Rat liver type I iodothyronine deiodinase is not identical to protein disulfide isomerase

This study was done to test the recent hypothesis (Boado et al. (1988) Biochem. Biophys. Res. Commun. 155, 1297-1304) that type I iodothyronine deiodinase (ID-I) is identical to protein disulfide isomerase (PDI). Autoradiograms of rat liver microsomal proteins, labeled with N-bromoacetyl-[125I]triiodothyronine (BrAc[125I]T3) and separated by SDS-PAGE, show predominantly 2 radioactive bands of Mr 27 and 56 kDa. Substrates and inhibitors of ID-I inhibited labeling of the 27 kDa band but not that of the 56 kDa band. Treatment of microsomes with trypsin abolished labeling of the 27 kDa protein and destroyed the activity of ID-I but did not prevent labeling of the 56 kDa protein. Following treatment of microsomes at pH 8.0-9.5 or with 0.05% deoxycholate (DOC) PDI content and labeling of the 56 kDa protein were strongly diminished but ID-I activity and labeling of the 27 kDa protein were not affected. The latter decreased in parallel after treatment at pH greater than or equal to 10. Rat pancreas microsomes contain high amounts of PDI but show no ID-I activity. Reaction of these microsomes with BrAc[125I]T3 results in extensive labeling of a 56 kDa protein but no labeling of a 27 kDa protein. Pure PDI (Mr 56 kDa) was readily labeled by BrAc[125I]T3 but showed no deiodinase activity. These results strongly suggest that the 27 kDa band represents (a subunit of) ID-I while the 56 kDa band represents PDI. From these and other data it is concluded that PDI and ID-I are not identical proteins.     

Selenouracil derivatives are potent inhibitors of the selenoenzyme type I iodothyronine deiodinase.

Type I iodothyronine deiodinase (ID-I) is a selenoenzyme, which is important for the conversion of the prohormone thyroxine (T4) to the bioactive thyroid hormone 3,3',5-triiodothyronine (T3). 2-Thiouracil derivatives inhibit ID-I by interaction with an enzyme form generated during catalysis. We have now tested the potential inhibitory effects of the selenocompounds 6-methyl- (MSU) and 6-propyl-2-selenouracil (PSU) in comparison with their thioanalogs 6-methyl- (MTU) and 6-propyl-2-thiouracil (PTU) on rat liver ID-I activity using 3,3',5-triiodothyronine (reverse T3, rT3) as substrate and dithiothreitol (DTT) as cofactor. All compounds showed dose-dependent inhibition of ID-I with IC50 values of 1, 0.5, 0.4 and 0.2 microM for MTU, MSU, PTU and PSU, respectively. Our results further suggest that these inhibitions are uncompetitive with substrate and competitive with cofactor. The high potency of selenouracils may be due to reaction with a substrate-induced enzyme selenenyl iodide intermediate under formation of a stable enzyme-selenouracil diselenide.     

In vitro metabolism of polychlorinated biphenyl congeners by beluga whale (Delphinapterus leucas) and pilot whale (Globicephala melas) and relationship to cytochrome P450 expression

We measured rates of oxidative metabolism of two tetrachlorobiphenyl (TCB) congeners by hepatic microsomes of two marine mammal species, beluga whale and pilot whale, as related to content of selected cytochrome P450 (CYP) forms. Beluga liver microsomes oxidized 3,3',4,4'-TCB at rates averaging 21 and 5 pmol/min per mg for males and females, respectively, while pilot whale samples oxidized this congener at 0.3 pmol/min per mg or less. However, rates of 3,3',4,4'-TCB metabolism correlated with immunodetected CYP1A1 protein content in liver microsomes of both species. The CYP1A inhibitor alpha-naphthoflavone inhibited 3,3',4,4'-TCB metabolism by 40% in beluga, supporting a role for a cetacean CYP1A as a catalyst of this activity. Major metabolites of 3,3',4,4'-TCB generated by beluga liver microsomes were 4-OH-3,3',4',5-TCB and 5-OH-3,3',4,4'-TCB (98% of total), similar to metabolites formed by other species CYP1A1, and suggesting a 4,5-epoxide-TCB intermediate. Liver microsomes of both species metabolized 2,2',5,5'-TCB at rates of 0.2-1.5 pmol/min per mg. Both species also expressed microsomal proteins cross-reactive with antibodies raised against some mammalian CYP2Bs (rabbit; dog), but not others (rat; scup). Whether CYP2B homologues occur and function in cetaceans is uncertain. This study demonstrates that PCBs are metabolized to aqueous-soluble products by cetacean liver enzymes, and that in beluga, rates of metabolism of 3,3',4,4'-TCB are substantially greater than those of 2,2',5,5'-TCB. These directly measured rates generally support the view that PCB metabolism plays a role in shaping the distribution patterns of PCB residues found in cetacean tissue.     

