previously tested, only the nitro-group containing 2-nitro-p-phenylenediamine, 3-nitro-o-phenylenediamine, 4-nitro-o-phenylenediamine and 4,4'-dinitro-2-biphenylamine caused base-pair reversion in the histidine locus of Salmonella typhimurium TA100. In order to determine the types of mutations involved, such as transversion or transition, these four nitro-group containing compounds were tested with S. typhimurium strains TA100, TA104, TA4001 and TA4006. Dose-mutagenicity relationships occurred with TA100 and TA104. However, the majority of revertants from TA100 and TA104 were insensitive to inhibition by histidine analogue, DL-1,2,4-triazole-3-alanine. These results suggested that the occurrence of histidine revertants was predominantly induced by base-pair (point) mutations and not by suppressor gene mutations. The CG-TA transition and CG-AT transversion are the major types of mutation induced by all these compounds in TA100. The TA-AT transversion also contributed to the mutagenicity of 4-nitro-o-phenylenediamine and 4,4'-dinitro-2-biphenylamine in TA104. These nitro-group containing compounds showed no mutagenicity in TA4001, but induced CG-GC transversion in TA4006.

Aminobiphenyl Compounds↗
PubMed1999

Deficient nucleotide excision repair increases base-pair substitutions but decreases TGGC frameshifts induced by methylglyoxal in Escherichia coli.

To investigate the mutation spectrum of a well-known mutagen, methylglyoxal, and the influence of nucleotide excision repair (NER) on methylglyoxal-induced mutations, we treated wild-type and NER-deficient (uvrA or uvrC) Escherichia coli strains with methylglyoxal, and analyzed mutations in the chromosomal lacI gene. In the three strains, the cell death and the mutation frequency increased according to the dose of methylglyoxal added to the culture medium. The frequencies of methylglyoxal-induced base-pair substitutions were higher in the NER-deficient strains than in the wild-type strain, in the presence and absence of mucAB gene. Paradoxically, the frequency of methylglyoxal-induced TGGC frameshifts was higher in the wild-type strain than in the NER-deficient strains. When the methylglyoxal-induced mutation spectra in the presence and absence of mucAB gene are compared, the ratios of base-pair substitutions to frameshifts were increased by the effects of mucAB gene. In the three strains, more than 75% of the base-pair substitutions occurred at G:C sites, independent of the mucAB gene. When the mucAB gene was present, G:C-->T:A transversions were predominant, followed by G:C-->A:T transitions. When the mucAB gene was absent, the predominant mutations differed in the three strains: in the wild-type and uvrC strains, G:C-->A:T transitions were predominant, followed by G:C-->T:A transversions, while in the uvrA strains, G:C-->T:A transversions were predominant, followed by G:C-->A:T transitions. These results suggest that NER may be involved in both the repair and the fixation of methylglyoxal-induced mutations.

Base Pair Mismatch↗
PubMed2003

Excited state complex formation between methyl glyoxal and some aromatic bio-molecules: a fluorescence quenching study.

Fluorescence quenching of some important aromatic bio-molecules (ABM) such as 3-aminophthalhydrazide (luminol), tryptophan (Try), phenylalanine and tyrosine (Tyr) by methyl glyoxal (MG) has been studied employing different spectroscopic techniques. The interaction of MG with ABM in the excited state has been analysed using Stern-Volmer (S-V) mechanism. In the case of MG-luminol system time correlated single photon counting (TCSPC) technique has also been applied to explain the S-V mechanism. The bimolecular rate constants obtained are found to be higher than the rate constant for diffusion controlled process. A plausible explanation of the quenching mechanism has been discussed on the basis of hydrogen bonding, charge transfer and energy transfer interaction between the colliding species.

Amino Acids↗
PubMed2003

Identification of N epsilon-(carboxyethyl)lysine, one of the methylglyoxal-derived AGE structures, in glucose-modified protein: mechanism for protein modification by reactive aldehydes.

