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61.
Eyal O Jadoun J Bitler A Skutelski E Sela S 《FEMS immunology and medical microbiology》2003,38(3):205-213
Streptococcus pyogenes utilizes multiple mechanisms for adherence to and internalization by epithelial cells. One of the molecules suggested of being involved in adherence and internalization is the M protein. Although strains of the M3 serotype form the second largest group isolated from patients with severe invasive diseases and fatal infections, not much information is known regarding the interactions of M3 protein with mammalian cells. In this study we have constructed an emm3 mutant of an invasive M3 serotype (SP268), and demonstrated that the M3 protein is involved in both adherence to and internalization by HEp-2 cells. Fibronectin promoted both adherence and internalization of SP268 in an M3-independent pathway. Utilizing speB and speB/emm3 double mutants, it was found that M3 protein is not essential for the maturation of SpeB, as was reported for the M1 protein. Increased internalization efficiency observed in both the speB and emm3/speB mutants suggested that inhibition of S. pyogenes internalization by SpeB is not related to the presence of an intact M3 protein. Thus, other proteins in SP268, which serve as targets for SpeB activity, have a prominent role in the internalization process. 相似文献
62.
Summary. Alterations in hepatic metabolism of S-amino acids were monitored over one week in male rats treated with a single dose of
ethanol (3 g/kg, ip). Methionine and S-adenosylhomocysteine concentrations were increased rapidly, but S-adenosylmethionine,
cysteine, and glutathione (GSH) decreased following ethanol administration. Activities of methionine adenosyltransferase,
cystathionine γ-lyase and cystathionine β-synthase were all inhibited. γ-Glutamylcysteine synthetase activity was increased from t = 8 hr, but GSH level did not return to control for 24 hr. Hepatic
hypotaurine and taurine levels were elevated immediately, but reduced below control in 18 hr. Changes in serum and urinary
taurine levels were consistent with results observed in liver. Cysteine dioxygenase activity was increased rapidly, but declined
from t = 24 hr. The results show that a single dose of ethanol induces profound changes in hepatic S-amino acid metabolism,
some of which persist for several days. Ethanol not only inhibits the cysteine synthesis but suppresses the cysteine availability
further by enhancing its irreversible catabolism to taurine, which would play a significant role in the depletion of hepatic
GSH.
Received April 26, 2002 Accepted June 12, 2002 Published online October 14, 2002
Authors' address: Young C. Kim, Ph.D., Professor of Toxicology, College of Pharmacy, Seoul National University, San 56-1 Shinrim-Dong, Kwanak-Ku,
Seoul, Korea, Fax: +82-2-872-1795, E-mail: youckim@snu.ac.kr
Abbreviations: CβS, cystathionine β-synthase; CDC, cysteine sulfinate decarboxylase; CDO, cysteine dioxygenase; CγL, cystathionine γ-lyase; GCS, γ-Glutamylcysteine synthetase; GSH, glutathione; MAT, methionine adenosyltransferase; SAH, S-adenosylhomocysteine; SAM, S-adenosylmethionine. 相似文献
63.
A previously unidentified extension of an open reading frame from the genomic DNA of Japonica rice (Oryza sativa L.) encoding oryzacystatin-I (OC-I; access. M29259, protein ID AAA33912.1) has been identified as a 5′ gene segment coding for the OC-I signal peptide. The signal peptide appears to direct a pre-protein (SPOC-I; Accession No. AF164378) to the endoplasmic reticulum,
where it is processed into the mature form of OC-I. The start codon of SPOC-I begins 114 bp upstream from that previously published for OC-I. A putative proteolytic site, which may yield a mature OC-I approximately 12 residues larger than previously described, has
been identified within SPOC-I between Ala-26 and Glu-27. The signal peptide sequence was amplified by polymerase chain reaction
using genomic DNA from O. sativa seedlings and ligated to the 5′ end of the truncated OC-I gene at the endogenous SalI site. Partially purified protein extracts from Escherichia coli expressing SPOC-I reacted with polyclonal antibodies raised against OC-I and revealed a protein of the expected molecular weight (15,355 Da).
In-vitro translation of SPOC-I in the presence of microsomal membranes yielded a processed product approximately 2.7 kDa smaller than the pre-protein. Nicotiana tabacum L. cv. Xanthi plants independently transformed with the SPOC-I gene processed SPOC-I and accumulated the mature form of OC-I (approximately 12.6 kDa), which co-migrated with natural, mature
OC-I extracted from rice seed when separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.
Received: 29 July 1999 / Accepted: 25 August 1999 相似文献
64.
