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Novel thyroid specific transcripts identified by SAGE: implication for congenital
hypothyroidism
Moreno Navarro, J.C.
Publication date
2003
Link to publication
Citation for published version (APA):
Moreno Navarro, J. C. (2003). Novel thyroid specific transcripts identified by SAGE:
implication for congenital hypothyroidism.
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CHAPTERR 4
Cloningg and characterization of the human iodotyrosine
dehalogenase. .
Joséé C. Moreno ', Remco Keijser 1; Saida Aaraas 1
, Lisa Gilhuijs-Pederson 2,
Davidd Gestel 3, Antoine H. C.van Kampen 2, Ed E. Moret 3. Jann J.M. de Vijlder \ Carolyn Rts-Stalpers 1.
11
Laboratory of Pediatric Endocrinology. Emma Children's Hospital.
AcademicAcademic Medical Center. University of Amsterdam. The Netherlands.
22
Laboratory of Bioinformatics. Academic Medical Center. UniversityUniversity of Amsterdam. The Netherlands.
DepartmentDepartment of Medicinal Chemistry. Faculty of Pharmaceutical Sciences. UtrechtUtrecht University. The Netherlands.
Characterizationn of f r e h j r r a n tnyrrjid d f - a l c g e r a s e
A b s t r a c t t
Thee scarce halogen element iodine is the essential component of thyroid hormone whichh is critically required for brain development and maturation. In the thyroid gland,, two highly specialized systems evolved to ensure hormone biosynthesis. Onee accumulates the ingested dietary iodine in thyroid cells by active membrane transport.. The other (of unknown molecular nature) re-cycles iodine through the dehalogenationn of iodotyrosines, the iodinated byproducts of thyroid hormonogenesis. .
Heree we describe the identification of DEHAL1 from a thyroid SAGE library based onn expression in thyroid, kidney, liver and mammary gland. The main open reading framee (ORF) encodes a novel human enzyme with a conserved nitroreductase domainn that is FMN dependent and dehalogenates iodotyrosines. Alternative splicingg of the 6-exon gene generates a second ORF lacking 64 residues of the highlyy conserved nitroreductase domain that is inactive. Computer assisted modellingg predicts homodimer formation with FMN bound at the interface with an activee site in each monomer.
Too our knowledge. DEHAL1 is the first cloned human dehalogenase. providing the molecularr basis for the evolutionary conserved mechanism for iodine recycling and dehalogenationn of other compounds.
AA serial analysis of gene expression [1 ] (SAGE) library was constructed from normal humann thyroid tissue [2]. Out of more than 4000 "no-match" SAGE tags representingg unidentified thyroidal transcripts, the tag corresponding to the DEHAL1 genee faaaagtattai was selected using tne tissue prefential expression (TPE) computationall substraction method [3]. Using the SAGEmap program [4] the tag wass linked to a human expressed sequence tag (EST) sequence (AA100259). Screeningg of the human genomic GenBank database of the NCBI with the AA1002599 identified the corresponding genomic clone (AL031010) on chromosome 6.. Genefinder programs were applied to genomic sequences upstream from the SAGEE tag. mapping putative exons and predicted coding sequences were validated byy reverse transcription polymerase chain reaction (RTPCR) combined with 5 -rapidd amplification of complementary DNA ends (5'-RACE) on human thyroid complementaryy DNA.
Thee DEHAL 1 gene contains at least 6 exons and 5 introns and spans over 35 kb on humann chromosome 6p24 (Fig. 'a.;. The thyroidal mRNA transcripts contains a relativelyy small 5" untranslated region (UTR). open reading frames of 867 (ORFA) andd 879 (ORFB) basepairs due to alternative splicing of one unpredicted exon and aa relatively large 3" UTR of over 6 kb. The splice variants encode 2 protein isoforms off 288 and 292 amino acids respectively (Fig. 1b. c).
Northernn analysis shows a 7.3 kb mRNA transcript in thyroid, liver, kidney and mammaryy gland (Fig. 2a. 2c). In thyroid. DEHAL1 ORFA is expressed much higher comparedd to ORFB (Fig. 2b).
