0013-7227/78/0102-0002102.00/0 Endocrinology Copyright © 1978 by The Endocrine Society
Vol. 102, No. 2 Printed in U.S.A.
Conversion of L-Thyroxine to Triiodothyronine in Rat Kidney Homogenate* P. CHIRASEVEENUPRAPUND.f U. BUERGI, A. GOSWAMI, AND I. N. ROSENBERG Department of Medicine, Framingham Union Hospital, Framingham, Massachusetts, and Department of Medicine, Boston University School of Medicine, Boston, Massachusetts ABSTRACT. Rat kidney homogenates, in phosphateEDTA buffer, consistently catalyzed the formation of T.i from added L-thyroxine (T4). The formation of T:i was assessed by both paper chromatography and RIA of T.i. Conversion of T4 to T.-i appeared to be enzymatic, showing pH and temperature optima (pH 7.0 and 37 C, respectively) and tissue and time dependence. Formation of T.i was unaffected by azide, cyanide, or catalase, nor was it dependent upon oxygen; indeed, under anaerobic conditions conversion of T4 to T.i was enhanced. Dialyzed homogenate retained full activity, and no cofactor requirement was demonstrated. A' role of iron and thiol groups in the enzymatic formation of Ti from T4 was suggested by the inhibitory action of iron che-
lators and thiol-blocking reagents. The capacity of kidney for T;i formation was considerable and increased with increasing T4 concentrations, being approximately 2 nmol/g tissue/h at very high T4 levels. The apparent Km was estimated to be 3 X 10~(> M. The conversion of T4 to T.i was inhibited by propylthiouracil at micromolar concentrations whereas methimazole, iodide, and lithium salts were without effect. The enzymatic activity of the homogenates was associated with its particulate components, the readily sedimenting fractions corresponding to plasma membranes and mitochondria being most active, and was absent from nuclei and cytosol. (Endocrinology 102: 612, 1978)
E
XTRATHYROIDAL conversion of Lthyroxine (T4) to T 3 has been well documented in recent years (1-5). In vitro this process has been demonstrated in various intact cell systems (6-13); broken cell preparations were reported to be inactive in most studies (14). Recent studies in our laboratory indicated that kidney homogenates consistently converted T4 to T 3 (15), and similar findings have recently been reported by others in liver homogenates (16-18). The studies herein described were aimed at further characterization of the conversion of L-T4 to T 3 in the kidney homogenate system. Materials and Methods White male rats, 200-250 g, of the Charles River strain, maintained on Purina Laboratory chow, served as the source of the kidney tissue. L-[3',5'-
125
I]T, (SA, 40 mCi/mg) and L-[3'- 131 I]T 3 (SA, 20
mCi/mg) were obtained from Amersham Searle, and L-[125I]T3 (SA, 500 mCi/mg) was purchased from Abbott. The following substances were obReceived October 19, 1976. * This research was supported by Research Grant A2585, USPHS, NIAMDD. t Requests for reprints should be addressed to: Dr. I. N. Rosenberg, Framingham Union Hospital, 25 Evergreen Street, Framingham, Massachusetts 01701.
tained from Sigma: L-T4, 3',3,5-triiodo-L-thyronine, tetraiodothyroacetic acid (tetrac), triiodothyroacetic acid (triac), sodium azide, potassium cyanide, catalase, reduced glutathione, a-methyl tyrosine, Nethylmaleimide, DL-propranolol, glucose oxidase (type V), and bovine serum albumin; 2-mercaptoethanol was obtained from Bio-Rad Laboratories. 8-Anilino-l-naphthalenesulfonic acid (ANS), sodium salt, was purchased from Eastman Kodak. Methylmercaptoimidazole (methimazole) was a gift from Eli Lilly and 6-n-propylthiouracil was generously donated by Lederle. Under light ether anesthesia, rats were exsanguinated by cardiac puncture. The kidneys were removed, freed of their capsular investments, rinsed in ice-cold 0.1 M phosphate buffer (Na>HPO4/ KH2PO4, molar ratio 61:39), containing EDTA 5 mM, pH 7.0, blotted with gauze, and weighed. The kidneys were then minced with scissors in a beaker immersed in an ice-water bath and ground in a TenBroeck homogenizer (Kontes) in 3 vol phosphate buffer to provide a 25% (wt/vol) homogenate. Individual incubation vessels (1 X 10 cm test tubes) contained 0.3 ml kidney homogenate (75 mg tissue), L-T 4 , and test substances in a final volume of 0.5 ml phosphate buffer. The conversion of L-T4 to T 3 in the phosphate-EDTA buffer appeared to be consistently higher than in Krebs-Ringer phosphate, which had been used for homogenization and incubation in the earlier experiments. Incubation was carried out at 37 C for 1-2 h in a Dubnoff
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FORMATION OF T., FROM T4 metabolic incubator, shaken at 100 strokes/min. For experiments involving prolonged incubation, penicillin and streptomycin (0.1 mg of each substance) were added to each vessel. Two analytical techniques, paper chromatography and RIA, were used to assess the formation of T, from L-T 4 . Paper chromatography method In experiments using paper chromatography, L[3',5'-12r>I]T4, 1-2 jiiCi (32-64 pmol) was added to the incubation media; tissue-free vessels containing [12r'I]T4 served as blank controls. At the end of incubation, a large excess of L-T 4 and L-T:) (40 jug each) in 0.1 ml of 0.05 M methimazole was added to each vessel and also 0.1 ml pooled human serum. Contents were rapidly mixed (Vortex mixer) and immediately chilled in an ice-water bath. An aliquot of the mixture (10-15 JU.1) was then applied to paper (Whatman no. 3MM), dried in air, and subjected to descending chromatography in the £-amyl alcohol-2 N ammonia-hexane (5:6:1) solvent as described by Bellabarba et al. (19). The zones containing radioactive iodine compounds were localized by autoradiography, and the locations of T;i, tetrac, and triac were also determined by visualization under UV light of these substances which had been cochromatographed in carrier amounts on each paper strip. Each zone was excised and radioactivity in these zones, as well as in the intervening segments of the strip, was determined in an automatic y-counter. Radioactivity of each zone was expressed as a percentage of the total activity on the strip. In addition to the conventional paper chromatographic method for determining the proportion of [12r>I]T:t, an isotopic ratio method was also frequently used, involving addition of a trace quantity of [1;"I]T.|. In this procedure, after the addition of excess stable T4, T;J, methimazole, and serum at the end of incubation, 0.1-ml aliquots of these mixtures were transferred to individual new vessels, and a trace amount of [1;>1I]T;J (0.1 jtiCi) was added to each vessel and thoroughly mixed. Small aliquots of these final mixtures containing both isotopes were subjected to paper chromatography as described above. By measuring the 12SI and '"I in the final mixtures in each vessel, as well as in a segment of the Ti zone on the paper, the percentage of [12r'I]T,i in each sample could be determined as follows:
613
final mixtures, R2 = 12r>I (cpm)/'"I (cpm) in a segment from the T:t zone of the paper chromatogram; percentage of purity of [1:tlI]T.i (generally 93-95%) was determined by paper chromatography of an unincubated aliquot of [l:uI]T,i. The percentage of [12r>I]T:1 found by the ratio method agreed closely with values obtained by the standard chromatographic method involving determination of the radioactivity in all zones of the paper as well as the T;1 zone. The isotopic ratio method is a simple procedure, most useful in experiments in which quantitation of T.t formation alone (in contrast to other metabolites of T4) is of interest. In calculating the amounts of T4 metabolized and Ti formed in each experimental vessel, the percentage of each labeled compound found on the chromatogram was corrected by subtracting the corresponding value found in the concurrently incubated tissue-free mixtures. Since it is assumed that in commercially available [12r>I]T4, the label is randomly distributed between the 3' and 5' positions, the labeled T:1 found represents only half of the T,i formed during incubation. The quantity of T;t formed was therefore calculated by: T:t formed = 0.02 x percentage of [12'TJT:1 found (corrected by subtraction of blank values) X quantity of T4 present in the incubation mixture. T3 formation was expressed as picomoles per g tissue per h. Radioimmunoassay method
In these experiments, only stable T4 (usually 129 pmol/vessel) was used as substrate. At the end of incubation, 1 ml ice-cold absolute ethanol was added to each incubation mixture (0.5 ml) which was then vortexed and kept in an ice-water bath for 15 min before centrifugation at 2000 X g for 15 min in a refrigerated centrifuge. The ethanol extract (0.1 ml) was then assayed for T;1 by RIA using a highly specific antibody to T;i according to the method of Patel and Burger (20). In samples containing large amounts of T.i, the ethanol extracts were diluted with an ethanol-phosphate buffer solution (vol/vol, 2:1) and 0.1 ml of the final diluted extract was used. The antiserum to T,i was prepared by immunization of rabbits with T.i-bovine serum albumin complexes that had been prepared by coupling with carbodiimide (21) and injected intradermally after emulsification in complete Freund's adjuvant. The antibody to T:) cross-reacted approximately 0.001-0.002 with T4, 0.001 with tetrac, 0.5 12n 125 with triac, and less than 0.0005 with rT;t, ITyr, or [ I]T,(% of total I) I 2Tyr, compared on a molar basis with reactivity = Ri X R2 X % purity of [1:UI]T:1, of T;t as 1.00. Since the amounts of triac found in these experiments, as judged from the findings uswhere Ri = '"I (cpm)/125I (cpm) in an aliquot of
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Kndo • I»7K Vol 102 • No 2
CHIRASEVEENUPRAPUND ET AL.
