Senile hair graying: H2O2-mediated oxidative stress affects human hair color by blunting methionine sulfoxide repair.

Senile graying of human hair has been the subject of intense research since ancient times. Reactive oxygen species have been implicated in hair follicle melanocyte apoptosis and DNA damage. Here we show for the first time by FT-Raman spectroscopy in vivo that human gray/white scalp hair shafts accumulate hydrogen peroxide (H(2)O(2)) in millimolar concentrations. Moreover, we demonstrate almost absent catalase and methionine sulfoxide reductase A and B protein expression via immunofluorescence and Western blot in association with a functional loss of methionine sulfoxide (Met-S=O) repair in the entire gray hair follicle. Accordingly, Met-S=O formation of Met residues, including Met 374 in the active site of tyrosinase, the key enzyme in melanogenesis, limits enzyme functionality, as evidenced by FT-Raman spectroscopy, computer simulation, and enzyme kinetics, which leads to gradual loss of hair color. Notably, under in vitro conditions, Met oxidation can be prevented by L-methionine. In summary, our data feed the long-voiced, but insufficiently proven, concept of H(2)O(2)-induced oxidative damage in the entire human hair follicle, inclusive of the hair shaft, as a key element in senile hair graying, which does not exclusively affect follicle melanocytes. This new insight could open new strategies for intervention and reversal of the hair graying process.

H(2)O(2) increases de novo synthesis of (6R)-L-erythro-5,6,7,8-tetrahydrobiopterin via GTP cyclohydrolase I and its feedback regulatory protein in vitiligo.

Patients with vitiligo accumulate up to 10(-3) mol/L concentrations of H(2)O(2) in their epidermis, which in turn affects many metabolic pathways in this compartment, including the synthesis and recycling of the cofactor (6R)-L-erythro-5,6,7,8-tetrahydrobiopterin (6BH(4)). De novo synthesis of 6BH(4) is dependent on the rate-limiting enzyme GTP cyclohydrolase I (GTPCHI) together with its feedback regulatory protein (GFRP). This step is controlled by 6BH(4) and the essential amino acid L-phenylalanine. In the study presented here we wanted to investigate whether H(2)O(2) affects the GTPCHI/GFRP cascade in these patients. Our results demonstrated concentration-dependent regulation of rhGTPCHI where 100 micromol/L H(2)O(2) was the optimum concentration for the activation of the enzyme and >300 micromol/L resulted in a decrease in activity. Oxidation of GFRP and GTPCHI does not affect feedback regulation via L-phenylalanine and 6BH(4). In vitiligo a constant upregulation of 6BH(4) de novo synthesis results from epidermal build up of L-phenylalanine that is not controlled by H(2)O(2). Taking the results together, 6BH(4) de novo synthesis is controlled by H(2)O(2) in a concentration-dependent manner, but H(2)O(2)-mediated oxidation does not affect the functionality of the GTPCHI/GFRP complex.

In vivo and in vitro evidence for autocrine DCoH/HNF-1alpha transcription of albumin in the human epidermis.

The presence of albumin in the human epidermis has been reported more than a decade ago, but until now, it was assumed that this protein is synthesized in the liver and transported to the avascular skin. To our knowledge, transcription of albumin in the human epidermis was never considered. In this report, we present for the first time evidence for autocrine synthesis of albumin in the human epidermis in keratinocytes in situ and in vitro. Using double immunofluorescence labelling, we identified that albumin colocalized together with its transcription factor PCD/DCoH/HNF-1alpha in suprabasal keratinocytes in human full-thickness skin sections and in keratinocytes cultured in serum-free medium. Moreover, albumin and HNF-1alpha protein expression was confirmed by Western blotting in undifferentiated and differentiated keratinocytes as well as in human epidermal suction blister roof extracts. Reverse-transcriptase polymerase chain reaction analysis from human epidermal keratinocytes and epidermal suction blister roofs revealed the transcription of albumin. Using in vivo fluorescence excitation spectroscopy at the surface of human skin, we confirmed albumin as a major constituent yielding a lambda(max) at 295 nm, which was assigned to the single tryptophan 214 fluorophore in this protein. This in vivo result is in agreement with albumin concentrations of 10(-3) M, underlining the importance of this protein in epidermal homeostasis.

Molecular evidence that halo in Sutton's naevus is not vitiligo.

