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The stereospecific removal of iodine from thyroid hormones is an essential first step for T3 action and is catalyzed by three different deiodinases: D2 and D3 remove iodine only from the outer or inner ring, respectively, whereas D1 catalyzes both pathways. We used in silico predictions from vertebrate deiodinase sequences to identify two domains: the N-terminal variable region (VR) containing the transmembrane, hinge and linker domains, and the conserved or globular region (CR). Given the high sequence and structural identity of the CR among paralogs as well as of the VR among orthologs but not paralogs, we hypothesized that both the catalytic properties and the subcellular localization rely on the VR. We used shark D2 and D3 as templates to build the chimeric enzymes D2VR/D3CR and D3VR/D2CR. Biochemical characterization revealed that D3VR/D2CR has inner-ring deiodination activity and T3 as preferred substrate, whereas D2VR/D3CR showed no deiodinating activity. Also, D2VR/D3CR and D3VR/D2CR reside in the endoplasmic reticulum and plasmatic membrane, respectively, as do their D2 and D3 wild-type counterparts. We conclude that the VR determines the subcellular localization and is critical in defining the catalytic properties and activity of thyroid hormone deiodinases.

From an evolutionary perspective, deiodinases may be considered pivotal players in the emergence and functional diversification of both thyroidal systems (TS) and their iodinated messengers. To better understand the evolutionary pathway and the concomitant functional diversification of vertebrate deiodinases, in the present review we summarized the highlights of the available information regarding this ubiquitous enzymatic component that represents the final, common physiological link of TS. The information reviewed here suggests that deiodination of tyrosine metabolites is an ancient feature of all chordates studied to date and consequently, that it precedes the integration of the TS that characterize vertebrates. Phylogenetic analysis presented here points to D1 as the oldest vertebrate deiodinase and to D2 as the most recent deiodinase gene, a hypothesis that agrees with the notion that D2 is the most specialized and finely regulated member of the family and plays a key role in vertebrate neurogenesis. Thus, deiodinases seem to be major participants in the evolution and functional expansion of the complex regulatory network of TS found in vertebrates.

Thyroid hormone bioactivity is finely regulated at the cellular level by the peripheral iodothyronine deiodinases (D). The study of thyroid function in fish has been restricted mainly to teleosts, whereas the study and characterization of Ds have been overlooked in chondrichthyes. Here we report the cloning and operational characterization of both the native and the recombinant hepatic type 3 iodothyronine deiodinase in the tropical shark Chiloscyllium punctatum. Native and recombinant sD3 show identical catalytic activities: a strong preference for T3-inner-ring deiodination, a requirement for a high concentration of DTT, a sequential reaction mechanism, and resistance to PTU inhibition. The cloned cDNA contains 1298 nucleotides [excluding the poly(A) tail] and encodes a predicted protein of 259 amino acids. The triplet TGA coding for selenocysteine (Sec) is at position 123. The consensus selenocysteine insertion sequence (SECIS) was identified 228 bp upstream of the poly(A) tail and corresponds to form 2. The deduced amino acid sequence was 77% and 72% identical to other D3 cDNAs in fishes and other vertebrates, respectively. As in the case of other piscivore teleost species, shark expresses hepatic D3 through adulthood. This characteristic may be associated with the alimentary strategy in which the protection from an exogenous overload of thyroid hormones could be of physiological importance for thyroidal homeostasis.

SUMMARY The physiological role played by thyroid hormones (TH) in hydro-osmotic homeostasis in fish remains a controversial issue. Previous studies have shown that in Fundulus heteroclitus (killifish) hypo-osmotic stress increases liver iodothyronine deiodinase type 2 (D2) mRNA and D2 activity. In this study we identified two conserved osmotic response element (ORE) motifs in the promoter region of the killifish D2 gene (FhDio2) and examined their possible role in the transcriptional regulation of FhDio2during hypo-osmotic stress. As assessed by the electrophoretic mobility shift assay, results from in vivo and in vitro experiments demonstrate that exposure to an abrupt hyposmotic challenge triggers in the liver of killifish a strong nuclear recruitment of a putative osmotic response element binding protein (OREBP). This protein–DNA binding is time-dependent, attains a maximum within 2–8 h after the osmotic stress,and is followed by a significant increase in D2 activity. Furthermore,protein–DNA binding and the subsequent elevation in enzyme activity were blocked by the tyrosine kinase inhibitor genistein. Thus, during hypo-osmotic stress, a putative OREBP kinase-activated pathway stimulates FhDio2transcription and enzymatic activity. These data and the fact that D2 is the major enzyme providing local intracellular T3 suggest that TH plays a direct role in osmoregulation in fish, possibly by participating in hepatic ammonia metabolism. This study provides important insight into the physiological role of TH in hydro-osmotic homeostasis in fish.