614
ing the paper chromatography technique, were relatively small (0-0.3% of the total T., added) as compared with the amounts of T:) formed (3-6%), no correction of T;) values was made for possible presence of triac despite the high reactivity of the latter with the antibody. Each sample was assayed in triplicate against standards (0.02-0.8 ng T:t/vessel) which also contained a comparable concentration of ethanol (6.6%). Ethanol at this concentration was found to have no adverse effect on the RIA of T.t, results being quite similar with standard curves prepared in the presence and absence of ethanol. The measured T,t values were corrected for incompleteness of recovery of T.i in the ethanol extraction procedure, which was determined by adding known quantities of stable T.i, extracting with ethanol, and performing RIA of the extracts. Recovery was 50-55% in most experiments. Blank controls were included in each experiment: vessels containing homogenates without added T4 were incubated and at the end of the incubation, L-T4 in amounts equal to that initially present in the experimental vessels was added to each vessel; the contents were rapidly mixed and immediately extracted with ethanol. The ethanol extract was assayed for T;i as described above. Blank control values were generally small in comparison with the amounts of T.t formed in the experimental vessels; these values were subtracted from the corresponding experimental values. In experiments carried out to determine the intracellular localization of the activity responsible for conversion of T4 to T;t, 10% homogenates were prepared in 0.25 M sucrose, 0.02 M Tris buffer, pH 7.4, and subjected to subcellular fractionation by differential ultracentrifugation according to a standard procedure (22). Purified preparations of nuclei were made by the method of Chauveau et al. (23), and the procedure described by Neville (24) and modified by Amir et al. (25) was followed to obtain
plasma membranes. To determine the activity of the subcellular fractions, each was diluted with the sucrose-Tris buffer and amounts equivalent to 75 mg kidney tissue were used in the assays for T.t formation as described for the whole homogenates. Results In experiments using labeled L-T 4 , kidney homogenates were consistently active in converting T4 to T.j. Representative results are shown in Table 1. During a 90-min incubation, labeled T4 declined by 13.9%, accompanied by an increase of 3.4, 8.5, 1.2, and 0.7% (of total activity) in the zones corresponding to T;t, iodide, tetrac, and the origin. From the known quantity of T4 initially added to each incubation vessel, the amounts of T4 metabolized and T 3 formed could then be calculated as 39.5 and 19.3 pmol/g/h, respectively. Thus, approximately half of the T4 metabolized was accounted for by formation of T:i. Conversion of T4 to T;i in kidney homogenates was also readily demonstrated with the RIA method. The values of T a formation as assayed by the RIA method tended to be higher than by the paper chromatographic method. In three experiments in which Tu formation was determined by both methods in parallel incubations, the results were 20-28% higher by the RIA method in two experiments and were in close agreement in the third experiment (Table 2). The differences in results may in small part be due to the presence of triac formed during the incubation, which, as noted above, cross-reacted 50% with the T;j antiserum.
TABLE 1. Metabolism of L-[l2r>I]T4 in rat kidney homogenates Distribution of labeled constituents on paper chromatograms (% of total activity) Origin Kidney homogenates Tissue-free blanks
1.5" ±0.10 0.8'' ±0.09
I 12.1 ±0.20 3.6 ±0.12
T4
Tetrac
78.6 ± 0.27 92.5 ±0.18
2.0 ±0.06 0.8 ±0.07
5.4 ±0.13 2.0 ±0.04
Triac 0.4 ± 0.09 0.3 ±0.04
T4 metabolized (pmol/g/h) 39.5 ±0.77
T, formed /h) 19.3 ±0.74
Each incubation vessel contained kidney tissue, 75 mg, and 32 pmol [l2r'I]T4 in 0.5 ml phosphate-EDTA buffer solution (pH 7.0); incubated at 37 C for 90 min. " Mean ± SRM of four incubations. h Mean ± SRM of three incubations.
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FORMATION OF T;, FROM T4
Kinetics of formation of T:i from L-T4 Over a prolonged incubation of 16 h, there was a progressive disappearance of [125I]T4; the rate was relatively rapid in the first few TABLE 2. Comparison of RIA and paper chromatographic (PC) methods of determining formation of T.i from T4 in kidney homogenates Experimental method
T.i formed (pmol/g/h)
Exp A RIA 44.9 ± 1.8 (a) (43.7) PC 35.0 ± 1.4 (b) ExpB RIA 98.3 ± 4.3 (c) (95.7) PC 93.3 ± 1.3 (d) ExpC RIA 74.0 ± 2.4 (e) (70.6) PC 61.9 ± 1.2 (0 Incubation conditions were as described in Table 1; incubation time: 1 h. A, B, and C were three separate experiments using different pools of homogenates. Each experiment was done in parallel incubations using the same homogenate preparation with equal amounts of T4 (64 pmol in A and 129 pmol in B and C) added to the incubation medium. Each value is the mean ± SRM of four incubations. Values of the RIA method presented in parentheses are values corrected for the formation of triac as determined by the PC method in the corresponding vessels. Statistical analysis, P values: a vs. b and e us. f, 0.05.
615
hours as compared with later hours of incubation. After 3 and 8 h of incubation, labeled T;t generated from T4 constituted 4.8% and 7.3% of total activity, respectively, whereas the increment in labeled iodide tended to be 1- to 2-fold greater than T.i. There was a slight increase of activity at the origin on the paper, which rose from 1.5% at 30 min to 3% at 16 h (lower panel of Fig. 1, left). Radioactivity at the tetrac and triac regions was detectable but represented only small fractions of the total. The cumulative amounts of T4 metabolized and T;i formed at each time interval were calculated and are shown in Fig. 1, right; T.t formation accounted for approximately 40-50% of the T4 metabolized. In several experiments, where T:t formation was studied using shorter incubation times (5, 10, 15, 30, 45, 60, 90, and 120 min), the rate of T 3 formation was found to be linear over the initial 45-60 min. Effects of pH and temperature The conversion of T4 to T:J was dependent on pH and temperature. The optimal pH was found to be 7.0 (Fig. 2), and the optimal tem-
Origin Triac Tetrac 2
4
6 8 10 12 Incubation time (hrs)
14
16
18 Time of incubation (hrs)
FIG. 1. Kinetics of T4 metabolism in kidney homogenate. Each incubation vessel contained homogenized kidney tissue, 75 mg, and [I2TJT4, 32 pmol, in 0.5 ml Krebs-Ringer phosphate, supplemented with 0.5 mg penicillin and 0.5 mg streptomycin. T4 metabolites were determined by paper chromatography and expressed as percentages of total activity (left); amounts of T4 metabolized and T.i formed as a function of time are shown cumulatively on the right. Each value is the mean of triplicate incubations from the same pool of kidney homogenate.
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616
CHIRASEVEENUPRAPUND
perature was 37 C (Fig. 3). The formation of T 3 from T4 was completely abolished in homogenates placed in a boiling water bath for 5 min. Effects of incubation under anaerobic conditions Incubation under anaerobic conditions, as achieved by gassing the stoppered reaction mixtures throughly with nitrogen, consistently enhanced T3 formation by 120-150% and T4 metabolism by 40-80% (Table 3); the genera120 EXPERIMENT A
Kndo» 1978 Vol 102 • No 2
ET AL.
tion of labeled iodide was also increased in parallel with the increase in T.} formation, whereas the formation of tetrac was markedly diminished in N2. The increased T3 formation under anaerobic conditions was similarly recorded in experiments in which the assays were done by RIA. Incubation in presence of varying concentrations of L-T4 Increasing the quantity of T4 over a range of 250-13,000 pmol/vessel in the presence of a constant amount of kidney tissue resulted in formation of progressively larger amounts of T3 (Fig. 4). In a series of six experiments,
100 en
80
O (J5
60
UJ
EXPERIMENT B
40 20
FIG. 2. Effect of varying pH on formation of T.t from LT4. Each incubation vessel contained kidney homogenate (75 mg) and T4 (stable L-T4, 120 pmol in Exp A, or [125I]T4l 180 pmol in Exp B) in 0.5 ml 0.1 M phosphate buffer; incubated for 90 min at 37 C. T.-i formed was determined by RIA in Exp A and by paper chromatography in Exp B. Each value is the mean of triplicate incubations.
0
10
20 30 40 50 TEMPERATURE (C)
60
FIG. 3. Effect of temperature upon formation of T.t from L-T 4 . Each incubation vessel contained kidney tissue, 75 mg, and ['-5I]T4, 64 pmol, in 0.5 ml phosphate-EDTA buffer, pH 7.0, and incubated for 75 min. T.-i formed was determined by paper chromatography. Each value is the mean of triplicate incubations.
TABLE 3. The effect of anaerobiosis on the conversion of L-T4 to T:t by kidney homogenates Incubation conditions Exp A Tissue, in air Tissue, in N2
Distribution of labeled constituents on paper chromatograms (% of total activity) Origin 3.1
6.9
±0.02
±0.10 11.5 ±0.31
1.7
±0.002 Tissue-free blanks ExpB Tissue, in air Tissue, in N2 Tissue-free blanks
r
1.3
3.2
±0.04
±0.08
1.8
6.5
±0.17
±0.10
1.5
9.9
±0.06
±0.13
1.2
3.6
±0.07
±0.14
T4 83.6 ±0.14 79.0 ±0.72 93.4 ±0.07 86.8 ±0.34 81.9 ±0.16 92.7 ±0.42
Tetrac
T,
Triac
2.2
4.1
0.4
±0.10
±0.01
±0.09
0.7
7.3
0.3
±0.18
±0.15
±0.08
0.7
1.4
0.3
±0.11
±0.05
±0.03
1.3
3.1
0.5
±0.13
±0.07
±0.09
0.7
5.8
0.4
±0.10
±0.30
±0.03
0.7
1.3
0.3
±0.10
±0.04
T 4 metabolized (pmol/g/h)
T, formed (pmol/g /h)
112.4 ±1.61 165.1 ±8.26
61.9 ±0.23 135.3 ±3.44
101.5 ±5.85 185.8 ±2.75
61.9 ±1.2 154.8 ±5.16
±0.05 Individual vessels contained kidney homogenates, 75 mg, and labeled T4, 129 pmol, in 0.5 ml of phosphate-EDTA buffer solution (pH 7.4 in Exp A, and 7.0 in Exp B), incubated for 90 min (in Exp A) and 60 min (in Exp B). Each value is the mean ± SEM of four incubations.
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FORMATION OF T3 FROM T4
of kidney tissue; however, the increased activity was not always proportional to the increase in quantity of tissue. In some experiments, there was a linear relationship between quantity of tissue and activity; in other experiments, dilution of homogenate was associated with relatively less decline in activity than in tissue concentration (Fig. 5).
