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.     

Comparative studies on the distribution of rhodanese in different tissues of domestic animals.

1. The activity of rhodanese in different tissues of some domestic animals was measured. 2. Rhodanese was present in all tissues studied. 3. The activity of rhodanese in most tissues of sheep was higher than other animals studied. 4. In sheep and cattle the epithelium of rumen, omasum and reticulum were the richest sources of rhodanese. Significant activity of rhodanese was also present in liver and kidney. 5. In camel the liver contained the highest level of rhodanese followed by lung and rumen epithelium. Camel liver contained a third of the activity of sheep liver. 6. Equine liver had a third of the activity of sheep liver. Other tissues showed low levels of rhodanese activity. 7. Dog liver contained only 4% of the activity of sheep liver. In this animal, brain was the richest source of rhodanese. 8. The results are discussed in terms of efficacy of different tissues of animals in cyanide detoxification.     

Distribution of rhodanese in different parts of the urogenital systems of sheep at pre- and post-natal stages

The enzyme rhodanese (thiosulfate:cyanide sulfurtransferase) is a ubiquitous enzyme present in all living organisms, from bacteria to humans and plays a central role in cyanide detoxification. The purpose of this investigation is to determine and compare rhodanese activity in different parts of urogenital systems of male and female sheep fetuses at 2.5, 3, 3.5, 4, 4.5, and 5 months of age. The highest activity of rhodanese in male fetus was in kidney cortex, followed by medulla of the kidney. No significant difference was observed in other organs. In female fetus, the highest activity was in kidney cortex followed by oviduct and medulla of kidney. The enzyme activity of tissues increased with age. There was no significant difference (P > 0.05) between male and female fetuses in levels of rhodanese activity of different tissues except in urinary bladder at 2.5 and 3 months and in urethra at 4.5 months of age. The results of this study might indicate the involvement of rhodanese in cyanide detoxification in tissues which are more exposed to cyanide. On the other hand, rhodanese might perform other functions which are specific in these tissues.     

Regional and Subcellular Distribution of Cyanide Metabolizing Enzymes in the Central Nervous System

Activities of cyanide metabolizing enzymes were measured in various subcellular fractions and regions in the central nervous system. Brain rhodanese and liver beta-mercaptopyruvate sulfurtransferase showed a slight decrease in activity after death. The activity of beta-mercaptopyruvate sulfurtransferase was negligible in the rat brain, compared with that of rhodanese. A small amount of thiocyanate was produced from cyanide and beta-mercaptopyruvate in the human brain, probably due to contamination with red blood cells. Rhodanese activity was widely distributed in all the areas of nervous tissue examined. In the rat the olfactory bulb showed the highest rhodanese activity, and high activity was also observed served in the thalamus, septum, hippocampus, and dorsal part of the midbrain. Rhodanese activity was low in various parts of the cerebral cortex. The distribution pattern of rhodanese in post-mortem human brain was essentially similar to that in rat brain. The thalamus, amygdala, centrum semiovale, colliculus superior, and cerebellar cortex showed high rhodanese activity in the human brain. Rhodanese activity was detected in the spinal cord. Anterior horn showed the highest rhodanese activity in the cervical, thoracic, and lumbar cord. Most rhodanese activity in the rat brain was recovered in the mitochondrial fraction with the highest specific activity. Rhodanese activity was lower in spinal cords obtained from autopsied cases with amyotrophic lateral sclerosis than in those of control subjects. A significant decrease in rhodanese was observed in the posterior column of the cervical or thoracic cord, but the activity in the anterior horn did not differ significantly between the two groups.     

Mercaptopyruvate sulfurtransferase as a defense against cyanide toxication: molecular properties and mode of detoxification.

