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.     

Phosphorylation and dimerization regulate nucleocytoplasmic shuttling of mammalian STE20-like kinase (MST).

Mammalian STE20-like kinase (MST) is a member of the yeast STE20-related kinase family and proteolytically activated by caspase during apoptosis. However, its other cellular functions are not known, including its activation mechanism, substrate(s), and subcellular localization. In this report, using anti-MST monoclonal antibodies, we clearly show that endogenous MST is localized in cytoplasm in a leptomycin B-dependent manner. Analyses with serial deletions and point mutations show that MST has two functional nuclear export signals and, unexpectedly, another localization motif for nuclear import. When cells are treated with leptomycin, monomeric MST is accumulated more rapidly in the nucleus than dimeric MST, indicating that dimerization contributes to the cytoplasmic retention of MST. Okadaic acid, an inhibitor of phosphatase 2A, induces activation of MST and translocation into the nucleus. Using phosphopeptide-specific antibody, we directly show that okadaic acid induces phosphorylation in the activation loop of MST, and, once phosphorylated, MST is rapidly translocated to the nucleus. However, kinase-deficient MST does not enter the nucleus, indicating that phosphorylation and activation is required for okadaic acid-induced nuclear translocation. In apoptotic cells, the activation of MST does not require phosphorylation in the activation loop and occurs through the release of C-terminal regulatory domain by caspase-dependent cleavage. Kinase-deficient MST functions dominant-negatively and represses okadaic acid-induced morphological change indicating that MST plays a role in okadaic acid-induced cellular shrinkage. Our identification of cytoplasmic and nuclear localization motifs and phosphorylation-dependent translocation of MST suggests that regulation of localization is important to the biological function of MST, including its effects on cellular morphology.     

Immunocytochemical localization of a tartrate-resistant and vanadate-sensitive acid nucleotide tri- and diphosphatase

Purified rabbit antiserum to a tartrate-resistant and vanadate-sensitive acid phosphatase (nucleotide tri- and diphosphatase) prepared from rat bone was used in immunocytochemical studies. The antigen was localized in sections of fixed, decalcified tissue (head from rat) using the peroxidase-antiperoxidase bridge (PAP) or the avidin-biotin-peroxidase complex (ABC) technique. Both techniques resulted in similar and specific immunostaining in the following cells and tissues: osteoclasts situated in resorption lacunae, epithelium overlying enamel-free areas of tips of cusps of unerupted molars, cilia of respiratory epithelium, and tissue macrophages. This distribution corresponds to the cellular sites of tartrate-resistant acid phosphatase activity, as revealed by enzyme histochemistry. With the ABC method, staining in osteoclasts was obtained with antiserum dilutions of up to 1:10,000. Biochemical studies revealed that vanadate-sensitive acid ATPase activity in liver subcellular fractions was almost exclusively confined to lysosomes. Thus, the immunostaining has revealed the presence of the tartrate-resistant and vanadate-sensitive nucleotide phosphatase in many cells associated with tissue resorption and phagocytosis.     

Quantitation and tissue localization of the cellular retinoic acid-binding protein

The distribution of the cellular retinoic acid-binding protein (CRABP) in some rat tissues has been determined, and the protein has been localized by immunocytochemical techniques in sections from rat testis. In the testis CRABP was found in the seminiferous tubuli with Sertoli cells and the spermatogonia most intensely stained. All other cells of the germinal epithelium appeared largely devoid of CRABP. By use of an enzyme-linked immunosorbent assay CRABP was quantitatively estimated in several tissues and the highest levels were found in testis and eye. Comparisons of the tissue levels of CRABP and of the cellular retinol-binding protein (CRBP) did not reveal any apparent correlation.     

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.     

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.     