Both halo naevus and vitiligo are acquired leucodermas of unknown aetiology. To date a significant contribution of oxidative stress through accumulation of hydrogen peroxide (H2O2) has been documented in the pathomechanism of vitiligo but not in halo naevus. Both epidermal pterin-4a-carbinolamine dehydratase (PCD) and catalase are sensitive markers to follow H2O2 concentration-dependent deactivation of these proteins. In situ protein expression of PCD and catalase was examined in full-skin biopsies from skin phototype-matched controls (n=5), untreated and treated vitiligo patients (n=5) and patients with untreated halo naevus in association with vitiligo (n=3). Vitiligo was treated with pseudocatalase (PC-KUS) only. Catalase levels were determined in epidermal suction blister extracts using fast protein liquid chromatography (FPLC). In addition, epidermal H2O2 levels were followed in vivo by Fourier-transform Raman spectroscopy. The results of this study ruled out a contribution of H2O2 in the millimolar range in the depigmentation process of halo naevus as previously documented in vitiligo. Therefore, it can be concluded that both leucodermas exercise distinct concentration-dependent H2O2 signalling in their pathomechanisms.

Antiviral and hemolytic activities of surfactin isoforms and their methyl ester derivatives.

Inactivation of enveloped viruses (VSV, SFV, and SHV-1) by surfactin lipopeptides was dependent on the hydrophobicity, i.e. the number of carbon atoms of the fatty acid, and on the charge of the peptide moiety as well as on the virus species. Surfactins with fatty acid chains of 13 carbon atoms showed very low antiviral activity in comparison to C14 and C15 isoforms. C15 surfactin monomethyl ester also inactivated SFV which was resistant to the mixture of surfactin isoforms as produced by Bacillus subtilis. In contrast, the dimethyl ester showed no virus-inactivation capacity. Disintegration of viral structures as determined by electron microscopy after inactivation of VSV and SFV was comparable to the titer reduction. The effect of the surfactin isoforms and methyl esters on erythrocyte hemolysis correlated with the virus-inactivation capacity. Surfactins with a fatty acid chain moiety of 15 carbon atoms and one negative charge showed the highest antiviral activity.

Serial transplants of DMBA-induced mammary tumors in Fischer rats as a model system for human breast cancer. VI. The role of different forms of tumor-associated stress for the regulation of pineal melatonin secretion.

Previous studies on human breast cancer patients showed a decline in circulating melatonin levels corresponding to primary tumor growth and an increase when relapse occurred. The aim of the current investigation was to study in an experimental model possible mechanisms involved. Inbred female F344 Fischer rats were used for serial passages derived from a chemically induced mammary adenocarcinoma. Animals with slow-growing carcinosarcomas at passage 2 showed a significant elevation of nocturnal urinary melatonin (23. 00-07.00 h; +50%, p < 0.05) and a nominal increase in plasma melatonin (+41%; 02.00-03.00 h). By contrast, these parameters were significantly depressed in animals with fast-growing sarcomas (urinary melatonin: -22%, p < 0.025; plasma melatonin: -56%, p < 0. 01). At passage 2 nocturnal pineal N-acetylserotonin (02.00-03.00 h) was significantly enhanced (+62%, p < 0.05) probably due to an increased activity of serotonin-N-acetyltransferase (SNAT, +45%), the rate-limiting step of pineal melatonin biosynthesis converting serotonin to N-acetylserotonin. The activation of SNAT may be due to a stimulation of the sympathetic nervous system (urinary noradrenaline; NA: +243%, p < 0.005) when the cellular immune system responded towards tumor growth (urinary biopterin, +214%, p < 0.005). At passage 12 SNAT and N-acetylserotonin were unaffected but a depletion of plasma tryptophan (-34%, p < 0.0001), the precursor amino acid of melatonin, was found. The marginal decline in pineal serotonin (-18%, p < 0.05) disputes that the drastic depletion in circulating melatonin (-56%, p < 0.01) can be exclusively explained by a reduced availability of tryptophan. Therefore, the involvement of an additional mechanism has to be postulated, such as a degradation of melatonin via indoleamine 2,3-dioxygenase, an extrahepatic enzyme which has been detected in tumor tissue and is related to tryptophan 2,3-dioxygenase (TDO). TDO occurs only in the liver, is highly specific for L-tryptophan and is induced by glucocorticoids which would account for the observed depletion of plasma tryptophan resulting from a tumor-associated activation of the hypothalamo-pituitary-adrenal axis (urinary corticosterone +208%, p < 0.01). These findings present first explanations for the previously observed modulation of melatonin levels in cancer patients but also illustrate the high degree of complexity of mechanisms involved in the interactions between tumor growth and the immunoneuroendocrine system.