In cyanide poisoning, metalloproteins and carbonyl groups containing proteins are the main target molecules of nucleophilic attack by cyanide. To defend against this attack, cyanide is metabolized to less toxic thiocyanate via transsulfuration. This reaction is catalyzed by rhodanese and mercaptopyruvate sulfurtransferase (MST). Rhodanese is a well characterized mitochondrial enzyme. On the other hand, little was known about MST because it was unstable and difficult to purify. We first purified MST to homogeneity and cloned MST cDNA from rat liver to characterize MST. We also found that MST was an evolutionarily related enzyme of rhodanese. MST and rhodanese are widely distributed in rat tissues, and the kidney and liver prominently contain these enzymes. Immunohistochemical study revealed that MST is mainly distributed in proximal tubular epithelial cells in the kidney, pericentral hepatocytes in the liver, the perinuclear area of myocardial cells in the heart, and glial cells in the brain, and immunoelectron microscopical study concluded that MST was distributed in both cytoplasm and mitochondria, so that MST first detoxifies cyanide in cytoplasm and the cyanide which escapes from catalysis due to MST enters mitochondria. MST then detoxifies cyanide again in cooperation with rhodanese in mitochondria. Tissues other than the liver and kidney are more susceptible to cyanide toxicity because they contain less MST and rhodanese. Even in the same tissue, sensitivity to cyanide toxicity may differ according to the kind of cell. It is determined by a balance between the amount of proteins to be attacked and that of enzymes to defend.     

Distribution of rhodanese in tissues of goat (Capra hircus)

Rhodanese (thiosulfate: cyanide sulfurtransferase, EC. 2.8.1.1) is a ubiquitous enzyme present in all living organisms, from bacteria to humans and plays a central role in cyanide detoxification. The purpose of this investigation is to determine and compare rhodanese activity in different tissues of adult male and female goats (Capra hircus). The results showed that the specific activity of rhodanese in different tissues was significantly different (P<0.05). The highest activity of rhodanese was in epithelium of rumen, followed by epithelia of reticulum and omasum and liver. No significant difference was observed when tissues of male and female goats were compared. The lowest specific activity of rhodanese was observed in spleen, urinary bladder, lymph node, ovary, skeletal muscle and pyloric muscle of abomasum. The results of this study may indicate the involvement of rhodanese in cyanide detoxification in goat tissues that have greater potential to be exposed to higher levels of cyanide.     

Rhodanese as a thioredoxin oxidase

A major catalytic difference between the two most common isoforms of bovine liver mitochondrial rhodanese (thiosulfate: cyanide sulfurtransferase, EC 2.8.1.1) has been observed. Both isoforms were shown to be capable of using reduced thioredoxin as a sulfur-acceptor substrate. However, only the less negative form in common with the recombinant mammalian rhodanese expressed in E. coli, can also catalyze the direct oxidation of reduced thioredoxin evidently by reactive oxygen species. These activities are understood in terms of the established persulfide structure (R-S-SH) of the covalently substituted rhodanese in the sulfurtransferase reaction and an analogous sulfenic acid structure (R-S-OH) when the enzyme acts as a thioredoxin oxidase. The observations suggest a role for one rhodanese isoform in the detoxication of intramitochondrial oxygen free radicals.     

Comparative properties of bovine heart and liver rhodaneses and the regulatory role of the rhodaneses in energy metabolism

In previous studies on the rhodanese activity of bovine liver mitochondria, we have shown that in addition to activity observed in the soluble protein fraction, there is rhodanese activity that is bound to the mitochondrial membrane. The latter activity accounts for as much as 40% of the total and, in situ, is associated in a multiprotein complex that forms iron-sulfur centers. In the present studies, we have investigated the rhodanese activity of bovine heart muscle. We have found that the major part of this enzyme activity is localized in the mitochondria and, further, that at least 25% of the total rhodanese activity of heart mitochondria is membrane-bound. As in liver tissue, the heart activity at least in part is associated in a multiprotein complex that forms iron-sulfur centers. Upon purification of the heart rhodanese in the soluble protein fraction, there is a 10- to 30-fold decrease inK _m values for the standard assay substrates thiosulfate and cyanide ions. These observations are consistent with the interpretation that there are activated and deactivated (low activity) forms of the heart enzyme in crude extracts, but only the activated form survives purification. The present results, together with our recent finding that liver mitochondrial rhodanese is subject to phosphorylation, lend support to our proposal that the rhodaneses serve as converter enzymes which regulate the rate of electron transport through sulfuration of respiratory chain components. The rhodaneses, in turn, are controlled by protein kinases and the local ATP concentration.     