0.005 1
V 0.004 ^Sx-
0.003
^
617
0.979
^
0.002
Effect of antithyroid drugs
0.001 i
-0.3 xlO6 0
O.5xlO6
i
I.OxlO6
J_
i
l.5xlO6
I
2.0xl0 6
S
FIG. 4. T.t formation as a function of substrate (T4) concentration. Each vessel contained kidney tissue, 75 mg, and [l25I]T.t supplemented with varying amounts of stable TV incubated under anaerobic conditions for 30 min at 37 C. T.i formed was determined by paper chromatography. Each value is the mean of triplicate incubations. V = rate of T.t formation, pmol/g tissue/h; S = molar concentrations of T4.
the apparent Michaelis-Menten constant (Km) for T4 was estimated to be 2.5-3.2 X 10"6 M (average value, 3 X 10~6 M), and the apparent Vmax was 1.7-2.3 nmol (average value, 2.0 nmol) of T 3 formed/g tissue/h. Effects of L-T3 Supplementing the incubation mixture with varying amounts of stable L-T3 in the range of 7.6-190 pmol/vessel did not appreciably affect the rate of formation of T 3 from labeled T4 (32 pmol/vessel) at either 1 or 4 h of incubation. These findings suggested that there was little inhibition of T4 monodeiodination by the product T3. In separate experiments, incubation of labeled T 3 (150 pmol/vessel) in kidney homogenate for 1 h resulted in deiodination of less than 2% of the added T3, whereas concurrent experiments with T4 using the same homogenates and under the same conditions showed 12-15% of the T4 was deiodinated. These findings suggest relatively little further metabolism of the T 3 newly formed from T4 incubated with kidney homogenates. Effect of varying amounts of kidney tissue There was a progressive increase of T 3 formation in the presence of increasing amounts
The conversion of T4 to T 3 was greatly inhibited by 6-/i-propylthiouracil, whereas methimazole was found to have no effect; T 3 formation in homogenates incubated in a nitrogen atmosphere was much greater than in air, as previously noted (Table 3), and the inhibitory action of propylthiouracil was apparent in both nitrogen and air incubations (Fig. 6). In homogenates incubated in presence of 5, 10, and 50 fiM propylthiouracil, T 3 formation was inhibited by 40, 70, and 80%, respectively. Neither sodium iodide (2 HIM) nor lithium carbonate (5 mM) was inhibitory. Effect of varying conditions and agents Sonication of homogenates before incubation resulted in substantial loss of the activity (Table 4). The activity appeared to be labile and was consistently decreased in homogenates which were frozen and thawed prior to incubation as well as in homogenates prein100 -
20 40 60 80 KIDNEY TISSUE (mg/vessel)
FIG. 5. Effect of varying amounts of tissue on rate of formation of T.t from T4. Incubation conditions were as described in Tables 1 and 2. Values shown in broken lines were obtained by the paper chromatographic method; values shown in solid lines were obtained by the RIA method. T:) formation is expressed as a percentage of the values found in the vessels containing 75 mg tissue. Each value is the mean of triplicate incubations.
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CHIRASEVEENUPRAPUND ET AL.
618 ju/an
D Air
X 200
• N
II ' e •• 3 100 1• ! 50 E
•? 150 in 0> O
a>
1
Addition of
None
1I •• 1 • 1 _• MMI 5mM
PTU 5mM
FIG. 6. Effect of propylthiouracil (PTU) and methimazole (MMI) on conversion of T4 to T.t in kidney homogenate. Each vessel contained kidney tissue, 75 mg, stable 1.-T4, 128 pmol, and test substances where indicated, in 0.5 ml phosphate buffer (0.1 M)-EDTA (5 DIM) solution; incubated for 2 h. Amounts of T:t formed were determined by RIA. Each value is the mean of triplicate incubations. TABLE 4. Effects of sonication, freezing, and preincubation at varying temperatures on formation of T, and T.,
but high salt concentrations inhibited greatly (90% decrease in activity in 1.2 M NaCl). A number of agents were tested for possible effects on the conversion of T4 to T;J in this system; results are summarized in Table 5. Preincubation of kidney homogenates with azide, cyanide or catalase and incubation in presence of these substances was found to have no effect on T:i formation. Formation of T 3 was greatly impaired by H2O2 and an H2O2generating system (glucose-glucose oxidase); the amounts of T4 metabolized were diminished proportional to the decline in T;) formation. In the presence of EDTA (10 mM), T3 formation was stimulated by 90%. The activity was inhibited by specific iron chelators, TABLE 5. Effects of various agents on formation of T.i from T.
li)7H Vol 102 i No 2
None Sodium azide" (2 mM) Sodium cyanide" (2 mM) Catalase" (2000 units/ml) H2O2 0.3 mM 1.0 mM Glucose" (2 mg/ml) + glucose oxidase 2 units/ml 10 units/ml EDTA" (10 mM) o-Phenanthroline (10 mM) 2,2'-Dipyridyl (10 mM) Mercaptoethanol" (5 mM) Reduced glutathione" (5 mM) N-Ethylmaleimide (5 mM) Ascorbic acid" (5 mM) DL-a-tocopherol (5 mM) Normal human serum (protein 28 mg/ml) Bovine serum albumin (28 mg/ml) Sodium salicylate 1 mM 5 mM ANS, sodium salt 1 mM 5 mM a-Methyltyrosine" (5 mM) DL-Propranolol (2 mM)
Formation ofT,(%of control) 100 93 113 112 65 3 26 22 191 32 19 116 90 14 100 102 10 45 29 16 27 11 96 103
Ti formation is expressed as a percentage of the quantity of T.i formed in the group which had no test substances added. Values are the mean of two or three experiments, each done in triplicate for each substance. Incubation conditions were as described in Table 1; T.i formation was determined by paper chromatography. 0 Effect of T 4 to T 3 conversion checked also by RIA of T,), with results similar to those found using paper chromatography.
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FORMATION OF T3 FROM T4 o-phenanthroline and 2,2'-dipyridyl and also by iV-ethylmaleimide, a thiol-blocking agent. These findings suggest that non-heme iron and thiol groups may participate in the T4 to T.t conversion. Addition of reduced glutathione, mercaptoethanol, ascorbic acid, and atocopherol had no significant effect. DL-propranolol, a /?-adrenergic blocker commonly used in treating hyperthyroid patients, was found to have no effect. Tyrosine hydroxylase has been suggested to play a role in converting T4 to T;t in neuroblastoma cells (13) and amethyl tyrosine, an inhibitor of this enzyme, was therefore tested and was found to exert no inhibition on the formation of T.t from T4 in the kidney homogenate. The conversion of T4 to T;i was greatly inhibited by human serum and, to a lesser extent, by bovine serum albumin. Both salicylate and ANS, agents known to displace T4 from protein binding sites, were also inhibitory. The activity was unchanged by addition of reduced or oxidized pyridine nucleotides (NAD, NADH, NADP, NADPH), FAD, or FMN to a final concentration of 1 mM. Subcellular localization of the conversion of T4 to T:i Studies were made to determine the intracellular localization of the activity responsible for the conversion of T4 to T3 in kidney tissue. Results are summarized in Table 6. The activity was regularly present in the 100,000 X g pellet and absent from the supernatant, suggesting its particulate nature. The activity was distributed in all the particulate fractions, being greatest in the crude nuclear pellet, with lesser activity in the mitochondrial and least in the microsomal fractions. Addition of pyridine nucleotides, flavins, and a dialysate of fresh homogenate did not restore activity to the microsomal fraction. Purified nuclei were found to be inactive. Activity was regularly found at the interface between 30% and 40% sucrose, a fraction considered to be enriched in plasma membranes; the activity was about 25% of that found in the whole homogenate. However, comparable activity was also observed in material from the layer between 40%
619
TABLR 6. Subcellular localization of the activity converting T\ to T:t in kidney tissue Cell fractions
T.rforming activity
Exp A 100 Whole homogenate 87 100,000 x g x 90 min pellet 0 100,000 x g x 90 min supernate ExpB 48 (a) Crude nuclear pellet (800 X g X 10 min) 25 (b) Mitochondrial fraction (8,000 x g x 10 min) 7 (c) Microsomal fraction (100,000 x g x 60 min) ExpC 0 (a) Purified nuclei" 25 (b) Purified plasma membrane'1 Ten percent kidney homogenates were freshly prepared with a Dounce homogenizer (loose pestle) in 0.25 M sucrose, 0.02 M Tris, pH 7.4. In Exp A, pellet and supernatant resulting from centrifugation at 100,000 x g for 90 min were separated and assayed. In Exp B, pellets sedimenting at 800 x g x 10 min, 8,000 X g x 10 min, and 100,000 x g x 60 min, were prepared by successive centrifugation of the preceding supernatant. Each pellet was washed once by suspending in sucrose-Tris buffer and recentrifuged at the same force; the washed pellet was re-suspended in sucrose-Tris buffer, and an aliquot equivalent to 75 mg kidney tissue was assayed for activity. Informing activity was expressed as a percentage of the activity found in 75 mg tissue of the whole homogenate. Each value is the mean of three experiments done in triplicate with each fraction of each experiment. " Purified nuclei were prepared by the method of Chauveau et al. (23). h Plasma membranes were prepared by the procedure of Neville (24) as modified by Amir et al. (25) using a discontinuous sucrose gradient (30, 40, and 45%, wt/vol). The fraction between 30% and 40% sucrose was considered to be enriched in plasma membranes at the interface.
and 45% sucrose, generally considered to contain both mitochondria and plasma membranes.
Discussion The ability of kidney tissue to effect the conversion of T4 to T3, previously noted in kidney slices (6), does not depend upon intact cell structure and is readily demonstrated in kidney homogenates. Microscopic observation of both the fresh homogenates and of stained sections of the sediment obtained after centrifuging homogenates revealed very few unbroken cells. In addition, homogenates prepared in distilled water, in which the hypotonicity would be expected to result in cell disruption, effectively catalyzed the formation of T,}.
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CHIRASEVEENUPRAPUND ET AL.