Affinity labeling of rat liver and kidney type I 5'-deiodinase. Identification of the 27-kDa substrate binding subunit

Extrathyroidal production of 3,3',5-triiodothyronine from the thyroid secretory product, thyroxine, is catalyzed by tissue-specific iodothyronine 5'-deiodinases. Type I 5'-deiodinase (5'D-I) produces greater than 75% of the T3 found in the circulation and in thyroid hormone-responsive tissues and is most abundant in rat liver and kidney. In this study, we used the bromoacetyl derivatives of T4 (N-bromoacetyl-[125I]L-thyroxine, BrAcT4) and T3 (N-bromoacetyl-[125I]3,3',5-triiodothyronine, BrAcT3) as alkylating affinity labels to identify 5'D-I-related protein(s). BrAcT4 and BrAcT3 rapidly and irreversibly inactivated 5'D-I activity in liver and kidney microsomes. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of affinity labeled 5'D-I preparations showed that approximately 80% of the affinity label was incorporated into a protein with a Mr of 27,000 (p27). 5'D-I substrates and inhibitors specifically blocked affinity labeling of p27 with a rank order of potency (BrAcT4 greater than BrAcT3 greater than 3,5,3'-triiodothyronine (rT3) approximately flavone EMD 21388 greater than iodoacetate greater than N-acetyl-T4 (NAcT4) greater than N-acetyl-T3 (NAcT3] identical to that determined for inhibition of 5'-deiodination. Hyper- and hypothyroidism-induced increases and decreases in 5'D-I activity, respectively, were matched by comparable changes in the quantity of affinity labeled p27. BrAcT3 was a less effective affinity label for p27 and minor labeling of a new band with 53 kDa was observed. Molecular sieve chromatography of detergent-solubilized 5'D-I showed coincident peaks of p27 and 5'-deiodinating activity with an apparent Mr approximately 51,000. Two-dimensional gel electrophoresis showed that p27 was a single polypeptide with a pI of 6.1. Approximately 2-5 pmol of p27 were present per mg of liver microsomal protein, equal to previous estimates for 5'D-I content. Our results suggest that p27 represents the substrate binding subunit of type I 5'-deiodinase, the enzyme catalyzing the key reaction in the activation of T4 to the thyromimetically active T3.     

Analysis of photoaffinity label derivatives to probe thyroid hormone receptor in human fibroblasts, GH1 cells, and soluble receptor preparations

The regulation of growth hormone gene expression by thyroid hormone in cultured GH1 cells is mediated by a chromatin-associated receptor. We have previously described a photoaffinity label derivative of 3,5,3'-triiodo-L-thyronine (L-T3) in which the alanine side chain was modified to form N-2-diazo-3,3,3-trifluoropropionyl-L-T3 (L-[125I]T3-PAL). On exposure to 254 nm UV light, L-[125I]T3-PAL generates a carbene which covalently modifies two thyroid hormone receptor forms in intact GH1 cells; an abundant 47,000 Mr species and a less abundant 57,000 Mr form. We have now synthesized similar photoaffinity label derivatives of 3,5,3',5'-tetraiodo-L-thyronine (L-T4) and 3,3',5'-triiodo-L-thyronine (L-rT3). Both compounds identify the same receptor forms in intact cells and in nuclear extracts in vitro as L-[125I]T3-PAL. Labeling by L-[125I]rT3-PAL was low and consistent with the very low occupancy of receptor by L-rT3. Underivatized L-[125I]T3 and L-[125I]T4 labeled the same receptor forms at 254 nm but at a markedly lower efficiency than their PAL derivatives. In contrast, N-bromoacetyl-L-[125I]T3, a chemical affinity labeling agent, did not derivatize either receptor form in vitro. The relative efficiency of coupling to receptor at 254 nm was L-[125I]T4-PAL greater than L-[125I]T3-PAL greater than L-[125I]T4 greater than L-[125I]T3. Although L-[125I]T4-PAL has a lower affinity for receptor than L-[125I]T3-PAL, its coupling efficiency was 5-10-fold higher. This suggests that the alanine side chain of L-[125I]T4-PAL is positioned in the ligand binding region near a residue which is efficiently modified by photoactivation. With L-[125I]T4-PAL we were able to identify three different molecular weight receptor species in human fibroblast nuclei.     

