0 1985 by The American Society of Biological Chemists, Inc. THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 260, No. 10, Issue of May 25, pp. 6012-6019. 1985

Printed in U.S.A.

Purification and Characterization of a Cytosolic Protein Enhancing GSH-dependent Microsomal Iodothyronine 5’-Monodeiodination*

(Received for publication, July 25, 1984)

Ajit Goswami and Isadore N. Rosenberg From the Department of Medicine, Framinghnm Union Hospital, Framinghnm, Massachusetts 01 701 and the Department of Medicine, Boston University School of Medicine, Boston, Massachusetts 02118

A protein has been purified from rat liver cytosol which promoted GSH-responsive iodothyronine 5’- deiodinase activities in rat kidney microsomes. The factor behaved as a basic protein with an M, of 11,000. It was active as a GSH-disulfide transhydrogenase with 8-hydroxyethyl disulfide as an acceptor and was also active in stimulating calf thymus ribonucleotide reductase with one-third the potency of native calf thymus glutaredoxin. Another basic protein, which de- graded iodothyronines oxidatively, was also identified in the cytosolic preparations; this co-purified with sol- uble protein factor in the earlier purification stages and was partially separated from this factor by CM- cellulose chromatography. The glutaredoxin-like pro- tein present in rat liver and kidney cytosol could pro- vide a physiologic regulatory mechanism for GSH- dependent 5’-monodeiodination of iodothyronines.

The conversion of thyroxine (T.# to triiodothyronine (T3) and of reverse triiodothyronine (rT3) to 3,3‘-diiodothyronine (3,3’-T2) is mediated by an enzyme present principally in the microsomal fractions of the liver and kidney (1, 2). The enzyme has an apparent K, of 5 PM for Tg and can also utilize reverse T3 as an alternate substrate with a K,,, of approxi- mately 0.5 PM. The enzymatic reaction involves replacement of the I atom at the 5’ (or 3’) position of the iodothyronine molecule by a hydrogen atom. An important role of enzyme SH groups has been identified in this reductive deiodination; the microsomal enzyme in vitro requires added dithiols such as dithiothreitol for activity. Monothiols, such as mercapto- ethanol or glutathionine, are much less effective than dithio- threitol in sustaining the reaction. The identity of the thiol compound supporting the reaction in vivo is as yet uncertain. Although the naturally occurring dithiol, dihydrolipoamide, is about 20-fold as potent as dithiothreitol in activating the enzyme in vitro, it occurs only as a bound form in the mito- chondrial pyruvate dehyrogenase complex (3), and attempts to couple this bound lipoamide to the iodothyronine deiodi- nase in vitro have been unsuccessful (4). Another naturally occurring dithiol, thioredoxin, which is the natural cofactor for the enzyme ribonucleotide reductase, also proved to be

* This work was supported by United States Public Health Service Grant A-2585 from the National Institutes of Arthritis, Diabetes, and Digestive and Kidney Diseases. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The abbreviations used are: T4, thyroxine (3,5,3’-tetraiodothyro- nine); SPF, soluble protein factor; rT3, reverse T3 (3,3’,5’-triiodothy- ronine); PTU, 6-propyl-2-thiouracil; DTT, dithiothreitol; HPLC, high pressure liquid chromatography.

completely inactive with the deiodinase. Glutaredoxin is a recently described and characterized di-

thiol protein initially identified in Escherichia coli; glutare- doxin in combination with GSH can substitute for the thio- redoxin system (thioredoxin, thioredoxin reductase, and NADPH) in stimulating ribonucleotide reductase in some mutant strains of E. coli (5, 6). Glutaredoxin has since been identified also in calf thymus (7), and its similarity to a liver GSH-disulfide transhydrogenase has been emphasized (8). The present studies were undertaken to explore the possibility that, by analogy with the activation of ribonucleotide reduc- tase in calf thymus and E. coli, a glutaredoxin-glutathione system may also be involved in the activation of iodothyronine deiodinase. The current report identifies in rat liver cytosol a soluble protein factor (designated SPF) exhibiting GSH-di- sulfide transhydrogenase activity with artificial electron car- riers which is active as a glutaredoxin (in activating GSH- mediated reduction of ribonucleotides by calf thymus ribo- nucleotide reductase), and can also activate microsomal io- dothyronine deiodinase in presence of GSH.

EXPERIMENTAL PROCEDURES

Materials L-3’- or 5’-’l-labeled rTS or T, (750-1250 pCi/pg) were obtained

from New England Nuclear and were further purified by paper electrophoresis (9) just before use. Dihydrolipoamide was prepared by borohydride reduction of the oxidized form (obtained from Sigma) by the method of Reed et al. (10) as modified by Silver (11). DEAE- cellulose (DE52) was from Whatman. Glutathione, glutathione re- ductase, NADPH, and CM-cellulose were obtained from Sigma. 8- Hydroxyethyl disulfide was from Aldrich.

Rat Kidney Microsomes Kidney microsomes were obtained from male rats (200-300 g) of

Charles River strain kept on Purina rat chow. Rats were killed by exsanguination under light ether anesthesia, and the kidneys were homogenized in a medium containing 250 mM sucrose, 20 mM Tris- HCl (pH 7.4), 1 mM EDTA and 100 p M DTT. The microsomal fractions were obtained from the homogenates by differential centrif- ugation as described previously (12) and stored at -70 “C at a con- centration of 1 mg of protein/ml in a medium containing 250 mM sucrose, 20 mM Tris-HC1 (pH 7.4), 1 mM EDTA, and 10 p M dithio- threitol. Homogenization and storage in a medium containing dithio- threitol yielded preparations with higher specific activities than those obtained with the sucrose/EDTA/Tris buffer alone.