Substrate availability has been thought to be a major regulator of the outer-ring deiodinating pathway (ORD) in fish. However, current information strongly suggests that while fish iodothyronine deiodinase type 2 (D2) responds to iodothyronines in the same manner as its mammalian counterpart, fish deiodinase type 1 (D1) exhibits a distinct response. Furthermore, 3,5-T 2 , generally considered to be an inactive product of iodothyronine metabolism, has recently been described as bioactive, but its effects upon D1 and D2 are not yet known. We examined the effect that short-term immersion in T 4 , T 3 , and 3,5-T 2 (0.1 μM; 12 or 24 h) exerts on both D1 and D2 activities and on the levels of expression of D1 and D2 mRNAs in killifish liver. In agreement with previous reports in teleosts, no iodothyronine exerted a significant effect on D1 enzymatic activity. However, all three iodothyronines significantly decreased D2 activity. Furthermore, at 24 h post-immersion T 4 , T 3 , and 3,5-T 2 inhibited both D1 and D2 transcription. Together, the present results confirm the differential effect of iodothyronines upon the hepatic ORD pathway in fish and show that this effect can occur at a transcriptional level. Furthermore, we provide the first evidence that 3,5-T 2 can affect both activity and transcription of hepatic deiodinases in teleosts.

The presence of a type 1 deiodinase (D1) in the liver of teleosts has been a controversial issue. Recently we characterized the deiodinase activity in rainbow trout and killifish liver and found that the liver of both species co-expresses the two enzymes (D1 and D2) that catalyze the outer ring-deiodinating pathway. We here report the cloning and characterization of an mRNA from the liver of the killifish Fundulus heteroclitus that encodes a D1 (FhD1). The cDNA amplified by RT-PCR from F. heteroclitus liver is 1314 nt long and encodes a protein of 248 aa. It contains a TGA codon in its open reading frame and a selenocysteine insertion sequence in its 3′ untranslated region, consistent with the structure of a selenoenzyme mRNA. The deduced peptide sequence is 73% identical to that encoded by the tilapia D1 cDNA cloned from kidney and 46% identical to the D1s reported in other vertebrates. Northern blot analysis shows that FhD1 mRNA is expressed in F. heteroclitus liver, consistent with prior biochemical evidence for hepatic D1 activity. Furthermore, heterologous expression of the FhD1 cDNA resulted in a protein with properties similar to the D1-like activity in F. heteroclitus liver. The cloned enzyme, like the native species, is relatively insensitive to inhibition by PTU, but mutation of Ser-159 in FhD1 to the Pro residue found in D2 and D3 isoforms increased the sensitivity to PTU. Our results show that, under basal conditions, killifish liver indeed expresses a D1 enzyme that is homologous to mammalian D1s, establishing this as a useful model in which to study the regulation of D1 and D2 concurrently.

The kinetic characterization of the outer-ring deiodination pathway using rT(3) (rT(3)-ORD) in male, female, and pregnant female livers of an endemic lizard, Sceloporus grammicus, is reported. The ORD pathway does not have the characteristics of deiodinase type II; it is exclusively carried out by deiodinase type I (DI). DI enzymatic activity in lizard liver contains one of the highest activities reported in vertebrates. This activity is sexually dimorphic, with males presenting the highest activity during the reproductive season. The properties of this enzyme correspond to those described in mammals, such as specificity for rT(3), susceptibility to inhibition by 6-n-propyl-2-thiouracil and gold-thioglucose, cofactor requirement, and kinetic pattern. Unlike other vertebrates, the lizard DI exhibits conspicuous stability in the thermal range of 15 to 42 degrees C and in the pH range of 5.0 to 9.0. Male true kinetic constants exhibit a direct correlation with temperature. This is in agreement with short-term adaptation to microenvironmental changes and the feasible expression of enzymatic forms/variants which, together, endow this lizard species with a greater adaptation to natural daily ambient thermal fluctuations.