Production and purification of monoclonal T lymphocyte antigen binding molecules (TABM)

The thyroid hormone derivative N-bromoacetyl-3,3',5-triiodothyronine (BrAcT3) acts as an active site-directed inhibitor of rat liver iodothyronine deiodinase. Lineweaver Burk analysis of enzyme kinetic measurements showed that BrAcT3 is a competitive inhibitor of the 5'-deiodination of 3,3',5'-triiodothyronine (rT3) with an apparent Ki value of 0.1 nM. Preincubations of enzyme with BrAcT3 indicated that inhibition by this compound is irreversible. The inactivation rate obeyed saturation kinetics with a limiting inactivation rate constant of 0.35 min-1. Substrates and substrate analogs protected against inactivation by BrAcT3. Covalent incorporation of 125I-labeled BrAcT3 into "substrate-protectable" sites was proportional to the loss of deiodinase activity. The results suggest that BrAcT3 is a very useful affinity label for rat liver iodothyronine deiodinase.     

Bacterial and mammalian thioredoxin systems activate iodothyronine 5'-deiodination

The identity of a dithiol (designated DFB) of relative mass (Mr) = 13,000, reported previously to be present in fraction B of rat liver cytosol and to participate as a cofactor in the 5'-deiodination of iodothyronines, has been investigated. Substitution of highly purified thioredoxin from Escherichia coli for fraction B or of highly purified thioredoxin reductase from either E. coli or rat liver for cytosolic fraction A (containing DFB reductase) permits deiodination of 3,3',5'-[125I]triiodothyronine by rat liver microsomes to proceed. Addition of antibodies to highly purified rat-liver thioredoxin or thioredoxin reductase inhibits deiodination. Thus, the thioredoxin system largely accounts for the activity of the cytosolic cofactor system supporting 5'-deiodination of 3,3',5'-triiodothyronine in rat liver.     

Characterization of a thyroid-hormone-binding site on nuclear envelopes and nuclear matrices of the male-rat liver

Nuclear envelopes and nuclear matrices were isolated from the male-rat liver. Incubation of 125I-labelled 3,3',5-tri-iodothyronine (T3) with the nuclear-envelope fraction resulted in specific binding of T3 to the membranes. Maximum specific binding occurred at 30 degrees C after 2h incubation. Storage for 1 week at -80 degrees C resulted in no loss of binding. Scatchard analysis revealed a class of binding sites with KD 86 nM. 3,3',5'-Tri-iodothyronine was as effective a competitor of [125I]T3 binding to nuclear envelopes as was L-T3 itself, and tri-iodothyroacetic acid was 70% as potent as T3. L- and D-thyronine did not compete for [125I]T3 binding. Incubation of nuclear envelopes with 0.6 M-NaCl before addition of T3 resulted in the complete loss of specific binding sites, whereas exposure of the membranes to 2.0 M-NaCl after incubation with T3 did not extract binding sites. Nuclear matrices, after incubation with [125I]T3 under the same conditions, were shown to possess a class of binding sites with a similar KD but with approx. 30% of the maximum binding capacity. Nuclear envelopes from hypothyroid animals may possess slightly lower numbers of binding sites compared with nuclear envelopes from the intact animal, whereas nuclear matrices from hypothyroid animals have the same number of binding sites as do nuclear envelopes from the intact animal. In conclusion, nuclear envelopes and nuclear matrices have a class of binding sites with relatively high affinity for T3. It is distinct from nuclear and cytosolic binding sites.     

