The cDNA for the Type I Iodothyronine 5”Deiodinase Encodes an Enzyme Manifesting Both High Km and LOW Km Activity EVIDENCE THAT RAT LIVER AND KIDNEY CONTAIN A SINGLE ENZYME WHICH CONVERTS THYROXINE TO 3,5,3”TRIIODOTHYRONINE*

The enzymatic conversion of thyroxine (T4) to 3,6,3’-triiodothyronine (Ts) by iodothyronine 6’-deio-dinase(s) is an obligate step in the physiologic action of thyroid hormones in most extrathyroidal tissues. In the rat liver and kidney, S’-deiodinase processes having either high K , (micromolar range) or low K , (nano- mokr range) values for thyroid hormone substrates have been described. The number of enzymes media- ting these reactions, however, remains uncertain and controversial. To examine this question we have com- pared the 6’-deiodinase activity expressed in membrane preparations of Xenopus laevis oocytes after the injection of either rat liver poly(A)+ RNA or in vitro prepared RNA transcribed using the G21 full-length type I 6”deiodinase cDNA. In oocytes injected with rat liver poly(A)+ RNA, high K , (i.e. type I) activity was observed when 20 mM dithiothreitol was used as the thiol cofactor, whereas K,,, values in the nanomolar range were noted with 0.6 mM dithiothreitol, glutathione, or a reconstituted thioredoxin cofactor system. This complex pattern of 6’-deiodinase activity, which mimics that found in homogenates and subcellular frac- tions of rat liver ) , thioredoxin reductase (42 nM), and NADPH (0.5 mM). Due to the limited amount of oocyte material available, most experiments utilized single data points for each determination. When sufficient material was available to perform duplicate incubations the coefficient of variation of such determinztions was 41%. In some experiments ascending paper chromatography using a tertial amyl alcohol/2 N ammonium hydroxide (1:l) solvent system was also utilized to identify the reac- tion products (11). Hybrid Arrest Experiment-Hybrid arrest of translation was per- formed using the B2-21 type I 5”deiodinase cDNA and the irrelevant B2-14 cDNA as previously described (8). After incubation for 3 days, membranes from injected oocytes were prepared and assayed for 5’-deiodinase activity as described above. Other Determinatiom-Protein was quantitated by the method of Bradford (12) with reagents obtained from Bio-Rad. Kinetic data were analyzed by Lineweaver-Burk plots.

The cDNA for the Type I Iodothyronine 5"Deiodinase Encodes an Enzyme Manifesting Both High Km and LOW Km Activity EVIDENCE THAT RAT LIVER AND KIDNEY CONTAIN A SINGLE ENZYME WHICH CONVERTS THYROXINE TO 3,5,3"TRIIODOTHYRONINE* (Received for publication, December 2, 1991) Jahangir Sharifi and Donald L. St The enzymatic conversion of thyroxine (T4) to 3,6,3'-triiodothyronine (Ts) by iodothyronine 6'-deiodinase(s) is an obligate step in the physiologic action of thyroid hormones in most extrathyroidal tissues. In the rat liver and kidney, S'-deiodinase processes having either high K , (micromolar range) or low K , (nano-mokr range) values for thyroid hormone substrates have been described. The number of enzymes mediating these reactions, however, remains uncertain and controversial. To examine this question we have compared the 6'-deiodinase activity expressed in membrane preparations of Xenopus laevis oocytes after the injection of either rat liver poly(A)+ RNA or in vitro prepared RNA transcribed using the G21 full-length type I 6"deiodinase cDNA. In oocytes injected with rat liver poly(A)+ RNA, high K , (i.e. type I) activity was observed when 20 mM dithiothreitol was used as the thiol cofactor, whereas K,,, values in the nanomolar range were noted with 0.6 m M dithiothreitol, glutathione, or a reconstituted thioredoxin cofactor system. This complex pattern of 6'-deiodinase activity, which mimics that found in homogenates and subcellular fractions of rat liver and kidney, was reproduced exactly in oocytes by the microinjection of G21-derived RNA transcripts. Furthermore, hybrid arrest of translation in oocytes using a partial type I 6'-deiodinase cDNA completely inhibited the expression of both high and low K , activity after the injection of rat liver poly(A)+ RNA. These findings demonstrate that rat liver and kidney contain only a single 6"deiodinase which manifests either high or low K , activity depending on the reduced thiol cofactor utilized in the reaction.
