Association of Hydrogen Sulfide with Femoral Bone Mineral Density in Osteoporosis Patients: A Preliminary Study.

BACKGROUND Accumulated evidence has suggested that hydrogen sulfide (H2S) has a role in bone formation and bone tissue regeneration. However, it is unknown whether the H2S content is associated with bone mineral density (BMD) in patients with osteopenia/osteoporosis. MATERIAL AND METHODS In the present study, we aimed to explore the changes of serum H2S in osteopenia and osteoporosis patients. We analyzed femur expression of cystathionine ß synthase (CBS), cystathionine γ lyase (CSE), and 3-mercaptopyruvate sulfurtransferase (3-MST), which are key enzymes for generating H2S. RESULTS Sixteen (16%) patients had osteopenia, 9 (9%) had osteoporosis, and 75 (75%) had normal BMD. In comparison with patients with normal BMD (controls), the serum levels of H2S were unexpectedly increased in patients with osteopenia and osteoporosis. This increase was much higher in patients with osteoporosis than in those with osteopenia. Serum H2S levels were negatively correlated with femoral BMD, but not lumbar BMD. Interestingly, the expression of CBS and CSE were downregulated in femur tissues in patients with osteoporosis, whereas the expression of 3-MST remained unchanged. Serum phosphorus levels, alkaline phosphatase, hemoglobin, and triglycerides were found to be closely associated with CBS and CSE scores in femur tissues. CONCLUSIONS Serum H2S levels and femur CBS and CSE expression may be involved in osteoporosis pathogenesis.

Analysis of the Cellular Roles of MOCS3 Identifies a MOCS3-Independent Localization of NFS1 at the Tips of the Centrosome.

The deficiency of the molybdenum cofactor (Moco) is an autosomal recessive disease, which leads to the loss of activity of all molybdoenzymes in humans with sulfite oxidase being the essential protein. Moco deficiency generally results in death in early childhood. Moco is a sulfur-containing cofactor synthesized in the cytosol with the sulfur being provided by a sulfur relay system composed of the l-cysteine desulfurase NFS1, MOCS3, and MOCS2A. Human MOCS3 is a dual-function protein that was shown to play an important role in Moco biosynthesis and in the mcm5s2U thio modifications of nucleosides in cytosolic tRNAs for Lys, Gln, and Glu. In this study, we constructed a homozygous MOCS3 knockout in HEK293T cells using the CRISPR/Cas9 system. The effects caused by the absence of MOCS3 were analyzed in detail. We show that sulfite oxidase activity was almost completely abolished, on the basis of the absence of Moco in these cells. In addition, mcm5s2U thio-modified tRNAs were not detectable. Because the l-cysteine desulfurase NFS1 was shown to act as a sulfur donor for MOCS3 in the cytosol, we additionally investigated the impact of a MOCS3 knockout on the cellular localization of NFS1. By different methods, we identified a MOCS3-independent novel localization of NFS1 at the centrosome.

Biochemical Analyses of Human Iron-Sulfur Protein Biogenesis and of Related Diseases.

Maturation of Fe/S proteins in mammals is an intricate process mediated by two assembly systems located in the mitochondrial and cytosolic-nuclear compartments. Malfunction particularly of the mitochondrial system gives rise to severe neurological, metabolic, or hematological disorders, often with fatal outcome. In this chapter, we describe approaches for the differential biochemical investigation of cellular Fe/S protein maturation in mitochondria, cytosol, and nucleus. The analyses may also facilitate the identification of the affected Fe/S protein assembly step in diseased state. As Fe/S cluster insertion into target apoproteins is a frequent determinant of protein stability, examination of protein steady-state levels in biological samples frequently permits reliable first clues about the maturation process. In some specific cases, this approach allows the assessment of enzymatic or regulatory functions of Fe/S proteins, including the formation of lipoate cofactor by mitochondrial lipoic acid synthase or the posttranscriptional regulation of transferrin receptor and ferritin expression by the cytosolic iron regulatory proteins. More direct Fe/S protein maturation assays like enzymatic analyses may further validate the observed maturation defects. Here, we present a simple protocol for the determination of dihydropyrimidine dehydrogenase enzyme activity by thin-layer chromatography. In order to directly monitor Fe/S cluster insertion into target apoproteins, we have developed a 55Fe radiolabeling technique tracing the in vivo Fe/S cofactor formation in mammalian tissue culture. The combination of the presented techniques represents a comprehensive strategy to assess the multiple facets of Fe/S protein assembly for both mechanistic analyses and for the elucidation of specific defects in Fe/S diseases.