Some requirement of structure at a subcellular level is suggested by the impairment of activity after sonication of the homogenate. The quantity of T 3 formed as assessed by the RIA method tended to be somewhat higher than by the paper chromatographic method (Table 2). However, both analytical methods yielded results that were generally comparable, and there was close agreement between the methods with respect to the degree of stimulation or inhibition in response to changes in experimental conditions or to test substances. The 5'-monodeiodination of L-T4 in the homogenate appeared to be enzymatic in nature showing temperature and pH optima and tissue dependence. Activity increased with increasing tissue concentration although a linear relationship between these variables was not consistently observed. Indeed, the activity of diluted homogenates often exceeded what would be expected from the degree of dilution, raising the possibility that the kidney may contain inhibitors of T:i formation. Such putative inhibitors appeared to be non-dialyzable since dialysis of homogenates did not enhance the enzymatic activity. The data on the rate of conversion of T4 to Ta by the whole kidney homogenate, as the total T4 concentrations were altered, appeared to conform to Michaelis-Menten kinetics (Fig. 4), and on this basis the apparent Km for T4 was estimated to be 3 x 10~h M. It is known that T4-binding proteins are present in kidney cytosol (26, 27), and therefore it is likely that the concentration of the free T4 in kidney homogenates was substantially less than that of the total, and it is conceivable that free rather than bound T4 is the effective substrate in the T4 to T;i conversion. If this were the case, the Km of 3 X lCT6 M might be a considerable overestimation. The importance of free T4 was suggested by our observation that diluting the homogenates enhanced, and addition of serum containing thyroxine-binding globulin diminished the T4 to T 3 conversion. On the other hand, agents that displace T4 from T4-binding proteins, ANS, and salicylates, which would presumably increase free T4 in the kidney homogenates, did not in-
Kudo Vol 102
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crease T.i formation, and experiments using the pellet fraction (1000 x g), presumably depleted of cytosol T4-binding proteins, disclosed a similar value for Km (unpublished observation). At present, it is not clear whether T4 must be made available to the enzyme in the free or in the bound form; the apparent Km calculated on the basis of the total T4 concentrations may have quantitative usefulness until this aspect of the reaction mechanism is better characterized. There is a possibility that the temperature and pH optima noted for the enzymatic conversion of T4 to T;J in the kidney homogenates reflect, at least in part, altered binding of the substrate to tissue proteins. Studies of alteration in free T4 concentrations and reaction rates as affected by changes in pH and temperature would be needed to assess this point. Several published studies of the deiodination of T4 by tissues in vitro have suggested an oxidative or peroxidative mechanism (28-31), and have emphasized the oxygen dependence of the deiodinating process and its inhibition by reducing substances. Iodide and protein-bound iodine of incompletely defined nature (origin material on paper chromatography) have been the major products of oxidative T4 deiodination in most studies, very little or no T.t being found. Light and flavins, as well as oxygen, have been shown to accelerate T4 deiodination (32, 33). In the present study, by contrast, the formation of origin material and of iodide in excess of T.( formation was modest; T;) formation was not affected by the presence of azide, cyanide, and catalase, and, indeed, H2O2 greatly inhibited both Ta formation and T4 deiodination. Light and flavins had no effect. Moreover, the conversion of T4 to T.j proceeded more efficiently under anaerobic conditions than in the presence of air. The lack of effect of anaerobiosis on the conversion of T4 to T,} has been reported in studies using kidney and liver slices (34, 35). The mechanism of the potentiating effect of nitrogen is not clear; it is possible that anaerobically the degradation of T4 via other oxygen-dependent pathways is inhibited and this could facilitate a metabolic pathway, not requiring oxygen and leading to T,t for-
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FORMATION OF T3 FROM T4 mation. The absence in anaerobic experiments of products of oxidative metabolism of T4 such as tetrac and triac, is consistent with this idea. However, a possible inhibitory action of oxygen on T 3 formation should also be considered since in nitrogen, not only was T4 more efficiently converted to T3 but more T4 was metabolized as well, compared to the aerobic experiments. The findings suggest that the 5'monodeiodination of T4 by kidney is not mediated by a peroxidative process, and that the process may resemble the enzymatic reductive deiodination of iodotyrosines by the thyroid and liver iodotyrosine dehalogenase (36). No evidence of a dialyzable cofactor or inhibitor of T 3 formation in the homogenate was adduced, activity being unaffected by dialysis. Unlike the thyroid iodotyrosine dehalogenase, which requires NADPH as a cofactor, none of the adenine nucleotides (NAD, NADH, NADP, NADPH) tested was found to have a consistent stimulating effect on the conversion of T4 to T3. Both iron and thiol groups may be necessary for this conversion since the activity was greatly impaired in presence of specific iron chelators (o-phenanthroline and 2,2'-dipyridyl) and of a thiol-blocking agent (iV-ethylmaleimide). Propylthiouracil was found to inhibit the formation of T3 from T4 in the system, in agreement with published observations on the in vivo (37, 38), and the in vitro metabolism of T4 to T3. Inhibition of the T4 conversion to T 3 was also observed with thiouracil and methylthiouracil in kidney slices (6, 39). Other agents used for the treatment of hyperthyroidism, such as methimazole, iodide, lithium salts, and propranolol, were found to have no effect. Since a-methyltyrosine, a potent tyrosine hydroxylase inhibitor, was likewise without effect on this process, it is unlikely that the formation of T3 in the kidney homogenates is mediated by an aromatic amino acid hydroxylase as proposed by Dratman et al. (40). Although both human serum and bovine serum albumin inhibited conversion of T4 to T3, conceivably because of their thyroxinebinding capacity, salicylate and ANS, which displace T4 and T 3 from circulating thyroxinebinding proteins, also inhibited homogenate
621
enzymatic activity. It is possible that ANS and salicylate may compete with T4 for enzymatic binding sites as well as for transport proteins; however, the observations also raise the possibility that a bound form of T4 could be the substrate for the T4-metabolizing enzymes. The enzymatic activity in kidney tissue mediating the generation of T 3 from T4 was found in particulate and not in the soluble fractions. The most active particulate fractions corresponded to the easily sedimented pellets, 800 X g and 8000 X g x 10 min. No activity was found in the purified preparation of nuclei. The subcellular fractionation experiments suggest that plasma membranes may be relatively rich in the T3-forming activity. It should be pointed out, however, that mitochondria preparations are also active although the contamination of this fraction with plasma membranes has not been excluded. More precise characterization of the subcellular localization of the enzyme activity must await quantitative comparisons of enzyme activity with enzyme markers of specific subcellular fractions, as well as morphological study of the active particulate fractions. The conversion of T4 to T3 in the rat kidney homogenate appears to be an enzymatic reductive process, probably different from other described pathways which lead to oxidative or peroxidative deiodination of T4. The results of the kidney homogenate system described herein are in some aspects similar to those of the liver slice system recently described by Green (35), in which conversion of T4 to T 3 was found to be not dependent upon oxygen, not inhibited by ascorbate or by methimazole, and greatly inhibited by propylthiouracil. The kidney homogenate system is simple and reproducible; it might be useful in further characterization of the mechanism of the conversion of T4 to T 3 and in studying agents which may modify this process. References 1. Braverman, L. E., S. H. Ingbar, and K. Sterling, Conversion of thyroxine (T4) to triiodothyronine (T:i) in athyreotic human subjects, J Clin Invest 49: 855, 1970. 2. Sterling, K., M. A. Brenner, and E. S. Newman. Conversion of thyroxine to triiodothyronine in normal human subjects, Science 169: 1099, 1970.
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3. Pittman, C. S., J. B. Chambers, Jr., and V. H. Read, The extrathyroidal conversion rate of thyroxine to triiodothyronine in normal man, J Clin Invest 50: 187, 1971. 4. Schwartz, H. L., M. I. Surks, and J. H. Oppenheimer, Conversion rate of thyroxine to triiodothyronine in normal man, J Clin Invest 50: 1124, 1971. 5. Fischer, D. S., I. J. Chopra, and J. H. Dussault, Extrathyroidal conversion of thyroxine to triiodothyronine in sheep, Endocrinology 91: 1141, 1972. 6. Larson, F. C, K. Tomita, and E. C. Albright, Deiodination of thyroxine to triiodothyronine by kidney slices of rats with varying thyroid functions, Endocrinology 57: 338, 1955. 7. Green, W. L. Altered thyroxine metabolism by tissues from hypotliyroid rats, Program of the Fiftieth Annual Meeting of American Thyroid Association, St. Louis, 1974 (Abstract T14). 8. Glitzer, M. S., S. Symchowicz, and J. Gross, Metabolism of labeled thyroxine in perfused rabbit kidney in vitro, Fed Proc 15: 76, 1956. 9. Becker, D. V., and J. F. Prudden, The metabolism of 1:"Ilabeled thyroxine, triiodothyronine and diiodotyrosine by an isolated perfused rabbit liver, Endocrinology 64: 136, 1959. 10. Rabinowitz, J. L., and E. S. Herker, Thyroxine: conversion to triiodothyronine by isolated perfused rat heart, Science 173: 1242, 1971. 11. Refetoff, S., R. Matalon, and M. Bigazzi, Metabolism of Lthyroxine (T,,) and L-triiodothyronine (T:i) by human fibroblasts in tissue culture: evidence of cellular binding proteins and conversion of T4 to T.,, Endocrinology, 91: 934, 1972. 12. Sterling, K., M. A. Brenner, and V. F. Saldanha, Conversion of thyroxine to triiodothyronine by cultured human cells, Science 179: 1000, 1973. 13. Shenkman, L., V. Peck, A. Harary, C. Thaw, H. Nadel, and C. S. Hollander, Conversion of thyroxine to triiodothyronine by mouse neuroblastoma cells: evidence for possible role of tyrosine hydroxylase, J Clin Invest 53: 74a, 1974. 14. Stanbury, J. B., Deiodination of the iodinated amino acids, Ann NYAcad Sci 86: 417, 1960. 15. Chiraseveenuprapund, P., U. Buergi, A. Goswami, and I. N. Rosenberg, Formation of triiodothyronine from L-thyroxine in rat kidney homogenate, In Robbins, J., and L. E. Braverman (eds.), Thyroid Research, Excerpta Medica, Oxford, 1975, p. 244. 16. Hesch, R. P., G. Grunner, and H. D. Soling, Conversion of thyroxine (T.,) and triiodothyronine (T.i) and the subcellular localization of the converting enzyme, Clin Chim Acta 59: 209, 1975. 17. Visser, T. J., I. V. Der Does-Tobe, R. Docter, and G. Hennemann, Subcellular localization of a rat liver enzyme converting thyroxine into triiodothyronine and possible involvement of essential thiol groups, Biochem J 150: 489, 1975. 18. Chopra, I. J., Study of extrathyroidal conversion of T4 to T.-t in vitro: evidence that reverse Ti is a potent inhibitor of T.t production, Clin Res 24: 142 A, 1976. 19. Bellabarba, D., R. E. Peterson, and K. Sterling, An improved method for chromatography of iodothyronines, J Clin Endocrinol Metab 28: 305, 1968. 20. Patel, Y. C, and H. G. Burger, A simplified radioimmunoassay for triiodothyronine, J Clin Endocrinol Metab 36 187, 1973. 21. Lieblich, J., and R. D. Utiger, Triiodothyronine radioimmunoassay, J Clin Invest 51: 157, 1972.
