@Copyright 1987 by The Humana Press Inc. All fights of any nature, whatsoever, reserved.
0163-4984187/1400-0087502.60
Selenium-Mediated Biochemical Changes in Japanese Quails Tissue Uptake and Distribution of Injected VSSelenium Labeled Sodium Selenite in Relation to Dietary Selenium Status VASANTHY NARAYANASWAMI AND K. LALITHA*
Department of Chemistry, Indian Institute of Technology, /Vladras-600 036, India Received March 4, 1987; Accepted April 20, 1987
ABSTRACT The tissue uptake and distribution of injected [75Se]-sodium selenite as a variance with time and as influenced by dietary selenium status was followed in the tissues of Japanese quails, Coturnix coturnix japonica. Quails maintained on a low selenium semipurified (basal) diet and basal diets supplemented with 0.2 and 2.0 peru selenium as sodium selenite were injected intraperitonially with ~S" e as sodium selenite (2.8 microcuries). 1"he injected 75Se was monitored in blood, liver, kidney, heart, and testis at 24, 72, and 144 h after injection. Maximal uptake of the injected 7~Se was observed in tissues of quails maintained on basal diet. The uptake of 7~Se in tissues in general was determined by the dietary Se status. Among the organs studied, kidney had the maximal level of rSse, 0.2 ppm (tzg/g wet tissue) folk)wed by liver, testis, and heart, but testis had the maximal level when the level per milligram of protein was considered, about 3.0 ng/mg protein, followed by liver, kidney, and heart. About 10-20~ of the tissue 7~Se was located in the mitochondria and 50-60% in the post-mitochondrial supernatant fractions in all the organs at all dietary Se levels. Significant incorporation of >Se in the mitochondrial membrane was observed. The percent distribution ratio between the membrane and matrix fractions of the mitochondria remained constant at all diet*Author to whom all correspondence and reprint requests should be addressed. Biological Trace Element Research
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ary Se levels which, in liver was 65:35, in kidney 55:45, and in testis 75:25. However, in heart mitochondria, the distribution of 75Se between membrane and matrix varied with dietary Se status, the ratio being 82:18 in the basal group, and 72:28 and 41:59 in the 0.2 and 2.0 ppm Se-supplemented groups, respectively. This is indicative of a preferential uptake of 7SSe in the mitochondrial membrane in conditions of deficiency. About 40-60% of the mitochondrial membrane-associated 7:3Sewas released upon Triton treatment in all the organs. Of the membrane-bound 75Se, about 10-15% was acid-labile in liver and kidney and 25% in the heart tissue. Possibilities of tissue specific roles, especially in the heart mitochondrial membrane-related processes, are indicated for selenium. Index Entries: Selenium, distribution and uptake in tissues; 7SSelenium, subcellular distribution in Japanese quails; regulatory uptake of selenium; selenium in mitochondrial membrane; time-dependent uptake of selenium.
INTRODUCTION The relevance of Se in the mammalian systems has so far been chiefly attributed to its role in the enzyme glutathione peroxidase (GSHPx) (Glutathione: H202 oxidoreductase, EC 1.11.1.9), which acts to scavenge the potentially toxic products of hydroperoxide metabolism (1-3). The requirement of Se by the erythocyte GSHPx wasproved in the elegant study by Rotruck et al. who showed that injected 7SSe as sodium selenite coeluted with GSHPx in rats (4). A few selenoproteins of unidentified functions have also been isolated from mammalian systems. A selenoprotein of mol wt 10,000 D has been isolated from the muscle and heart of lambs (5) consisting of cytochrome C-type of chromophore (6). Burk and Gregory (7) have identified a selenoprotein through radiotracer studies in rat liver and plasma designated as 7SSe-P. Though a major fraction of tissue Se is located in GSHPx and the above-mentioned proteins, the other possible cellular targets for this element in the various tissues are yet unclear. The precise requirement of Se is in part a result of its role as a redox catalyst. Its unique chemical properties and the fact that it is a better nudeophile than sulfur offer exciting possibilities for various types of biochemical interactions in which Se could be involved. Our previous reports deal with the variation of tissue GSHPx in response to dietary Se in quails (8) and the involvement of Se in collagen metabolism (9). In view of the possible diverse interactions of Se, the tissue uptake, retention, distribution, and subcellular localization of injected 75Se in the various organs in Japanese quails maintained on different dietary Se status were followed and are reported here. The low Se semipurified diet earlier formulated in our laboratory (8) for creating an uncomplicated Se deficiency in quails was used in this study. Biological Trace Elernent Research
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MATERIALS AriD METHODS Acrylamide, methylene bisacrylamide, sodium selenite, N,N,N', N'-tetramethylethylenediamine (TEMED), ammonium persulfate, Tris, glycine, and Triton X-100 were obtained from Sigma Chemical Company, USA, and 75Se in the form of sodium selenite of specific activity 825 p,Ci was purchased from Bhaba Atomic Research Centre, Bombay. Other reagents were of AR grade from Merck or BDH. Deionized water was used throughout the studies.
Experimental
Setup
One-d-old Japanese quails weighing 6-8 g each were obtained from the Poultry Research Station, Madras, and divided at random into 3 groups of 18 each. One group was fed the low Se semipurified diet, to be referred to as the basal diet hereafter, while the other two groups were fed Se-supplemented diets at levels of 0.2 and 2.0 ppm. Deionized water was given ad libitum. At the end of 45 d, 7~Se in the form of sodium selenite (2.8 i~Ci, about 0.04 Ixg/g body wt) in isotonic saline was injected intraperitoneally to quails in all the groups. Six quails from each group were sacrified at the end of 24, 72, and 144 h after injection.
Preparation of Tissue Fractions The quails were decapitated and blood collected in heparinised tubes. The radioactivity in 1 mL fractions of blood was measured. Organs such as liver, kidney, heart, and testis were quickly excised, washed thoroughly in ice cold sucrose solution (0.25M), dried between folds of filter paper, weighed, and processed for mitochondrial and postmitochondrial supernatant fractions by differential centrifugation, as described earlier (8). The mitochondrial pellets were washed twice to remove adhering radioactivity and suspended uniformly in 0.25M sucrose, to give 1 mL suspension/g of tissue. Portions of homogenate, mitochondrial, and post-mitochondrial supernatant fractions (henceforth referred to as supernatant or soluble fractions) were measured for radioactivity.
Sonication of /qitochondfia The mitochondria were sonicated at 50 W and 4~ for 3 min with a 1-min interval, and centrifuged at 12,000g for 30 min. The supernatant, constituting the mitochondrial matrix released during sonication, and the membrane fragments in the pellet were separated and measured for radioactivity.
