Phagocytic clearance of electric field induced 'apoptosis-mimetic' cells.

Cells undergoing apoptosis lose lipid asymmetry that is often manifested by the exposure of phosphatidylserine (PS) to the outer surface of the cell membrane. Macrophages and other cell types recognize externalized PS to signal phagocytosis, thereby eliciting a non-inflammatory response. PS exposure is obligatory in the recognition and clearance of apoptotic cells. Here, we find that externally applied moderate electric field induces PS externalization in a mouse B-cell (FOX-NY) membrane without procaspase-3 activation, a major characteristic of apoptotic cells. The field-induced PS inversion is caused as a result of electroporation and/or a process involving membrane reorganizations and recovery that ensues following field exposure. Using a mouse macrophage cell line (J7444A.1) from the same strain, we show phagocytic clearance of PS expressing B-cells and demonstrate that this is in part due to the apoptosis mimicry of the field exposed cells.

Selective field effects on intracellular vacuoles and vesicle membranes with nanosecond electric pulses.

Electric pulses across intact vesicles and cells can lead to transient increase in permeability of their membranes. We studied the integrity of these membranes in response to external electric pulses of high amplitude and submicrosecond duration with a primary aim of achieving selective permeabilization. These effects were examined in two separate model systems comprising of 1), a mixed population of 1,2-di-oleoyl-sn-glycero-3-phosphocholine phospholipid vesicles and in 2), single COS-7 cells, in which large endosomal membrane vacuoles were induced by stimulated endocytosis. It has been shown that large and rapidly varying external electric fields, with pulses shorter than the charging time of the outer-cell membrane, could substantially increase intracellular fields to achieve selective manipulations of intracellular organelles. The underlying principle of this earlier work is further developed and applied to the systems studied here. Under appropriate conditions, we show preferential permeabilization of one vesicle population in a mixed preparation of vesicles of similar size distribution. It is further shown that large endocytosed vacuoles in COS-7 cells can be selectively permeabilized with little effect on the integrity of outer cell membrane.

Reversible glutathionylation regulates actin polymerization in A431 cells.

In response to growth factor stimulation, many mammalian cells transiently generate reactive oxygen species (ROS) that lead to the elevation of tyrosine-phosphorylated and glutathionylated proteins. While investigating EGF-induced glutathionylation in A431 cells, paradoxically we found deglutathionylation of a major 42-kDa protein identified as actin. Mass spectrometric analysis revealed that the glutathionylation site is Cys-374. Deglutathionylation of the G-actin leads to about a 6-fold increase in the rate of polymerization. In vivo studies revealed a 12% increase in F-actin content 15 min after EGF treatment, and F-actin was found in the cell periphery suggesting that in response to growth factor, actin polymerization in vivo is regulated by a reversible glutathionylation mechanism. Deglutathionylation is most likely catalyzed by glutaredoxin (thioltranferase), because Cd(II), an inhibitor of glutaredoxin, inhibits intracellular actin deglutathionylation at 2 microM comparable with its IC(50) in vitro. Moreover, mass spectral analysis showed efficient transfer of GSH from immobilized S-glutathionylated actin to glutaredoxin. Overall, this study revealed a novel physiological relevance of actin polymerization regulated by reversible glutathionylation of the penultimate cysteine mediated by growth factor stimulation.

Mitochondria play no roles in Mn(II)-induced apoptosis in HeLa cells.

Manganese(II) has been shown to exhibit catalase-like activity under physiological conditions. In the course of studies to test the antioxidant activity of Mn(II) on HeLa cells, it was observed at high concentrations (1-2 mM) that Mn(II) also induced apoptosis, as judged by changes in cell morphology, caspase-3 activation, cleavage of poly(ADP) ribose, and DNA condensation. However, in contrast to established mechanisms, the Mn(II)-induced apoptosis is associated with an increase rather than a decrease in mitochondrial inner-membrane potential, as monitored by the fluorescent probe tetramethylrhodamine ethyl ester. Based on immunochemical analysis, Mn(II)-induced apoptosis does not lead to the release of cytochrome c into the cytosol. These and other measurements show that treatment with Mn(II) leads to enhancement of the mitochondrial "membrane mass," has no effect on mitochondrial volume, and does not affect the permeability transition pore. Together, these results support the view that Mn(II)-induced apoptosis occurs by a heretofore unrecognized mechanism. In addition, it was demonstrated that Mn(II) treatment leads to an increase in the production of reactive oxygen species (peroxides) and to the induction of the manganese superoxide dismutase and catalase activities but has no effect on the Cu,Zn-superoxide dismutase level.

