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Metabolism in vivo of connective-tissue mucopolysaccharides
BIOCHIMICA ET BIOPHYSICA ACTA
459
BBA 3 9 5 I
METABOLISM
IN
VIVO
OF
CONNECTIVE-TISSUE
MUCOPOLYSACCHARI IiI. CHONDROITIN
SULFATE
AND
DES KERATOSULFATE
OF
CARTILAGE E. A. DAVIDSON AXD WILLIAM SMAIA. Department of Biochemistry and Center f o r the Study of .4giJ2g, Duke University 3tledical Ce,t/er, Durham, N . C. ( U . S . A . )
(Received September 24th, 1962)
SUMMARY T h e i n c o r p o r a t i o n of glucose i n t o t h e h e x o s a m i n e m o i e t i e s of c a r t i l a g e m u c o p o l y s a c c h a r i d e s h a s b e e n s t u d i e d . I s o t o p e a p p e a r s in t h e g a l a c t o s a m i n e m o i e t y of t h e light m u c o p r o t e i n f r a c t i o n first a n d r a p i d l y d i s a p p e a r s f r o m t h i s f r a c t i o n w i t h a n a c c o m p a n y i n g a p p e a r a n c e of i s o t o p e in t h e i n s o l u b l e residue. C h o n d r o i t i n s u l f a t e a c c o u n t s for a b o u t 80 % of t h e t o t a l p o l y s a c c h a r i d e a n d a p p e a r s t o be r e l a t i v e l y i n e r t m e t a b o l i c a l l y . A p p e a r a n c e of label in c h o n d r o i t i n s u l f a t e p r i o r t o k e r a t o s u l f a t e s u g g e s t s t h a t t h e s e p o l y s a c c h a r i d e s do n o t r e p r e s e n t p a r t of t h e s a m e m a c r o m o l e c u l a r s t r u c t u r e . T h i s l a t t e r p o i n t is n o t e s t a b l i s h e d . T h e effect of s e v e r a l h o r m o n e s on t h e i n c o r p o r a t i o n a n d t u r n o v e r of i s o t o p e in +h~,. ~,,o ~: . s ~. . . ~. . ::'as also r e p o r t e d .
INTRODUCTION C a r t i l a g e h a s b e e n one of t h e m o s t e x t e n s i v e l y s t u d i e d of all c o n n e c t i v e tissues. It h a s b e e n u s e d as a s o u r c e of c h o n d r o i t i n s u l f a t e since t h e I g t h c e n t u r y a n d a l t h o u g h t h e c o m p o s i t i o n of t h e p o l y s a c c h a r i d e f r a c t i o n w a s o r i g i n a l l y t h o u g h t t o be exclusively c h o n d r o i t i n 4 - s u l f a t e , k e r a t o s u l f a t e a n d c h o n d r o i t i n 6 - s u l f a t e h a v e also b e e n d e t e c t e d 1. S t u d i e s in s e v e r a l l a b o r a t o r i e s on h u m a n m a t e r i a l h a v e r e v e a l e d a g e - d e p e n d e n t c o m p o s i t i o n c h a n g e s 2-5. I n p a r t i c u l a r , t h e r e is a c o n t i n u o u s i n c r e a s e in t h e k e r a t o s u l f a t e c o n t e n t t h r o u g h t h e s e c o n d d e c a d e in life a n d t h e c h o n d r o i t i n 4s u l f a t e is p r o g r e s s i v e l y r e p l a c e d b y c h o n d r o i t i n 6-sulfate. Q u a l i t a t i v e s t u d i e s s u g g e s t t h a t k e r a t o s u l f a t e also i n c r e a s e s in s h a r k a n d s q u i d cartilage% C a r t i l a g e h a s b e e n u s e d in s t u d i e s of r a d i o - s u l f a t e f i x a t i o n a n d t h e effect of h o r m o n e s on s u c h p r o c e s s e s h a s b e e n reporte&-X% T h e p h y s i c a l s t a t e of t h e h e t e r o p o l y s a c c h a r i d e s in t h i s t i s s u e h a s b e e n t h e s u b j e c t of c o n s i d e r a b l e i n v e s t i g a t i o n . T h e c h o n d r o i t i n s u l f a t e m a y be e x t r a c t e d f r o m c a r t i l a g e in t h e f o r m of a p r o t e i n c o m p l e x w h o s e p r o p e r t i e s s u g g e s t s o m e t h i n g m o r e t h a n s i m p l e e l e c t r o s t a t i c a t t r a c t i o n n-a4. Tile h o m o g e n e i t y of t h i s c o m p l e x h a s n o t b e e n Biochim. Bioph3,s, Acta, 69 (I963) 459-463
460
E.A.