Production of rhodanese by bacteria present in bio-oxidation plants used to recover gold from arsenopyrite concentrates

Considerably larger quantities of cyanide are required to solubilize gold following the bio-oxidation of gold-bearing ores compared with oxidation by physical-chemical processes. A possible cause of this excessive cyanide consumption is the presence of the enzyme rhodanese. Rhodanese activities were determined for the bacteria most commonly encountered in bio-oxidation tanks. Activities of between 6.4 and 8.2 micromol SCN min(-1) mg protein(-1) were obtained for crude enzyme extracts of Thiobacillus ferrooxidans, Thiobacillus thiooxidans and Thiobacillus caldus, but no rhodanese activity was detected in Leptospirillum ferrooxidans. Rhodanese activities 2-2.5-fold higher were found in the total mixed cell mass from a bio-oxidation plant. T. ferrooxidans synthesized rhodanese irrespective of whether it was grown on iron or sulphur. With a PCR-based detection technique, only L. ferrooxidans and T. caldus cells were detected in the bio-oxidation tanks. As no rhodanese activity was associated with L. ferrooxidans, it was concluded that T. caldus was responsible for all of the rhodanese activity. Production of rhodanese by T. caldus in batch culture was growth phase-dependent and highest during early stationary phase. Although the sulphur-oxidizing bacteria were clearly able to convert cyanide to thiocyanate, it is unlikely that this rhodanese activity is responsible for the excessive cyanide wastage at the high pH values associated with the gold solubilization process.     

Interspecies differences in rhodanese (thiosulfate sulfurtransferase, EC 2.8.1.1) activity in liver, kidney and plasma

Rhodanese levels have been measured in liver, kidney and plasma from a number of species. Liver activity was low in marmosets, pigeons and beagle bitches. Levels were high in rats and somewhat lower in hamsters and guinea pigs while levels in two strains of rabbits were intermediate between guinea pigs and marmosets. The relationship between hepatic and plasma rhodanese and cyanide sensitivity is discussed.     

NADH: Nitrate reductase activity restoration by rhodanese

The NADH: nitrate reductase from durum wheat leaves was inactivated by cyanide and its activity restored by thiosulphate and beef kidney rhodanese. Rhodanese and thiosulphate, added to NADH-nitrate reductase before cyanide treatment protected NADH-nitrate reductase activity. No oxidizing agent was required for the protection or restoration of cyanide treated NADH-nitrate reductase.     

Acceptor substrate-potentiated inactivation of bovine liver rhodanese

The interaction of bovine liver rhodanese (thiosulfate:cyanide sulfurtransferase, EC 2.8.1.1) with the acceptor substrates, dithiothreitol or cyanide, was studied. When incubated in the presence of cyanide or dithiothreitol, rhodanese was inactivated in a time-dependent process. This inactivation was detectable only at low enzyme concentrations; the rate and degree of inactivation could be modulated by varying the substrate concentration or the system pH. Activity measurements and fluorescence spectroscopy techniques were used in examining the inactivation phenomenon. Sulfur transfer to dithiothreitol was measured by direct assay and was shown to involve the dequenching of enzymic intrinsic fluorescence that had been previously observed only with cyanide as the acceptor substrate. Substrate-potentiated inactivation of rhodanese (with cyanide) has been reported before, but the cause and nature of this interaction were unexplained. The results presented here are consistent with an explanation invoking oxidation of rhodanese in the course of inactivation.     

