The Journal of Biochemistry,Vol. 62,No. 6,1967

Deiodination of Iodinated Amino Acids by Pig Thyroid Microsomes

By SHIGERU MATSUZAKI and MITSUO SUZUKI

(From the Department of Physiology,Institute of Endocrinology, Gunma University, Maebashi)

(Received for publication,June 26,1967)

Studies were carried out on the metabolism of iodinated amino acids in the pig thyroid with the following results: 1.•@ It was confirmed that NADPH-dependent,iodotyrosine-deiodi- nating activity is localized mainly in the microsomal fractions. 2.•@ L-Monoiodotyrosine was revealed to be present as an intermediate during the deiodination of L-diiodotyrosine.This fact,together with oxygen independency and requirement of reduced nicotinamide nucleo- tide for this reaction,indicates that the deiodination of iodotyrosines is of a reductive nature. 3.•@ Iodotyrosine deiodinase is highly specific for L-iodotyrosines.At the substrate concentration of 1•~10-6M,the rate of deiodination of L- diiodotyrosine was about one fifth the rate of deiodination of L-mono- iodotyrosine.At the optimal pH of 8.0,the K,for L-diiodotyrosine was

greater than that for L-monoiodotyrosine,the maximal velocity being the same for each iodotyrosine. 4.•@ Some other enzymatic properties of the enzyme such as cofactor requirement,and sensitivity toward inhibitors were investigated. 5.•@ L-Tyrosine,and keto acid analogues of monoiodotyrosine and diiodotyrosine were demonstrated to act as competitive inhibitors of the deiodination reaction. 6.•@ Peroxidase[EC 1.11.1.7],NADPH-cytochrome c reductase and DT-diaphorase[EC 1.6.99.2],each derived from pig thyroid,do not appear to be concerned in the deiodination of iodotyrosines.

It is well known that intrathyroidal free defect in this thyroidal deiodinating system iodotyrosines are deiodinated by a microsomal (5-9). enzyme which requires NADPH as its cofactor It has generally been recognized that the (1).Since the enzyme catalizes also bromo- main metabolic pathway of free iodotyrosines tyrosine dehalogenation,it is also called de- in the thyroid is deiodination.No deaminating halogenase(2).The thyroidal deiodination activity for iodotyrosines has been demonstrated of free iodotyrosines has been generally ac- in the thyroid(1,10).On the oher hand, cepted to be of physiological importance, iodothyronines are not deiodinated by thyroid because iodide released from iodotyrosines by tissue(2).Although iodohistidines(11,12), this enzyme is conserved in the so-called keto acid analogues of iodotyrosines(13),and "second iodide pool"(3 ,4)and re-utilized other iodinated compounds have been found by the thyroid to form thyroid hormones. in the gland,little is known about the meta- Several investigators have reported patients bolic fate of these compounds. with goiter which was caused by a congenital Deiodinating activity is also found in the 746 Thyroidal Iodotyrosine Deiodinase 747 liver,kidney and other tissues(10,14,15). total volume of 3.0ml,The reaction was started by However,the enzyme in the thyroid is the the addition of glucose,Incubation was carried out most important and interesting to investigate, at 37.5•Ž for 60 minutes,After the incubation,6ml, in view of its physiological function and of of absolute ethanol was added and the mixture was centrifuged at 3,000 r.p.m,for 10 minutes to remove the importance in the metabolism of iodo- tyrosines in the gland,In this paper we de- proteins,The supernatant was subjected to column chromatography on Dowex-1X2 in the acetate form scribe various properties of the enzyme and (17),and L-MIT and L-DIT were eluted by 0.025% the metabolism of some iodinated amino acids and 1%acetic acid,respectively. other than iodotyrosines. The radioiodination by the use of Chloramine T

was a modification of the method of Reith and MATERIALS AND METHODS Tampion (18),A mixture of Na'"I (0.5-1.0mc),

