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Thyroidal iodide transport
58 It E. l~ R . ta F . l? y . t* M . t, R . 20 R . ,* j. ~ A. ~s C. •4 D. • s A. ~S E .
T. W I N N I C K ,
R. E. WI~NS(IK,
r4v_.EH.~AN,N
~ I . G A L , 1 ). A . D R E W E S AND ,_N. F . T A Y L O R , HrC$~.,~[ttgdd,a ~ . H i o ~ I i 3 , s , , 93 (1961) I . j. "~VILLIAM,% R . E . E A K I N AND E . E . S N ~ t - t , J_2~m.cU/v~'nz.:S~c..0z: ( I 9 4 o ) 1 2 o 4 . L Y N E N , E . R E I C H E R T AND L . R U E F F , A n n . : C l , e m . 1 2 ~ 5 . ~ - - - " ~ . (I~-x) I. A v I - D o R AND J . ~ % G E R , Biockem. J., 6 3 (tgYS)'f~33L " B . C H E N O W E T H , ] . Pharmacol. Exp'.l. T t w r a p . , * O T ~ g r ~ ) ~ R . F R A Z E R AND F . L . M . PATTISON, Nature, : r 7 6 ~xrqiSiS)c~mE . ~,VINNICK A N D T . "~VINNICK, B u l l . . q o c . Chim.LI:ii#l.,44~(~r~h-~B trT"._ G R A F F A N D H . D . H O B E R M A N , J . B i o l . C h e m . . ~1-8~ g l t A ~ ) 3 i ~ , I ~ H L AND P . FRtTZSON, ] . Biol. Chem., z ~ 3 ~ ) 3 3 b ~ . F. B A X T E R A N D E . R O B E R T S , J . B i o l . Chem.,__oz3fi ¢I~ft~)2~L~l,. B . MCCORMICK AND E . E . SNELL, .~. Biol. Cke, m.,---~6(~t*!-.xo83, F . D E B O N S AND J . A. PITTMAN, Federation t a r o c . , _ ~ I ( ~ ! X ~ M. G A L AND R . A. P E T E R S , Biochem. J . , 64 (:x~56) .*mr.
BBA
THYRO!DAL
I OD~
IV. T H E
3849
WIi~~ORT
ROLE 'OF ~(f~ ~Iv~ ~
J. WOLFF
AND J . ~R. ~ ' ~ d ~ t l ' [ I ~
N a t i o n a l Institute of Arthritis and Metabolic L O ~ . _ ~ i ~ t o ~ . Bethesda, M d . (U.:S:t411 (Received
I n s t i t u t e s o f Health,
J u l y , 6 d ' h , :--~.e_~
SI,]'ffM~
x. T h e a b i l i t y of v a r i o u s a n i o n s t o ~ e a c t ",v~-Ithtti~. i o d i d e - t r a n s p o r t s y s t e m of s h e e p - t h y r o i d slices h a s b e e n i n v e s t i g a t e d . ~ a ~ a m a f l / ~ l ~ r a n g e d from 3-5" Io-7 M t o 2 . 1 o -= M to give a series of i n c r e a s i n g FK ~xaihm~,: "ITQ(!)~- ~< CIO~- <: R e 0 4 - < B F a- < S e C N - -~ S O , F - < S C N - < I - < ~gIO~.- << ~ <: O C N - ~-~ B r - . 2. T h e s e w e r e c o m p a r e d w i t h t h e partiglrmdM]iimiirx~olixmes a t infinite d i l u t i o n , Oo, w h i c h s h o w s t h a t t h i s series also :f0Hnws aa dimmasihg= o r d e r of ~ 0 (with t h e e x c e p t i o n s of R e O 4 - , S O a F - a n d SeCN-)..-'¢ Llixemarr~h~n~ exists b e t w e e n t h e p K v a l u e s a n d t h e p a r t i a l m o l a l ionic volumes, c~xazr0itmmaag~off~-46 ml/mole. A l t h o u g h t h e p K ' s decline w i t h l a r g e r volumes, a d e a r * a m mmeiimma~ w a s n o t observed. N o similar c o r r e l a t i o n exists b e t w e e n t h e p K : m l f l , ~ r m i i m ~ t ~ x - a m e t e r s . 3 - ' A l l of t h e a n i o n s (except T c 0 4 - . =~Riidh ~va~ nott tested) were s h o w n to be c o m p e t i t i v e i n h i b i t o r s of iodide t r a n s p o r t ! h y < f i n t i t i h e ~ e M i p l o t analysis. 4- T h e i m p o r t a n c e of size, u n i v a t e n c y : m a f l ~ f f r t r a n i 0 n ~ t r a n s p o r t in t h y r o i d t i s s u e a r e briefly discussed in r e l a t i o n t o cem~ha ~ l ~ p e r t i e s of t h e ions,
IN~FR OlIUCr~]~A"
T h e role of ionic size in anion transpox't ihy ~ t~iiimm~!i~s been suspected ever since it w a s found that the Br- ion is £one~tku~mLq tn~l~sser extent• than iodideI. I t w a s s u b s e q u e n t l y f o u n d t h a t t h e halide, ~ x ~ : , , ~ w~flE c o n c e n t r a t e d b y th3Toid g l a n d s ~, w h e r e a s t h e s m a l l e r ions Cl- a n d .=~m--.~auremm~.]Em,a o ~ l e x a n i o n s of periodic ~B~tiim~ Hiop~~,~. .4cta, 69 (z963) 48-58
THYROIDAL IODIDE TRANSPORT
59
g r o u p V I I A , TcO 4- a n d ReO4-, are also c o n c e n t r a t e d b y t h y r o i d tissuea, 4. Certain o t h e r complex anions h a d been found to i n h i b i t iodide c o n c e n t r a t i o n b y t h y r o i d tissue e.g. SCN-, CIO4-, N O a - (see ref. 5)- I t was p o s t u l a t e d t h a t this i n h i b i t o r y p o t e n t i a l was, in some m a n n e r , a function of t h e position of these ions in t h e Hofmeister or lyotropic series. More r e c e n t l y ANBAR el al. 6 suggested t h a t t h e i n h i b i t o r y properties of t h e anions were related to their size. T h e y showed t h a t c e r t a i n complex anions, B F 4 - , S O a F - a n d PO~Fa-, chosen for t h e i r size, could i n h i b i t i o d i d e t r a n s p o r t . F u r t h e r m o r e , CIO 4a n d B F 4- were shown to be c o n c e n t r a t e d b y t h y r o i d tissueL I o n sizes were calculated according to H0CKEL a, a p p a r e n t l y as multiples of t h e oxygen or fluorine v o l u m e s at the corners of t h e b o d y - c e n t e r e d t e t r a h e d r a . Such size e s t i m a t e s were not, however, in a g r e e m e n t w i t h our finding 4 t h a t TcO 4- a n d ReO4-, are more a v i d l y c o n c e n t r a t e d b y t h y r o i d tissue t h a n I - , despite t h e fact t h a t t h e i r v o l u m e s would be of approxim a t e l y t h e same size (4" IO-e~ cmS) in the above calculations. I n order to decide w h a t sort of size p a r a m e t e r would best describe t h e size r e q u i r e m e n t of t h e a n i o n - t r a n s p o r t i n g s y s t e m of t h e t h y r o i d , it is n e c e s s a r y to h a v e some q u a n t i t a t i v e m e a s u r e of t h e a n i o n effects on t h y r o i d tissue. We h a v e , therefore, m e a s u r e d " c o n s t a n t s " of t h e Michaelis t y p e , Km or Kt, t h a t describe h a l f - s a t u r a t i o n of t h e t r a n s p o r t s y s t e m in t h e s t e a d y state, for a n u m b e r of u n i v a l e n t anions. These h a v e been c o m p a r e d w i t h c e r t a i n size p a r a m e t e r s t a k e n from t h e l i t e r a t u r e or det e r m i n e d b y us. By far t h e best correlation b e t w e e n K values a n d size exists w i t h p a r t i a l molat ionic volumes. A p r e l i m i n a r y report of t h e s e findings h a s a p p e a r e d ~. MATERIALS AND METHODS A n a l y t i c a l r e a g e n t s were used unless otherwise indicated. KSeCN (Bios) was purified b y acetone e x t r a c t i o n a t room t e m p e r a t u r e a n d crystallization from acetone. H o t acetone a n d e v a p o r a t i o n u n d e r v a c u u m led to decomposition to free selenium in our hands. N H 4 B F 4 was m a d e according to BOOTH AND REHMAR1°. Crude N H 4 S O a F was o b t a i n e d from t h e Chemicals P r o c u r e m e n t L a b o r a t o r i e s , N.Y. I t w a s purified b y m e t h a n o l e x t r a c t i o n a n d crystallization from e t h a n o l to yield a p r o d u c t w i t h a melting point in a g r e e m e n t w i t h t h e l i t e r a t u r e (245°). E l e m e n t a l analysis of t h e a m m o n i u m salt yielded: found: N, I 1 . 8 8 % " F, 16.36°,/o; S, 0_7.o30/0 • theoretical" N, 11.96 %; F, 16.22 %; S, 27.38 %. R a d i o a c t i v e iodate was m a d e by oxidation of 25-3o/~C of m l I - in t h e absence or presence of carrier (15 t~g KI) in H~O w i t h C12 gas. This m a t e r i a l was t h e n c h r o m a tograpl-,edl-. :3 on "~Vixatman No. 3MM p a p e r b y t h e a s c e n d i n g technique. T h e position of the b a n d was identified b y brief e x p o s u r e to X - r a y film. T h e a p p r o p r i a t e s t r i p was cut out, e l u t e d w i t h H~O, a n d c o n c e n t r a t e d . T h e yield was essentially q u a n t i t ative. Control strips with stable iodate were s t a i n e d w i t h d i p h e n y l a m i n e - H n P O 4 (see refs. 11-13). P a r t i a l molal ionic volumes were t a k e n from t h e l i t e r a t u r e t4' or, w h e n n o t available, t h e y were derived from t h e densities of t h e i r solutions d e t e r m i n e d in R i c h a r d s o n p y c n o m e t e r s a t ~5.o °. Volumes at infinite dilution were d e t e r m i n e d g r a p h i c a l l y from a m i n i n m m of five points per curve according to t h e empirical e q u a t i o n of MASSON15: = ,too + sv/ Biochirn, Biophy~. Acta, 69 (I963) 58-67
6~O
J. W O L F F ,
J. R. MAURE'Y"