Assay of SPF SPF was determined by its ability to stimulate renal microsomal

deiodination of rT3 or T4 in the presence of 5 mM GSH. The incu- bation mixture was as described below and contained, in addition, 100 p~ propylthiouracil (PTU). PTU was added because it abolished the basal microsomal deiodinating activity observed with 5 mM GSH alone at low (nM) substrate concentrations (see Table IA), but had little effect on SPF-stimulated microsomal activity. SPF activity was calculated by subtracting, from the total deiodinating activity ob-

6012

Cytosolic Factor Enhancing GSH-dependent Deiodination 6013

tained in the presence of microsomes plus SPF, the "blank" deiodi- nating activity obtained with 1) SPF in the absence of microsomes and 2) microsomes in the absence of SPF. One unit of SPF was taken to be the quantity that increased rT3 degradation by microsomes by 1 pmol/h/mg of microsomal protein.

Assay of Microsomal Iodothyronine 5'-Deiodinase Iodothyronine 5"deiodination was assayed by measurement of '=I-

liberated from outer ring-labeled '=I-rTS or '=I-T4 (13). For rT3 deiodinase assays, the reaction mixture contained, in a total volume of 250 pl: K-phosphate buffer (pH 7.0), 25 pmol; EDTA, 250 nmol; renal microsomal protein, 50 pg; dithiothreitol or GSH, 1.25 pmol, and varying amounts of '"I-rT3 as indicated in legends. T4 deiodinase assays were performed in a similar reaction mixture with specified concentrations of '=I-T4 using 100 pg of microsomal protein in a total volume of 100 pl. Incubation was at 37 'C and, unless otherwise indicated, the incubation time was 3 min for rTI and 60 min for T,. The reaction was started by the addition of appropriate amounts of the substrate. The incubation was terminated by the addition of 0.1 ml of 0.2% bovine serum albumin, followed immediately by 0.5 ml of ice-cold trichloroacetic acid. An aliquot was then applied to a Bio- Rad AG 5OW-XS (H+) (mesh size 100-200) Econo-Column (Bio-Rad) previously equilibrated with 10% acetic acid. The I- was eluted with two successive washes with 5 ml of 10% acetic acid and the radioac- tivity determined in a Gamma Counter (Searle). Concurrent paper chromatographic runs (in hexane, t-amyl alcohol, 2 N ammonia (1:lOll); descending) were also frequently performed for the quan- titative identification of the iodothyronine product. After subtraction of the blank activity with SPF alone, I- liberated was found to be equimolar with 3,3'-T2 or T3 formed.

For anaerobic incubation, the reaction mixture containing all com- ponents except the substrate was placed in the main chamber of a Thunberg tube, with the side arm containing 0.1 ml of the appropriate radioactive substrate. The tubes were placed in ice and repeatedly evacuated and filled with Oz-free Nz. The contents of the side arm and main chamber were then thoroughly mixed and incubated at 37 "C for appropriate time intervals. The I- liberated was then deter- mined by procedures as described above.

Assay of Thioltransferase Thioltransferase activity was determined by measurements of the

rate of disappearance of NADPH absorbance at 340 nm in the presence of P-hydroxyethyl disulfide in a Cary 219 spectrophotometer equipped with a cell programmer, using the protocol of Nagai and Black (14). One unit of enzymatic activity is defined as 1 pmol of NADPH oxidized per min under these conditions.

Assay of Ribonucleotide Reductase Calf thymus ribonucleotide reductase activity was assayed by the

spectrophotometric procedure of Brown et ai. (15); the rate of NADPH oxidation in the presence of cytidine diphosphate (CDP) (0.5 mM), GSH (1 mM), and glutathione reductase was taken as a measure of enzyme activity, with either SPF or calf thymus glutare- doxin (50-100 pg) being substituted for thioredoxin and thioredoxin reductase. Activities in reaction cells containing: (a) all the compo- nents save for the test sample and (b) all components except for CDP served as blanks. Glutaredoxin activity in the sample was measured by the degree of stimulation of NADPH oxidation in presence of CDP.

Purification of SPF The procedure employed was based on the methods described by

Luthman and Holmgren (8) for the preparation of calf thymus glutaredoxin, and by Axelsson et ai. (16) for purification of rat liver cytoplasmic thiol transferase. All steps were carried out a t 4 "C. Prior to all chromatographic steps, the extiacts were preincubated with 100 pM DTT at 4 "C for 1 h.

Step I-The livers from 12-15 rats (200-250 g body weight) of Charles River strain were minced, suspended in 3 volumes of 0.05 M Tris/acetate buffer (pH 7.5), and homogenized in a Brinkmann polytron homogenizer with three 5-5 exposures a t a setting of 5. The homogenate was centrifuged at 15,000 X g for 20 min, and the supernatant (Fraction F1) adjusted to pH 5 with ice-cold 10% acetic acid. The precipitate formed was removed by centrifueation at 15.000 X g for 10 min and the supernatant readjusted to <H 7.5 with 1 M NH40H (Fraction F2).

