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M K Cha

Publications and source records attributed to M K Cha.

At least 19 recordsLinked to original sources
PubMed2000

Activation of thiol-dependent antioxidant activity of human serum albumin by alkaline pH is due to the B-like conformational change.

Antioxidant activity of human serum albumin (HSA) increased steeply as the reaction mixture was shifted from neutral to alkaline pH. The antioxidant activity was also remarkably increased by Ca(2+) or a cationic detergent (cetyltrimethylammonium chloride). Carboxyl group modification of HSA resulted in about 40-fold increase of the antioxidant activity. The chemical modification study indicated that in addition to functional cysteine(s), cationic amino acid residues such as histidine, arginine and lysine appeared to involve in the antioxidant reaction. HSA also exhibited alkaline-pH dependent peroxidase activity to remove fatty acid hydroperoxide. At neutral pH, only two thiols of Cys-289 and free Cys-34 of HSA were modified by a thiol-specific modification reagent, 5-((((2-iodoacetyl)amino)ethy)amino)naphthalene-1-sulfonic acid (I14), regardless of the presence or absence of dithiothreitol (DTT), and the resultant antioxidant activity was not decreased, suggesting that Cys-289 and Cys-34 did not participate in the antioxidant reaction. At alkaline pH, I14 modified several additional HSA thiols in the presence, but did not in the absence of DTT. The antioxidant activity of the modified HSA was remarkably decreased to as much as 30% of the antioxidant activity given by the unmodified HSA in the absence of DTT. The HPLC pattern for tryptic peptides containing modified cysteine(s) derived from the I14-treated c-HSA (carboxyl group-modified HSA) at pH 7.0 with DTT was very similar to that of the I14-modified HSA at pH 8.0 with DTT. Taken together, these results suggest that activation of thiol-dependent antioxidant activity of HSA at alkaline pH is due to the conformational change favorable for the functional cysteine(s)-mediated catalysis.

Antioxidants↗
PubMed2000

Interaction of human thiol-specific antioxidant protein 1 with erythrocyte plasma membrane.

During the purification from human erythrocytes, human thiol-specific antioxidant protein 1 (hTSA1), one human member of the TSA/alkyl hydroperoxide reductase subunit C (AhpC) family, was fragmented to a molecular mass of 20 323.9300. The fragmented form, in contrast to the intact form, did not bind to the C-terminal peptide (Gln-185-Gln-197) antibody. On the basis of the molecular mass of the fragmented form, the cleavage site was calculated to be between Val-186 and Asp-187. The C-terminal region of hTSA1 appeared to be unnecessary for the antioxidant reaction. In addition to hTSA1, two isoenzymes (hORF06 and hTSA2) were detected in the soluble fraction, whereas only hTSA1 was detected in the membrane fraction. A membrane binding study shows that the intact form binds to erythrocyte plasma membrane but the fragment does not, which suggests that the deleted C-terminal legion (Asp-187-Gln-197) is required for the membrane binding. A model membrane study using phospholipid vesicle showed a strong association of hTSA1 with the phospholipid. Human TSA1 exhibited high catalytic activity for the reduction of the fatty acid hydroperoxide as indicated by K(m) and V(max) (89.9 microM for linoleic acid hydroperoxide, 28.64 micromol(-1) min(-1) mg(-1), respectively). In this paper, we are making the first report of the involvement of the C-terminal region of hTSA1 in membrane binding as evidence supporting the existence of the membrane-associated forms in the erythrocyte. On the basis of our observations, we suggest that hTSA1 can act as a very effective antioxidant to remove oxidative stresses not only in matrix as a free form but also in the membrane surface of red blood cells (RBC) as a membrane-associated form.

Antioxidants↗
PubMed2000

Distinct physiological functions of thiol peroxidase isoenzymes in Saccharomyces cerevisiae.