Thyroxine (TJ,' the principal secretory product of the thyroid gland, functions primarily as a prohormone and undergoes a single deiodination of the phenolic ring in extrathyroidal tissues to form the metabolically more active hormone 3,5,3'-triiodothyronine (T3) (1). The biochemical characterization of the iodothyronine 5"deiodinases which catalyze this reaction has been hampered by the inability to purify * These studies were supported by National Institutes of Health Grant DK-42271 (to D. L. S.) and Norris Cotton Cancer Center Core Grant CA 23108. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "aduertkement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The abbreviations used are: T,, thyroxine; T3, 3,5,3'-triiodothyronine; DTT, dithiothreitol; HRL, hyperthyroid rat liver; IOP, iopanoic acid; PTU, 6-n-propyl-2-thiouracil; rT3, 3,3',5'-triiodothyronine. these proteins to homogeneity in an active form. Thus, considerable uncertainty and controversy exists as to the number of enzymes which mediate 5"deiodination (2, 3). Based on kinetic studies, tissue distribution, and susceptibility to inhibition by 6-n-propyl-2-thiouracil (PTU), two principle types of 5"deiodinase activity have been defined in various tissue homogenates when assayed using high concentrations of dithiothreitol (DTT, e.g. 20 mM) as cofactor (1). Type I 5'deiodinase is found primarily in the liver, kidney, and thyroid gland, manifests a high K , for thyroid hormone substrates, and is sensitive to inhibition by PTU. In contrast, type I1 activity is relatively resistant to inhibition by PTU, manifests much lower substrate K , values, and is present almost exclusively in brown adipose tissue, the pituitary gland, and the central nervous system.
In addition to the type I activity in the liver and kidney, "low K," 5"deiodinase processes have also been described in these tissues when thiol cofactors such as glutathione (4), thioredoxin (5,6), or low concentrations of DTT (0.5 mM) (7) have been used to support deiodination. Whether or not such low K , activity is secondary to the type I enzyme or mediated by other 5"deiodinases is uncertain, but is of considerable importance; the high K,,, values of 4 and 0.1 PM of the type I enzyme for T, and 3,3',5'-triiodothyronine (reverse TS, rT3), respectively, far exceed their intracellular concentrations. Thus, the low K , renal and hepatic 5"deiodinase processes, which manifest K , values for these same substrates in the nanomolar range and can potentially utilize naturally occurring thiols as cofactors to support deiodination, are likely to be of more physiologic relevance.
The recent isolation of cDNAs for the type I 5"deiodinase by ourselves (8) and Berry et al. (9) has provided a means of studying the patterns of deiodination in the rat liver and kidney in greater detail and in a more definitive fashion. In particular, the availability of a full-length cDNA for this enzyme (9) affords the opportunity to express the type I 5'deiodinase in an in vivo model system and to study its properties under conditions of varying substrate and cofactors. For such studies, we chose to use the Xenopus laevis oocyte expression system so that the properties of the enzyme encoded by the recombinant type I 5"deiodinase cDNA could be compared directly with the activity induced by the injection of native rat liver poly(A)+ RNA. Our findings provide strong evidence that rat liver and kidney contain only a single iodothyronine 5"deiodinase which manifests both type I and low K,,, activity.

EXPERIMENTAL PROCEDURES
General Method~-['~~I]rT~ (specific activity -1200 pCi/pg) or [lZ5I] Tq (specific activity -150 pCi/pg) were obtained from Du Pont-New England Nuclear and purified by Chromatography using Sephadex LH-20 (Sigma) prior to use. Recombinant Escherichia coli thioredoxin was purchased from Calbiochem (San Diego, CA) in lyophilized form and dissolved in 0.1 M potassium phosphate, pH 7.0, 1.0 mM EDTA. Recombinant E. coli thioredoxin reductase was generously provided by Dr. James A. Fuchs (University of Minnesota, St. Paul, MN). Male Sprague-Dawley rats (200 g, Charles River Laboratories, Wilmington, MA) were rendered hyperthyroid by the daily injection of T, (12 pg/100 g body weight, subcutaneously), andpoly(A)+ RNA was prepared from liver tissue as previously described (10). Capped RNA transcripts were synthesized in vitro using T3 RNA polymerase according to instructions provided by the supplier (Stratagene, La Jolla, CA). The transcripts were purified by phenol-chloroform extraction and ethanol precipitation prior to injection into oocytes.