Bruton's tyrosine kinase is essential for hydrogen peroxide-induced calcium signaling.

Using Btk-deficient DT40 cells and the transfectants expressing wild-type Btk or Btk mutants in either kinase (Arg(525) to Gln), Src homology 2 (SH2, Arg(307) to Ala), or pleckstrin homology (PH, Arg(28) to Cys) domains, we investigated the roles and structure-function relationships of Btk in hydrogen peroxide-induced calcium mobilization. Our genetic evidence showed that Btk deficiency resulted in a significant reduction in hydrogen peroxide-induced calcium response. This impaired calcium signaling is correlated with the complete elimination of IP3 production and the significantly reduced tyrosine phosphorylation of PLCgamma2 in Btk-deficient DT40 cells. All of these defects were fully restored by the expression of wild-type Btk in Btk-deficient DT40 cells. The data from the point mutation study revealed that a defect at any one of the three functional domains would prevent a full recovery of Btk-mediated hydrogen peroxide-induced intracellular calcium mobilization. However, mutation at either the SH2 or PH domain did not affect the hydrogen peroxide-induced activation of Btk. Mutation at the SH2 domain abrogates both IP3 generation and calcium release, while the mutant with the nonfunctional PH domain can partially activate PLCgamma2 and catalyze IP3 production but fails to produce significant calcium mobilization. Thus, these observations suggest that Btk-dependent tyrosine phosphorylation of PLCgamma2 is required but not sufficient for hydrogen peroxide-induced calcium mobilization. Furthermore, hydrogen peroxide stimulates a Syk-, but not Btk-, dependent tyrosine phosphorylation of B cell linker protein BLNK. The overall results, together with those reported earlier [Qin et al. (2000) Proc. Natl. Acad. Sci. U.S.A. 97, 7118], are consistent with the notion that functional SH2 and PH domains are required for Btk to form a complex with PLCgamma2 through BLNK in order to position the Btk, PLCgamma2, and phosphatidylinositol 4,5-bisphosphate in close proximity for efficient activation of PLCgamma2 and to maximize its catalytic efficiency for IP3 production.

Asymmetric pore distribution and loss of membrane lipid in electroporated DOPC vesicles.

An externally applied electric field across vesicles leads to transient perforation of the membrane. The distribution and lifetime of these pores was examined using 1,2-di-oleoyl-sn-glycero-3-phosphocholine (DOPC) phospholipid vesicles using a standard fluorescent microscope. The vesicle membrane was stained with a fluorescent membrane dye, and upon field application, a single membrane pore as large as approximately 7 microm in diameter was observed at the vesicle membrane facing the negative electrode. At the anode-facing hemisphere, large and visible pores are seldom found, but formation of many small pores is implicated by the data. Analysis of pre- and post-field fluorescent vesicle images, as well as images from negatively stained electron micrographs, indicate that pore formation is associated with a partial loss of the phospholipid bilayer from the vesicle membrane. Up to approximately 14% of the membrane surface could be lost due to pore formation. Interestingly, despite a clear difference in the size distribution of the pores observed, the effective porous areas at both hemispheres was approximately equal. Ca(2+) influx measurements into perforated vesicles further showed that pores are essentially resealed within approximately 165 ms after the pulse. The pore distribution found in this study is in line with an earlier hypothesis (E. Tekle, R. D. Astumian, and P. B. Chock, 1994, Proc. Natl. Acad. Sci. U.S.A. 91:11512--11516) of asymmetric pore distribution based on selective transport of various fluorescent markers across electroporated membranes.