Hydrogen sulfide ameliorates learning memory impairment in APP/PS1 transgenic mice: A novel mechanism mediated by the activation of Nrf2.

Beta-amyloid (Aß) plaques and oxidative stress are associated with the pathogenesis of Alzheimer's disease (AD). Hydrogen sulfide (H2S) has been recognized as a cytoprotectant, which improves learning memory impairment and exerts antioxidant effects in neurodegenerative disorders, including AD. The experiment was projected to explore the effects of H2S on cognitive deficits, Aß levels and possible antioxidant mechanisms. Here, APP/PS1 transgenic mice were injected sodium hydrosulfide (NaHS, a H2S donor, 2.8mg/kg) once a day for three months. It was found that APP/PS1 transgenic mice exhibited cognitive deficits and a large number of senile plaques, along with neurons decrease and Aß increase. However, intraperitoneal (i.p.) injection of NaHS improved learning memory deficits, decreased the number of senile plaques, Aß1-40 and Aß1-42 levels, suppressed neurons loss, together with up-regulated the levels of cystathionine-ß-synthase (CBS) and 3-mercaptopyruvate-sulfurtransferase (3MST). Furthermore, the protein levels of beta-amyloid precursor (APP) and beta-secretase 1 (BACE1) were dramatically restrained after administration of H2S. In addition, H2S exerted antioxidant effects via up-regulation nuclear factor erythroid-2-related factor 2 (Nrf2), heme oxygenase-1(HO-1) and glutathione S-transferase (GST). Taken together, these findings suggest that H2S ameliorates learning memory impairment, decreases the number of senile plaques in APP/PS1 mice possibly through inhibition of Aß production and activation of Nrf2/antioxidant response element (ARE) pathway.

Cysteine Metabolism and Oxidative Processes in the Rat Liver and Kidney after Acute and Repeated Cocaine Treatment.

The role of cocaine in modulating the metabolism of sulfur-containing compounds in the peripheral tissues is poorly understood. In the present study we addressed the question about the effects of acute and repeated (5 days) cocaine (10 mg/kg i.p.) administration on the total cysteine (Cys) metabolism and on the oxidative processes in the rat liver and kidney. The whole pool of sulfane sulfur, its bound fraction and hydrogen sulfide (H2S) were considered as markers of anaerobic Cys metabolism while the sulfate as a measure of its aerobic metabolism. The total-, non-protein- and protein- SH group levels were assayed as indicators of the redox status of thiols. Additionally, the activities of enzymes involved in H2S formation (cystathionine γ-lyase, CSE; 3-mercaptopyruvate sulfurtransferase, 3-MST) and GSH metabolism (γ-glutamyl transpeptidase, γ-GT; glutathione S-transferase, GST) were determined. Finally, we assayed the concentrations of reactive oxygen species (ROS) and malondialdehyde (MDA) as markers of oxidative stress and lipid peroxidation, respectively. In the liver, acute cocaine treatment, did not change concentrations of the whole pool of sulfane sulfur, its bound fraction, H2S or sulfate but markedly decreased levels of non-protein SH groups (NPSH), ROS and GST activity while γ-GT was unaffected. In the kidney, acute cocaine significantly increased concentration of the whole pool of sulfane sulfur, reduced the content of its bound fraction but H2S, sulfate and NPSH levels were unchanged while ROS and activities of GST and γ-GT were reduced. Acute cocaine enhanced activity of the CSE and 3-MST in the liver and kidney, respectively. Repeatedly administered cocaine enhanced the whole pool of sulfane sulfur and reduced H2S level simultaneously increasing sulfate content both in the liver and kidney. After repeated cocaine, a significant decrease in ROS was still observed in the liver while in the kidney, despite unchanged ROS content, a marked increase in MDA level was visible. The repeated cocaine decreased 3-MST and increased γ-GT activities in both organs but reduced GST in the kidney. Our results show that cocaine administered at a relatively low dose shifts Cys metabolism towards the formation of sulfane sulfur compounds which possess antioxidant and redox regulatory properties and are a source of H2S which can support mitochondrial bioenergetics.