Fig.. 1 The DEHAL1 gene and transcripts, a, Genomic organization of the DEHAL1
genegene and transcripts A and B. generated by alternative splicing of exon 5. b, c
ComplementaryComplementary nucleotide and deduced ammo-acid sequences of human DEHAL1-ORFA (b)(b) and DEHAL1-ORFB (c) cDNAs. Amino-acid sequences are shown belovs the nucleotide
sequencessequences The signal anchor the ntt'nr&ductase domain and the SAGE tag for the gene are shadedshaded in nine. >~ed and green, respectively. Polyadenylation signal in the 3' untranslated domaindomain is underlined. Amino acids involved in binding of the 2 FMN molecules in the DEHAL1DEHAL1 homodimer are indicated with filled circles (FMN-1! and filled rhombs (FMN-2). 3-untranslateduntranslated regions of transcripts A <b; and B (c) are comprised between nucleotides 49.697 andand respectively 56.090 and 56.190 in the AL031010.1 genomic clone. (* p. 180)
Characterizationn of the human thyroid d e h a l o g e n a s e Exons s 85,433 3 1 1 ATG G
b b
33 4 5 6 TGAA TAG-"--"- — I — I — F
alternative e splicing g Genomicc clone AL031010 0 Genome e (Chr.. 6q24) DEHAL1.DEHAL1. A (ORF:: 867 Dp) DEHAL1.B DEHAL1.B (ORF:: 876 bp) tt » r s e- . .
..
-Chapterr 4 7.55 k b . 1.355 kb CC C E E o o w w "C C u u
> >
--TJ J U U
--Q. . C/J J 0 0
z z
E E _ J J---C C
-- h--c h--c X X
n n
"ra a~ ~
< <
5 5 c c t t 5 5 0 0 cc c— —
cc c o o X X--X --X r r
;.: :
_ _
ra ra L_ _ c c 2 2 CD D C C cz cz Ü_ _ D E H A L 1 1 G A P D H H 1 22 3 4 5 6 7 3966 bp 2200 bp Breast t Thyroid d D E H A L 1 1 A T P 6 6F i g .. 2 Expression of DEHAL1 mRNA. a, Northern blot analysis of DEHAL1 mRNA
expressionexpression in 15 human tissues. Upper panels were probed with DEHAL1 mRNA and the lowerlower with GAPDH mRNA. DEHAL1 signal in the trachea lane probably corresponds to thyroidthyroid RNA contamination during the isolation of anatomically close tissues, b, Relative expressionexpression of DEHAL1-ORFA and DEHAL1-ORFB splice variants by RT-PCR in thyroid tissue,tissue, c, Semiquantitative RT-PCR of DEHAL1 and ATP6 on 4 mammary gland cDNA samplessamples (lanes 1-4) and thyroid cDNA samples (lanes 5-8). DEHAL1 was amplified from exonsexons 2 to 4. common to ORFs A and B.
Ühsr
3Ctenz;itiünn the h j m a r th
yro'd clehaicq&rase
Proteinn sequence analysis reveals that DEHAL1 belongs to the Nitroreductase (NR) family.. Members of this family utilise FMN as a cofactor to reduce a broad range of nitroaromaticc compounds [5]. Comparative protein structure modelling and docking studiess suggest that the monomers of the homodimeric DEHAL1A enzyme adopt an (// + [I fold and bind one FMN cofactor per monomer at the dimer interface (Fig. 3a). Furthermore,, DEHAL1A residues that are predicted to interact with FMN correspondd to FMN-binding residues in known structures of homologues (Fig. 3c). Outsidee the NR domain, DEHAL1A shares little similarity to bacterial nitroreductases,, diverging in the amino-terminal region with an uncleaved localizationn signal (Fig. 3b). High similarity is maintained over the whole amino acid sequencee with its eukaryotic homologues (Fig. 3b).
Basedd on the reductive and FMN-dependent activity of the enzyme responsible for iodotyrosinee dehalogenation in the thyroid [12]. we tested the potential of DEHAL1 forr 3.5-di-iodotyrosine (DIT) and mono-iodotyrosine (MIT) dehalogenation in CHO cellss stably expressing ORFA. ORFB or the combination of both. CHO cells expressingg DEHAL1 ORFA show more than 30-fold increase of DIT deiodination overr background (Fig. 4a) Since no MIT is detectable during this assay, this stronglyy suggests a rapid conversion from MIT to iodide. The enzyme activity is completelyy inhibited by the specific competitor 3-nitro-tyrosine [13] (Fig. 4a).
DEHAL1DEHAL1 ORFB. lacking 64 amino acids of the nitroreductase consensus domain at
thee carboxy-termmal tail, is not able to dehalogenate DIT and also does not influencee the activity of ORFA (Fig. 4a). Dehalogenation rates of the enzyme display saturationn kinetics (Fig. 4b). The apparent Km for DIT is 37 pM (Vmax: 0.04 uM/ min.),, in the same order of magnitude as described for bovine thyroid homogenate [12].. Activity is strongly dependent on FMN (Fig. 4c).
