A C T A U N I V E R S I T A T I S L O D Z I E N S I S FOLIA BIOCHIMICA KT BIOPHYSICA 6, 1988
J a dw i g a G n i ot - S z ul i yc k a, A nn a J a k ub o w s k a
O E S T R O N E S U L P HA T E S U L P HO H Y D R O L AS E
AN D A R Y L S U L P H A T A S E C A C T I VI T I E S OF HU M A N P L A C E N T A CE LL NU CLE I A N D N U C L E A R S U B F RA C TI O NS
The procedure of isolation of cell nuclei and nuclear envelopes from human placenta was described. The nuclear preparations consist of 56.4% of protein, 37% of DNA, 5.02% of phospholipid and 1.42% of RNA. The protein to DNA ratio varies from 1.20-1.94; the E23()/®260 an(* E 260^E280 rati-os are 0.689, respectively. The purified cell nuclei were subfractionated into: nuclear sap, histone rich fraction and chromatin-membrane fraction. Successive digestion with deoxyribonuclease of the chromatinmembrane fraction lead to getting the n u -clear envelopes which contained: 59.4% of protein, 1-12% of DNA and 28.6% of phospholipid.
There is evidence that oestrone sulphohydrolase and arylsulphatase C are integral parts of human placenta cell nuclei. The enzyme acti-vities are associated with the nuclear envelopes and with non-histone chromatin proteins tightly bound with DNA.
INTRODUCTION
T he s u bd i v i s i o n of a r yl s u l p h a t e h y d ro l y s i n g e n zy m e s int o type I (mic rosomal) an d t yp e II (l ysosomal) o u t co m e s f ro m ex p er i me n ts on s u b c e l lu l a r d i s t r i b u t i o n of the e nz y me ac ti vi ti es . T he u se of 2 - h y dr o x y - 5 - n i t r o p h e n y l s u l ph a te (NCS) at a ci d ic p H i n d i ca t ed the a r y l s u l p h at a s e s as ly s os o ma l e nz y me s ( R o y [27], P e r u- m a 1 e t a l . [22]), w h i l e th e u se of p - n i tr o p h e n y l s u l ph a te (NPS), as s ub s tr a t e p o i n t e d to m i c r o s o m a l l o c a l i z a ti o n of a r y l s u l p h a t a -ses (D o g s o n e t al. [6], D o l l y et al. [7]). Th e p r e s en c e of a r y ls u l p h a t a s e a c t i vi t i e s of type II in the nu cl ear
f r a c t i o n w a s a n n o u n c e d b y P o k r o v s k y e t a l . ,[24 ] M a * k i t a , S a n d b o r n [17] an d L e w i c k i , T r z e- c i a k [15]. T h e a r y l s u l p h a t a s e a c t i v i ti e s of t yp e I an d ste- r o l s u l p h o h y d r o l a s e a c t i v i t i e s w e r e s ho w n in n u c l ea r f ra c t i on s in s e v e ra l l a b o r a t o r i e s ( D o l l y et al. [7], K i s h i m o t o, S o s t e k [14] , B a l a s u b r a m a n i a n [2] G n i o t- - S z u l z y c k a , J a k u b o w s k a [11, 12]). A c c o r d i n g to the p r e l i m i n a r y d a t a p r e s e n t e d in ou r p r ev i ou s pape r, t he a r y l s u l h p a t a s e C a n d o e s t r o n e s u l p h a te s u l p h oh y d r o l a se a c t i v i t i e s w e r e a s s o c i a t e d w i t h th e c h r o m a t i n - m e m b r a n e f r a ct i on (G n i o t -S z u l i y c k a , J a k u b o w s k a [12]). T h e p r e s e n t w o r k ha s b e e n a i m e d at i s o l a ti n g of h i g h l y p u r i -f i ed c e l l n uc l e i a n d n u c le a r s u b f r a c t io n s f ro m h um a n p l a c e n t a as w e l l as to t es t t h e m for s u l p h o h y d r o l a s e a c ti vi ti es . It is not u n l i k e l y t ha t s u l p h o h y d r o l a s e p l a y an i mp o rt a n t rol e in n ucl e o- - c y t o p l a s m i c e x c h a n g e of v a r i o u s com po un ds.