Synthetic flavonoids cross the placenta in the rat and are found in fetal brain.

The synthetic flavonoid EMD-49209 is a potent inhibitor of the in vivo and in vitro binding of thyroxine (T4) to transthyretin (TTR). We studied the distribution of 125I-labeled EMD-49209 in maternal tissues, intestinal contents, and fetal tissues in rats that were 20 days pregnant (from 1 to 24 h after intravenous injection). The percent dose of EMD decreased quickly with time. In maternal brain no radioactive flavonoid could be detected. EMD was excreted very rapidly from the intestines. In the fetal compartment the percent dose of EMD increased with time; after 24 h it contained 17% of the EMD. The flavonoid was found in all fetal tissues investigated and also in the fetal brain. Because TTR concentrations are high in the fetal rat, especially in the brain, the transfer of flavonoid to the fetal brain might be linked to TTR expression. The presence of flavonoid in the fetal brain raises the possibility of an essential interference of flavonoids with the availability of T4 in the fetal compartment.

Correlations between endotoxin, interferon-gamma, biopterin and serum phospholipase A2-activities during lethal gram negative sepsis in rats.

OBJECTIVE: To establish a standardised reproducible animal model of intraperitoneal sepsis, and to investigate early immunoserological responses to find a mediator-based system for evaluation and grading of diffuse peritonitis in patients DESIGN: Prospective experimental study SETTING: 4 Teaching hospitals, Germany and Austria MATERIAL: 42 LEW. 1W rats, 12 of which acted as controls INTERVENTIONS: Gram negative sepsis was induced by intraperitoneal injection of 6 ml of a mixture of Escherichia coli (K1:H+) 10(10) organisms/ml, autogenous haemoglobin 2.9 ml (haemoglobin concentration 3%), 0.9% sodium chloride 3 ml, and suspension 0.1 ml. Control rats were given physiological saline 6 ml alone. MAIN OUTCOME MEASURES: Concentrations of endotoxin, interferon gamma (IFN-gamma), and biopterin, and serum phospholipase A2 (PLA2) activity. RESULTS: There were significant differences between the septic and control rats in concentrations of endotoxin (EU/ml) (median (interquartile range) 21.85 (2.02-159.5) compared with 0, p < 0.0001; IFN-gamma (pg/ml) 1263.0 (271.0-7575.0) compared with 101.0 (89.0-141.0), p < 0.0001; biopterin (nmol/L) 111.0 (66.4-156.3) compared with 53.7 (38.3-67.6), p < 0.001; and PLA2 (U/L) 163.0 (125.8-209.0) compared with 112.5 (88.5-126.5) p < 0.01. CONCLUSIONS: Measurements of concentrations of endotoxin, IFN-gamma, pteridines, and PLA2 activity may well be adequate markers for early recognition of sepsis, and perhaps for grading it during the first 6 hours after induction. The allow a clear distinction to be made between septic and non-septic disorders in 87% of cases.

Different tissue distribution, elimination, and kinetics of thyroxine and its conformational analog, the synthetic flavonoid EMD 49209 in the rat.

The synthetic flavonoids EMD 23188 and EMD 49209, developed as T4 analogs, displace T4 from transthyretin, and in vitro they inhibit 5'-deiodinase activity. In vivo EMD 21388 causes tissue-specific changes in thyroid hormone metabolism. In tissues that are dependent on T3 locally produced from T4, total T3 was diminished. It was not known whether it was the presence of EMD interfering with 5'-deiodinase type II in tissues or the decreased T4 (substrate) availability that caused the lowered T3. To study whether the flavonoids enter tissues and, if this were the case, whether they enter tissues similarly, [125I]EMD 49209 together with [131I]T4 were injected into female rats and rats pretreated with EMD 21388. Tissues were extracted and submitted to HPLC. [125I]EMD 49209 disappeared quickly from plasma and enters peripheral tissues; peak values were reached after 0.25-0.5 h. Then [125I]EMD 49209 appeared in the intestines (after 6 h 40% of the dose). Tissue uptake of [131I]T4 was very rapid. EMD 21388 pretreatment caused an increase in the excretion of [125I]EMD 49209 into the intestines (40% after 0.25 h). The uptake of [131I]T4 increased, but not high enough to ensure normal tissue T4 concentrations. In the 5'-deiodinase type II-expressing tissues, no [125I]EMD 49209 could be detected. We conclude that the decrease in T3 locally produced from T4 is caused by the shortage of T4 as substrate and not to a direct effect of EMD on the activity of 5'-deiodinases I and II.