Antibody against nuclear thyroid hormone receptors

Rat liver nuclear thyroid hormone receptor was purified to 700-1600 pmol T3 binding capacity/mg protein by sequentially using hydroxylapatite column, ammonium sulfate precipitation, Sephadex G-150 gel filtration, DNA-cellulose column, DEAE-Sephadex A-50 column, and heparin-Sepharose column. Serum from a mouse immunized using this purified receptor preparation caused a shift of [125I]T3-receptor peak on glycerol density gradient sedimentation from 3.4 S to approximately 7 S. [125I]T3-receptor complex was immunoprecipitated using this serum and goat anti-mouse IgG. The serum showed reduced ability to immunoprecipitate the globular T3 binding fragment with Stokes radius of 22 A produced by trypsin digestion, a receptor fragment which has core histone and hormone binding but not DNA binding activity. These data indicate the production of anti-nuclear thyroid hormone receptor antibody which mainly recognized epitopes unrelated to hormone and core histone binding domain.     

Kinetics of enzymic reductive deiodination of iodothyronines. Effect of pH.

5'-Deiodination of thyroxine (yielding 3,3',5-tri-iodothyronine; reaction I) and of 3,3',5'-tri-iodothyronine (yielding 3,3'-di-iodothyronine; reaction II) and 5-deiodination of thyroxine (yielding 3,3',5'-tri-iodothyronine; reaction III) and of 3,3',5-tri-iodothyronine (yielding 3,3'-di-iodothyronine; reaction IV) as catalysed by rat liver microsomal fraction were studied at pH 6.5, 7.2 and 8.0 It was found that: (1) the Km of reaction I was relatively independent of pH (approx. 3 microM), whereas V was highest at pH 6.5 (63 pmol of 3,3',5-tri-iodothyronine/min per mg of protein); (2) the Km of reaction II was lowest at pH 6.5 (0.035 microM), but V was highest at pH 8.0 (829 pmol of 3,3'-di-iodothyronine/min per mg of protein); (3) thyroxine inhibited reaction II competitively; Ki values were identical at pH 6.5 and 8.0 (1 microM); (4) for both reactions III and IV Km was lowest and V was highest at pH 8.0. The results are compatible with the view that reactions I and II are mediated by a single enzyme (iodothyronine 5'-deiodinase) and that reactions III and IV are catalysed by a second enzyme (iodothyronine 5-deiodinase).     

Glucocorticoids,thyroid hormones,and iodothyronine deiodinases in embryonic saltwater crocodiles

We investigated the relationship between glucocorticoids, thyroid hormones, and outer ring and inner ring deiodinases (ORD and IRD) during embryonic development in the saltwater crocodile (Crocodylus porosus). We treated the embryos with the synthetic glucocorticoid dexamethasone (Dex), 3,3',5-triiodothyronine (T(3)), and a combination of these two hormones (Dex + T(3)). The effects of these treatments were specific in different tissues and at different stages of development and also brought about changes in plasma concentrations of free thyroid hormones and corticosterone. Administration of Dex to crocodile eggs resulted in a decrease in 3,3',5,5'-tetraiodothyronine (T(4)) ORD activities in liver and kidney microsomes, and a decrease in the high-K(m) rT(3) ORD activity in kidney microsomes, on day 60 of incubation. Dex treatment increased the T(4) ORD activity in liver microsomes, but not kidney microsomes, on day 75 of incubation. Dex administration decreased T(3) IRD activity in liver microsomes. However, this decrease did not change plasma-free T(3) concentrations, which suggests that free thyroid hormone levels are likely to be tightly regulated during development.     