MATERIAL AND METHODS
R ea g en t s: T he f o l l o w i n g r e a g e n t s w e r e p u r c h a s e d f r om Se rv a H ei d e l b er g , Ge rma ny : T r i t o n X -1 00 , p - n i t r o p h e n y l s u l p h a t e d i p o -t a s s i u m sal-t; 2 - d e ox y ri b os e ; d e o x y r i b o n u c l e i c a c i d f ro m calf t h y m u s ; 2 - ( 4 - i o d o p h e n y l ) - 3- ( - 4 - n i t r o p h e n y l )- 5 - p h e n y l t e t r a z o l i u m c h l o r i d e (INT). G l u c o s e - 6 - p h o s p h a t e , b a r i u m s al t a nd o e s t r on e s ul ph ate , s o d i u m s al t w e r e f r o m S i gm a Chem. Co., St. L ou i s U SA D e o x y r i b o n u c l e a s e (amorp hous ) fr o m o x b o vi n e p a nc r e a s wa s from S c h u ch a rd t , M ün ch e n, Ge r ma n y. p - N i t r o p h e n y l p h o s p h a t e d i p o t a s s i u m sal t w a s f ro m K o c h L i g h t Labs. , C o l n b r o o k - B u c k s , Eng land . R ib o se w a s fr o m B D H B i o c h e m i c a l D i v i s i o n LTD, P o o l e Engl and. E n z y m e a c t i v i t y d e t e r m i n at i o n . A r y l s u l p h a t a s e C wa s d e t e r m i n e d in th e p r e s e n c e of 10 m M p - n i t r o p h e n y l s u l ph a t e (NPS) or 10 m M 2 - h y d r o x y - 5 - n i t r o p h e n y l s u lp h a t e (NCS) in 0.2 5 m M T r i s - H C l bu ff er p H 7.5. T h e r e a c t i o n w a s t e r m i n a t e d af t er a p p r o p r i a t e tim e of i n c u b a t i o n at 37 °C b y th e a d d i t i o n of 3 m l of 5% NaOH. T h e l i b e -ra t e d p - n i t r o p h e n o l or 4 - n i t r o c a t e c h o l w as m e a s u r e d at 405 and 510 nm. T h e s t e r o i d s u l p h o h y d r o l a s e a c t i v i t y w as d e t e r m i n e d at 0. 25 m M o e s t r o n e s u l p h a t e c o n c e n t r a t i o n and 0.25 m M T r i s H C l b u f -fer p H 7.5. T he u n h y d r o l y s e d s u b s t r a t e w as e s t i m a t e d b y the p r o -c e d u r e of R o y [26].
G l u c os e - 6 - p h o s p h a t a s e a c t i v i t y d e t er m i n a t i o n wa s p er f or m ed a c c o r di n g to the p r o c e d u re of S w a n s o n [29]. S u c c i na t e d e h y dr o g e n a s e w as d e t e r m i n e d a cc o rd i n g to the p r o -c e du r e d e s -c r i b e d b y L e f c n i c k i et al. [16]. A ci d p h o s p h a ta s e a c t iv i t y w as d e t e r m in e d in the p r e se n ce of 10 m M c o n c e n t r a t i o n of p - n i tr o p h e n y l p ho s p h at e in 0.5 M ac et at e bu f fe r pH 5.0. Aft er 5 min. in c ub a t i o n the r ea c t i o n wa s t e r m i n a -te d by the a d di t i o n of 3 ml of 5% NaOH. Th e l ib e ra t ed p- n it r op h e- nol w as m e a s u r e d at 405 nm. D N A a nd R N A w e r e s e p a r a t ed A c c o r di n g to M u n r o , F l e c k [21] D N A w as e s t i m a t e d b y the D i sc h e p ro c ed u re ( S c h n e i d e r [28]). R N A w as d e t e r m i n e d s p e c t r o p h o t o m e t r i c a l l y a cc o r di n g to M u n r o , F l e c k [21] . P r o t e i n c o n te n t w a s d e t e r m i n e d by the b i u r et p r oc e du r e 11 9 1 or, if the i nt e r f er i n g m a t e r i a l w as p re s ent ( Trit on X- 100), the p r o t e i n c o nt e nt w as d e t e r m i n e d by the B e n s a d o w n , W e i n s t e i n p r o ce d u r e [3] . P h o s p h o l i p i d Pi w as e s t i m a t e d b y the p r o c e d ur e of F i s k e, S u b b a r o w [8]. For e x t r a c t i o n of p h o s p h o li p i d s a mi xt u re of e t a n o i - e th y l e th er (1:3} w as used. Th e p h o s p h o l i p i d co nt en t w as c a l c u l a t e d b y m u l t i p l y i n g the d e t e r m i n e d a m o un t of p ho sp h or u s by [26] .