NADH,NADPH,13-tyrosine,cytochrome c,horse- 1 ƒÊmole of KI,and 2 pmoles of amino acid in 0.1M radish peroxidase[EC 1,11,1,7],catalase [EC 1,11,1,6] phosphate buffer(pH7.4)in a total volume of 3.0ml, and glucose oxidase[EC 1,1,3,4]were obtained from were incubated at 20•Ž for 60 seconds after adding Sigma Chemical Company,Authentic samples of 3- 25 ƒÊmoles of freshly prepared Chloramine T,After iodo-4-hydroxyphenylpyruvic acid(MIHPPA)and 3,5- incubation,excess iodine was reduced by the addition diiodo-4-hydroxyphenylpyruvic acid(DIHPPA)were of 50 ƒÊmoles of metabisulfite,Iodinated amino acids generously supplied by Dr,M,Nakano and highly prepared by this method were L-MIT,D-MIT,L-DIT purified thyroid peroxidase by Dr,T,Hosoya of this and L-monoiodohistidine (L-MIH),Iodotyrosines were University. purified by column chromatography as stated above, Preparation of Thyroid Cytoplasmic Fractions-Thyroids L-MIH was isolated by paper chromatography as were obtained freshly from slaughtered pigs at the local follows:After incubation,the reaction mixture was abattoir and brought immediately to the laboratory, applied on a filter paper(40cm,X40cm.)and run in

Small portions from at least ten glands were trimmed n-butanol-acetic acid-water(5:1:4,v/v)for 18 hours to remove connective tissue,cut into small pieces and at 20•Ž,After radioautography,a paper strip corre- sponding to L-MIH was cut off,eluted with 0.01 N ground with the use of a glass homogenizer with a loosely fitted Teflon pestle in 0.25 M sucrose,in some NaOH in an atmosphere of NH3 and the eluate was cases in 0.154M KCl buffered with 0.01M Tris-HC1 evaporated in vacuo. buffer (pH7.3),to make a 20% homogenate,Unbroken When analyzed by paper chromatography,more cells,nuclei,and cell debris were removed by cen- than 96%of the radioactivity was found in the desired trifugation in a refrigerated Sakuma centrifuge at iodoamino acids,The concentration of stable iodo- 700Xg for 10 minutes,The supernatant was centrifuged amino acids was determined by the method of Barker at 5,000•~g for 10 minutes to obtain mitochondria, (19),The specific activity was approximately 0.5mc/ The supernatant so obtained was subjected to cen- pmole just before use. trifugation at 20,000•~g for 30 minutes and 105,000Xg Determination of Deiodinase Activity-The activity for 60 minutes in the RP40 rotor of a Hitachi prepara- was determined by a slight modification of the method tive centrifuge to obtain heavy and light microsomes, of Stanbury and Morris(20),The incubation respectively,The pellets collected at each centrifuga- mixture contained "I-labeled amino acid(1.0pc), tion were redispersed in the same volume of 0.25M 0.1 pmole of NADPH,6 pmoles of nicotinamide,100 sucrose and recentrifuged,The sediments were re- pmoles of Tris-HCI buffer (pH7.3)and 0.1ml,of suspended in 0.25M sucrose to make the volume to thyroid tissue preparation as an enzyme source in total one fifth of the initial volume. volume of 1.0 ml,One pmole of thiourea was added Preparation of 1311-Labeled lodoamino Acids-181I- to the reaction mixture to prevent reutilization of Labeled iodoamino acids were prepared either by an en- released iodide,The incubation vessels were agitated zymatic iodination or by a Chloramine T iodination,The gently in a constant temperature bath at 37.5•Ž for procedure for enzymatic radioiodination was as follows: 10 minutes,unless otherwise noted,The reaction was L-Tyrosine or 3-iodo-L-tyrosine(L-MIT)was employed stopped by adding an excess of the substrate iodoamino as a substrate for iodination to synthesize "I-i.-MIT acid and heating at 100•Ž for 90 seconds,Since or 131I-3,5-diiodo-L-tyrosine(131I-L-DIT),The reaction iodinated amino acids were found to be deiodinated mixture contained 1 pmole of the substrate(L-tyrosine non-enzymatically to some extent upon exposure to or L-MIT),1 gmole of KI,0.5-1.0mc of Na181I, light,all the incubation was carried out in brown glass 0.05ml,of partially purified thyroid iodinase[EC tubes and in the dark, 1,11,1.8]prepared as previously reported(16),100 An aliquot of 50 pl,of the reaction mixture was pg,of glucose oxidase and 10 pmoles of glucose in a applied to Toyo Roshi No,50 filter paper(2cm,X40 748 S.MATSUZAKI and M.SUZUKI cm.)and was developed ascendingly using a solvent activity resided mainly in the microsomal system of n-butanol-acetic acid-water(5;1:4,v/v),or fractions,when either L-MIT or L-DIT was n-butanol-ethanol-2 N ammonium hydroxide(5:1:2, employed as a substrate. When thyroids were v/v).Radioactive compounds were identified and ground in 0.25M sucrose,higher enzyme acti- determined as previously described(21).The de- vity was observed in heavy microsomes than iodinating activity was measured from the amount of in light microsomes,whereas if homogenized iodide liberated from iodinated compounds. in buffered KC1 solution by Waring blender, Protein was determined by the method of Lowry et al.(22),using bovine albumin as a standard. the highest activity was in light microsomes. Determination of Activity and Purification of Other Only a little activity was present in the mito- Enzymes-Peroxidase activity was measured by a guai- chondrial and soluble fractions. acol assay method(23).NADPH-cytochrome c re- Time Course of Deiodination of L-Iodotyrosines ductase was determined spectrophotometrically(16), -Since deiodinating activity was shown to be and DT-diaphorase by the method of Ernsteretal. concentrated in heavy microsomes when the (24). thyroid glands were homogenized in 0.25M NADPH-cytochrome c reductase was partially puri- sucrose,this fraction was used as the enzyme fied from pig thyroid microsomes as described previously source in the remainder of the study.Heavy (16).DT-Diaphorase was purified partially from pig microsomes could be stored at -20•Ž for one thyroid soluble fraction with the use of carboxymethyl Sephadex(CM-50)after precipitating between 45% week without appreciable loss of activity.In and 85% ammonium sulfate saturation.*This prepa- most experiments,heavy microsomes of 0.2- ration had an activity 18 times that of the original 0.4 mg.protein were employed per sample. soluble fraction. The time course of L-DIT deiodination is shown in Fig.1.As a deiodination product, RESULTS L-MIT was demonstrated along with iodide. Intracellular Distribution of Iodotyrosine De- L-MIT increased up to 60 minutes and then iodinase As shown in Table I,deiodinating gradually decreased.This indicates that L- MIT is an intermediate during deiodination TABLEI of L-DIT. Deiodination of L-Iodotyrosines by Thyroid The rate of liberation of iodide from L- Cell Fractions MIT and L-DIT was measured as a function