A new type of peroxidase ("thiol peroxidase"; TPx) having cysteine as the primary site of catalysis has been discovered from prokaryotes to eukaryotes. In addition to two yeast TPx isoforms (TSA I and TSA II/AHPC1) previously described, three additional TPx homologues were identified by analysis of the open reading frame data base for Saccharomyces cerevisiae. Three novel isoforms showed a distinct thiol peroxidase activity supported by thioredoxin, and appeared to be distinctively localized in cytoplasm, mitochondria, and nucleus. Each isoform was named after its subcellular localization such as cytoplasmic TPx I (cTPx I or TSA I), cTPx II, cTPx III (TSA II/AHPC1), mitochondrial TPx (mTPx), and nuclear TPx (nTPx). Their transcriptional activities suggest that cTPx I and cTPx III are the most predominant isoforms among the five type isoforms. Transcriptional activities of TPx isoenzymes during yeast life span were quite different from each other. Unlike other TPx null mutants, cTPx I null mutant was hypersensitive to various oxidants except for 4-nitroquinoline N-oxide. The null mutant was more resistant toward 4-nitroquinoline N-oxide and acidic culture than its wild type. The severe growth retardation of cTPx II mutant resulted in accumulation of G(1)-phased cells. Based on kinetic properties of five isoforms, their subcellular localizations, and distinct physiology of each null mutant, we discussed the physiological functions of five types of TPx isoenzymes in yeast throughout the full growth cycle.

Amino Acid Sequence↗
PubMed2000

Thioredoxin-dependent hydroperoxide peroxidase activity of bacterioferritin comigratory protein (BCP) as a new member of the thiol-specific antioxidant protein (TSA)/Alkyl hydroperoxide peroxidase C (AhpC) family.

Escherichia coli bacterioferritin comigratory protein (BCP), a putative bacterial member of the TSA/AhpC family, was characterized as a thiol peroxidase. BCP showed a thioredoxin-dependent thiol peroxidase activity. BCP preferentially reduced linoleic acid hydroperoxide rather than H(2)O(2) and t-butyl hydroperoxide with the use of thioredoxin as an in vivo immediate electron donor. The value of V(max)/K(m) of BCP for linoleic acid hydroperoxide was calculated to be 5-fold higher than that for H(2)O(2), implying that BCP has a selective capability to reduce linoleic acid hydroperoxide. Replacement of Cys-45 with serine resulted in the complete loss of thiol peroxidase activity, suggesting that BCP is a new bacterial member of TSA/AhpC family having a conserved cysteine as the primary site of catalysis. BCP exists as a monomer, and its functional Cys-45 appeared to exist as cysteine sulfenic acid. The expression level of BCP gradually elevated during exponential growth until mid-log phase growth, beyond which the expression level was decreased. BCP was induced 3-fold by the oxidative stress given by changing the growth conditions from the anaerobic to aerobic culture. Bcp null mutant grew more slowly than its wild type in aerobic culture and showed the hypersensitivity toward various oxidants such as H(2)O(2), t-butyl hydroperoxide, and linoleic acid hydroperoxide. The peroxide hypersensitivity of the null mutant could be complemented by the expression of bcp gene. Taken together, these data suggest that BCP is a new member of thioredoxin-dependent TSA/AhpC family, acting as a general hydroperoxide peroxidase.

Amino Acid Sequence↗
PubMed2000

Effects of conventional and new peritoneal dialysis solutions on human peritoneal mesothelial cell viability and proliferation.