Glycogen synthase (casein) kinase-1: tissue distribution and subcellular localization

The distribution of glycogen synthase (casein) kinase-1 (CK-1) among different rat tissues and subcellular fractions was investigated. Using casein, glycogen synthase and phosphorylase kinase as substrates, CK-1 activity was detected in kidney, spleen, liver, testis, lung, brain, heart, skeletal muscle and adipose tissue. The distribution of CK-1 among different subcellular fractions of rat liver was; cytosol (72.1%), microsome (17.6%), mitochondria (9.6%) and nuclei (0.7%). CK-1 from rat tissues was shown to have a similarly wide substrate specificity as highly purified CK-1 from rabbit skeletal muscle. Such wide substrate specificity and distribution among different mammalian tissues and subcellular organelles indicate that CK-1 may be involved in the regulation of diverse cellular functions.     

Sulfurtransferases and the content of cysteine, glutathione and sulfane sulfur in tissues of the frog Rana temporaria

L-cysteine desulfuration was examined in tissues of Rana temporaria, in October and January. The activities of 3-mercaptopyruvate sulfurtransferase (MPST), cystathionine gamma-lyase (CST) and rhodanese were primarily concentrated in frog liver and kidney. The values of CST and rhodanese activity, as well as sulfane sulfur compounds levels fell in the range characteristic of rat. For each of the investigated tissues changes noted in the enzymatic activities and in the level of glutathione (GSH), protein-bound cysteine (PbCys) and sulfane sulfur compounds were dependent on the month in which the determination was performed, and on the character of the tissue. In such tissues as the liver or gonads, high GSH levels and high activities of MPST (in the liver) or MPST and rhodanese (in the gonads) seemed to accompany protein biosynthesis during hibernation. PbCys, the level of which was consequently diminished in all tissues in January, compensated the absence of exogenous cysteine. A significantly reduced GSH level in the brain in January seemed to be correlated with decreased requirements of the tissue for this important natural antioxidant at diminished thyroid hormones levels in the serum and minimal oxygen consumption during the hibernation. In the kidney, the possible participation of sulfane sulfur compounds in detoxification processes requires elucidation, similarly as in protection against cellular oxidative stress at extremely low levels of GSH.     

Subcellular localization of fumarase in mammalian cells and tissues

Fumarase, a mitochondrial matrix protein, is previously indicated to be present in substantial amounts in the cytosol as well. However, recent studies show that newly synthesized human fumarase is efficiently imported into mitochondria with no detectable amount in the cytosol. To clarify its subcellular localization, the subcellular distribution of fumarase in mammalian cells/tissues was examined by a number of different methods. Cell fractionation using either a mitochondria fraction kit or extraction with low concentrations of digitonin, detected no fumarase in a 100,000 g supernatant fraction. Immunoflourescence labeling with an affinity-purified antibody to fumarase and an antibody to the mitochondrial Hsp60 protein showed identical labeling pattern with labeling seen mainly in mitochondria. Detailed studies were performed using high-resolution immunogold electron microscopy to determine the subcellular localization of fumarase in rat tissues, embedded in LR White resin. In thin sections from kidney, liver, heart, adrenal gland and anterior pituitary, strong and specific labeling due to fumarase antibody was only detected in mitochondria. However, in the pancreatic acinar cells, in addition to mitochondria, highly significant labeling was also observed in the zymogen granules and endoplasmic reticulum. The observed labeling in all cases was completely abolished upon omission of the primary antibody indicating that it was specific. In a western blot of purified zymogen granules, a fumarase-antibody cross-reactive protein of the same molecular mass as seen in the mitochondria was present. These results provide evidence that fumarase in mammalian cells/tissues is mainly localized in mitochondria and significant amounts of this protein are not present in the cytosol. However, these studies also reveal that in certain tissues, in addition to mitochondria, this protein is also present at specific extramitochondrial sites. Although the cellular function of fumarase at these extramitochondrial locations is not known, the appearance/localization of fumarase outside mitochondria may help explain how mutations in this mitochondrial protein can give rise to a number of different types of cancers.     