Dual targeting of Nfs1 and discovery of its novel processing enzyme, Icp55.

In eukaryotes, each subcellular compartment harbors a specific group of proteins that must accomplish specific tasks. Nfs1 is a highly conserved mitochondrial cysteine desulfurase that participates in iron-sulfur cluster assembly as a sulfur donor. Previous genetic studies, in Saccharomyces cerevisiae, have suggested that this protein distributes between the mitochondria and the nucleus with biochemically undetectable amounts in the nucleus (termed "eclipsed distribution"). Here, we provide direct evidence for Nfs1 nuclear localization (in addition to mitochondria) using both alpha-complementation and subcellular fractionation. We also demonstrate that mitochondrial and nuclear Nfs1 are derived from a single translation product. Our data suggest that the Nfs1 distribution mechanism involves at least partial entry of the Nfs1 precursor into mitochondria, and then retrieval of a minor subpopulation (probably by reverse translocation) into the cytosol and then the nucleus. To further elucidate the mechanism of Nfs1 distribution we determined the N-terminal mitochondrial sequence of Nfs1 by Edman degradation. This led to the discovery of a novel mitochondrial processing enzyme, Icp55. This enzyme removes three amino acids from the N terminus of Nfs1 after cleavage by mitochondrial processing peptidase. Intriguingly, Icp55 protease (like its substrate Nfs1) appears to be dual distributed between the nucleus and mitochondria.

[Development of assay methods for endogenous inorganic sulfur compounds and sulfurtransferases and evaluation of the physiological functions of bound sulfur].

Inorganic sulfur compounds, such as S(2-), SO(3)(2-) and S(2)O(3)(2-), are produced from sulfur- containing amino acids as intermediary metabolites in mammalian tissues through complex pathways and are ultimately incorporated into sulfate. Reduced sulfur is also produced via the desulfuration of cysteine by several sulfurtransferases present in mammalian tissues; these enzymes include gamma-cystathionase (gamma-CST), and 3-mercaptopyruvate sulfurtransferase (3-MST). This reduced sulfur is then incorporated into pools of active reduced sulfur (sulfane sulfur; polysulfides, polythionates, thiosulfate, thiosulfonates and elemental sulfur) that are involved in the detoxication of cyanide and in the biosynthesis of iron-sulfur cluster. Sulfane sulfur is labile and is reduced to H(2)S by reducing agents. The physiological function of these sulfur species is less clear. We have found that a reduced sulfur species is commonly present in mammalian sera and tissues as a high molecular weight material and as both a high and a low molecular weight material, respectively; we designated this sulfur species as "bound sulfur." Bound sulfur can be easily liberated as sulfide by reduction with DTT. This review describes sensitive and specific assay method for determining the presence of inorganic sulfur compounds as well as bound sulfur and related sulfurtransferases in biological samples. The physiological functions of bound sulfur in rat tissues were also evaluated using these assay methods. Bound sulfur was found to be located primarily in the rat liver cytosolic fraction in the form of high molecular weight components. The capacity of bound sulfur production was enriched in the cytosol fraction and depended on gamma-CST. Bound sulfur also affected redox regulation by modifying active thiol residues in some liver cytosol enzymes and effectively inhibited cytochrome P-450-dependent lipid peroxidation induced by CCl(4) and t-BuOOH.

Characterization of the cofactor composition of Escherichia coli biotin synthase.