~~s~t~a,pparent molal v o l u m e ; ff~ois t h e molal v o l u m e e x t r a p o l a t e d to infinite , ~ ~ i ~ t t ~ slope; a n d c is t h e c o n c e n t r a t i o n . ~v~mTtes were s u b t r a c t e d according to t h e c o n v e n t i o n of FAJA~S A,'CD j j I m ~ e ~ . . E o t a s s h x m iodide, NaSCN a n d NH4SCN were used to check t h e m e t h o d a~giim~x~iim~itx t h e literature. Iodide ion gave a value of 36.8 ml/rnole (literature 3~677~)aaKl S C N - ion was 4I.o ml/mole as c o m p a r e d to 4o.6 ml/mole t*. A ~i~ktd~sdi~'oxt of t h e s e d a t a will be r e p o r t e d elsewhere t~. / ~ m ~ m d : ~ v a l u e s were d e t e r m i n e d in sheep t h y r o i d slices b y m e t h o d s previously .,t t c m c i t t t ~ , ' m r T h e v o l u m e of m e d i u m w a s increased to 5 ml to r e d u c e c o n c e n t r a t i o n ,:~sii~tli~medium a t t h e s t e a d y state. E q u i l i b r a t i o n t i m e for iodide in t h e anioni l e ~ r d i ~ z ~ t t ~ w a s c h e c k e d w i t h S C N - a n d CIO4- a n d w a s found to be well w i t h i n t h e ~3o-nmmr~i~iim~bation period. Kt v a l u e s were d e t e r m i n e d at t h r e e levels of iodide a a m t : V i ~ f i tfliii~or. All p o i n t s were in d u p l i c a t e so t h a t a single Kt value was o b t a i n e d ~-.N3. ~ 3o flasks on t h e same tissue. Generally, this procedure was followed ,mnf~i~7~thyroid g l a n d s for e a c h compound. I t was a s s u m e d t h a t t h e s y s t e m ~x, e s a ~ s i m p l e s a t u r a t a b l e s y s t e m xg. A l t h o u g h this m a y be too simple ,~ view 2°, r t ~ [ ~ K ~ l m ~ s ~ ,obtained are useful for c o m p a r a t i v e purposes. No corrections were ,q~m~~e~cr~e~|ar (inulin) space as t h e g r a d i e n t s ( T I M I X - ] ) were usually quite !i~ge,~A~of'o.75 w a s s u b t r a c t e d from t h e T I M [ X - ] for t h e anion t h a t e n t e r e d b y '~'nT:/i:~ v a l u e s were o b t a i n e d from plots of t h e reciprocal of t h e s t e a d y s t a t e ~.~ttfitece~m~on in t h e slice vs. t h e i n h i b i t o r c o n c e n t r a t i o n . T h e Km values were , ~ m i i m e E ~ D l f i c a l l y from double reciprocal plots as p r e v i o u s l y described~, ~. T h e ~ ~ z e ~ . fmmt applied to t h y r o i d a l t r a n s p o r t problems b y WOLLMA~"~-x. YIx~clteel~: for.,, protein b i n d i n g of a n i o n s in t h e m e d i u m , slices were i n c u b a t e d ~~~tmrtiealiconditions b u t w i t h a tissue c o n c e n t r a t i o n t h i r t y - f o l d t h a t usually , . ~ ~ e k t _ ~ ( ~ , ~ s l i e e s / x o ml m e d i u m ) . After 9o rain t h e m e d i u m w a s c e n t r i f u g e d to rem~eece~ T i m c a l c u l a t e d protein c o n c e n t r a t i o n (on t h e basis of thyroglobulin) ~msI~.~~ T h i s solution was dialyzed a g a i n s t e i t h e r 3" IO-~ M I - or 4" IO-~ M ~--(i0mlttm~rsolution ) a t 4 ~ w i t h i n t e r n a l a n d e x t e r n a l stirring. Similar experi~ ~ w z z ~ c c ~ _ v r i _ ' e d out w i t h a 5 % solution of " p u r e " beef thyroglobulin (95% I9-S fft~iomnam~sF/o zs-S fraction). W i t h t h e m e m b r a n e s used, > 9 9 % equilibration h a d
RESULTS AND DISCUSSION
Z l t ~ a ~ . i u t x ~ t h e c u r v e s o b t a i n e d b y p l o t t i n g the reciprocal of t h e iodide ion concenL~__~~t~m~n t h e slices w h e n t h e s t e a d y s t a t e is a t t a i n e d a g a i n s t t h e concentration ~tI~tmedlis s h o w n in Fig. x. Iodide values are c a l c u l a t e d from t h e concen~ofi'm~lP a&ded a n d t h e fraction of r a d i o a c t i v i t y r e m a i n i n g in solution w h e n ~]m_~occurred ( 9 o - I o o min). I t was a s s u m e d t h a t t h e ioa,adde c o n t r i b u t e d ~ ~ ~ s H c e was not i m p o r t a n t at t h e iodide c o n c e n t r a t i o n s used. P r e l i m i n a r y ~ t h e x~I- released into t h e m e d i u m ~2 suggest t h i s to be t h e case. ~ ~ m t h i of t h e e x p e r i m e n t s t h e d a t a o b t a i n e d did n o t yield lines s a t i s f a c t o r y f ~ m r ~ i ~ m m ~ i .l~0~ analysis. T h e d e v i a t i o n s of t h e points did n o t show a n y consistent _qm0ztt~~er;, a n d t h e reason for t h i s is, at present, u n e x p l a i n e d . Such d a t a were rmmmm~. iha~-l~i axe listed t h e Km values for I - , TcO 4- a n d ReO4-, t a k e n from our Biochim. Biophys. Acta, 69 (x963) 5 8 - 6 7
THYROIDAL IODIDE TRANSPORT
v~2[
previous work4, ~v, as well as /Q values for iodide t r a n s p o r t of t h e sev~al~umitm.c h o s e n for this s t u d y because of t h e i r size. I t is a p p a r e n t t h a t t h e r a n g e ,0frtl~h~s~, " c o n s t a n t s " e x t e n d s over n e a r l y five orders of m a g n i t u d e , t h e smaller anionsflxa~mz/: larger c o n s t a n t s t h a n m o s t of t h e large, t e t r a h e d r a l ions. Of interest is t h e - f i n ~ ~ a ~ for ReOa-, t h e Km value, using ~ R e was identical to a Kl v a l u e determinedu~gaias~ r. . . . . . . . . . . . . . . . . . . . . . . W"
.2~6"10'6M ["
t/ ~.01
;
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,~ ,.,..,.4,,e
~
6
~
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7.5"10"° M I -
-. "X
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4 0 4
pK
8 12 16 20 24
3
[SCN-1,IO-SM
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NOi- J ° ~ O ; 2
'
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--"
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i
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7.~6"10"MI" .05 L
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3
4
m~m.m.m~i !
'- , . _ ~ - - " ~ o ~ 4 6 e ~o ~2 [SeCN-~,IO'SM
5
8
1
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24
28
32 3: 40 czT]o, ml/MOLE
4~
48
:~52
F i g . 2. T h e r e l a t i o n b e t w e e n t h e p / ( F - , l ~ g / t 7 ~ , t ~ r Ki) f o r a n i o n t r a n s p o r t in s h e e p .*.hyroid~*iliems~',L, t h e p a r t i a l m o l a l i o n i c v o l u m e a t infztxite, ifliltitimm. ~ , o f t h e s e a n i o n s . I o d i d e a n d TcOa--.vialel~sAz~rt • d e r i v e d f r o m t h e K m v a l u e s , R e O 4 - .~rom>i~2ttlh/~, o r K l , a n d all o t h e r a n i o n p K valttes:~u-trtntitht. Kl values.
i
0 4
B ~2 182024
[ReOT~I,IO'~M [GIO~},IO-TM F i g . i. T h e e f f e c t of v a r i o u s a n i o n s o n t h e a c c u m u l a t i o n o f i n t r a t h y r o i d a l iodide.~ 'l-?l~tmttro,lbt t h e r e c i p r o c a l o f t h e c o r r e c t e d i o d i d e c o n c e n t r a t i o n in t h e slice in t h e s t e a d y s t a t e x ~ ] ~ - ~ ' x , - , the concentration of inhibiting anion. Curves for three different steady state eonc~,~tti~s~bl i o d i d e i n t h e m e d i u m a r e g i v e n . T h e i r i n t e r s e c t i o n d e t e r m i n e s t h e 1£1 v a l u e .
iodide ion (Table I). T h e s e values agree well w i t h t h e in vivo finetings:!~r-iduni_4s poorly c o n c e n t r a t e d ~, N O a - is a poor inhibitor, w h e r e a s C104- h a s b e e n 4 o m i d m ~ very p o t e n t 5. T h e y are in s a t i s f a c t o r y a g r e e m e n t also w i t h t h e c o n c e n t r a t i t m ~ for 50% inhibition of iodide a c c u m u l a t i o n in t h e choroid plexus o f : - ~ b h i ~ 3 : e ~ . , . ,. C104-, 2 " 1 o -e M; B F , -, , 5 . 1 o .6 M", oc ~~- - , - , 3 " i o -s M a n d NO3-, 3:zo~m~M;amatdifin m o u s e salivary glands: CIO4-, 2" I o -6 M; I - , 3" xo-5 M ; a n d SCN-, 3'-'~o*-~M(~(eee ref. 24). T h e S C N - values s h o u l d a l s o be c o m p a r e d w i t h a Kt of 6 . i o - 5 : , M , 0 ~ b y WOLLMAN in t h e t h y r o i d s of i n t a c t mice 2x. T h e slight d i s c r e p a n c y c a z z ~ ~ be explained, at least in p a r t , b y b i n d i n g of S C N - to s e r u m p r o t e i n s ~=-Jthdfimaaet animals. W h e n t h e Km or Kt values are p l o t t e d (as t h e pK's) vs. t h e ~ ~ h l ionic volumes, ~0, of t h e .anions, a h i g h l y significant correlation was o b t a i m ~ 1 0 F ~ ,-f_). T h e m a x i m u m p K value was n e a r 3" zo-T M w i t h TcO4-. Since technetitml~e:x~tsmt~. in t h e r a d i o a c t i v e form it was n o t p r a c t i c a l t o o b t a i n a p a r t i a l ~ m o l a l ' , ~ f d m T TcO4-, a n d we h a v e used t h e m e a n b e t w e e n t h e MnO4- a n d ReO 4- v o h m m s . ~ ~ Biochim. Biophys. Ac,~, 69 ~ 5 ) ~
v ka 0o
g
A
e~
a,
t~
i--,. Jo -3
Nitrate (NOa-)
' Km values in italics from WOLFF AND MAUREY 4.17. +° From FAJANS AND JOHNSON14. " ° ' Determined for this study. t Taken as the mean of M n O ( and R e O / ( s e e ref. t4).
3-5' Io-7
4" to-7
Perchlorate (004-)
Pertechnetate (TcO()
x.xo -e, z. lo -~
3' Io-~
Perrhenate ( R e O ( )
Tetrafluoroborate ( B F ( )
i. io -n
I-2' IO-5
Monofluorosulfonate (SOsF-)
Selenocyanate (SeCN-)
z- 3. I o - '
Thiocyanate (SCN-)
3" lo -s
4' m-3
Nitrite (NO2-)
Iodide (I-)
i-2. m -a
__
Cyanate (OCN-}
-. 2. m -z
Iodate ( I O f )
Transport h'i or Kra* (51)
Bromide (Br-)
?¢am~
I
44.5'* 46.0 t
48.7'*"
44.o'"
5o.3""
47.8'**
40.6 't
3O.7 ° '
29.4 ' *
25 * t , 2 0 °
20.7''*
25.I -)
~-5. i ' "
Parlial molal volu)~ ¢, at :5.0* (mtlmote)
Volumeso/
3.94 (4.05) §
4.1o
~4
~4
4.Z3
3,a3
(to-"cm*)
ASBAael al.'
