Identification of Rack1, EF-Tu and Rhodanese as aging-related proteins in human colonic epithelium by proteomic analysis.

The aging process of human colonic epithelium involves a slow decline in physiological vigor and an increasing susceptibility to age-related diseases, especially, colon cancer, but the mechanisms still remain to be elucidated. To reveal the molecular bases of colonic epithelial aging, a proteomic approach was used to screen for differential proteins in the human normal colonic epithelial tissues from young and old people. As a result, 17 differential proteins were identified by two-dimensional electrophoresis and mass spectrometry, and the partial differential proteins were confirmed by immunohistochemistry. Rack1, EF-Tu and Rhodanese, three validated differential proteins, were further investigated for their role in the in vitro cell senescence. Western blot showed that the expression of all the three proteins was downregulated in the senescent NIH/3T3 cells induced by D-galactose as compared to the control cells. Furthermore, knockdown of Rack1 by siRNA could promote NIH/3T3 cell senescence. Taken together, our results suggest that Rack1, EF-Tu and Rhodanese are aging-related proteins in human colonic epithelium, and injury of mitochondrial function and decline of antioxidant capability are important reasons for the aging of human colonic epithelium.

Physicochemical and kinetic characteristics of rhodanese from the liver of African catfish Clarias gariepinus Burchell in Asejire lake.

Two forms of rhodanese were purified from the liver of Clarias gariepinus Burchell, designated catfish rhodanese I (cRHD I) and rhodanese II (cRHD II), by ion-exchange chromatography on a CM-Sepharose CL-6B column and gel filtration through a Sephadex G-75 column. The apparent molecular weight obtained for cRHD I and cRHD II was 34,500 +/- 707 and 36,800 +/- 283 Da, respectively. The subunit molecular weight determined by sodium dodecyl sulphate-polyacrylamide gel electrophoresis was 33,200 +/- 283 and 35,100 +/- 141 Da for cRHD I and cRHD II, respectively. Atomic absorption spectrophotometric analysis revealed that cRHD II contained a high level of iron (Fe), which presumably was responsible for the brownish colour of the preparation. In contrast, no Fe was identified in cRHD I, and its preparation was colourless. Further characterization of cRHD II gave true Michaelis-Menten constant (K(m)) values of 25.40 +/- 1.70 and 18.60 +/- 1.68 mM for KCN and Na(2)S(2)O(3), respectively, an optimum pH of 6.5 and an optimum temperature of 40 degrees C. The Arrhenius plot of the effects of temperature on the reaction rate consisted of two linear segments with a break occurring at 40 degrees C. The apparent activation energy values from these slopes were 7.3 and 72.9 kcal/mol. Inhibition studies on the cRHD II enzyme showed that the activity of the enzyme was not affected by Mn(2+), Co(2+), Sn(2+), Ni(2+) and NH(4) (+), but Zn(2+) inhibited the enzyme considerably.

Thiosulfate Oxidation and mixotrophic growth of Methylobacterium goesingense and Methylobacterium fujisawaense.

The mixotrophic growth with methanol plus thiosulfate was examined in nutrient-limited mixotrophic condition for Methylobacterium goesingense CBMB5 and Methylobacterium fujisawaense CBMB37. Thiosulfate oxidation increased the growth and protein yield in mixotrophic medium that contained 150 mM methanol and 20 mM sodium thiosulfate, at 144 h. Respirometric study revealed that thiosulfate was the most preferable reduced inorganic sulfur source, followed by sulfite and sulfur. M. goesingense CBMB5 and M. fujisawaense CBMB37 oxidized thiosulfate directly to sulfate, and intermediate products of thiosulfate oxidation such as polythionates, sulfite, and sulfur were not detected in spent medium and they did not yield positive amplification for tested soxB primers. Enzymes of thiosulfate oxidation such as rhodanese and sulfite oxidase activities were detected in cell-free extracts of M. goesingense CBMB5, and M. fujisawaense CBMB37, and thiosulfate oxidase (tetrathionate synthase) activity was not observed. It indicated that both the organisms use the "non-S4 intermediate" sulfur oxidation pathway for thiosulfate oxidation. It is concluded from this study that M. goesingense CBMB5, and M. fujisawaense CBMB37 exhibited mixotrophic metabolism in medium containing methanol plus thiosulfate and that thiosulfate oxidation and the presence of a "Paracoccus sulfur oxidation" (PSO) pathway in methylotrophic bacteria are species dependant.

Functional sulfurtransferase is associated with mitochondrial complex I from Yarrowia lipolytica, but is not required for assembly of its iron-sulfur clusters.