Step 2: Ammonium Sulfate Fractionation-F2 was adjusted to 40% saturation by the addition of solid ammonium sulfate with continuous stirring at 4 "C and centrifuged at 15,000 X g for 20 min. The pellet was discarded, and the supernatant was brought to 90% saturation with solid ammonium sulfate. The pellet obtained from this prepa- ration after centrifugation at 100,000 X g for 60 min was dissolved in Tris/acetate buffer and dialyzed overnight against 0.05 M Tris/acetate buffer (pH 7 4 , and then applied to a Sephadex G-50 (coarse) column (1.6 X 25 cm) previously equilibrated with the same buffer. The SPF activity emerged in fractions corresponding to 10,000-15,OOO daltons (with cytochrome c as marker).

Step 3: DEAE-cellulose Chromatography-The active fractions from the G-50 column in Step 2 were combined, adjusted to pH 8, and passed through a column of DE52 (2.6 X 15 cm), equilibrated with 50 mM Tris-HC1 (pH 8). SPF was not bound to the ion exchange cellulose at this ionic strength and was eluted with the same buffer.

Step 4: CM-cellulose Chromatography-The pooled effluent from Step 3 was adjusted to pH 6.1 with 0.2 M acetic acid and dialyzed overnight against 10 mM sodium phosphate buffer (pH 6.1) containing 1 mM EDTA. The extract was then applied to a column of CM- cellulose (2.6 X 15 cm), previously equilibrated with 10 mM sodium phosphate (pH 6.1) containing 1 mM EDTA. After washing the column with 150 ml of the starting buffer, the column was eluted with a linear gradient formed by mixing 150 ml of the starting buffer with 150 ml of the same buffer containing 250 mM sodium chloride.

Step 5: High Pressure Liquid Chromatography-The peak fractions containing SPF activity emerging from the CM-cellulose column were pooled, adjusted to pH 7.0, and concentrated to approximately 10 ml on an Amicon UM-2 filter. A portion of the material was then subjected to high pressure gel filtration on an 1-125 column (Waters Associates) using a Waters Associates Model 6000 solvent delivery system. After equilibration of the column with 50 mM sodium phos- phate buffer (pH 7.0), 100 pl of the sample were injected, followed by elution with the same buffer a t a flow rate of 1 ml/min until the last peak was eluted (approximately 15 rnin). 250-pl fractions of the eluates were collected in a GiIson microfractionator. The procedure was repeated 4-5 times with distribution of the eluate among the same tubes in the fractionator. The fractions containing SPF activity were combined, freeze-dried, and dissolved in a small volume (200- 300 pl) of deionized water.

Other Methods

Partially purified preparations of thioredoxin and thioredoxin re- ductase were obtained from rat liver by following the method of Engstrom et al. (17), described for calf liver, with omission of the heat-treatment step. The procedure was followed up to, and including, the Sephadex G-50 chromatography step and the fractions containing thioredoxin reductase and thioredoxin were collected as fractions T5- 1 and T5-2, respectively. Calf thymus glutaredoxin was prepared by the method described by Luthman and Holmgren (8). A partially purified preparation of ribonucleotide reductase, containing both B1 and B2 subunits, was obtained from calf thymus by following the procedure of Brown et al. (151, described for E. coli, up to, and including, the first DEAE-cellulose stage. Polyacrylamide gel electro- phoresis was performed on 2-mm slab gels (7.5%, pH 7.0) on an LKB Multiphor system. About 20-100 pg of protein was applied per slot. Running times were for approximately 3 h, and after the run, the proteins were detected by staining with Coomassie Blue. Isoelectric focusing was carried out on 2-mm slab gels using both broad (pH 3.5- 10) and narrow (pH 7.0-9.0) range ampholines (obtained from LKB). Gels were prerun for 30 min before application of the samples (50- 100 pg of protein/slot). The run was for approximately 1.5 h for the narrow range and 3 h for the broad range ampholines. The proteins were detected by staining with Coomassie Blue. The pH gradient was determined immediately after the run by measurements in water homogenates of 1-cmz gel slices obtained from an edge of the gel slab in the direction of the voltage gradient. Protein was determined by the method of Lowry et al. (18). For solubilized preparations, the method of Bradford (19) was also frequently used.

RESULTS

In the experiments described below, liver cytosol was the source of SPF because of higher yields, and iodothyronine 5'- deiodinase activity was assayed with kidney microsomes be- cause of its higher specific activity compared to liver micro-

6014 Cytosolic Factor Enhancing GSH-dependent Deiodination

somes. Experiments using liver cytosol with liver microsomes and kidney cytosol with kidney or liver microsomes yielded essentially similar results.

Thiol Activation: The Thioredoxin System-In agreement with earlier observations (4), the synthetic dithiols, dithio- threitol and dihydrolipoamide, were found to be the most effective cofactors for 5"deiodinase (Table IA). The mono- thiol, glutathione (at 5 mM), had little activity in assays where 0.5 p~ rT3 was used as substrate, although at lower rT, concentration, GSH was stimulatory (Table IA). A combina- tion of partially purified thioredoxin and thioredoxin reduc- tase and NADPH failed to activate the deiodinase enzyme. During these experiments it was noted, however, that a rela- tively crude liver cytosolic preparation, obtained from the earlier stages in the preparation of thioredoxin and thiore- doxin reductase (Fraction T-3: desalted 0-90% (NH4)2SOI precipitate), was quite active in stimulating the iodothyronine deiodinase activity in the presence of 5 mM GSH (Table IB). This suggested the presence in this preparation of a soluble protein factor that was capable of reconstituting GSH-de- pendent microsomal iodothyronine deiodinase activity and was separable from thioredoxin on DEAE-cellulose. Further studies revealed that SPF was not retained on DEAE at 50 mM Tris-HC1 (while thioredoxin was retained, requiring 0.1- 0.15 M NaCl for elution). SPF thus resembled glutaredoxin (7.8) and the ligandins (20) in being a basic substance, and a method was devised for the purification of SPF using proce- dures patterned after those which have been reported for purifying these basic proteins.