T he p r e p a r a t i o n of n u cl e ar f r a ct i on fr om h u m a n pla cent a. The p r o c e du r e of P h i l p o t , S t a n i e r [23] w a s a dd a pt e d for the p r e p a r a t i o n of h u m a n p l a c e n ta c el l nuclei. T h e p la c e n ta tissue, s h o r tl y a ft e r d e li ve ry , w a s w a s h e d w i t h 0.15 M N aC l and h o m o g e n i z e d in a m e d i u m c o n s i s t i n g of 0.3 M sucrose, 0.0 01 M M g C l 2 at p H 7.0. T he p l a c e n ta t i ss u e w as d i l u t e d 1:10 (w/v) for h o m o g e n i z a t i o n an d ho mo ge n iz e d. Th e r es u l ti n g s us p e ns i o n w as f i l -t er ed -t h r ou g h a n y lo n clo-th. T h e f il tr at e w as d i l u t e d w i t h an eq u al v o lu m e of the h o m o g e n i z i n g m e d i u m c o n t a i n i n g 80% glycerol. Al l the s ol u ti o ns u s ed w e r e ke pt at 0-5°C. T he c e n t r i f u g a t i o n at 700 x g for 10 m i n r e s u l t e d in p r e c i p i t a t i o n of cr ud e nu cl ea r frac tion , w h i c h c o n ta i n s 72% of D N A p r es e nt in the honiogenate. Th e p e ll e t w a s s us p e nd e d ^n an eq u al v ol u m e (w/v) of the h dm o ge ni - z at io n m ediu m. 15 m l a li q uo t es w e r e la ye re d o ve r 70 m l of the h o -m o g e n i z a t i o n -m e d i u -m c o n t a i n i n g 40% g l y c e ro l a nd c e n t r i f u g e d for 10 m i n at 700 x g in H e i n tz - J a n e t z k i r e f r i g e r at e d ce ntri fu ge . The p e ll e t s w e r e r e s us p e n d e d in h o m o ge n i z a t i u n m e d i u m an d the l a -y e r i n g a nd c e n t r i f u g a t i o n o p e r at i o n w as r e p ea t e d o nce again. Th e p u r i f i e d n u c l e a r f r a c ti o n c o n t a i n e d 53% of the D N A p r es e nt in the
ho m og en at e. A d d i ti o n a l p u r i f i c a t i o n of the n uc le ar fr a ct i o n wa s do n e b y r e p e at i n g the la y er i n g an d the c e n t r i f u g a t i o n ope ra ti on. T he f in al p r e p a r a t i o n c o n t a i n e d 46% of the D NA p r e s en t in the h o -mo ge n at e . T he p r e c i p i t a t e of t he n ucl e ar p r e p a r a t i o n w a s s u s p e n -d e-d in the h o m o g e n i z a t i o n m e d i u m c o n t a i n i n g 40% g l y c e r o l and st o r e d at - 15°C. T h e s u b f r a c t i o n a t i o n of t he n u cl e i a nd p r e p a r a t i o n of n ucl e ar m e m b r a n e s . 9> E
Fig. 1. DNA and protein content and sulphohydrolase activities in purified n u -clear fraction, chromatin-membrane fraction and envelope fractions after
deoxyribonuclease digestion
N - nuclear fraction, 1 - chromatin-membrane fraction, 2 envelopes after the first DN-ase digestion, 3 - envelopes after the second DN-ase digestion, 4 -
envelopes after the third DN-ase digestion
Rys. 1. Zawartość DNA i białka oraz aktywność sulfohydrolaz w oczyszczonych j ą -drach komórkowych, frakcji chromatyczno-błonowej oraz frakcjach otoczek
jądro-wych po trawieniu dezoksyrybonukleazą
N - frakcja jądrowa, 1 - frakcja chromatynowo-błonowa, 2 - otoczki jądrowe po pierwszym trawieniu DN-azą, 3 - otoczki jądrowe po drugim trawieniu DN-azą, 4 -
T he n u c le a r f ra c ti o n w as s u b j et e d to o s m ot i c s we ll in g 'follo-w e d by m e c h a n i c a l shrin king , g e n e r al l y a cc o r di n g to the B u s c h and M a u r i t z e n p r o c e d u re [5]. The w h o le nuc lei w er e s w ol l en by r es u s p e nd i n g the nu cl e ar pe ll e ts in 0.-14 M NaC l and 0.05 M Tris-|HC1 b uf f er p H 7.6 c on t a i ni n g 1 m M M g C l 2 and a f t e r -wa r ds t he y u n d e r we n t a c a r ef u l l sh e ar i ng in P ot t er gl as s h omog e- nizer. Th e s he ar ed nu cl ei we r e c e n t r if u g e d at 40 000 x g for 1 h for s e p a r at i o n of the n u cl e ar sap. The deb ri s f rom the c e n t r i f u -ga t i o n wa s e x tr a c t e d w i t h 2 M Na C l at pH 7.6, ke pt in ice bath for 1 h, an d c e n t ri f u g e d at 16 000 x g for 20 min.