OBJECTIVE: To investigate the biocompatibility of "new" peritoneal dialysis (PD) solutions with bicarbonate/lactate buffer, non glucose osmotic agents (icodextrin or amino acids), neutral pH, and low levels of glucose degradation products (GDPs). DESIGN: Using M199 culture medium as a control, we compared conventional and new PD solutions with respect to their effects on the viability of human peritoneal mesothelial cells (HPMCs) [using lactate dehydrogenase (LDH) release], on DNA damage in HPMCs [using single-cell gel electrophoresis (Comet assay)], and on HPMC proliferation (using [3H]-thymidine incorporation). The experiments were performed after cell growth was synchronized by incubation with serum-free media for 24 hours. The PD solutions tested included commercial 1.5% glucose and 4.25% glucose solutions with 40 mmol/L lactate (D 1.5 and D 4.25, respectively), 7.5% icodextrin (E), 1.1% amino acid (N), 1.5% glucose solution in a triple-chambered bag (Bio 1.5), 1.5% glucose solution in a dual-chambered bag with neutral pH (Bal 1.5), and 1.5% glucose and 4.25% glucose solution containing 25 mmol/L bicarbonate and 15 mmol/L lactate (P 1.5 and P 4.25, respectively). RESULTS: When HPMCs were continuously exposed to undiluted PD solutions, D 1.5, D 4.25, P 4.25, and E increased LDH release by more than 60% at 24 hours. All PD solutions tested increased LDH release by more than 75% at 96 hours. With 2-fold diluted PD solutions, only D 4.25 significantly increased LDH release at 96 hours, though not at 24 hours. When cells were exposed to undiluted PD solutions for 60 min and allowed to recover in M199 for up to 96 hours, LDH release was significantly higher at 24-96 hours in E (55%-69%) and D 1.5 (48%-72%) as compared with control [M199 (18%)]. Release of LDH was significantly lower with PD solutions containing lower levels of GDPs than those in D 1.5, suggesting that GDPs may have a role in cell viability. The D solutions (D 1.5 and D 4.25) and E solution also induced significant DNA damage. Both LDH release and DNA damage by D and E were significantly attenuated by adjusting the solution pH to 7.4, suggesting that low pH may be implicated in PD solution-induced DNA damage and cell death. When diluted 2-fold, D 1.5, D 4.25, and P 4.25 decreased [3H]-thymidine incorporation to 43%, 34%, and 41% of control, respectively, at 24 hours and to 45%, 26%, and 35% of control, respectively, at 96 hours. When cells were exposed to undiluted PD solutions for 5 minutes and allowed to recover in M199 for up to 96 hours, D1.5 and P 4.25--but not D 4.25--significantly inhibited cell proliferation at 24 hours. This effect was sustained up to 96 hours. CONCLUSIONS: The present in vitro data demonstrate that PD solutions with low pH, or high levels of GDPs, or both, promote HPMC death and DNA damage, and that PD solutions with high osmolality inhibit cell proliferation. Solutions with neutral pH, amino acids, and "low GDPs" appear to be more biocompatible than conventional PD solutions. These results require confirmation in in vivo animal and clinical studies.

Cell Division↗
PubMed1999

Ceruloplasmin has a distinct active site for the catalyzing glutathione-dependent reduction of alkyl hydroperoxide.

Ceruloplasmin, a blue multi-copper alpha(2)-glycoprotein found in the plasma of all vertebrates, is capable of oxidizing aromatic amines and ferrous iron. Here, we report that human ceruloplasmin exhibits an alkyl hydroperoxide peroxidase activity, which is independent of the oxidase activity. The site-specific modification of the sulfhydryl of cysteine at position 699 in ceruloplasmin completely abolished the antioxidant activity, suggesting that ceruloplasmin is a peroxidase with a cysteinyl thiol as a functional nucleophile. The crystal structure of human ceruloplasmin reveals that the domain containing Cys-699 is apart from the multi-copper complex domains. Taken together, these data suggest that ceruloplasmin has a distinct active site for a glutathione-linked peroxidase activity apart from the copper complex site exerting ferroxidase activity.

Antioxidants↗
PubMed1998

Effect of prolonged subcutaneous implantation of peritoneal catheter on peritonitis rate during CAPD: a prospective randomized study.

We conducted a prospective randomized controlled study to confirm our earlier observation that prolonged subcutaneous implantation of peritoneal catheter reduced peritonitis rate when compared to retrospective data from patients with catheters placed by conventional access technique. A total of 60 patients were randomized into two groups: 30 patients had catheters left implanted subcutaneously for 6 weeks (I) and the other 30 patients had catheters inserted by conventional technique and had 6 weeks of break-in period (C). Subgroups of 15 patients each with new and conventional techniques used Y-connector (IY, CY) and remaining patients used standard spikes (IS, CS). Mean age was 47.7 years (range 16-71); 61.0% were male and 44.1% diabetics. Peritonitis, exit site infection, simultaneous peritonitis and exit site infection, and complication related to Staphylococcus or Pseudomonas infections were observed for up to 2 years in each patient after initiation of bag exchange or until termination of CAPD by transfer to hemodialysis or by death. Total duration of observation was 493.2 patient-months for new access technique and 409.6 patient-months for conventional technique. Patients in IY group had the lowest incidence of peritonitis (1/14.9 patient-months) and exit site infection (1/16.8 patient-months) among four subgroups. Peritonitis rate in IY was significantly lower compared to CY or CS. The total peritonitis-free period in those patients who did not experience peritonitis during the observation period was also significantly longer in IY (120 patient-months) than in CY (26 patient-months), IS (10.6 patient-months), or CS (10.4 patient-months). Simultaneous peritonitis and exit site infection was observed in none of IY group but 3 episodes in CY, 4 episodes in IS, and 3 episodes in CS. The rates of complications related to Staphylococcus aureus and Pseudomonas infections were also significantly lower in IY than in CY, IS, or CS. Technique survival did not differ between the two groups. The present results confirm our previous observation that the new access technique reduces the incidence of peritonitis probably by reducing infection via periluminal route. The Y-connector system further reduces peritonitis rate by reducing infection via intraluminal route.