Here, we report that in the obligate aerobic yeast Yarrowia lipolytica, a protein exhibiting rhodanese (thiosulfate:cyanide sulfurtransferase) activity is associated with proton pumping NADH:ubiquinone oxidoreductase (complex I). Complex I is a key enzyme of the mitochondrial respiratory chain that contains eight iron-sulfur clusters. From a rhodanese deletion strain, we purified functional complex I that lacked the additional protein but was fully assembled and displayed no functional defects or changes in EPR signature. In contrast to previous suggestions, this indicated that the sulfurtransferase associated with Y. lipolytica complex I is not required for assembly of its iron-sulfur clusters.

Effect of sub-acute oral cyanide administration in rats: protective efficacy of alpha-ketoglutarate and sodium thiosulfate.

Chronic toxicity of cyanide in humans and animals has been previously described. Alpha-ketoglutarate (alpha-KG) and sodium thiosulfate (STS) are known to confer remarkable protection against acute cyanide poisoning in rodents. Their efficacy against sub-acute or chronic cyanide exposure is not known. The objective of the present study was to assess the sub-acute toxicity of potassium cyanide (KCN) in female rats following oral administration of 7.0 mg/kg (0.5 LD50) for 14 d. The effect of alpha-KG (oral; 1.0 g/kg) and/or STS (intraperitoneal, 1.0 g/kg) on cyanide toxicity was also evaluated. Various hematological and biochemical indices were determined after 7 d of treatment and additional parameters like organ-body weight index (OBI) and histology of brain, heart, lung, liver, kidney and spleen were performed after 14 and 21 d (recovery group) of cyanide exposure. Sub-acute exposure of KCN did not produce any significant change in body weight of the animals, OBI, hematology and the levels of blood urea, creatinine, aspartate aminotransferase, triiodothyronine (T3) and tetraiodothyronine (T4). The levels of temporal glutathione disulfide (GSSG) and hepatic malondialdehyde (MDA), reduced glutathione (GSH) and GSSG were unaffected. However, in KCN treated animals elevated levels of blood glucose and reduced levels of alanine aminotransferase were observed. Activities of cytochrome c oxidase in the brain and rhodanese in the liver were diminished. Reduced levels of GSH and enhanced levels of MDA in brain were observed. Increased levels of blood thiocyanate were observed in all the treatments of KCN. Additionally, KCN also produced various histological changes in the brain, heart, liver and kidney. Although, treatment of alpha-KG and STS alone significantly blunted the toxicity of KCN, concomitant use of both interventions afforded to maximum protection. This study indicates a promising role of alpha-KG and STS for the treatment of prolonged cyanide exposures.

Rhodanese activity: a simple and reliable taxonomic tool for gram-negative bacteria.

The thiosulphate: cyanide sulphurtransferase (rhodanese) test of Vandenbergh, Bawdon and Berk (1979) has been simplified and 2469 strains from a wide variety of sources representing different biochemical, serological or phage-pattern entities were tested. The percentages of rhondanese-producing strains were: Escherichia coli 98%, Shigella flexneri serovars 1-5%, X and Y 0%, other shigellae 73-100%, Yersinia spp. 0%, Salmonella subgenera I-IV 0%, Citrobacter freundi 16%, Klebsiella 37%, Enterobacter 4%, Hafnia alvei 61%, Proteus spp. 0%, Pseudomonas spp. 98-100%. Rhondanese production by S. flexneri serovar 6 supports the view that this group of bacteria should be removed from S. flexneri and placed in another species of Shigella.

Determination of rhodanese enzyme activity by capillary zone electrophoresis.

A new sensitive method has been developed for the determination of rhodanese activity. The enzymatic reactions were carried out directly in thermostatted autosampler vials and the formation of SCN- was monitored by sequential capillary zone electrophoretic runs. The determinations were performed in a 75-micron fused-silica capillary using 0.1 M beta-alanine-HCl (pH 3.50) as a background electrolyte, a separation voltage of 18 kV (negative polarity), a capillary temperature of 25 degrees C and direct detection at 200 nm. Short-end injection or long-end injection procedures were used for sample application. The method is rapid, able to be automated and requires only small amounts of sample and substrates, which is especially important in the case of highly toxic cyanide. The developed capillary electrophoretic method also has great potential for thiocyanate determinations in other applications.

The cyanide-metabolizing enzyme rhodanese in rat nasal respiratory and olfactory mucosa.

Hydrogen cyanide is a commonly occurring and highly toxic air pollutant. Inhalation of hydrogen cyanide would expose the nasal tissues to its toxic affects unless a detoxicating mechanism were available. Experiments with rat nasal tissues showed that the cyanide-metabolizing enzyme, rhodanese, is present in high concentrations, particularly in the olfactory region. The olfactory tissues had nearly 7-fold more rhodanese on a per mg mitochondrial protein basis than did the liver. These experiments show that nasal metabolism of cyanide may have an important influence on the toxicity of inhaled cyanide and cyanogenic materials.