Properties of cysteinyl residues in the vesicular acetylcholine transporter (VAChT) of synaptic vesicles isolated from Torpedo californica were probed. Cysteine-specific reagents of different size and polarity were used and the effects on [3H]vesamicol binding determined. The vesamicol dissociation constant increased 1,000-fold after reaction with p-chloromercuriphenylsulfonate or phenylmercury acetate, but only severalfold after reaction with relatively small methylmercury chloride or methylmethanethiosulfonate (MMTS). Methylmercury chloride, but not MMTS, protected binding from phenylmercury acetate. Thus, two classes of cysteines react to affect vesamicol binding. Class 1 reacts with only organomercurials, and class 2 reacts with both organomercurials and MMTS. Quantitative analysis of the competition between p-chloromercuriphenylsulfonate and VAChT ligands was possible after defining second-order reaction conditions. The results indicate that each cysteinyl class probably contains a single residue. Acetylcholine protects cysteine 1, but apparently does not protect cysteine 2. Vesamicol, which binds to a different site than acetylcholine does, apparently protects both cysteines, suggesting that it induces a conformational change. The relatively large reagent glutathione removes a substituent from cysteine 1, but not cysteine 2, suggesting that cysteine 2 is deeper in the transporter than cysteine 1 is. The complete sequence of T. californica VAChT is given, and possible identities of cysteines 1 and 2 are discussed. 相似文献
65.
Kidney bean (Phaseolus vulgaris L.) ornithine carbamoyltransferase (OCT; EC 2.1.3.3) was purified to homogeneity from leaf homogenates in a single-step procedure,
using δ-N-(phosphonoacetyl)-l-ornithine-Sepharose 6B affinity chromatography. The 8540-fold-purified OCT exhibited a specific activity of 526 micromoles
citrulline per minute per milligram of protein at 35 °C and pH 8.0. The enzyme represents approximately 0.01% of the total
soluble protein in the leaf. The molecular mass of the native enzyme was approximately 109 kDa as estimated by Sephacryl S-200
gel filtration chromatography. The purified protein ran as a single band of molecular mass 36 kDa when subjected to sodium
dodecyl sulfate-polyacrylamide gel electrophoresis and at a single isoelectric point of 6.6 when subjected to denaturing isoelectric
focusing. These results suggest that the enzyme is a trimer of identical subunits. Among the tested amino acids, l-cysteine and S-carbamoyl-l-cysteine were the most effective inhibitors of the enzyme. The OCT of kidney bean showed a very low activity towards canaline.
The OCTs of canavanine-deficient plants have very low canaline-dependent activities, but the OCTs of canavanine-containing
plants showed high canaline-dependent activities. It was assumed that the substrate specificity of this enzyme determines
the canavanine synthetic activity of the urea cycle.
Received: 22 July 1997 / Accepted: 4 December 1997 相似文献
66.
The field of redox proteomics focuses to a large extent on analyzing cysteine oxidation in proteins under different experimental conditions and states of diseases. The identification and localization of oxidized cysteines within the cellular milieu is critical for understanding the redox regulation of proteins under physiological and pathophysiological conditions, and it will in turn provide important information that are potentially useful for the development of novel strategies in the treatment and prevention of diseases associated with oxidative stress. Antioxidant enzymes that catalyze oxidation/reduction processes are able to serve as redox biomarkers in various human diseases, and they are key regulators controlling the redox state of functional proteins. Redox regulators with antioxidant properties related to active mediators, cellular organelles, and the surrounding environments are all connected within a network and are involved in diseases related to redox imbalance including cancer, ischemia/reperfusion injury, neurodegenerative diseases, as well as normal aging. In this review, we will briefly look at the selected aspects of oxidative thiol modification in antioxidant enzymes and thiol oxidation in proteins affected by redox control of antioxidant enzymes and their relation to disease. [BMB Reports 2015; 48(4): 200-208] 相似文献
67.
《FEBS letters》2014,588(23):4382-4389
rCNT2 is a purine-preferring concentrative nucleoside transporter implicated in the regulation of extracellular adenosine levels and purinergic signaling. This study addressed the analysis of the CNT2 C-terminus tail as a domain likely to be implicated in transporter sorting. The topological mapping of this segment revealed that Cys615 and Cys649 are important residues for the proper trafficking of CNT2 to the plasma membrane. The inhibition of protein disulfide isomerase (PDI) and ER glycosidase I and II impaired rCNT2 trafficking to the cell surface, similarly to Cys615 and Cys649 mutants. The present work suggests these two cysteine residues are relevant for the proper sorting of the transporter and its functional performance. 相似文献
68.
