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Deiodination of Iodinated Amino Acids by Pig Thyroid Microsomes

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Deiodination of Iodinated Amino Acids by Pig Thyroid Microsomes 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.
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