Purifkution of SPF-Table I1 summarizes the results of the purification. The step involving 0-40% (NH4),SO4 saturation was frequently omitted without any significant effects on the characteristics of the final preparation. The SPF activity was not retained on DEAE at 50 mM salt concentration, and batch elution with DEAE at 50 mM Tris-HC1 was found to be a quick and convenient way for separation of SPF from thio- redoxin and other more acidic proteins. On gradient elution of the DEAE-purified material on CM-cellulose the SPF activity emerged with a peak at 0.11 M NaCl (Fig. 1). This fraction stimulated microsomal deiodinase activity in the presence of 5 mM GSH. This activation was only slightly inhibited by 0.1-1 mM PTU. Stimulation of deiodination was improved 10-20% in Nz. The products of rT3 deiodination were identified by paper chromatography as I- and 3,3'-T2 which were equimolar after correcting for the blank activity in the absence of microsomes.

SPF thus resembles the liver thiol transferase (elution at 0.1 M NaCl from CM-cellulose (16)) more than calf thymus glutaredoxin (elution at 30 mM Na-acetate from CM-Sepha- rose (8)) in its chromatographic behavior. The similarity between SPF and thiol transferase was further borne out by the correspondence of SPF activity, on CM-cellulose, with a single peak of GSH-disulfide transhydrogenase activity with 8-hydroxyethyl disulfide as acceptor (Fig. 1). This disulfide has previuosly been shown also to be a suitable substrate for yeast thiol disulfide transhydrogenase (14) and calf thymus glutaredoxin (8). Glutathione S-transferases (ligandins) ap-

TABLE I Iodothyronine 5'-deiodinose in rat kidney microsomes (A) and GSH-responsive wdothyronine 5'-dewdinose in rat

kidney microsomes in the presence of partially processed liver cytosol (B) For A, iodothyronine deiodinase activities were assayed with 50 (for rT3) or 100 (for T4) pg of microsomal

protein with indicated concentrations of or T,, with or without indicated cofactors, in 0.1 M K-phosphate, 1 mM EDTA buffer (pH 7.0) in a final volume of 250 (rT3) or 100 (T,) pl. '%I liberated was determined by ion exchange chromatography as described under "Experimental Procedures." For B, experimental conditions were as described for A.

Substrate 5'-Deiodinase activity

nnwl substrate degraded. pmol substrate degraded. mg protein". h" mg protein". h"

Additions A

1. 2. 3. 4. 5. 6. 7.

B 1. 2. 3. 4. 5. 6. 7.

None Dithiothreitol (5 mM) Lipoamide (100 p ~ ) (reduced) GSH (5 mM) NADPH (1 mM) Fraction T5-2' (100 pug) Fraction T5-2* (100 Ng) + Fraction T5-lb (100 pg)

+ NADPH (1 mM)

Microsomes Microsomes + dithiothreitol (5 mM) Microsomes + GSH (5 mM) Fraction T-3' (200 pg) Fraction T-3' + GSH Microsomes + T-3' Microsomes + T-3' + GSH

1.95 ND" 1.10 ND 23.75 0.95 91.1 1.71 28.15 1.02 95.6 2.10 2.15 ND 35.3 0.89 2.10 ND 1.14 ND 2.05 ND 1.11 ND 2.2 0.11 1.08 ND

nmol substrate degraded. mg microsomalprotein".

h"

ND ND 25.8 1.05

ND ND 0.071 ND 0.115 ND 0.08 ND

13.7 0.53 ' ND, not detectable. 'These two preparations were obtained during the fifth stage of purification of thioredoxin and thioredoxin

reductase from rat liver cytosol by the procedure of Engstrom et al. (17). See text. Preparation after the third stage of purification of thioredoxin and thioredoxin reductase from rat liver cytosol

by the procedure of Engstrom et al. (17). This crude preparation was found to have substantial GSH-disulfide transhydrogenase activity.

Cytosolic Factor Enhancing GSH-dependent Deiodination TABLE I1

Purification of SPF from rat liver: co-purification of GSH-disulfide transhydrogenase Rat kidney microsomes were assayed for iodothyronine 5”deiodinase activity with 0.5 nM ’261-rT3 in the presence

of 5 mM GSH, 100 PM PTU with or without 50-500 pg of SPF. SPF activation of the microsomes were determined by subtracting, from the t o t a l deiodinating activity, blank activities due to SPF alone in the absence of microsomes and microsomes alone in the absence of SPF. One unit is 1 pmol of rTs degraded per mg of micrdsomal protein. h-l. GSH-disulfide transhydrogenase activity was determined by following oxidation of NADPH spectrophoto- metricallv under assav conditions as described under “Emerimental Procedures.”