The reaction mixture contained 0.1ml.of thyroid tissue preparation,labeled iodotyrosine, 0.1 umole of NADPH,6 umoles of nicotinamide, 1 umole of thiourea and 100 umoles of Tris-HC1 buffer(pH 7.3)in a total volume of 1.0 ml.Incu- bation time was 60 minutes.As a substrate,0.31 and 0.79 mpmoles of L-MIT were employed in experiments I and II, respectively.L-DIT used was 1.48 mpmoles.Deiodinating activity was ex- FIG.1.•@ Time course of L-DIT deiodination by thyroid heavy microsomes. pressed as percentage deiodination of iodotyrosines Reaction was carried out as described in the per hour per 0.1 mg.protein. text.3.0 mƒÊmoles of L-DIT and thyroid heavy *Suzuki,M.,unpublished data. microsomes of 2.5 mg.protein were employed. Thyroidal Iodotyrosine Deiodinase 749

FIG.2.•@ Time course of liberation of iodide

from L-DIT and L-MIT. The experimental conditions were the same as in Fig.1. of time(Fig.2).Iodide liberation from L-MIT was several times greater than that from L-DIT. Though L-MIT was formed as a product of L-DIT monodeiodination,the amount of L- MIT was almost negligible(Fig.1).This FIG.3.Effect of pH on deiodinase activity. indicates that L-MIT is readily deiodinated as Each flask contained 0.1ml.of thyroid heavy microsomes,1mƒÊmole of 13I-Iabeled iodotyrosine, soon as it is formed.Therefore,the rate of 0.1ƒÊmole of NADPH,6ƒÊmoles of nicotinamide, monodeiodination of L-DIT is approximately 1ƒÊmole of thiourea and 100ƒÊmoles of potassium estimated as one half of that of iodide phosphate buffer in a total volume of 1.0ml. liberation from L-DIT.The velocity of de- Incubation time was 10minutes.The enzyme iodination of both iodotyrosines showed an activity was expressed as msmole of iodide liberated almost linear relation to time during the first per hour. 10 minutes(Fig.2). TABLE II Effectof Hydrogen Ion Concentrationand Ionic Substrate Specificity of the Deiodinase Strength on Deiodinase-Fig.3 shows the de- pendency of the microsomal deiodinase upon pH. Liberation of iodide from L-MIT was greater than that from L-DIT at any pH's. The enzyme activity increased sharply at pH's above 6.5 to reach a maximum at pH of 8.0. At pH6.0 or below,no enzyme activity was observed.Deiodination was not observed with boiled microsomes at any pH's.