Description of cDNAs-The full-length type I 5"deiodinase G21 cDNA in the Bluescript vector was kindly provided by Drs. Marla J.
Berry and P. Reed Larsen (Harvard Medical School, Boston, MA).
The partial length B2-21 cDNA, previously isolated in this laboratory (8) and encompassing nucleotides 32-256 of the coding region of the G21 clone, was used in the hybrid arrest experiment.
Oocyte Injection and Harvesting-Stage V-VI X . laevis oocytes were isolated and microinjected as previously described (10) with 10-25 ng of rat liver poly(A)+ RNA or 0.5 ng of in vitro prepared G21 RNA transcripts. Control oocytes were either not injected or were injected with an equal volume (50 nl) of water. After injection oocytes were incubated in L-15 medium a t 18 "C for 3-6 days prior to harvesting. Pools of approximately 60 injected oocytes were then homogenized and subjected to differential centrifugation for the preparation of crude nuclei (1,000 X g pellet), mitochondria (16,000 X g pellet), microsomes (100,000 X g pellet), and cytosolic (100,000 X g supernatant) fractions as previously described (10). In most experiments, the infranatant from the 1,000 X g initial centrifugation was used to prepare a combined mitochondrial/microsomal ("membrane") subcellular fraction. For this preparation the infranatant was centrifuged at 100,000 X g for 1 h in an Airfuge ultracentrifuge (Beckman Instruments, Palo Alto, CA), the pellet rinsed twice in buffer, then resuspended in buffer and centrifuged again a t 100,000 X g for 1 h, followed by another rinse and final resuspension in buffer using a Dounce homogenizer. Several buffers were used in different experiments and included 5'-Deiodinase Assay--5'-Deiodinase activity was assayed in a total reaction volume of 50 pl as the amount of lZ51 released from ['"I]rT, or [12sI]T4 as previously described (10). Reaction mixtures typically contained 20-40 pg of membrane protein, and incubation times ranged from 10 min to 3 h. Thiol cofactors used in the assay included D T T (20 or 0.5 mM final concentration), GSH (5 mM), or a reconstituted thioredoxin system which included thioredoxin (42 p~) , thioredoxin reductase (42 nM), and NADPH (0.5 mM). Due to the limited amount of oocyte material available, most experiments utilized single data points for each determination. When sufficient material was available to perform duplicate incubations the coefficient of variation of such determinztions was 4 1 % . I n some experiments ascending paper chromatography using a tertial amyl alcohol/2 N ammonium hydroxide (1:l) solvent system was also utilized to identify the reaction products (11). Hybrid Arrest Experiment-Hybrid arrest of translation was performed using the B2-21 type I 5"deiodinase cDNA and the irrelevant B2-14 cDNA as previously described (8). After incubation for 3 days, membranes from injected oocytes were prepared and assayed for 5'deiodinase activity as described above.
Other Determinatiom-Protein was quantitated by the method of Bradford (12) with reagents obtained from Bio-Rad. Kinetic data were analyzed by Lineweaver-Burk plots.