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22. Mahler, H. R., and E. H. Cordes, Biological Chemistry, ed. 2, Harper and Row, New York, 1971, p. 448. 23. Chauveau, J., Y. Moule, and C. Rouiller, Isolation of pure and unaltered liver nuclei morphology and biochemical composition, Exp Cell Res 11: 317, 1956. 24. Neville, D. M., Jr., The isolation of a cell membrane fraction from rat liver, J Biophys Biochem Cytol 8: 413, 1960. 25. Amir, S. M., T. F. Carraway, Jr., and L. D. Kohn, The binding of thyrotropin to isolated bovine thyroid plasma membranes, JBiol Chem 248: 4092, 1973. 26. Dillman, W., M. I. Surks, and J. H. Oppenheimer, Quantitative aspects of iodothyronine binding by cytosol proteins of rat liver and kidney, Endocrinology 95: 492, 1974. 27. Sterling, K., V. F. Saldanha, M. A. Brenner, and P. O. Milch, Cytosol-binding protein of thyroxine and triiodothyronine in human and rat kidney tissue, Nature 259: 661, 1974. 28. Dawber, N. A., V. A. Gallon, and S. H. Ingbar, Degradation of thyroxine by a thyroidal peroxidase, Endocrinology 88: 144, 1971. 29. Klebanoff, S. J., and W. L. Green, Degradation of thyroid hormone by phagocytosing human leukocytes, J Clin Invest 52: 60, 1973. 30. Woeber, K. A., and S. H. Ingbar, Metabolism of L-thyroxine by phagocytosing human leukocytes, J Clin. Invest 52: 1706, 1973. 31. Wynn, J., and R. Gibbs, Thyroxine degradation. II. Products of thyroxine degradation by rat liver microsomes, J Biol Chem 237: 3499, 1962. 32. Galton, V. A., and S. H. Ingbar, A photoactivated flavininduced degradation of thyroxine and related phenols, Endocrinology 70: 210, 1962. 33. Reinwein, D., and J. E. Rail, Nonenzymatic deiodination of thyroid hormones by flavin mononucleotide and light, J Biol Chem 241: 1636, 1966. 34. Lardy, H., in Wolstenholme, G. E. W., and E. C. P. Millar (eds.), Ciba Foundation Colloquia in Endocrinology, vol. 10, Regulation and Mode of Action of Thyroid Hormones, Little, Brown, Boston, 1951, p. 154. 35. Green, W. L., Thyroxine metabolism by rat liver slices: evidence for a specific T,-forming pathway, In Robbins, J., and L. E. Braverman (eds.), Thyroid Research, Excerpta Medica, Oxford, 1976, p. 239. 36. Rosenberg, I. N., and C. S. Ahn, Enzymatic deiodination of diiodotyrosine; possible mediation by reduced flavin nucleotide, Endocrinology 84: 727, 1969. 37. Oppenheimer, J. H., H. L. Schwartz, and M. I. Surks, Propylthiouracil inhibits the conversion of L-thyroxine to i.-triiodothyronine, J Clin Invest 51: 2493, 1972. 38. Geffner, D. L., M. Azukizawa, and J. M. Hershman, Propylthiouracil blocks extrathyroidal conversion of thyroxine to triiodothyronine and augments thyrotropin secretion in man, J Clin Invest 55: 244, 1975. 39. Cruchaud, S., A. Vannotti, C. Mahaim, and J. Deckelmann, In vitro effect of methylthiouracil and oestradiol monophosphate on conversion of thyroxine to triiodothyronine by kidney slices, Lancet 2: 906, 1955. 40. Dratman, M. A. F. L. Crutchfield, J. Axelrod, C. Bond, and E. Marsh, Alpha methyl paratyrosine, a potent tyrosine hydroxylase inhibitor, inhibits T4 deiodination in vivo, Program of the Fiftieth Annual Meeting of American Thyroid Association, St. Louis, 1974 (Abstract T-9).
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Vol. 102, No. 2 Printed in U.S.A.
Conversion of L-Thyroxine to Triiodothyronine in Rat Kidney Homogenate* P. CHIRASEVEENUPRAPUND.f U. BUERGI, A. GOSWAMI, AND I. N. ROSENBERG Department of Medicine, Framingham Union Hospital, Framingham, Massachusetts, and Department of Medicine, Boston University School of Medicine, Boston, Massachusetts ABSTRACT. Rat kidney homogenates, in phosphateEDTA buffer, consistently catalyzed the formation of T.i from added L-thyroxine (T4). The formation of T:i was assessed by both paper chromatography and RIA of T.i. Conversion of T4 to T.-i appeared to be enzymatic, showing pH and temperature optima (pH 7.0 and 37 C, respectively) and tissue and time dependence. Formation of T.i was unaffected by azide, cyanide, or catalase, nor was it dependent upon oxygen; indeed, under anaerobic conditions conversion of T4 to T.i was enhanced. Dialyzed homogenate retained full activity, and no cofactor requirement was demonstrated. A' role of iron and thiol groups in the enzymatic formation of Ti from T4 was suggested by the inhibitory action of iron che-
lators and thiol-blocking reagents. The capacity of kidney for T;i formation was considerable and increased with increasing T4 concentrations, being approximately 2 nmol/g tissue/h at very high T4 levels. The apparent Km was estimated to be 3 X 10~(> M. The conversion of T4 to T.i was inhibited by propylthiouracil at micromolar concentrations whereas methimazole, iodide, and lithium salts were without effect. The enzymatic activity of the homogenates was associated with its particulate components, the readily sedimenting fractions corresponding to plasma membranes and mitochondria being most active, and was absent from nuclei and cytosol. (Endocrinology 102: 612, 1978)
E
XTRATHYROIDAL conversion of Lthyroxine (T4) to T 3 has been well documented in recent years (1-5). In vitro this process has been demonstrated in various intact cell systems (6-13); broken cell preparations were reported to be inactive in most studies (14). Recent studies in our laboratory indicated that kidney homogenates consistently converted T4 to T 3 (15), and similar findings have recently been reported by others in liver homogenates (16-18). The studies herein described were aimed at further characterization of the conversion of L-T4 to T 3 in the kidney homogenate system. Materials and Methods White male rats, 200-250 g, of the Charles River strain, maintained on Purina Laboratory chow, served as the source of the kidney tissue. L-[3',5'-
125
I]T, (SA, 40 mCi/mg) and L-[3'- 131 I]T 3 (SA, 20
mCi/mg) were obtained from Amersham Searle, and L-[125I]T3 (SA, 500 mCi/mg) was purchased from Abbott. The following substances were obReceived October 19, 1976. * This research was supported by Research Grant A2585, USPHS, NIAMDD. t Requests for reprints should be addressed to: Dr. I. N. Rosenberg, Framingham Union Hospital, 25 Evergreen Street, Framingham, Massachusetts 01701.
tained from Sigma: L-T4, 3',3,5-triiodo-L-thyronine, tetraiodothyroacetic acid (tetrac), triiodothyroacetic acid (triac), sodium azide, potassium cyanide, catalase, reduced glutathione, a-methyl tyrosine, Nethylmaleimide, DL-propranolol, glucose oxidase (type V), and bovine serum albumin; 2-mercaptoethanol was obtained from Bio-Rad Laboratories. 8-Anilino-l-naphthalenesulfonic acid (ANS), sodium salt, was purchased from Eastman Kodak. Methylmercaptoimidazole (methimazole) was a gift from Eli Lilly and 6-n-propylthiouracil was generously donated by Lederle. Under light ether anesthesia, rats were exsanguinated by cardiac puncture. The kidneys were removed, freed of their capsular investments, rinsed in ice-cold 0.1 M phosphate buffer (Na>HPO4/ KH2PO4, molar ratio 61:39), containing EDTA 5 mM, pH 7.0, blotted with gauze, and weighed. The kidneys were then minced with scissors in a beaker immersed in an ice-water bath and ground in a TenBroeck homogenizer (Kontes) in 3 vol phosphate buffer to provide a 25% (wt/vol) homogenate. Individual incubation vessels (1 X 10 cm test tubes) contained 0.3 ml kidney homogenate (75 mg tissue), L-T 4 , and test substances in a final volume of 0.5 ml phosphate buffer. The conversion of L-T4 to T 3 in the phosphate-EDTA buffer appeared to be consistently higher than in Krebs-Ringer phosphate, which had been used for homogenization and incubation in the earlier experiments. Incubation was carried out at 37 C for 1-2 h in a Dubnoff
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FORMATION OF T., FROM T4 metabolic incubator, shaken at 100 strokes/min. For experiments involving prolonged incubation, penicillin and streptomycin (0.1 mg of each substance) were added to each vessel. Two analytical techniques, paper chromatography and RIA, were used to assess the formation of T, from L-T 4 . Paper chromatography method In experiments using paper chromatography, L[3',5'-12r>I]T4, 1-2 jiiCi (32-64 pmol) was added to the incubation media; tissue-free vessels containing [12r'I]T4 served as blank controls. At the end of incubation, a large excess of L-T 4 and L-T:) (40 jug each) in 0.1 ml of 0.05 M methimazole was added to each vessel and also 0.1 ml pooled human serum. Contents were rapidly mixed (Vortex mixer) and immediately chilled in an ice-water bath. An aliquot of the mixture (10-15 JU.1) was then applied to paper (Whatman no. 3MM), dried in air, and subjected to descending chromatography in the £-amyl alcohol-2 N ammonia-hexane (5:6:1) solvent as described by Bellabarba et al. (19). The zones containing radioactive iodine compounds were localized by autoradiography, and the locations of T;i, tetrac, and triac were also determined by visualization under UV light of these substances which had been cochromatographed in carrier amounts on each paper strip. Each zone was excised and radioactivity in these zones, as well as in the intervening segments of the strip, was determined in an automatic y-counter. Radioactivity of each zone was expressed as a percentage of the total activity on the strip. In addition to the conventional paper chromatographic method for determining the proportion of [12r>I]T:t, an isotopic ratio method was also frequently used, involving addition of a trace quantity of [1;"I]T.|. In this procedure, after the addition of excess stable T4, T;J, methimazole, and serum at the end of incubation, 0.1-ml aliquots of these mixtures were transferred to individual new vessels, and a trace amount of [1;>1I]T;J (0.1 jtiCi) was added to each vessel and thoroughly mixed. Small aliquots of these final mixtures containing both isotopes were subjected to paper chromatography as described above. By measuring the 12SI and '"I in the final mixtures in each vessel, as well as in a segment of the Ti zone on the paper, the percentage of [12r'I]T,i in each sample could be determined as follows:
613
final mixtures, R2 = 12r>I (cpm)/'"I (cpm) in a segment from the T:t zone of the paper chromatogram; percentage of purity of [1:tlI]T.i (generally 93-95%) was determined by paper chromatography of an unincubated aliquot of [l:uI]T,i. The percentage of [12r>I]T:1 found by the ratio method agreed closely with values obtained by the standard chromatographic method involving determination of the radioactivity in all zones of the paper as well as the T;1 zone. The isotopic ratio method is a simple procedure, most useful in experiments in which quantitation of T.t formation alone (in contrast to other metabolites of T4) is of interest. In calculating the amounts of T4 metabolized and Ti formed in each experimental vessel, the percentage of each labeled compound found on the chromatogram was corrected by subtracting the corresponding value found in the concurrently incubated tissue-free mixtures. Since it is assumed that in commercially available [12r>I]T4, the label is randomly distributed between the 3' and 5' positions, the labeled T:1 found represents only half of the T,i formed during incubation. The quantity of T;t formed was therefore calculated by: T:t formed = 0.02 x percentage of [12'TJT:1 found (corrected by subtraction of blank values) X quantity of T4 present in the incubation mixture. T3 formation was expressed as picomoles per g tissue per h. Radioimmunoassay method
In these experiments, only stable T4 (usually 129 pmol/vessel) was used as substrate. At the end of incubation, 1 ml ice-cold absolute ethanol was added to each incubation mixture (0.5 ml) which was then vortexed and kept in an ice-water bath for 15 min before centrifugation at 2000 X g for 15 min in a refrigerated centrifuge. The ethanol extract (0.1 ml) was then assayed for T;1 by RIA using a highly specific antibody to T;i according to the method of Patel and Burger (20). In samples containing large amounts of T.i, the ethanol extracts were diluted with an ethanol-phosphate buffer solution (vol/vol, 2:1) and 0.1 ml of the final diluted extract was used. The antiserum to T,i was prepared by immunization of rabbits with T.i-bovine serum albumin complexes that had been prepared by coupling with carbodiimide (21) and injected intradermally after emulsification in complete Freund's adjuvant. The antibody to T:) cross-reacted approximately 0.001-0.002 with T4, 0.001 with tetrac, 0.5 12n 125 with triac, and less than 0.0005 with rT;t, ITyr, or [ I]T,(% of total I) I 2Tyr, compared on a molar basis with reactivity = Ri X R2 X % purity of [1:UI]T:1, of T;t as 1.00. Since the amounts of triac found in these experiments, as judged from the findings uswhere Ri = '"I (cpm)/125I (cpm) in an aliquot of
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Kndo • I»7K Vol 102 • No 2
CHIRASEVEENUPRAPUND ET AL.