Antioxidant activity of Ferrozine-iron-amino acid complexes.

Amino acid-Fe(II)-chelator complexes exhibit strong antioxidant activity. Taking advantage of the unique spectral characteristics of the complexes formed when Ferrozine (Fz) is used as the chelator, we now show that the primary blue complex (epsilon(max) at 632 nm) decomposes by two independent pathways: (i) a nonoxidative pathway involving dissociation of the amino acid component and formation of a purple complex (epsilon(max) at 562 nm) and (ii) an oxidative pathway leading to Fe(III) and colorless products. Quantitative conversion of the blue to purple complex yields an isosbestic point (i.p.) at 601 nm, whereas no i.p. is formed during quantitative oxidation of the blue complex. However, under some experimental conditions, decomposition of the blue product occurs by both pathways, leading to occurrence of a clean i.p. at wavelengths varying from 601 to 574 nm. Results of simulation experiments, confirmed by direct analysis, demonstrate that shifts in the i.p. reflect differences in the fractions of blue compound that decompose by the oxidative and nonoxidative pathways. Indeed, the fraction of blue that is converted to the purple complex is readily deduced from the wavelength of the i.p. These results suggest that identification of a physiological chelator that can replace Ferrozine in amino acid-iron complexes might have important physiological and pharmacological applications.

Protein glycation: creation of catalytic sites for free radical generation.

In a glycation reaction, alpha-dicarbonyl compounds such as deoxyglucosone, methylglyoxal, and glyoxal are more reactive than the parent sugars with respect to their ability to react with amino groups of proteins to form inter- and intramolecular cross-links of proteins, stable end products called advanced Maillard products or advanced end products (AGEs). The AGEs, which are irreversibly formed, accumulate with aging, atherosclerosis, and diabetes mellitus, and are especially associated with long-lived proteins such as collagens, lens crystallins, and nerve proteins. It was suggested that the formation of AGEs not only modifies protein properites but also induces biological damage in vivo. In this report, we summerize results obtained from our studies for (1) identifying the structure of the cross-linked radical species formed in the model system-the reaction between alpha-dicarbonyl methylglyoxal with amino acids, and (2) the reactivity of the radical center of the protein created by the similar reaction. These results indicate that glycation of protein generates active centers for catalyzing one-electron oxidation-reduction reactions. This active center, which exhibits enzyme-like character, is suggested to be the cross-linked Schiff-based radical cation of the protein. It mimics the characteristics of the metal-catalyzed oxidation system. These results together indicate that glycated proteins accumulated in vivo provide stable active sites for catalyzing the formation of free redicals.

Enzyme-like activity of glycated cross-linked proteins in free radical generation.

The structure and property of cross-linked amino acids and proteins produced by a three- carbon alpha-dicarbonyl methylglyoxal in glycation reaction were investigated. Our results showed that these reactions generated yellow fluorescent products and several free radical species. From the reaction with alanine, three types of free radicals were identified by EPR spectroscopy: 1) the cross-linked radical cation, methylglyoxal diaklylimine cation radical; 2) the methylglyoxal radical anion as the counterion; 3) the superoxide radical anion produced only in the presence of oxygen. Glycation of bovine serum albumin by methylglyoxal also generated the protein-bound, cross-linked free radical, probably the cation radical of the cross-linked Schiff base as observed with alanine. The glycated protein reduced ferricytochrome c to ferrocytochrome c in the absence of oxygen or added metal ions. This reduction of cytochrome c was accompanied by a large increase in the amplitude of the electron paramagnetic resonance signal originated from the protein-bound free radical. In addition, the glycated protein catalyzed the oxidation of ascorbate in the presence of oxygen while the protein-free radical signal disappeared. These results indicate that glycation of protein generates active centers for catalyzing one-electron oxidation-reduction reactions. This active center, which exhibits enzyme-like character, was suggested to be the cross-linked Schiff base/the cross-linked Schiff base radical cation of the protein. It mimics the characteristics of metal-catalyzed oxidation system. These results together indicate that glycated proteins accumulated in vivo provide stable active-sites for catalyzing the formation of free radicals.