Effect of septicaemia on the plasma levels of biopterin and nitric oxide metabolites in rats and rabbits.

Live Escherichia coli decreased mean arterial blood pressure in rabbits from 67 to 20 mmHg. E. coli did not affect blood pressure in rats but did significantly increase heart rate by 29%. To related the cardiovascular effects with putative relevant biochemical pathways, the plasma levels of nitrate + nitrite (NOx) and biopterin, representing the main metabolites of nitric oxide and tetrahydrobiopterin, respectively, were determined in conscious rats and rabbits after treatment with live E. coli. In rats, E. coli induced a rapid 43% increase in the plasma level of biopterin preceding the 7- to 26-fold increase in NOx level. In rabbits, no increase in the NOx level was observed despite a 3- to 5-fold increase in the biopterin level at 6-10 hr posttreatment. It is concluded that the synthesis of tetrahydrobiopterin precedes nitric oxide synthesis after induction of septicaemia in the rat. After the induction of septicaemia, rabbits show a clear hypotensive response and an increase in biopterin level but no concomitant increase in NOx. Biopterin apparently represents a more appropriate biochemical marker of septic shock than does NOx.

Serial transplants of DMBA-induced mammary tumors in Fischer rats as model system for human breast cancer. IV. Parallel changes of biopterin and melatonin indicate interactions between the pineal gland and cellular immunity in malignancy.

Nocturnal (23.00-07.00 h) urinary melatonin and total biopterin (tBI; after acidic oxidation of reduced biopterins) were analyzed during the growth of two passages of a mammary tumor line in female F344 Fischer rats. In addition, nocturnal (02.00-03.00 h) peak concentrations of pineal melatonin in plasma were analyzed when tumors had reached comparable average tumor volumes of 25-30 cm3. Since tetrahydrobiopterin (BH4) is produced by murine macrophages in response to interferon-gamma released by activated T lymphocytes, measurements of tBI can serve to estimate the state of cellular immunity. At passage 2, a slow-growing localized carcinosarcoma, tBI showed a progressing increase during tumor growth reaching more than 200% (p < 0.05-0.005) of controls by the end of the experiment. Urinary and plasma melatonin were elevated by 30-50% (p < 0.05) and 42% respectively. At passage 12, a fast-growing metastasizing sarcoma, a depression of about 20-30% was found for tBI (p < 0.05) and urinary melatonin (p < 0.025); plasma melatonin was depleted by 70% (p < 0.005). Parallel changes of both parameters at each tumor passage indicate a close link between the pineal hormone melatonin and cellular immunity. The opposite trends observed at the two passages indicate a clear stimulation of the immune system and the pineal gland at early but inhibition at advanced stages of cancer.

3,5-Di-iodo-L-thyronine suppresses TSH in rats in vivo and in rat pituitary fragments in vitro.

3,5-Di-iodo-L-thyronine (T2) is a naturally occurring metabolite of thyroxine (T4). Contrary to earlier findings, T2 has recently been shown to have rapid effects in rat liver and in mononuclear blood cells. In the experiments described here, T2 was tested to determine whether it has a TSH suppressive effect in rats in vivo and in rat pituitary fragments in vitro. In experiments over 2 weeks in rats in vivo, low doses of T2 (20-200 micrograms/100 g body weight per day) had no significant influence on body and organ weights, but significantly decreased TSh and T4 serum concentrations. At 200 micrograms/100 g per day, T2 suppressed TSH to 43% and T4 to 29% of control levels. At 1-15 micrograms/100 g per day, 3,5,3'-tri-iodo-L-thyronine (T3), used as a comparison to T2, had significant effects on TSH and T4 levels, and also on body weight. Fifteen micrograms T3/100 g per day decreased TSH to 44%, T4 to 25%, and body weight to 59% of control levels. In experiments over 3 months in rats in vivo, a low dose (25 micrograms/100 g per day) of T2 suppressed TSH to 60% and T4 to 57% of control levels and had no significant influence on other parameters. Conversely, 0.1 microgram/100 g per day T3 had significant effects on body and organ weights as well as pellet intake, but a less pronounced TSH suppressive effect: TSH concentrations were unchanged and T4 concentrations were down to 80% of control values.(ABSTRACT TRUNCATED AT 250 WORDS)

Serum biopterin--a novel marker for immune activation during pre-diabetes in the BB rat.