6015

SPF activity Total protein GSH-disulfide trans-

Total units” Specific activity hydrogenase

w 1. Supernatant 22,100 2. pH 5 supernatant 11,200 3. (NH&SO, (40-90% saturation) 4,100 4. Sephadex G-50 205 5. DEAE-cellulose (flow-through) 88 6. CM-cellulose (0.11 M NaC1) 30 7. HPLC (1-125; 0.05 M NAPi) 9.4

a Apmol of rT3 degraded per mg of protein. h-l. * pmol NADPH oxidized per mg of protein. h-l.

Fraction No.

FIG. 1. Elution patterns of SPF and GSH-disulfide trans- hydrogenase activity from CM-cellulose column during step 4 of the purification. 5-ml fractions were collected and iodothyro- nine monodeiodinase activities were assayed on 0.2-ml aliquots using 0.5 nM rTs and 5 mM GSH in the presence or absence of 50 pg of rat kidney microsomes. GSH-disulfide transhydrogenase assays were car- ried out on 100-pl aliquots with 8-hydroxyethyl disulfide as acceptor. 0, Am; 0 and 0, activity profiles in the absence or presence of microsomes; A, GSH-disulfide transhydrogenase activity.

pear to be inactive as SPF. In two separate experiments, the fractions were monitored for glutathione S-transferase activ- ity with 5,5’-dithiobis-(2-nitrobenzoate). The active fractions were eluted as three distinct peaks below 75 mM salt solutions. None of the individually pooled and concentrated 5,5’-di- thiobis-(2-nitrobenzoate)-active peaks were active in promot- ing deiodination of rT3 in the presence of GSH, either in the presence or absence of microsomes.

The CM-cellulose elution profile (Fig. 1) also showed a GSH-dependent deiodinating activity which was active in the absence of microsomes; this eluted at 0.18 M NaC1, overlap- ping the SPF zone, and caused high blanks in the CM- cellulose-purified SPF preparations. This deiodinating activ- ity was destroyed by heating at 80 “C for 4 min, and the deiodination appeared to be 02-dependent, as it was dimin- ished by 60-70% under anaerobic conditions (Thunberg tubes). This activity was not inhibited by PTU (1 mM). It was over 80% inhibited by 1 mM NaCN, NaN3, or dipyridyl. It was inhibited by 50% when incubated with normal rat kidney microsomes, but was unaffected by heat-inactivated (boiled) microsomes. Paper chromatography of the reaction mixture

unitslmg 0.

5824 0.52 5330 1.3 2255 11.0 1848 21.0 993 33.1 510 54.2

units/mg 0.12 0.2 1.9

18 34 54

105

using an alkaline solvent system showed the reaction products to be I- and an unidentified origin material in approximately equal parts; neither 3-3’-T2 (with rT3 as a substrate) nor T, (with T, as a substrate) were detected as reaction products. The material in this zone was devoid of thiol transferase activity.

High pressure gel filtration on an 1-125 column resolved the CM-cellulose-purified SPF material essentially into three protein peaks. Of these, the early eluting peak (Peak l), with a retention time (R,) of 7.21 min, was inactive. The second peak (Peak 2), with an Rt of 8.42 was only mildly active. Almost all (over 90%) of the SPF activity, along with the major peak of GSH-disulfide transhydrogenase, was eluted in the third peak with an R, of 9.98 (Peak 3).

Properties of the Purified Enzyme-Peak 3 was nearly homogeneous on rechromatography on HPLC (Fig. 2). An M, of 10,715 was estimated for the HPLC-purified protein from a plot of K., values of marker proteins on an 1-125 column (Fig. 3). Gel electrophoresis of the HPLC-purified enzyme under nondenaturing conditions at pH 7 revealed a single cationic protein component (Fig. 4), both in the presence and absence of 2 mM DTT. The isoelectric point was determined to be at pH 8.5 by isoelectric focusing on 5% polyacrylamide gels on an ampholine pH gradient.

The purified SPF was unstable and lost its activity within weeks on storage, even at -25 “C. Freezing and thawing in air destroyed its activity. The oxidative deiodinating activity in the absence of microsomes was, however, stable when stored at -25 “C.

Characteristics of the Enzymatic Reaction-Table I11 shows the activity of purified SPF in stimulating microsomal deio- dination of rT3 and T,. With 500 pg of the HPLC-purified preparation and 5 mM GSH, microsomal deiodination was at a level of about 80 and 60% of that obtained with 5 mM DTT for rT3 (0.5 pM) and T4 (5 WM), respectively. The products were identified by paper chromatography, as 3,3’-T2 and TS with rT3 and T, as substrates, respectively. The activation was eliminated by heat treatment or Pronase digestion of SPF.

As mentioned earlier and as shown in Fig. 1 and Table 111, SPF fractions from the earlier stages of purification contained a GSH-dependent enzymatic activity which did not require microsomes and which catalyzed the oxidative deiodination of iodothyronines and thus resulted in high blank values in the SPF assays. This “intrinsic” (ie. microsome-independent)

6016 Cytosolic Factor Enhancing GSH-dependent Deiodination * rn

2

FIG. 2. High pressure gel filtration of purified SPF. 30 pg of the purified sample was applied on an 1-125 column and eluted with 50 RIM Na-phosphate buffer. The abscissa is retention time (R,) and the ordinate is optical density a t 254 nm.

4.9-

4.8-

5 4.7-

2 4.5-

$ 4.4-

c.