DAVIDSON,
,XV..q~aT~
estaot]snett'-' 't,u, ~.~,: . . . ., ,. ,.p c u,~,,~ . . . s .u g. g.e s.t . c,,,coutt~,~**~:~---~ . . . ~,,,.,~m~ox'~ ~ r~ ~.~ at.. lem~t p a r t of t h e p r o t e i n m o i e t y a n d t h e p o l y s a c c h a r i d e chain. T h e p r e s e n t s t u d y r e p o r t s t h e c o m p o s i t i o n ,o~ ~[pa~r~eiira~ ~.ornp[exes i s o l a t e d f r o m r a b b i t Iib c a r t ilage, t h e i n c o r p o r a t i o n of c a r b o h y ~ a ~ e it~.~um-~o~- i!nto t h e h e x o s a m i n e c o m p o n e n t s of t h e pol3;saccharides a n d t h e effect ,¢¢ff,~:an6i0r~ lhgrTnonal a g e n t s on t h i s process. P r e l i m i n a r y r e p o r t s h a v e b e e n preserrtedaZ-a~.. T h e g e n e r a l e x p e r i m e n t a l design, age distribtrdi0m ,0¢/fttt~e.-,-mimals, a n a l y t i c a l a n d c o u n t i n g t e c h n i q u e s h a v e b e e n p r e v i o u s l y d e s c r / b e d ~ . tlr~0m~_~=m r e l a t i v e to specific e x p e r i m e n t s is d e s c r i b e d in t h e p e r t i n e n t figure ileg~n~l. C a r t i l a g e m u c o p r o t e i n w a s p r e p a r e d f r o m :ral-d~'t ~b, t t ~ a g e by the m e t h o d of SCHUBERT et al. ~s. M a t e r i a l i n s o l u b l e in w a t e r ~ a s ~e-x~,~.~edl b3: s h a k i n g w i t h IO vol. of 2 % K O H in t h e c o l d for 24 h. T h e IKOt-] ,e~-twa¢~t v~;~. v e p e a t e d t w i c e a n d a f t e r n e u t r a l i z a t i o n t h e soluble p o r t i o n w a s d i a l y z e d :to a~cm0m~• t,m:e.~s salt. H e x o s a m i n e s w e r e l i b e r a t e d f r o m p o l y m e r i c m a t e r i a l b y h v d r 0 / , v ~ ~ ;a ~::eti~¢~ vessel w i t h 4 N HCI for 16 h. A f t e r r e m o v a l of t h e acid, t h e h y d r o l y s a ~ e ,am~. (4fi+.m,)tx:ed in w a t e r a n d t h e g l u c o s a r n i n e a n d g a t a c t o s a m i n e r e s o l v e d b y Ct~.<,~a~0gIratpNy: ~n Dowex--5o. H+ resin *'. Q u a n t i t a t i o n of h e x o s a m i n e c o n t e n t a n d ~za~0mCmJ~,i~rg w a s c a r r i e d o u t as previously deseribedlL Neutral sugars were e ~ i m ~ t ~aff~,~ t~ydrolx-sis for 4 h w i t h r N H ~ S O , . T h e h y d r o l y s a t e w a s n e u t r a l i z e d w i t h ~a'~0)lNli...,:md', f u r t h e r d e i o n i z e d b3; treatment with Dowex-5o, H + and Dowex-~, ~0@C n-~r~. _-~er concentration, s u i t a b l e aliquot.~ w e r e e h r o m a t o g r a p h e d in t h e f,Lloxvihr~.,dl~,,m~+: b u t a n o l - p y r i d i n e w a t e r (6: 4: 3), e t h y l a c e t a t e - p y r i d i n e - w a t e r (-~::~:: ~_I)rc~ne.rr p[mse, b u t a n o l - acetic acid-water (5o : x5" 35), i s o p r o p a n o l - w a t e r , (9::,.-r,).. INtdk~diag c o m p o u n d s w e r e v i s u a l i z e d w i t h a silver n i t r a t e r e a g e n t ~-°. RESI;I.T.~
AND
DISC[~.%IO~'N
T h e c o m p o s i t i o n of t h e h e a v y a n d light m u c o p r o t e i n ~atttii0m~md! t h e residue i.~ s h o w n in T a b l e I. I n a d d i t i o n to t h e h e x o s a r n i n e componerrt.~, tb0~tt~ ~r'~t~u.'tose a n d fucose were d e m o n s t r a b l e in all f r a c t i o n s b y p a p e r chromatogra~ra-;a+dh.~.~i/Sed a b o v e . Q u a n t i t a tion was not attempted. .~
T A I3LE
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HEXOSA.'VlI~H C O N T E N T OF R A B B I T RII3~C'ARTILAG/E BII[TC~tI~0~FI~'~,x~ .-t-~,D~ RE.q|D(TE F H A C T I O N S Values are expressed or galactosamine.
a s [ , m o l e s / g d r y w t . F i g u r e s i n p a r ~ [ l : h t . ~ e , ; t r ~ v [~eE t-t;rlt o f t o t a l g l u c o s a m i n c ~ E a c h f i g u r e is t h e a v e r a g e o f ~¢ 'lu~.=rt :l~l~ttt. ~ m i m a l v t t 5 m o n t h s of age. Control Glta~xa m i tie
Light
mucoprotein fraction
Heavy m ucoprotein fractiun Residue
" Decreases "" Increases
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l~ilgli:3~s._ A:cta,
69
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459-4¢)3
CARTILAGE MUCOPOLYSACCHARIDE METABOLISM
4.6r
Tim a p p e a r a n c e of r a d i o a c t i v i t y in t h e g a l a c t o s a m i n e c o m p o n e n t of t h e m u c o p r o t e i n a n d r e s i d u e f r a c t i o n s is i l l u s t r a t e d i n Fig. I. T h e effect of v a r i o u s h o r m o n e s on t h e r a t e of a p p e a r a n c e of r a d i o a c t i v i t y f r o m gluc os e i n t h e g a l a c t o s a m i n e a n d glucosa m i n e c o m p o n e n t s of t h e l i g h t m u c o p r o t e i n f r a c t i o n is i l l u s t r a t e d in Figs. 3 a n d 4. T h e d i f f e r e n t r a t e s of i n c o r p o r a t i o n of gluc os e i n t o g l u c o s a m i n e a n d g a l a c t o s a m i n e r e s p e c t i v e l y of t h e l i g h t m u c o p r o t e i n of c o n t r o l a n i m a l s is i l l u s t r a t e d i n Fig. 2. T h e o c c u r r e n c e of g l u c o s a m i n e a n d g a l a c t o s a m i n e i n b o t h m u c o p r o t e i n f r a c t i o n s as well as t h e r e s i d u e c o n f i r m s r e s u l t s r e p o r t e d b y GREGORY AND RODEN 2' a n d b y PARTRIDGE a n d c o w o r k e r s x3. A l t h o u g h t h e s e f r a c t i o n s a r e q u a l i t a t i v e l y h o m o g e n e o u s , s a t i s f a c t o r y e v i d e n c e h a s n o t b e e n p r e s e n t e d to e s t a b l i s h t h i s p o i n t . S t u d i e s in cell-free s y s t e m s h a v e s h o w n t h a t u r i d i n e d i p h o s p h o - N - a c e t y l glucosa m i n e s e r v e s as t h e i m m e d i a t e p r e c u r s o r of t h e c o r r e s p o n d i n g g a l a c t o s a m i n e n u c l e o tide-°2, ~3. T h e a p p e a r a n c e of t h e g l u c o s e c a r b o n c h a i n in g a l a c t o s a m i n e p r i o r to glucosa m i n e in t h i s s y s t e m s u g g e s t s t h a t t h e r e is n o t a l i n k e d s y n t h e s i s of t h e k e r a t o s u l f a t e
3
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Fig. 2 I
4
9
Fig. r. Appearance of radioactivity in the galactosamine moiety of rabbit mucoprotein and residue fractions following an injection of uniformly labeled rt4C~glucosel~. Each point is the average of at least two animals, x5 months of age. A, • . light mucoprotein fraction; B, II, heavy mucoprotein fraction; C, A, redidue.