Lucero A. Ramón‐Luing Francisco J. Rendón‐Gandarilla Rosa E. Cárdenas‐Guerra Norma A. Rodríguez‐Cabrera Jaime Ortega‐López Leticia Avila‐González Claudia Angel‐Ortiz Carmen N. Herrera‐Sánchez Manuela Mendoza‐García Rossana Arroyo 《Proteomics》2010,10(3):435-444
Trichomonas vaginalis, a sexually transmitted parasite, has many cysteine proteinases (CPs); some are involved in trichomonal pathogenesis, express during infection, and antibodies against CPs have been detected in patient sera. The goal of this study was to identify the antigenic proteinases of T. vaginalis as potential biomarkers for trichomonosis. The proteases detected when T. vaginalis protein extracts are incubated without protease inhibitors, the trichomonad‐active degradome, and the immunoproteome were obtained by using 2‐DE, 2‐D‐zymograms, 2‐D‐Western blot (WB) assays with trichomonosis patient sera, and MS analysis. Forty‐nine silver‐stained spots were detected in the region of 200–21 kDa of parasite protease‐resistant extracts. A similar proteolytic pattern was observed in the 2‐D zymograms. Nine CPs were identified in the 30 kDa region (TvCP1, TvCP2, TvCP3, TvCP4, TvCP4‐like, TvCP12, TvCPT, TvLEGU‐1, and another legumain‐like CP). The major reactive spots to T. vaginalis‐positive patient sera by 2‐D‐WB corresponded to four papain‐like (TvCP2, TvCP4, TvCP4‐like, TvCPT), and one legumain‐like (TvLEGU‐1) CPs. The genes of TvCP4, TvCPT, and TvLEGU‐1 were cloned, sequenced, and expressed in Escherichia coli. Purified recombinant CPs were recognized by culture‐positive patient sera in 1‐D‐WB assays. These data show that some CPs could be potential biomarkers for serodiagnosis of trichomonosis. 相似文献
69.
CopA, a thermophilic ATPase from Archaeoglobus fulgidus, drives the outward movement of Cu+ across the cell membrane. Millimolar concentration of Cys dramatically increases (≅ 800%) the activity of CopA and other PIB-type ATPases (Escherichia coli ZntA and Arabidopsis thaliana HMA2). The high affinity of CopA for metal (≅ 1 μM) together with the low Cu+-Cys KD (< 10− 10M) suggested a multifaceted interaction of Cys with CopA, perhaps acting as a substitute for the Cu+ chaperone protein present in vivo. To explain the activation by the amino acid and further understand the mechanism of metal delivery to transport ATPases, Cys effects on the turnover and partial reactions of CopA were studied. 2-20 mM Cys accelerates enzyme turnover with little effect on CopA affinity for Cu+, suggesting a metal independent activation. Furthermore, Cys activates the p-nitrophenyl phosphatase activity of CopA, even though this activity is metal independent. Cys accelerates enzyme phosphorylation and the forward dephosphorylation rates yielding higher steady state phosphoenzyme levels. The faster dephosphorylation would explain the higher enzyme turnover in the presence of Cys. The amino acid has no significant effect on low affinity ATP Km suggesting no changes in the E1 ↔ E2 equilibrium. Characterization of Cu+ transport into sealed vesicles indicates that Cys acts on the cytoplasmic side of the enzyme. However, the Cys activation of truncated CopA lacking the N-terminal metal binding domain (N-MBD) indicates that activation by Cys is independent of the regulatory N-MBD. These results suggest that Cys is a non-essential activator of CopA, interacting with the cytoplasmic side of the enzyme while this is in an E1 form. Interestingly, these effects also point out that Cu+ can reach the cytoplasmic opening of the access path into the transmembrane transport sites either as a free metal or a Cu+-Cys complex. 相似文献
70.
Cloning and functional characterization of an O-acetylserine(thiol)lyase-encoding gene in wild soybean (Glycine soja) 总被引:2,自引:0,他引:2
The terminal step of soybean cysteine synthesis is catalyzed by O-acetylserine(thiol)lyase (OAS-TL, EC 2.5.1.47). In this study, we isolated and characterized an OAS-TL gene from a wild soybean material (designated as GsOAS-TL1). GsOAS-TL1 cDNA sequence showed strict conservation at both nucleotide and amino acid levels compared with that from cultivated soybean.
Genomic structure analysis of GsOAS-TL1 indicated that it contained 10 exons and 9 introns in the coding region with conserved exon sizes and intron locations compared
with Arabidopsis thaliana
OAS-TL-like genes. Among the complete GsOAS-TL1 cDNA and three part-deletion fragments, only expression of the full-length cDNA could rescue the NK3 cys−
Escherichia coli auxotroph, which was coherent with the assayed enzyme activity of purified fusion proteins. For RT-PCR analysis in different
wild soybean tissues, GsOAS-TL1 showed lower expression in roots and developing seeds, whereas total OAS-TL activity of corresponding tissues showed significantly
higher level in seeds than other tissues. To our knowledge, this is the first report on cloning and characterization of an
OAS-TL gene from wild soybean. Our results are informative to further elucidate the function and evolution of OAS-TL in soybean. 相似文献