The cofactor content of in vivo, as-isolated, and reconstituted forms of recombinant Escherichia coli biotin synthase (BioB) has been investigated using the combination of UV-visible absorption, resonance Raman, and Mössbauer spectroscopies along with parallel analytical and activity assays. In contrast to the recent report that E. coli BioB is a pyridoxal phosphate (PLP)-dependent enzyme with intrinsic cysteine desulfurase activity (Ollagnier-deChoudens, S., Mulliez, E., Hewitson, K. S., and Fontecave, M. (2002) Biochemistry 41, 9145-9152), no evidence for PLP binding or for PLP-induced cysteine desulfurase or biotin synthase activity was observed with any of the forms of BioB investigated in this work. The results confirm that BioB contains two distinct Fe-S cluster binding sites. One site accommodates a [2Fe-2S](2+) cluster with partial noncysteinyl ligation that can only be reconstituted in vitro in the presence of O(2). The other site accommodates a [4Fe-4S](2+,+) cluster that binds S-adenosylmethionine (SAM) at a unique Fe site of the [4Fe-4S](2+) cluster and undergoes O(2)-induced degradation via a distinct type of [2Fe-2S](2+) cluster intermediate. In vivo Mössbauer studies show that recombinant BioB in anaerobically grown cells is expressed exclusively in an inactive form containing only the as-isolated [2Fe-2S](2+) cluster and demonstrate that the [2Fe-2S](2+) cluster is not a consequence of overexpressing the recombinant enzyme under aerobic growth conditions. Overall the results clarify the confusion in the literature concerning the Fe-S cluster composition and the in vitro reconstitution and O(2)-induced cluster transformations that are possible for recombinant BioB. In addition, they provide a firm foundation for assessing cluster transformations that occur during turnover and the catalytic competence of the [2Fe-2S](2+) cluster as the immediate S-donor for biotin biosynthesis.

Tissue and subcellular distribution of mercaptopyruvate sulfurtransferase in the rat: confocal laser fluorescence and immunoelectron microscopic studies combined with biochemical analysis.

In our previous study, we found that mercaptopyruvate sulfurtransferase (MST) was evolutionarily related to mitochondrial rhodanese. To elucidate the difference between MST and rhodanese, the tissue, cellular, and subcellular distribution of rat MST was determined biochemically and immunohistochemically by using anti-MST antibody raised in rabbit. In an immunohistochemical study, tetramethyl rhodamine isothiocyanate-conjugated phalloidin against F-actin and fluorescein isothiocyanate-conjugated goat anti-rabbit immunoglobulin as a secondary antibody to the anti-MST antibody were used for double fluorescent staining. They were detected by confocal laser fluorescence microscopy. In the immunoelectron microscopic study of hepatocyte and renal tubular epithelium, a postembedding immunogold method was used. Biochemical studies including western blot analyses of various tissues and subcellular fractions of the liver were also performed. MST was widely distributed in rat tissues but the cellular distribution was found to be different in each tissue. MST was predominantly localized in proximal tubular epithelium in the kidney, pericentral hepatocytes in the liver, cardiac cells in the heart, and neuroglial cells in the brain. This immunocytochemical study also found that MST was localized in both mitochondria and cytoplasm.

Estrogen and hydroxysteroid sulfotransferases in guinea pig adrenal cortex: cellular and subcellular distributions.