Immunocytochemistryy of CHO cells transiently expressing DEHAL1 ORFA carboxy-terminallyy tagged with a myc epitope showed cytosolic staining, fitting with assays donee on thyroid homogenates [12] (Fig, 5).
Halogenss (chlonne; bromine, iodine) are elements with important functions in
physiology.. In dehalogenating bacteria, chlorinated compounds are used as the solee source of carbon and energy [14] or as electron acceptors in the process of halorespirationn [15]. In vertebrates, iodine is an essentia! component of thyroid hormonee required for the correct development and maturation of the brain [16,17]. Iodinee is a scarce element in the terrestrial environment. In 1990. 29% of the world populationn was reported to suffer from endemic iodine deficiency [18]. World-wide
Chapterr 4
544 million people are affected by iodine deficiency and suffer some form of mental impairmentt as a consequence [19].
Inn order to guarantee iodine availability for hormone synthesis, two highly specializedd systems have evolved in the thyroid gland. The plasma membrane sodium-iodidee symporter. that accumulates ingested circulating iodine in the gland byy active transport, has already been elucidated at the molecular level [20], The other,, that recycles iodide from the mam lodinated side products of thyroid hormone synthesis,, was described as enzyme activity decades ago. but its molecular nature hass been sofar elusive [21].
Thee characterization of DEHAL1 will be invaluable for the analysis of the molecular basiss of iodotyrosine dehalogenase deficiency [22-24], and to investigate the geneticc determinants of iodine deficiency disorders [25], including the impact of alternativee splicing defects on the disease [26], The expression of DEHAL1 in liver, kidneyy and mammary giand suggests a role in solubilization of (toxic) halogenatcd compoundss into urine and bile, or the protection of breast-fed babies against iodine deficiencyy during lactation. Medical implications of DEHAL1 activity on pharmacokineticss of widely used anti-cancer agents [27.28], and applications in biotechnologyy of pro-drug conversion [29] or in environmental bioremediation [30.31]] are open for research
Fig.. 3 DEHAL1 belongs to nitroreductase (NR) family, a Model of the NR domains of
thethe homodimonc DEHAL1A enzyme mth monomers coloured in blue and green and the 2 FMNFMN cofactor molecules in yellow. Comparative protein modelling was done with the MODELLERMODELLER [6] programme using the templates 1VFR (Vibrio fischen). 1ICR (Ec P38489). andand 1F5V (Ec P17117). Cofactor FMN was flexibly docked into the active site of the model
withwith AUTODOCK 3.0 [7] Image generated with MOLMOL [8]. b. Unrooted tree and NR domaindomain arrangement of DEHAL1A and homologues. The NR domain definitions as in c. Tree waswas buut with the MEGA programme [9J using the neighbour-joining method. Species are: Hs.Hs. Homo sapiens: Mm. Mus musculus: Dm. Drosophila melanogaster: Sc. Strcptomyces coelicolor:coelicolor: Pf. Pyrococcus funosus: Tt. Thermus thermophilus: Ec. Escherichia coli. Signal anchorsanchors were predicted using SignaiP ri 0] . c Pioteiu sequence alignment of the NR domain ofof DEHAL1A and homologues us:ng the structural alignment information of Tt Q60049 (1NOX).(1NOX). Ec P38489 (1ICR) and Ec P17117 (1F5V). Multiple structure alignment was done withwith StAMP [11] . Residues in blue and green boxes interact vjith one FMN1 and FMN2 moleculesmolecules respectively (> p. 181)