A comparable enzyme activity was ob- The reaction mixture contained 0.1ml.of served when either Tris-HC1,potassium phos- thyroid heavy microsomes,131 I-labeled iodoamino phate,Krebs-Ringer phosphate,or Krebs- acid 0.1ƒÊmole of NADPH,6ƒÊmoles of nicotin- Ringer bicarbonate buffer(pH7.3)was em- amide,1ƒÊpmole of thiourea and 100ƒÊmoles of Tris ployed.There was no significant difference buffer at pH7.3 in a total volume of 1ml.Incu- in the enzyme activity when the ionic strength bation time was 10minutes.Relative deiodination of potassium phosphate buffer(pH7.3)was was expressed as the percentage of iodide liberated 0.06,0.6 or 1.2.These results showed that the from each iodoamino acid versus that liberated deiodinase differed from thyroid microsomal from L-MIT. NADPH-cytochrome c reductase,which has Substrate Specificity Cofactor Requirementand been reported to have an optimal pH of 7.5 Oxygen Dependency-As shown in Table II,L- and to be activated in higher ionic strength(16). MIT was deiodinated most rapidly.Iodide 750 S.MATSUZAKI and M.SUZUKI liberation from L-DIT was approximately 40% of that from L-MIT.D-MIT was deiodinated to an extent,but L-MIH and L-thyroxine TABLEV (L-T4)were not. Effectof Various Agents on Deiodinase At optimal pH of 8.0,apparent Michaelis constants(Km)of the enzyme for iodotyrosines were 4.9•~10-6M for L-MIT and 6.7•~10-6M for L-DIT.At pH 7.3 Km,values were 6.2•~ 10-6M for L-MIT and 1.1•~10-5M for L-DIT. The maximal velocity at pH 8.0 was about 1.1 mƒÊmoles per minute per mg.protein for either iodotyrosine.At the concentration of 10-5M or above,substrate inhibition of L-DIT was observed. It seemed that NADH could serve as a cofactor,though deiodination of L-MIT was about one eighth of that with NADPH(Table III). In the presence of NADPH or NADH, no deiodination of L-T4 was observed. No attempt was made to determine Km values for NADPH and NADH,because oxi- dation of the reduced nicotinamide nucleotides by the deiodinase was too slight to permit the

TABLE III CofactorReguirement of the Deiodinase

The experiment was performed as described

under Table II,except 0.8 mƒÊmoles of L-MIT and

T4 were employed.Deiodination activity was ex-

pressed as percentage deiodination in 10 minutes.

TABLEIV Oxygen Dependency of the Enzyme

The experiment was performed as described under Table II,except 1.0 mƒÊmole of L-MIT was The experiment was performed as described employed.Enzyme activity was expressed in under Table II,except that NADPH was added percentage deiodination of the control. as indicated.As a substrate,0.3 mƒÊmoles of L-MIT was employed. Thyroidal iodotyrosine Deiodinase 751