Treatment of/vb'tochondfial Membrane with Triton-X- 1 O0 The pellet consisting of the mitochondrial membrane was washed, suspended in 0.01M potassium phosphate buffer, pH 7.4, and measured Biological Trace Element Research
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for radioactivity. A portion of the membrane fraction was then incubated with Triton X-100 (0.1%) for 3 h at 4~ to release the membrane-bound 75Se, centrifuged at 12,000g for 30 rain and the pellet dispersed in phosphate buffer. The pellet and supernatant fractions were measured for radioactivity.
Acid Lability A portion of the membrane fraction was treated with conc. HC] to release the acid-labile 75Se and measured for radioactivity before and after treatment.
Ammonium Sulfate Precipitation The sonicated supernatant of mitochondrial fractions of each organ in the respective groups were pooled and ammonium sulfate was added to 80% saturation level with constant stirring in cold. The precipitated pellet obtained after centrifugation was dissolved in 0.02M phosphate buffer pH 7.4 and dialyzed overnight against 0.001M phosphate buffer pH 7.4 The dialysate from the three dietary Se groups were subjected to polyacrylamide gel electrophoresis (PAGE) and processed for autoradiography. The PAGE was performed by the method of Davis (10) with modifications (11) in a non-denaturing discontinuous system at alkaline pH in 7.5% resolving gel and 2.5% stacking gel with a current of 40 mA/ slab in a Biorad "Protean" model electrophoretic system with LKB model 2197 power supply. The gels were stained for protein in 0.25% Coomassie blue in acetic acid : methanol: water (10: 40: 50). The gels were fixed and dried in a slab gel dryer and exposed to X-ray film (Agfa Gaevert Curix RP! 50 AFW) in the dark. The films were subsequently developed and photographed. Radioactivity measurements were done with a gammaray spectrometer (Single Channel Analyzer, SC 60.4B) with NaI (TI) crystal detector at 630 V and expressed as counts per minute (cpm) per g tissue or per mg protein. Protein was measured by the method of Lowry et al. (12).
RESULTS AND DISCUSSION Tissue
Uptake and Distribution of 75Se
The tissue uptake, distribution, and cellular localization of injected
75Se in quails were assessed as a function of the dietary Se status and time. The level of 7SSe in blood, liver, kidney, heart, and testis in quails maintained on basal, 0.2 ppm, and 2.0 ppm Se-supplemented diets at 24, 72, and 144 h after injection is presented graphically as percentage of injected dose (Fig. 1). At 24 h after injection, maximal level of 75Se, about Biological Trace Element Research
Vol. 14, 1987
I,
.~
0
~
5.o
! V
"6
"r" .
"6
5.0
#_
o
B~
L
K
H
T
Fig. 1. Influence of dietary selenium on the distribution of injected 7SSelenium in different tissues at different time intervals. The different groups on basal, 0.2 p p m Se and 2.0 p p m Se supplemented diets are represented as A, B, and C, respectively. B*--blood, % uptake/mL); L--liver; K--kidney; H--heart; and T--testis. Level of 7SSelenium at 24, 72, and 144 h after injection are represented in graphs 1, 2, and 3, respectively. Biological Trace Element Research
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18% of injected dose was retained by blood and other tissues of the basal group of quails, while in the 0.2 ppm Se group, the amount was 13%, and in the 2.0 ppm Se group, about 4% of the injected dose. The total level of 75Se measured in these organs at 72 h decreased to 16, 12, and 3.8% of injected dose in basal, 0.2, and 2.0 ppm Se groups, respectively, while in 144 h, the corresponding levels were 14.6, 11.6, and 2.8%. The levels of 75Se in blood, liver, and kidney of the 0.2 ppm Se group were about 80% of the corresponding levels in basal group of quails, in heart it was nearly 65%, and in the testis about 70%. On the other hand, in the 2.0 ppm Se group, the level in blood was only 36% of that in basal group with liver, kidney, heart, and testis having about 15-25% of the level in basal group. The higher uptake of 75Se in the basal group is reflective of a higher tissue demand. The retention of the injected dose of 7SSe appeared to decrease when the tissue demand was satisfied by the dietary Se. The observed marginal difference in uptake between the basal and the 0.2 ppm Se group is indicative of the insufficiency of this dietary level to balance the requirement for Se in quails. On the other hand, the relatively low level of uptake of 75Se in the 2.0 ppm group, about 25% of that in basal, is probably the result of adequate maintenance levels in the tissue. The tissue level of Se thus mediates control on the uptake by the system, though the mode of regulation of this uptake is not clear.
Tissue Levels of
75Se
The level of 75Se in the various organs of quails at different dietary Se status is represented in Table 1. Considering the basal grou~, the order of decrease in the tissue level of 7~Se when expressed as t~g 7 Se/g wet tissue was kidney > liver > testis > heart, the levels being 0.2, 0.12, 0.07, and 0.035 ppm, respectively; when expressed as ng 7s# - ae]mg protein, the order was testis > liver > kidney > heart, the levels being 3.0, 1.34, 0.95, and 0.58 ng/mg protein, respectively. However, the tissue distribution of the selenoenzyme glutathione peroxidase that is considered to be the major target for Se did not correlate with the level of 75Sein these tissues. This enzyme activity was in the decreasing order of liver > kidney > heart > testis. From this trend, it could be surmised that there are other possible functions for Se besides its well-established role at the catalytic center of GSHPx, which would be more relevant in kidney as it appears to have a tendency to accumulate Se. A recent report that appeared during the course of the present investigation indicates that there are high levels of Se in the kidney of pony stallions also (13). The greater demand for Se in testis, observed during the present study, is consistent with previous reports where Se was found to accumulate, especially in conditions of its deficiency (14,15). In testis, 75Se has been observed to be selectively incorporated into a specific polypeptide -
Biological Trace Element Research
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TABLE 1 Uptake of 7sSE in Tissues of Quails in Relation to Dietary Selenium Levels ~
Uptake level of 75Se in different groups~' Tissue
Basal
Liver Kidney Heart Testis
0.130 0.200 0.038 0.072
Liver Kidney Heart Testis
1.34 0.95 0.58 3.10
0.2 ppm Se
2.0 ppm Se
I~g/g tissue 0.110 0.150 0.024 0.040
0.020 0.030 0.009 0.008
ng/mg protein 1.04 0.76 0.32 1.30
0.21 0.19 0.12 0.20
~ are expressed as average of three sets of experiments with six quails in each group. ~Level of 75Se measured 72 h after single injection of (75Se) sodium selenite.
of 17,000 D, not related to GSHPx (16). Among other aspects of the role of Se in testis is its function in sperm motility, as shown by studies on Se-deficient rats, which produce sperms with impaired motility and characteristic midpiece damage (17), and which is also revealed by investigations of the associated ultrastructural changes (18).