Adolescent↗
PubMed1996

Glutathione-linked thiol peroxidase activity of human serum albumin: a possible antioxidant role of serum albumin in blood plasma.

A 65-kDa molecular mass of thiol-specific antioxidant protein was purified from human plasma and identified as human serum albumin (HSA) by the analysis of amino-terminal amino acid sequence. This protein exhibited the preventive effects against the inactivation of glutamine synthetase activity and the peroxidation of lipid by a metal-catalyzed oxidation system. These antioxidant activities were supported by a thiol-reducing equivalent such as DTT and reduced glutathione. The thiol-specific antioxidant activity of HSA was greatly activated by halide ion, especially by chloride ion. HSA showed a significant capability to destroy H2O2 in the presence of reduced glutathione, resulting in the production of oxidized glutathione. Both the preventive activity against the glutamine synthetase inactivation and the peroxidase activity were completely abolished by the reactions of HSA with N-ethylmaleimide and iodoacetate, chemical modification agents for sulfhydryl of protein, only in the presence of thiol-reducing equivalent such as DTT. These results suggest that serum albumin acts as a major and predominate antioxidant exerting a glutathione-linked thiol peroxidase activity which removes reactive oxygen species such as H2O2 within blood plasma.

Amino Acid Sequence↗
PubMed1996

Identification of promoter in the 5'-flanking region of the E. coli thioredoxin-linked thiol peroxidase gene: evidence for the existence of oxygen-related transcriptional regulatory protein.

E. coli thiol peroxidase (Tpx) linked to the thioredoxin as an in vivo thiol regenerating system acts as an antioxidant enzyme removing peroxides and H2O2. In order to elucidate the mechanism regulating tpx gene expression in E. coli in response to oxygen stress, we made 5' progressive deletions of upstream region from tpx gene, and fused to lacZ gene. LacZ activity was increased 6-fold by oxygen stress and inverted repeat sequence located between -47 and -33 nt was proven to be essential for the oxygen response of tpx promoter. Primer extension experiment and analysis of upstream sequence revealed transcription start point, -10, and -35 regions, which are in good agreements with the consensus sequences recognized by E sigma 70. Northern hybridization showed that expression of tpx gene is regulated at the transcriptional level. DNA binding assays using inverted repeat sequence including -35 region provides preliminary evidence that expression of tpx requires additional transcriptional factor in response to oxygen stress.

Amino Acid Sequence↗
PubMed1996

Mutation and Mutagenesis of thiol peroxidase of Escherichia coli and a new type of thiol peroxidase family.

A novel thioredoxin-linked thiol peroxidase (Px) from Escherichia coli has been reported previously (M. K. Cha, H. K. Kim, and I. H. Kim, J. Biol. Chem. 270:28635-28641, 1995). In an attempt to perform physiological and biochemical characterizations of the thiol Px, a thiol Px null (tpx) mutant and a functional-residue mutant of thiol Px were produced. The tpx mutant was viable in aerobic culture but grew more slowly than the wild-type cells. The difference in growth rate became more pronounced when oxidative-stress-inducing reagents, such as peroxides and paraquat, were added to the cultures. The viability of the individual tpx mutant under oxidative stress was much lower than that of wild-type cells. tpx mutants growing aerobically respond to paraquat with a sixfold greater induction of Mn-superoxide dismutase than that of the wild-type cells. The deduced amino acid sequence of the thiol Px was found to be from 42 to 72% identical to the sequences of proteins from Haemophilus influenzae (ToxR regulon), Vibrio cholerae (ToxR regulon), and three kinds of streptococci (coaggregation-mediating adhesins), suggesting that they all belong to a new thiol Px family. Alignment of the amino acid sequences of the thiol Px family members showed that one cysteine, which corresponds to Cys-94 in E. coli thiol Px, is perfectly conserved. The substitution of serine for this cysteine residue resulted in complete loss of Px activity. These results suggest that the members of the thiol Px family, including E. coli thiol Px, have a functional cysteine residue and function in vivo as peroxidases.