Th is p r o c ed u r e w as su p po s e d to s epa ra te mo s t of the hi ston e p r o t ei n s in s up e r n at a n t a nd c h r o m a t in - m e m b r an e s in pellets. The p e ll e ts of the c h r o m a t in - m e m b r an e s (Fig. 1, f r a ct i on 1) w e re s u s -p e nd e d in the h o m o g e n i z i n g b uf f er to a fin al p r o te i n c o n c e n t r a -tio n of ab ou t 5 mg pe r ml; d e o x y r i b o n u c l e a s e wa s ad d ed (100 yg per ml) and the d i g e s t i o n w as c o n t i nu e d for 16 h at 2°C. The s u s p en s i o n was c e nt r i f u g e d at 36 000 x g for 15 min. The p e l -let r e p r es e n t e d nu cl ea r e n ve l op e fraction, d e s i g n a t e d as fra cti on 2; the s u p e r n a ta n t r e p r e s en t e d c h r o m at i n f ra c ti o n 2. Th e se con d an d th ir d ad d i ti o n a l d i g e s t i o n w i t h d eo x y r i b o n u c l e a s e ma de p o ss i b l e to o b t a i n the n uc l e a r e nv e lo p e s w i t h low D N A content, d e si g n a t e d as f r a ct i on 3 a nd 4 and the r es p ec t iv e ch r om a t i n f r a -ct i on s . RESULTS E n z y m at i c a ct i v i ti e s a nd the d at a o n p r o t e i n a nd D NA c on te nt s in the c ru d e (A), p u r i f i e d (B) an d a d d i t i o na l l y p u r if i e d (C) n u -cl e ar fr a ct i o n s are p r e s e n t e d in Tab. 1.
T he n u cl e a r p r e p a r at i o n s A, B, an d C c o n t a i n e d resp ecti vely : 72%, 53% a nd 45.7% of the D N A a nd 13%, 7.8% a nd 4% of the p r o -t ei n p r es e n -t in -the o r i gi n a l h om o ge na te . Th e r at io of protein: DNA, r e v e a li n g the va l ue of 4.14 in tha less p u r i f ie d nu cl ea r p r ep a ra t io n s, w as lo we re d to 1. 94 -1 .5 4 in the p u r i f i e d nu cle ar p r e p a r a t i o n s (Tab. 1). A c c o r d i n g to R o o d y n [25j the v a -lue of pro te in : D NA r at io v ar i e s f ro m 2.4-22; t he h i g h l y p u r if i ed n u cl e ar p r e p a r a t io n s r e v e al e d low er v al ue s (2.4-5.8) w h i l e ha evi - ly c o n t a m i n a t e d n u cl e ar p r e p a r a t io n s s ho w ed r e l at i v e l y h ig h p r o -tein: D NA r ati os (12-22).
T a b l e 1 DNA and protein content as well as marker enzyme and sulphohydrolase activities
in the nuclear fraction from human placenta
Zawartość DNA i białka oraz aktywność enzymów markerowych i sulfohydrolaz we frakcji jąder komórkowych z łożyska ludzkiego
Specification A B C
DNA recovery (%) 72.00 53.25 45.70
DNA mg per g of fresh tissue 1.77 ± 0.31 1.31 ± 0.41 1.12 ± 0.69
Protein recovery (%) 13.31 7.76 4.02
Protein mg per g of fresh tissue 7. 31+ 0.86 4.27 t 0.95 2.21 t 0.53
Protein: DNA ratio 4. 14 3.26 1.94
Succinate dehydrogenase umole of substrate • min 1 .