28 Time
(cloys)
Fig. i 3
...........
Fig. 2. Appearance oi radioactivity in galactosamine and glucosamine of the light mucoprotein fraction. Each point is the average of at least two animals, x5 months oi age. • - - 0 , galactosamine, light mueoprotein fraction ; i l - - II, glucosamine, light mucoprotein fraction.
?_;a~ tJ
i~
:~ g~2i
l
3
6
9
Time (doys}
Fig. 3
28
Fig. 3. Effect of growth hormone and cortisone on appearance of radioactivity in the galactosamine moiety of the light mucoprotein fraction, Isotope and hormone administered as previously described~L Each point is the average of at least two animals, I5 months oi age. • 10, growth hormone; • - - • , control; A--~k, cortisone.
Biochim. Biophys. Acta, 69 (I963) 459-463
462
E . A . DAVIDSON, W. SMALL
a n d c h o n d r ~ i d n st~lfate m o i e t i e s p r e s e n t in t h e l i g h t m u c o p r o t e i n f r a c t i o n . I t is p o s s i b l e t h a t . a s w~th c e r t a i n b a c t e r i a l l i p o p o ! y s a c c h a r i d e s , t h e a c c e p t e r s i t e for t h e k e r a t ¢ ~ f l f f a t e m u s t b e p r e s e n t b e f o r e i t s s~nnthesis t a k e s p l a c e e*. T h u s , o n e m a y en,~ision t h a t t h e c h o n d r o i t i n s u l f a t e f o r m s t h e i m m e d i a t e c o v a l e n t l i n k a g e t o p r o t e i n a n d u n t i l t h i s is s y n t h e s i z e d , n o a p p r e c i a b l e s y n t h e s i s of k e r a t o s t f l f a t e m a y t a k e p l a c e . T h e a c t i v e m e t a b o N e b e h a v i o r of t h i s f r a c t i o n c o u p l e d w i t h t h e c o n s i d e r a b l e d e l a v in a p p e ~ ¢ e o f i s o t o p e in k e r a t o s u l f a t e m a k e s t h i s p o s s i b i l i t y e x t r e m e l y f a r f e t c h e d . j~
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Fig. 4. Effect of growth hormone and TSH on appearance of radioactivity in cartilage galactosamine following a single in~ection of uniformly labeled 2l~C~glucose. Hexosamine isolated after direct alkali extraction of tissue Note how mixing "pools" distorts data. Each point average of four animals, ! 5 months off age. A, control g r o u p B, TSH-treated group- C, growth hormone-treated group.
r~ ME (DAYSI
The slow a ~ n c e of i s o t o p e in t h e r e s i d u e f r a c t i o n a n d t h e l o w specific a c t i v i t i e s m a k e a n a c c u r a t e d e t e r m i n a t i o n of p o l y s a c c h a r i d e " h a l f - l i f e ' " difficult. lch'elirninar$_- e_x-periments h a v e b e e n c a r r i e d o u t a n d s u g g e s t a t u r n o v e r t i m e for t h i s f r a c t i o n in e x c e s s e l 4 m o n t h s , i n t h i s t i s s u e , a s in s k i n , t h e p o l y s a c c h a r i d e " p o o l " is n o t h o m Q g e n e o o s a n d a p p a r e n t l y u n d e r g o e s s o m e c h a n g e e i t h e r in p h y s i c a l s t a t e or linkage to protein following s~mthesis from simpler precursors. It would appear that the light mu¢oprotein fraction, operationally defined as being more readily extracta b l e , r e p r e s e n t s t h e first a p p e a r a n c e of n e w l y s ~ n t h e s i z e d p o l y s a c c h a r i d e . S i n c e t h e p r o t e i n moiel3" o f t h e m u c o p r o t e i n c o m p l e x d o e s n o t y i e l d a h o m o g e n e o u s p r o t e i n f r a c t i o n -alRer r e m o v a l o f t h e m a j o r i t y of t h e p o l y s a c c h a r i d e b y e x h a u s t i v e h y a l u r o n i d a s e d i g e s t i o n , i t is e x t r e m e l y d i f f i c u l t t o a.~sign a n y c o h e r e n t s t r u c t u r e t o t h e m u c o protein itselL T h e effe~zts o f c o r t i s o n e a r e in k e e p i n g w i t h p r e v i o u s r e p o r t s of t h e a b i l i t y of t h i s h o r n l o n e t o i n h i b i t s n l f a t e f i x a t i o n a n d a l s o in a g r e e m e n t w i t h t h e g e n e r a I effect of cortisone on other heteropclysaccharide systems ~-~. It would appear that the o b s e r v e d phenolt~er~a a r e n o t d u e to a d i r e c t i n f l u e n c e o n s u l f a t e f i x a t i o n o r c h a i n s~nvathesis b u t r a t h e r t o a n i n f l u e n c e o n t h e a v a i l a b i l i t y of s i m p l e p r e c u r s o r s for t h e fonglation of t h e lpOlysaccharide chain ; this m a v be related to capilla~, p e r m e a b i l i t y , d i f f u s i o n o f n n t r i e n t s t o t h e cell.