I.o9
1.34
1.12
I.II
0.90
I.O3
1.O2
0.94
Relative hydcatcd ske K .... l.od ~
THE RELATION' OF h ' m AND K I TO ANION SIZE
TABLE
46.,1
46.9
36.6
29.3
a6,5
a5.O
(ml/mole)
AND LAIDLERza
Volumes oI CoI:TCaE
Tetrahedral
Tetrahedral
Tetrahedral
"l'etrahedral
Linear
Tetrahedral
Linear
Spherical
Planar
Angular
Linear
Pyramidal
Spherical
Str~IU?~)a~
o~
,~
~,
,tl .
.~ O
tO
TH~OID3A~ ~i~:
THA~SI~ORT
63
t h e r e is a decline in t h e p K ~ a s t t h e ~ i d d m g l i d ] ionic v o l u m e s e x c e e d 46 m l / m o l e (as s u g g e s t e d b y R e O 4 - , s O ~ F - z a n t t ~ - ) ) omxld3 n o t be a d e q u a t e l y t e s t e d for lack of a n i o n s of s u i t a b l e v o l u m e . ~ h e d ~ s ~ ~ t s w i t h S O s F - are u n e x p l a i n e d a t p r e s e n t . Similarly, a "minimum",~izeiis~fffmxltxto ,obtain b e c a u s e t h e c o n c e n t r a t i o n s r e q u i r e d are so large as t o demmaticti~bttimnofffadln*ge f r a c t i o n of C1- from t h e m e d i u m t o m a i n t a i n o s m o l a r i t y . Non-~pecqfic efffim~wcml~ t h u s b e likely t o occur. T a b l e I also lists various, rrther.~met]mmaaett~, r n a d d i t i o n t o t h e p a r t i a l m o l a l ionic volumes, w e h a v e listed, ~ o r c e ~ . t t i e - v o l u m e s g i v e n b y ANBAR el al. 6, r e l a t i v e h y d r a t e d sizes f r o m : c ~ n ~ ~ e n t s ~, a n d t h e " e f f e c t i v e " radii as c a l c u l a t e d b y COUTURE A-ND I L ~ m _ m t ~ . , ~ ~ the values calculated according t o H0CKEL n o r t h o s e o b t a i n e d f i r o m l l i i n ~ i x : m m m u r e m e n t s show a c o m p a r a b l e correl a t i o n w i t h t h e K m or K t v a l u e s . l ~ t i i s i i s i i n a o n t t a ~ t u a n i o n p e r m e a b i l i t y as e x h i b i t e d , for e x a m p l e , in c a t neurones~',vYhexetm0m~l~irna.wiflx k i n e t i c r a d i i is found. I t is d e a r , h o w e v e r , t h a t t h e e m p i r i c a l . e q u a t i m a d t ~ q r g e ~ t ] t i y C,,,UTUR~ Arid LAIDLER2e, w h i c h is b a s e d on t h e n u m b e r ofligantis~anHttize"0at/mtive'" r a d i u s (sum of t h e b o n d l e n g t h a n d V a n d e r W a a l s r a d i u s of, oxygen), ~yidittsv~lttmes t h a t show close c o r r e l a t i o n w i t h m o s t of t h e p a r t i a l m o l a l :ionic w d h m e ~ ~ e d i e x p e r i m e n t a l l y a n d h e n c e also w i t h t h e Km or/ISi values. I t s h o u l d be p o i n t e d o u t ~ h ~ t t h e q ~ m f l i m ~ w e r e m e a s u r e d a t 2,5 ° w h e r e a s K m or Kt v a l u e s were o b t a i n e d a t 37--.387. ?~stiitemaiim~volumes s h o w c o n s i d e r a b l e t e m p e r a t u r e d e p e n d e n c e n o t directly~rdla~lflttDtiteev-~li~__~of~ Oo x*, it m i g h t be s u p p o s e d t h a t t h e o r d e r of sizes differs a t 37 9.~ited~li~6CtS/7liviit~ttie o r d e r CIO4- :> N O a - :> I - > B r b u t a m o u n t s t o o n l y o . 5 - x . 5 _ m l p r n d i e f f m ~ i ~ t t m a g e z a t u r e difference, h e n c e t h e o r d e r of T a b l e i or Fig. 2 w o u l d n o t i b e i i r ~ m n n w B . T h e r e are s e v e r a l u n c e r t a i n l i ~ s i i n ~ t t i a t deal w i t h (a) possible d e c o m p o sition of t h e a n i o n s in o u r - s y ~ e m , amta (~k))t~ssibte b i n d i n g of ions b y p r o t e i n t h a t h a s l e a k e d o u t of t h e slices, kBoth,~fftflemew~ultEtend t o lower t h e c o n c e n t r a t i o n of a n i o n a n d h e n c e give falsely ihi~41/K~,aitm~. z. T h e possibility t h a t iE[F~--,~rr~]F-we~m,irthibitory on t h e b a s i s of h y d r o l y s i s to F - ,~n ~ , , , d n o t be e n t i x e l y ~ m m n t t e g d . X ~ x ~ t l i e r e f o r e m e a s u r e d t h e effect of F on iodide t r a n s p o r t in t h e sheeprlixymith~!iime.TI~ier~ ~ a s n o significant effect on iodide t r a n s p o r t w i t h m e d i u m F - t e v e t s ~ s t t i ~ h ~ s 3 ~ - m ~ , l ~ L T h u s t h e i n h i b i t o r y effects of t h e s e ions c a n n o t be aceourrteflttxrrklay _tO~"rr~rtm~al c o n t e n t of F - e v e n if c o m p l e t e h y d r o l y s i s occurred. I t h a s : b e e n a ~ [ p ~ t l i a ~ t t i e - l i r e a ! . l o w n of B~SF, - is negligible v. ,2. ~VvN~AARI)V,r~ e:" al:r'~tl~p~r~tltfla~ I I ~ - ; - w a s a n effective i n h i b i t o r of iodide a c c u m u l a t i o n a n d also s t i r n u l a t e I f l ~ o f f i 0 d i d e from the gland. We therefore t e s t e d t h e a c c u m u l a t i o n of ~ x a ~ I O ~ - - i _ i n ~ ~ d ~ s t i c e s . W h i l e c o n s i d e r a b l e radioa c t i v i t y d i d a c c u m u l a t e in .-the ~ g l i e ~ i x l g g a m n a t ~ a r e n t , t h o u g h s o m e w h a t s k e w e d , s a t u r a t i o n curve, it w a s f o u n t l t i t m t atll ~ m d i 0 a c t i v i t y in t h e t h y r o i d b e h a v e d c h r o m a t o g r a p h i c a l l y 1 1 - 1 B i l i k e i i o H _i_l_ ~ _ . ~ ~ n o , i o d a t e . I n t h e m e d i u m different p r o p o r t i o n s of t h e radioaefi-eity~meeiintlt~f~__~a_ ~oI~ i o d a t e or iodide, d e p e n d i n g on t h e i n i t i a l c o n c e n t r a t i o n of ~iottm~ ~ ] 5 . l~t ~ . ~ likely, therefore, t h a t i o d a t e is r e d u c e d (as also s h o w n b y ~ ~ . ~ a ~ - ~ , a n d l t h a t t h e a c c u m u l a t i o n of radioa c t i v i t y of ~-Oa-. a_n_d pro_~_~!yiitsiifl_tit!~ttr~. , ~ a s wall, r e s u l t from t h e iodide so formed. 3. C h l o r a t e a n d b r o m a t e ~,eer~iixtfitfittm~, h~rE~ t r a n s p o r t w i t h 5 ° % p o i n t s i n t h e r a n g e of zo-a-xo-~:'M. \ W e ~ v ~ m m t i t ~ t O , o b t a i n ~ r e p r o d u c i b l e results, a n d ~ h i g h B i e , h i m . Biophys. A cta. 69 (x963) 58-67
64
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levels caused o x i d a t i o n of t h e tissue p i g m e n t s . F r o m their o x i d a t i o n p o t e n t i a l s , w h i c h are higher t b a n those of IO 3- (seeref. 28) •*' ~ e v_ w o u l d not be e x p e c t e d t o persist" however, we were u n a b l e t o p r o v e t h a t t h e y were r e d u c e d because o u r c h r o m a t o graphic t e c h n i q u e s were n o t sensitive e n o u g h u-~a. T h e fact t h a t o t h e r s t r o n g oxidizing agents, such as Cr±O~9- or CrO~ ~-, were s t r o n g l y i n h i b i t o r y suggests t h a t t h e effect of CIO~- or B r O 3- m a y be non-specific. W e find it difficult, however, to e x p l a i n t h e inhibition in vivo r e p o r t e d for CIO 8- (see ref. 5)4. A c e r t a i n degree of u n c e r t a i n t y applies t o N O g - a n d O C N - . T h e former was s l i g h t l y less a c t i v e t h a n N O 3- b u t as t h e r e was obvious i n t e r a c t i o n w i t h t h e p i g m e n t of t h e slices, it m a y be t h a t t h e effective: c o n c e n t r a t i o n differed f r o m t h e s t a t e d one. S o d i u m c y a n a t e i n h i b i t e d 5 0 % a t 1 - 2 . zo -~ M, b u t t h e odor of H C N was easily d e t e c t e d in this s y s t e m , a n d no a c c u r a c y can be c l a i m e d for t h e s e d e t e r m i n a t i o n s .
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E .,,M-' F i g . 3- T h e c o m p e t i t i v e n a t u r e of a n i o n i n h i b i t i o n of i o d i d e accumL, l a t i o n i n s h e e p t h y r o i d slices. D o u b l e r e c i p r o c a l ~ ! o t s o f t h e ~v,, ~+~d~ s+~a ~ ' ' .,., ~ s t a t e i o d i d e c o n c e n t r a t i o n i n t h e slice I / r l l-~!e (ordi-• h a t e s ) vs. t h a t o f t h e m e d i u m t / r I0- ~e (abscissas). C o n t r o l i n d i c a t e s t h e a b s e n c e of i n h i b i t i n g a n i o n s .