Regulation of oxidative stress-induced calcium release by phosphatidylinositol 3-kinase and Bruton's tyrosine kinase in B cells.

Hydrogen peroxide stimulates a tyrosine kinase-dependent calcium release from intracellular stores, which is assumed to be achieved through the activation of phospholipase Cgamma2 (PLCgamma2) via a tyrosine phosphorylation mechanism in B cells. Here we show that H(2)O(2) induces both tyrosine phosphorylation on PLCgamma2 and the activation of phosphatidylinositol 3-kinase (PI3K) in B cells, and that the phosphatidylinositol 3-kinase inhibitor, Wortmannin, partially inhibited the H(2)O(2)-induced calcium release without affecting tyrosine phosphorylation on PLCgamma2. Overexpression of human Bruton's tyrosine kinase (Btk), which was activated by H(2)O(2), almost completely overcame the inhibition of calcium release by Wortmannin. The reversal of Wortmannin's inhibition by enhancing Btk concentration seemed unique to the H(2)O(2)-mediated effect, because Btk failed to overcome the inhibition of Wortmannin on B cell receptor-triggered calcium mobilization. Immunoblot analysis revealed that Btk formed stable complexes with several tyrosine-phosphorylated proteins, including PLCgamma2, only in Btk-overexpressed cells on H(2)O(2) stimulation. Together, our data are consistent with the notion that PIP3 and/or a high concentration of Btk target the activated PLCgamma2 to its substrate site for maximal catalytic efficiency.

Inositol tetrakisphosphate as a frequency regulator in calcium oscillations in HeLa cells.

Cellular signaling mediated by inositol (1,4,5)trisphosphate (Ins(1, 4,5)P(3)) results in oscillatory intracellular calcium (Ca(2+)) release. Because the amplitude of the Ca(2+) spikes is relatively invariant, the extent of the agonist-mediated effects must reside in their ability to regulate the oscillating frequency. Using electroporation techniques, we show that Ins(1,4,5)P(3), Ins(1,3,4, 5)P(4), and Ins(1,3,4,6)P(4) cause a rapid intracellular Ca(2+) release in resting HeLa cells and a transient increase in the frequency of ongoing Ca(2+) oscillations stimulated by histamine. Two poorly metabolizable analogs of Ins(1,4,5)P(3), Ins(2,4,5)P(3), and 2,3-dideoxy-Ins(1,4,5)P(3), gave a single Ca(2+) spike and failed to alter the frequency of ongoing oscillations. Complete inhibition of Ins(1,4,5)P(3) 3-kinase (IP3K) by either adriamycin or its specific antibody blocked Ca(2+) oscillations. Partial inhibition of IP3K causes a significant reduction in frequency. Taken together, our results indicate that Ins(1,3,4,5)P(4) is the frequency regulator in vivo, and IP3K, which phosphorylates Ins(1,4, 5)P(3) to Ins(1,3,4,5)P(4), plays a major regulatory role in intracellular Ca(2+) oscillations.

Transcriptional activation of the human manganese superoxide dismutase gene mediated by tetradecanoylphorbol acetate.