Mammary deiodinase type I (M-D1) is present only during lactation and exhibits a clear direct correlation with lactation intensity (size of litters). The present work shows that M-D1 is suckling dependent and that intervals between suckling periods no longer than 12 h are essential to maintain this activity. Moreover, we find that with only 15 min of resuckling in 12-h nonsuckled mothers, the 50% decrease in both M-D1 messenger RNA and enzymatic activity could be restored to control values. This restorative effect by suckling may involve pre- and posttranscriptional mechanisms in which norepinephrine and PRL play important roles. Norepinephrine elicits a potent stimulatory effect on M-D1 messenger RNA and enzyme activities, whereas PRL only increases M-D 1 activity and may modulate the enzyme response to norepinephrine. Oxytocin and GH had no effect. These data suggest that the adrenergic nervous system and PRL could directly participate in mammary energetic expenditure, regulating the local T3 supply.

Recent studies in our laboratory have shown that in some teleosts, 3,5-di-iodothyronine (T2 or 3,5-T2) is as bioactive as 3,5,3′-tri-iodothyronine (T3) and that its effects are in part mediated by a TRβ1 (THRB) isoform that contains a 9-amino acid insert in its ligand-binding domain (long TRβ1 (L-TRβ1)), whereas T3 binds preferentially to a short TRβ1 (S-TRβ1) isoform that lacks this insert. To further understand the functional relevance of T2 bioactivity and its mechanism of action, we used in vivo and ex vivo (organotypic liver cultures) approaches and analyzed whether T3 and T2 differentially regulate the S-TRβ1 and L-TRβ1s during a physiological demand such as growth. In vivo, T3 and T2 treatment induced body weight gain in tilapia. The expression of L-TRβ1 and S-TRβ1 was specifically regulated by T2 and T3 respectively both in vivo and ex vivo. The TR antagonist 1–850 effectively blocked thyroid hormone-dependent gene expression; however, T3 or T2 reversed 1–850 effects only on S-TRβ1 or L-TRβ1 expression, respectively. Together, our results support the notion that both T3 and T2 participate in the growth process; however, their effects are mediated by different, specific TRβ1 isoforms.

The ontogeny of 5'-monodeiodinase activity (5'-MA) was analyzed in chick embryo brain and liver tissue. The enzymatic type predominant in this path and the activity of the deactivating pathway (5 MA-III) were determined during certain periods of neural development in both organs. Results show that T3 is predominantly formed in both organs during the first third of embryogenesis (from day 5) until neuroblast proliferation (day 13). Within this lapse, the slow type II enzyme (insensible to propylthiouracil) is present in the brain, whereas in the liver the predominating enzyme is type I (the rapid enzyme). Further along the synaptogenesis period (day 14-17), 5' deiodination virtually disappears in the brain, the hepatic type I enzyme switches to the slow autoconsume enzyme (type II), and 5 MA-III levels increase significantly in both organs. Finally, on days 18-20 (perinatal period) the 5' pathway reaches the highest levels observed throughout the study in both tissues. Associated to this increase, liver enzymatic activity returns to type I. During this period, 5 MA-III is reduced by 40% in the brain and disappears from the liver. Together, these data strengthen the notion of a protective mechanism against brain overexposure to T3 during synaptogenesis and suggest that the protective mechanism also involves the regulation of extraneural deiodinases.

Iodide is a trace element and a key component of thyroid hormones (TH). The availability of this halogen is the rate-limiting step for TH synthesis; therefore, thyroidal iodide uptake and recycling during TH synthesis are of major importance in maintaining an adequate supply. In the rat, the thyroid gland co-expresses a distinctive pair of intrathyroidal deiodinating enzymes: the thyroid iodotyrosine dehalogenase (tDh) and the iodothyronine deiodinase type 1 (ID1). In the present work, we studied the activity of these two dehalogenases in conditions of hypo- and hyperthyroidism as well as during acute and chronic iodide administration in both intact and hypophysectomized (HPX) rats. In order to confirm our observations, we also measured the mRNA levels for both dehalogenases and for the sodium/iodide symporter, the protein responsible for thyroidal iodide uptake. Our results show that triiodothyronine differentially regulates tDh and ID1 enzymatic activities, and that both acute and chronic iodide administration significantly decreases rat tDh and ID1 activities and mRNA levels. Conversely, both enzymatic activities increase when intrathyroidal iodide is pharmacologically depleted in TSH-replaced HPX rats. These results show a regulatory effect by iodide on the intrathyroidal dehalogenating enzymes and suggest that they contribute to the iodide-induced autoregulatory processes involved in the Wolff-Chaikoff effect.