5. I t is k n o w n t h a t a l b u m i n , a n d p r o b a b l y o t h e r proteins, can b i n d these anions. T h e o r d e r ot affirfities seems t o be similar t o t h e p r e s e n t series n. I t was t h e r e f o r e possible t h a t e n o u g h t h y r o g l o b u l i n or o t h e r p r o t e i n s l e a k e d o u t of t h e slices t o b i n d t h e a n i o n s a n d t h u s lower t h e c o n c e n t r a t i o n of free anions. A l t h o u g h t h e chloride c o n c e n t r a t i o n of t h e m e d i u m w o u l d t e n d t o d i m i n i s h t h i s effect, e x p e r i m e n t s were c a r r i e d o u t t o t e s t t h i s directly. T h e largest g r a d i e n t in f a v o r of t h e p r o t e i n c o m p a r t m e n t was z.o5 for iodide a n d z.z2 for TcO4-. Since t h e p r o t e i n c o n c e n t r a t i o n s were Biochim. Biophys. Acla, 69 ( t 9 6 3 ) .58-67
THYROIDAL IODIDE TRANSPORT
65
inle~cess (thirty-fold) of w h a t is usually found in t h e m e d i u m u n d e r our conditions, iS seems unlikely t h a t t h e c o n c e n t r a t i o n s of free anions was significantly lowered b y l~Votein binding. l:* seems probable from t h e evidence so far available t h a t u n i v a l e n c y is also a~vequirement of the system. Sulfate (16.4 ml/mole) a n d sulfite (9.5 ml/mole) c a u s e d no intiibition of iodide c o n c e n t r a t i o n at 2o mM. As these ions are of small p a r t i a l molal ii}nm volume, decision b e t w e e n v a l e n c y a n d size is not possible in this system. Howe~el% WO42- (26.3 ml/mole),. MoO42- (29.5 ml/mole) a n d S~O32- ( ~ 34 ml/mole), do falll w i t h i n t h e size r a n g e of t h e smaller i n h i b i t o r y u n i v a l e n t anions. None of these d i v a l e n t ions i n h i b i t e d iodide t r a n s p o r t at a c o n c e n t r a t i o n of 2o raM. [~: has been a s s u m e d b y m a n y a u t h o r s t h a t t h e ions s t u d i e d here c o m p e t e for t i e s a m e site on a h y p o t h e t i c a l carrier. I n general, no evidence h a s been p r e s e n t e d sazce t h a t (a) S C N - -goiter is p r e v e n t e d b y a n increased iodide i n t a k e ~ a n d (b) t h a t It-, T.'cO 4- a n d ReO4- are m u t u a l l y inhibitory, i.e. a n y one of t h e m will i n h i b i t t h e tmansport of a n y o t h e r i n t o t h e t h y r o i d a, 4. T h e only rigorous d e m o n s t r a t i o n for comp e t i t i v e inhibition prior t o this s t u d y is t h a t for S C N - ion i n vivoaL Similar criteria (no,inhibition, i.e. t h e s a m e m a x i m u m c a p a c i t y , at infinite " s u b s t r a t e " c o n c e n t r a t i o n ) lim~e been applied in t h e p r e s e n t i n vitro system. Fig. 3 shows t h a t in d o u b l e reciprocal lflbts t h e O C N - , NO3-, SCN-, B F 4 - , R e O 4- a n d C10 4- ions i n h i b i t iodide t r a n s p o r t in~ ~ compeCAtive m a n n e r . N o t depictcd, b u t also c o m p e t i t i v e i n h i b i t o r s b y t h i s criterion, are t h e B r - , NO2-, S O 3 F - a n d S C N - ions.
COMMENTS
qIhe concept of ionic size is an o p e r a t i o n a l one w h i c h depends on t h e c o n d i t i o n s u n d e r ~ h i c h it is m e a s u r e d 31. Since t h e conditions a t t h e site at which t h y r o i d tissue concenImams anions are u n k n o w n , we h a v e a t t e m p t e d to look a t several t y p e s of ion "sizes" t ~ find t h e best correlation w i t h a biological t r a n s p o r t p a r a m e t e r , in this case t h e ~ m or Kt. S a t i s f a c t o r y c o r r e s p o n d e n c e b e t w e e n t h e t r a n s p o r t p a r a m e t e r a n d size is o b t a i n e d only when t h e l a t t e r is expressed as t h e p a r t i a l molal volume, ~0, according t o e i t h e r MASSON 15 o r COUTURE AND L A I D L E R ~6, b u t not w i t h o t h e r expressions of anionic size. T h e a n i o n s s t u d i e d here fit a series of increasing K v a l u e s : TcO a- ~< C10~7 < R e O 4 - < B F , - < S e C N - ---~ S O 3 F - < S C N - < I - < NO a- < N O , - < OCN. ---~ B r - . Such a series will also describe a n u m b e r of chemical p r o p e r t i e s of t h e s e ions as far as t h e y h a v e been s t u d i e d : (a) t h e p a r t i a l molal ionic v o l u m e s at infinite dilution (except for S O a F - , ReO 4- a n d S e C N - as discussed a b o v e ) ; (b) t h e Etbfmeister series; (c) t h e h y d r a t i o n energies, i.e. t h e H o f m e i s t e r series follows t h e inverse order of t h e h y d r a t i o n energies a2. T h e s e can, in t u r n , be r e l a t e d t o t h e ionic vadii~; (d) t h e order of t h e selectivity coefficients of certain q u a r t e r n a r y a m m o n i u m i o n e x c h a n g e resins, w h i c h can also be correlated w i t h t h e p a r t i a l molal ionic volumes of~ t h e e x c h a n g i n g anions a4. I t is realized t h a t t h i s size p a r a m e t e r includes effects on t h e solvent molecules, t h a t this relationship m a y c h a n g e d u r i n g t r a n s p o r t of t h e anion across t h e cell m e m b r a n e (e.g. zolvation b y t h e m e m b r a n e or carrier), a n d t h a t interionic effects m a y n o t b e negligible a t a site of c o n c e n t r a t i o n . F o r example, MULLINSa5 h a d p o s t u l a t e d t h a t , fo~ cations at least, a close fitting pore could replace t h e h y d r a t i o n of t h e ions, t h e Biochim. Biophys. Aeta, 69 (x963) 58--67
66
j. WOLFF,
J . R. M A U ! ~ Y
size d e t e r m i n a n t t h e n b e c o m i n g t h e c r y s t a l r a d i u s r a t h e r ~ h a m c b h e ~ ~ : r a ~ u s o b t a i n e d from c o n d u c t a n c e d a t a . I t m u s t be e m p h a s i z e d t h a t while u n i v a l e n c y a~-~d proper~siz~ru~_yb~e~.r~eessax?¢, t h e y are n o t sufficient c o n d i t i o n s for i n h i b i t o r s of iodide _ - t - t ~ a ~ r t . [ t ~ t h ~ series S O a N H ~ - , S O a F - , C H a S O a- a n d CeH~SOa-, only S O a F - ~ i s ~ ~ 3 - i / ~ t ~ i ~ i , t o r y for I - t r a n s p o r t , a n d it is o u t of line w i t h t h e r e m a i n i n g p K ~ i ~ , o ~ ~ _ ~ a ~ m a t e L~/~ {41.4 ml/mole) a n d p r o b a b l y also m e t h a n e s u l f o n a t e f n t ~ ! ~ ~ ~ ~ ~ DtLt~ -:-a n d u n i t charge, b u t n e i t h e r c o m p o u n d (nor p h e n y l s u l f o n a t e , ~ r m l m ~ i n f i ~ n c e d iodide t r a n s p o r t a t c o n c e n t r a t i o n s as h i g h as 30 mM. F ~ , ~ f ~ m m ~ e (26.3 ml/mole) n o r a c e t a t e (40.5 ml/mole) were i n h i b i t o r y ~ a t ~ c ~ ~ ~ i o n s . I t is of i n t e r e s t t h a t t h e c o r r e l a t i o n b e t w e e n ~0 a n d t h e se~ecti~3_- c ~ e r ~ on n o w e x ~ 2 for t h e s e t w o a n i o n s does not hold as it does f o r : i n ~ t : ~ . ~ : ; T h e a b o v e c o r r e l a t i o n b e t w e e n p K a n d ~0 neglects s t r n c t m ~ ~ e ~ : l s e t w e e n t h e ions (Table I) w h i c h m a y be ~)f c o n s i d e r a b l e i m p o r t a n c e , ~ ' ~ L t m ~ _ y ~ the lack of c o n c e n t r a t i o n of soma of t h e m . T h u s , it h a s b e e n - s h o m m r t ~ - - ~ . - s ~ e r i e a ! h a l i d e s B r - (see ref. x), I - a n d A t - (see ref. I), a n d t h e ~ E a i a ~ d m k l i ~ B E ~ y (see ref. 7), CIO4- (see ref. 7), T c O a - (see ref. 4), a n d R e O a - (see:~f.44)a~eec~nce~itra~ed b y t h y r o i d t i s s u e as well as b e i n g i n h i b i t o r s of i o d i d e ~ m a s p o r t A ~ r t ~ _ ~ hand S C N - , a l i n e a r a n i o n , is n o t c o n c e n t r a t e d t o a n y s i g n i f i c a n ~ ~ ~ n , , ~ o u g h it is a p o t e n t i n h i b i t o r of iodide t r a n s p o r t . T h e d e t e r m i n a t i o n 0 f ~ L - ~ - - e ~ ~ o n will be a n i m p o r t a n t t e s t of t h i s c o n c e p t of a different b e ~ v d ~ f 6 o r i i / m ¢ ~ amions. W h e t h e r N O a - a n d N O , - will also fall i n t o t h i s g r o u p r e m a ~ g v t x ~ c d ~ e c n ~ e d . I t is of i n t e r e s t in t h i s r e g a r d t h a t t h e p l a n a r complexes o f ~ ~ ~ & ~ u ~ S C N ) ~ a n d AuCI~-, as well as t h e l i n e a r A u ( C N ) o - a r e n o t c o n c e n t r ~ ~li~3xft3_.~-o~itiss~e a~. T h e role of t h e s h a p e of t h e a n i o n s s t u d i e d here will t h e r e f o r e ~ ~ - s t u d y . ACKNOWLEDGEMENTS
W e s h o u l d like t o t h a n k Dr. C. G. LEWALLEN for p o ~ e~rtrti~~es e x c h a n g e resin b e h a v i o r a n d Dr. S. H. WOLLMAN for ver~" l t S e ~ l ~ ] , _ - ~ ~ .
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REFERENCES t I. P E R L M A N , M. E . MORTON AND I. L. C H A I K O F F , A m . J. Physiol.., ~i34 ~ I 4 7 ~ q 7 , a C. J . SItELLABARGER AND J. T . GODWIN. J . Clin. Endocrinol., I4 ( ~ S - r 4 ) ' , t ~ G. E. BOYD. J . Chem. Education, 36 (1959) 3. J . W O L F F AND J . R . MAUREY, Biochim. Biophys. Acta, 57 ( t 9 6 z ) . ~ _ 2 . 5 j . B. WYNGAARDEN, B. M. WRIGHT AND P. ~VAYS, Endovrinol.,-50 ([~2~).553"7:57~:~'t~53~ 56~. e M. ANBAR, S. GUTXMANN AND Z. LEWITUS, Nature, ~83 (I959) I 5 r 7 v M ANBAR, S GraTrMAN AND Z LEWXTUS, Intern. J. A p p l . R ~ l . lso~ape,s.77r~5~ ~7: Endocrinology, 6 6 (x96o) 888. s ~ - . HOCKEL, Structural Chemistry of Inorgan;~c Compounds, E l s e v i e r , - , ~ s ~ ,t ~5o~, i j . WOLFF ANO J . R . MAVREY, f . Clin. Invest., 4 t (t96z} z4x3. te H . S. BOOTH AND S. REHMAR, Inorganic Syntheses, I I ( t 9 4 6 ) ~ - 3 . zt E . LEDI~RER AND M, LF, D~--RER. Ch:omatography, E!scv,~cr, _~_~a_ffi~e~,~_~77 ,~.-'..5.g~ zffi K . YAMAGUCHI, J . Pharm. Soc. J a p a n , 73 (I953) I : 8 5 . z~ ~ . S E v l G N ~ , Compt. Rend., 239 ([954} ~-72. ti K . F ~ J ~ N S AND 0 . JonssoN, y. ~ m . Chem. Sot., 64 ( ] 9 4 z) 668. ~" D. O. Ni~sso~, Phil. N1ag., 8 (x929} z~8. ~ J. R. M~uR~ A s v J . w o i . ~ , ~ , J . lnorg. Nu~l. Chem., in t h e p r e s s , t~ J . ~VOLFF AND J . R . M A u . ~ - v , Biochim. Biophys. Acta, 47 ( t 9 6 ~ ) ~ 6 7 . ~s j . WOLFF, Bioehim. Biophys..4eta, 38 ( t 9 6 o ) 316.