Variation of Tissue 75Se with Time The level of 75Se as a variance with time in tissues of quails maintained on different dietary Se status is represented in Fig. 2. In the basal group, the initial high uptake of 75Se in 24 h was followed by a decrease of 18% in 72 h in blood and other organs except liver, where the decrease was about 12%; the decline in tissue level in 144 h was negligible, reflecting a tendency to stabilize in 72-144 h. In the marginall~, supplemented 0.2 ppm Se group, a gradual trend of decrease in tissue 73Se by about 5% in 72 h and about 10% in 144 h was observed both in blood and liver, whereas the decrease in kidney and heart was to a greater extent, about 20 and 35% in 72 and 144 h, respectively. A similar pattern of elimination of 75Se was exhibited by the 2.0 ppm Se group also. The initial high uptake of 75Se by the basal group indicates a higher demand by the depleted tissues, which later stabilizes in 72-144 h. Retention and elimination patterns of injected 7-~Se vary with each organ and depend on the dietary Se status to a large extent. The tissue level of Se is also determined by the dose and mode of administration, and by the form of dietary Se (19). A faster turnover and higher requirement of Se in testis are indicated from the present study. Biological Trace Element Research
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Harayanaswami and Lafitha
94 BLOOD
HEART
0.8
0.81
0.6
0.61
LIVER
-o
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0.4~
0.4
2.4 0.2t
0.2
oI
0
I
t
t
t
01
2.0
I
1
TESTIS
KIDNEY
4.0
16
~ 1.6
3.0
1.2
1.2
2.0
0-8
0.8
0.4
0-4
A
(L
I
o
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1.0
I
24
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I
+
I
72 TIME IN HOURS
I
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L
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72 TIME IN HOURS
[
144
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--M I
24
l
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72 TIME IN HOURS
1
144
Fig. 2. Tissue uptake and retention of 75Selenium with time in different tissues. Dietary groups A, B, and C are as described in Fig. 1 and are indicated by 9 A; X, B; and G, C.
Subcellular Dist#bution of 75Se The distribution of 75Se in the mitochondrial and post-mitochondrial supernatant fractions of the tissues of quails reared on different dietary Se status was assessed. A general pattern of distribution emerged, with about 12-20% of the9 cellular 7 59Se located in the mitochondria, and about 50-60% associated with post-mitochondrial supernatant fractions; the nuclear fractions and cell debris had about 10% of the cellular 75Se. It is estimated that the yield of mitochondria in the present study was about 60% only; thus the actual level of 5Se in this organelle is likely to be higher. This uniform subcellular distribution was observed with respect to all the organs and at all dietary Se levels. In rats maintained on chow diet, it has been reported (20) that about 58% of the injected 75Se in liver was located in the cytosol, 15% in the mitochondria, and about 8% in the microsomes. A major fraction of the liver cytosolic 75Se could be associated with glutathione peroxidase. Selenium i n d e p e n d e n t glutathione peroxidases have also been reported in liver of rats (21). Selenium has also been shown to be located in the mitochondrial form of GSHPx (22). In microsomes, it has been suggested Biological Trace Element Research
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Subceilular Distribution of 75Sein Tissues
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that Se could be associated with the electron transport systems (23,24). From the reports of the occurrence of Se in different organelles, it is possible that specific mechanisms are operative for the uptake and functionalization of Se in different compartments.
Intramitochondrial Distribution of 75Selenium The intramitochondrial distribution of 75Se in various tissues of quails is represented in Table 2. The mitochondria were sonicated in order to release the contents of the matrix. In liver, mitochondrial membrane of the basal group had the maximal level of incorporated 75Se, followed by the 0.2 ppm Se group having 40%, and the 2.0 ppm Se group about 28% of that in basal. However, the ratio of distribution between the membrane and matrix remained unaltered in all dietary Se groups, the membrane having about 65% and the matrix about 35% of the total amount of incorporated 75Se in mitochondria. Similarly, in kidney the distribution ratio of 75Se in the mitochondria remained constant, being about 55% in the membrane and 45% in matrix. The basal group had maximal level of incorporated Se in mitochondrial membrane; the level in the 0.2 ppm Se group was about 61% of that in the basal group, whereas that in the 2.0 ppm Se group was only about 12%. TABLE 2 Intra-mitochondrial Distribution of 75Seleniumin Tissues of Quails Distribution of 75Se in mitochondria/' percentage of basal' Organ Liver Kidney Heart Testis
Group" A B C A B C A B C A B C
Membrane fraction 66 63 66 59 53 62 82 72 41 76 74 75
(40) (27) (61) (12)
(47) (7) (84) (20)
Matrix 34 37 34 41 47 38 18 28 59 24 26 25
(47) (28) (77) (11)
(85) (45) (100) (21)
"Groups A, B, and C represent the quails maintained on basal, 0.2 ppm Se, and 2.0 ppm Se-supplemented diet, respectively. ~Percent distribution ratio in mitochondrial fractions of tissues (having the sum of counts in membrane and matrix as 100) at 72 h after injection. q~he numbers indicated in parentheses represent the percent of actual counts per rain in the fractions in the respective groups. Biological Trace Element Research
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In testis also, the percent distribution ratio of 75Se between the mitochondrial membrane and matrix was constant at 75:25, irrespective of the dietary Se status. From the observed consistent pattern of the distribution ratio elicited by mitochondria of various tissues, a distinct deviation was noted in the case of heart mitochondria of quails wherein this ratio altered with dietary Se level. In the basal group, about 82% of the total mitochondrial 75Se was located in the membrane, the remaining 18% in the matrix, whereas the percent distribution ratio in the 0.2 p p m and 2.0 ppm Se groups were 72: 28 and 41 : 59, respectively. A highly specific requirement of Se in the heart mitochondrial membrane-related processes is strongly implied from high level of 75Se incorporation in this fraction in the quails of the basal group. Efforts were made to release the mitochondrial membrane bound 75Se by Triton treatment. The results are presented in Table 3. In the mitochondrial membrane of liver, kidney, and heart about 40-60% of the m e m b r a n e bound 75Se was released upon Triton treatment whereas in testis 60-70% was released. In liver and kidney mitochondria, about 15% of the membrane bound 75Se was found to be acid labile, whereas in heart the lability was significantly higher, being about 25%. Gel electrophoretic studies on the a m m o n i u m sulfate precipitated proteins from mitochondrial matrix fractions of all the tissues from quails of different dietary Se groups were carried out. Upon autoradiography (Fig. 3), a single zone due to 7SSe labeled protein (indicated by arrow) TABLE 3 Effect of Triton Treatment on 75SE Associated With Mitochondrial Membrane Fractions of Tissues Percent of 7SSe~ Organ
Group
Liver
A
Triton released Membrane bound
Kidney
C A
Heart
C A
43 41 34 43 34 60 33
B
28
B
B
57 59 66 57 66 40 67 62
C A
52 48 Testis 71 29 B 67 33 C 57 43 ~After Triton treatment, 7SSereleased in solution and remaining bound to membrane are presented as percent of total counts per rain in the mitochondrial membrane before treatment. Details as described in Table 2. Biological Trace Element Research
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Fig. 3. Electrophoretic detection of 7~Selenoprotein in heart mitochondria. 3A: Protein profile. 3B: Autoradiographic pattern. Lane 1--basal group; lane 2---2.0 ppm Se supplemented group. Zone of 7~Selenium containing moiety is indicated by arrow. Other details as described in text. appeared in the heart samples of the basal group only. (The protein profiles from samples of other organs are not shown.) The probable occurrence of different seleno-moieties and a faster turnover of Se would account for the nonappearance of a concentrated 7SSe zone in the other tissues examined. In the heart, it appears that the requirement of Se by a single protein is significantly high, and is especially enhanced in deficiency conditions, thereby resulting in concentration of 75Se in this protein when given to the Se-deficient group. This has made the detection possible after autoradiography. This unidentified Se-containing protein needs to be studied in greater detail. The nature of the membrane-bound 75Se moiety and the matrix protein containing 75Se are not clear from the present studies, also, the possible partial extraction of membrane components by treatment with Triton and other solubilizing agents need further investigation.