Rhodanese distribution in porcine (Sus scrofa) tissues.

The enzyme rhodanese (thiosulfate/cyanide sulfurtransferase) is an ubiquitous enzyme and its activity is present in all living organisms from bacteria to man. Evidence has been accumulated to indicate that this enzyme plays a central role in cyanide detoxification. A comparison was made of rhodanese activity in different tissues of young male and adult male and female pig (Sus scrofa). The highest activity of rhodanese was in liver and kidney cortex of all animals. Among the remaining tissues examined, the kidney medulla and the stomach epithelium tended to have higher levels than other tissues, although this was not significant (P>0.05). The rhodanese activity of heart ventricle tissue of 6-month-old male animals was higher than 7-week-old male animals (P<0.05), and 6-month-old male animals had higher rhodanese activity in lung tissue, compared to 6-month-old female pigs (P<0.05). Medulla and spleen of younger male animals exhibited higher levels of activity (P<0.10) compared to older male pigs. The results of this study may indicate the involvement of rhodanese in cyanide detoxification in pig tissues, which have greater potential to be exposed to higher levels of cyanide.

An enzymatic lysis procedure for the assay of enzymes in Thiobacillus A2.

A micromethod is described for the production of lysed preparations of Thiobacillus A2 following treatment with lysozyme and EDTA. These may be used for the assay of intra-cellular enzymes including rhodanese, hexokinase, glucose 6-phosphate dehydrogenase and phosphoglucoseisomerase. The procedure is useful for assaying enzymes in samples too small to be treated by conventional mechanical methods, but gives comparable recoveries of enzyme activities.

Methods for in situ visualization and assay of sulfurtransferases.

The dansyl derivative 5-dimethylamino-1-naphthalene thiosulfonate (DANTS) can serve as a sulfane sulfur-donor substrate for several of the sulfurtransferases, the reaction being dependent on the acceptor substrates supplied. Enzymatic cleavage of the sulfur-sulfur bond of DANTS releases the intrinsic fluorescence of the molecule, with an emission maximum of 500-510 nm (excitation at 325 nm). This process permits selective visualization of active sulfurtransferase enzymes separated in nondenaturing polyacrylamide gels, even from impure preparations. This technique was used to locate rhodanese (thiosulfate: cyanide sulfurtransferase, EC 2.8.1.1), thiosulfate reductase (EC unassigned), and a recently isolated prokaryotic enzyme that has been called sulfane sulfurtransferase. In addition, a refinement of the thiosulfate reductase assay technique is reported.

The cyanide-metabolizing enzyme rhodanese in human nasal respiratory mucosa.

The cyanide-metabolizing enzyme rhodanese is present in rat nasal epithelium at high activity levels. Cyanide is a common environmental pollutant. It is both toxic and an odorant. The high rhodanese activity in rat nasal epithelium may provide a mechanism for detoxicating inhaled hydrogen cyanide and may also play a role in olfaction by limiting the concentrations of cyanide in the nasal epithelium. The objective of this study was to determine whether high levels of rhodanese activity are also present in human nasal epithelium. On a per milligram mitochondrial protein basis, the rhodanese in human nasal tissue exhibited both a lower affinity (higher Km) for cyanide and a lower maximum velocity (Vmax) for cyanide metabolism than did rhodanese from rat nasal tissue. As in human liver, the human nasal enzyme appeared to exhibit substrate activation by cyanide. Rhodanese activity in the maxilloturbinates of nonsmokers was statistically higher than in smokers although only three samples per group were available. The Vmax/Km ratios for rhodanese from the nasal tissue of nonsmokers were consistently greater, thus suggesting the possibility of higher rates of cyanide metabolism in nonsmokers than in smokers.

Chromogenic substrates for sulfurtransferases.

The azo dye 4-(dimethylamino)-4'-azobenzene (DAB) thiosulfonate anion can serve as a sulfur-donor substrate for rhodanese (thiosulfate: cyanide sulfurtransferase, EC 2.8.1.1) and for thiosulfate reductase (EC unassigned) with cyanide anion and GSH, respectively, as acceptor substrates. In either case, the dye product is DAB sulfinate, which differs substantially in light absorption at 500 nm. Moreover, DAB sulfinate can serve as a sulfur-acceptor substrate for rhodanese with either inorganic thiosulfate or a colorless thiosulfonate anion as donor, and this reaction provides a second chromogenic assay procedure.

The sulfurtransferases.