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TRANSPORT
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n H . AKEDO AND H . N . C H R I S T E N S E N , J. Biol. Chem., z 3 7 ( t 9 6 2 ) I t 8 . 20 S. H . WOLLMAN'AND F . E . I{EED, ~m. J. Physiol., t 9 6 ( I 9 5 9 ) I 1 3 . H . ~,VOLLMAN, A m . J. Physiol., x 8 6 (x956) 453211 p . ]~ S C H N E I D E R AND J . W O L F F , u n p u b l i s h e d . 23 B . BECKER, A m . J. Physiol., 2 o l ( t 9 6 I ) x x49. 24 K2 F L E T C H E R , A. J . S O N O U R AND E . N . R O W L A N D S , Biochem. J., 63 ( [ 9 5 6 ) x94. 25 T. ARAKI, i . ITO AND O . OSCARSON, J. Physiol. (London), I 5 9 ( I 9 6 I ) 4 I O . 26 A . M . C O U T U R E AND K . J . L A I D L E R , Can. J. Chem., 35 (1957) 2 0 7 . '~7 C. •'. L E B L O N D AND P . S / ) E , A~q'/. J . Physiol., x34 ( I 9 4 ~ ) 5 4 9 . 2s "W. C. LATI~tER, Oxidation Potentials, P r e n t i c e - H a l l I n c . , N . Y . , I 9 5 2 . 29 G. SCATCHARD AND E . S. BLACK, J. Phys. chem., 53 (x949) 88. z0 E . B. ASTWOOD, J . Pharm. Exptl. Therap., 78 (x943) 79. st K . H . STERN AND E . S. AMIS, Chem. Revs., 59 (1959) x. z2 A. VOE'r, Chem. Revs., 2o ( I 9 3 7 ) x69. ~s W . D . ROBERrSON, J . Chem. Phys., x8 (195o) x365. a, H . P . GREGOR, J. BELLE AND R . A. MARCUS, J . A m . Chem. Sue., 76 ( t 9 5 4 ) t 9 8 4 ; 77 (~955) 2 7 1 3 • 35 L . J. MULLZNS, J . Gen. Physiol., 42 ( I 9 5 9 J xox3. n6 j . L. WOOD a N D N . K1NGSLA~rD. J. Biol. Chem., t 8 5 (x95o) 8 3 3 . a~ S, H . "~VOt.LMAN, J. C. RE1D AND F . E . R E E D , A m . J . Physiol., z93 ( t 9 5 8 ) 83. as M. ANBAR, q u o t e d b y N . S. HAt.MI, Vitamins and Hormones, x962, p. t 3 3 . N . V. SIDGWICK, The Chemical Elements and their Compounds, O x f o r d U n i v . P r e s s , L o n d o n , I 9 5 o .
Biochim. Biophys. Acta, 6 9 (x963) 5 8 - 6 7
T. W I N N I C K ,
R. E. WI~NS(IK,
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~ I . G A L , 1 ). A . D R E W E S AND ,_N. F . T A Y L O R , HrC$~.,~[ttgdd,a ~ . H i o ~ I i 3 , s , , 93 (1961) I . j. "~VILLIAM,% R . E . E A K I N AND E . E . S N ~ t - t , J_2~m.cU/v~'nz.:S~c..0z: ( I 9 4 o ) 1 2 o 4 . L Y N E N , E . R E I C H E R T AND L . R U E F F , A n n . : C l , e m . 1 2 ~ 5 . ~ - - - " ~ . (I~-x) I. A v I - D o R AND J . ~ % G E R , Biockem. J., 6 3 (tgYS)'f~33L " B . C H E N O W E T H , ] . Pharmacol. Exp'.l. T t w r a p . , * O T ~ g r ~ ) ~ R . F R A Z E R AND F . L . M . PATTISON, Nature, : r 7 6 ~xrqiSiS)c~mE . ~,VINNICK A N D T . "~VINNICK, B u l l . . q o c . Chim.LI:ii#l.,44~(~r~h-~B trT"._ G R A F F A N D H . D . H O B E R M A N , J . B i o l . C h e m . . ~1-8~ g l t A ~ ) 3 i ~ , I ~ H L AND P . FRtTZSON, ] . Biol. Chem., z ~ 3 ~ ) 3 3 b ~ . F. B A X T E R A N D E . R O B E R T S , J . B i o l . Chem.,__oz3fi ¢I~ft~)2~L~l,. B . MCCORMICK AND E . E . SNELL, .~. Biol. Cke, m.,---~6(~t*!-.xo83, F . D E B O N S AND J . A. PITTMAN, Federation t a r o c . , _ ~ I ( ~ ! X ~ M. G A L AND R . A. P E T E R S , Biochem. J . , 64 (:x~56) .*mr.
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x. T h e a b i l i t y of v a r i o u s a n i o n s t o ~ e a c t ",v~-Ithtti~. i o d i d e - t r a n s p o r t s y s t e m of s h e e p - t h y r o i d slices h a s b e e n i n v e s t i g a t e d . ~ a ~ a m a f l / ~ l ~ r a n g e d from 3-5" Io-7 M t o 2 . 1 o -= M to give a series of i n c r e a s i n g FK ~xaihm~,: "ITQ(!)~- ~< CIO~- <: R e 0 4 - < B F a- < S e C N - -~ S O , F - < S C N - < I - < ~gIO~.- << ~ <: O C N - ~-~ B r - . 2. T h e s e w e r e c o m p a r e d w i t h t h e partiglrmdM]iimiirx~olixmes a t infinite d i l u t i o n , Oo, w h i c h s h o w s t h a t t h i s series also :f0Hnws aa dimmasihg= o r d e r of ~ 0 (with t h e e x c e p t i o n s of R e O 4 - , S O a F - a n d SeCN-)..-'¢ Llixemarr~h~n~ exists b e t w e e n t h e p K v a l u e s a n d t h e p a r t i a l m o l a l ionic volumes, c~xazr0itmmaag~off~-46 ml/mole. A l t h o u g h t h e p K ' s decline w i t h l a r g e r volumes, a d e a r * a m mmeiimma~ w a s n o t observed. N o similar c o r r e l a t i o n exists b e t w e e n t h e p K : m l f l , ~ r m i i m ~ t ~ x - a m e t e r s . 3 - ' A l l of t h e a n i o n s (except T c 0 4 - . =~Riidh ~va~ nott tested) were s h o w n to be c o m p e t i t i v e i n h i b i t o r s of iodide t r a n s p o r t ! h y < f i n t i t i h e ~ e M i p l o t analysis. 4- T h e i m p o r t a n c e of size, u n i v a t e n c y : m a f l ~ f f r t r a n i 0 n ~ t r a n s p o r t in t h y r o i d t i s s u e a r e briefly discussed in r e l a t i o n t o cem~ha ~ l ~ p e r t i e s of t h e ions,
IN~FR OlIUCr~]~A"
T h e role of ionic size in anion transpox't ihy ~ t~iiimm~!i~s been suspected ever since it w a s found that the Br- ion is £one~tku~mLq tn~l~sser extent• than iodideI. I t w a s s u b s e q u e n t l y f o u n d t h a t t h e halide, ~ x ~ : , , ~ w~flE c o n c e n t r a t e d b y th3Toid g l a n d s ~, w h e r e a s t h e s m a l l e r ions Cl- a n d .=~m--.~auremm~.]Em,a o ~ l e x a n i o n s of periodic ~B~tiim~ Hiop~~,~. .4cta, 69 (z963) 48-58
THYROIDAL IODIDE TRANSPORT
59
g r o u p V I I A , TcO 4- a n d ReO4-, are also c o n c e n t r a t e d b y t h y r o i d tissuea, 4. Certain o t h e r complex anions h a d been found to i n h i b i t iodide c o n c e n t r a t i o n b y t h y r o i d tissue e.g. SCN-, CIO4-, N O a - (see ref. 5)- I t was p o s t u l a t e d t h a t this i n h i b i t o r y p o t e n t i a l was, in some m a n n e r , a function of t h e position of these ions in t h e Hofmeister or lyotropic series. More r e c e n t l y ANBAR el al. 6 suggested t h a t t h e i n h i b i t o r y properties of t h e anions were related to their size. T h e y showed t h a t c e r t a i n complex anions, B F 4 - , S O a F - a n d PO~Fa-, chosen for t h e i r size, could i n h i b i t i o d i d e t r a n s p o r t . F u r t h e r m o r e , CIO 4a n d B F 4- were shown to be c o n c e n t r a t e d b y t h y r o i d tissueL I o n sizes were calculated according to H0CKEL a, a p p a r e n t l y as multiples of t h e oxygen or fluorine v o l u m e s at the corners of t h e b o d y - c e n t e r e d t e t r a h e d r a . Such size e s t i m a t e s were not, however, in a g r e e m e n t w i t h our finding 4 t h a t TcO 4- a n d ReO4-, are more a v i d l y c o n c e n t r a t e d b y t h y r o i d tissue t h a n I - , despite t h e fact t h a t t h e i r v o l u m e s would be of approxim a t e l y t h e same size (4" IO-e~ cmS) in the above calculations. I n order to decide w h a t sort of size p a r a m e t e r would best describe t h e size r e q u i r e m e n t of t h e a n i o n - t r a n s p o r t i n g s y s t e m of t h e t h y r o i d , it is n e c e s s a r y to h a v e some q u a n t i t a t i v e m e a s u r e of t h e a n i o n effects on t h y r o i d tissue. We h a v e , therefore, m e a s u r e d " c o n s t a n t s " of t h e Michaelis t y p e , Km or Kt, t h a t describe h a l f - s a t u r a t i o n of t h e t r a n s p o r t s y s t e m in t h e s t e a d y state, for a n u m b e r of u n i v a l e n t anions. These h a v e been c o m p a r e d w i t h c e r t a i n size p a r a m e t e r s t a k e n from t h e l i t e r a t u r e or det e r m i n e d b y us. By far t h e best correlation b e t w e e n K values a n d size exists w i t h p a r t i a l molat ionic volumes. A p r e l i m i n a r y report of t h e s e findings h a s a p p e a r e d ~. MATERIALS AND METHODS A n a l y t i c a l r e a g e n t s were used unless otherwise indicated. KSeCN (Bios) was purified b y acetone e x t r a c t i o n a t room t e m p e r a t u r e a n d crystallization from acetone. H o t acetone a n d e v a p o r a t i o n u n d e r v a c u u m led to decomposition to free selenium in our hands. N H 4 B F 4 was m a d e according to BOOTH AND REHMAR1°. Crude N H 4 S O a F was o b t a i n e d from t h e Chemicals P r o c u r e m e n t L a b o r a t o r i e s , N.Y. I t w a s purified b y m e t h a n o l e x t r a c t i o n a n d crystallization from e t h a n o l to yield a p r o d u c t w i t h a melting point in a g r e e m e n t w i t h t h e l i t e r a t u r e (245°). E l e m e n t a l analysis of t h e a m m o n i u m salt yielded: found: N, I 1 . 8 8 % " F, 16.36°,/o; S, 0_7.o30/0 • theoretical" N, 11.96 %; F, 16.22 %; S, 27.38 %. R a d i o a c t i v e iodate was m a d e by oxidation of 25-3o/~C of m l I - in t h e absence or presence of carrier (15 t~g KI) in H~O w i t h C12 gas. This m a t e r i a l was t h e n c h r o m a tograpl-,edl-. :3 on "~Vixatman No. 3MM p a p e r b y t h e a s c e n d i n g technique. T h e position of the b a n d was identified b y brief e x p o s u r e to X - r a y film. T h e a p p r o p r i a t e s t r i p was cut out, e l u t e d w i t h H~O, a n d c o n c e n t r a t e d . T h e yield was essentially q u a n t i t ative. Control strips with stable iodate were s t a i n e d w i t h d i p h e n y l a m i n e - H n P O 4 (see refs. 11-13). P a r t i a l molal ionic volumes were t a k e n from t h e l i t e r a t u r e t4' or, w h e n n o t available, t h e y were derived from t h e densities of t h e i r solutions d e t e r m i n e d in R i c h a r d s o n p y c n o m e t e r s a t ~5.o °. Volumes at infinite dilution were d e t e r m i n e d g r a p h i c a l l y from a m i n i n m m of five points per curve according to t h e empirical e q u a t i o n of MASSON15: = ,too + sv/ Biochirn, Biophy~. Acta, 69 (I963) 58-67