We have developed a separation system for N(epsilon)-(carboxyethyl)lysine (CEL) and N(epsilon)-(carboxymethyl)lysine (CML) by HPLC equipped with a styrene-divinylbenzene copolymer resin coupled with sulfonic group cation-exchange column and examined whether CEL is formed from proteins modified by glucose via the Maillard reaction. CEL was generated by incubating bovine serum albumin (BSA) with glucose, a reaction inhibited by aminoguanidine, but enhanced by phosphate. Although several aldehydes were detected during incubation of N(alpha)-acetyllysine with glucose, incubation of BSA with methylglyoxal alone generated CEL. These results indicate that methylglyoxal is responsible for CEL formation on protein in vitro.

Aldehydes↗
PubMed1994

Stereospecificity of substrate usage by glyoxalase 1: nuclear magnetic resonance studies of kinetics and hemithioacetal substrate conformation.

The specificity of glyoxalase 1 for the diastereomers of its hemithioacetal substrate [which forms spontaneously between an alpha-keto aldehyde and reduced glutathione (GSH)] was investigated by exploiting the differences between their 1H NMR spectra at pH* 4.4. The 1H NMR spectra of the hemithioacetals of glutathione with phenylglyoxal or methylglyoxal were assigned with the aid of conventional decoupling and two-dimensional NMR spectroscopic techniques. The rate of interconversion of the diastereomers was determined at 30 degrees C from the results of an inversion-transfer technique and found to be 0.30 +/- 0.04 s-1 (+/- sd) in the case of phenylglyoxal and 0.15 +/- 0.02 s-1 in the case of methylglyoxal. Stereopreference of the enzyme was tested by the addition of large amounts of yeast glyoxalase 1 to a reaction mixture; glyoxalase 1 preferentially operated on one diastereomer of the phenylglyoxal hemithioacetal but the diastereomers of methylglyoxal appeared to be operated upon indiscriminately. From computer models of the kinetics of possible reaction schemes, a mechanism involving glyoxalase 1 catalysis of both diastereomers of the hemithioacetals was shown to be the most consistent with the experimental data. Estimates of internuclear distances in the diastereomers, obtained from 2D NMR spectra were used in "dynamical simulated annealing" calculations to generate likely structures of the substrates. Relative ring-current shifts obtained from 1D NMR spectra were used, together with a ring-current shift algorithm, to select structures with compatible conformations. We conclude that the rate of conversion of substrate by the enzyme is dependent upon the overall conformation of the substrate molecule, rather than merely its stereochemical configuration (R or S).

Glutathione↗
PubMed1991

Kinetic parameters for the elimination reaction catalyzed by triosephosphate isomerase and an estimation of the reaction's physiological significance.

Kinetic parameters for triosephosphate isomerase catalysis of the elimination reaction of an equilibrium mixture of dihydroxyacetone phosphate (DHAP) and D-glyceraldehyde-3-phosphate (DGAP) to form methylglyoxal and phosphate ion are reported for the enzyme from rabbit muscle. Pseudo-first-order rate constants for the disappearance of substrate (kelim) were determined for reactions at [Enzyme] much greater than [Substrate]. The second-order rate constant kEnz = 10.1 M-1 s-1 was determined from a plot of kelim against enzyme concentration. The kinetic parameters, determined from a steady-state kinetic analysis at [Substrate] much greater than [Enzyme], are kcat = 0.011 s-1, Km = 0.76 mM, and kcat/Km = 14 M-1 s-1. The estimated rate-constant ratio for partitioning of the enzyme-bound intermediate between protonation at carbon 2 and elimination, 1,000,000, is much larger than the ratio of 6.5 determined for the reaction of the enediolate phosphate in a loose complex with quinuclidinonium cation, a small buffer catalyst. There is a 10(5)-10(8)-fold decrease in the rate constant for the elimination reaction of the enediolate phosphate when this species binds to triosephosphate isomerase. The kinetic parameters for the elimination reaction catalyzed by the native triosephosphate isomerase and for the reaction catalyzed by a mutant form of the enzyme, which is missing a segment that forms hydrogen bonds with the phosphate group of substrate [Pompliano, D. L., Peyman, A., & Knowles, J. R. (1990) Biochemistry 29, 3186-3194] are similar.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1988

Optimization of efficiency in the glyoxalase pathway.