GENERAL SUMMARY AND CONCLUSIONS The higher retention of injected 7SSe by the basal group was clearly indicative of a greater tissue demand in conditions of deprivation. The Biological Trace Elernent Research
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Narayanaswami and Lalitha
minimal retention in the 2.0 ppm Se supplemented group substantiates this view. A regulated uptake and distribution of the injected 7SSe as a function of dietary Se was thus observed. It has emerged from the present study that a dietary Se level of 0.2 ppm was insufficient to meet the tissue demand for Se, while 2.0 ppm Se level was found adequate to meet the requirement of this element in quails. The cellular and organelle localization of 7SSe also appeared to be constant at all dietary Se levels. The high level of tissue Se in kidney and testis could have interesting implications as to their functional role. The significant incorporation of 75Se in the mitochondrial membrane could indicate possible involvement of Se in energy-related processes in the membranes of this organelle. This is interesting in view of the key roles of sulfhydryl moieties in the mitochondrial respiratory phenomenon (25,26) and the possible targets for Se in the membrane aiding or accelerating electron transport and oxygen utilization. Incidentally, it has been shown in our laboratory that in Se deficiency the mitochondrial energy metabolism is subdued, both in terms of oxygen uptake and energy-dependent ion uptake (27). Acid lability of membrane-bound 75Se in mitochondria could be explained by its possible association with nonheme iron proteins. Core substitution of Se for sulphur in the iron-sulfur centers of ferrodoxins from parsley has been reported, and such derivatized molecules have been found to possess biological activity (28). The stoichiometric substitution of Se in the core or ligands of iron-sulfur proteins, in vivo in the mitochondrial membrane, though being an attractive possibility, is not substantiated by the actual amount of Se present and requires further consideration, especially since other acid-labile seleno-moieties could also occur. It has also been reported that Se was found in muscle selenoprotein in lambs (5,6) with a cytochrome C-type of chromophore. The abundance of such redox centers in the membranes appears to offer ideal locations for the multifaceted transient interactions of Se which albeit kinetically unstable are thermodynamically feasible.
REFERENCES 1. L. Flohe, (1982) Free Radicals in Biology, Vol. 5, Academic, NY, pp. 223-249. 2. L. Flohe, (1979), Oxygen Free Radicalsand Tissue Damage, Ciba Found. Syrup., no. 65, Excerta Med. Found., Amsterdam, p. 95. 3. A. Wendel, (1980), Enzymatic Basis of Detoxication, vol. 1, Academic, NY, pp. 333-353. 4. J. T. Rotruck, A. L. Pope, H. E. Ganther, A. B. Swanson, D. G. Hafeman, and W. G. Hoekstra, (1973), Science, 179, 588. 5. N. D. Pedersen, P. D. Whanger, P. H. Weswig, and O. H. Muth, (1972), Bioinorg. Chem. 2, 33. 6. P. D. Whanger, N. D. Pedersen, and P. H. Weswig, (1973), Biochem. Biophys. Res. Commun. 53, 1031. 7. R. F. Burk, and P. E. Gregory, (1982), Arch. Biochem. Biophys. 213, 73. Biological Trace Element Research
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8. N. Vasanthy, R. Padma Bai, M. Babu, and K. Lalitha, (1986), Biol. Trace Elem. Res. 10, 79. 9. M. Babu, R. Padma Bai, N. Vasanthy, K. Lalitha, and K. T. Joseph, (1986), Biol. Trace Elem. Res. 10, 317. 10. B. J. Davis, (1964), Ann. N Y Acad. Sci., 121, 404. 11. P. J. Blackshear, (1984), Meth. Enzymol. 104, 237. 12. O. H. Lowry, N. J. Rosebrough, A. L. Farr, and R. J. Randals, (1951), J. Biol. Chem. 193, 265. 13. E. D. Heimann, J. S. Morris, and W. E. Loch, (1985), Nutr. Rep. Inter., 32, 1153. 14. D. Behne, and T. Hofer, (1982), J. Nutr. 112, 1082. 15. W. G. Hoekstra, D. Hafeman, S. H. Oh, and H. E. Ganther, (1973), Fed. Proc. 32, 385. 16. H. I. Calvin, (1978), J. Exp. Zool., 204, 445. 17. A. S. M. Wu, J. E. Oldfield, L. R. Shull, and P. F. Cheeke, (1979), Biol. Reprod. 20, 793. 18. E. Wallace, H. I. Calvin, and G. W. Cooper, (1983), Gamete Research, 4, 377. 19. J. C. Hansen, and P. Kristensen, (1979), J. Nutr., 109, 1223. 20. F. H. Stults, J. W. Forstrom, D. T. Y. Chiu, and A. L. Tappel, (1977), Arch. Biochem. Biophys. 183, 490. 21. R. D. Lawrence, and R. F. Burk, (1975), Biochem. Biophys. Res. Commun., 71, 952. 22. J. J. Zakowski, and A. L. Tappel, (1976), Biochim. Biophys. Acta. 445, 558. 23. C. P. J. Caygill, and A. T. Diplock, (1973), FEBS Lett., 33, 172. 24. C. P. J. Caygill, A. T. Diplock, and E. H. Jeffery, (1973), Biochem. J. 136, 851. 25. N. Haugaard, N. H. Lee, R. Kostrzewa, R. S. Horn, and E. S. Haugaard, (1969), Biochim. Biophys. Acta., 172, 198. 26. S. Minakami, F. Schindler, and R. W. Estabrook, (1964), J. Biol. Chem., 239, 2042. 27. N. Vasanthy, K. Lalitha, and B. Chance, Arch. Biochem. Biophys. (Communicated). 28. J. A. Fee, and G. Palmer, (1971), Biochim. Biophys. Acta., 245, 175.