Characterizationn of the human thyroid dehalogenase HsHs DEHAL1A 289 aa H s D E H A U BB 292 aa MmMm NP_081667 285 aa —— Dm AAM11009 287 aa DmDm NP_64843 757 aa ScSc NP_629466 226 aa PfNPPfNP 579385 187 aa TtTt Q60049 205 aa £cP384899 217 aa £FcP171177 240 aa Signall a n c h o r N i t r o r e d u c t a s ee dc HsHs D E H A L 1 A H ss D E H A L 1 B MmMm N P _ 0 8 1 6 6 7 DmDm A A M 1 1 0 0 9 DmDm NP 6 4 8 4 3 ScSc N P 6 2 9 4 6 6 PfPf N P _ 5 7 9 3 8 5 TtTt Q 6 0 0 4 9 EcEc P 3 8 4 8 9 EcEc P 1 7 1 1 7 -- S N E O V P M E V EE F V E I I N K 0 R 0 V jjj E F Y E L L N K R R S V R f II S N E Q V P M E V E F Y E L L N K R R S V R F I - S S E H V P M E V V R L Y E L M R G R R S I R S F S S H P K P D L S V V R L Y E L M R G R R S I R S F N S H P K P D L S V V DD N V I R T A G T i A l j y H A H]T E P|WT F V V V D N VV t R T A G T A P S G A H T E P W T F V V V E N VV I K A A G T A P S G A H T E P W T F V V V E D C I R A A G T A P S G A H T E P W T Y C V V V E D C I R A A G T A P S G A H T E P W T Y C V V V D F V S F V D R R R S V R F F - S D E P V P R E C L D L A V A A A N T A P S G A H F Q P W K F A V I I R V L E L A K R R K T V R K FF - S K D R P P I E L V F K A I E T A K E A P S G M N A Q P W K F V V I DD A K T A A I. K E R H I fij R Y R K D P V P E G L L R E I L E A A L R A P S A W N I Q P W R I V V V DD I I S V A L K R H M T y A r D A S K K L T P E Q A E Q I K T L L Q Y S P I P T I E L I C G G 1 H F - T D E P I S E A Q R E A II I N S A R A T ! ISS Q PJWH F I V A ! SS S Q L O C ' S S I I R I KK D P KK D P K DD P QQ E P Q E P P GG 0 A DD N P RR D P ss T'E T D K K H ss D E H A L 1 A H ss D E H A L 1 B MmMm N P _ 0 8 1 6 6 7 DmDm A A M 1 1 0 0 9 DmDm N P _ 6 4 8 4 3 ScSc N P _ 6 2 9 4 6 6 PfPf N P _ 5 7 9 3 8 5 TtTt Q 6 0 0 4 9 EcEc P 3 8 4 8 9 EcEc P 1 7 1 1 7 H ss D E H A L 1 A H ss D E H A L 1 B MmMm N P _ 0 8 1 6 6 7 DmDm A A M 11009 DmDm N P 6 4 8 4 3 ScSc N P _ 6 2 9 4 6 6 PfPf N P _ 5 7 9 3 8 5 TtTt Q 6 0 0 4 9 EcEc P 3 8 4 8 9 E cc P 1 7 1 1 7 II E E E E E I N Y M K R I M G • H R W V T D L K K L R T N W II E E E E E I N Y I KK E Y L D TIA P I L I L I F DD V K H K ! R K D V K H K I R K I I E E E E E I N Y M K R M G - H R W V T D L K K L R T N W I K E Y L D " T A P I L I L I F F D M K H KK I R E I I E E E E E I N Y M K R M G - K R W V T D L K K L R T N W I K E Y L D T A P V L I L I F E L K R SS I R E I V E O E E L V N Y S O R M H - P Q W V T D L R P L Q T N H V K E Y L T E A P Y L I L I F E L K R SS I R E I V E Q E E L V N Y S Q R M H - P Q W V T D L R P L Q T N H V K E Y L T E A P Y L I L I F A T K H RR I R V A A E E E E R V N Y E G G R I P P E W R A A L A R L E T D S D K G F L D V V P W I V V C F E L KK A R I R E V C E V E E E K F Y E R I K G - - E L K E W L V E N E F T P E K P F L T E A P Y L V L V F AA T K R A L R E A|A - - - F G E | A [ Ï Ï V E E|A P;V V L V L Y E G K A R V A K S A A G N YY - - V F £ ] L R JJ] M L U A S H V V V F C AA L R E E L V T L[ T G G E J K ^ H V A Q A A E F W V F C A C G I L L A A L Q N A G G ss v s S V SS I A C G I L L A A L Q V N N S V SS I A C G L L L A A L Q N A G S T S I A A G I L L C A L Q A A G G S T S I A A G II L L C A L Q A A G KQ|VV H G F A A N G K | K K V H Y Y N E K Q V H G F AA A N G K K K V H Y Y N E K Q V H G F AA A N G K K K V H Y Y N E K Q T Y G L SS E N G K R M R R H Y Y N E K Q T Y G L SS E N G K R M R R H Y Y N E A E K S T A LL P D G S L R K N Y Y V N E S V G I A C G L F I T A L H A M G GG Y - T K A P Y W L Q S V W I A V G Y F L L A L E E V E A|D|LL E P A L A J H l [24] A A M G Q E A R K A W A S G