614
ing the paper chromatography technique, were relatively small (0-0.3% of the total T., added) as compared with the amounts of T:) formed (3-6%), no correction of T;) values was made for possible presence of triac despite the high reactivity of the latter with the antibody. Each sample was assayed in triplicate against standards (0.02-0.8 ng T:t/vessel) which also contained a comparable concentration of ethanol (6.6%). Ethanol at this concentration was found to have no adverse effect on the RIA of T.t, results being quite similar with standard curves prepared in the presence and absence of ethanol. The measured T,t values were corrected for incompleteness of recovery of T.i in the ethanol extraction procedure, which was determined by adding known quantities of stable T.i, extracting with ethanol, and performing RIA of the extracts. Recovery was 50-55% in most experiments. Blank controls were included in each experiment: vessels containing homogenates without added T4 were incubated and at the end of the incubation, L-T4 in amounts equal to that initially present in the experimental vessels was added to each vessel; the contents were rapidly mixed and immediately extracted with ethanol. The ethanol extract was assayed for T;i as described above. Blank control values were generally small in comparison with the amounts of T.t formed in the experimental vessels; these values were subtracted from the corresponding experimental values. In experiments carried out to determine the intracellular localization of the activity responsible for conversion of T4 to T;t, 10% homogenates were prepared in 0.25 M sucrose, 0.02 M Tris buffer, pH 7.4, and subjected to subcellular fractionation by differential ultracentrifugation according to a standard procedure (22). Purified preparations of nuclei were made by the method of Chauveau et al. (23), and the procedure described by Neville (24) and modified by Amir et al. (25) was followed to obtain
plasma membranes. To determine the activity of the subcellular fractions, each was diluted with the sucrose-Tris buffer and amounts equivalent to 75 mg kidney tissue were used in the assays for T.t formation as described for the whole homogenates. Results In experiments using labeled L-T 4 , kidney homogenates were consistently active in converting T4 to T.j. Representative results are shown in Table 1. During a 90-min incubation, labeled T4 declined by 13.9%, accompanied by an increase of 3.4, 8.5, 1.2, and 0.7% (of total activity) in the zones corresponding to T;t, iodide, tetrac, and the origin. From the known quantity of T4 initially added to each incubation vessel, the amounts of T4 metabolized and T 3 formed could then be calculated as 39.5 and 19.3 pmol/g/h, respectively. Thus, approximately half of the T4 metabolized was accounted for by formation of T:i. Conversion of T4 to T;i in kidney homogenates was also readily demonstrated with the RIA method. The values of T a formation as assayed by the RIA method tended to be higher than by the paper chromatographic method. In three experiments in which Tu formation was determined by both methods in parallel incubations, the results were 20-28% higher by the RIA method in two experiments and were in close agreement in the third experiment (Table 2). The differences in results may in small part be due to the presence of triac formed during the incubation, which, as noted above, cross-reacted 50% with the T;j antiserum.
TABLE 1. Metabolism of L-[l2r>I]T4 in rat kidney homogenates Distribution of labeled constituents on paper chromatograms (% of total activity) Origin Kidney homogenates Tissue-free blanks
1.5" ±0.10 0.8'' ±0.09
I 12.1 ±0.20 3.6 ±0.12
T4
Tetrac
78.6 ± 0.27 92.5 ±0.18
2.0 ±0.06 0.8 ±0.07
5.4 ±0.13 2.0 ±0.04
Triac 0.4 ± 0.09 0.3 ±0.04
T4 metabolized (pmol/g/h) 39.5 ±0.77
T, formed /h) 19.3 ±0.74
Each incubation vessel contained kidney tissue, 75 mg, and 32 pmol [l2r'I]T4 in 0.5 ml phosphate-EDTA buffer solution (pH 7.0); incubated at 37 C for 90 min. " Mean ± SRM of four incubations. h Mean ± SRM of three incubations.
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FORMATION OF T;, FROM T4
Kinetics of formation of T:i from L-T4 Over a prolonged incubation of 16 h, there was a progressive disappearance of [125I]T4; the rate was relatively rapid in the first few TABLE 2. Comparison of RIA and paper chromatographic (PC) methods of determining formation of T.i from T4 in kidney homogenates Experimental method
T.i formed (pmol/g/h)
Exp A RIA 44.9 ± 1.8 (a) (43.7) PC 35.0 ± 1.4 (b) ExpB RIA 98.3 ± 4.3 (c) (95.7) PC 93.3 ± 1.3 (d) ExpC RIA 74.0 ± 2.4 (e) (70.6) PC 61.9 ± 1.2 (0 Incubation conditions were as described in Table 1; incubation time: 1 h. A, B, and C were three separate experiments using different pools of homogenates. Each experiment was done in parallel incubations using the same homogenate preparation with equal amounts of T4 (64 pmol in A and 129 pmol in B and C) added to the incubation medium. Each value is the mean ± SRM of four incubations. Values of the RIA method presented in parentheses are values corrected for the formation of triac as determined by the PC method in the corresponding vessels. Statistical analysis, P values: a vs. b and e us. f, 0.05.
615
hours as compared with later hours of incubation. After 3 and 8 h of incubation, labeled T;t generated from T4 constituted 4.8% and 7.3% of total activity, respectively, whereas the increment in labeled iodide tended to be 1- to 2-fold greater than T.i. There was a slight increase of activity at the origin on the paper, which rose from 1.5% at 30 min to 3% at 16 h (lower panel of Fig. 1, left). Radioactivity at the tetrac and triac regions was detectable but represented only small fractions of the total. The cumulative amounts of T4 metabolized and T;i formed at each time interval were calculated and are shown in Fig. 1, right; T.t formation accounted for approximately 40-50% of the T4 metabolized. In several experiments, where T:t formation was studied using shorter incubation times (5, 10, 15, 30, 45, 60, 90, and 120 min), the rate of T 3 formation was found to be linear over the initial 45-60 min. Effects of pH and temperature The conversion of T4 to T:J was dependent on pH and temperature. The optimal pH was found to be 7.0 (Fig. 2), and the optimal tem-
Origin Triac Tetrac 2
4
6 8 10 12 Incubation time (hrs)
14
16
18 Time of incubation (hrs)
FIG. 1. Kinetics of T4 metabolism in kidney homogenate. Each incubation vessel contained homogenized kidney tissue, 75 mg, and [I2TJT4, 32 pmol, in 0.5 ml Krebs-Ringer phosphate, supplemented with 0.5 mg penicillin and 0.5 mg streptomycin. T4 metabolites were determined by paper chromatography and expressed as percentages of total activity (left); amounts of T4 metabolized and T.i formed as a function of time are shown cumulatively on the right. Each value is the mean of triplicate incubations from the same pool of kidney homogenate.
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616
CHIRASEVEENUPRAPUND
perature was 37 C (Fig. 3). The formation of T 3 from T4 was completely abolished in homogenates placed in a boiling water bath for 5 min. Effects of incubation under anaerobic conditions Incubation under anaerobic conditions, as achieved by gassing the stoppered reaction mixtures throughly with nitrogen, consistently enhanced T3 formation by 120-150% and T4 metabolism by 40-80% (Table 3); the genera120 EXPERIMENT A
Kndo» 1978 Vol 102 • No 2
ET AL.
tion of labeled iodide was also increased in parallel with the increase in T.} formation, whereas the formation of tetrac was markedly diminished in N2. The increased T3 formation under anaerobic conditions was similarly recorded in experiments in which the assays were done by RIA. Incubation in presence of varying concentrations of L-T4 Increasing the quantity of T4 over a range of 250-13,000 pmol/vessel in the presence of a constant amount of kidney tissue resulted in formation of progressively larger amounts of T3 (Fig. 4). In a series of six experiments,
100 en
80
O (J5
60
UJ
EXPERIMENT B
40 20
FIG. 2. Effect of varying pH on formation of T.t from LT4. Each incubation vessel contained kidney homogenate (75 mg) and T4 (stable L-T4, 120 pmol in Exp A, or [125I]T4l 180 pmol in Exp B) in 0.5 ml 0.1 M phosphate buffer; incubated for 90 min at 37 C. T.-i formed was determined by RIA in Exp A and by paper chromatography in Exp B. Each value is the mean of triplicate incubations.