A quantitative kinetic model for the glutathione-dependent conversion of methylglyoxal to D-lactate in mammalian erythrocytes has been formulated, on the basis of the measured or calculated rate and equilibrium constants associated with (a) the hydration of methylglyoxal, (b) the specific base catalyzed formation of glutathione-(R,S)-methylglyoxal thiohemiacetals, (c) the glyoxalase I catalyzed conversion of the diastereotopic thiohemiacetals to (S)-D-lactoylglutathione, and (d) the glyoxalase II catalyzed hydrolysis of (S)-D-lactoylglutathione to form D-lactate and glutathione. The model exhibits the following properties under conditions where substrate concentrations are small in comparison to the Km values for the glyoxalase enzymes: The overall rate of conversion of methylglyoxal to D-lactate is primarily limited by the rate of formation of the diastereotopic thiohemiacetals. The hydration of methylglyoxal is kinetically unimportant, since the apparent rate constant for hydration is (approximately 500-10(3))-fold smaller than that for formation of the thiohemiacetals. The rate of conversion of methylglyoxal to (S)-D-lactoylglutathione is near optimal, on the basis that the apparent rate constant for the glyoxalase I reaction (kcatEt/Km congruent to 4-20 s-1 for pig, rat, and human erythrocytes) is roughly equal to the apparent rate constant for decomposition of the thiohemiacetals to form glutathione and methylglyoxal [k(obsd) = 11 s-1, pH 7]. The capacity of glyoxalase I to use both diastereotopic thiohemiacetals, versus only one of the diastereomers, as substrates represents a 3- to 6-fold advantage in the steady-state rate of conversion of the diastereomers to (S)-D-lactoylglutathione.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1981

Liberation of the triosephosphate isomerase reaction intermediate and its trapping by isomerase, yeast aldolase, and methylglyoxal synthase.

When a mixture of triosephosphate isomerase (rabbit muscle) and dihydroxyacetone phosphate (DHAP) is quenched with acid, a compound is liberated, presumed to be the cis-enediol 3-phosphate, that decomposes to inorganic phosphate (Pi) and methylglyoxal [Iyengar, R., & Rose, I.A. (1981) Biochemistry (preceding paper is this issue)]. The decomposition can be prevented by rapid neutralization if a catalytic amount of fresh isomerase is present. Varying the time between acidification and rescue gave a half-life of the liberate compound of approximately 12-17 ms. Varying the concentration of enzyme used for rescue gave a minimum second-order rate constant for trapping of 10(9)M(-1)s(-1). These results add further evidence favoring a stepwise mechanism for the aldose-ketose isomerase reactions in which a chemically defined enzyme-bound intermediate is found. The high rate of trapping over a wide pH range indicates that the enediol phosphate, not the enediolate phosphate, is the intermediate. One property of the enzyme is to stabilize the intermediate with respect to its fragmentation in solution by greater than 1000-fold. Yeast aldolase is also able to rescue all of the isomerase intermediate, though higher concentrations of enzyme are required. Although different enantiotopic protons of DHAP are abstracted by isomerase and aldolase, both enzymes use the same enediol phosphate intermediate. Methylglyoxal synthase at a 50-fold greater concentration was unable to compete with triosephosphate isomerase for cis-enediol phosphate. Either the synthetase has a low V/K for the cis isomer or it uses the trans-enediol phosphate form specifically. A new strategy for the chemical and enzymological characterization of enzyme reaction intermediates is proved here based on the liberation of the intermediate from the reaction equilibrium and its recovery by fresh enzyme or another enzyme species.

Animals↗
PubMed1975

Substrate activity of structural analogs of isocitrate for isocitrate dehydrogenases from bovine heart.