.- p 4.6-

- 3

- 4.3-

4.2- 0)

4.1-

4.0-

3.9- 0 0.1 0.2 0.3 0.4 0.5 0.6

Kav FIG. 3. Estimation of SPF M, by high pressure gel filtration.

The K.,, values were calculated by fitting elution volume (V,) data into the equation K., = (V , - V,)/V, - Vo), where the void volume (V,) was experimentally determined and the total bed volume (V,) was calculated from the data supplied by the manufacturer. BSA, bovine serum albumin.

deiodinating activity was concentration-dependent, being more pronounced at higher substrate concentrations and, as shown in Fig. 5, A and B, constituted a much smaller fraction of the total deiodinating activity in mixtures of microsomes plus GSH incubated with low (nM), as opposed to high ( p ~ ) , substrate concentrations. In order to avoid high blanks, 0.5

+

ORIGIN

1 2 3 FIG. 4. Polyacrylamide gel electrophoretic analysis of SPF

after selected steps of the purification. Slot I received 20 pg of HPLC-purified material, while slots 2 and 3 received 100 pg of material purified by CM-cellulose and DEAE-cellulose, respectively. Methodological details are described under "Experimental Proce- dures."

TABLE 111 Renal microsomal iodothyronine B'-deiodinase

Effects of propylthiouracil on activation by dithiothreitol and SPF. The assay conditions were as described under Table I.

5"Deiodinase Activity

Substrate Addition Concentration

rT3 T, (0.5 MM) (5 I" nmol substrate de-

soma1 protein". h" graded. mg micro-

None ND" ND Dithiothreitol 5 mM 26.80 1.15 Dithiothreitol + 5 mM

1 mM 0.088 ND GSH 5 mM ND ND SPF 500 pg 2.2 0.18 SPF +

GSH 5 mM 21.65' 0.68 SPF + GSH + PTU

5 mM 1 mM 19.95 0.59

PTU

500 pg

500 pg

ND, not detectable. *Activity without microsomes: 4.85 nmol/mg of protein/h.

nM rT3 was therefore routinely used in the microsomal-deio- dinating system for monitoring SPF activity during its puri- fication and characterization. There is evidence2 that rat kidney microsomes contain not only a high K,,, (K, (rT3) = 0.5 p ~ ) but also a low K, (K , (rT3) = 2.5 nM) iodothyronine 5"deiodinase; the latter may be predominant at the 0.5 nM rT3 concentrations routinely used in SPF assays. As shown in Fig. 6, SPF has an activating effect on both enzymes. SPF enhanced the reaction velocities of the 5"deiodinases in the

* Goswami, A., and Rosenberg, I. N. (1984) J. Clin. Invest. 74, 2097-2106.

Cytosolic Factor Enhancing GSH-dependent Deiodination 6017

A ;!F 4

2

100 200 300 100 500 600 :E 100 200 300 100 500 600 L d e 5 z I Z 100 200 300 400 500 000

CB SPF CB SPF

FIG. 5. Demonstration of SPF as a promoter of GSH-de- pendent microsomal rTs-deiodlnase activity with 0.6 pM (A) and 0.5 UM (B) substrate. Assay conditions were as described under the “Experimental Procedures.” Assay medium for the lower substrate concentration contained 100 PM propylthiouracil. Under these conditions, microsomal deiodinase activity with GSH alone was negligible. 0 and 0, activity with and without microsomes, respec- tively.

1.4-

1.2-

1.0-

.€I-

.6 - / 0 2 4 6 8 1 0 0 2 4 6 10

rT3 (pM)” 1Ts (nM)” FIG. 6. Lineweaver-Burk plots of SPF-mediated micro-

somal rTs deiodinase activities at micromolar (A) and nana- molar (B) substrate (rTs) concentrations. Incubation conditions were as described under the “Experimental Procedures.”

presence of GSH without affecting the K,,, values. Lineweaver- Burk plots of reaction velocities in the presence of SPF and 5 mM GSH at p~ and nM substrate concentrations (Fig. 6, A and B ) yielded K , values of 0.53 PM and 2.86 nM for rT3, as compared to the previously reported values of 0.5 MM and 2.5 nM in the presence of 5 mM D m ?

Inhibition-Fig. 7 shows the effects of three well-known iodothyronine deiodinase inhibitors, propylthiouracil, iopa- noic acid, and dicoumarol, on SPF activation of the micro- somal enzyme at 0.5 nM rT3. In sharp contrast to the DTT- supported deiodination which has been shown to be inhibited by PTU at these substrate concentrations with a Ki of ap- proximately 100 PM? the SPF-mediated deiodination was quite insensitive, with about 20% inhibition at 1 mM PTU. As with DTT at this substrate concentration,2 dicoumarol was also an ineffective inhibitor. Iopanoate was a strong inhibitor and, as with the DTT-supported reaction, the inhi- bition was competitive with rTT3 in SPF-mediated deiodina- tion, with a Ki of approximately 0.9 mM; this was about 100- fold higher than the Ki reported for the DTT-activated mi- crosomal deiodination at high substrate concentrations (21). Sample preparations, obtained by preincubation of SPF with 1 mM N-ethylmaleimide (4 “C for 10 min) and subsequent

100 -I

.E 60 Y \ SPF: 50 pg

GSH: 5mM rT3: 0.5 nM

0 , I I I I I f i 0.6 1.4 2.2 3 .O

Inhibitor (mM) FIG. 7. Concentration-response curves for the inhibition of

the SPF-activated microsomal rTs deiodinase by dicoumarol, propylthiouracil, and iopanoic acid. Incubation conditions were as described under “Experimental Procedures.” rTB was at 0.5 nM.

dialysis to remove excess N-ethylmaleimide, were completely devoid of both SPF activity and the activity in the absence of microsomes, indicating dependence on intact sulfhydryl groups for both these enzymatic processes.