0
10
20 30 40 50 TEMPERATURE (C)
60
FIG. 3. Effect of temperature upon formation of T.t from L-T 4 . Each incubation vessel contained kidney tissue, 75 mg, and ['-5I]T4, 64 pmol, in 0.5 ml phosphate-EDTA buffer, pH 7.0, and incubated for 75 min. T.-i formed was determined by paper chromatography. Each value is the mean of triplicate incubations.
TABLE 3. The effect of anaerobiosis on the conversion of L-T4 to T:t by kidney homogenates Incubation conditions Exp A Tissue, in air Tissue, in N2
Distribution of labeled constituents on paper chromatograms (% of total activity) Origin 3.1
6.9
±0.02
±0.10 11.5 ±0.31
1.7
±0.002 Tissue-free blanks ExpB Tissue, in air Tissue, in N2 Tissue-free blanks
r
1.3
3.2
±0.04
±0.08
1.8
6.5
±0.17
±0.10
1.5
9.9
±0.06
±0.13
1.2
3.6
±0.07
±0.14
T4 83.6 ±0.14 79.0 ±0.72 93.4 ±0.07 86.8 ±0.34 81.9 ±0.16 92.7 ±0.42
Tetrac
T,
Triac
2.2
4.1
0.4
±0.10
±0.01
±0.09
0.7
7.3
0.3
±0.18
±0.15
±0.08
0.7
1.4
0.3
±0.11
±0.05
±0.03
1.3
3.1
0.5
±0.13
±0.07
±0.09
0.7
5.8
0.4
±0.10
±0.30
±0.03
0.7
1.3
0.3
±0.10
±0.04
T 4 metabolized (pmol/g/h)
T, formed (pmol/g /h)
112.4 ±1.61 165.1 ±8.26
61.9 ±0.23 135.3 ±3.44
101.5 ±5.85 185.8 ±2.75
61.9 ±1.2 154.8 ±5.16
±0.05 Individual vessels contained kidney homogenates, 75 mg, and labeled T4, 129 pmol, in 0.5 ml of phosphate-EDTA buffer solution (pH 7.4 in Exp A, and 7.0 in Exp B), incubated for 90 min (in Exp A) and 60 min (in Exp B). Each value is the mean ± SEM of four incubations.
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FORMATION OF T3 FROM T4
of kidney tissue; however, the increased activity was not always proportional to the increase in quantity of tissue. In some experiments, there was a linear relationship between quantity of tissue and activity; in other experiments, dilution of homogenate was associated with relatively less decline in activity than in tissue concentration (Fig. 5).
0.005 1
V 0.004 ^Sx-
0.003
^
617
0.979
^
0.002
Effect of antithyroid drugs
0.001 i
-0.3 xlO6 0
O.5xlO6
i
I.OxlO6
J_
i
l.5xlO6
I
2.0xl0 6
S
FIG. 4. T.t formation as a function of substrate (T4) concentration. Each vessel contained kidney tissue, 75 mg, and [l25I]T.t supplemented with varying amounts of stable TV incubated under anaerobic conditions for 30 min at 37 C. T.i formed was determined by paper chromatography. Each value is the mean of triplicate incubations. V = rate of T.t formation, pmol/g tissue/h; S = molar concentrations of T4.
the apparent Michaelis-Menten constant (Km) for T4 was estimated to be 2.5-3.2 X 10"6 M (average value, 3 X 10~6 M), and the apparent Vmax was 1.7-2.3 nmol (average value, 2.0 nmol) of T 3 formed/g tissue/h. Effects of L-T3 Supplementing the incubation mixture with varying amounts of stable L-T3 in the range of 7.6-190 pmol/vessel did not appreciably affect the rate of formation of T 3 from labeled T4 (32 pmol/vessel) at either 1 or 4 h of incubation. These findings suggested that there was little inhibition of T4 monodeiodination by the product T3. In separate experiments, incubation of labeled T 3 (150 pmol/vessel) in kidney homogenate for 1 h resulted in deiodination of less than 2% of the added T3, whereas concurrent experiments with T4 using the same homogenates and under the same conditions showed 12-15% of the T4 was deiodinated. These findings suggest relatively little further metabolism of the T 3 newly formed from T4 incubated with kidney homogenates. Effect of varying amounts of kidney tissue There was a progressive increase of T 3 formation in the presence of increasing amounts
The conversion of T4 to T 3 was greatly inhibited by 6-/i-propylthiouracil, whereas methimazole was found to have no effect; T 3 formation in homogenates incubated in a nitrogen atmosphere was much greater than in air, as previously noted (Table 3), and the inhibitory action of propylthiouracil was apparent in both nitrogen and air incubations (Fig. 6). In homogenates incubated in presence of 5, 10, and 50 fiM propylthiouracil, T 3 formation was inhibited by 40, 70, and 80%, respectively. Neither sodium iodide (2 HIM) nor lithium carbonate (5 mM) was inhibitory. Effect of varying conditions and agents Sonication of homogenates before incubation resulted in substantial loss of the activity (Table 4). The activity appeared to be labile and was consistently decreased in homogenates which were frozen and thawed prior to incubation as well as in homogenates prein100 -
20 40 60 80 KIDNEY TISSUE (mg/vessel)
FIG. 5. Effect of varying amounts of tissue on rate of formation of T.t from T4. Incubation conditions were as described in Tables 1 and 2. Values shown in broken lines were obtained by the paper chromatographic method; values shown in solid lines were obtained by the RIA method. T:) formation is expressed as a percentage of the values found in the vessels containing 75 mg tissue. Each value is the mean of triplicate incubations.
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CHIRASEVEENUPRAPUND ET AL.
618 ju/an
D Air
X 200
• N
II ' e •• 3 100 1• ! 50 E
•? 150 in 0> O
a>
1
Addition of
None
1I •• 1 • 1 _• MMI 5mM
PTU 5mM
FIG. 6. Effect of propylthiouracil (PTU) and methimazole (MMI) on conversion of T4 to T.t in kidney homogenate. Each vessel contained kidney tissue, 75 mg, stable 1.-T4, 128 pmol, and test substances where indicated, in 0.5 ml phosphate buffer (0.1 M)-EDTA (5 DIM) solution; incubated for 2 h. Amounts of T:t formed were determined by RIA. Each value is the mean of triplicate incubations. TABLE 4. Effects of sonication, freezing, and preincubation at varying temperatures on formation of T, and T.,
but high salt concentrations inhibited greatly (90% decrease in activity in 1.2 M NaCl). A number of agents were tested for possible effects on the conversion of T4 to T;J in this system; results are summarized in Table 5. Preincubation of kidney homogenates with azide, cyanide or catalase and incubation in presence of these substances was found to have no effect on T:i formation. Formation of T 3 was greatly impaired by H2O2 and an H2O2generating system (glucose-glucose oxidase); the amounts of T4 metabolized were diminished proportional to the decline in T;) formation. In the presence of EDTA (10 mM), T3 formation was stimulated by 90%. The activity was inhibited by specific iron chelators, TABLE 5. Effects of various agents on formation of T.i from T.
li)7H Vol 102 i No 2
None Sodium azide" (2 mM) Sodium cyanide" (2 mM) Catalase" (2000 units/ml) H2O2 0.3 mM 1.0 mM Glucose" (2 mg/ml) + glucose oxidase 2 units/ml 10 units/ml EDTA" (10 mM) o-Phenanthroline (10 mM) 2,2'-Dipyridyl (10 mM) Mercaptoethanol" (5 mM) Reduced glutathione" (5 mM) N-Ethylmaleimide (5 mM) Ascorbic acid" (5 mM) DL-a-tocopherol (5 mM) Normal human serum (protein 28 mg/ml) Bovine serum albumin (28 mg/ml) Sodium salicylate 1 mM 5 mM ANS, sodium salt 1 mM 5 mM a-Methyltyrosine" (5 mM) DL-Propranolol (2 mM)
Formation ofT,(%of control) 100 93 113 112 65 3 26 22 191 32 19 116 90 14 100 102 10 45 29 16 27 11 96 103
Ti formation is expressed as a percentage of the quantity of T.i formed in the group which had no test substances added. Values are the mean of two or three experiments, each done in triplicate for each substance. Incubation conditions were as described in Table 1; T.i formation was determined by paper chromatography. 0 Effect of T 4 to T 3 conversion checked also by RIA of T,), with results similar to those found using paper chromatography.
