Abstract
The enzyme rhodanese (EC 2.8.1.1) is an ubiquitous enzyme which is present in all living organisms, from bacteria to man. It is speculated that this enzyme plays a central role in cyanide detoxification. However, its wide tissue distribution suggests this enzyme might perform other functions beside cyanide detoxification. Although the distribution of rhodanese in different tissues of human and domestic animals has been studied, little is known about the pattern of distribution and physiological roles of this enzyme in the cat. The purpose of this investigation was to determine the enzyme levels and compare the distribution of this enzyme in different tissues of the cat. A selection of tissue samples was assayed for rhodanese activity. The protein content of tissue extracts and enzymatic activities were calculated as units per gram tissue and units per milligram protein of the tissue. Results showed that in terms of units per milligram protein of the tissue (specific activity of the enzyme), colon and rectum mucosal layers and testis were the richest sources of the enzyme followed by ovary, mucosal layer of jejunum and liver. With respect to units/gram tissue, liver followed by testis, colon and rectum mucosal layers, ovary and mucosa of jejunum exhibited highest activities. The results of this study will allow one to speculate on the involvement of rhodanese in several biochemical and physiological functions in different tissues and organs of this species.
The enzyme rhodanese (EC 2.8.1.1., thiosulphate: cyanide sulphurtransferase) is an ubiquitous enzyme present in all living organisms from bacteria to man. 1–7 Rhodanese catalyses the cyanide-dependent cleavage of thiosulphate to form thiocyanate and sulphite. 8 The enzyme reacts with anions containing the sulphane-sulphur group, transferring the sulphur to thiophylic anions via a persulphide intermediate on sulphydryl group of the cysteine-247 residue at its active site. 9,10 It is believed that the primary function of rhodanese is cyanide detoxification, but its wide distribution in different tissues of domestic animals and human suggests that there may be other functions. 8,11–13 The other functions could include formation of iron sulphur center in proteins, 14 participation in energy metabolism, 15 a thioredoxin oxidase 16 and detoxification of hydrogen sulphide. 17 A physiological role has been suggested for a rhodanese-like protein in the biosynthesis of molybdenum cofactor in humans. 18,19 The presence of rhodanese has been detected in many tissues of animals and human. 3,5,6,8,11,20–24 No information is available on the presence and distribution of this multi-functional enzyme in different tissues of the domestic cat. The aim of this study is to determine and compare the pattern of distribution of rhodanese in various tissues of the adult male and female cat (Felis catus).
Materials and Methods
All chemicals were of analytical grade and were purchased from commercial sources. Organs and tissues samples were obtained at post mortem from 12 adult male and non-pregnant female domestic cats, killed by trauma and referred to the veterinary hospital within 0–1 h after death. All samples were transferred within 30 min to the laboratory on ice. Tissues were separated, stripped from fat and extraneous materials, washed with physiological saline and then blotted. Tissue extract were prepared by freezing in liquid nitrogen, homogenising with hand homogeniser and diluting the homogenate in nine parts of 0.025 M sodium phosphate buffer (pH=7.2). The suspensions were centrifuged for 15 min at 4000 × g in a high-speed refrigerated centrifuge (MSE, UK) and supernatants were used as the source of enzyme. Rhodanese was assayed by the modified method of Sorbo. 25 The reaction mixture contained 16.8 mM sodium thiosulphate, 40 mM glycine buffer, pH 9.2, 167 mM KCN and 50 μl enzyme solution in a final volume of 4.0 ml. The reaction was carried out for 15 min at 37°C and stopped by adding 0.5 ml 38% formaldehyde. In control tubes, formaldehyde was added before the addition of enzyme solution. The concentration of thiocyanate was determined as follows. 25 Samples were mixed with 1 ml ferric nitrate solution containing 0.025 g Fe(NO3)3·9H2O in 0.74 ml water and 0.26 ml concentrated nitric acid. After centrifuging the mixture to remove the interfering turbidity, the absorbence was measured at 460 nm against a blank containing all reagents. Instead of enzyme solution for the blank, 50 μl of water was used. The concentration of thiocyanate formed was obtained from a standard curve produced by treating solutions containing different concentrations of thiocyanate as described above. Samples whose rhodanese activity was too high or whose protein was too concentrated were appropriately diluted with 25 mM phosphate buffer. The units of enzyme activity were defined as micromole of thiocyanate formed per minute at pH 9.2 and 37°C. Total protein was assayed according to Lowry et al 26 using crystalline bovine serum albumin as a standard. Statistical analysis was performed by one-way analysis of variance and Duncan test using SPSS software for multiple comparisons of the means of rhodanese activity of different tissues.