Biological Trace Element Research
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0163-4984187/1400-0087502.60
Selenium-Mediated Biochemical Changes in Japanese Quails Tissue Uptake and Distribution of Injected VSSelenium Labeled Sodium Selenite in Relation to Dietary Selenium Status VASANTHY NARAYANASWAMI AND K. LALITHA*
Department of Chemistry, Indian Institute of Technology, /Vladras-600 036, India Received March 4, 1987; Accepted April 20, 1987
ABSTRACT The tissue uptake and distribution of injected [75Se]-sodium selenite as a variance with time and as influenced by dietary selenium status was followed in the tissues of Japanese quails, Coturnix coturnix japonica. Quails maintained on a low selenium semipurified (basal) diet and basal diets supplemented with 0.2 and 2.0 peru selenium as sodium selenite were injected intraperitonially with ~S" e as sodium selenite (2.8 microcuries). 1"he injected 75Se was monitored in blood, liver, kidney, heart, and testis at 24, 72, and 144 h after injection. Maximal uptake of the injected 7~Se was observed in tissues of quails maintained on basal diet. The uptake of 7~Se in tissues in general was determined by the dietary Se status. Among the organs studied, kidney had the maximal level of rSse, 0.2 ppm (tzg/g wet tissue) folk)wed by liver, testis, and heart, but testis had the maximal level when the level per milligram of protein was considered, about 3.0 ng/mg protein, followed by liver, kidney, and heart. About 10-20~ of the tissue 7~Se was located in the mitochondria and 50-60% in the post-mitochondrial supernatant fractions in all the organs at all dietary Se levels. Significant incorporation of >Se in the mitochondrial membrane was observed. The percent distribution ratio between the membrane and matrix fractions of the mitochondria remained constant at all diet*Author to whom all correspondence and reprint requests should be addressed. Biological Trace Element Research
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ary Se levels which, in liver was 65:35, in kidney 55:45, and in testis 75:25. However, in heart mitochondria, the distribution of 75Se between membrane and matrix varied with dietary Se status, the ratio being 82:18 in the basal group, and 72:28 and 41:59 in the 0.2 and 2.0 ppm Se-supplemented groups, respectively. This is indicative of a preferential uptake of 7SSe in the mitochondrial membrane in conditions of deficiency. About 40-60% of the mitochondrial membrane-associated 7:3Sewas released upon Triton treatment in all the organs. Of the membrane-bound 75Se, about 10-15% was acid-labile in liver and kidney and 25% in the heart tissue. Possibilities of tissue specific roles, especially in the heart mitochondrial membrane-related processes, are indicated for selenium. Index Entries: Selenium, distribution and uptake in tissues; 7SSelenium, subcellular distribution in Japanese quails; regulatory uptake of selenium; selenium in mitochondrial membrane; time-dependent uptake of selenium.
INTRODUCTION The relevance of Se in the mammalian systems has so far been chiefly attributed to its role in the enzyme glutathione peroxidase (GSHPx) (Glutathione: H202 oxidoreductase, EC 1.11.1.9), which acts to scavenge the potentially toxic products of hydroperoxide metabolism (1-3). The requirement of Se by the erythocyte GSHPx wasproved in the elegant study by Rotruck et al. who showed that injected 7SSe as sodium selenite coeluted with GSHPx in rats (4). A few selenoproteins of unidentified functions have also been isolated from mammalian systems. A selenoprotein of mol wt 10,000 D has been isolated from the muscle and heart of lambs (5) consisting of cytochrome C-type of chromophore (6). Burk and Gregory (7) have identified a selenoprotein through radiotracer studies in rat liver and plasma designated as 7SSe-P. Though a major fraction of tissue Se is located in GSHPx and the above-mentioned proteins, the other possible cellular targets for this element in the various tissues are yet unclear. The precise requirement of Se is in part a result of its role as a redox catalyst. Its unique chemical properties and the fact that it is a better nudeophile than sulfur offer exciting possibilities for various types of biochemical interactions in which Se could be involved. Our previous reports deal with the variation of tissue GSHPx in response to dietary Se in quails (8) and the involvement of Se in collagen metabolism (9). In view of the possible diverse interactions of Se, the tissue uptake, retention, distribution, and subcellular localization of injected 75Se in the various organs in Japanese quails maintained on different dietary Se status were followed and are reported here. The low Se semipurified diet earlier formulated in our laboratory (8) for creating an uncomplicated Se deficiency in quails was used in this study. Biological Trace Elernent Research
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Subceilular Distribution of 75Sein Tissues
89
MATERIALS AriD METHODS Acrylamide, methylene bisacrylamide, sodium selenite, N,N,N', N'-tetramethylethylenediamine (TEMED), ammonium persulfate, Tris, glycine, and Triton X-100 were obtained from Sigma Chemical Company, USA, and 75Se in the form of sodium selenite of specific activity 825 p,Ci was purchased from Bhaba Atomic Research Centre, Bombay. Other reagents were of AR grade from Merck or BDH. Deionized water was used throughout the studies.
Experimental
Setup
One-d-old Japanese quails weighing 6-8 g each were obtained from the Poultry Research Station, Madras, and divided at random into 3 groups of 18 each. One group was fed the low Se semipurified diet, to be referred to as the basal diet hereafter, while the other two groups were fed Se-supplemented diets at levels of 0.2 and 2.0 ppm. Deionized water was given ad libitum. At the end of 45 d, 7~Se in the form of sodium selenite (2.8 i~Ci, about 0.04 Ixg/g body wt) in isotonic saline was injected intraperitoneally to quails in all the groups. Six quails from each group were sacrified at the end of 24, 72, and 144 h after injection.
Preparation of Tissue Fractions The quails were decapitated and blood collected in heparinised tubes. The radioactivity in 1 mL fractions of blood was measured. Organs such as liver, kidney, heart, and testis were quickly excised, washed thoroughly in ice cold sucrose solution (0.25M), dried between folds of filter paper, weighed, and processed for mitochondrial and postmitochondrial supernatant fractions by differential centrifugation, as described earlier (8). The mitochondrial pellets were washed twice to remove adhering radioactivity and suspended uniformly in 0.25M sucrose, to give 1 mL suspension/g of tissue. Portions of homogenate, mitochondrial, and post-mitochondrial supernatant fractions (henceforth referred to as supernatant or soluble fractions) were measured for radioactivity.
Sonication of /qitochondfia The mitochondria were sonicated at 50 W and 4~ for 3 min with a 1-min interval, and centrifuged at 12,000g for 30 min. The supernatant, constituting the mitochondrial matrix released during sonication, and the membrane fragments in the pellet were separated and measured for radioactivity.
Treatment of/vb'tochondfial Membrane with Triton-X- 1 O0 The pellet consisting of the mitochondrial membrane was washed, suspended in 0.01M potassium phosphate buffer, pH 7.4, and measured Biological Trace Element Research
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Narayanaswami and Lalitha
90
for radioactivity. A portion of the membrane fraction was then incubated with Triton X-100 (0.1%) for 3 h at 4~ to release the membrane-bound 75Se, centrifuged at 12,000g for 30 rain and the pellet dispersed in phosphate buffer. The pellet and supernatant fractions were measured for radioactivity.