• mg protein * 0.025 + 0.01 0.014 ± 0.001 0.012 + 0.007
Glucose-6-phosphatase *
umoles of substrate • min ' •
• mg protein 1 0 . 1 63 + 0.06 0.129 ± 0.040 0.031 ± 0.020 Acid phosphatase
umoles of substrate • min ^ •
• mg protein ^ 14.74 ± 5.44 17.59 ± 5.62 13.99 ± 1.23 Arylsulphatase C
nmoles of substrate • min 1 •
■ mg protein ^ 4 . 7 1 + 0.48 5.79 t 1.24 9.06 + 2.74 Oestrone sulphate
sulphohydro-lase
nmoles of substrate • min 1 •
• mg protein * 2 . 4 5 + 0.32 2.04 + 0.51 3.33 ± 1^62 N o t e : A - crude nuclear fraction, B - purified nuclear fraction, C -additionally purified nuclear faction. The data are mean of 4-5 series of experiment.
T h e s p e c i f i c a c t i v i t y of s u c c i n i c a c id d eh y d r o g e na s e , w h i c h is th e m a r k e r e n zy m e of the m i t o c h o n d r i a l fr ac tion , is v e r y low; an d is b e e i n g l o v er e d d u r i n g t he c o u r s e of the p u r i f i c a t i o n of the n u c l e a r f r a c t i o n f ro m th e v a l u e of 0. 02 5 to 0.012. T hi s i n -d i c a t e s t ha t th e c o n t a m i n a t i o n w i t h m i t o c h o n d r i a l p a r t i c l e s of the p u r i f i e d n uc l e a r p r e p a r a t i o n s (C) is v e r y low. Si n c e th e n u cl e a r m e m b r a n e is c o nt i n e o u s in c e r t a i n re gi on s w i t h e n d o p l a s m i c r e t i c u l u m ( M i r s k y , O s a w a [20] ;
F r a n k e , S c h e e r [9] ; A r i o n et al. [1] ) it is qu i t e l ike ly that c er t a i n en z y m e s in the n u cl e ar fr ac t io n are of m i c r o s o m a l origin. Th e mo s t li k ely "ma rker " for c on t a m i n at i o n w i t h m i cr o s o m e s is g lu c o s e - 6 -p h o s p h a ta s e . Th e d a ta p r e s en t e d in Tab. 1, s ho w that the s p e ci f ic a c t i v i t y of the g lu c o s e- 6 - p h o s ph a - tas e in the p u r if i e d n u cl e ar fr ac t io n is ve r y low. Th e p u r i f i c a -ti on p r o c ed u r e r e su l t e d in a f i v e- f o l d d i m i n i s h i n g of the s p e c i -fic a c t i v i t y of the enzyme.
In c o n t ra s t to the d a ta for s uc ci ni c a ci d d e h y d r o g e na s e and g l u c o s e - 6 -p h o s p h a ta s e , the sp e ci f ic a c t i v i t y of aci d p h o sp h at a se wa s not re d u c e d d ur i n g the co u r s e of the p u r i f i c a t i o n proced ure, w h i c h s u g ge s te th at the e n zy m e is an i nt eg r al c o m p o ne n t of the n u -cl ea r fra ction. T hi s st a te m e n t is in a c c o r d i n g w i t h the d at a giv en in the litera ture , i n di c a t i n g tha t aci d p h o s p ha t a s e (a low m o l e -cu lar form) is p r es e nt in the n u cl e a r p r e p a r at i o n s ( G a ł k a et
al. [10]; S z e s z a k , P i n n a [30]).
Fig. 2. DNA and protein content and sulphohydrolase activities in subnuclear fractions
1 - purified nuclei, 2 - nuclear sap, 3 - histone rich fraction, 4 - chromatin* -membrane fraction, 5 - recovery
Rys. 2. Zawartość DNA i białka oraz aktywność sulfohydrolaz we frakcjach subjądrowych
1 - jądra oczyszczone, 2 - sok jądrowy, 3 - frakcja bogata w histony, A - chro- matynowo-membranowa frakcja, 5 - odzysk
T h e s pe c if i c a c t i v i ti e s of s u l p ho h y d r o l a s e C an d oe s tr o ne s u l p h a t e s u l ph o h y d r o l a s e w e r e b e e i n g r ai s e d af t er p u r i f i c a t i o n of the n u cl e a r f r a c ti o n (Tab. 1). T he r es p e ct i v e d at a of s pe ci fi c a c t i v i t i e s in n u c l e a r p r e p a r a t i o n s A, B a nd C are: 4.75, 5.79 and 9.06 in th e p r e s e n c e of N PS as s ub s tr a t e an d 2.45, 2.04 and 3.33 in the p r e s e n c e of o e st r o n e s u l ph a t e as su bst rate . T h e s e re sult s i n d i c a t e the s u l p h o h y d r o l a s e s as the t rue n u c l ea r en zyme s.
