Sulfotransferases of ocular tissue

Exlotl Eye Rcs. (1970) 10, 156-1,58

L E t t E R TO T H E I~DITO]~.S

S u l f o t r a n s f e r a s e s o f O c u l a r Tissue* T h e conversion o£ inorganic sulfate to a n o r g a n i c a l l y b o u n d sulfate deriv~tive is a reag.tion of i m p o r t a n c e in detoxifieation a n d in t h e synthesis of steroid sulfates, stflfa.ted glyc~osa.minoglycans, a n d su]folipids (Bostr6m and "Vestermurk, I961; Gregory a n d lRobbins, 1960; ~ o y , 1960; Bos~rSm a n d R e d a n , 1966). I n t h e ca.so o£ ocular tissue, o n l y in the cornea, has tlm conversion of inorganic '~'~S-labeled sulfate to ester slfl/'~te been d e m o n s t r a t e d , m o s t l y w i t h regard to the synthesis of p h e n y l stfl£ate a n d sulfated g l y c o s a m i n o g l y c a n s (for a revimv of t h e presence 'rod m e t a b o l i s m of g l y c o s a m i n o g l y c a n s iu ocular tissue, see B~dazs, ] 965). The prese:~t c o m m u n i c a t i o n represent~s a p r e l i m i n a r y survey o£ t,he a b i l i t y o:f various ocular tissues to c o n v e r t inorganic sulf~t,e to a b o l m d fm'm. The studies of ~Vengtc a.nd Bost~r6m (1963). -Wengle (1963), BostrSm a n d Wengle (1964) a n d Wengle (1964a,b) have d e a l t in detail wit].l t h e c a p a c i t y of e x t r a c t s of m a n y m ~ m m a l i a n tissues to transfer sn]fat:e from r a d i o a c t i v e PA_Ps~SI- to accepters such as phenol and a v a r i e t y of st,e.roh]s. All m e t h o d s utilized in t,hese studies h a v e been previously described (see e.~pc,.:ial]y Wengl(;, 1964a). P h e n o l mid two steroids have been employed as sulfate accepters in these e.xperimen~s. The following tissues were collected ~'rom calf eyes a n d kept frozen a n t i l use : iris; ciliary b o d y ; conjunct.ira; cornea; lens; r e t i n a ; limlms; the sedhue,~.t, t'rom [.}te occipital vitreous (hy~locytes) a n d :h'ontal vitreous (hyalocytes ~md ciliary cpib!,elium). W i t h t h e exception of t h e corneal ~nd vitreous p r e p a r a t i o n s , ,~.xtracts of each ~issue wm'e prepared by homogenization in fmtr volumes of 0-].5 _~t Kfi'!-0-00! ~ ~:ersene, p I [ 7-0, using an all glass P o t t c r - E l v e h j e m homogmdzer, t~ollowing ccnt,rifl~gabkm at 105,000 g fox" 60 rain, the clear s u p e r t m t a n t was used as enzyme source. AK.hough some sulfotrm~sferase a c t i v i t y m ~ y tmve been present in the. 105,000 g smlime.nt, ouly tile suI~ernatant, w h i c h is k n o w n to contai~ phenol su!fot.ransf~ra.~e a c t i v i t y ('~Vengle a n d Bostzr6m, 1963) was assayed in the present studies. I n t.he case. of t h e corneal and vitreous preparations, t h e tissues were t~rst £roze~ iu liquid n i t r o g e n and. g r o u n d in a m o l a r a t liquid nil;rogen temperature, before homogenization, a n d cent.rifugation. :Pz*k_~as8was prepared from a yea,st extra,or a,nd 1--2 ,no o£ c~rrie.r-~¥ce Rv~e~SOa as desoribect b y Wengle (1964a). ~kliquots of this reaction mix*,ure, cont a i n i n g :pApa~s were t,hen i n c u b a t e d with t h e same. volume (50/~1) of tissue e:vtraces a n d each accept.or. Since mos~ tissue e x t r a c t s assayed b y ]3ostrSm and ~Vengle showed t h e presence of endogenous sulfa,re accepters, an i n c u b a t i o n with no added a c c e p t e r was also izcluded in each series. _4_fret i n c u b a t i o n , a n y remairdng i norga.nic sulfate, _4~P8, a n d ]?A_PS were r e m o v e d quantit-0.tively b y p r e c i p i t a t i o n ~ i t h Ba.(OH)e as described b y Wengle (196.4a). All i n c u b a t i o n s were r u n .;n dui01icate a n d a control c o n t a i n i n g boiled e x t r a c t wa.s also carried tl~rough the entire procedure. The radioa~tive p r o d u c t in t h e s , t p e r n a t ~ n t solution wz~s t h e n assayed. R a d i o a c t i v i t y determina~ions were r u n in" duplicate in a l u m i n i u m p l a n e h e t s counted in a Geiger-~I~I]o.r * T h i s w o r k w a s auppor~.~cl in p a r s b y ~ :Public H e a l t h Serv-ica~ ~ r a u t IN-o. ~N3303370 f r o m t h e k'~ationM I n s t i t u t e o f ~¢t~rological Diseases a n d :Blindness, aacl G r a n ~ lxTo. I-~E-05472. ~-:PAk~S: 3' phespho~t.tenosine-5" phosphost~lfate; AYES: ~.denosine 5' p h o s p h o a u l f a t e ; :DO0: 21h y d ~ x y - A * p r e g n e n e - 3 , 20-d[one (11-deoxycor~icoeterono) ; DI~DX: 3fl-bydroxy-A" - a n d r o s t o n u - 1 7 - o n o ( d e h y ~ r rmpiandrost~ron o).