Acid Lability A portion of the membrane fraction was treated with conc. HC] to release the acid-labile 75Se and measured for radioactivity before and after treatment.
Ammonium Sulfate Precipitation The sonicated supernatant of mitochondrial fractions of each organ in the respective groups were pooled and ammonium sulfate was added to 80% saturation level with constant stirring in cold. The precipitated pellet obtained after centrifugation was dissolved in 0.02M phosphate buffer pH 7.4 and dialyzed overnight against 0.001M phosphate buffer pH 7.4 The dialysate from the three dietary Se groups were subjected to polyacrylamide gel electrophoresis (PAGE) and processed for autoradiography. The PAGE was performed by the method of Davis (10) with modifications (11) in a non-denaturing discontinuous system at alkaline pH in 7.5% resolving gel and 2.5% stacking gel with a current of 40 mA/ slab in a Biorad "Protean" model electrophoretic system with LKB model 2197 power supply. The gels were stained for protein in 0.25% Coomassie blue in acetic acid : methanol: water (10: 40: 50). The gels were fixed and dried in a slab gel dryer and exposed to X-ray film (Agfa Gaevert Curix RP! 50 AFW) in the dark. The films were subsequently developed and photographed. Radioactivity measurements were done with a gammaray spectrometer (Single Channel Analyzer, SC 60.4B) with NaI (TI) crystal detector at 630 V and expressed as counts per minute (cpm) per g tissue or per mg protein. Protein was measured by the method of Lowry et al. (12).
RESULTS AND DISCUSSION Tissue
Uptake and Distribution of 75Se
The tissue uptake, distribution, and cellular localization of injected
75Se in quails were assessed as a function of the dietary Se status and time. The level of 7SSe in blood, liver, kidney, heart, and testis in quails maintained on basal, 0.2 ppm, and 2.0 ppm Se-supplemented diets at 24, 72, and 144 h after injection is presented graphically as percentage of injected dose (Fig. 1). At 24 h after injection, maximal level of 75Se, about Biological Trace Element Research
Vol. 14, 1987
I,
.~
0
~
5.o
! V
"6
"r" .
"6
5.0
#_
o
B~
L
K
H
T
Fig. 1. Influence of dietary selenium on the distribution of injected 7SSelenium in different tissues at different time intervals. The different groups on basal, 0.2 p p m Se and 2.0 p p m Se supplemented diets are represented as A, B, and C, respectively. B*--blood, % uptake/mL); L--liver; K--kidney; H--heart; and T--testis. Level of 7SSelenium at 24, 72, and 144 h after injection are represented in graphs 1, 2, and 3, respectively. Biological Trace Element Research
VoL 14, 1987
blarayanaswami and Lafitha
92
18% of injected dose was retained by blood and other tissues of the basal group of quails, while in the 0.2 ppm Se group, the amount was 13%, and in the 2.0 ppm Se group, about 4% of the injected dose. The total level of 75Se measured in these organs at 72 h decreased to 16, 12, and 3.8% of injected dose in basal, 0.2, and 2.0 ppm Se groups, respectively, while in 144 h, the corresponding levels were 14.6, 11.6, and 2.8%. The levels of 75Se in blood, liver, and kidney of the 0.2 ppm Se group were about 80% of the corresponding levels in basal group of quails, in heart it was nearly 65%, and in the testis about 70%. On the other hand, in the 2.0 ppm Se group, the level in blood was only 36% of that in basal group with liver, kidney, heart, and testis having about 15-25% of the level in basal group. The higher uptake of 75Se in the basal group is reflective of a higher tissue demand. The retention of the injected dose of 7SSe appeared to decrease when the tissue demand was satisfied by the dietary Se. The observed marginal difference in uptake between the basal and the 0.2 ppm Se group is indicative of the insufficiency of this dietary level to balance the requirement for Se in quails. On the other hand, the relatively low level of uptake of 75Se in the 2.0 ppm group, about 25% of that in basal, is probably the result of adequate maintenance levels in the tissue. The tissue level of Se thus mediates control on the uptake by the system, though the mode of regulation of this uptake is not clear.
Tissue Levels of
75Se
The level of 75Se in the various organs of quails at different dietary Se status is represented in Table 1. Considering the basal grou~, the order of decrease in the tissue level of 7~Se when expressed as t~g 7 Se/g wet tissue was kidney > liver > testis > heart, the levels being 0.2, 0.12, 0.07, and 0.035 ppm, respectively; when expressed as ng 7s# - ae]mg protein, the order was testis > liver > kidney > heart, the levels being 3.0, 1.34, 0.95, and 0.58 ng/mg protein, respectively. However, the tissue distribution of the selenoenzyme glutathione peroxidase that is considered to be the major target for Se did not correlate with the level of 75Sein these tissues. This enzyme activity was in the decreasing order of liver > kidney > heart > testis. From this trend, it could be surmised that there are other possible functions for Se besides its well-established role at the catalytic center of GSHPx, which would be more relevant in kidney as it appears to have a tendency to accumulate Se. A recent report that appeared during the course of the present investigation indicates that there are high levels of Se in the kidney of pony stallions also (13). The greater demand for Se in testis, observed during the present study, is consistent with previous reports where Se was found to accumulate, especially in conditions of its deficiency (14,15). In testis, 75Se has been observed to be selectively incorporated into a specific polypeptide -
Biological Trace Element Research
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Subcellular Distribution of 75Sein Tissues
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TABLE 1 Uptake of 7sSE in Tissues of Quails in Relation to Dietary Selenium Levels ~
Uptake level of 75Se in different groups~' Tissue
Basal
Liver Kidney Heart Testis
0.130 0.200 0.038 0.072
Liver Kidney Heart Testis
1.34 0.95 0.58 3.10
0.2 ppm Se
2.0 ppm Se
I~g/g tissue 0.110 0.150 0.024 0.040
0.020 0.030 0.009 0.008
ng/mg protein 1.04 0.76 0.32 1.30
0.21 0.19 0.12 0.20
~ are expressed as average of three sets of experiments with six quails in each group. ~Level of 75Se measured 72 h after single injection of (75Se) sodium selenite.
of 17,000 D, not related to GSHPx (16). Among other aspects of the role of Se in testis is its function in sperm motility, as shown by studies on Se-deficient rats, which produce sperms with impaired motility and characteristic midpiece damage (17), and which is also revealed by investigations of the associated ultrastructural changes (18).