Identification of monocarboxylate transporter 8 as a specific thyroid hormone transporter

Transport of thyroid hormone across the cell membrane is required for its action and metabolism. Recently, a T-type amino acid transporter was cloned which transports aromatic amino acids but not iodothyronines. This transporter belongs to the monocarboxylate transporter (MCT) family and is most homologous with MCT8 (SLC16A2). Therefore, we cloned rat MCT8 and tested it for thyroid hormone transport in Xenopus laevis oocytes. Oocytes were injected with rat MCT8 cRNA, and after 3 days immunofluorescence microscopy demonstrated expression of the protein at the plasma membrane. MCT8 cRNA induced an approximately 10-fold increase in uptake of 10 nM 125I-labeled thyroxine (T4), 3,3',5-triiodothyronine (T3), 3,3',5'-triiodothyronine (rT3) and 3,3'-diiodothyronine. Because of the rapid uptake of the ligands, transport was only linear with time for <4 min. MCT8 did not transport Leu, Phe, Trp, or Tyr. [125I]T4 transport was strongly inhibited by L-T4, D-T4, L-T3, D-T3, 3,3',5-triiodothyroacetic acid, N-bromoacetyl-T3, and bromosulfophthalein. T3 transport was less affected by these inhibitors. Iodothyronine uptake in uninjected oocytes was reduced by albumin, but the stimulation induced by MCT8 was markedly increased. Saturation analysis provided apparent Km values of 2-5 microM for T4, T3, and rT3. Immunohistochemistry showed high expression in liver, kidney, brain, and heart. In conclusion, we have identified MCT8 as a very active and specific thyroid hormone transporter.     

Specific binding of iodothyronines with apolipoprotein A-1 in high density lipoprotein particle isolated from human serum by affinity chromatography on thyroxine-sepharose

The kinetic and equilibrium characteristics of interaction of thyroxine (T4) and its structural analogs with a high density lipoprotein (HDL) fraction isolated from human serum by T4-Sepharose affinity chromatography and containing apolipoprotein A-I (apo A-I) as a sole protein component, were studied. The binding of [125I]T4 to apo A-I-HDL reached a maximum after 40 min and did not change during the next 80 min of incubation at 0 degrees--22 degrees C. Dissociation of [125I]T4 induced by the addition of excess unlabeled T4 to the complex solution proceeded more intensely on a time scale at 0--2 degrees C than at 22 degrees C. Incubation of apo A-I-HDL with increasing concentrations of T4 showed that the binding is saturable. The data analysis using different computer programs revealed the presence in apo A-I-HDL of a single class of binding sites with K alpha = (4.0 +/- 2.1).10(-7) M- and Bmax = 1.7 +/- 0.8 nmol T4/mg of protein. Naturally occurring iodothyronines, their analogs and D-isomers of thyroid hormones competed with [125I]T4 for the binding sites on apo A-I-HDL with the following inhibitory potencies: L-T4 = D-T4 greater than or equal to 3,3',5-triiodo-L-thyronine = 3,3',5-triiodo-D-thyronine greater than 3,5-diiodo-L-thyronine = 3,3',5- triiodothyroacetic acid greater than 3,3',5-triiodothyropropionic acid greater than or equal to 3,5-diiodo-L-thyrosine.(ABSTRACT TRUNCATED AT 250 WORDS)     

Carrier-Mediated Transport of Thyroid Hormones into Rat Glial Cells in Primary Culture

The uptake of 3,3',5-[3'-125I]triiodo-L-thyronine ([125I]L-T3) and of L-[3',5'-125I]thyroxine ([125I]L-T4) by cultured rat glial cells was studied under initial velocity (Vi) conditions. Uptake of both hormones was carrier mediated and obeyed simple Michaelis-Menten kinetics. The following respective values of Km (microM) and Vmax (fmol/min/microgram of DNA) were obtained at 25 degrees C: 0.52 +/- 0.09 and 727 +/- 55 for L-T3 and 1.02 +/- 0.21 and 690 +/- 85 for L-T4. Ki values (microM) for the inhibition of [125I]L-T3 uptake by unlabeled analogues were as follows: L-T4, 0.88; 3,3',5'-triiodo-L-thyronine, 1.4; 3,3'-diiodo-L-thyronine, 2.9; 3,3',5-triiodo-D-thyronine, 4.8; and triiodothyroacetic acid, 5.3. These values indicate that the uptake system is stereospecific. Unlabeled L-T3 was a better competitor than unlabeled L-T4 for the uptake of [125I]L-T4, an observation suggesting that both hormones were taken up by a common carrier system. L-T3, and L-T4 uptake was pH dependent, a finding suggesting that the phenolic unionized form of the hormones was preferentially taken up. L-T3 uptake was studied in the presence of various inhibitors; the results suggest that uptake was independent of the transmembrane Na+ gradient and of the cellular energy. Compounds that inhibited cellular uptake but were without effect on L-T3 binding to isolated nuclei also inhibited L-T3 nuclear binding in intact cells, an observation suggesting that uptake could be rate limiting for the access of L-T3 to nuclear receptors when transport is severely inhibited.     