6~O
J. W O L F F ,
J. R. MAURE'Y"
~~s~t~a,pparent molal v o l u m e ; ff~ois t h e molal v o l u m e e x t r a p o l a t e d to infinite , ~ ~ i ~ t t ~ slope; a n d c is t h e c o n c e n t r a t i o n . ~v~mTtes were s u b t r a c t e d according to t h e c o n v e n t i o n of FAJA~S A,'CD j j I m ~ e ~ . . E o t a s s h x m iodide, NaSCN a n d NH4SCN were used to check t h e m e t h o d a~giim~x~iim~itx t h e literature. Iodide ion gave a value of 36.8 ml/rnole (literature 3~677~)aaKl S C N - ion was 4I.o ml/mole as c o m p a r e d to 4o.6 ml/mole t*. A ~i~ktd~sdi~'oxt of t h e s e d a t a will be r e p o r t e d elsewhere t~. / ~ m ~ m d : ~ v a l u e s were d e t e r m i n e d in sheep t h y r o i d slices b y m e t h o d s previously .,t t c m c i t t t ~ , ' m r T h e v o l u m e of m e d i u m w a s increased to 5 ml to r e d u c e c o n c e n t r a t i o n ,:~sii~tli~medium a t t h e s t e a d y state. E q u i l i b r a t i o n t i m e for iodide in t h e anioni l e ~ r d i ~ z ~ t t ~ w a s c h e c k e d w i t h S C N - a n d CIO4- a n d w a s found to be well w i t h i n t h e ~3o-nmmr~i~iim~bation period. Kt v a l u e s were d e t e r m i n e d at t h r e e levels of iodide a a m t : V i ~ f i tfliii~or. All p o i n t s were in d u p l i c a t e so t h a t a single Kt value was o b t a i n e d ~-.N3. ~ 3o flasks on t h e same tissue. Generally, this procedure was followed ,mnf~i~7~thyroid g l a n d s for e a c h compound. I t was a s s u m e d t h a t t h e s y s t e m ~x, e s a ~ s i m p l e s a t u r a t a b l e s y s t e m xg. A l t h o u g h this m a y be too simple ,~ view 2°, r t ~ [ ~ K ~ l m ~ s ~ ,obtained are useful for c o m p a r a t i v e purposes. No corrections were ,q~m~~e~cr~e~|ar (inulin) space as t h e g r a d i e n t s ( T I M I X - ] ) were usually quite !i~ge,~A~of'o.75 w a s s u b t r a c t e d from t h e T I M [ X - ] for t h e anion t h a t e n t e r e d b y '~'nT:/i:~ v a l u e s were o b t a i n e d from plots of t h e reciprocal of t h e s t e a d y s t a t e ~.~ttfitece~m~on in t h e slice vs. t h e i n h i b i t o r c o n c e n t r a t i o n . T h e Km values were , ~ m i i m e E ~ D l f i c a l l y from double reciprocal plots as p r e v i o u s l y described~, ~. T h e ~ ~ z e ~ . fmmt applied to t h y r o i d a l t r a n s p o r t problems b y WOLLMA~"~-x. YIx~clteel~: for.,, protein b i n d i n g of a n i o n s in t h e m e d i u m , slices were i n c u b a t e d ~~~tmrtiealiconditions b u t w i t h a tissue c o n c e n t r a t i o n t h i r t y - f o l d t h a t usually , . ~ ~ e k t _ ~ ( ~ , ~ s l i e e s / x o ml m e d i u m ) . After 9o rain t h e m e d i u m w a s c e n t r i f u g e d to rem~eece~ T i m c a l c u l a t e d protein c o n c e n t r a t i o n (on t h e basis of thyroglobulin) ~msI~.~~ T h i s solution was dialyzed a g a i n s t e i t h e r 3" IO-~ M I - or 4" IO-~ M ~--(i0mlttm~rsolution ) a t 4 ~ w i t h i n t e r n a l a n d e x t e r n a l stirring. Similar experi~ ~ w z z ~ c c ~ _ v r i _ ' e d out w i t h a 5 % solution of " p u r e " beef thyroglobulin (95% I9-S fft~iomnam~sF/o zs-S fraction). W i t h t h e m e m b r a n e s used, > 9 9 % equilibration h a d
RESULTS AND DISCUSSION
Z l t ~ a ~ . i u t x ~ t h e c u r v e s o b t a i n e d b y p l o t t i n g the reciprocal of t h e iodide ion concenL~__~~t~m~n t h e slices w h e n t h e s t e a d y s t a t e is a t t a i n e d a g a i n s t t h e concentration ~tI~tmedlis s h o w n in Fig. x. Iodide values are c a l c u l a t e d from t h e concen~ofi'm~lP a&ded a n d t h e fraction of r a d i o a c t i v i t y r e m a i n i n g in solution w h e n ~]m_~occurred ( 9 o - I o o min). I t was a s s u m e d t h a t t h e ioa,adde c o n t r i b u t e d ~ ~ ~ s H c e was not i m p o r t a n t at t h e iodide c o n c e n t r a t i o n s used. P r e l i m i n a r y ~ t h e x~I- released into t h e m e d i u m ~2 suggest t h i s to be t h e case. ~ ~ m t h i of t h e e x p e r i m e n t s t h e d a t a o b t a i n e d did n o t yield lines s a t i s f a c t o r y f ~ m r ~ i ~ m m ~ i .l~0~ analysis. T h e d e v i a t i o n s of t h e points did n o t show a n y consistent _qm0ztt~~er;, a n d t h e reason for t h i s is, at present, u n e x p l a i n e d . Such d a t a were rmmmm~. iha~-l~i axe listed t h e Km values for I - , TcO 4- a n d ReO4-, t a k e n from our Biochim. Biophys. Acta, 69 (x963) 5 8 - 6 7
THYROIDAL IODIDE TRANSPORT
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previous work4, ~v, as well as /Q values for iodide t r a n s p o r t of t h e sev~al~umitm.c h o s e n for this s t u d y because of t h e i r size. I t is a p p a r e n t t h a t t h e r a n g e ,0frtl~h~s~, " c o n s t a n t s " e x t e n d s over n e a r l y five orders of m a g n i t u d e , t h e smaller anionsflxa~mz/: larger c o n s t a n t s t h a n m o s t of t h e large, t e t r a h e d r a l ions. Of interest is t h e - f i n ~ ~ a ~ for ReOa-, t h e Km value, using ~ R e was identical to a Kl v a l u e determinedu~gaias~ r. . . . . . . . . . . . . . . . . . . . . . . W"
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F i g . 2. T h e r e l a t i o n b e t w e e n t h e p / ( F - , l ~ g / t 7 ~ , t ~ r Ki) f o r a n i o n t r a n s p o r t in s h e e p .*.hyroid~*iliems~',L, t h e p a r t i a l m o l a l i o n i c v o l u m e a t infztxite, ifliltitimm. ~ , o f t h e s e a n i o n s . I o d i d e a n d TcOa--.vialel~sAz~rt • d e r i v e d f r o m t h e K m v a l u e s , R e O 4 - .~rom>i~2ttlh/~, o r K l , a n d all o t h e r a n i o n p K valttes:~u-trtntitht. Kl values.
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[ReOT~I,IO'~M [GIO~},IO-TM F i g . i. T h e e f f e c t of v a r i o u s a n i o n s o n t h e a c c u m u l a t i o n o f i n t r a t h y r o i d a l iodide.~ 'l-?l~tmttro,lbt t h e r e c i p r o c a l o f t h e c o r r e c t e d i o d i d e c o n c e n t r a t i o n in t h e slice in t h e s t e a d y s t a t e x ~ ] ~ - ~ ' x , - , the concentration of inhibiting anion. Curves for three different steady state eonc~,~tti~s~bl i o d i d e i n t h e m e d i u m a r e g i v e n . T h e i r i n t e r s e c t i o n d e t e r m i n e s t h e 1£1 v a l u e .
iodide ion (Table I). T h e s e values agree well w i t h t h e in vivo finetings:!~r-iduni_4s poorly c o n c e n t r a t e d ~, N O a - is a poor inhibitor, w h e r e a s C104- h a s b e e n 4 o m i d m ~ very p o t e n t 5. T h e y are in s a t i s f a c t o r y a g r e e m e n t also w i t h t h e c o n c e n t r a t i t m ~ for 50% inhibition of iodide a c c u m u l a t i o n in t h e choroid plexus o f : - ~ b h i ~ 3 : e ~ . , . ,. C104-, 2 " 1 o -e M; B F , -, , 5 . 1 o .6 M", oc ~~- - , - , 3 " i o -s M a n d NO3-, 3:zo~m~M;amatdifin m o u s e salivary glands: CIO4-, 2" I o -6 M; I - , 3" xo-5 M ; a n d SCN-, 3'-'~o*-~M(~(eee ref. 24). T h e S C N - values s h o u l d a l s o be c o m p a r e d w i t h a Kt of 6 . i o - 5 : , M , 0 ~ b y WOLLMAN in t h e t h y r o i d s of i n t a c t mice 2x. T h e slight d i s c r e p a n c y c a z z ~ ~ be explained, at least in p a r t , b y b i n d i n g of S C N - to s e r u m p r o t e i n s ~=-Jthdfimaaet animals. W h e n t h e Km or Kt values are p l o t t e d (as t h e pK's) vs. t h e ~ ~ h l ionic volumes, ~0, of t h e .anions, a h i g h l y significant correlation was o b t a i m ~ 1 0 F ~ ,-f_). T h e m a x i m u m p K value was n e a r 3" zo-T M w i t h TcO4-. Since technetitml~e:x~tsmt~. in t h e r a d i o a c t i v e form it was n o t p r a c t i c a l t o o b t a i n a p a r t i a l ~ m o l a l ' , ~ f d m T TcO4-, a n d we h a v e used t h e m e a n b e t w e e n t h e MnO4- a n d ReO 4- v o h m m s . ~ ~ Biochim. Biophys. Ac,~, 69 ~ 5 ) ~
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i--,. Jo -3
Nitrate (NOa-)
' Km values in italics from WOLFF AND MAUREY 4.17. +° From FAJANS AND JOHNSON14. " ° ' Determined for this study. t Taken as the mean of M n O ( and R e O / ( s e e ref. t4).