RESULTS
We have previously demonstrated that the injection of poly(A)+ RNA from rat liver into X . laevis oocytes induces type I 5"deiodinase activity (as measured using 20 mM DTT as cofactor) in a subcellular distribution similar to that noted in rat liver and kidney. Highest specific activity is found in the microsomal fraction (100,000 X g pellet) with lesser, but significant amounts of activity in the mitochondria (16,000 x g pellet) (10). In an initial experiment, we sought to determine if GSH could support 5'-deiodination in oocytes injected with rat liver poly(A)+ RNA. As shown in Fig. 1, GSH stimulated 5"deiodinase activity in subcellular fractions from oocytes injected with poly(A)+ RNA from hyperthyroid rat liver, whereas no activity was found in non-injected or water injected oocytes (data not shown). The distribution of specific activity using GSH as cofactor was identical to that of type I activity (lo), being highest in the microsomal fraction. However, the K,,, for rT3 was considerably lower (0.2-0.3 nM) than the value of approximately 0.2 p~ previously determined for the type I process (10) and was thus consistent with values reported in rat liver and kidney microsomes using GSH as a cofactor (4, 13). These results demonstrated that a low K,,, 5 'deiodinase process could be induced in X . laevis oocytes by the injection of rat liver poly(A)+ RNA. Because significant amounts of low K,,, activity were present in the crude mitochondrial fraction (16,000 X g pellet) we utilized a combined membrane preparation containing both crude mitochondria and microsomes for all subsequent experiments (see "Experimental Procedures").
The time course of i n vitro deiodination of rTg in membrane preparations from control (non-injected or water-injected) oocytes or oocytes injected with hyperthyroid rat liver poly(A)+ RNA or i n uitro prepared G21 transcripts is shown in Fig. 2. In control oocytes (and buffer only reaction mixtures (data not shown)), little or no deiodination was detected during the 130-min incubation period in the absence or presence of thiol cofactors. Significant deiodination was noted, however, in the membrane preparation from hyperthyroid rat liver poly(A)+-injected oocytes (Fig. 2 A ) in the absence of added cofactors (-13% substrate deiodinated after 130 min).
In the presence of GSH or the thioredoxin cofactor system, 5"deiodinase activity increased approximately 2-fold during the incubation period from 40 to 130 min. Using 0.5 mM DTT as cofactor, activity was increased %fold.
In oocytes injected with G21 transcripts, considerable activity (20-30%) was also noted in the absence of cofactors, and this activity was stimulated with GSH, DTT, and the thioredoxin system from 2.5-to 7-fold in both Tris-sucrose (  PTU is a potent inhibitor of type I and the low K, 5'deiodinating processes in liver and kidney microsomes (1-7). As demonstrated in Fig. 3, PTU (100 KM) inhibited by 99% the GSHand thioredoxin system-stimulated 5"deiodinase activity in oocytes injected with either hyperthyroid rat liver poly(A)+ RNA or G21 transcripts.
Kinetic analysis, using rT3 as substrate, of the 5"deiodinase activity induced in oocytes by the injection of hyperthyroid rat liver poly(A)+ RNA or G21 transcripts was performed without and with a variety of cofactors and buffers ( Fig. 4 and Table I). In the absence of cofactors, 5"deiodinase activity followed simple saturable kinetics with K,,, values of 1.0-1.6 nM in membrane preparations of oocytes injected with either RNA preparation. Similar K,,, values were noted when  Table I were dependent on a number of factors including: (a) the thiol cofactor used, ( b ) the activity of various poly(A)+ RNA and G21 transcript preparations used during the course of these studies, and (c) inherent batch-to-batch variation in oocyte expression. For these reasons comparison of V,,, values between different cofactor groups in Table I is difficult. Iopanoic acid is a competitive inhibitor of both type I 5'deiodinase as well as the low K,,, processes determined using various cofactors in rat liver and kidney (1-7). Using 0.5 mM DTT in oocyte membrane preparations, iopanoic acid inhibited activity induced by both hyperthyroid rat liver poly(A)+ RNA and G21 transcripts in a competitive fashion with a K , value of 0.5 PM (Fig. 5). Using 20 mM DTT as cofactor, the K; value with a membrane preparation from G21 transcriptinjected oocytes was considerably higher (100 PM, data not shown). These Ki values are essentially identical to those previously determined by others in rat liver microsomes using these cofactor concentrations (7). Experiments were also performed with various cofactors using T4 as substrate (Fig. 6). In uninjected oocytes deiodination of T4 was minimal (51.2%). In oocytes injected with hyperthyroid rat liver poly(A)+ RNA or G21 transcripts, T4 deiodination was clearly demonstrated in the absence of cofactors and was stimulated by DTT, GSH, and the thioredoxin system. Under all conditions, however, the extent of deiodi-  nation was considerably less than when rTB was used as substrate. This low level of 5'-deiodination precluded accurate kinetic analysis using T 4 as substrate. In one experiment the efficiency of 5'-deiodination of T4 compared to rT3 was determined in the same membrane preparation from G21 transcript-injected oocytes using equal concentrations of both substrates (4 nM) and the thioredoxin system as cofactor. The reaction velocity with rT, was at least 60-fold greater than the velocity using T4, a value similar to that noted in a control experiment using rat kidney microsomes with the same substrate and cofactor system (data not shown). The marked substrate preference for rT3 demonstrated in these studies has been observed previously by other investigators in liver microsomes (5-7, 14). In other experiments using membrane preparations from oocytes injected with hyperthyroid rat liver poly(A)+ RNA or G21 transcripts, PTU (100 PM) inhibited 5'-deiodinase activity by 32-73% when assayed using T4 as substrate and 0.5 mM DTT, 5 mM GSH, or the thioredoxin system as cofactor.