Peroxidatic degradation and ether link cleavage of thyroxine in a particulate fraction of human thyroid

The present study was undertaken to investigate degradation of thyroxine (T4) mediated by thyroid peroxidase in man. A particulate fraction (1,000-100,000 x g) of normal human thyroid tissue was prepared and used as crude enzyme. 125I-T4 and unlabeled T4 were incubated with the particulate fraction in buffer containing glucose and glucose oxidase for generation of H2O2. After incubation, iodoamino acids were extracted with ethanol and the products of T4 degradation were analyzed by thin layer chromatography. In this system, T4 was degraded in time-, temperature- and pH-dependent manners, but not in the absence of the H2O2-generating system. The rate of degradation was related to concentration of the particulate fraction. The reaction was inhibited by methimazole, propylthiouracil and catalase. When [3',5'-125I] T4 was used as a tracer, major labeled products of T4 degradation were inorganic iodide and ethanol-unextracted fraction and no detectable labeled 3,5,3'-triiodothyronine (T3) or 3,3',5'-triiodothyronine (rT3) was generated. From a kinetic study by adding various doses of unlabeled T4, the apparent Km value for T4 was 30 microM and the Vmax value was 230 pmol/mg protein/min. When [3,5-125I] T4 was incubated with enzyme preparation, one third of degraded T4 was recovered as diiodotyrosine (DIT) and half of 125I-DIT was degraded in parallel incubation. No formation of radiolabeled DIT was observed in incubation with Na- 125I done in tandem. These findings suggest that thyroid hormones can be metabolized by peroxidase in human thyroid by pathways that include cleavage of ether linkage.     

Identification of type I iodothyronine 5'-deiodinase as a selenoenzyme

A 27.8 kDa membrane selenoprotein was previously identified in rat thyroid, liver and kidney, the tissues with the highest activities of type I iodothyronine 5'-deiodinase. This membrane enzyme catalyzes the deiodination of L-thyroxine to the biologically active thyroid hormone 3,3',5-triiodothyronine. A decrease in the activity of this enzyme, observed here in the liver of selenium-deficient rats, was found to be due to the absence of a selenium-dependent membrane-bound component. By chemical and enzymatic fragmentation of the 75Se-labeled selenoprotein and of the 27 kDa substrate binding type I 5'-deiodinase subunit, affinity-labeled with N-bromoacetyl-[125I]L-thyroxine, and comparison of the tracer distribution in the peptide fragments the identity of the two proteins was shown. The data indicate that the deiodinase subunit contains one selenium atom per molecule and suggest that a highly reactive selenocysteine is the residue essential for the catalysis of 5'-deiodination. From the results it can be concluded that type I iodothyronine 5'-deiodinase is a selenoenzyme.     

Purification and characterization of N-acetylglucosaminyl sulfotransferase from chick corneas

Sulfation is an important conjugation pathway in deactivating thyroid hormones, keeping the proper hormonal balance, and increasing the rate of thyroid hormone metabolism. We have identified, cloned, and characterized a sulfotransferase (SULT) that is capable of thyroid hormone conjugation in the dog. This enzyme, designated cSULT1B1, displays a strong identity (>84%) to the human ST1B2 enzyme. However, cSULT1B1 displays less identity, about 73%, to mouse and rat orthologs. In addition, the canine enzyme is three amino acids shorter than the rodent ones but has the same length as the human ortholog, 296 amino acids. The bacterial expressed and partial purified cSULT1B1 enzyme sulfates p-nitrophenol and 1-naphtol, but not dopamine. The thyroid hormones 3,3'-diiodothyronine and 3,5,3'-triiodothyronine are efficiently sulfated. 3,3',5'-Triiodothyronine is sulfated to lesser degree while sulfation of 3,5'-diiodothyronine and 3,3',5,5'-tetraiodothyronine cannot be detected. The cSULT1B1 is found in the colon (highest level), kidney and small intestine in dogs, but surprisingly not in the male dog liver although low levels of immunoreactivity were detected in the female dog liver. The male dog expresses more of SULT1B1 enzyme in the lower part of the small intestine while the female dog displays an opposite pattern of expression. These results describe the cloning and characterization of a canine thyroid hormone sulfating enzyme that is more closely related to the human ortholog than to the rodent thyroid sulfating enzymes.     