T h e d a t a o n D N A a nd p r o t e i n c on te nt , as w e l l as p -n i t r o ph e - n y l s u l p h a t e a nd o e s t r o n e s u l p h a t e h y d r o l y s i n g a c t i v it i e s in sub- n u cl e a r f r a c t io n s are p r e s e n t e d in Fig. 2. T h e o v e r a l r e c o v e r y of D N A an d p r o t e i n af t er s u b fr a c t i o n a t i o n of h i g h l y p u r i f i e d c el l n uc l ei is 78% a nd 1 0 4 % , r es p ec ti ve ly . The o e s t r o n e s u l p h at e s u l p h o h y d r o l a s e a nd a r y l s u l p h a t a s e C are r e c o -v e r e d in t he ra n ge of 80-97%. It is w o r t h m e n t i o n i n g that s u b -f r a c t i o n a t i o n o-f the n u c le a r f r a c t i o n w i t h b i o c h e m i c a l c h a r a c t e -r i st i cs simila-r- to the n u c l e a r f r a c t i o n B g av e low er r e c o v e r y of o e s t r o n e s u l p h o h y d r o l a s e a cti vi ty . A l m o s t all of the s u l p h o h y d r o -la se a c t i v i t i e s a p p e a r e d in the " c h r o ma t i n - m e m b r a n e " f ra c ti o n (Fig. 2). In the n u c l ea r s ap s om e p - n i t r o p h e n y l s u l p h at e h y d r o -l ys i ng a c t i v i t y w a s d e t e c t e d b u t n o ne of o e s t r o n e s u l p h at e s u l -p h o h y d r o l a s e a c t i v i t y w as found. N e i t h e r a r y l s u l p h a t a s e C nor 0S- -a se a c t i v i t y w a s d e t e c t e d in h i s t o n e r i ch f r a c t i o n (2 M NaCl). T h r e e s u c c e s s i v e d i g e s t i o n of the c h r o m a t i n - m e m b r a n e f r ac t i on w i t h d e o x y r i b o n u c l e a s e r e s u l t e d in e n v e l o p e f r a c ti o ns d e s i g n a t e d as f r a c t io n s 2, 3 a nd 4 a n d t he r e s p e c t i v e c h r o m a t i n p r o t e i n f r a c -ti o ns t i g h t l y b o u n d w i t h DNA. T h e d a t a c o n c e r n i n g D N A a n d p r o t e i n c o n t e n t as w e l l as s u l -p h o h y d r o l a s e a c t i v i t i e s i n t he n u c l e a r f r a c t i o n (N), c h r o m a t i n- - m e m b r a n e f r a c t i o n (1) a n d e n v e l o p e f r a c ti o n s (2, 3 a n d 4) are p r e s e n t e d o n Fig. 1. T h e D N A c o n t e n t in t he e n v e l o p e s u b f r a c t io n s a f te r the first, s e c o n d a n d t h i r d d i g e s t i o n w i t h D N a s e w a s d i m i -n i s h e d f r o m 12% to 0-1%. T h e s u l p h o h y d r o l a s e a c t i v i t i e s in the e n v e l o p e f r a c t i o n s w e r e l o w e r e d a ft e r th e fi r st D N- a s e d ig es ti on . N o c h a n g e of s u l p h o h y d r o l a s e a c t i v i t i e s a p p e a r e d a ft e r th e s ec on d a nd t h i r d s u c c e s s i v e d i g e s t i o n w i t h D N-as e. T h e s e r e s ul t s s u g -g e s t t ha t s u l p h o h y d r o l a s e a re b o t h i n t e gr a l p a rt of th e n u cl e ar e nv e lo p e, a n d are c o n s t i t u e n t s of t he c h r o m a t i n p r o t e i n s f ra c t io n t i g h t l y b o u n d w i t h DNA. T h e o v e r a l r e c o v e r y of the s u l p h o h y d r o l a -se a c t i v i t i e s in the e n v e l o p e f r a c t i o n a nd c h r o m a t i n f ra c ti o ns t i g h t l y b o u n d w i t h D N A are f o u n d in the r a n ge of 91%-96%. C o m p o s i t i o n a n a l y s i s of t he n u c le a r p r e p a r a t i o n s (C) and
nu cle ar e n ve l o p e p r e p a r at i o n s (after si ng le d i ge s t i o n w i t h DN -a se of the c h r o m a t i n - m e m b r a n e fr action) is p r e s e n t e d in Tab. 2 and 3.