~ r e s p o n s i b l e for s y n t h e s i s , etc. T h e failmre o f t h e ~C~t:~ERT t e c h n i q u e t o s o l u b i l i z e m o r e t h a n 2o % of t h e t o t a l mue, o p o ~ - s a c c h a r i d e h e x o s a m i n e p r e s e n t in c a r t i l a g e is s o m e w h a t d i s c r e p a n t f r o m r e t o r t e d r e s u l t s o n bo~-ine n a s a l s e p t a 27. T h e a b o v e d a t a a r e for r a b b i t r i b c a r t i l a g e of the ~ age and generalizations to other animals are not valid. The relatively constant ~ i i o n of t h e s o l u b l e z,erstts t h e i n s o l u b l e p o r t i o n s is s o m e w h a t a f f e c t e d b y g r o w t h h e r m o n e a n d b y t h e a g e of t h e a n i m a l . H o w e v e r , sm'ficient s t u d i e s h a v e n o t been carried out to de,he any consistent trend. Biochim. Biopkys. ,4cta, 6x2 ,-'9631 459-463
CARTILAGE
MUCOPOLYSACCHARIDE
463
METABOLISM
T h e p h e n o m e n o n in h u m a n c a r t i l a g e of t h e r e p l a c e m e n t of c h o n d r o i t i n 4 - s u l f a t e b y c h o n d r o i t i n 6 - s u l f a t e w i t h t h e p a s s a g e of t i m e is of c o n s i d e r a b l e i n t e r e s t . T h i s c o n v e r s i o n i n v o l v e s n o t h i n g m o r e c o m p l e x t h a n a t r a n s f e r of s u l f a t e f r o m t h e 4 p o s i t i o n of t h e g a l a c t o s a m i n e m o i e t y t o t h e 6 p o s i t i o n . T h i s m a y r e q u i r e b r e a k d o w n .,nd r e s y n t h e s i s of t h e p o l y s a c c h a r i d e c h a i n , de novo s y n t h e s i s of t h e c h o n d r o i t i n s u l f a t e C f r o m g l u c o s e , or a s i m p l e s u l f o t r a n s f e r a s e m e c h a n i s m . Since s i m i l a r phenom-. e n a h a v e n o t b e e n e s t a b l i s h e d for o t h e r a n i m a l s , it is q u i t e i m p o s s i b l e t o t e s t t h e s e hypotheses experimentally. Considerable evidence has been adduced to strengthen the original proposal by DAVIDSON AND MEYER 28 t h a t h e t e r o p o l y s a c c h a r i d e s u l f a t i o n t a k e s p l a c e s u b s e q u e n t t o c h a i n s y n t h e s i s ~ - a L N e v e r t h e l e s s , d e f i n i t i v e e v i d e n c e a t t h i s p o i n t is m i s s i n g . I t is e s p e c i a l l y t o be n o t e d t h a t all of t h e sulfo t r a n s f e r a s e d a t a r e p o r t e d l e a v e 9 ° % or m o r e of t h e t h e o r e t i c a l h y d r o x y l a c c e p t o r s u n e s t e r i f i e d . I n t h i s i n s t a n c e , a l t h o u g h t h e s u l f a t e g r o u p q u a l i t a t i v e l y m i r r o r s t h e m e t a b o l i c b e h a v i o r of t h e h e x o s e c a r b o n s , q u a n t i t a t i v e s t u d i e s h a v e n o t b e e n c a r r i e d o u t . T h e t u r n o v e r of t h e s u l f a t e g r o u p in t h i s t i s s u e will be t h e s u b j e c t of a s u b s e q u e n t r e p o r t . ACKNOWLEDGEMENTS
T h i s w o r k w a s s u p p o r t e d b y g r a n t s A z9o 3 (C-2), A 4315 (C-I) a n d H - 3 5 8 2 a n d M - 2 t o 9 f r o m t h e N a t i o n a l I n s t i t u t e s of H e a l t h , B e t h e s d a , Md. (U.S.A.}. REFERENCES t |4. M E Y E R , P . HOFFMAN AND A . L I N K E R , Science, I 2 8 (1958) 8 9 6 .
2 D. KAPLAr¢ AND K. MZVER, Nature. I83 (1959) I 2 6 7 . a R. Kt:H,'~ AND H. J. LEVPELMAN, A n n . Chem., 61I (I958) 254. a M. R. SrtErLAR ANn Y. F. MASTERS, Proc. Soc. E x p t l . Biol. ~'tled., 90 (1955) 31. s j. IV. LAsH AriD M. IV. ~VHtTEHOUSE, Biochem. J . , 74 (I96O) 35 I. J. vf. LASH AND M. V¢. VCHITEttOUSE, Arch. Biochem. Biophys., 90 (I96o) I59. D. ~,V. DZIEWIATr~OWSKI, J. Biol. Chem., I89 (I95I) 187. a H. BOSTROM AND B. MANSSON, .4rhiv K e m i , 6 (I953) 23. 9 l-]. I~OSTRO-M AND E . (.)DEBLAD, o4rhill I ( e m i , 6 (1953) 39I.. L . LAYTON, Proc. Soc. E x p l l . Biol. Meal., 76 (195 I) 5 9 6 . 11 j . SHATTON AND i . SCHUBERT, J . Biol. Chem., 2 I I (1954) 505. to
~2 H. MUIR, Biochem. J . , 69 (1958) 195. la S. M . P A R T R I D G E , H . F . DAVIS AND G- A D A I R , Biochem. J . , 79 ( I 9 6 1 ) I5 14 ( ; . B E R N A O I , B i o c h i m . B i o p h y s . Acta, 26 (1957) 4715 E . A. DAVIDSON, ~,~,'. SMALL, P . P E R C H E M L I D E S A N n IV. BAXLEY, B i o c h i m .