determination and because microsomes contain the enzyme.The enzyme was found to be other enzymes which consume the nicotin- insensitive to antimyosin A,rotenone and amide nucleotides. amytal,but was inhibited by dicumarol at a Oxygen dependency of iodotyrosine de- high concentration of 1•~10-4M. iodinase is shown in Table IV.Anaerobiosis The deiodination was not significantly caused no diminution in the deiodinating affected by phenylalanine,histidine and L- and activity with the addition of NADPH.At a D-tyrosine.Among iodinated amino acids and concentration of 2•~10-6M,deiodinating acti- their derivatives,L-T4,3,5,3'-L-triiodothyronine vity under anaerobic condition was even (L-T3),L-diiodohistidine(L-DIH)and L-MIH greater than under aerobic condition.This showed no noticeable effect.On the other suggested that NADPH available for the de- hand,MIHPPA and DIHPPA,keto acid iodination was partially consumed by thyroid analogues of MIT and DIT,clearly inhibited microsomes in the presence of molecular the activity.KI was found to be inhibitory oxygen(16). only at a high concentration of 1•~10-1M. Effects of Respiratory Inhibitors,Metal Ions, Inhibition of L-tyrosine on deiodination Amino Acidsand Iodinated Compounds-The effect of L-MIT was so slight at pH 7.3(Table V), of various agents on the deiodination of L- but as shown in Fig.4,further study confirmed MIT by thyroid heavy microsomes is sum- that L-tyrosine depressed L-MIT deiodination marized in Table V.Sodium azide and po- by competitive inhibition at the optimal pH tassium cyanide,which were inhibitors of heme of 8.0. MIHPPA(Fig.5)and DIHPPA were protein,exhibited no inhibitory effect on the also found to act as competitive inhibitors on deiodinase activity.Catalase,as much as 1 L-MIT deiodination. mg.did not inhibit the activity.At the Deiodination of Iodotyrosinesby Other Enzymes -It was possible that iodotyrosines might be concentration of 2•~10-5M,Cu++was inhibitory but other cations had little effect,if any,on deiodinated by thyroid peroxidase,because the enzyme is present in thyroid microsomes and because H2O2 is expected to be generated by thyroid microsomes in the presence of NADPH and molecular oxygen(16).An attempt was made to examine this possibility. Table VI shows the results.In the absence

Fro.4.•@ Effect of L-tyrosine on deiodination of L-MIT. FIG.5.•@ Effect of MIHPPA on L-MIT deio- The experimental conditions were the same as dination. described in Fig.3,except that phosphate buffer The experimental conditions were the same of pH 8.0 was employed. as in Fig.4. 752 S.MATSUZAKI and M.SUZUKI

TABLE VI

Deiodination Catalized by Peroxidase and Other Enzymes

Reaction mixture contained 2 mpmoles of"I-labeled L-MIT,6 pmoles of nicotinamide, 1 pmole of thiourea and 100 pmoles of Tris buffer(pH7.3)in a total volume of 1.0ml Microsomes1),2.5ƒÊg.of horse-

radish peroxidase and thyroid peroxidase had the same peroxidase activity when determined by guaiacol assay.The activity was approximately 0.007G.U.as defined by Hosoya et al.(23).NADPH-cyto-

chrome c reductase applied here had the enzyme activity of reducing 0.3pmole of cytochrome c per minute

at 30•Ž and DT-diaphorase had the potency of reducing 2 pmoles of dichlorophenolindophenol per minute at 30•Ž.

1)•@Microsomes containes 2.1mg.protein. 2)•@Microsomes containes 0.2mg.protein.