Variation of Tissue 75Se with Time The level of 75Se as a variance with time in tissues of quails maintained on different dietary Se status is represented in Fig. 2. In the basal group, the initial high uptake of 75Se in 24 h was followed by a decrease of 18% in 72 h in blood and other organs except liver, where the decrease was about 12%; the decline in tissue level in 144 h was negligible, reflecting a tendency to stabilize in 72-144 h. In the marginall~, supplemented 0.2 ppm Se group, a gradual trend of decrease in tissue 73Se by about 5% in 72 h and about 10% in 144 h was observed both in blood and liver, whereas the decrease in kidney and heart was to a greater extent, about 20 and 35% in 72 and 144 h, respectively. A similar pattern of elimination of 75Se was exhibited by the 2.0 ppm Se group also. The initial high uptake of 75Se by the basal group indicates a higher demand by the depleted tissues, which later stabilizes in 72-144 h. Retention and elimination patterns of injected 7-~Se vary with each organ and depend on the dietary Se status to a large extent. The tissue level of Se is also determined by the dose and mode of administration, and by the form of dietary Se (19). A faster turnover and higher requirement of Se in testis are indicated from the present study. Biological Trace Element Research
Vol. 14, 1987
Harayanaswami and Lafitha
94 BLOOD
HEART
0.8
0.81
0.6
0.61
LIVER
-o
_•
bJ
0.4~
0.4
2.4 0.2t
0.2
oI
0
I
t
t
t
01
2.0
I
1
TESTIS
KIDNEY
4.0
16
~ 1.6
3.0
1.2
1.2
2.0
0-8
0.8
0.4
0-4
A
(L
I
o
&,-
1.0
I
24
+
I
+
I
72 TIME IN HOURS
I
144
0
I
24
I
I
L
I
72 TIME IN HOURS
[
144
0
--M I
24
l
I
J
l
72 TIME IN HOURS
1
144
Fig. 2. Tissue uptake and retention of 75Selenium with time in different tissues. Dietary groups A, B, and C are as described in Fig. 1 and are indicated by 9 A; X, B; and G, C.
Subcellular Dist#bution of 75Se The distribution of 75Se in the mitochondrial and post-mitochondrial supernatant fractions of the tissues of quails reared on different dietary Se status was assessed. A general pattern of distribution emerged, with about 12-20% of the9 cellular 7 59Se located in the mitochondria, and about 50-60% associated with post-mitochondrial supernatant fractions; the nuclear fractions and cell debris had about 10% of the cellular 75Se. It is estimated that the yield of mitochondria in the present study was about 60% only; thus the actual level of 5Se in this organelle is likely to be higher. This uniform subcellular distribution was observed with respect to all the organs and at all dietary Se levels. In rats maintained on chow diet, it has been reported (20) that about 58% of the injected 75Se in liver was located in the cytosol, 15% in the mitochondria, and about 8% in the microsomes. A major fraction of the liver cytosolic 75Se could be associated with glutathione peroxidase. Selenium i n d e p e n d e n t glutathione peroxidases have also been reported in liver of rats (21). Selenium has also been shown to be located in the mitochondrial form of GSHPx (22). In microsomes, it has been suggested Biological Trace Element Research
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Subceilular Distribution of 75Sein Tissues
95
that Se could be associated with the electron transport systems (23,24). From the reports of the occurrence of Se in different organelles, it is possible that specific mechanisms are operative for the uptake and functionalization of Se in different compartments.
Intramitochondrial Distribution of 75Selenium The intramitochondrial distribution of 75Se in various tissues of quails is represented in Table 2. The mitochondria were sonicated in order to release the contents of the matrix. In liver, mitochondrial membrane of the basal group had the maximal level of incorporated 75Se, followed by the 0.2 ppm Se group having 40%, and the 2.0 ppm Se group about 28% of that in basal. However, the ratio of distribution between the membrane and matrix remained unaltered in all dietary Se groups, the membrane having about 65% and the matrix about 35% of the total amount of incorporated 75Se in mitochondria. Similarly, in kidney the distribution ratio of 75Se in the mitochondria remained constant, being about 55% in the membrane and 45% in matrix. The basal group had maximal level of incorporated Se in mitochondrial membrane; the level in the 0.2 ppm Se group was about 61% of that in the basal group, whereas that in the 2.0 ppm Se group was only about 12%. TABLE 2 Intra-mitochondrial Distribution of 75Seleniumin Tissues of Quails Distribution of 75Se in mitochondria/' percentage of basal' Organ Liver Kidney Heart Testis
Group" A B C A B C A B C A B C
Membrane fraction 66 63 66 59 53 62 82 72 41 76 74 75
(40) (27) (61) (12)
(47) (7) (84) (20)
Matrix 34 37 34 41 47 38 18 28 59 24 26 25
(47) (28) (77) (11)
(85) (45) (100) (21)
"Groups A, B, and C represent the quails maintained on basal, 0.2 ppm Se, and 2.0 ppm Se-supplemented diet, respectively. ~Percent distribution ratio in mitochondrial fractions of tissues (having the sum of counts in membrane and matrix as 100) at 72 h after injection. q~he numbers indicated in parentheses represent the percent of actual counts per rain in the fractions in the respective groups. Biological Trace Element Research
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Narayanaswarniand Lafitha
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In testis also, the percent distribution ratio of 75Se between the mitochondrial membrane and matrix was constant at 75:25, irrespective of the dietary Se status. From the observed consistent pattern of the distribution ratio elicited by mitochondria of various tissues, a distinct deviation was noted in the case of heart mitochondria of quails wherein this ratio altered with dietary Se level. In the basal group, about 82% of the total mitochondrial 75Se was located in the membrane, the remaining 18% in the matrix, whereas the percent distribution ratio in the 0.2 p p m and 2.0 ppm Se groups were 72: 28 and 41 : 59, respectively. A highly specific requirement of Se in the heart mitochondrial membrane-related processes is strongly implied from high level of 75Se incorporation in this fraction in the quails of the basal group. Efforts were made to release the mitochondrial membrane bound 75Se by Triton treatment. The results are presented in Table 3. In the mitochondrial membrane of liver, kidney, and heart about 40-60% of the m e m b r a n e bound 75Se was released upon Triton treatment whereas in testis 60-70% was released. In liver and kidney mitochondria, about 15% of the membrane bound 75Se was found to be acid labile, whereas in heart the lability was significantly higher, being about 25%. Gel electrophoretic studies on the a m m o n i u m sulfate precipitated proteins from mitochondrial matrix fractions of all the tissues from quails of different dietary Se groups were carried out. Upon autoradiography (Fig. 3), a single zone due to 7SSe labeled protein (indicated by arrow) TABLE 3 Effect of Triton Treatment on 75SE Associated With Mitochondrial Membrane Fractions of Tissues Percent of 7SSe~ Organ
Group
Liver
A
Triton released Membrane bound
Kidney
C A
Heart
C A
43 41 34 43 34 60 33
B
28
B
B
57 59 66 57 66 40 67 62
C A
52 48 Testis 71 29 B 67 33 C 57 43 ~After Triton treatment, 7SSereleased in solution and remaining bound to membrane are presented as percent of total counts per rain in the mitochondrial membrane before treatment. Details as described in Table 2. Biological Trace Element Research
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Subcellular Distribution of 75Se in Tissues
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Fig. 3. Electrophoretic detection of 7~Selenoprotein in heart mitochondria. 3A: Protein profile. 3B: Autoradiographic pattern. Lane 1--basal group; lane 2---2.0 ppm Se supplemented group. Zone of 7~Selenium containing moiety is indicated by arrow. Other details as described in text. appeared in the heart samples of the basal group only. (The protein profiles from samples of other organs are not shown.) The probable occurrence of different seleno-moieties and a faster turnover of Se would account for the nonappearance of a concentrated 7SSe zone in the other tissues examined. In the heart, it appears that the requirement of Se by a single protein is significantly high, and is especially enhanced in deficiency conditions, thereby resulting in concentration of 75Se in this protein when given to the Se-deficient group. This has made the detection possible after autoradiography. This unidentified Se-containing protein needs to be studied in greater detail. The nature of the membrane-bound 75Se moiety and the matrix protein containing 75Se are not clear from the present studies, also, the possible partial extraction of membrane components by treatment with Triton and other solubilizing agents need further investigation.