B i o # h y s . Acla,
46 (t961) 189.
16 E . A. DAVIDSON, IV. S.~IALL AND P . PI~-RCHEMLIDES, Federation l:~roc., 2o ( I 9 6 1 ) I 6 2 . 17 E . A. DAVIDSON AND ~,lv'. SMALL, Biochiat. B i o p h y s . Acta, 6 9 (1963) 445~s B . I(. G E R B E R , E . C. F R A N K L I N AND M. SCHUBERT, J . Biol. Chem., 235 (196o) 287o.
19 S. GARDELL, Acta Chem. Scand., 7 (1953) 207. t0 V~r. TREVELYAN AND S. IIARRISON, Biochem. J . , 5 ° ( I 9 5 2 ) -'98. zt j . GREGORY A N n L . R O O E N , Biochem. B i o p h y s . Res. C o m m u n . , 5 (1961) 43 ° . -"2L. G L A S E R , J . Biol. Chem., 234 (1959) z8ot.
2a B. JACOBSON AND E. A. DAVIDSON. Biochim. Biophys. Acta, in the press. ** H. NIKAIDO, Biochim. Biophys. Acta, 48 (196I) 46o. 25 S. S C H I L L E R AND A. DORFMAN, E::drocrinology, 6 0 (1957) 370.
ea E. A. DAVtDSON AND \V. S.~iALL, Biochim. Biophys. Acta, 69 (1965) 453,7 I. ~iAI.AXVISTA AND M . SCHUBERT, J . Biol. Cheat., 2250 (1958) 535-
z~ E, z~ j. a, E. at S.
A. DAv~!~soN AND K. MEYER. J . Biol. Chem., 211 (I954) 605. B. ADA.~tS, Nature, I86 (190o) 555. A. I)AVlOSON AND J. G. RILEY, J . Biol. Chem., 235 (I96o) 3367. SUZVKt AriD J. L. STRO,~itSG~R, J . Biol. Chem., 235 (t96o) 267. Bioahim. Biophys. Acla, 69 (1963) 459-463
459
BBA 3 9 5 I
METABOLISM
IN
VIVO
OF
CONNECTIVE-TISSUE
MUCOPOLYSACCHARI IiI. CHONDROITIN
SULFATE
AND
DES KERATOSULFATE
OF
CARTILAGE E. A. DAVIDSON AXD WILLIAM SMAIA. Department of Biochemistry and Center f o r the Study of .4giJ2g, Duke University 3tledical Ce,t/er, Durham, N . C. ( U . S . A . )
(Received September 24th, 1962)
SUMMARY T h e i n c o r p o r a t i o n of glucose i n t o t h e h e x o s a m i n e m o i e t i e s of c a r t i l a g e m u c o p o l y s a c c h a r i d e s h a s b e e n s t u d i e d . I s o t o p e a p p e a r s in t h e g a l a c t o s a m i n e m o i e t y of t h e light m u c o p r o t e i n f r a c t i o n first a n d r a p i d l y d i s a p p e a r s f r o m t h i s f r a c t i o n w i t h a n a c c o m p a n y i n g a p p e a r a n c e of i s o t o p e in t h e i n s o l u b l e residue. C h o n d r o i t i n s u l f a t e a c c o u n t s for a b o u t 80 % of t h e t o t a l p o l y s a c c h a r i d e a n d a p p e a r s t o be r e l a t i v e l y i n e r t m e t a b o l i c a l l y . A p p e a r a n c e of label in c h o n d r o i t i n s u l f a t e p r i o r t o k e r a t o s u l f a t e s u g g e s t s t h a t t h e s e p o l y s a c c h a r i d e s do n o t r e p r e s e n t p a r t of t h e s a m e m a c r o m o l e c u l a r s t r u c t u r e . T h i s l a t t e r p o i n t is n o t e s t a b l i s h e d . T h e effect of s e v e r a l h o r m o n e s on t h e i n c o r p o r a t i o n a n d t u r n o v e r of i s o t o p e in +h~,. ~,,o ~: . s ~. . . ~. . ::'as also r e p o r t e d .
INTRODUCTION C a r t i l a g e h a s b e e n one of t h e m o s t e x t e n s i v e l y s t u d i e d of all c o n n e c t i v e tissues. It h a s b e e n u s e d as a s o u r c e of c h o n d r o i t i n s u l f a t e since t h e I g t h c e n t u r y a n d a l t h o u g h t h e c o m p o s i t i o n of t h e p o l y s a c c h a r i d e f r a c t i o n w a s o r i g i n a l l y t h o u g h t t o be exclusively c h o n d r o i t i n 4 - s u l f a t e , k e r a t o s u l f a t e a n d c h o n d r o i t i n 6 - s u l f a t e h a v e also b e e n d e t e c t e d 1. S t u d i e s in s e v e r a l l a b o r a t o r i e s on h u m a n m a t e r i a l h a v e r e v e a l e d a g e - d e p e n d e n t c o m p o s i t i o n c h a n g e s 2-5. I n p a r t i c u l a r , t h e r e is a c o n t i n u o u s i n c r e a s e in t h e k e r a t o s u l f a t e c o n t e n t t h r o u g h t h e s e c o n d d e c a d e in life a n d t h e c h o n d r o i t i n 4s u l f a t e is p r o g r e s s i v e l y r e p l a c e d b y c h o n d r o i t i n 6-sulfate. Q u a l i t a t i v e s t u d i e s s u g g e s t t h a t k e r a t o s u l f a t e also i n c r e a s e s in s h a r k a n d s q u i d cartilage% C a r t i l a g e h a s b e e n u s e d in s t u d i e s of r a d i o - s u l f a t e f i x a t i o n a n d t h e effect of h o r m o n e s on s u c h p r o c e s s e s h a s b e e n reporte&-X% T h e p h y s i c a l s t a t e of t h e h e t e r o p o l y s a c c h a r i d e s in t h i s t i s s u e h a s b e e n t h e s u b j e c t of c o n s i d e r a b l e i n v e s t i g a t i o n . T h e c h o n d r o i t i n s u l f a t e m a y be e x t r a c t e d f r o m c a r t i l a g e in t h e f o r m of a p r o t e i n c o m p l e x w h o s e p r o p e r t i e s s u g g e s t s o m e t h i n g m o r e t h a n s i m p l e e l e c t r o s t a t i c a t t r a c t i o n n-a4. Tile h o m o g e n e i t y of t h i s c o m p l e x h a s n o t b e e n Biochim. Bioph3,s, Acta, 69 (I963) 459-463
460
E.A.