The sulfurtransferases are a group of proteins that catalyze the formation, interconversion and reactions of compounds containing sulfane sulfur atoms. Serum albumin has properties that implicate it as a major potential sulfur carrier/transferase. The relevance of the sulfane pool system as a whole to cyanide detoxication appears clear. The mechanisms of action of the various components at the molecular level are still under investigation.

Oxidation increases the proteolytic susceptibility of a localized region in rhodanese.

For the first time, the enzyme rhodanese has been proteolytically cleaved to give species that most likely correspond to individual domains. This indicates cleavage can occur in the interdomain tether. Further, the conditions for cleavage show that availability of the susceptible bond(s) depends on conformational changes triggered by oxidative inactivation. Rhodanese, without persulfide sulfur (E), was oxidized consequent to incubation with phenylglyoxal, NADH, or hydrogen peroxide. The oxidized enzyme (Eox) was probed using the proteolytic enzymes endoproteinase glutamate C (V8), trypsin, chymotrypsin, or subtilisin. The proteolytic susceptibility of Eox, formed using hydrogen peroxide, was compared with that of E and the form of the enzyme containing transferred sulfur, ES. ES was totally refractory to proteolysis, while E was only clipped to a small extent by trypsin or V8 and not at all by chymotrypsin or subtilisin. Eox was susceptible to proteolysis by all the proteases used, and, although there were some differences among the proteolytic patterns, there was always a band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis corresponding to Mr = 16,500. This was the only band observed in addition to the parent species (Mr = 33,000) when Eox was digested with chymotrypsin, and conservation of total protein was observed after digestion up to 90 min. No additional species were observable on silver staining, although there was some indication that the band at 16,500 might be a doublet. The results are consistent with the occurrence of a conformational change after oxidation that results in increased exposure and/or flexibility of the interdomain tether which contains residues that meet the specificity requirements of the proteases used.

Rhodanese isozymes in human tissues.

An investigation of a range of tissue homogenates by various electrophoretic methods, followed by staining for specific enzyme activity, has revealed a series of isozymes of human rhodanese. Polyacrylamide gel isoelectric focusing provided the most data and rhodanese activity was found in all of the tissues examined. The simplest isozyme pattern was found in red cell lysates; liver homogenates generated the most complex pattern which included the 'red cell' forms together with a set of more basic 'tissue' isozymes. Variation in isozyme patterns thought to be attributable to storage changes affecting reactive sulphydryl residues was observed in 'red cell' rhodanese but no genetic variants of either 'red cell' or 'tissue' rhodanese were encountered in a study of material from the European population. We conclude that 'red cell' and 'tissue' rhodanese are determined by separate genes but more than one locus may be concerned with the synthesis of the heterogeneous 'tissue' isozymes.

Histochemical localization of rhodanese activity in rat liver and skeletal muscle.

A previously described histochemical technique was applied to the localization of rhodanese (thiosulfate sulfurtransferase, EC 2.8.1.1) activity in rat skeletal muscle and liver. The physiological function of rhodanese is controversial, but it and other sulfurtransferases can catalyze the conversion of cyanide to the much less toxic thiocyanate. The volume of distribution of cyanide in human and dog is said to correspond roughly to the blood volume. Because of this and other observations, it was hypothesized that sulfurtransferase activity associated with the vascular endothelium on smooth muscle layers of blood vessels might play a role in cyanide detoxification. However, little enzyme activity as identified histochemically was associated with those sites in comparison with others examined. As expected, high activity was found in the liver and moderately high levels were present in skeletal muscle. In muscles sectioned longitudinally, points of rhodanese staining occurred in linear arrays along the lengths of the muscle fiber corresponding to the location of mitochondria within the fiber. The original technique called for incubation of tissue sections with both thiosulfate and cyanide. When thiosulfate was omitted, staining for rhodanese activity was still clearly identifiable in both liver and muscle sections with cyanide alone. In muscle sections the inclusion of both thiosulfate and cyanide resulted in a preferential staining of type I fibers presumably because of their higher content of mitochondria. Thus, this technique is a potential alternative to the NADH dehydrogenase stain for distinguishing between type I and type II muscle fibers. Incubation of tissue sections with only thiosulfate produced results that did not appear to differ from those obtained when both substrates were omitted. From these observations it may be inferred that the endogenous pool of sulfane-sulfur available to sulfurtransferases is larger than any alleged endogenous pool of cyanide. Although sulfurtransferase activity in muscle appeared to be lower than that in liver, the total body muscle mass is greater than the liver mass. Thus, these results support other evidence that skeletal muscle may make a significant contribution to total cyanide biotransformation in the absence of exogenously added thiosulfate.