T a b l e 2 Chemical composition of nuclear fraction and nuclear envelope fraction (in %)
Skład chemiczny frakcji jąder komórkowych i frakcji otoczek jądrowych (w %)
Specification Protein DNA RNA Phospholipid RNA : DNA
Cell nuclei3 56.37 37.04 1.41 5.02 0.04
Envelopes 59.43 12.12 0 28.59
-a The d-at-a -are me-an of 8 series of experiment. The data are mean of 3 series of experiment.
T a b l e 3 Chemical composition and sulphohydrolase activities of nuclear fraction
and nuclear envelope fraction
Skład chemiczny i aktywność sulfohydrolaz we frakcji jąder komórkowych i frakcji otoczek jądrowych
Specification Nuclear fraction Nuclear envelopes Protein
mg per g of fresh tissue 3.600 ± 1.00 0.454 t 0.104 DNA mg/mg protein 0.554 ± 0.262 0.154 ± 0.039 RNA mg/mg of protein 0.034 ± 0.020 0 Phospholipid mg/mg of protein 0.093 ± 0.031 0.484 ± 0.047 E230/E260 1.337 ± 0.135 2.019 ± 0.332 E260/E280 0.689 ± 0.051 0.820 ± 0.102 Aryisulphatase C
nmoles • min 1 • mg protein 1 10.38 ± 3.56 14.32 f 4.69 nmoles • min 1 • mg DMA 1 18.53 ± 6.43 92.98 ± 30.45 Oestrone sulphate sulphohydrolase
nmoles • min * • mg protein * 3.53 ± 1.08 5.46 ± 1.58 nmoles • min 1 • mg DNA * 6.37 ± 1.95 35.45 ± 10.26 N o t e : The data are mean of 5 series of experiments.
W i t h th e a s s u m p t i o n th at % p r o t e i n + % D N A + % R NA + % p h o s p h o l i -p i d = 100%, th e n u c le a r f r a c t i o n c on ta in ed : 56.4% of protein, 37% of DNA, 5. 02 % of p h o s p h o l i p i d an d 1.42 % of RNA. T he protei n: DNA, p h o s p h o l i p i d : DNA, R N A : D N A r a ti o s as w e l l as the r ati os of E 2 30 ^ E 260 a nd E 2 6 0 ^ E 280 (T a b ‘ 3 *< are c o m p a r a b l e w i t h th os e of the m o s t h i g h l y p u r i f i e d n u c l e a r p r e p a r a t i o n s (R o o d y n [25])* T h e n u c le a r e n v e l o p e f r a c t i o n c on ta in ed : 59.4 % of pr otein, 12 .1 2% of DN A an d 28 .58 % of p h o sp h o li p id . N o R NA wa s d e te c te d in t he e n v e l o p e fra ctio n. T h e d a ta of p r o t e i n a nd p h o s p h o l i p i d c o nt e n t (Tab. 2 an d 3) are v e r y c h a r a c t e r i s t i c for n uc l e a r e n v e -lo pe p r e p a r a t i o n s ( B e r e z n y e t a l . [4], M a l e e [18], K a s p e r [13]), b ut th e D N A c o n t e n t af t er the s in gl e d i g e -s ti o n w i t h D N - a s e is r e l a t i v e l y h i g h (9-12%). T h e s e r e su l ts pro- o ve d th e g e n e r a l s t a t e m e n t t h at the m o s t s i g n i f i ca n t d i f f er e n c e s in e n v e l o p e c o m p o s i t i o n fr o m d i f f e r e n t s o ur c es is the v a ri a t i o n in D N A c o nt e n t w h i c h va r i e s f r o m 0- 8 % (Ka sper [13]).
DISCUSSION AND CONCLUSIONS
T h e a n a li t y c a l d a t a c o n c e r n i n g DNA, pro tein , R N A a nd p h o s p h o -li p i d c o n c e n t r at i o n , as w e l l as the d a t a on th e m a r k e r en z ym e a c t i v i t i e s s ho w th at the n u c l e a r f r a ct i o n i s o l a t e d f ro m h um an p l a c e n t a b y t he p r o c e d u r e d e s c r i b e d in t hi s pa p er ar e of h ig h p u -r it y (R o o d y n [25] ).