Subcellular and organ distribution of cholesterol epoxide hydrolase in the rat

The subcellular and organ distributions of microsomal epoxide hydrolases measured with cis-stilbene oxide and cholesterol 5,6 alpha-epoxide as substrates have been investigated. These two enzyme activities were found to have essentially the same subcellular distribution, with the highest total and specific activities localized in rough and smooth endoplasmic reticulum. Among the tissues studied (i.e., liver, kidney, lung, testis, spleen, brain and intestinal epithelium), the highest specific activities were recovered in liver microsomes, where the activities were at least 5-fold greater than in any of the other microsomal preparations.     

Cell-specific subcellular localization of soluble epoxide hydrolase in human tissues.

Soluble epoxide hydrolase (sEH) is a phase-I xenobiotic metabolizing enzyme having both an N-terminal phosphatase activity and a C-terminal epoxide hydrolase activity. Endogenous hydrolase substrates include arachidonic acid epoxides, which have been involved in regulating blood pressure and inflammation. The subcellular localization of sEH has been controversial. Earlier studies using mouse and rat liver suggested that sEH may be cytosolic and/or peroxisomal. In this study we applied immunofluorescence and confocal microscopy using markers for different subcellular compartments to evaluate sEH colocalization in an array of human tissues. Results showed that sEH is both cytosolic and peroxisomal in human hepatocytes and renal proximal tubules and exclusively cytosolic in other sEH-containing tissues such as pancreatic islet cells, intestinal epithelium, anterior pituitary cells, adrenal gland, endometrium, lymphoid follicles, prostate ductal epithelium, alveolar wall, and blood vessels. sEH was not exclusively peroxisomal in any of the tissues evaluated. Our data suggest that human sEH subcellular localization is tissue dependent, and that sEH may have tissue- or cell-type-specific functionality. To our knowledge, this is the first report showing the subcellular localization of sEH in a wide array of human tissues.     

Quantification of neutral cysteine protease bleomycin hydrolase and its localization in rat tissues

A neutral cysteine protease, bleomycin hydrolase (BH), was found to be present in the range 3.7-131.1 ng per mg of rat tissues by enzyme-lined immunosorbent assay (ELISA). Newborn rat skin contained the highest amount of BH, and relatively high levels of BH were detected in the kidney and liver of 6-week-old male rats. The tissue distribution of BH in female rats was similar to that in male rats. Moreover, BH was detected in the extracts of erythrocytes and leukocyte-rich cells as well as in those of rat hemo-lymphocytic lineage cell lines by Western blotting. The BH level was increased at 6 weeks after birth and then slightly decreased. By immunohistochemistry, BH was localized as granular staining in the distal and proximal tubular cells of the kidney, and it was also detected in hepatocytes of the liver, in the red pulpy region of the spleen and in neurons of the brain. An immunoelectron microscopic study showed that BH-immunoreactivity was essentially located in the cytoplasm and at the outer membrane of the rough endoplasmic reticulum of epithelial cells of the kidney, as well as in that of hepatocytes of the liver. These results suggest that BH may play ubiquitous and unique roles in rat tissues.     

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.     

Cellular localization of insulin-degrading enzyme in rat liver using monoclonal antibodies specific for this enzyme

Although insulin-degrading enzyme (IDE) has been implicated in the intracellular degradation of insulin, the cellular localization of this enzyme is still controversial. In the present study, we have examined the cellular localization of IDE in the rat liver by three different techniques using monoclonal antibodies. First, direct immunohistochemical staining of rat liver with one of the monoclonal antibodies revealed that IDE immunoreactivity mainly exists in parenchymal cells, especially in the vicinity of the portal tract and also in the epithelium of the bile duct under light microscopy. In the electron microscopic study, IDE immunoreactivity was found in the cytoplasm near the rough endoplasmic reticulum but not in the plasma membrane, nucleus, or mitochondria. Second, immunoblotting analysis of the subcellular fraction in rat liver showed that the monoclonal antibody specifically reacted with a single polypeptide in the cytosolic fraction, of apparent Mr 110,000, which was consistent with the Mr of IDE. However, a polypeptide band corresponding to IDE could not be observed in the plasma membrane, mitochondrial, or lysosomal fraction. Third, IDE was only detectable in the cytosolic fraction by sandwich radioimmunoassay using two monoclonal antibodies. These results all suggest that IDE is a cytosolic enzyme.     