Q S Y I L L G Y L L L L L E A Y G AA K T A M D D V VV L [32] H R K D L H O / D A E W M A K Q V A DD FIN R H L Q IJC P D A Q L G L A E Q L L L G V L V T V V G I T M R R LL V T V T LL A S L V LL A S L V LL S T L T LL G T V T LL G S V PP L -HH Q T A R TT T P L TT T P L TT T P L -H I PP • • YY T P -HsHs D E H A L 1 A H ss D E H A L 1 B MmMm N P _ 0 8 1 6 6 7 DmDm A A M 1 1 0 0 9 DmDm N P _ 6 4 8 4 3 ScSc N P 6 2 9 4 6 6 PfPf N P _ 5 7 9 3 8 5 TtTt Q 6 0 0 4 9 EcEc P 3 8 4 8 9 EcEc P17117 N C G J PP R L R V L L | G R P - A H[E K L t E | L L P V G Y P S K E • A T V P O L K R 0 P L J D Q I M V T V H R H L I E G P G R S S E A C S K L S S Q G R P E C R S G D C H Y H S S Q L W P S T E G A P G P P R T T N C G P R L R V L L G R P - S H E K L L V L L P V G Y P S R D A T V P D L K R K A L D Q I M V T V V N C G P A L R N L L G R PP - V N E K L L N C G P A L R N L L G R PP - V N E K L L N P M A FF L T R I C A R P • G N E R P Y L L P V G Y P K D G G L L P V G Y P K D G G L F P V G Y A A P D D P N T R PP I E E L L Q V P - R E W R L Q V I L P V G Y P D D P F D P E R V R A I L G L P - S R A A I P A L V A L G Y P A E --C T V P D L A R K N L S NN I M V T F C T V P D L A R K N L S NN I M V T F C E V P D L A R K P L A E AA I T E P - - K P K L K R K S I E EE V V^S F N -- • E G Y P S H B L P I L E R V V L W FF D A A I L D A E F G L K E K G Y T S L V V V P V G H H S V E D F N A l T L P I N N T I E A V T K L L K L P - O H V L P L F G L C L G W P A D -- • - • N P D l. I S Q LL P Q N l T L T E P Q LL P A S I L V H E S e c o n d a r yy s t r u c t u r e 3-helix x FMN1 1
Chapterr 4 1000 0 CC (/) oo — -- Q> njj o ^ O O
aa
0)) Lnx
T33 ~-T33 5 K K . : ; .. j 2'-:: 2'-::ORFA A ORFB B ORFA+B B MOCK K
re re cc c oo •= aii E oo ^
II I
1)) 3 . TJJ —' H H Q Q QQ o • • 00 u. 0 . 0 1 --jj :;fi.•• /
y '' 1000 750 0 > > —— 500 250 0 0 0 S* S* t t 00 0.2 0.4 0.6 0.8 1 1/[S] ] 11 1• •
—— T_ D I T ( u M ) ) Z~Z~ 15 C C O O oo HI > > c c o o o o F M NN a d d e d (\iM)O r r a c t e rr zaticn of t i e nu'^-ar thyroid dehaiogenase
Methods s
GeneGene expression studies.
NorthernNorthern blots. Human multiple tissue northern blot I and III membranes (ClonTech)
weree incubated at 68 C in hybridization solution for 1 hour and hybridized
(68 C, 4 h) with a radiolabelled DEHAL1 exons 2 to 4 probe, amplified from thyroid cDNAA and labelled by random priming with dATP [32P] (10 uCi) using the Klenow fragmentt of the Taq polymerase. Blots were washed following the instructions of manufacturers,, exposed for 24 h in an Imager system and visualized using the AIDAA software (Raytest). Semiquantitavive RT-PCR. DEHAL1 and ATP6 genes weree amplified at 28 and 22 cycles respectively, both within the exponential phase off amplification in 8 cDNA samples from thyroid and mammary gland.
Transfecttons. Transfecttons.
DEHAL1DEHAL1 open reading frames A and B were PCR-amplified from thyroid cDNA,
purified.. TA-cloned in pGEM-T-Easy (Promega) and shuttled into the pCDNA3 expressionn vector (Invitrogen). Chinese hamster ovary (CHO) cells were stably transfectedd with 3 ug DNA p C D N A - D E H ^ M - O R F A . pCDNA-DEH/\M-ORFB. both plasmidss or empty pCDNA3 vector as a control, using Fugene 6 (Boehringer Mannheim).. Cells maintained in DMEM containing 10% fetal calf serum were split afterr 48 hours and selected with 500 (jg/ml geneticin (Roche Molecular Biochemicals).. Cells surviving in selection media for 1 month were tested for foreign DNAA integration by RT-PCR and further used for functional assays.