D-Garcinia acid (D-threo-1,2-dihydroxy-1,2,3-propanetricarboxylate), like D-isocitrate, has an alpha-DS-hydroxyl group and a beta-LS configuration of the second carboxyl group. The maximal velocity of pyridine nucleotide reduction with D-garcinia acid is 8 and 21% of D-threo-isocitrate with the DPN-linked and TPN-linked isocitrate dehydrogenase from bovine heart, respectively. The other stereoisomers of hydroxycitrate [L-garcinia acid, D- and L-hibiscus acid (D- and L-erythro-1,2-dihydroxy-1,2,3-propanetricarboxylate)] are inactive. DL-threo-Homoisocitrate (DL-threo-1-hydroxy-1,2,4-butanetricarboxylate) supports DPN+ reduction at 10-15% of the rate observed for isocitrate with the DPN-specific enzyme, but is not a substrate for TPN-linked isocitrate dehydrogenase. The values of apparent S0.5 for total isocitrate and total garcinia acid are similar with both enzymes; the apparent S0.5 of total homoisocitrate is two- to threefold higher than that of total isocitrate with the DPN-linked enzyme. Enzymatic oxidative decarboxylation of garcinia acid and homoisocitrate leads to formation of alpha-keto-beta-hydroxyglutarate and alpha-ketoadipate, respectively. DL-Methylmalate (DL-1-hydroxy-2-methylsuccinate) is inactive as a substrate for either dehydrogenase as are the newly synthesized compounds: DL-threo-gamma-isocitrate amide (DL-threo-1-hydroxy-3-carbamy01,2-propanedicarboxylate), beta-methyl-DL-isocitrate (DL-1-hydroxy-2-methyl-1,2,3-propanetricarboxylate), beta-methyl-DL-garcinia acid (DL-threo-1-hydroxyl-2-methoxy-1,2,3-propanetricarboxylate), DL-1-hydroxyl-1,2,2-ethanetricarboxylate, and DL-1,4-dihydroxy-1,2-butanedicarboxylate.

2,6-Dichloroindophenol↗
PubMed2002

Catalysis and stability of triosephosphate isomerase from Trypanosoma brucei with different residues at position 14 of the dimer interface. Characterization of a catalytically competent monomeric enzyme.

In homodimeric triosephosphate isomerase from Trypanosoma brucei (TbTIM), cysteine 14 of each the two subunits forms part of the dimer interface. This residue is central for the catalysis and stability of TbTIM. Cys14 was changed to the other 19 amino acids to determine the characteristics that the residue must have to yield catalytically competent stable enzymes. C14A, C14S, C14P, C14T, and C14V TbTIMs were essentially wild type in activity and stability. Mutants with Asn, Arg, and Gly had low activities and stabilities. The other mutants had less than 1% of the activity of TbTIM. One of the latter enzymes (C14F) was purified to homogeneity. Size exclusion chromatography and equilibrium sedimentation studies showed that C14F TbTIM is a monomer, with a k(cat) approximately 1000 times lower and a K(m) approximately 6 times higher than those of TbTIM. In C14F TbTIM, the ratio of the elimination (methylglyoxal and phosphate formation) to isomerization reactions was higher than in TbTIM. Its secondary structure was very similar to that of TbTIM; however, the quantum yield of its aromatic residues was lower. The analysis of the data with the 19 mutants showed that to yield enzymes similar to the wild type, the residue must have low polarity and a van der Waals volume between 65 and 110 A(3). The results with C14F TbTIM illustrate that the secondary structure of TbTIM can be formed in the absence of intersubunit contacts, and that it has sufficient tertiary structure to support catalysis.

Animals↗
PubMed2003

Enhancement of chaperone function of alpha-crystallin by methylglyoxal modification.

The molecular chaperone function of alpha-crystallin in the lens prevents the aggregation and insolubilization of lens proteins that occur during the process of aging. We found that chemical modification of alpha-crystallin by a physiological alpha-dicarbonyl compound, methylglyoxal (MG), enhances its chaperone function. Protein-modifying sugars and ascorbate have no such effect and actually reduce chaperone function. Chaperone assay after immunoprecipitation or with immunoaffinity-purified argpyrimidine-alpha-crystallin indicates that 50-60% of the increased chaperone function is due to argpyrimidine-modified protein. Incubation of alpha-crystallin with DL-glyceraldehyde and arginine-modifying agents also enhances chaperone function, and we believe that the increased chaperone activity depends on the extent of arginine modification. Far- and near-UV circular dichroism spectra indicate modest changes in secondary and tertiary structure of MG-modified alpha-crystallin. LC MS/MS analysis of MG-modified alpha-crystallin following chymotryptic digestion revealed that R21, R49, and R103 in alphaA-crystallin were converted to argpyrimidine. 1,1'-Bis(4-anilino)naphthalene-5,5'-disulfonic acid binding, an indicator of hydrophobicity of proteins, increased in alpha-crystallin modified by low concentrations of MG (2-100 microM). MG similarly enhances chaperone function of another small heat shock protein, Hsp27. Our results show that posttranslational modification by a metabolic product can enhance the chaperone function of alpha-crystallin and Hsp27 and suggest that such modification may be a protective mechanism against environmental and metabolic stresses. Augmentation of the chaperone function of alpha-crystallin might have evolved to protect the lens from deleterious protein modifications associated with aging.