Transcriptional activation of human manganese superoxide dismutase (MnSOD) mRNA induced by a phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA), was examined to identify the responsive transcriptional regulator. The effect of various deletions and mutations within the 5'-flanking region of the human MnSOD gene promoter was evaluated using the luciferase reporter system in A549 human lung carcinoma cells. Deletion of a region between -1292 and -1202 nucleotides upstream of the transcription start site abolished TPA-responsive induction, whereas deletion of the putative binding sequence for NF-kappaB or AP-1 did not. The region between -1292 and -1202 contains a cAMP-responsive element-like sequence, TGACGTCT, which we identified as the manganese superoxide dismutase TPA-responsive element, MSTRE. Site-specific mutation of the MSTRE abolished the TPA-responsive induction, validating the critical role of this sequence. We detected specific MSTRE activity from nuclear extracts and demonstrated by antibody supershift assay that this activity is closely related to CREB-1/ATF-1. TPA treatment rapidly induced phosphorylation of the CREB-1/ATF-1-like factor via the protein kinase C pathway. These results led us to conclude that the human MnSOD gene having the promoter construct used in this study is induced by TPA via activation of a CREB-1/ATF-1-like factor and not via either NF-kappaB or AP-1. In addition, we found that this induction was blocked by inhibitors of flavoproteins and NADPH oxidases, indicating involvement of enhanced generation of superoxide radical anion as an upstream signal.

Degradation of alpha-synuclein by proteasome.

Mutations in alpha-synuclein are known to be associated with Parkinson's disease (PD). The coexistence of this neuronal protein with ubiquitin and proteasome subunits in Lewy bodies in sporadic disease suggests that alterations of alpha-synuclein catabolism may contribute to the pathogenesis of PD. The degradation pathway of alpha-synuclein has not been identified nor has the kinetics of this process been described. We investigated the degradation kinetics of both wild-type and A53T mutant 6XHis-tagged alpha-synuclein in transiently transfected SH-SY5Y cells. Degradation of both isoforms followed first-order kinetics over 24 h as monitored by the pulse-chase method. However, the t((1)/(2)) of mutant alpha-synuclein was 50% longer than that of the wild-type protein (p < 0.01). The degradation of both recombinant proteins and endogenous alpha-synuclein in these cells was blocked by the selective proteasome inhibitor beta-lactone (40 microM), indicating that both wild-type and A53T mutant alpha-synuclein are degraded by the ubiquitin-proteasome pathway. The slower degradation of mutant alpha-synuclein provides a kinetic basis for its intracellular accumulation, thus favoring its aggregation.

Roles of superoxide radical anion in signal transduction mediated by reversible regulation of protein-tyrosine phosphatase 1B.

Growth factors induce intracellular production of reactive oxygen species in non-phagocytic cells and elevation of their phosphorylated protein tyrosine level. The latter can be achieved by activating protein-tyrosine kinases and/or inactivating protein-tyrosine phosphatases (PTPs). A highly abundant PTP, PTP-1B, is known to be inactivated by oxidation of its catalytic site Cys-215. We show that O-(2) is kinetically more efficient and chemically more specific oxidant than H(2)O(2) for inactivating PTP-1B. The second-order rate constant for the O-(2)- and H(2)O(2)-mediated inactivation is 334 +/- 45 M(-1) s(-1) and 42.8 +/- 3.8 M(-1) s(-1), respectively. PTP-1B oxidized by H(2)O(2) exhibits significantly more oxidized methionine residues and shows a lower degree of reversibility. The initial oxidative product, the Cys-215 sulfenic derivative, can easily be oxidized further to its irreversible sulfinic and sulfonic derivatives. This step is prevented by glutathionylation of the sulfenic derivative to form a S-glutathionylated PTP-1B, which can be reactivated by dithiothreitol or thioltransferase. Thus, a signal transduction mechanism mediated by the O-(2) and the participation of glutathione is proposed for the regulation of PTP-1B. This mechanism is supported by the in vivo demonstration that glutathionylated PTP-1B at Cys-215 is formed in A431 cells when they were treated with epidermal growth factor.

Peroxynitrite-mediated modification of proteins at physiological carbon dioxide concentration: pH dependence of carbonyl formation, tyrosine nitration, and methionine oxidation.