We have previously demonstrated in hybrid arrest experiments that the B2-21 type I 5"deiodinase cDNA prevents expression of type I activity in oocytes injected with hyperthyroid rat liver poly(A)+ RNA (8). Thus, a similar experiment was conducted to determine if this cDNA could also impair the expression of low K,,, activity. As shown in Fig. 7, expression of both type I and low K,,, (i.e. GSH-stimulated) 5'deiodinase activity was inhibited >95% by hybrid arrest with the B2-21 cDNA, whereas no inhibition was noted in oocytes injected with a hybridization mixture containing no cDNA (mock) or cDNA from an irrelevant clone (B2-14).

DISCUSSION
In rat liver and kidney homogenates, a complex pattern of 5"deiodination is noted, with the kinetic properties of the reaction dependent on the substrates and thiol cofactors employed (1, 2). These markedly different kinetic patterns of deiodinase activity have given rise to considerable uncertainty concerning the number of enzymes capable of, and responsible for, catalyzing thyroid hormone deiodination in these tissues (2, 3). The seminal finding of the present study is that the injection of rat liver poly(A)+ RNA into X. laeuis oocytes results in a pattern of 5"deiodinase expression that exactly mimics that found in homogenates and subcellular fractions of rat liver and kidney, and that this complex pattern of 5'deiodination can be reproduced in its entirety by the injection of RNA transcripts prepared in vitro using a full-length type I 5"deiodinase cDNA as template.
Following the injection of rat liver poly(A)+ RNA, both high and low K, 5'-deiodinase processes were readily detected in membrane preparations from oocyte homogenates. Using high concentrations of DTT as cofactor, typical type I reaction kinetics were noted with K, values for rTB of approximately 0.1 pM. When lower DTT concentrations (0.5 mM) were utilized, K , values of approximately 20 nM were demonstrated, similar to that previously described by Boada and Chopra (7) in rat liver microsomes. Using GSH or a thioredoxin cofactor system, K, values for rT3 of approximately 1 nM were noted in injected oocytes. Such values are in complete agreement with both our own experience and that of others using these cofactors to support 5"deiodination in liver and kidney subcellular fractions (4-6, 13).
Furthermore, the marked sensitivity of these low K, processes to inhibition by PTU as well as the Ki values of 0.5 p~ for competitive inhibition by IOP are entirely analogous to the published experience of others (4-7). Of importance, T4 can also serve as a substrate for the induced low K , processes. Thus, the pattern of 5"deiodinase activity seen in native rat liver and kidney are mimicked in their entirety in X. lueuis oocytes by the injection of rat liver poly(A)+ RNA. The finding that the injection of G21 transcripts reproduces this pattern exactly strongly suggests that the type I enzyme encoded by this cDNA is the sole mediator of 5"deiodination in these organs.