Tetrahydrobiopterin (BH4) is a pteridine product which is released by rodent macrophages on activation by cytokines. We have used serial pancreatic biopsy, and measurement of serum biopterin at 30, 60, 90 and 120 days in the BB/S rat to relate histological change to macrophage activation during the course of pre-diabetes. Using immunohistochemistry, and an arbitrary scoring system read blind and standardised against day 30, we found that pancreatic MHC class I, MHC class II and infiltrating macrophage staining were up-regulated in the BB/S diabetes-prone rats (n = 17) at day 60, markedly so at day 90, and less so at day 120. Staining for resident pancreatic macrophages remained unchanged throughout in diabetes prone, diabetes resistant and Wistar (n = 28) control animals. Serum biopterin fell progressively and identically with age in BB diabetes resistant rats (n = 11) and Wistar controls. No change in weight gain or biopterin levels was observed in the biopsied animals. Mean serum biopterin levels in diabetes prone rats (of which 13 of 17 became diabetic at median 85 days) were the same as in diabetes resistant and Wistar rats at days 30, 60 and 120, but showed a striking and highly significant elevation (p < 0.001) at day 90. Although macrophages infiltrate the islet early in pre-diabetes, the timing of their activation is unknown. The rise in biopterin we observed is a potentially important immunological event which occurred late in the progression of pre-diabetes. This acute terminal event has not been reported previously, and may modify current concepts concerning the tempo of cell destruction during pre-diabetes.

Structural requirements of iodothyronines for the rapid inactivation and internalization of type II iodothyronine 5'-deiodinase in glial cells.

3,3'5,5'-Tetraiodothyronine (T4), but not 3,3'5-triiodothyronine (T3), acutely regulates the activity of the plasma membrane-bound enzyme, type II iodothyronine 5'-deiodinase (5'D-II), by inducing internalization of the enzyme through an extranuclear, energy-dependent mechanism that requires an intact actin cytoskeleton. The affinity label, N-bromoacetyl-L-T4, binds to 5'D-II and irreversibly inhibits the enzyme but does not initiate internalization. To determine the structural elements of T4 which are required for enzyme internalization, T4 analogs were modified in the alanine side chain and were then evaluated for their ability to induce enzyme internalization, to inhibit enzyme activity, and to promote actin polymerization in hypothyroid cells. The analogs studied showed marked variability in their ability to inactivate 5'D-II. The rank order of potency for enzyme inactivation was T4 > COOH-blocked analogs > NH3 and COOH blocked analogs >> NH3 blocked analogs (EC50 values range from 1 to > 1000 nM). In contrast, all T4 analogs tested and T4 were excellent competitive inhibitors of 5'D-II with respect to substrate (Ki values ranged from 4 to 27 nM). The differential capability of iodothyronines to inactivate the enzyme was not related to their ability to enter the cell, since Ki values measured in intact glial cells were equivalent to those measured in cell sonicates. The power of the T4 analogs to inactivate 5'D-II was paralleled by their ability to polymerize actin in hypothyroid cells and to induce 5'D-II binding to F-actin. The data show that modification of the alanine side chain markedly alters the ability of T4 analogs to induce 5'D-II inactivation and actin polymerization. A net negative charge on the alanine side chain of T4 is detrimental for the hormone-dependent inactivation of 5'D-II and polymerization of actin, whereas uncharged or positively charged molecules retain significant activity.

Selenium-dependent regulation of type I 5'-deiodinase expression.

Selenite concentration regulates activity and expression of the p27 substrate-binding subunit of type I 5'deiodinase (5'-D) and of a protein labeled with bromoacetylthyroxin (BrAcT4), or p30, with yet unknown function in a porcine-kidney epithelial cell line (LLC-PK1) cultured in serum-free medium. p27 is metabolically labeled by 75-selenite and affinity labeled by BrAc[125I]T4. Compared with glutathione peroxidase, expression of the p27 5'D subunit (5'-DI) is observed at 10-fold-lower concentrations of selenium in the growth medium, suggesting an intracellular hierarchy of selenite utilization. Selenium deficiency retards cell growth and prevents 5'-DI expression and may thus impair thyroid hormone action in vivo.