3-5' Io-7
4" to-7
Perchlorate (004-)
Pertechnetate (TcO()
x.xo -e, z. lo -~
3' Io-~
Perrhenate ( R e O ( )
Tetrafluoroborate ( B F ( )
i. io -n
I-2' IO-5
Monofluorosulfonate (SOsF-)
Selenocyanate (SeCN-)
z- 3. I o - '
Thiocyanate (SCN-)
3" lo -s
4' m-3
Nitrite (NO2-)
Iodide (I-)
i-2. m -a
__
Cyanate (OCN-}
-. 2. m -z
Iodate ( I O f )
Transport h'i or Kra* (51)
Bromide (Br-)
?¢am~
I
44.5'* 46.0 t
48.7'*"
44.o'"
5o.3""
47.8'**
40.6 't
3O.7 ° '
29.4 ' *
25 * t , 2 0 °
20.7''*
25.I -)
~-5. i ' "
Parlial molal volu)~ ¢, at :5.0* (mtlmote)
Volumeso/
3.94 (4.05) §
4.1o
~4
~4
4.Z3
3,a3
(to-"cm*)
ASBAael al.'
I.o9
1.34
1.12
I.II
0.90
I.O3
1.O2
0.94
Relative hydcatcd ske K .... l.od ~
THE RELATION' OF h ' m AND K I TO ANION SIZE
TABLE
46.,1
46.9
36.6
29.3
a6,5
a5.O
(ml/mole)
AND LAIDLERza
Volumes oI CoI:TCaE
Tetrahedral
Tetrahedral
Tetrahedral
"l'etrahedral
Linear
Tetrahedral
Linear
Spherical
Planar
Angular
Linear
Pyramidal
Spherical
Str~IU?~)a~
o~
,~
~,
,tl .
.~ O
tO
TH~OID3A~ ~i~:
THA~SI~ORT
63
t h e r e is a decline in t h e p K ~ a s t t h e ~ i d d m g l i d ] ionic v o l u m e s e x c e e d 46 m l / m o l e (as s u g g e s t e d b y R e O 4 - , s O ~ F - z a n t t ~ - ) ) omxld3 n o t be a d e q u a t e l y t e s t e d for lack of a n i o n s of s u i t a b l e v o l u m e . ~ h e d ~ s ~ ~ t s w i t h S O s F - are u n e x p l a i n e d a t p r e s e n t . Similarly, a "minimum",~izeiis~fffmxltxto ,obtain b e c a u s e t h e c o n c e n t r a t i o n s r e q u i r e d are so large as t o demmaticti~bttimnofffadln*ge f r a c t i o n of C1- from t h e m e d i u m t o m a i n t a i n o s m o l a r i t y . Non-~pecqfic efffim~wcml~ t h u s b e likely t o occur. T a b l e I also lists various, rrther.~met]mmaaett~, r n a d d i t i o n t o t h e p a r t i a l m o l a l ionic volumes, w e h a v e listed, ~ o r c e ~ . t t i e - v o l u m e s g i v e n b y ANBAR el al. 6, r e l a t i v e h y d r a t e d sizes f r o m : c ~ n ~ ~ e n t s ~, a n d t h e " e f f e c t i v e " radii as c a l c u l a t e d b y COUTURE A-ND I L ~ m _ m t ~ . , ~ ~ the values calculated according t o H0CKEL n o r t h o s e o b t a i n e d f i r o m l l i i n ~ i x : m m m u r e m e n t s show a c o m p a r a b l e correl a t i o n w i t h t h e K m or K t v a l u e s . l ~ t i i s i i s i i n a o n t t a ~ t u a n i o n p e r m e a b i l i t y as e x h i b i t e d , for e x a m p l e , in c a t neurones~',vYhexetm0m~l~irna.wiflx k i n e t i c r a d i i is found. I t is d e a r , h o w e v e r , t h a t t h e e m p i r i c a l . e q u a t i m a d t ~ q r g e ~ t ] t i y C,,,UTUR~ Arid LAIDLER2e, w h i c h is b a s e d on t h e n u m b e r ofligantis~anHttize"0at/mtive'" r a d i u s (sum of t h e b o n d l e n g t h a n d V a n d e r W a a l s r a d i u s of, oxygen), ~yidittsv~lttmes t h a t show close c o r r e l a t i o n w i t h m o s t of t h e p a r t i a l m o l a l :ionic w d h m e ~ ~ e d i e x p e r i m e n t a l l y a n d h e n c e also w i t h t h e Km or/ISi values. I t s h o u l d be p o i n t e d o u t ~ h ~ t t h e q ~ m f l i m ~ w e r e m e a s u r e d a t 2,5 ° w h e r e a s K m or Kt v a l u e s were o b t a i n e d a t 37--.387. ?~stiitemaiim~volumes s h o w c o n s i d e r a b l e t e m p e r a t u r e d e p e n d e n c e n o t directly~rdla~lflttDtiteev-~li~__~of~ Oo x*, it m i g h t be s u p p o s e d t h a t t h e o r d e r of sizes differs a t 37 9.~ited~li~6CtS/7liviit~ttie o r d e r CIO4- :> N O a - :> I - > B r b u t a m o u n t s t o o n l y o . 5 - x . 5 _ m l p r n d i e f f m ~ i ~ t t m a g e z a t u r e difference, h e n c e t h e o r d e r of T a b l e i or Fig. 2 w o u l d n o t i b e i i r ~ m n n w B . T h e r e are s e v e r a l u n c e r t a i n l i ~ s i i n ~ t t i a t deal w i t h (a) possible d e c o m p o sition of t h e a n i o n s in o u r - s y ~ e m , amta (~k))t~ssibte b i n d i n g of ions b y p r o t e i n t h a t h a s l e a k e d o u t of t h e slices, kBoth,~fftflemew~ultEtend t o lower t h e c o n c e n t r a t i o n of a n i o n a n d h e n c e give falsely ihi~41/K~,aitm~. z. T h e possibility t h a t iE[F~--,~rr~]F-we~m,irthibitory on t h e b a s i s of h y d r o l y s i s to F - ,~n ~ , , , d n o t be e n t i x e l y ~ m m n t t e g d . X ~ x ~ t l i e r e f o r e m e a s u r e d t h e effect of F on iodide t r a n s p o r t in t h e sheeprlixymith~!iime.TI~ier~ ~ a s n o significant effect on iodide t r a n s p o r t w i t h m e d i u m F - t e v e t s ~ s t t i ~ h ~ s 3 ~ - m ~ , l ~ L T h u s t h e i n h i b i t o r y effects of t h e s e ions c a n n o t be aceourrteflttxrrklay _tO~"rr~rtm~al c o n t e n t of F - e v e n if c o m p l e t e h y d r o l y s i s occurred. I t h a s : b e e n a ~ [ p ~ t l i a ~ t t i e - l i r e a ! . l o w n of B~SF, - is negligible v. ,2. ~VvN~AARI)V,r~ e:" al:r'~tl~p~r~tltfla~ I I ~ - ; - w a s a n effective i n h i b i t o r of iodide a c c u m u l a t i o n a n d also s t i r n u l a t e I f l ~ o f f i 0 d i d e from the gland. We therefore t e s t e d t h e a c c u m u l a t i o n of ~ x a ~ I O ~ - - i _ i n ~ ~ d ~ s t i c e s . W h i l e c o n s i d e r a b l e radioa c t i v i t y d i d a c c u m u l a t e in .-the ~ g l i e ~ i x l g g a m n a t ~ a r e n t , t h o u g h s o m e w h a t s k e w e d , s a t u r a t i o n curve, it w a s f o u n t l t i t m t atll ~ m d i 0 a c t i v i t y in t h e t h y r o i d b e h a v e d c h r o m a t o g r a p h i c a l l y 1 1 - 1 B i l i k e i i o H _i_l_ ~ _ . ~ ~ n o , i o d a t e . I n t h e m e d i u m different p r o p o r t i o n s of t h e radioaefi-eity~meeiintlt~f~__~a_ ~oI~ i o d a t e or iodide, d e p e n d i n g on t h e i n i t i a l c o n c e n t r a t i o n of ~iottm~ ~ ] 5 . l~t ~ . ~ likely, therefore, t h a t i o d a t e is r e d u c e d (as also s h o w n b y ~ ~ . ~ a ~ - ~ , a n d l t h a t t h e a c c u m u l a t i o n of radioa c t i v i t y of ~-Oa-. a_n_d pro_~_~!yiitsiifl_tit!~ttr~. , ~ a s wall, r e s u l t from t h e iodide so formed. 3. C h l o r a t e a n d b r o m a t e ~,eer~iixtfitfittm~, h~rE~ t r a n s p o r t w i t h 5 ° % p o i n t s i n t h e r a n g e of zo-a-xo-~:'M. \ W e ~ v ~ m m t i t ~ t O , o b t a i n ~ r e p r o d u c i b l e results, a n d ~ h i g h B i e , h i m . Biophys. A cta. 69 (x963) 58-67
64
j. WOLFF,
i
J. R. 3IAUI~EY
levels caused o x i d a t i o n of t h e tissue p i g m e n t s . F r o m their o x i d a t i o n p o t e n t i a l s , w h i c h are higher t b a n those of IO 3- (seeref. 28) •*' ~ e v_ w o u l d not be e x p e c t e d t o persist" however, we were u n a b l e t o p r o v e t h a t t h e y were r e d u c e d because o u r c h r o m a t o graphic t e c h n i q u e s were n o t sensitive e n o u g h u-~a. T h e fact t h a t o t h e r s t r o n g oxidizing agents, such as Cr±O~9- or CrO~ ~-, were s t r o n g l y i n h i b i t o r y suggests t h a t t h e effect of CIO~- or B r O 3- m a y be non-specific. W e find it difficult, however, to e x p l a i n t h e inhibition in vivo r e p o r t e d for CIO 8- (see ref. 5)4. A c e r t a i n degree of u n c e r t a i n t y applies t o N O g - a n d O C N - . T h e former was s l i g h t l y less a c t i v e t h a n N O 3- b u t as t h e r e was obvious i n t e r a c t i o n w i t h t h e p i g m e n t of t h e slices, it m a y be t h a t t h e effective: c o n c e n t r a t i o n differed f r o m t h e s t a t e d one. S o d i u m c y a n a t e i n h i b i t e d 5 0 % a t 1 - 2 . zo -~ M, b u t t h e odor of H C N was easily d e t e c t e d in this s y s t e m , a n d no a c c u r a c y can be c l a i m e d for t h e s e d e t e r m i n a t i o n s .