This report describes for the first time the cellular and subcellular localization of estrogen sulfotransferase (EST) as well as the subcellular localization of hydroxysteroid sulfotransferase (HST) in the mammalian adrenal cortex. A 34-kilodalton EST and two HSTs with 3 alpha- and 3 beta-hydroxysteroid substrate specificities (32 and 33 kilodaltons, respectively) were previously purified from guinea pig adrenal cortex and characterized. Western blots were used to establish that two antisera generated against EST were highly specific for EST, whereas three antisera generated against the HSTs were highly specific for the HSTs, but did not distinguish between the 3 alpha- and 3 beta HSTs. Light and electron microscopic immunoperoxidase labeling with these antisera revealed that the sulfotransferases were expressed only within the ACTH-responsive layers of the guinea pig adrenal cortex, with EST localized to zona fasciculata and zona reticularis cells, and the HSTs confined to the zona reticularis. No labeling was detected in the zona glomerulosa or adrenal medulla. EST was concentrated in cell nuclei; sparse labeling was distributed throughout the cytoplasm. HST labeling was intense in smooth endoplasmic reticulum of zona reticularis cells, but was absent from nuclei. Ovoid inclusions about 1-4 microns in diameter, with no limiting membrane, were observed in zona reticularis cells; these inclusions were strongly labeled for both EST and HSTs. No gender-specific differences in distribution or labeling intensity were apparent. The high concentration of EST immunoreactivity in nuclei suggests that EST may play a role in modulating the ability of active estrogens to regulate gene expression in ACTH-responsive cells. The distribution of HST labeling suggests that sulfonation of adrenocortical 3-hydroxysteroids takes place largely within smooth endoplasmic reticulum in the zona reticularis in adult guinea pigs.

Spatio-temporal expression of estrogen sulfotransferase within the hepatic lobule of male rats: implication of in situ estrogen inactivation in androgen action.

Estrogen sulfotransferase (EST) catalyzes transfer of the sulfate group from phosphoadenosine phosphosulfate to estrogenic steroids. Since estrogen sulfates do not bind to the estrogen receptor with high affinity, EST can control the intracellular level of the receptor-active estrogens. Androgen action in the rat liver, as indicated by the androgenic induction of alpha 2u-globulin, is inhibited by low levels of estrogens. Thus, in situ estrogen inactivation by EST is expected to increase hepatic androgen sensitivity. During the lifespan of the animal, rat liver undergoes three distinct phases of androgen sensitivity, i.e. prepubertal androgen insensitivity, androgen sensitivity after approximately 40 days of age, and androgen insensitivity during senescence (greater than 750 days). EST in the liver is expressed only after puberty, when the liver becomes androgen sensitive. Furthermore, localization of EST and its corresponding mRNA within the lobular unit of the liver demonstrates that only androgen-responsive hepatocytes located around the central vein contain immunoreactive EST and its corresponding mRNA. These temporal and spatial correlations of EST expression and hepatic androgen sensitivity support the concept that steroid-inactivating enzymes play important roles in sex hormone action.

Estrogen sulfotransferase distribution in tissues of mouse and guinea pig: steroidal inhibition of the guinea pig enzyme.

The highest total activity of estrogen sulfotransferase in guinea pig is in liver and the highest specific activities are in the adrenal and the midgestational chorion. Guinea pig gonads contain scarcely detectable activities. In CD-1 mice the highest specific activity is in testis and the highest total activity is in late placenta. Adrenals from both sexes and ovaries contain minimal activities, while liver and fetal membrane activities are remarkably low. In CD-1, DBA, C57BL, and BALB mice, qualitative patterns are similar. Purified or partially purified estrogen sulfotransferase from guinea pig adrenal and chorion were used to study the effect of a number of possible steroidal inhibitors. Considerable structural specificity is evident within a range of steroids, even among some which are not substrates. Pregnenolone is the most effective 21-carbon inhibitor and, in general, more highly hydroxylated forms are less inhibitory. Within a series of 21-, 19- and 18-carbon steroids, the structure of the A ring appears to be extremely important in regard to inhibitory effects.

Comparison of estrogen sulfotransferase and pregnenolone sulfotransferase of guinea pig.