Fig.. 4 Kinetics of di-iodotyrosine dehalogenation by DEHAL1. a. Dehalogenation
activityactivity of DEHAL1 protein isoforms. in a typical experiment, iodide formation is measured in thethe medium of CHO cells transfected with DEHAL1-A (ORF A). DEHAL1-B (ORF B) piasmids.piasmids. both (ORF A+B). and empty pCDNA3 vector (MOCK) (see Methods). Activity was assayedassayed in the absence (grey bars) and presence (white bars) of the specific competitor 3-nitro-L-tyrosinenitro-L-tyrosine (10 uM). b. kinetics of DIT dehalogenation. Velocities of DEHAL1 activity werewere determined for various DIT concentrations (0.1-100 uM) at 2 hours assay. lnset:doubie
reciprocalreciprocal plot. c. Dependency of DEHAL1A activity to FMN. FMN was added to the culture mediummedium in a range of concentrations (0-4mM) and the DEHAL1 activity assayed for 2 h.Activityh.Activity was calculated as reduction of area under the curve of the DIT peak at chromatographychromatography (% DIT conversion).
Chapterr 4
Fig.. 5 Subcellular localization of the recombinant DEHAL1 A-myc/His protein in two
endocrineendocrine cell lines. Both in Chinese hamster ovary (CHO) cells (a) and in human thyroid
cellscells (H-TORI 3) (b). the DEHAL1A protein shows cytosolic location, in contrast with the
C^arac'enzaticrr c' the h u " i a r tnyroic denalogenase
lodotyrosinelodotyrosine dehalogenation assay.
Cellss were plated on a 162 cm2 culture flask. At 80% confluency. medium was removedd and cells were washed with phosphate buffered saline. Ten uM di-iodotyrosinee (DIT) containing tracer amounts of 125iodine-labeled DIT (Amersham) andd 4 mM FMN were added in DMEM to cells. The inhibitory effect of 3-nitro-L-tyrosinee on the enzyme activity was tested by the addition of equimolar amounts of DITT and the putative inhibitor (10 uM) to the culture medium. Medium was harvestedd after 2 hours incubations at 37 C.
Iodide,, mono- and di-iodotyrosine in medium samples (pH 4.8) were separated usingg 30 cm glass columns (Biorad) filled with Sephadex G-25 fine (Pharmacia) bufferedd with 0.2 M ammonium acetate (pH 4.8). Samples were subjected to a flow ratee of 0.5 ml/min using a peristaltic pump ( P - 1 , Pharmacia Biotech) and collected in 0.55 ml fractions using a fraction collector (LBK-FRAC-100, Pharmacia) Iodide. MIT andd DIT were respectively purified in 50-60 ml. 75-85 ml and 85-115 ml eluation volumes.. Counts per minute in every 0.5 ml fraction were determined using a gammaa detector (y Auto-gamma 5000. Minaxi). Dehalogenation activity was defined ass per cent increase of area under the curve of the iodide peak over background.
EnzymeEnzyme kinetics.
Thee Michaelis Menten constant {Km) was calculated from a series of 10 assays with 0.11 to 100 uM DIT. Lineweaver-Burk curve was calculated from plotted dots using thee GraphPad Prism 3.0. FMN-dependency of enzyme activity was tested with 0 to 44 mM FMN.
SubcellularSubcellular localization.
DEHAL1ADEHAL1A cDNA was subcloned in the pCDNA3.1 expression vector, attaching a
mycc epitope carboxyterminally. The construct was transfected to both human thyroidall HTORI3 and the Chinese hamster ovary (CHO) cell lines and recombinant proteinss were detected by a mouse anti-myc antibody and goat FITC-conjugated secondd antibody. Fluorescent signal was visualized by confocal microscopy, using a laserr microscope (Coherent Enterprise II DM IRBE, Leica) and the Leica Confocal.2 software. .
Acknowledgements s
Wee thank Dr. L.J. van 't Veer (The Netherlands Cancer Institute) for supplying mammaryy gland cDNA samples. J . C M . was supported partially by an ESPE
Chapte-- 4
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