of NADPH,microsomes were not effective on siderable amount of iodide was liberated.In L-MIT deiodination even when H202 was this system,not only iodide but also MIHPPA added at concentrations up to 1 •~ 10-3M.But and other unknown iodinated compounds were a slight deiodination was observed with 1 •~ detected on paper-chromatograms.With thy- 10-2M H2O2 added to boiled microsomes. roid peroxidase,an MIHPPA-like substance This might be due to non-enzymatic deiodina- was also found in a small amount. tion caused by H2O2. Thyroid microsomes have been found to Horseradish peroxidase(2.5pg.)and puri- contain NADPH-cytochrome c reductase(16). fied thyroid peroxidase showed only a negligible However,when tested in the presence of deiodination in the presence of H2O2 at con- NADPH,partially purified NADPH-cyto- centrations between 3•~10-5M and 1•~10-8M. chrome c reductase of the thyroid(16)did not In this experiment,thyroid microsomes,horse- show any deiodinating activity(TableVI). radish peroxidase and thyroid peroxidase with Addition of the reductase to the microsomal the same peroxidase activity based on guaiacol deiodinating system resulted in no activation assay method(23),were used.When a larger of deiodination but rather an inhibition. amount(1mg.)of horseradish peroxidase was The existence of DT-diaphorase in thyroid used together with 1•~10-2M H2O2,a con- soluble fractions has been reported(25).Slight Thyroidal Iodotyrosine Deiodinase 753 but consistent deiodinating activity was found. deiodination of iodotyrosines is a reductive in the thyroid supernatant(Table I).How- reaction. ever,this deiodinase activity was not due to From the result shown in Table II,the DT-diaphorase,since partially purified thy- rate of conversion of L-DIT to L-MIT is con- roidal DT-diaphorase had no deiodinating sidered to be about one fifth the rate of de- activity(TableVI).Most soluble deiodinase iodination of L-MIT.L-MIT has a greater was salted out between 20%and50%ammo- affinity to the deiodinase than L-DIT,since nium sulfate,while DT-diaphorase between the Km value for L-MIT was smaller than 45%and 85%ammonium sulfate.Employing that for L-DIT.Although,Stanbury and Sephadex G-200column chromatography,the Morris(1,20)reported that the K.of sheep soluble deiodinase was separated from DT- thyroid deiodinase for L-DIT was smaller(3.7 diaphorase activity. x10-7M) than that for L-MIT(9.2KIx10-7M), Several attempts were made to solubilize under nearly the same conditions as those and purify the microsomal deiodinase.Thyroid used in the present experiments.It is not heavy microsomes were subjected to sonic likely since L-MIT was more readily deiodi- oscillation for 10minutes at 10,000cycles and nated than L-DIT. centrifuged at 105,000xg for 60minutes.In The deiodinase of thyroid microsomes is the resultant supernatant,only a little deiodi- highly specific for L-iodotyrosines.Though nase activity was detected.Sodium cholate D-MIT was deiodinated slightly,L-MIH and was added at a final concentration of1%. L-T4 were not deiodinated.MIHPPA and After centrifugation at 105,000xg for 60 DIHPPA were reported not to be enzymati- minutes,the supernatant was fractionated by cally deiodinated(14),but were found to be salting-out with ammonium sulfate.The potent competitive inhibitors of the enzyme. enzyme lost its activity almost entirely during L-Tyrosine was confirmed to be a competitive this procedure. inhibitor.Tong et al.(10) observed that N-acetyldiiodotyrosine was not deiodinated by DISCUSSION the thyroid and that it interfered with the Although Roche and his coworkers deiodination of L-DIT.Iodohistidines have (2,15)suggested that L-MIT might be an no inhibitory effect on the deiodinase.These intermediate in the deiodination of L-DIT, results suggest that both the free a-amino Slingerland and Josephs( 26)failed to group and the phenol group are essential for detect L-MIT during L-DIT deiodination.The enzymatic deiodination of iodinated amino present study has made it clear that L-MIT acids,except iodothyronines,and that iodo- was really present as an intermediate when phenol compounds without a-amino group can L-DIT was deiodinated by thyroid heavy act as competitive inhibitors of iodotyrosine microsomes.van Zyl et al.