LETTElZ

TO

TI~[F, E D I T O R S

I57

counter (mica end window tube, 1-9 rag/era"-). The synthesis of radioactive p h e n y i sul:t~rte was confirmed b y two-dimensional paper c h r o m a t o g r a p h y in phenol: water (4 : 1) and butanol-acetic acid-water (12 : 3 : 5) followed b y a u t o r a d i o g r a p h y for 1 week. As can be seen from the d a t a in Table I, all of the ocuJar tissues stud.ied, w i t h the exception of the vitreous preparations, were able to catalyze the s3mthesis of phenyt sulfate from .PAPasS. The failure of the vitreous prep~rations to catalyze sulfate t n m s f e r ma.y have been due to the v e r y s~.all a m o u n t s of m a t e r i a l available..&[1 extracts contained endogmmus accepters of ra, lioaetivity and, of the extract~ as.~yed, only the lens contained a n y significant aot.i~,-iby, above the value for the endogenous accepter, in transferring sulfate to D 0 C and t.he l i m b u s preparation possessed some T ~ I

Sutfotransfera.~e activity of tissue extracts

I ria Ciliary body lletim~ Lens IAmbus Conjunetiv,x Cornc,~ Occil3it,al v i t r e o u s Anterior vitreous

]Phenol

DOC

5031 1850 5189 1172 3875 97 ° 3331 24 S0

1072 2,tl) 257 325 330 127 498 S 30

ct.s/min ])HA

1126 278 301 254

492 I98 58.1. 14

37

:Endogenous

1193 215 321 187 327 136 557

1 S

S_peeifie a c t i v i t y * Phenol Endogenm, s

2676 1011 1059 15 10,5'64 1451 2562 -~

635 117 66 2 908 201 428 ---

I n c u b a t i o n s cont~tincd 3-5 ml~ m o l e s :P.~P~aS, ID t u ~ m o l e s .~ecepbor, 50 ~1 tL~sue e x t r a c t . , a n d 50/~} o f ~ b u f f ~ r c o n s i s t i n g o f e q u a l p a r t s o f 3IgClz (0-005 3z), K,,SO~(0-03 ~t), l ~ I t z l ) O 4 ( 0 - 3 ~I), p/c[ 6-N. I n c u b a . t i o n w a s c a m ' l e d o u t a~ 37 ° f o r 60 rain. A f t e r h e a t i ~ g for 1 m i t t e.g I 0 0 °, t h e r m m t i o n m i x t l z r e s ~,-ere a s s a y e d for es~.er s u l f a t e , a f t e r r e m o v a l o f i n o ~ g a n l c sulfuric, A P S ~ n d I'~kPS b y t h e m e ~ h o d o f ~iVengle ( 1 9 g 4 a ) . * cts/min]mg protein (calculated t~om Kjcldahl dcterminationN