The inhibition of rhodanese by lipoate and iron-sulfur proteins

A study was made on the effects of DL-dihydrolipoate, lipoate and iron-sulfur proteins on the activity of rhodanese (EC 2.8.1.1) with dihydrolipoate or cyanide as acceptors. DL-Dihydrolipoate inactivates rhodanese, lipoate does not, and the opposite occurs with the sulfur-free form of the transferase. The observed effects vary with the sulfane sulfur acceptor from rhodanese (i.e., dihydrolipoate or cyanide) and depend on intramolecular oxidation of the catalytic sulfhydryl or on formation of a mixed disulfide with dihydrolipoate. Thiosulfate protects against inactivation by reloading the active-site cysteine with persulfide sulfur. The inhibition of sulfur transfer by iron-sulfur proteins appears related to the amount of native iron-sulfur structure interacting with rhodanese. The implications of the results for a possible biological role of rhodanese are considered.     

Binding of metal cyanide complexes to bovine liver rhodanese in the crystalline state

Bovine liver rhodanese, which catalyzes the transfer of sulfur atoms between a variety of sulfur donor and sulfur acceptor substrates, is inhibited by metal cyanide complexes [Volini, M., Van Sweringen, B., & Chen, F.-Sh. (1978) Arch. Biochem. Biophys. 191, 205-215]. Crystallographic studies are described which reveal the binding mode of four different metal cyanides to bovine liver rhodanese: Na[Au(CN2], K2[Pt(CN)4], K2[Ni(CN)4], and K2[Zn(CN)4]. It appears that these complexes bind at one common site at the entrance of the active site pocket, interacting with the positively charged side chains of Arg-186 and Lys-249. This observation explains the inhibition of rhodanese by this class of compounds. For the platinum and nickel cyanide complexes virtually no other binding sites are observed. The gold complex binds, however, to three additional cysteine residues, thereby also displacing the extra sulfur atom which was bound to the essential Cys-247 in the sulfur-rhodanese complex. The zinc complex binds to completely different additional sites and forms complexes with the side chains of Asp-101 and His-203. Possible reasons for these different binding modes are discussed in terms of the preference for "hard" and "soft" ligands of these four metal ions.     

Purification of bovine liver rhodanese by low-pH column chromatography

We report a purification of bovine liver rhodanese (thiosulfate:cyanide sulfurtransferase, EC 2.8.1.1) using column chromatography under conditions that take advantage of recent information regarding the structure and stability of this enzyme. At low pH (e.g., pH 4-6), rhodanese is stabilized against inactivation processes. By maintaining rhodanese at low pH, column chromatography, and especially ion-exchange chromatography, becomes practical, without loss of enzymatic activity. A purification method involving the sequential use of cation-exchange, size-exclusion, and hydrophobic-interaction chromatography was developed, and rhodanese was purified with good yield to electrophoretic purity and high specific activity. Previous methods for purifying bovine liver rhodanese employ repeated ammonium sulfate fractionations and crystallization of the rhodanese. In these methods, it is difficult to separate rhodanese from yellow-brown contaminants in the final stages of the procedures. Here, yellow-brown contaminants, which copurify with rhodanese on the first two columns, are completely resolved by hydrophobic interaction chromatography. This method can be readily scaled up, requires no special equipment, eliminates the variability inherent in previous methods, and is less dependent upon experience.     

Inhibition of rat liver rhodanese by di-, tricarboxylic, and alpha-keto acids.

Rat liver rhodanese [EC 2.8.1.1] purified by ammonium sulfate fractionation, CM-cellulose and Sephadex G-200 chromatography yielded two active fractions (I & II). Their molecular weights were estimated to be 1.75 X 10(4) (I) and 1.26 X 10(4) (II) by the gel filtration method. Kinetic studies revealed that Fraction I rat liver rhodanese catalyzes thiocyanate formation from thiosulfate and cyanide by a double displacement mechanism. Carboxylic acids such as DL-isocitric, citric malic, pyruvic, and oxaloacetic acid were competitive inhibitors with respect to thiosulfate, whereas fumaric, succinic, and alpha-ketoglutaric acids were noncompetitive inhibitors with respect ot thiosulfate. Incubation of mitochondria with sulfate and alpha-ketoglutaric acid caused a significant decrease in rhodanese activity.     