The ability of peroxynitrite to modify amino acid residues in glutamine synthetase (GS) and BSA is greatly influenced by pH and CO2. At physiological concentrations of CO2 (1.3 mM), the generation of carbonyl groups (0.2-0.4 equivalents/subunit) is little affected by pH over the range of 7.2-9.0, but, in the absence of CO2, carbonyl formation increases (from 0.1- 1.2 equivalents/subunit) as the pH is raised from 7.2 to 10.5. This increase is attributable, in part but not entirely, to the increase in peroxynitrite (PN) stability with increasing pH. Of several amino acid polymers tested, only those containing lysine residues yielded carbonyl derivatives. In contrast, the nitration of tyrosine residues of both GS and BSA at pH 7.5 almost completely depends on the presence of CO2. However, the pH profiles of tyrosine nitration in GS and BSA are not the same. With both proteins, nitration decreases approximately 65% with increasing pH over the range of 7.2-8.4, but, then in the case of GS only, there is a 3.4-fold increase in the level of nitration over the range pH 8.4-8.8. The oxidation of methionine residues in both proteins and in the tripeptide Ala-Met-Ala was inhibited by CO2 at both high and low pH values. These results emphasize the importance of controlling the pH and CO2 concentrations in studies involving PN and indicate that PN is not likely to contribute appreciably to carbonyl formation or oxidation of methionine residues of proteins at physiological pH and CO2 concentrations.

Regulation of PTP1B via glutathionylation of the active site cysteine 215.

The reversible regulation of protein tyrosine phosphatase is an important mechanism in processing signal transduction and regulating cell cycle. Recent reports have shown that the active site cysteine residue, Cys215, can be reversibly oxidized to a cysteine sulfenic derivative (Denu and Tanner, 1998; Lee et al., 1998). We propose an additional modification that has implications for the in vivo regulation of protein tyrosine phosphatase 1B (PTP1B, EC 3.1.3.48): the glutathionylation of Cys215 to a mixed protein disulfide. Treatment of PTP1B with diamide and reduced glutathione or with only glutathione disulfide (GSSG) results in a modification detected by mass spectrometry in which the cysteine residues are oxidized to mixed disulfides with glutathione. The activity is recovered by the addition of dithiothreitol, presumably by reducing the cysteine disulfides. In addition, inactivated PTP1B is reactivated enzymatically by the glutathione-specific dethiolase enzyme thioltransferase (glutaredoxin), indicating that the inactivated form of the phosphatase is a glutathionyl mixed disulfide. The cysteine sulfenic derivative can easily oxidize to its irreversible sulfinic and sulfonic forms and hinder the regulatory efficiency if it is not converted to a more stable and reversible end product such as a glutathionyl derivative. Glutathionylation of the cysteine sulfenic derivative will prevent the enzyme from further oxidation to its irreversible forms, and constitutes an efficient regulatory mechanism.

Enhanced free radical generation of FALS-associated Cu,Zn-SOD mutants.

Familial amyotrophic lateral sclerosis (FALS) is an inherited disorder of motor neurons, which is associated with missense mutations in the Cu,Zn-superoxide dismutase (Cu,Zn-SOD) gene. Mice from the G93A transgenic line were reported to develop a syndrome of FALS. The fact that the symptoms occurred against a background of normal mouse Cu,Zn-SOD activity suggests that dominant, gain-of-function mutations in SOD play a role in the pathogenesis of FALS. We investigated the nature of this gain-of-function of FALS mutants. We have previously reported that Cu,Zn-SOD has the free radical-generating function in addition to normal dismutation activity. These two enzymic activities were compared by using mutants (G93A and A4V) and the wild-type Cu,Zn-SOD prepared by recombinant method. Our results showed that the wild-type, G93A, and A4V enzymes have identical dismutation activity. However, the free radical-generating function of the G93A and A4V mutants, as measured by the spin trapping and EPR method, is enhanced relative to that of the wild-type enzyme (wild type < G93A < A4V), particularly at lower H(2)O(2) concentrations. This is due to the decrease in the K(m) value for H(2)O(2), wild-type > G93A > A4V. The catalytic activity to generate free radicals is correlated to the clinical severity of the disorder induced by these mutant enzymes. Furthermore, we found that intact FALS mutants failed to enhance tyrosine nitration. Together our results indicate that the amyotrophic lateral sclerosis symptoms are not caused by the reduction of Cu,Zn-SOD dismutation activity with the mutant enzymes; rather, it is induced in part by enhancement of the free radical-generating function.