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E .,,M-' F i g . 3- T h e c o m p e t i t i v e n a t u r e of a n i o n i n h i b i t i o n of i o d i d e accumL, l a t i o n i n s h e e p t h y r o i d slices. D o u b l e r e c i p r o c a l ~ ! o t s o f t h e ~v,, ~+~d~ s+~a ~ ' ' .,., ~ s t a t e i o d i d e c o n c e n t r a t i o n i n t h e slice I / r l l-~!e (ordi-• h a t e s ) vs. t h a t o f t h e m e d i u m t / r I0- ~e (abscissas). C o n t r o l i n d i c a t e s t h e a b s e n c e of i n h i b i t i n g a n i o n s .
5. I t is k n o w n t h a t a l b u m i n , a n d p r o b a b l y o t h e r proteins, can b i n d these anions. T h e o r d e r ot affirfities seems t o be similar t o t h e p r e s e n t series n. I t was t h e r e f o r e possible t h a t e n o u g h t h y r o g l o b u l i n or o t h e r p r o t e i n s l e a k e d o u t of t h e slices t o b i n d t h e a n i o n s a n d t h u s lower t h e c o n c e n t r a t i o n of free anions. A l t h o u g h t h e chloride c o n c e n t r a t i o n of t h e m e d i u m w o u l d t e n d t o d i m i n i s h t h i s effect, e x p e r i m e n t s were c a r r i e d o u t t o t e s t t h i s directly. T h e largest g r a d i e n t in f a v o r of t h e p r o t e i n c o m p a r t m e n t was z.o5 for iodide a n d z.z2 for TcO4-. Since t h e p r o t e i n c o n c e n t r a t i o n s were Biochim. Biophys. Acla, 69 ( t 9 6 3 ) .58-67
THYROIDAL IODIDE TRANSPORT
65
inle~cess (thirty-fold) of w h a t is usually found in t h e m e d i u m u n d e r our conditions, iS seems unlikely t h a t t h e c o n c e n t r a t i o n s of free anions was significantly lowered b y l~Votein binding. l:* seems probable from t h e evidence so far available t h a t u n i v a l e n c y is also a~vequirement of the system. Sulfate (16.4 ml/mole) a n d sulfite (9.5 ml/mole) c a u s e d no intiibition of iodide c o n c e n t r a t i o n at 2o mM. As these ions are of small p a r t i a l molal ii}nm volume, decision b e t w e e n v a l e n c y a n d size is not possible in this system. Howe~el% WO42- (26.3 ml/mole),. MoO42- (29.5 ml/mole) a n d S~O32- ( ~ 34 ml/mole), do falll w i t h i n t h e size r a n g e of t h e smaller i n h i b i t o r y u n i v a l e n t anions. None of these d i v a l e n t ions i n h i b i t e d iodide t r a n s p o r t at a c o n c e n t r a t i o n of 2o raM. [~: has been a s s u m e d b y m a n y a u t h o r s t h a t t h e ions s t u d i e d here c o m p e t e for t i e s a m e site on a h y p o t h e t i c a l carrier. I n general, no evidence h a s been p r e s e n t e d sazce t h a t (a) S C N - -goiter is p r e v e n t e d b y a n increased iodide i n t a k e ~ a n d (b) t h a t It-, T.'cO 4- a n d ReO4- are m u t u a l l y inhibitory, i.e. a n y one of t h e m will i n h i b i t t h e tmansport of a n y o t h e r i n t o t h e t h y r o i d a, 4. T h e only rigorous d e m o n s t r a t i o n for comp e t i t i v e inhibition prior t o this s t u d y is t h a t for S C N - ion i n vivoaL Similar criteria (no,inhibition, i.e. t h e s a m e m a x i m u m c a p a c i t y , at infinite " s u b s t r a t e " c o n c e n t r a t i o n ) lim~e been applied in t h e p r e s e n t i n vitro system. Fig. 3 shows t h a t in d o u b l e reciprocal lflbts t h e O C N - , NO3-, SCN-, B F 4 - , R e O 4- a n d C10 4- ions i n h i b i t iodide t r a n s p o r t in~ ~ compeCAtive m a n n e r . N o t depictcd, b u t also c o m p e t i t i v e i n h i b i t o r s b y t h i s criterion, are t h e B r - , NO2-, S O 3 F - a n d S C N - ions.
COMMENTS
qIhe concept of ionic size is an o p e r a t i o n a l one w h i c h depends on t h e c o n d i t i o n s u n d e r ~ h i c h it is m e a s u r e d 31. Since t h e conditions a t t h e site at which t h y r o i d tissue concenImams anions are u n k n o w n , we h a v e a t t e m p t e d to look a t several t y p e s of ion "sizes" t ~ find t h e best correlation w i t h a biological t r a n s p o r t p a r a m e t e r , in this case t h e ~ m or Kt. S a t i s f a c t o r y c o r r e s p o n d e n c e b e t w e e n t h e t r a n s p o r t p a r a m e t e r a n d size is o b t a i n e d only when t h e l a t t e r is expressed as t h e p a r t i a l molal volume, ~0, according t o e i t h e r MASSON 15 o r COUTURE AND L A I D L E R ~6, b u t not w i t h o t h e r expressions of anionic size. T h e a n i o n s s t u d i e d here fit a series of increasing K v a l u e s : TcO a- ~< C10~7 < R e O 4 - < B F , - < S e C N - ---~ S O 3 F - < S C N - < I - < NO a- < N O , - < OCN. ---~ B r - . Such a series will also describe a n u m b e r of chemical p r o p e r t i e s of t h e s e ions as far as t h e y h a v e been s t u d i e d : (a) t h e p a r t i a l molal ionic v o l u m e s at infinite dilution (except for S O a F - , ReO 4- a n d S e C N - as discussed a b o v e ) ; (b) t h e Etbfmeister series; (c) t h e h y d r a t i o n energies, i.e. t h e H o f m e i s t e r series follows t h e inverse order of t h e h y d r a t i o n energies a2. T h e s e can, in t u r n , be r e l a t e d t o t h e ionic vadii~; (d) t h e order of t h e selectivity coefficients of certain q u a r t e r n a r y a m m o n i u m i o n e x c h a n g e resins, w h i c h can also be correlated w i t h t h e p a r t i a l molal ionic volumes of~ t h e e x c h a n g i n g anions a4. I t is realized t h a t t h i s size p a r a m e t e r includes effects on t h e solvent molecules, t h a t this relationship m a y c h a n g e d u r i n g t r a n s p o r t of t h e anion across t h e cell m e m b r a n e (e.g. zolvation b y t h e m e m b r a n e or carrier), a n d t h a t interionic effects m a y n o t b e negligible a t a site of c o n c e n t r a t i o n . F o r example, MULLINSa5 h a d p o s t u l a t e d t h a t , fo~ cations at least, a close fitting pore could replace t h e h y d r a t i o n of t h e ions, t h e Biochim. Biophys. Aeta, 69 (x963) 58--67
66
j. WOLFF,
J . R. M A U ! ~ Y
size d e t e r m i n a n t t h e n b e c o m i n g t h e c r y s t a l r a d i u s r a t h e r ~ h a m c b h e ~ ~ : r a ~ u s o b t a i n e d from c o n d u c t a n c e d a t a . I t m u s t be e m p h a s i z e d t h a t while u n i v a l e n c y a~-~d proper~siz~ru~_yb~e~.r~eessax?¢, t h e y are n o t sufficient c o n d i t i o n s for i n h i b i t o r s of iodide _ - t - t ~ a ~ r t . [ t ~ t h ~ series S O a N H ~ - , S O a F - , C H a S O a- a n d CeH~SOa-, only S O a F - ~ i s ~ ~ 3 - i / ~ t ~ i ~ i , t o r y for I - t r a n s p o r t , a n d it is o u t of line w i t h t h e r e m a i n i n g p K ~ i ~ , o ~ ~ _ ~ a ~ m a t e L~/~ {41.4 ml/mole) a n d p r o b a b l y also m e t h a n e s u l f o n a t e f n t ~ ! ~ ~ ~ ~ ~ DtLt~ -:-a n d u n i t charge, b u t n e i t h e r c o m p o u n d (nor p h e n y l s u l f o n a t e , ~ r m l m ~ i n f i ~ n c e d iodide t r a n s p o r t a t c o n c e n t r a t i o n s as h i g h as 30 mM. F ~ , ~ f ~ m m ~ e (26.3 ml/mole) n o r a c e t a t e (40.5 ml/mole) were i n h i b i t o r y ~ a t ~ c ~ ~ ~ i o n s . I t is of i n t e r e s t t h a t t h e c o r r e l a t i o n b e t w e e n ~0 a n d t h e se~ecti~3_- c ~ e r ~ on n o w e x ~ 2 for t h e s e t w o a n i o n s does not hold as it does f o r : i n ~ t : ~ . ~ : ; T h e a b o v e c o r r e l a t i o n b e t w e e n p K a n d ~0 neglects s t r n c t m ~ ~ e ~ : l s e t w e e n t h e ions (Table I) w h i c h m a y be ~)f c o n s i d e r a b l e i m p o r t a n c e , ~ ' ~ L t m ~ _ y ~ the lack of c o n c e n t r a t i o n of soma of t h e m . T h u s , it h a s b e e n - s h o m m r t ~ - - ~ . - s ~ e r i e a ! h a l i d e s B r - (see ref. x), I - a n d A t - (see ref. I), a n d t h e ~ E a i a ~ d m k l i ~ B E ~ y (see ref. 7), CIO4- (see ref. 7), T c O a - (see ref. 4), a n d R e O a - (see:~f.44)a~eec~nce~itra~ed b y t h y r o i d t i s s u e as well as b e i n g i n h i b i t o r s of i o d i d e ~ m a s p o r t A ~ r t ~ _ ~ hand S C N - , a l i n e a r a n i o n , is n o t c o n c e n t r a t e d t o a n y s i g n i f i c a n ~ ~ ~ n , , ~ o u g h it is a p o t e n t i n h i b i t o r of iodide t r a n s p o r t . T h e d e t e r m i n a t i o n 0 f ~ L - ~ - - e ~ ~ o n will be a n i m p o r t a n t t e s t of t h i s c o n c e p t of a different b e ~ v d ~ f 6 o r i i / m ¢ ~ amions. W h e t h e r N O a - a n d N O , - will also fall i n t o t h i s g r o u p r e m a ~ g v t x ~ c d ~ e c n ~ e d . I t is of i n t e r e s t in t h i s r e g a r d t h a t t h e p l a n a r complexes o f ~ ~ ~ & ~ u ~ S C N ) ~ a n d AuCI~-, as well as t h e l i n e a r A u ( C N ) o - a r e n o t c o n c e n t r ~ ~li~3xft3_.~-o~itiss~e a~. T h e role of t h e s h a p e of t h e a n i o n s s t u d i e d here will t h e r e f o r e ~ ~ - s t u d y . ACKNOWLEDGEMENTS
W e s h o u l d like t o t h a n k Dr. C. G. LEWALLEN for p o ~ e~rtrti~~es e x c h a n g e resin b e h a v i o r a n d Dr. S. H. WOLLMAN for ver~" l t S e ~ l ~ ] , _ - ~ ~ .
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