Alcohol Dehydrogenase↗
PubMed2004

Entropy effects on protein hinges: the reaction catalyzed by triosephosphate isomerase.

Many proteins utilize segmental motions to catalyze a specific reaction. The Omega loop of triosephosphate isomerase (TIM) is important for preventing the loss of the reactive enediol(ate) intermediate. The loop opens and closes even in the absence of the ligand, and the loop itself does not change conformation during movement. The conformational changes are localized to two hinges at the loop termini. Glycine is never observed in native TIM hinge sequences. In this paper, the hypothesis that limited access to conformational space is a requirement for protein hinges involved in catalysis was tested. The N-terminal hinge was mutated to P166/V167G/W168G (PGG), and the C-terminal hinge was mutated to K174G/T175G/A176G (GGG) in chicken TIM. The single-hinge mutants PGG and GGG had k(cat) values 200-fold lower than that of the wild type and K(m) values 10-fold higher. The k(cat) of double-hinge mutant P166/V167G/W168G/K174G/T175G/A176G was reduced 2500-fold; the K(m) was 10-fold higher. A combination of primary kinetic isotope effect measurements, isothermal calorimetric measurements, and (31)P NMR spectroscopic titration with the inhibitor 2-phosphoglycolate revealed that the mutants have a different ligand-binding mode than that of the wild-type enzyme. The predominant conformations of the mutants even in the presence of the inhibitor are loop-open conformations. In conclusion, mutation of the hinge residues to glycine resulted in the sampling of many more hinge conformations with the consequence that the population of the active-closed conformation is reduced. This reduced population results in a reduced catalytic activity.

Animals↗
PubMed2006

Effect of site-directed mutagenesis of methylglyoxal-modifiable arginine residues on the structure and chaperone function of human alphaA-crystallin.

We reported previously that chemical modification of human alphaA-crystallin by a metabolic dicarbonyl compound, methylglyoxal (MGO), enhances its chaperone-like function, a phenomenon which we attributed to formation of argpyrimidine at arginine residues (R) 21, 49, and 103. This structural change removes the positive charge on the arginine residues. To explore this mechanism further, we replaced these three R residues with a neutral alanine (A) residue one at a time or in combination and examined the impact on the structure and chaperone function. Measurement of intrinsic tryptophan fluorescence and near-UV CD spectra revealed alteration of the microenvironment of aromatic amino acid residues in mutant proteins. When compared to wild-type (wt) alphaA-crystallin, the chaperone function of R21A and R103A mutants increased 20% and 18% as measured by the insulin aggregation assay and increased it as much as 39% and 28% when measured by the citrate synthase (CS) aggregation assay. While the R49A mutant lost most of its chaperone function, R21A/R103A and R21A/R49A/R103A mutants had slightly better function (6-14% and 10-14%) than the wt protein in these assays. R21A and R103A mutants had higher surface hydrophobicity than wt alphaA-crystallin, but the R49A mutant had lower hydrophobicity. R21A and R103A mutants, but not the R49A mutant, were more efficient than wt protein in refolding guanidine hydrochloride-treated malate dehydrogenase to its native state. Our findings indicate that the positive charges on R21, R49, and R103 are important determinants of the chaperone function of alphaA-crystallin and suggest that chemical modification of arginine residues may play a role in protein aggregation during lens aging and cataract formation.

Arginine↗