The three distinct but related isotypes of the iodothyronine deiodinase family: D1, D2, and D3, have been amply studied in vertebrate homeotherms and to a lesser extent in ectotherms, particularly in reptiles. Here, we report the molecular and kinetic characteristics of both the native and the recombinant hepatic D3 from the pine snake Pituophis deppei (PdD3). The complete PdD3 cDNA (1680 bp) encodes a protein of 287 amino acids (aa), which is the longest type 3 deiodinase so far cloned. PdD3 shares 78% identity with chicken and 71% with its other orthologs. Interestingly, the hinge domain in D3s, including PdD3, is rich in proline. This structural feature is shared with D1s, the other inner-ring deiodinases, and deserves further study. The kinetic characteristics of both native and recombinant PdD3 were similar to those reported for D3 in other vertebrates. True K(m) values for T(3) IRD were 9 and 11 nM for native and recombinant PdD3, respectively. Both exhibited a requirement for a high concentration of cofactor (40 mM DTT), insensitivity to inhibition by PTU (2 mM), and bisubstrate, sequential-type reaction kinetics. In summary, the present data demonstrate that the liver of the adult pine snake P. deppei expresses D3. Furthermore, this is the first report of the cloning and expression of a reptilian D3 cDNA. The finding of hepatic D3 expression in the adult pine snake P. deppei is consistent with results in adult piscine species in which the dietary T(3) content seems to regulate liver deiodinase expression. Thus, our present results support the proposal that hepatic D3 in adult vertebrates plays a sentinel role in avoiding an inappropriate overload of exogenous T(3) secondary to feeding in those species that devour the whole prey.

Circulating levels of T4, T3, corticosterone, noradrenaline, and adrenaline, as well as 5'-monodeiodinase activity (5'-MA) were measured in control and hypophysectomized rats acutely exposed to cold environment (15-120 min, 4 C). In addition to the well known activation of the sympathoadrenomedullary system and the hypothalamic-pituitary-adrenal and-thyroid axes, cold exposure was followed by a rapid and sustained increase of 5'-MA in the hypothalamus, and a byphasic course of activation in the adrenal gland in control rats. The adrenal rapid activation (30 min) corresponded to the medulla and the slower activation (120 min) to the cortex. Both, the basal adrenal 5'-MA and the response to cold in adrenal and hypothalamus were 2-fold higher in hypophysectomized rats compared to control. The time course of enzyme activation in these structures suggests that: 1) organ-specific increases in 5'-MA may be associated to a simultaneous rise in their metabolic and/or functional activity, 2) the triggering mechanisms involves an immediate sympathetic signal activating the hypothalamic-adrenal medulla response and a pituitary signal eliciting a slower adrenocortical response, and 3) the compensatory sympathetic hyperactivity after panhypopituitarism contribute to enhance both the adrenal enzyme basal activity and the hypothalamic and adrenal hyperresponse to cold stress.

Until recently, 3,5-diiodothyronine (3,5-T2) has been considered an inactive by-product of triiodothyronine (T3) deiodination. However, studies from several laboratories have shown that 3,5-T2has specific, nongenomic effects on mitochondrial oxidative capacity and respiration rate that are distinct from those due to T3. Nevertheless, little is known about the putative genomic effects of 3,5-T2. We have previously shown that hyperthyroidism induced by supraphysiological doses of 3,5-T2inhibits hepatic iodothyronine deiodinase type 2 (D2) activity and lowers mRNA levels in the killifish in the same manner as T3and T4, suggesting a pretranslational effect of 3,5-T2(Garcia-G C, Jeziorski MC, Valverde-R C, Orozco A. Gen Comp Endocrinol 135: 201–209, 2004). The question remains as to whether 3,5-T2would have effects under conditions similar to those that are physiological for T3. To this end, intact killifish were rendered hypothyroid by administering methimazole. Groups of hypothyroid animals simultaneously received 30 nM of either T3, reverse T3, or 3,5-T2. Under these conditions, we expected that, if it were bioactive, 3,5-T2would mimic T3and thus reverse the compensatory upregulation of D2 and tyroid receptor β1 and downregulation of growth hormone that characterize hypothyroidism. Our results demonstrate that 3,5-T2is indeed bioactive, reversing both hepatic D2 and growth hormone responses during a hypothyroidal state. Furthermore, we observed that 3,5-T2and T3recruit two distinct populations of transcription factors to typical palindromic and DR4 thyroid hormone response elements. Taken together, these results add further evidence to support the notion that 3,5-T2is a bioactive iodothyronine.