Activity as Clutaredorin-Purified rat liver SPF prepara- tions were found to be active in the reduction of calf thymus ribonucleotide reductase. Under standard assay conditions, the activity of one representative CM-cellulose-purified SPF preparation was 1.47 nmol of dCDP formed per mg of protein/ h compared with a value of 4.08 obtained with a calf thymus glutaredoxin preparation at a similar stage of purification. An HPLC-purified SPF showed a 2-fold increase in specific ac- tivity. Similarly, a purified preparation of calf thymus gluta- redoxin was also effective in promoting GSH-dependent rT3 deiodinating activity of rat kidney microsomes; CM-cellulose preparations, containing approximately 300 units of thiol transferase (with @-hydroxyethyl disulfide) per mg of protein, had an average of 5-8 units of SPF activity per mg of protein, compared with values of 20-30 units/mg of protein observed with rat liver cytosolic preparations at similar stages of puri- fication. Moreover, the calf thymus glutaredoxin preparations were also found to be contaminated with the CN--sensitive, 02-dependent iodothyronine-degrading activity analogous to that observed during the preparation of rat liver SPF.

DISCUSSION

This report describes the purification of a low molecular weight protein (SPF) from rat liver cytosol that can promote GSH activation of rat kidney microsomal iodothyronine 5’- deiodinase. The elution profile (CM-cellulose and HPLC) of the stimulatory activity for microsomal deiodination corre- sponded well with that of GSH-disulfide transhydrogenase activity (using @-hydroxyethyl disulfide as acceptor) and was the basis for the purification procedure employed for the studies reported here. The intracellular localization (i.e. cy- tosolic localization) and the purification procedure, as well as the molecular properties (such as the molecular size and isoelectric points) of SPF, are very similar to those described for a GSH-disulfide oxidoreductase in rat liver cytosol by Axelsson et al. (16), and it is possible that SPF and the GSH-

6018 Cytosolic Factor Enhancing GSH-dependent Deiodinatwn

disulfide transhydrogenase of Axelsson et al. may be identical. The similarity between the rat liver enzyme and calf thymus glutaredoxin has previously been emphasized (8), and the “glutaredoxin-like” activity of SPF in supporting calf thymus ribonucleotide reductase, and the “SPF-like” activity of calf thymus glutaredoxin in promoting GSH-activation of rat kidney iodothyronine deiodinase, as reported here, suggests that SPF and glutaredoxin may be closely related in structural and functional characteristics. To our knowledge, this is’the first demonstration of the presence of a glutaredoxin-like activity in rat liver cytosol.

The molecular weight and subcellular localization of SPF distinguishes it from a number of other thiol transferases such as the glutathione S-transferases which are characterized by much higher molecular weights (40,OOO-45,000) (22) and the glutathione-insulin transhydrogenase, which is predomi- nantly a microsomal enzyme (23). Moreover, SPF showed very little activity as glutathione-disulfide transhydrogenase with insulin as an acceptor and, in two experiments, a crude rat liver cytosolic glutathione S-transferase preparation was found to be inactive as SPF.

Washed microsomal preparations are completely devoid of iodothyronine deiodinating capacity, and reconstitution ex- periments in several laboratories have indicated the require- ment for a cytosolic cofactor (24-26). Further experiments revealed that the cytosolic preparations could be effectively replaced by various thiols, and it was suggested that glutathi- one may be the endogenous cofactor (24, 25). This idea was supported by the moderate stimulatory effects of GSH, often observed in homogenates (26) and isolated cell preparations (27), that was substantially inhibited by the GSH-depleting agents, diamide and t-butylhydroperoxide (28), and indirectly by in vivo experiments which showed a substantial fall of hepatic T4-deiodinase activity during starvation (24,29). The in vivo observations have variously been explained on the basis of cellular depletion of GSH (24,30) or of NADPH (24), the role of the latter cofactor being the regeneration of GSH, oxidized during the deiodinase reaction, by interaction with NADPH-glutathione reductase (28). These explanations, however, do not appear to be entirely satisfactory because: 1) there was no correlation between glutathione concentration and 5”deiodinating activity in rat livers (31) nor was the enzymatic activity affected by substantial decrease in intra- cellular glutathione concentrations in primary cultured rat hepatocytes (32); 2) cytosolic NADPH concentrations in the liver do not show any significant fall during fasting (33), and 3) one would expect little alteration of cytosolic GSH levels (4 mM (34)) and NADPH/NADP ratio (100 (35)) which are high relative to the expected rate of iodothyronine 5“deiodi- nation at physiologic concentrations of iodothyronines, ren- dering rather unlikely the possibility that GSH regeneration by NADPH could be a rate-limiting step in the enzymatic reaction. Moreover, the concentrations of glutathione needed to stimulate T4-deiodination (at 5 p~ T4) in uitro have been found to be 5-10-fold higher than the cellular level of GSH (36). These observations suggest that, if glutathione is indeed the endogenous cofactor for the iodothyronine 5’-deiodinase, another controlling mechanism may exist in vivo for the interaction of GSH with the deiodinase enzyme. The data presented in this paper suggest that a low molecular weight cytosolic protein with thiol transferase activity may provide such a mechanism.