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FORMATION OF T3 FROM T4 o-phenanthroline and 2,2'-dipyridyl and also by iV-ethylmaleimide, a thiol-blocking agent. These findings suggest that non-heme iron and thiol groups may participate in the T4 to T.t conversion. Addition of reduced glutathione, mercaptoethanol, ascorbic acid, and atocopherol had no significant effect. DL-propranolol, a /?-adrenergic blocker commonly used in treating hyperthyroid patients, was found to have no effect. Tyrosine hydroxylase has been suggested to play a role in converting T4 to T;t in neuroblastoma cells (13) and amethyl tyrosine, an inhibitor of this enzyme, was therefore tested and was found to exert no inhibition on the formation of T.t from T4 in the kidney homogenate. The conversion of T4 to T;i was greatly inhibited by human serum and, to a lesser extent, by bovine serum albumin. Both salicylate and ANS, agents known to displace T4 from protein binding sites, were also inhibitory. The activity was unchanged by addition of reduced or oxidized pyridine nucleotides (NAD, NADH, NADP, NADPH), FAD, or FMN to a final concentration of 1 mM. Subcellular localization of the conversion of T4 to T:i Studies were made to determine the intracellular localization of the activity responsible for the conversion of T4 to T3 in kidney tissue. Results are summarized in Table 6. The activity was regularly present in the 100,000 X g pellet and absent from the supernatant, suggesting its particulate nature. The activity was distributed in all the particulate fractions, being greatest in the crude nuclear pellet, with lesser activity in the mitochondrial and least in the microsomal fractions. Addition of pyridine nucleotides, flavins, and a dialysate of fresh homogenate did not restore activity to the microsomal fraction. Purified nuclei were found to be inactive. Activity was regularly found at the interface between 30% and 40% sucrose, a fraction considered to be enriched in plasma membranes; the activity was about 25% of that found in the whole homogenate. However, comparable activity was also observed in material from the layer between 40%
619
TABLR 6. Subcellular localization of the activity converting T\ to T:t in kidney tissue Cell fractions
T.rforming activity
Exp A 100 Whole homogenate 87 100,000 x g x 90 min pellet 0 100,000 x g x 90 min supernate ExpB 48 (a) Crude nuclear pellet (800 X g X 10 min) 25 (b) Mitochondrial fraction (8,000 x g x 10 min) 7 (c) Microsomal fraction (100,000 x g x 60 min) ExpC 0 (a) Purified nuclei" 25 (b) Purified plasma membrane'1 Ten percent kidney homogenates were freshly prepared with a Dounce homogenizer (loose pestle) in 0.25 M sucrose, 0.02 M Tris, pH 7.4. In Exp A, pellet and supernatant resulting from centrifugation at 100,000 x g for 90 min were separated and assayed. In Exp B, pellets sedimenting at 800 x g x 10 min, 8,000 X g x 10 min, and 100,000 x g x 60 min, were prepared by successive centrifugation of the preceding supernatant. Each pellet was washed once by suspending in sucrose-Tris buffer and recentrifuged at the same force; the washed pellet was re-suspended in sucrose-Tris buffer, and an aliquot equivalent to 75 mg kidney tissue was assayed for activity. Informing activity was expressed as a percentage of the activity found in 75 mg tissue of the whole homogenate. Each value is the mean of three experiments done in triplicate with each fraction of each experiment. " Purified nuclei were prepared by the method of Chauveau et al. (23). h Plasma membranes were prepared by the procedure of Neville (24) as modified by Amir et al. (25) using a discontinuous sucrose gradient (30, 40, and 45%, wt/vol). The fraction between 30% and 40% sucrose was considered to be enriched in plasma membranes at the interface.
and 45% sucrose, generally considered to contain both mitochondria and plasma membranes.
Discussion The ability of kidney tissue to effect the conversion of T4 to T3, previously noted in kidney slices (6), does not depend upon intact cell structure and is readily demonstrated in kidney homogenates. Microscopic observation of both the fresh homogenates and of stained sections of the sediment obtained after centrifuging homogenates revealed very few unbroken cells. In addition, homogenates prepared in distilled water, in which the hypotonicity would be expected to result in cell disruption, effectively catalyzed the formation of T,}.
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620
CHIRASEVEENUPRAPUND ET AL.
Some requirement of structure at a subcellular level is suggested by the impairment of activity after sonication of the homogenate. The quantity of T 3 formed as assessed by the RIA method tended to be somewhat higher than by the paper chromatographic method (Table 2). However, both analytical methods yielded results that were generally comparable, and there was close agreement between the methods with respect to the degree of stimulation or inhibition in response to changes in experimental conditions or to test substances. The 5'-monodeiodination of L-T4 in the homogenate appeared to be enzymatic in nature showing temperature and pH optima and tissue dependence. Activity increased with increasing tissue concentration although a linear relationship between these variables was not consistently observed. Indeed, the activity of diluted homogenates often exceeded what would be expected from the degree of dilution, raising the possibility that the kidney may contain inhibitors of T:i formation. Such putative inhibitors appeared to be non-dialyzable since dialysis of homogenates did not enhance the enzymatic activity. The data on the rate of conversion of T4 to Ta by the whole kidney homogenate, as the total T4 concentrations were altered, appeared to conform to Michaelis-Menten kinetics (Fig. 4), and on this basis the apparent Km for T4 was estimated to be 3 x 10~h M. It is known that T4-binding proteins are present in kidney cytosol (26, 27), and therefore it is likely that the concentration of the free T4 in kidney homogenates was substantially less than that of the total, and it is conceivable that free rather than bound T4 is the effective substrate in the T4 to T;i conversion. If this were the case, the Km of 3 X lCT6 M might be a considerable overestimation. The importance of free T4 was suggested by our observation that diluting the homogenates enhanced, and addition of serum containing thyroxine-binding globulin diminished the T4 to T 3 conversion. On the other hand, agents that displace T4 from T4-binding proteins, ANS, and salicylates, which would presumably increase free T4 in the kidney homogenates, did not in-
Kudo Vol 102
l!)78 No 2
crease T.i formation, and experiments using the pellet fraction (1000 x g), presumably depleted of cytosol T4-binding proteins, disclosed a similar value for Km (unpublished observation). At present, it is not clear whether T4 must be made available to the enzyme in the free or in the bound form; the apparent Km calculated on the basis of the total T4 concentrations may have quantitative usefulness until this aspect of the reaction mechanism is better characterized. There is a possibility that the temperature and pH optima noted for the enzymatic conversion of T4 to T;J in the kidney homogenates reflect, at least in part, altered binding of the substrate to tissue proteins. Studies of alteration in free T4 concentrations and reaction rates as affected by changes in pH and temperature would be needed to assess this point. Several published studies of the deiodination of T4 by tissues in vitro have suggested an oxidative or peroxidative mechanism (28-31), and have emphasized the oxygen dependence of the deiodinating process and its inhibition by reducing substances. Iodide and protein-bound iodine of incompletely defined nature (origin material on paper chromatography) have been the major products of oxidative T4 deiodination in most studies, very little or no T.t being found. Light and flavins, as well as oxygen, have been shown to accelerate T4 deiodination (32, 33). In the present study, by contrast, the formation of origin material and of iodide in excess of T.( formation was modest; T;) formation was not affected by the presence of azide, cyanide, and catalase, and, indeed, H2O2 greatly inhibited both Ta formation and T4 deiodination. Light and flavins had no effect. Moreover, the conversion of T4 to T.j proceeded more efficiently under anaerobic conditions than in the presence of air. The lack of effect of anaerobiosis on the conversion of T4 to T,} has been reported in studies using kidney and liver slices (34, 35). The mechanism of the potentiating effect of nitrogen is not clear; it is possible that anaerobically the degradation of T4 via other oxygen-dependent pathways is inhibited and this could facilitate a metabolic pathway, not requiring oxygen and leading to T,t for-
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FORMATION OF T3 FROM T4 mation. The absence in anaerobic experiments of products of oxidative metabolism of T4 such as tetrac and triac, is consistent with this idea. However, a possible inhibitory action of oxygen on T 3 formation should also be considered since in nitrogen, not only was T4 more efficiently converted to T3 but more T4 was metabolized as well, compared to the aerobic experiments. The findings suggest that the 5'monodeiodination of T4 by kidney is not mediated by a peroxidative process, and that the process may resemble the enzymatic reductive deiodination of iodotyrosines by the thyroid and liver iodotyrosine dehalogenase (36). No evidence of a dialyzable cofactor or inhibitor of T 3 formation in the homogenate was adduced, activity being unaffected by dialysis. Unlike the thyroid iodotyrosine dehalogenase, which requires NADPH as a cofactor, none of the adenine nucleotides (NAD, NADH, NADP, NADPH) tested was found to have a consistent stimulating effect on the conversion of T4 to T3. Both iron and thiol groups may be necessary for this conversion since the activity was greatly impaired in presence of specific iron chelators (o-phenanthroline and 2,2'-dipyridyl) and of a thiol-blocking agent (iV-ethylmaleimide). Propylthiouracil was found to inhibit the formation of T3 from T4 in the system, in agreement with published observations on the in vivo (37, 38), and the in vitro metabolism of T4 to T3. Inhibition of the T4 conversion to T 3 was also observed with thiouracil and methylthiouracil in kidney slices (6, 39). Other agents used for the treatment of hyperthyroidism, such as methimazole, iodide, lithium salts, and propranolol, were found to have no effect. Since a-methyltyrosine, a potent tyrosine hydroxylase inhibitor, was likewise without effect on this process, it is unlikely that the formation of T3 in the kidney homogenates is mediated by an aromatic amino acid hydroxylase as proposed by Dratman et al. (40). Although both human serum and bovine serum albumin inhibited conversion of T4 to T3, conceivably because of their thyroxinebinding capacity, salicylate and ANS, which displace T4 and T 3 from circulating thyroxinebinding proteins, also inhibited homogenate
621
enzymatic activity. It is possible that ANS and salicylate may compete with T4 for enzymatic binding sites as well as for transport proteins; however, the observations also raise the possibility that a bound form of T4 could be the substrate for the T4-metabolizing enzymes. The enzymatic activity in kidney tissue mediating the generation of T 3 from T4 was found in particulate and not in the soluble fractions. The most active particulate fractions corresponded to the easily sedimented pellets, 800 X g and 8000 X g x 10 min. No activity was found in the purified preparation of nuclei. The subcellular fractionation experiments suggest that plasma membranes may be relatively rich in the T3-forming activity. It should be pointed out, however, that mitochondria preparations are also active although the contamination of this fraction with plasma membranes has not been excluded. More precise characterization of the subcellular localization of the enzyme activity must await quantitative comparisons of enzyme activity with enzyme markers of specific subcellular fractions, as well as morphological study of the active particulate fractions. The conversion of T4 to T3 in the rat kidney homogenate appears to be an enzymatic reductive process, probably different from other described pathways which lead to oxidative or peroxidative deiodination of T4. The results of the kidney homogenate system described herein are in some aspects similar to those of the liver slice system recently described by Green (35), in which conversion of T4 to T 3 was found to be not dependent upon oxygen, not inhibited by ascorbate or by methimazole, and greatly inhibited by propylthiouracil. The kidney homogenate system is simple and reproducible; it might be useful in further characterization of the mechanism of the conversion of T4 to T 3 and in studying agents which may modify this process. References 1. Braverman, L. E., S. H. Ingbar, and K. Sterling, Conversion of thyroxine (T4) to triiodothyronine (T:i) in athyreotic human subjects, J Clin Invest 49: 855, 1970. 2. Sterling, K., M. A. Brenner, and E. S. Newman. Conversion of thyroxine to triiodothyronine in normal human subjects, Science 169: 1099, 1970.
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CHIRASEVEENUPRAPUND ET AL.
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