We review the experimental evidence accumulated within the past decade regarding the physiologic, biochemical, and molecular characterization of iodothyronine deiodinases (IDs) in piscine species. Agnathans, chondrichthyes, and teleosts express the three isotypes of IDs: ID1, ID2, and ID3, which are responsible for the peripheral fine-tuning of thyroid hormone (TH) bioactivity. At the molecular and operational level, fish IDs share properties with their corresponding vertebrate counterparts. However, fish IDs also exhibit discrete features that seem to be distinctive for piscine species. Indeed, teleostean ID1 is conspicuously resistant to propylthiouracil (PTU) inhibition, and its response to thyroidal status differs from that exhibited by other ID1s. Moreover, both the high level of ID2 activity and its expression in the liver of teleosts are unique among vertebrates. The physiologic role of iodothyronine deiodination in functions regulated by TH in fish is not entirely clear. Nevertheless, current experimental evidence suggests that IDs may coordinate and facilitate, in a tissue-specific fashion, the action of iodothyronines and other hormones involved in such processes.

The initial characterization of a thyroid iodotyrosine dehalogenase (tDh), which deiodinates mono-iodotyrosine and di-iodotyrosine, was made almost 50 years ago, but little is known about its catalytic and kinetic properties. A distinct group of dehalogenases, the so-called iodothyronine deiodinases (IDs), that specifically remove iodine atoms from iodothyronines were subsequently discovered and have been extensively characterized. Iodothyronine deiodinase type 1 (ID1) is highly expressed in the rat thyroid gland, but the co-expression in this tissue of the two different dehalogenating enzymes has not yet been clearly defined. This work compares and contrasts the kinetic properties of tDh and ID1 in the rat thyroid gland. Differential affinities for substrates, cofactors and inhibitors distinguish the two activities, and a reaction mechanism for tDh is proposed. The results reported here support the view that the rat thyroid gland has a distinctive set of dehalogenases specialized in iodine metabolism.

Recently, we reported the cloning of a cDNA fragment from Fundulus heteroclitus liver encoding the open reading frame of type 2 deiodinase (FhD2). We here report the cloning of 14 kb of genomic sequence from F. heteroclitus that includes the previously reported coding region of the F. heteroclitus Dio2 gene (FhDio2), the 5(') and 3(') untranslated regions, and flanking regions and introns. This FhDio2 gene comprises two exons divided by a 4.8-kb intron. The position of the intron is similar to that of introns in other Dio2 genes. The analysis of approximately 1.3 kb of genomic sequence upstream of the mRNA start site revealed that, in contrast to mammalian Dio2 genes, there were no apparent TATA or CRE sequences. Nevertheless, a putative Sp1 site was found, similar to that in other F. heteroclitus TATA-less promoters. We have also cloned the complete FhD2 cDNA, which spans 4652 bp and contains a sequence adjacent to its poly(A) tail that is highly similar to the selenocysteine insertion sequence (SECIS) found in human D2 cDNA. The expression of a construct containing the FhD2 ORF plus the native SECIS resulted in a protein with deiodinase activity similar to that of the native FhD2. Analysis of the regulation of this gene, combined with ongoing studies of the F. heteroclitus D1 gene, will allow us to elucidate the functions of the colocalized deiodinases in teleost liver.

Conversion of T4 to T3 is the first step in TH action and deiodinases are the major determinants of TH tissue availability and disposal. We here report the kinetic characterization of the outer-ring deiodinating (ORD) enzymes in the liver, gill and retina of sea water-adapted killifish, by using both rT3 and T4 as substrates. In liver, by using rT3, we detected a high Km (84 nM) and a low Km (1.3 nM) component with kinetic characteristics similar to mammalian deiodinases DI and DII. In contrast, T4-ORD only generated a low Km (0.5 nM) component. As judged by its Vmax (920 fmol 125I/mg per h) this DII enzyme is very abundant, approximately five and 20 times higher than that found in trout liver and hypothyroid rat, respectively. Kinetic analysis in killifish gill showed only one enzymatic component, with a high rT3 Km (430 nM) and a relatively low Vmax (4.3 pmol 125I/mg per h). Our results in killifish retina show the expression of a T4-low Km (0.6 nM) deiodinase with high cofactor requirements akin to the mammalian DII. The Vmax value for this enzyme is 182 fmol 125I/mg per h, five times lower than the one found in killifish liver, but comparable to that in hypothyroid rat pituitary. The biochemical similarities between fish and mammalian deiodinases could reflect their high conservation during vertebrate evolution and thus their importance in the regulation of thyroid hormone action.