(I-25)

a.et.ivity in sulfate transfer to DHA. A_tthmkgh lens exit-tact possessed a c t i v i t y in catalyzing sulfate t~ramafer, the specific activivy is low due to the large ammmt.s of protein in the extract diluting the a m o u n t of sulfotransfe.ro,se e n z y ~ e present. Of the ocular tissues studied, regina a n d cornea are k n o w n to contain sulfaf,ed glycosaminoglycans, while histochemieal evidence indicates t h a t the hatercellular space o.f the nonpig~nented e p i t h e l i u m of the ciliary b o d y a d j a c e n t to the vitreous contains gIyeosaminoglyca~s (Be,lees, 1965; Berman, 1968), therefore, the endogenous accepter in some of these preparations m a y have been glycosaminoglyc~n. ~ r o r t m ~ n (.1961) has shown t h a t cornea,1 e p i t h e l i u m contains phenol a,nd glycosaminoglycan sulf.otrausferase activity. Thd presence of sulfated glyeosaminoglyc,~ns in other tiss.~es listed in Table I has ~o~ been studied arid further experim.enta are necessary in order to characterize the endogenous accepter in the~e e~-tr~at~, t t is of interest, t h a t the tissues with the lowest suffotran~erase a c t i v i t y were the cell preparations from the vibreous. These ceils (hyaloc)~es) produce the noz~sulfat~d gtyeoc~minoglycan hyah~rGnJc acid (~)sterIin and J a c o b s e n , 1968a,b).

I58

L E T T E R , TO T.~IE E D I T O R S

The results reported herein, although indicative only of the presence of suIfotransferase activity in ocular tissues, may pave the w~y for further studies on the physiological sulfate acceptors i~l the tissues studied. ACKNOWLEDGMENT We would like to express our gratitude to Dr ]3o Wengle whose experience iu this field was of invahmble aid in these experiments.

DeTartme~g of Connective Tissue Research, Boston Biomedical Research Institute, 20 Staniford Street Boston, Mass. 02111, U.S.A.

BJ,:a~'a~v JAcoI~so.~" E~Dez A. BALAZS

St. Erik Hospital, Stockholm, Sweden

{-IAI~RY :BosTI~031

Received 17 September 1969, Boston I%EFER, ENCE S Balazs, E, A. (1965). in The Amino 5'ugar8, vol. IIA, p. 401. (Ed. by Balazs, E. A. and Jeatdoz, R.). Academic Press, ~'ow York. Bernmn, E. ~,. (1968). Biochem. J. 108, 75. Bos~rSm, ~. and Vestermark, A. (196t). Biochem, Phar~acol. 6, 72. Bostx61a, H. and Wenglc, B. (1964). Acla Soc. Med. UT,.salicn. 69, 41. Bostr6m, H. and t~o,.ldn, L. (1966). In The Amino Sugars. voI. 1113, p. 46. (Ed. by Balazs, E. A. and ,leanloz, R.). Academic Press, New York. Grego~T, J. D. and ~obbins, P. W. (1960). Ann. l?ev. Biochem. 29, 347. Osterlin, S. and Jacobson, B. (I968a). Exptl Eye Pea. 7, 497. 0sterlin, S. and Jacobson, B. (1968b). ~a:pil Eye R~s, 7, 51 ]. l ~ y , A. B. (I960). Advan. Euzymol. 22, 205. WengIe, B. and Bostr6m, H. (I963). Acta Chem.. So,tend. 17, 1203. Wer~le, B. (1993). AcAa 8o0. ,Ved. Up.s~l~en68, :154. Wengle, ]3. (1964a). Acta, Chem. 5~car~l. 18, 65. Weng]o, B., (1964b). Acta ~?oc. Med. Upsalien 69, t05. Wortman, B. (1961). J. Biol. Chem. 236, 974.