As c o n c l u d e d f ro m t he s p e c i fi c a c t i v i t i e s of s u c ci n ic a ci d d e h y d r o g e n a s e a nd g luc ose- -6-p hosp hata se (Tab. 1), t he fi rs t st ep of t he p u r i f i c a t i o n p r o c e d u r e m a d e p o s s i b l e to s e p a r at e m o s t of th e m i t o c h o n d r i a l i m p ur i ti e s, w h i l e the se c o n d a d di t i o n a l ste p a l l o w e d to e l i m i n a t e th e m i c r o s o m a l c on t a m i n a ti o n s . It is also u n l i k e l y t ha t t he o ut e r l e a fl e t of th e e n ve lo pe , t ha t r e v ea l ed c o n s i d e r a b l e a c t i v i t y of g l u c o s e - 6 - p h o s p h a t a s e , w as c l e a r e d a wa y af t e r th e t h i r d a d d i t i o n a l s t e p of p u r i f i c a t i o n (A r i o n et al. [1]). In c o nt r a s t to this, n e i t h e r the fi r st nor the s ec ond a n d t h i r d s te p of th e p u r i f i c a t i o n p r o c e d u r e e f f e c t e d in lo w er i ng of t he o e s t r o n e s u l p h a t e s u l p h o h y d r o l a s e a nd a r y l s u l p h a t a s e C ac t iv i ti e s: t he s p e ci f i c a c t i v i t i e s of b o t h e n z y m e s a re r ai s ed a ft e r e a c h s te p of p u r i f i c a ti o n ; th is i n d i ca t e s , t h at t he two s u l p h o h y d r o l a s e s m a y be c o n s i d e r e d as tru e p l a c e n t a l c e l l n uc l ea r
en zymes. An o pe n q u e s t i o n at p re se n t is, d i d the en zy me s r e -pr es e nt a ct i v i ti e s c o mm o n to the nu cle i o r i g i n at e d f rom all ti ss u es or w e t h e r are t h ey c ha r a c t e r is t i c o nl y for cel l nuc le i of v e ry d i s t i nc t t is sue s or o rg an s ? It s h ou l d be s tr e ss e d that the sp e ci f ic a c ti v it i es of a r yl s u l p h a ta s e C an d o e s tr o ne su lph at e s u lp h o h y d r ol a s e of the p u r i fi e d p la c e nt a l ce ll n uc le i and e n v e l o -pes are of su ch m a g ni t u d e that th e y ca n not be ignored.
Th e p r e l i m i n a r y d a t a p r e s e n t e d in this pa p er s ho wed that s u lp h o h y d r ol a s e a ct i v i ti e s are a ss o c i at e d w i t h the nu cl ea r e n v e -lope an d n on hi st o ne c h r om a t i n pr o te i ns t i g h t l y b ou n d w i t h DNA.
Th e b i o c h em i c a l s ig n i f ic a nc e on the p re s e nc e of OS -h y dr o ly - s in g a c t i vi t y an d a r yl s u l p h a ta s e C a c t iv i t y is not, at present, clear; it m i g h t not be ru l ed out tha t t he y pl a y an d im po rta nt ro le in n u c l eo - c y t o p l a s m i c e x c ha n ge of v a ri o us c omp ound s.
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Department of Biochemistry Institute of Biological University N. Copernicus in Toruri
Jadwiga Gniot-Szulżycka, Anna Jakubowska
AKTYWNOŚĆ SULFOHYDROLAZY SIARCZANU ESTRONU I ARYLOSULFATAZY C W JĄDRACH KOMÓRKOWYCH Z ŁOŻYSKA LUDZKIEGO I SUBFRAKCJACH JĄDROWYCH
Opisano metodą otrzymywania jąder komórkowych i otoczek jądrowych z tkanki łożyska ludzkiego. Preparaty jąder komórkowych zawierały: 56,4% białka, 37% DNA 5,02% fosfolipidów i 1,42% RNA. Stosunek białka do DNA wahaf się w granicach 1,20-1,94; E230^E260 1 E 260^E280 wynosiły odpowiednio 1,34 i 0,689. Z oczy-szczonych jąder komórkowych uzyskiwano: sok jądrowy, frakcją bogatą w białka histonowe i frakcją chromatynowo-błonową. Trawienie tej ostatniej dezoksyrybo- nukleazą pozwoliło na otrzymanie frakcji otoczek jądrowych o składzie: 59,4% białka, 1-12% DNA, 28,6% fosfolipidów.
Przedstawiono dane świadczące, iż sulfohydrolaza C i sulfohydrolaza siar-czanu estrone są integralnymi składnikami jąder komórkowych łożyska ludzkiego. Aktywność enzymatyczną stwierdzono w otoczkach jądrowych i frakcji białek chro- matynowych silnie związanych z DNA i częściowo pozbawionych białek histonowych.