Immunocytochemical detection of ornithine decarboxylase.

Ornithine decarboxylase (ODC), a regulatory enzyme of polyamine biosynthesis, is involved in cell growth and differentiation. Lack of information about the exact cellular and subcellular localization of ODC is one of the main obstacles to precise interpretation of the biological roles of the ODC/polyamine system. Here we describe the development and optimization of an immunocytochemical method to detect ODC in cells and tissues. For this purpose a monoclonal antibody (MP16-2) against a defined epitope of ODC protein was developed. Specificity of the antibody for ODC was substantiated by Western blotting and ELISA analysis using cell and tissue homogenates. In cultured cells, optimal staining results were obtained after fixation with crosslinking fixatives followed by permeabilization with methanol. In rat tissues, ODC immunoreactivity was best preserved in paraffin sections fixed with Bouin's fixative. Antigen retrieval using SDS and citrate buffer substantially increased ODC immunostaining and decreased background staining. Localization studies of ODC in different cell lines showed that strongest staining for ODC was found in the nucleoplasm of mitotic cells, whereas confluent cells showed moderate perinuclear staining. Immunocytochemical studies of various rat tissues showed high cytoplasmic immunostaining of ODC in epithelial cells of kidney, prostate, and adrenal medulla of testosterone-treated rats, in glandular epithelium of small intestine, and in pancreas of neonatal and adult rats. (J Histochem Cytochem 47:1395-1404, 1999)     

The hyaluronate receptor is preferentially expressed on proliferating epithelial cells

In the present study, we have examined the distribution of the hyaluronate receptor as well as hyaluronate itself in a variety of adult tissues. The hyaluronate receptor was localized with a monoclonal antibody, termed K-3, while hyaluronate was localized using proteolytic fragments of cartilage proteoglycan. Staining with the K-3 monoclonal antibody revealed that the hyaluronate receptor was present in a variety of epithelia including the skin, cheek, tongue, esophagus, vagina, intestines, oviduct, and bladder. However, it was notably absent from epithelial cells of the cornea and stomach as well as from endothelial cells of blood vessels. When present, the hyaluronate receptor was preferentially located in regions of active cell growth, such as in the basal layers of stratified epithelium and at the base of the crypts of Lieberkuhn in intestinal epithelium. A similar phenomenon was observed in cultured 3T3 cells. Cultures of 3T3 cells that were actively proliferating were found to have greater amounts of the receptor than their nonproliferating counterparts. When the various tissues were examined for hyaluronate, it was found to have a widespread distribution, being present in most of the basement membranes and between the cells in stratified epithelium. Indeed, in many cases, the distribution of hyaluronate closely paralleled that of the hyaluronate receptor. These results suggest that the interaction between hyaluronate and its receptor is involved in cell-to-substratum adhesion.     

Distribution of phosphodiesterase I in normal human tissues

Phosphodiesterase I (PDE I) is an exonuclease capable of hydrolyzing a variety of phosphate ester and pyrophosphate bonds. Cell fractionation and histochemical studies in animal tissues have localized PDE I in the plasma membrane of various epithelia. This suggests a role for the enzyme in active transport. Distribution of PDE I in human tissues has not previously been studied. We have produced a polyclonal antiserum to bovine intestinal PDE I and have demonstrated crossreactivity with the human intestinal enzyme. This polyclonal antiserum was used in PAP immunocytochemistry to localize immunoreactive PDE I in a variety of human tissues. Localization was prominent in the gastrointestinal tract, including the cytoplasm of gastric mucosa parietal cells, cytoplasm of surface epithelium and isolated crypt cells in small intestine, and the colonic epithelial cytoplasm and brush border. Parotid gland acinar cells and scattered ductal cells showed positive cytoplasmic staining. Acinar and scattered pancreatic islet cells contained immunoreactive PDE I, as did Kupffer cells of the liver sinusoids. Immunoreactive PDE I was found in all vascular endothelia. The epithelium of the urinary tract showed extensive immunoreactivity. This included the distal convoluted and collecting tubules of the kidney, and ureteral and bladder urothelium. In previous histochemical studies of animal tissues, no evidence of PDE I activity was noted in male or female reproductive tract. In this study, immunoreactive PDE I was localized to human Sertoli cells and to basal epithelium of the epididymis and prostate acini. Fallopian tube epithelium of female reproductive tract also demonstrated immunoreactive PDI I, as did several cell types in term placenta. Our immunocytochemical results with human tissues differ significantly from previous histochemical studies in animal tissues, principally in the genitourinary system. This may be due in part to the different detection systems employed as well as the higher sensitivity of the immunoperoxidase technique. This underscores the importance of adjunct techniques in tissue surveys. The widespread epithelial distribution of immunoreactive PDE I detected by this polyclonal antibody implies an integral role in cell function, probably in active transport.     