(27)and deiodinase. Bastomsky and Rosenberg(28)found It has been reported that NADPH can a relatively large amount of L-MIT employing not be replaced by NADH in the deiodination carrier-free131I-L-DIT and thyroid homoge- reaction(1),but the present study demonstrated nate.In the present study,however,only a that NADH can stimulate this reaction,though slight amount of L-MIT was found.Since its effect was far less than that of NADPH. deiodination of L-MIT was much faster than NADH might activate deiodination,at least that of L-DIT,it is conceivable that the amount partially,by yielding NADPH through trans- of L-MIT as an intermediate of L-DIT de- hydrogenation,though it has been noted that iodination is small. transhydrogenase activity is relatively low in Oxygen did not appear to be required for the thyroid(29). the deiodinase.Even an activation was ob- Only a negligible deiodination of L-MIT served under anaerobic conditions.This fact, was brought about by purified thyroid perox- together with that L-MIT was produced during idase.Since deiodination by thyroid heavy the course of L-DIT deiodination,indicate that micromsomes was not inhibited by KCN, 754 S.MATSUZAKI and M. SUZUKI catalase or anaerobiosis,it may be concluded that the thyroid peroxidase is not involvedin REFERENCES the physiological deiodination of free iodo- tyrosines in the thyroid.An amount of in- (1) Stanbury, J. B., J. Biol. Chem., 228, 801 (1957) organic iodide was detected on chromatograms, (2) Roche, J., Michel, O., Michel, R., Gorbman, A., and Lissitzky, S., Biochim. et Biophys. Acta, when a large amount of horseradish perox- 12, 570 (1953) idase was employed.In this case,however, (3) Halmi, N. S., and Pitt-Rivers, R., Endocrinology, MIHPPA and other unidentified iodinated 70, 660 (1962) compounds were also found.MIHPPA thus (4) Nagataki, S., and Ingbar, S. H., Endocrinology, 73, formed may be deiodinated spontaneously,as 479 (1963) suggested by Hartman(14).It may be (5) Stanbury, J. B., Kassenaar, A. A. H., Meijer, possible that MIHPPA and DIHPPA in the J. W. A., and Terpstra, J., J. Clin. Endocrinol. thyroid is formed by thyroid peroxidase,since Metab., 15, 1216 (1955) L-amino-acid oxidase [EC 1.4.3.7]or trans- (6) Stanbury, J. B., and Litvak, J., J. Clin. Endocrinol. Metab., 17, 654 (1957) aminase[EC 2.6.1 group]for iodotyrosines, (7) Mosier, H. D., Blizzard, R. M., and Wilkins, L., which may form MIHPPA and DIHPPA,has Pediatrics, 21, 248 (1958) not been demonstrated in the thyroid(1,10). (8) Kusakabe, T., and Miyake, T., J. Clin. Endocrinol., NADPH-cytochrome c reductase found in 23, 132 (1963) thyroid microsomes seems to differ from the (9) Kusakabe, T., and Miyake, T. , J. Clin. Endocrinol., microsomal deiodinase.The reductase has 24, 456 (1964) no deiodinating activity.The addition of the (10) Tong, W., Taurog, A., and Chaikoff, I. L., J. reductase to microsomes rather inhibited the Biol. Chem., 207, 59 (1954) deiodination.This is presumably because (11) Block, R. J., Mandl, R.H., and Keller, S., Arch. NADPH available for the deiodination was Biochem. Biophys., 75, 508 (1958) (12) Roche, J., Lissitzky, S., and Michel, R., Biochim. partially oxidized by the reductase.NADPH- et Biophys. Acta, 8, 339 (1952) cytochrome c reductase is activated in higher (13) Haney, J. F. B., and Lissitzky, S., Biochim. et ionic strength(16),while microsomal de- Biophys. Acta, 63, 557 (1962) iodinase is not affected by changes in ionic (14) Hartman, N., Hoppe-Seyler's Z. Physiol. Chem., strength. 285, 1 (1950) Recently Kusakabe and Miyake(30) (15) Roche, J., Michel, R., Michel, O., and Lissitzky, reported that deiodinating activity was also S., Biochim. et Biophys. Acta, 9, 161 (1952) present in the soluble fraction.They de-, (16) Suzuki, M., Gunma Symposia on Endocrinology, 3, monstrated that particulate deiodinase mig- 85 (1966) rated electrophoretically towards the cathode (17) Pitt-Rivers, R., and Sacks, B. I., Biochem. J., 82, 111 (1962) and that the soluble deiodinase migrated towards the anode.We also found some (18) Reith, W. S., and Tampion, W., Nature, 197, 180 deiodinase activity in the supernatant fraction. (1963) (19) Barker, S. B., J. Biol. Chem., 173, 715 (1948) Relatively low specific activity of the soluble (20) Stanbury, J .B., and Morris, M. L., J. Biol. Chem., fraction might be due to the presence of a 233, 106 (1958) large amount of thyroglobulin. (21) Suzuki, M., Nagashima, M., and Yamamoto, K., It was proved that the activity of soluble Gen. Comp. Endocrinol., 1, 103 (1961) deiodinase is not due to DT-diaphorase.Fur- (22) Lowry, O.H., Rosebrough, N. J., Farr, A. L., and ther studies will be needed to determine Randall, R. J., J. Biol. Chem., 193, 265 (1951) whether soluble deiodinase is different from (23) Hosoya, T., Kondo, Y., and Ui, N., J. Biochem., the particulate deiodinase.Our attempts to 52, 180 (1962) (24) Ernster, L., Danielson, L., and Ljunggren, M., purify the deiodinase was unsuccessful. Biochim. et Biophys. Acta, 58, 171 (1962) (25) DeGroot, L. J., Dechene, R., and Thompson, J., The authors wish to express thanks to Prof.K. Biochim. et Biophys. Acta, 92, 223 (1964) Yamamoto for the encouragement during the course (26) Slingerland, D. W., and Josephs, R.K., Endo- of this study. crinology, 62, 853 (1958) Thyroidal Iodotyrosine Deiodinase 755

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