Guinea pig adrenal estrogen sulfotransferase from either sex was eluted as a single peak, irrespective of buffer salt concentration, when subjected to fast protein liquid chromatography on gel filtration columns. The same enzyme was consistently eluted in two distinct peaks during chromatofocusing. Adrenal pregnenolone sulfotransferase was eluted during gel filtration in a heterogeneous pattern, dependent on salt concentration. These properties have made possible almost complete separation of the two sulfotransferases in one step, although adrenal estrogen sulfotransferase may possess a minute intrinsic ability to catalyze sulfation of pregnenolone. Pregnenolone sulfotransferase had no measurable activity toward estrone. Pregnenolone sulfotransferase from both sexes yielded variable elution patterns during chromatofocusing. Estrogen sulfotransferase from the adrenal, as well as that of guinea pig chorion, was strongly inhibited by N-ethylmaleimide and to a lesser degree by iodoacetamide and iodoacetate. Adrenal and chorion estrogen sulfotransferases were thermolabile and were activated, although not protected from the effect of heat, by binding to 3'-phosphoadenosine 5'-phosphosulfate. Adrenal pregnenolone sulfotransferase was inhibited only by high concentrations of N-ethylmaleimide and not at all by iodoacetamide or iodoacetate. It was more thermostable than the estrogen sulfotransferase and was not activated by binding to 3'-phosphoadenosine 5'-phosphosulfate.

Immunochemical characterization of developmental changes in rat hepatic hydroxysteroid sulfotransferase.

A major isoenzyme of hepatic androsterone-sulfating sulfotransferase (AD-ST) was purified from adult female rats. The activity was purified 122-fold over that found in the cytosol and showed a single protein band with a subunit molecular mass of 30 kDa after sodium dodecyl sulfate polyacrylamide gel electrophoresis. The purified enzyme exhibited four isoelectric variants of subunits on denaturing isoelectrofocusing gels (pI = 5.8, 6.1, 6.7 and 7.2). Rabbit antiserum raised against the enzyme specifically detected AD-ST polypeptide in rat liver cytosol. Immunoblot analysis of liver cytosol from female and male rats at various ages showed good correlation between the levels of AD-ST activity and AD-ST polypeptide. Significant levels of AD-ST activity and polypeptide were detected in senescent male rats, though normal adult male rats have very low levels of AD-ST activity and protein. The relative content of the isoelectric variants of AD-ST were different in liver cytosol of weanling and adult females, indicating that age- and gender-related alterations of hepatic AD-ST activity are primarily determined by the levels of AD-ST polypeptide and the relative amounts of the four isoelectric variants of the enzyme.

An assay method for ganglioside synthase using anion-exchange chromatography.

A rapid procedure which is based on combined ion-exchange chromatography and solubility was established for determination of the activity of ganglioside synthases and cerebroside sulfotransferase. The procedure consists of selective elution of radiolabeled reaction products (acidic glycolipids) freed from labeled precursors and breakdown products on a DEAE-Sephadex column and of direct radioassay of the products in the eluate. Monosialogangliosides were eluted from the column with 40 mM ammonium acetate (AcONH4) in methanol, cerebroside sulfate with 90 mM AcONH4 in methanol, and disialogangliosides with 40 mM AcONH4 in isopropanol/n-hexane/water (55/20/19, v/v/v). The established procedure is simple, reproducible, and economical. Using rat Golgi membrane as enzyme source the recovery rate of the products was over 95%.

Determination of the isoelectric point value of 3-mercaptopyruvate sulfurtransferase and its shift by treatment with oxidized glutathione.

The isoelectric point (pI) value of 3-mercaptopyruvate sulfurtransferase (MST) from human erythrocytes was determined to be 6.3 at 10 degrees C by isoelectric focusing in horizontal slab polyacrylamide gel containing 2% carrier ampholyte (pH 3-10). The value was determined by comparison with the electrofocused bands of bovine pancreatic ribonuclease A-glutathione mixed disulfides (RNase-SG), which were composed of 8 species containing 1 (RNase-SG1) through 8 (RNase-SG8) moles of glutathione per mole of ribonuclease A with different pI values ranging from 5.3 (RNase-SG8) to 8.8 (RNase-SG1). The pI value of the same enzyme in a 110,000 X g supernatant of rat liver was 5.9, which was the same as that of rat erythrocyte enzyme. Treatments of rat hemolysate with oxidized glutathione or diamide resulted in a shift of the pI of MST to a lower value, 5.7-5.5. This shift was inhibited when these treatments were performed in the presence of dithiothreitol. These results indicate that the treatment of the enzyme with oxidized glutathione results in the formation of enzyme-glutathione mixed disulfide.

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.

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.