DAVIDSON,
,XV..q~aT~
estaot]snett'-' 't,u, ~.~,: . . . ., ,. ,.p c u,~,,~ . . . s .u g. g.e s.t . c,,,coutt~,~**~:~---~ . . . ~,,,.,~m~ox'~ ~ r~ ~.~ at.. lem~t p a r t of t h e p r o t e i n m o i e t y a n d t h e p o l y s a c c h a r i d e chain. T h e p r e s e n t s t u d y r e p o r t s t h e c o m p o s i t i o n ,o~ ~[pa~r~eiira~ ~.ornp[exes i s o l a t e d f r o m r a b b i t Iib c a r t ilage, t h e i n c o r p o r a t i o n of c a r b o h y ~ a ~ e it~.~um-~o~- i!nto t h e h e x o s a m i n e c o m p o n e n t s of t h e pol3;saccharides a n d t h e effect ,¢¢ff,~:an6i0r~ lhgrTnonal a g e n t s on t h i s process. P r e l i m i n a r y r e p o r t s h a v e b e e n preserrtedaZ-a~.. T h e g e n e r a l e x p e r i m e n t a l design, age distribtrdi0m ,0¢/fttt~e.-,-mimals, a n a l y t i c a l a n d c o u n t i n g t e c h n i q u e s h a v e b e e n p r e v i o u s l y d e s c r / b e d ~ . tlr~0m~_~=m r e l a t i v e to specific e x p e r i m e n t s is d e s c r i b e d in t h e p e r t i n e n t figure ileg~n~l. C a r t i l a g e m u c o p r o t e i n w a s p r e p a r e d f r o m :ral-d~'t ~b, t t ~ a g e by the m e t h o d of SCHUBERT et al. ~s. M a t e r i a l i n s o l u b l e in w a t e r ~ a s ~e-x~,~.~edl b3: s h a k i n g w i t h IO vol. of 2 % K O H in t h e c o l d for 24 h. T h e IKOt-] ,e~-twa¢~t v~;~. v e p e a t e d t w i c e a n d a f t e r n e u t r a l i z a t i o n t h e soluble p o r t i o n w a s d i a l y z e d :to a~cm0m~• t,m:e.~s salt. H e x o s a m i n e s w e r e l i b e r a t e d f r o m p o l y m e r i c m a t e r i a l b y h v d r 0 / , v ~ ~ ;a ~::eti~¢~ vessel w i t h 4 N HCI for 16 h. A f t e r r e m o v a l of t h e acid, t h e h y d r o l y s a ~ e ,am~. (4fi+.m,)tx:ed in w a t e r a n d t h e g l u c o s a r n i n e a n d g a t a c t o s a m i n e r e s o l v e d b y Ct~.<,~a~0gIratpNy: ~n Dowex--5o. H+ resin *'. Q u a n t i t a t i o n of h e x o s a m i n e c o n t e n t a n d ~za~0mCmJ~,i~rg w a s c a r r i e d o u t as previously deseribedlL Neutral sugars were e ~ i m ~ t ~aff~,~ t~ydrolx-sis for 4 h w i t h r N H ~ S O , . T h e h y d r o l y s a t e w a s n e u t r a l i z e d w i t h ~a'~0)lNli...,:md', f u r t h e r d e i o n i z e d b3; treatment with Dowex-5o, H + and Dowex-~, ~0@C n-~r~. _-~er concentration, s u i t a b l e aliquot.~ w e r e e h r o m a t o g r a p h e d in t h e f,Lloxvihr~.,dl~,,m~+: b u t a n o l - p y r i d i n e w a t e r (6: 4: 3), e t h y l a c e t a t e - p y r i d i n e - w a t e r (-~::~:: ~_I)rc~ne.rr p[mse, b u t a n o l - acetic acid-water (5o : x5" 35), i s o p r o p a n o l - w a t e r , (9::,.-r,).. INtdk~diag c o m p o u n d s w e r e v i s u a l i z e d w i t h a silver n i t r a t e r e a g e n t ~-°. RESI;I.T.~
AND
DISC[~.%IO~'N
T h e c o m p o s i t i o n of t h e h e a v y a n d light m u c o p r o t e i n ~atttii0m~md! t h e residue i.~ s h o w n in T a b l e I. I n a d d i t i o n to t h e h e x o s a r n i n e componerrt.~, tb0~tt~ ~r'~t~u.'tose a n d fucose were d e m o n s t r a b l e in all f r a c t i o n s b y p a p e r chromatogra~ra-;a+dh.~.~i/Sed a b o v e . Q u a n t i t a tion was not attempted. .~
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HEXOSA.'VlI~H C O N T E N T OF R A B B I T RII3~C'ARTILAG/E BII[TC~tI~0~FI~'~,x~ .-t-~,D~ RE.q|D(TE F H A C T I O N S Values are expressed or galactosamine.