Amino Acid Sequence↗
PubMed1996

Dialysis in patients with diabetic nephropathy: CAPD versus hemodialysis.

Diabetic nephropathy has emerged as a major cause of ESRD over the past decade, being the most prevalent cause of ESRD requiring dialysis in North America (United States and Canada) and the second highest in the incidence rate in Europe, Japan, Korea, Australia, and New Zealand. A greater proportion of older patients and of patients with diabetic nephropathy and other comorbid conditions has been treated with CAPD. Despite the preferential use of CAPD to treat a high-risk group of patients, the overall and/or selection-adjusted mortality was similar between HD and CAPD groups. Among diabetic patients, selection-adjusted mortality was similar between HD and CAPD or lower in CAPD than in HD, the difference being greatest among younger patients and significant through the age of 52, or higher in CAPD than in HD with higher risk of death for older diabetics (age > or = 50 years), but with similar risk among younger diabetics (age < 50 years). Technique survival was also variably reported as similar between HD and CAPD, lower, or higher with CAPD compared to HD. Diabetic CAPD patients had more hospital admissions and more days in the hospital and higher withdrawal rates from dialysis compared to diabetic HD patients. These disparate results of patient and technique survival between HD and CAPD in diabetic patients may have resulted from patient selection criteria with different comorbid conditions on entrance to dialysis, quantity of dialysis, and other unrecognized factors. Prospective randomized studies are needed to assign a cause-and-effect relationship between the choice of dialysis modality and patient and technique survival among patients with diabetes mellitus as well as with all other diagnostic categories.

Diabetic Nephropathies↗
PubMed1995

Thioredoxin-linked peroxidase from human red blood cell: evidence for the existence of thioredoxin and thioredoxin reductase in human red blood cell.

A thiol-dependent antioxidant protein (HRPRP) was previously reported as a predominant antioxidant protein in human red blood cell (RBC). The analysis of amino acid sequence of HRPRP with those of human PRP-like gene products indicates that HRPRP is identical to brain PRP (HPRP). This protein act as a peroxidase linked to thioredoxin (Trx)/thioredoxin reductase (TR). Until now, there was no evidence for Trx/TR system in RBC. The existence of the Trx/TR system in RBC was immunologically determined. A 58-kDa protein showing TR activity was partially purified from human RBC and characterized. Our results reveal that HRPRP act as a new type of peroxidase supported by Trx/TR system in human RBC.

Amino Acid Sequence↗
PubMed1995

Thioredoxin-linked "thiol peroxidase" from periplasmic space of Escherichia coli.

Three different molecular masses (24, 22, and 20 kDa) of antioxidant proteins were purified in Escherichia coli. These proteins exhibited the preventive effects against the inactivation of glutamine synthetase activity and the cleavage of DNA by a metal-catalyzed oxidation system capable of generating reactive oxygen species. Their antioxidant activities were supported by a thiol-reducing equivalent such as dithiothreitol. Analysis of the amino-terminal amino acid sequences and the immunoblots between 24- and 22-kDa proteins indicates that the 24-kDa protein is an intact form of the 22-kDa protein that was previously identified 22-kDa subunit (AhpC) of E. coli alkyl hydroperoxide reductase (AhpC/AhpF). We isolated and sequenced an E. coli genomic DNA fragment that encodes 20-kDa protein. Comparison of the deduced amino acid sequence of the 20-kDa protein with that of AhpC revealed no sequence homology. A search of a data bank showed that the 20-kDa protein is a new type of antioxidant enzyme. The synthesis of this novel 20-kDa protein was increased in response to oxygen stress during growth. The 20-kDa protein resides mainly in the periplasmic space of E. coli, whereas the 24-kDa AhpC resides mainly in the matrix. The 20-kDa protein was functionally linked to the thioredoxin as an in vivo thiol-regenerating system and exerted a peroxidase activity. This 20-kDa protein is thus named "thiol peroxidase," which could act as an antioxidant enzyme removing peroxides or H2O2 within the catalase- and peroxidase-deficient periplasmic space of E. coli.

Amino Acid Sequence↗