Albumin and collagen mRNA expression in normal and analbuminemic rodent liver: analysis by in situ hybridization using biotinylated probes

We used in situ nucleic acid hybridization cytochemistry to examine cell types and subcellular sites expressing albumin (alb) or pro alpha 2 collagen (col) mRNA in livers from normal and analbuminemic rodents. Biotinylated cDNA or RNA probes were applied to aldehyde-fixed, non-frozen sections and the resulting DNA-RNA or RNA-RNA hybrids were subsequently visualized by enzymatic detection of either peroxidase or alkaline phosphatase conjugated to anti-biotin IgG or streptavidin. In normal rat liver, alb mRNA was expressed in all hepatocytes and was localized to discrete subcellular structures distributed as aggregates in the cytoplasm and in specific structures encircling the nucleus; these subcellular structures most likely represent the endoplasmic reticulum and nuclear envelope. In mouse liver, pro alpha 2 col mRNA was identified in a subpopulation of sinusoidal lining cells which have the morphological appearance of lipocytes. In liver from analbuminemic rats, a small number of hepatocytes, distributed throughout the hepatic lobule, expressed alb mRNA at high levels; the subcellular distribution of this alb mRNA was essentially identical to that observed in normal rat hepatocytes. Since non-radioactive in situ hybridization detected mRNA within the boundaries of individual cells and showed its precise subcellular location under conditions in which there was excellent preservation of tissue morphology, this procedure should be useful for a wide variety of histopathologic studies.     

Covalent affinity labeling, radioautography, and immunocytochemistry localize the glucocorticoid receptor in rat testicular Leydig cells

The presence and distribution of glucocorticoid receptors in the rat testis were examined by using 2 approaches: in vivo quantitative radioautography and immunocytochemistry. Radioautographic localization was made possible through the availability of a glucocorticoid receptor affinity label, dexamethasone 21-mesylate, which binds covalently to the glucocorticoid receptor, thereby preventing dissociation of the steroid-receptor complex. Adrenalectomized adult rats were injected with a tritiated (3H) form of this steroid into the testis and the tissue was processed for light-microscope radioautography. Silver grains were observed primarily over the Leydig cells of the interstitial space and to a lesser extent, over the cellular layers which make up the seminiferous epithelium, with no one cell type showing preferential labeling. To determine the specificity of the labeling, a 25- or 50-fold excess of unlabeled dexamethasone was injected simultaneously with the same dose of (3H)-dexamethasone 21-mesylate. In these control experiments, a marked reduction in label intensity was noted over the Leydig as well as tubular cells. Endocytic macrophages of the interstitium were non-specifically labeled, indicating uptake of the ligand possibly by fluid-phase endocytosis. A quantitative analysis of the label confirmed the presence of statistically significant numbers of specific binding sites for glucocorticoids in both Leydig cells and the cellular layers of the seminiferous epithelium; 86% of the label was found over Leydig cells, and only 14% over the cells of the seminiferous epithelium. These binding data were confirmed by light-microscope immunocytochemistry using a monoclonal antibody to the glucocorticoid receptor.(ABSTRACT TRUNCATED AT 250 WORDS)