Lactating rat mammary gland expresses a deiodinating activity that, on the basis of kinetic characteristics, corresponds to the so-called 5'-deiodinase type I (D1). In the present study we amplified and sequenced several D1 complementary DNA (cDNA) fragments from rat lactating mammary gland. The mammary cDNA was found to be identical to the previously reported rat liver cDNA in the coding region, but 465 nucleotides shorter on its 3'-untranslated region, suggesting that the D1 is the same in both tissues. D1 messenger RNA (mRNA) was also detected by reverse transcriptase-PCR in mammary glands from puberal and late pregnant rats, but not in virgin animals. Densitometric analysis showed a close and direct correlation between mRNA content and enzyme specific activity in mammary gland. Our results also show that rat liver contains both D1 mRNA forms and that the large form may respond to the thyroid status. These data suggest a differential and organ-specific expression of these mRNA forms, which could play a role in the functional regulation of D1 activity.

Deiodinases are major determinants of thyroid hormone tissue availability and disposal. The knowledge of the expression of these enzymes in lower species is important to understand evolutionary and ontogenetic aspects of thyroid hormone action and metabolism. Here we have studied outer ring deiodination in the trout liver using both reverse T3 (rT3) and T4 as substrates. The use of rT3 disclosed two enzymatic components with the characteristics of mammalian types I and II 5'-deiodinases. The high rT3-K(m) type I 5'-deiodinase activity (180 nM) has a low cofactor requirement (5 mM dithiothreitol) and is relatively sensitive to propylthiouracil inhibition, whereas the low rT3-K(m) activity was akin to the outer ring deiodination of T4 in these regards. The use of T4 exhibited only a single type of activity with a low K(m) (0.63 nM), a relatively high cofactor requirement (25 mM dithiothreitol), and propylthiouracil-resistance. Teleosts constitute a unique example of type II activity expression in the liver of an adult vertebrate. Furthermore, the Vmax of this enzyme is as high as that found in comparable homogenates from hypothyroid mammalian tissues, whereas the Vmax of the type I activity is lower than that of mammalian liver. These findings are in consonance with the peculiar kinetics of T3 in trout liver, kinetics remarkably similar to those of the mammalian pituitary, cerebral cortex, and brown adipose tissue, which also preferentially express type II deiodinase.

The well known metabolic and endocrine adaptive responses accompanying lactation include a change in circulating thyronines that resembles the so-called euthyroid sick syndrome. To analyze the role played by tissue monodeiodination in this state, circulating levels of thyronines as well as hepatic and mammary 5'-monodeiodinative activity (5'MA) were assessed during lactation in rats. Results show that the serum iodothyronine changes that accompany lactation are associated with a significant decrease in hepatic 5'MA and a simultaneous increase in mammary 5'MA. These changes begin within the first postpartum day, are proportional to lactation intensity (litter size), and disappear 48 h after either precocious (1st postpartum day) or natural (21st postpartum day) weaning. These data demonstrate that the compartmentalized change in energy expenditure characteristic of lactation is accompanied by organ-specific and opposite adjustments in hepatic and mammary monodeiodinative pathways.

Mammary homogenates from lactating, weaned, pregnant, and nonpregnant rats were analyzed for 5'-monodeiodinase activity (5'-MA). Only lactating glands exhibited significant enzyme activity. Competitive analysis showed that mammary 5'-MA is not inhibited by high (10 microM) concentrations of T4 or T3, but is highly sensitive to prophythiouracil (5 mM). Kinetic parameters demonstrate an acompetitive bisubstrate enzymatic mechanism known as the ping-pong type. The Km and maximum velocity for rT3 were 0.40 microM and 1.42 nmol/mg.min, respectively. These results provide the first evidence that 5'-MA is present in the lactating mammary gland and suggest that this enzymatic activity corresponds to type I enzyme.