Neopterin as parameter of cell-mediated immunity response in thermally injured patients.

Serum neopterin levels have been determined retrospectively in 22 patients with burn injuries. Neopterin, which is produced by monocytes/macrophages following stimulation by interferons, is regarded as a marker for the activation of the cellular immune response. In most patients neopterin levels were initially in the normal range. All patients had their first operation and skin transplantation during the first week; mean neopterin levels increased significantly thereafter. Further skin transplantations or infections did not significantly influence the elevated neopterin level. This result may be due to continuous stimulation of the cellular immune system, as indicated by the elevated neopterin levels during most of the follow-up. In four low risk patients, neopterin levels always remained in the normal range. Two patients with inhalation trauma showed highly elevated neopterin levels (30-70 nmol/l) already upon admission and for 3 days thereafter. No correlation of neopterin levels with the burned body surface area was observed. However, mean serum neopterin levels were higher in the group of non-survivors (five patients) compared to survivors during the first 2 weeks after the trauma; rising neopterin levels were observed during the last 5 days before death. Although elevated neopterin levels could not be attributed either to the burn itself or to later events, the cellular immune system of burned patients was shown to be highly activated.

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.

Selective affinity labeling of a 27-kDa integral membrane protein in rat liver and kidney with N-bromoacetyl derivatives of L-thyroxine and 3,5,3'-triiodo-L-thyronine.

125I-Labeled N-bromoacetyl derivatives of L-thyroxine and L-triiodothyronine were used as alkylating affinity labels to identify rat liver and kidney microsomal membrane proteins which specifically bind thyroid hormones. Affinity label incorporation was analyzed by ethanol precipitation and individual affinity labeled proteins were identified by autoradiography after separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions. Six to eight membrane proteins ranging in size from 17 to 84 kDa were affinity labeled by both bromoacetyl-L-thyroxine (BrAcT4) and bromoacetyl-L-triiodothyronine (BrAcT3). Affinity labeling was time- and temperature-dependent, and both reduced dithiols and detergents increased affinity labeling, predominantly in a 27-kDa protein(s). Up to 80% of the affinity label was associated with a 27-kDa protein (p27) under optimal conditions. Affinity labeling of p27 by 0.4 nM BrAc[125I]L-T4 was blocked by 0.1 microM of the alkylating ligands BrAcT4, BrAcT3, or 100 microM iodoacetate, by 10 microM concentrations of the non-alkylating, reversible ligands N-acetyl-L-thyroxine, 3,3',5'-triiodothyronine, 3,5-diiodosalicylate, and EMD 21388, a T4-antagonistic flavonoid. Neither 10 microM L-T4, nor 10 microM N-acetyltriiodothyronine or 10 microM L-triiodothyronine blocked affinity labeling of p27 or other affinity labeled bands. Affinity labeling of a 17-kDa band was partially inhibited by excess of the alkylating ligands BrAcT4, BrAcT3, and iodoacetate, but labeling of other minor bands was not blocked by excess of the competitors. BrAc[125I]T4 yielded higher affinity label incorporation than BrAc[125I]T3, although similar banding patterns were observed, except that BrAcT3 affinity labeled more intensely a 58,000-Da band in liver and a 53,000-55,000-Da band in kidney. The pattern of other affinity labeled proteins with p27 as the predominant band was similar in liver and kidney. Peptide mapping of affinity labeled p27 and p55 bands by chemical cleavage and protease fragmentation revealed no common bands excluding that p27 is a degradation product of p55. These data indicate that N-bromoacetyl derivatives of T4 and T3 affinity label a limited but similar constellation of membrane proteins with BrAcT4 incorporation greater than that of BrAcT3. One membrane protein (p27) of low abundance (2-5 pmol/mg microsomal protein) with a reactive sulfhydryl group is selectively labeled under conditions identical to those used to measure thyroid hormone 5'-deiodination. Only p27 showed differential affinity labeling in the presence of noncovalently bound inhibitors or substrates on 5'-deiodinase suggesting that p27 is likely to be a component of type I 5'-deiodinase in rat liver and kidney.