GENERAL SUMMARY AND CONCLUSIONS The higher retention of injected 7SSe by the basal group was clearly indicative of a greater tissue demand in conditions of deprivation. The Biological Trace Elernent Research
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Narayanaswami and Lalitha
minimal retention in the 2.0 ppm Se supplemented group substantiates this view. A regulated uptake and distribution of the injected 7SSe as a function of dietary Se was thus observed. It has emerged from the present study that a dietary Se level of 0.2 ppm was insufficient to meet the tissue demand for Se, while 2.0 ppm Se level was found adequate to meet the requirement of this element in quails. The cellular and organelle localization of 7SSe also appeared to be constant at all dietary Se levels. The high level of tissue Se in kidney and testis could have interesting implications as to their functional role. The significant incorporation of 75Se in the mitochondrial membrane could indicate possible involvement of Se in energy-related processes in the membranes of this organelle. This is interesting in view of the key roles of sulfhydryl moieties in the mitochondrial respiratory phenomenon (25,26) and the possible targets for Se in the membrane aiding or accelerating electron transport and oxygen utilization. Incidentally, it has been shown in our laboratory that in Se deficiency the mitochondrial energy metabolism is subdued, both in terms of oxygen uptake and energy-dependent ion uptake (27). Acid lability of membrane-bound 75Se in mitochondria could be explained by its possible association with nonheme iron proteins. Core substitution of Se for sulphur in the iron-sulfur centers of ferrodoxins from parsley has been reported, and such derivatized molecules have been found to possess biological activity (28). The stoichiometric substitution of Se in the core or ligands of iron-sulfur proteins, in vivo in the mitochondrial membrane, though being an attractive possibility, is not substantiated by the actual amount of Se present and requires further consideration, especially since other acid-labile seleno-moieties could also occur. It has also been reported that Se was found in muscle selenoprotein in lambs (5,6) with a cytochrome C-type of chromophore. The abundance of such redox centers in the membranes appears to offer ideal locations for the multifaceted transient interactions of Se which albeit kinetically unstable are thermodynamically feasible.
REFERENCES 1. L. Flohe, (1982) Free Radicals in Biology, Vol. 5, Academic, NY, pp. 223-249. 2. L. Flohe, (1979), Oxygen Free Radicalsand Tissue Damage, Ciba Found. Syrup., no. 65, Excerta Med. Found., Amsterdam, p. 95. 3. A. Wendel, (1980), Enzymatic Basis of Detoxication, vol. 1, Academic, NY, pp. 333-353. 4. J. T. Rotruck, A. L. Pope, H. E. Ganther, A. B. Swanson, D. G. Hafeman, and W. G. Hoekstra, (1973), Science, 179, 588. 5. N. D. Pedersen, P. D. Whanger, P. H. Weswig, and O. H. Muth, (1972), Bioinorg. Chem. 2, 33. 6. P. D. Whanger, N. D. Pedersen, and P. H. Weswig, (1973), Biochem. Biophys. Res. Commun. 53, 1031. 7. R. F. Burk, and P. E. Gregory, (1982), Arch. Biochem. Biophys. 213, 73. Biological Trace Element Research
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8. N. Vasanthy, R. Padma Bai, M. Babu, and K. Lalitha, (1986), Biol. Trace Elem. Res. 10, 79. 9. M. Babu, R. Padma Bai, N. Vasanthy, K. Lalitha, and K. T. Joseph, (1986), Biol. Trace Elem. Res. 10, 317. 10. B. J. Davis, (1964), Ann. N Y Acad. Sci., 121, 404. 11. P. J. Blackshear, (1984), Meth. Enzymol. 104, 237. 12. O. H. Lowry, N. J. Rosebrough, A. L. Farr, and R. J. Randals, (1951), J. Biol. Chem. 193, 265. 13. E. D. Heimann, J. S. Morris, and W. E. Loch, (1985), Nutr. Rep. Inter., 32, 1153. 14. D. Behne, and T. Hofer, (1982), J. Nutr. 112, 1082. 15. W. G. Hoekstra, D. Hafeman, S. H. Oh, and H. E. Ganther, (1973), Fed. Proc. 32, 385. 16. H. I. Calvin, (1978), J. Exp. Zool., 204, 445. 17. A. S. M. Wu, J. E. Oldfield, L. R. Shull, and P. F. Cheeke, (1979), Biol. Reprod. 20, 793. 18. E. Wallace, H. I. Calvin, and G. W. Cooper, (1983), Gamete Research, 4, 377. 19. J. C. Hansen, and P. Kristensen, (1979), J. Nutr., 109, 1223. 20. F. H. Stults, J. W. Forstrom, D. T. Y. Chiu, and A. L. Tappel, (1977), Arch. Biochem. Biophys. 183, 490. 21. R. D. Lawrence, and R. F. Burk, (1975), Biochem. Biophys. Res. Commun., 71, 952. 22. J. J. Zakowski, and A. L. Tappel, (1976), Biochim. Biophys. Acta. 445, 558. 23. C. P. J. Caygill, and A. T. Diplock, (1973), FEBS Lett., 33, 172. 24. C. P. J. Caygill, A. T. Diplock, and E. H. Jeffery, (1973), Biochem. J. 136, 851. 25. N. Haugaard, N. H. Lee, R. Kostrzewa, R. S. Horn, and E. S. Haugaard, (1969), Biochim. Biophys. Acta., 172, 198. 26. S. Minakami, F. Schindler, and R. W. Estabrook, (1964), J. Biol. Chem., 239, 2042. 27. N. Vasanthy, K. Lalitha, and B. Chance, Arch. Biochem. Biophys. (Communicated). 28. J. A. Fee, and G. Palmer, (1971), Biochim. Biophys. Acta., 245, 175.
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