Antioxidant capacity of edible plants: extraction protocol and direct evaluation by cyclic voltammetry.

Reactive oxygen-derived species are produced in cells under physiological conditions and in response to stress. Among the various antioxidant systems responsible for protection against these species, the low-molecular-weight antioxidants (LMWA), such as ascorbate, play an important role. Cyclic voltammetry (CV) has been proposed as a tool for quantitation of the total antioxidant capacity of plasma. It has also been shown that biological oxidation potentials, as determined from the anodic current waves of the CV tracings, are specific characteristics of the various LMWA components, and that the amplitude of each wave can be used for quantitation of the specific component. The adaptation of CV for evaluation of the total antioxidant capacity of edible plants is demonstrated here. The area under the anodic current wave is proposed as a better indicator for the content of LMWA, compared with the amplitude. This distinction could prove valuable when more than a single molecule contributes toward a specific anodic wave and when the identities of the components of a wave are not known. Vegetables and fruits that are commonly consumed in the U.S. diet were used. They were extracted with either water, aqueous acetic acid (30%), or a mixture of water, acetic acid, and acetonitrile (40:30:30). The LMWA contents were evaluated by CV. In three to five steps the LMWAs were completely extracted from the edible foods, and their amounts were translated into equivalents of ascorbate.

Identification of the oxidation states of the active site cysteine in a recombinant protein tyrosine phosphatase by electrospray mass spectrometry using on-line desalting.

The oxidation state of the cysteine residue at the active site of human protein tyrosine phosphatase (PTP-1B) greatly affects its enzymatic activity. We wished to examine peroxide-treated preparations for modifications of this enzyme with electrospray mass spectrometry in order to determine the locations and oxidation states of the cysteines or other residues involved in the process. Since these reaction products contained large amounts of salts and buffers, they required desalting prior to analysis. Existing on- and off-line methods presented certain difficulties in handling and sample usage. Based on recent experience with direct syringe admission of sample, we developed a procedure as a simple, inexpensive alternative to full high-performance liquid chromatography systems that provides on-line desalting using only a few microL of sample. The method was applied to the analysis of oxidized PTP-1B preparations where conversion of cysteine 215 to both sulfinic and sulfonic acid residues was demonstrated.

Oxidation-reduction properties of methylglyoxal-modified protein in relation to free radical generation.

Oxidation-reduction properties of methylglyoxal-modified protein in relation to free radical generation were investigated. Glycation of bovine serum albumin by methylglyoxal generated the protein-bound free radical, probably the cation radical of the cross-linked Schiff base, as observed in the reaction of methylglyoxal with L-alanine (Yim, H.-S., Kang, S.-O., Hah, Y. C., Chock, P. B., and Yim, M. B. (1995) J. Biol. Chem. 270, 28228-28233) or with Nalpha-acetyl-L-lysine. The glycated bovine serum albumin showed increased electrophoretic mobility suggesting that the basic residues, such as lysine, were modified by methylglyoxal. The glycated protein reduced ferricytochrome c to ferrocytochrome c in the absence of oxygen or added metal ions. This reduction of cytochrome c was accompanied by a large increase in the amplitude of the electron paramagnetic resonance signal originated from the protein-bound free radical. In addition, the glycated protein catalyzed the oxidation of ascorbate in the presence of oxygen, whereas the protein free radical signal disappeared. These results indicate that glycation of protein generates active centers for catalyzing one-electron oxidation-reduction reactions. This active center, which exhibits enzyme-like characteristic, was suggested to be the cross-linked Schiff base/the cross-linked Schiff base radical cation of the protein. It mimics the characteristics of the metal-catalyzed oxidation system. The glycated bovine serum albumin cross-linked further to the cytochrome c in the absence of methylglyoxal. The cross-linked cytochrome c maintains its oxidation-reduction properties. These results together indicate that glycated proteins accumulated in vivo provide stable active sites for catalyzing the formation of free radicals.