a s [ , m o l e s / g d r y w t . F i g u r e s i n p a r ~ [ l : h t . ~ e , ; t r ~ v [~eE t-t;rlt o f t o t a l g l u c o s a m i n c ~ E a c h f i g u r e is t h e a v e r a g e o f ~¢ 'lu~.=rt :l~l~ttt. ~ m i m a l v t t 5 m o n t h s of age. Control Glta~xa m i tie
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Tim a p p e a r a n c e of r a d i o a c t i v i t y in t h e g a l a c t o s a m i n e c o m p o n e n t of t h e m u c o p r o t e i n a n d r e s i d u e f r a c t i o n s is i l l u s t r a t e d i n Fig. I. T h e effect of v a r i o u s h o r m o n e s on t h e r a t e of a p p e a r a n c e of r a d i o a c t i v i t y f r o m gluc os e i n t h e g a l a c t o s a m i n e a n d glucosa m i n e c o m p o n e n t s of t h e l i g h t m u c o p r o t e i n f r a c t i o n is i l l u s t r a t e d in Figs. 3 a n d 4. T h e d i f f e r e n t r a t e s of i n c o r p o r a t i o n of gluc os e i n t o g l u c o s a m i n e a n d g a l a c t o s a m i n e r e s p e c t i v e l y of t h e l i g h t m u c o p r o t e i n of c o n t r o l a n i m a l s is i l l u s t r a t e d i n Fig. 2. T h e o c c u r r e n c e of g l u c o s a m i n e a n d g a l a c t o s a m i n e i n b o t h m u c o p r o t e i n f r a c t i o n s as well as t h e r e s i d u e c o n f i r m s r e s u l t s r e p o r t e d b y GREGORY AND RODEN 2' a n d b y PARTRIDGE a n d c o w o r k e r s x3. A l t h o u g h t h e s e f r a c t i o n s a r e q u a l i t a t i v e l y h o m o g e n e o u s , s a t i s f a c t o r y e v i d e n c e h a s n o t b e e n p r e s e n t e d to e s t a b l i s h t h i s p o i n t . S t u d i e s in cell-free s y s t e m s h a v e s h o w n t h a t u r i d i n e d i p h o s p h o - N - a c e t y l glucosa m i n e s e r v e s as t h e i m m e d i a t e p r e c u r s o r of t h e c o r r e s p o n d i n g g a l a c t o s a m i n e n u c l e o tide-°2, ~3. T h e a p p e a r a n c e of t h e g l u c o s e c a r b o n c h a i n in g a l a c t o s a m i n e p r i o r to glucosa m i n e in t h i s s y s t e m s u g g e s t s t h a t t h e r e is n o t a l i n k e d s y n t h e s i s of t h e k e r a t o s u l f a t e
3
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Fig. r. Appearance of radioactivity in the galactosamine moiety of rabbit mucoprotein and residue fractions following an injection of uniformly labeled rt4C~glucosel~. Each point is the average of at least two animals, x5 months of age. A, • . light mucoprotein fraction; B, II, heavy mucoprotein fraction; C, A, redidue.
28 Time
(cloys)
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...........
Fig. 2. Appearance oi radioactivity in galactosamine and glucosamine of the light mucoprotein fraction. Each point is the average of at least two animals, x5 months oi age. • - - 0 , galactosamine, light mueoprotein fraction ; i l - - II, glucosamine, light mucoprotein fraction.
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Fig. 3. Effect of growth hormone and cortisone on appearance of radioactivity in the galactosamine moiety of the light mucoprotein fraction, Isotope and hormone administered as previously described~L Each point is the average of at least two animals, I5 months oi age. • 10, growth hormone; • - - • , control; A--~k, cortisone.
Biochim. Biophys. Acta, 69 (I963) 459-463
462
E . A . DAVIDSON, W. SMALL
a n d c h o n d r ~ i d n st~lfate m o i e t i e s p r e s e n t in t h e l i g h t m u c o p r o t e i n f r a c t i o n . I t is p o s s i b l e t h a t . a s w~th c e r t a i n b a c t e r i a l l i p o p o ! y s a c c h a r i d e s , t h e a c c e p t e r s i t e for t h e k e r a t ¢ ~ f l f f a t e m u s t b e p r e s e n t b e f o r e i t s s~nnthesis t a k e s p l a c e e*. T h u s , o n e m a y en,~ision t h a t t h e c h o n d r o i t i n s u l f a t e f o r m s t h e i m m e d i a t e c o v a l e n t l i n k a g e t o p r o t e i n a n d u n t i l t h i s is s y n t h e s i z e d , n o a p p r e c i a b l e s y n t h e s i s of k e r a t o s t f l f a t e m a y t a k e p l a c e . T h e a c t i v e m e t a b o N e b e h a v i o r of t h i s f r a c t i o n c o u p l e d w i t h t h e c o n s i d e r a b l e d e l a v in a p p e ~ ¢ e o f i s o t o p e in k e r a t o s u l f a t e m a k e s t h i s p o s s i b i l i t y e x t r e m e l y f a r f e t c h e d . j~
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Fig. 4. Effect of growth hormone and TSH on appearance of radioactivity in cartilage galactosamine following a single in~ection of uniformly labeled 2l~C~glucose. Hexosamine isolated after direct alkali extraction of tissue Note how mixing "pools" distorts data. Each point average of four animals, ! 5 months off age. A, control g r o u p B, TSH-treated group- C, growth hormone-treated group.