Results
The rhodanese activity in the different cat tissues is shown in Table 1. All of the tissues studied contain rhodanese activity. In terms of specific activity, colon and rectum mucosal layer and testis were found to be the richest sources of this enzyme (P<0.05) followed by ovary, mucosal layer of jejunum and liver. When calculated in terms of unit/g tissue, liver followed by testis, mucosal layer of rectum, colon and jejunum, and ovary contain significantly higher rhodanese than other tissues. Other tissues with relatively high rhodanese activity were duodenal mucosa, tongue (whole tissue) and stomach mucosa. In rectum, colon, jejunum and duodenum, specific activity of rhodanese was significantly higher in mucosal layer than submucosal layers (P<0.05) but the differences were not significant in mucosal and submucosal layers of other parts of gastrointestinal tract. In the large intestine, specific activities of rhodanese were significantly higher than those of small intestine. Very low rhodanese activity was observed in diaphragm, skeletal muscle, lung, pancreas, kidney, spleen, heart and submucosal layers of jejunum and duodenum.
Mean (±SD) activity (units/mg protein) rhodanese activity in different tissues of cat *
Tissues were weighted after washing with normal saline and blotting with blotter paper.
Values with different superscripts are significantly different (P<0.05).
Discussion
Rhodanese activity showed a widespread distribution in all tissues studied but in comparision with other domestic animals, 7 rhodanese activities were lowest in cat. Rectum and colon mucosal layers had the highest level of the enzyme activity in terms of units/mg protein of the tissue.
During past 20 years, research in this laboratory has established a species-specific pattern of distribution of rhodanese in tissues of domestic animals. Depending on the tissues, rhodanese may perform different functions including cyanide detoxification. 5,11,17,27,28
In most animals studied the liver appears to be the richest source of rhodanese. 3,20 However, in ruminants the epithelial layer of different parts of the digestive tract contain high levels of rhodanese activity which in some cases exceeds that of the liver. 7 Both dogs 21 and cats have very high rhodanese activity in the mucosal layer of large intestine. The clinical significance of these findings can be discussed in following aspects:
A major source of cyanide in nature is cyanogenic glycosides. More than 2000 species of plants are known to contain cyanogenic glycoside. 27 The enzymatic action of microbial flora of digestive tract on these glycosides ingested through foodstuff liberates toxic hydrogen cyanide. 2,29 It has been suggested that the level of rhodanese in different tissues of animals is correlated with the level of exposure to cyanide. 2,30 In ruminants, high rhodanaese activity in the mucosa of gastrointestinal tract ensures detoxification of liberated HCN. In cats, however, this pattern of distribution is different. In the cat, part of cyanide detoxification is probably performed by rhodanese concentrated in the mucosa of small and large intestine. On the other hand, as an obligate carnivore, the cat is less likely to get exposed to cyanide through foodstuff than herbivorous animals, hence very low activity of rhodanese in these tissues of cat as compared with similar tissues of herbivores. Main detoxification is likely to take place in liver. Cat liver rhodanese activity reported in this study is approximately 1/130, 1/120- 1/70, 1/40, 1/40 1/15, 1/4 and 1/3 of that reported for sheep, cattle, goat, camel, horse, donkey, pig, dog and human, respectively. 7 Based on units/gram tissue calculations and considering the mass of liver, however, this organ contains large amount of rhodanese, and probably is more involved in cyanide toxicity than other organs.
It is reported that rhodanese in the large intestine is the principal enzyme involved in hydrogen sulphide detoxification which is produced normally in the intestine. 17 Therefore, this enzyme may have an important role in detoxifying hydrogen sulphide in the large intestine of cat and dog.
This study also demonstrated very high activity of rhodanese in testis and ovary of cat. High activity of this enzyme in these tissues might reflect the activity of rhodanese in formation of iron-sulphur proteins. 14 The high activity of rhodanese in testis and ovary of cat might be the consequence of its role in steroidogenesis via formation of iron-sulphur proteins. The components of cholesterol side-chain cleavage (SCC) enzyme complex which is involved in steroidogenesis are cytochrome P-450, the iron-sulphur proteins adrenodoxin, and NADPH:ISP reductase. 28 It also has been demonstrated that similar iron-sulphur proteins are involved in the oxidative cleavage of the cholesterol side-chain occurring in ovary and testis during steroidogenesis process. 31
Blood rhodanese activity in sheep and dog is elevated after experimentally-induced liver necrosis. 32 Therefore, it was suggested that serum rhodanese determination be used as a liver function test in animals. This test might be useful in clinic for following liver function in cat.
In summary, this study showed a widespread distribution of rhodanese activity in hepatic and extrahepatic tissues of the cat. The presence of rhodanese in various extrahepatic tissues suggests that this enzyme may be functional in many physiological activities in the cat. Future studies are needed to clarify the involvement of rhodanese in various physiological processes and pathophysiological conditions in cat.
Footnotes
Acknowledgement
This research was financially supported by grant number 86-GR-VT-11 of Shiraz University Research Council.