Rabbit liver microsomal lipid peroxidation. The effect of lipid on the rate of peroxidation

Rat and rabbit liver microsomes catalyze an NADPH-cytochrome P-450 reductase-dependent peroxidation of endogenous lipid in the presence of the chelate, ADP-Fe3+. Although liver microsomes from both species contain comparable levels of NADPH-cytochrome P-450 reductase and cytochrome P-450, the rate of lipid peroxidation (assayed by malondialdehyde and lipid hydroperoxide formation) catalyzed by rabbit liver microsomes is only about 40% of that catalyzed by rat liver microsomes. Microsomal lipid peroxidation was reconstituted with liposomes made from extracted microsomal lipid and purified protease-solubilized NADPH-cytochrome P-450 reductase from both rat and rabbit liver microsomes. The results demonstrated that the lower rates of lipid peroxidation catalyzed by rabbit liver microsomes could not be attributed to the specific activity of the reductase. Microsomal lipid from rabbit liver was found to be much less susceptible to lipid peroxidation. This was due to the lower polyunsaturated fatty acid content rather than the presence of antioxidants in rabbit liver microsomal lipid. Gas-liquid chromatographic analysis of fatty acids lost during microsomal lipid peroxidation revealed that the degree of fatty acid unsaturation correlated well with rates of lipid peroxidation.     

Hormone and DNA binding activity of a purified human thyroid hormone nuclear receptor expressed in Escherichia coli

Using a T7 expression system, large amounts of the human placental c-erbA protein (h-TR beta 1) were expressed. From 1 liter of Escherichia coli culture, approximately 50-100 micrograms of purified h-TR beta 1 were obtained. Analysis of the binding data indicated that the purified h-TR beta 1 binds to 3,3',5-triiodo-L-thyronine (T3) with a Ka = 2.8 x 10(9) M-1. It binds to 3,3',5-triiodo-L-thyropropionic acid, 3,3',5-triiodo-L-thyroacetic acid, D-T3, L-thyroxine (T4), and 3',5',3-triiodo-L-thyronine with 475, 120, 39, 7, and 0.1%, respectively, of the activity of L-T3. This order of binding activity to T3 analogs is similar to that reported for the T3 nuclear receptor identified in tissues or cultured cells. Furthermore, the purified h-TR beta 1 binds to the T3 response element of the rat growth hormone gene. Thus, the purified h-TR beta 1 is active. To identify the hormone binding domain, the purified h-TR beta 1 was affinity labeled with underivatized [3',5'-125I]T4. A partial digestion by trypsin yielded a 125I-labeled 25-kDa fragment which was identified to be the domain Phe240-Asp456 by amino acid sequencing. Thus, the purified h-TR beta 1 appears suitable for other structural and functional studies.     

Hepatic iodothyronine 5''-deiodinase. The role of selenium.

Selenium (Se) deficiency decreased by 8-fold the activity of type 1 iodothyronine 5'-deiodinase (ID-I) in hepatic microsomal fractions from rats. Solubilized hepatic microsomes from rats injected with 75Se-labelled Na2SeO3 4 days before killing were found by chromatography on agarose gels to contain a 75Se-containing fraction with ID-I activity. PAGE of this fraction under reducing conditions, followed by autoradiography, revealed a single 75Se-containing protein (Mr 27,400 +/- 300). This protein could also be labelled with 125I-bromoacetyl reverse tri-iodothyronine, an affinity label for ID-I. The results suggest that hepatic ID-I is a selenoprotein or has an Se-containing subunit essential for activity.