r~ ME (DAYSI
The slow a ~ n c e of i s o t o p e in t h e r e s i d u e f r a c t i o n a n d t h e l o w specific a c t i v i t i e s m a k e a n a c c u r a t e d e t e r m i n a t i o n of p o l y s a c c h a r i d e " h a l f - l i f e ' " difficult. lch'elirninar$_- e_x-periments h a v e b e e n c a r r i e d o u t a n d s u g g e s t a t u r n o v e r t i m e for t h i s f r a c t i o n in e x c e s s e l 4 m o n t h s , i n t h i s t i s s u e , a s in s k i n , t h e p o l y s a c c h a r i d e " p o o l " is n o t h o m Q g e n e o o s a n d a p p a r e n t l y u n d e r g o e s s o m e c h a n g e e i t h e r in p h y s i c a l s t a t e or linkage to protein following s~mthesis from simpler precursors. It would appear that the light mu¢oprotein fraction, operationally defined as being more readily extracta b l e , r e p r e s e n t s t h e first a p p e a r a n c e of n e w l y s ~ n t h e s i z e d p o l y s a c c h a r i d e . S i n c e t h e p r o t e i n moiel3" o f t h e m u c o p r o t e i n c o m p l e x d o e s n o t y i e l d a h o m o g e n e o u s p r o t e i n f r a c t i o n -alRer r e m o v a l o f t h e m a j o r i t y of t h e p o l y s a c c h a r i d e b y e x h a u s t i v e h y a l u r o n i d a s e d i g e s t i o n , i t is e x t r e m e l y d i f f i c u l t t o a.~sign a n y c o h e r e n t s t r u c t u r e t o t h e m u c o protein itselL T h e effe~zts o f c o r t i s o n e a r e in k e e p i n g w i t h p r e v i o u s r e p o r t s of t h e a b i l i t y of t h i s h o r n l o n e t o i n h i b i t s n l f a t e f i x a t i o n a n d a l s o in a g r e e m e n t w i t h t h e g e n e r a I effect of cortisone on other heteropclysaccharide systems ~-~. It would appear that the o b s e r v e d phenolt~er~a a r e n o t d u e to a d i r e c t i n f l u e n c e o n s u l f a t e f i x a t i o n o r c h a i n s~nvathesis b u t r a t h e r t o a n i n f l u e n c e o n t h e a v a i l a b i l i t y of s i m p l e p r e c u r s o r s for t h e fonglation of t h e lpOlysaccharide chain ; this m a v be related to capilla~, p e r m e a b i l i t y , d i f f u s i o n o f n n t r i e n t s t o t h e cell.~ r e s p o n s i b l e for s y n t h e s i s , etc. T h e failmre o f t h e ~C~t:~ERT t e c h n i q u e t o s o l u b i l i z e m o r e t h a n 2o % of t h e t o t a l mue, o p o ~ - s a c c h a r i d e h e x o s a m i n e p r e s e n t in c a r t i l a g e is s o m e w h a t d i s c r e p a n t f r o m r e t o r t e d r e s u l t s o n bo~-ine n a s a l s e p t a 27. T h e a b o v e d a t a a r e for r a b b i t r i b c a r t i l a g e of the ~ age and generalizations to other animals are not valid. The relatively constant ~ i i o n of t h e s o l u b l e z,erstts t h e i n s o l u b l e p o r t i o n s is s o m e w h a t a f f e c t e d b y g r o w t h h e r m o n e a n d b y t h e a g e of t h e a n i m a l . H o w e v e r , sm'ficient s t u d i e s h a v e n o t been carried out to de,he any consistent trend. Biochim. Biopkys. ,4cta, 6x2 ,-'9631 459-463
CARTILAGE
MUCOPOLYSACCHARIDE
463
METABOLISM
T h e p h e n o m e n o n in h u m a n c a r t i l a g e of t h e r e p l a c e m e n t of c h o n d r o i t i n 4 - s u l f a t e b y c h o n d r o i t i n 6 - s u l f a t e w i t h t h e p a s s a g e of t i m e is of c o n s i d e r a b l e i n t e r e s t . T h i s c o n v e r s i o n i n v o l v e s n o t h i n g m o r e c o m p l e x t h a n a t r a n s f e r of s u l f a t e f r o m t h e 4 p o s i t i o n of t h e g a l a c t o s a m i n e m o i e t y t o t h e 6 p o s i t i o n . T h i s m a y r e q u i r e b r e a k d o w n .,nd r e s y n t h e s i s of t h e p o l y s a c c h a r i d e c h a i n , de novo s y n t h e s i s of t h e c h o n d r o i t i n s u l f a t e C f r o m g l u c o s e , or a s i m p l e s u l f o t r a n s f e r a s e m e c h a n i s m . Since s i m i l a r phenom-. e n a h a v e n o t b e e n e s t a b l i s h e d for o t h e r a n i m a l s , it is q u i t e i m p o s s i b l e t o t e s t t h e s e hypotheses experimentally. Considerable evidence has been adduced to strengthen the original proposal by DAVIDSON AND MEYER 28 t h a t h e t e r o p o l y s a c c h a r i d e s u l f a t i o n t a k e s p l a c e s u b s e q u e n t t o c h a i n s y n t h e s i s ~ - a L N e v e r t h e l e s s , d e f i n i t i v e e v i d e n c e a t t h i s p o i n t is m i s s i n g . I t is e s p e c i a l l y t o be n o t e d t h a t all of t h e sulfo t r a n s f e r a s e d a t a r e p o r t e d l e a v e 9 ° % or m o r e of t h e t h e o r e t i c a l h y d r o x y l a c c e p t o r s u n e s t e r i f i e d . I n t h i s i n s t a n c e , a l t h o u g h t h e s u l f a t e g r o u p q u a l i t a t i v e l y m i r r o r s t h e m e t a b o l i c b e h a v i o r of t h e h e x o s e c a r b o n s , q u a n t i t a t i v e s t u d i e s h a v e n o t b e e n c a r r i e d o u t . T h e t u r n o v e r of t h e s u l f a t e g r o u p in t h i s t i s s u e will be t h e s u b j e c t of a s u b s e q u e n t r e p o r t . ACKNOWLEDGEMENTS
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