By virtue of its GSH-disulfide transhydrogenase activity and its ability to interact with the microsomal iodothyronine deiodinase, SPF may be a physiologically important cofactor in the deiodination of iodothyronines. The requirement for a

specific cytosolic cofactor for GSH-activation of the micro- somal iodothyronine deiodination may account for the failure in many laboratories, including our own, to activate washed microsomal preparations from liver and kidneys at high sub- strate concentrations of T4 and rT3 by physiological concen- trations of GSH alone (4).

Recent observations in our laboratory indicate that, in addition to the well-characterized “high K,” enzyme, rat kidney microsomes may also contain a “low K,” iodothyro- nine deiodinase; the latter is moderately responsive to phys- iological concentrations (1-5 mM) of GSH.2 Our results, as presented in this paper, show that a cytosolic protein factor is not only capable of activating this low K, enzyme further, but is also effective in making the microsomal high K, enzyme responsive to physiological concentrations of GSH. In this capacity, this factor may conceivably provide a regulatory mechanism for GSH-dependent iodothyronine deiodination in vivo. Moreover, while the thiol-stimulated deiodinating activities at both high and low substrate concentrations are sensitive to inhibition by PTU, the corresponding SPF-stim- ulated activities are only slightly affected by PTU. In light of the claims that PTU-insensitive (type 11) pathways of outer ring-iodothyronine monodeiodination, such as have been dis- covered in the brain (37), pituitary (38), and brown adipose tissue (39) do not occur in the liver and kidney (40), and in view of the generally held opinion (41) that the liver and kidney supply most of the circulating (as opposed to local cellular) T3, the striking PTU insensitivity of SPF stimulation of GSH-dependent microsomal deiodination suggests that this pathway could have an important role in T3 generation in vivo in the presence of PTU and may provide a basis for the persistence, in the PTU-treated rat maintained on thyroxine, of appreciable circulating T3 concentrations even at very high dosages of PTU (42).

It is of interest that native hepatic cytosol (presumably containing both SPF and GSH), failed to activate the 5’- deiodinase enzyme in washed microsomal preparations. A possible explanation may be the presence of an inhibitor that is removed during the purification procedure. The studies of Leonard and Rosenberg (43), where they observed consider- able enhancement of T4-deiodinating activity in kidney ho- mogenates and postnuclear supernatants upon dilution, pre- sumably due to concommitant dilution of an inhibitor, would support this possibility. Another curious observation is the presence in rat liver cytosol of a deiodination activity, active with both rT3 and T4, which differed from the SPF- or dithiothreitol-stimulated microsomal deiodinase in being 0 2 -

dependent, CN--sensitive with the reaction products being I- and some unidentified material staying at the origin on paper chromatography in the hexanelt-amyl alcohol/ammonia sys-

Hypothesis:

PTU sensitiviw:

Dithiothmitol fl o-dithmne

high at high [SI low at low IS]

lodothyronine 6’ daiodiner

SPF T. IrTd - Ta (3. TI) + I-

high and low [SI None at both

N A D P A NADPH*H+ Glutathione nductaM

FIG. 8. A hypothetical model for the activation of micro- somal iodothyronine deiodinase by SPF in the presence of GSH.

Cytosolic Factor Enhancing GSH-dependent Deiodination 6019

tern rather than I- and 3,3’-T2 or T3. The 0 2 dependence and formation of “origin material” suggest similarities of this activity to the oxidative T4-deiodinating systems described by Stanbury (44) and by Lissitzky et al. (45).

A hypothetical model for the mechanism of SPF activation of the deiodinase enzyme is shown in Fig. 8. In this scheme, dithiothreitol can directly reduce the disulfide groups in the enzyme for its activation, but the transfer of the reductive potential of GSH requires the intermediation of a specific GSH-disulfide transhydrogenase (designated SPF). The di- thiothreitol-mediated reaction is strongly inhibited by PTU, presumably through the formation of a mixed disulfide with an enzyme-sulfenyl iodide, believed to be the central complex in the two-step transfer (ping-pong) mechanism postulated for the enzymatic reaction (12, 46). Support for such an interaction also derives from the studies of Cunningham and Jirousek (47,48) on the mechanism of PTU interaction with some iodinated thiol proteins. However, as has recently been pointed out (49, 50), mixed disulfides of protein ”SH and some thiocarbamides may also be formed at physiological pH by an interaction of the SH with a sulfenyl derivative of the thiocarbamide, generated through an NADPH and O,-de- pendent S-oxygenation of the thiocarbamide by a microsomal monooxygenase. Such a mechanism, although a distinct pos- sibility in the crude systems, would, however, appear to be unlikely for the potent PTU inhibition of the DTT-activated enzymatic reaction observed in washed microsomal prepara- tions which require neither NADPH nor 02 (Refs. 12, 13, and the present studies). In contrast, the SPF-mediated activation is only mildly sensitive to PTU, suggesting the formation of a reactive complex in which the oxidized form of the enzyme is unavailable for interaction with PTU. The reduced form of glutathione, oxidized during the deiodinase reaction, is pre- sumably regenerated by NADPH through the cytosolic NADPH/GSH oxidoreductase.

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