This conclusion is strengthened further by the results of the hybrid arrest experiment. Solution hybridization of rat liver poly(A)+ RNA with the B2-21 cDNA prior to injection into oocytes results in essentially complete inhibition of expression of both high and low K,,, 5'-deiodinase processes, indicating that the type I mRNA is solely responsible for these expressed patterns of activity. Thus, the disparate reaction kinetics noted in liver and kidney homogenates under different reaction conditions appear to be attributable to variances in the interaction of the type I enzyme with specific thiol cofactors. The biochemical basis for such differences remains uncertain. Of note, however, is that in the presence of any of these cofactors, several properties of the reaction are the same (1-7): ( a ) rT3 is a more efficient substrate than T4 as determined by the higher V,,, and lower K, values for the former, ( b ) the reaction is sensitive to inhibition by PTU, (c) compounds such as iopanoic acid act as competitive inhibitors, and ( d ) activity decreases in tissue homogenates from experimental animals rendered hypothyroid and increases in the hyperthyroid state. Such physiologic alterations in activity are paralleled by changes in type I 5-deiodinase mRNA levels in both tissues (8,9). Taken together, these similarities further underscore the basic thesis of this report.
In studies reported previously, Goswami and Rosenberg (5) noted that a reconstituted thioredoxin cofactor system would support the 5'-deiodination of rT3 but not of T4 in liver microsomes. This led these investigators to suggest that a separate 5"deiodinase enzyme may be present in the liver and kidney which specifically deiodinates T4 to TB. The present studies do not support this speculation; using the thioredoxin system the 5'-deiodination of T4 was observed in oocytes injected with rat liver poly(A)+ RNA or G21 RNA transcripts as well as in kidney microsomes (data not shown). These latter results in kidney microsomes, although different from those of Goswami and Rosenberg ( 5 ) , are in agreement with the findings of others (6).
Of note in the present studies is our observation that the washed mitochondrial-microsomal membrane preparations from oocytes injected with either rat liver poly(A)+ RNA or G21 RNA transcripts manifested considerable low K , 5'deiodinating capability in the absence of added cofactors. This finding may be of considerable physiologic importance as it suggests that in the native state, the enzyme functions in a low K, mode. Deiodination in the absence of exogenous cofactors has been noted previously in crude, washed mitochondrial preparations from rat liver (15), although the kinetics of such a process has not been previously reported. Whether this activity is attributable to an endogenous cofactor present in our membrane preparation is uncertain, but is suggested by the finding that product formation continued (albeit nonlinearly) throughout the 130-min incubation period of our time course experiments. Dihydrolipoamide, which is present in mitochondria and has been shown to support 5'deiodination, represents such a potential endogenous cofactor (15, 16). Alternatively, a cytosolic cofactor could have been present in our membrane preparations in spite of the extensive wash protocol that we utilized, or the activity may be secondary to enzyme present in a reduced, active form at the time of membrane isolation and which subsequently undergoes a single round of catalysis during the in vitro assay.
Recent advances have provided important insights into the structure and function of the type I 5"deiodinase. The molecular cloning of a full-length cDNA (9), as well as affinity labeling studies followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (17,18), have demonstrated that this enzyme (or at least its catalytic subunit) is a selenoprotein of approximately 27 kDa in size. Others, however, have recently reported the purification of an approximately 56-kDa protein(s) from rat liver microsomes which is purported to have 5"deiodinase activity (19,20). Of note is that the specific activity of these preparations is quite low despite purification to apparent homogeneity as assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the 5"deiodinase activity has not yet been demonstrated to reside in the 56-kDa protein band. Thus, the significance of these reports remains unclear.
In summary, the results of the present studies serve to clarify the uncertainty concerning the number of enzymes responsible for the 5"deiodination of thyroid hormones. In the rat liver and kidney, a single enzyme, the type I 5'deiodinase, appears to be responsible for all of the activity observed in homogenates and subcellular fractions from these tissues. There appears to be little doubt, however, that the type I and I1 5"deiodinases are different proteins based on their kinetic behavior, PTU sensitivity, tissue distribution, differences in size and patterns of proteolytic digestion as determined by affinity labeling followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and finally the recent evidence that the type I1 enzyme is probably not a selenoprotein (1, 21-24). The structural and functional relationship between these two proteins awaits their purification and/or the isolation of a cDNA for the type I1 enzyme.