Application of cyanide-metabolizing enzymes to environmental control; enzyme thermistor assay of cyanide using immobilized rhodanese and injectase

The application of the enzyme thermistor in the analysis of cyanide in standard solutions as well as in blast furnace waste water is described. The heat signal is generated in the conversion of cyanide, catalyzed by the immobilized enzymes rhodanese (E.C. 2.8.1.1) and injectase (E.C. 4.4.19). Using the combination of cyanide-metabolizing enzymes and the enzyme thermistor unit, assays down to 20μM cyanide can be carried out. Linear relationships were obtained at 20–600μM cyanide for injectase and 20–1000μM for rhodanese. The stability at 27°C of the heat response was initially decreased, but soon stabilized at about 80% of the initial value and remained so for at least 200 hr. The technique was easily adapted to continuous analysis, applicable to environmental control (e.g., a “cyanide guard”) with a response time at present within 2–3 min after a sudden change in cyanide concentration has appeared.     

Localization of Membrane-Bound Catechol-O-Methyltransferase

Abstract: Previous studies of the distribution of catechol-O-methyltransferase (COMT) have concentrated on the soluble enzyme activity. In this study the activity of the membrane-bound enzyme was determined in different brain regions and peripheral tissues of the rat. Membrane-bound COMT, like the soluble enzyme, has a general distribution with high levels in liver, kidney, and vas deferens. However, the ratio of membrane-bound to soluble activity varies almost 30-fold in different tissues, with the highest ratio in brain. Membrane-bound activity varies twofold within brain. In view of their different localization and kinetic properties, it seems likely that the two forms of COMT have different functions in vivo.     

The chaperonin assisted and unassisted refolding of rhodanese can be modulated by its N-terminal peptide

Thein vitro refolding of the monomeric, mitochondrial enzyme rhodanese (thiosulfate: cyanide sulfurtransferase, EC 2.8.1.1), which is assisted by theE. coli chaperonins, is modulated by the 23 amino acid peptide (VHQVLYRALVSTKWLAESVRAGK) corresponding to the amino terminal sequence (1–23) of rhodanese. In the absence of the peptide, a maximum recovery of active enzyme of about 65% is achieved after 90 min of initiation of the chaperonin assisted folding reaction. In contrast, this process is substantially inhibited in the presence of the peptide. The maximum recovery of active enzyme is peptide concentration-dependent. The peptide, however, does not prevent the interaction of rhodanese with the chaperonin 60 (cpn60), which leads to the formation of the cpn60-rhodanese complex. In addition, the peptide does not affect the rate of recovery of active enzyme, although it does affect the extent of recovery. Further, the unassisted refolding of rhodanese is also inhibited by the peptide. Thus, the peptide interferes with the folding of rhodanese in either the chaperonin assisted or the unassisted refolding of the enzyme. A 13 amino acid peptide (STKWLAESVRAGK) corresponding to the amino terminal sequence (11–23) of rhodanese does not show any significant effect on the chaperonin assisted or unassisted refolding of the enzyme. The results suggest that other sequences of rhodanese, in addition to the N-terminus, may be required for the binding of cpn60, in accord with a model in which cpn60 interacts with polypeptides through multiple binding sites.     

Selenium binding to beef-kidney rhodanese.

The reaction of beef kidney rhodanese with selenosulfate was studied. The selenium-treated enzyme shows an absorption spectrum with a maximum at 375 nm attributable to a sulfoselenide group. This absorption is bleached by addition of cyanide. After cyanide treatment stoichiometric amount of selenocyanate can be found. The intrinsic fluorescence of rhodanese is quenched by addition of stoichiometric selenosulfate. This effect can be reversed by cyanide or sulfite but not by selenite or glutathione. By comparison with model complexes the selenium-rhodanese intermediate was identified as a cysteinyl-selenium derivative.