Relative amount of HMG 1 and HMG 2 proteins in different Syrian hamster tissues

A C T A U N I V E R S l i A T I S L O D Z I E N S I S

FOLIA BIOCHIMICA ET BIOPHYSICA 6, 1988

Ha n n a Mo dr z ej e ws k a, G ra ż y n a Gałązka, Ilon a Ko wa l sk a

R E L A T I V E A M O U N T O F H M G 1 A N D H M G 2 P R O T E I N S I N D I F F E R E N T S Y R I A N H A M S T E R T I S S U E S

Relative amount of HMG 1 and HMG 2 fractions have hfven established in different Syrian hamster tissues. It has been detected that in c e-rebral hemispheres, the liver of healthy animals and in Kirkman-Rob- bins hepatoma as well as in the liver of tumor bearing animals, the amounts of HMG 1 and HMG 2 have been basically the same. A significant increase of HMG 2 fraction have been found in Syrian hamster testes.

INTRODUCTION

A m o n g the m o r e a b un d a nt n o n- h i s t on e p r ot e i ns th er e is the h ig h m o b i l i t y g r o up (HMG). In m amm als , th e four m a j o r H M G c a n be d i -vi d ed i nt o two ca te go ri es : th e p r o t ei n s H MG 1 a nd H M G 2 as w ell as H MG 14 a nd H MG 17. H M G 14 and H M G 17 ha ve b ee n i n v ol v ed in the m a i n t e n a n c e of the c h r o m a t i n c o n f i g u r a t i o n n e ce s s a r y for t r a n s c r ip t i o n [8]. H M G 1 and H M G 2 are of the same size an d show h o m o l o g y in seq uence. F un c ti o n s of HM G 1 ah d H M G 2 h ave no t be en d e m o n s t r a t e d yet. S tu d ie s c a rr i e d ou t in di c at e tha t H M G 1 and HM G 2 r e pl a ce h is t o n e H I in n u c l e os o m e s [5]. S e y e d i n an d K i s t 1 e r [12, 13] a ss u m e d that H MG 2 a nd H 1° h i s t o -ne p e r f o r m a l t e r n at i v e fu n ct i o n s in n u c l eo s o m e linkers. H 1° is c h ar a c t e r i s t i c of n o n- r e p l i c a t i v e tis sues, w h er e a s HM G 2 ap pear s in the p r o l i f e r a t i n g tis sues. L ev e l s of c h r o m os o m a l pr o t e i n HM G 2 are p a r al l e l w i t h the p r o l i f e r a t i v e a c t iv i t y of t est is cells, s ke l et a l m u s e l e a nd o t h er o rgans. In o u r t is su e m o d e l an i n t e -r e s ti n g f act b e en found, i.e. in K i r k m an - R o b b i ns h e p a t om a an e x -tra s u b f r a c t io n of H 1 h i s t o n e ap pear ed, c a ll e d H 1 "slow" [4].

T he p u r p o s e of th is w o r k w a s th e s ea r ch for c o r r e l a t i o n b e t we e n t he t i s su e p r o l i f e r a t i o n a ct ivit y, H 1 s u b f r a c t i on s u bs e t and on t he o t he r hand, t he a m o un t of H M G 1 a n d H MG 2 p ro tein s.

MATERIAL AND METHODS

T h e t r a n s p l a n t a b l e h a m s t er h e p a t o m a d e r i v e d f ro m the line o r i g i n a l l y i n d u ce d b y K i r k m a n a nd . R o b b i n s [6]. T he n e o p l a s t i c m a t e r i a l w as c o l l e c t e d o n the e ig h t d a y a ft er the t r a n s p l a n t a t i o n of th e tumor. T h e H MG 1 an d H MG 2 p r o te i n s w er e i s o l at e d f r o $ t he t um or a nd o t h e r t i ss u es a c c o r d i n g to S e y e- d i n a n d K i s t 1 e r [12] t h r ou g h e x t r a c t i o n b y m ea ns of 0,2 M H 2S 0 4 , 10 m M P M S F , 10 m M 0 -m e rk a pt o e t h a no l . H M G 1 an d H M G 2 w e r e i s o l at e d f r om t ot a l f r a c t i o n b y f r a c t i o n a t e d p r e c i p i -t a -t i o n w i -t h -t r i c h l o r o a c e -t i c acid. H M G p r o t ei n s w e r e s e p a r a t e d b y p o l y a c r y l a m i d e g el e l e c t r o p h o r e s i s a c c o r d i n g to P a n y i m an C h a l k l e y [10]. In 25% a c ry l a m i d e ge l sl a bs c o nt a i n i n g 2.5 M ure a, 30 c m l ong w e r e r u n for 72 h at 4°C a nd 210 V. Th e g e ls w e r e s t a i n e d w i t h Araido-Black 10 B a n d a ft e r d e s t a i n i n g t he y w e r e s t a i n e d o nc e a g a i n w i t h C o m a s s i e b l u e R-250. H M G 1 an d H M G 2 i s o l a t e d f r o m c al f t h ym u s w e r e u s e d as s t a n d a r ds [2]. T h e d e ns i - t o m e t r i c p a t t e r n s of th e g el f r a g m e n t s c o n t a i n i n g H M G p r o te i ns w e r e o b t a i n e d w i t h t he u s e of a d e n s i to m et r . T h e a m o u n t s of H M G 1 a n d H M G 2 w e r e e s t i m a t e d b y c a l c u l a t i n g t he r a ti o s of tne ar eas u n d e r t he p r o p e r peak s. T h e p r e p a r a t e s c h b s e n for t h e s t u di e s w e -re t ho s e w h o s e b a n d a b s o r p t i o n w a s in th e l in e ar d e p e n d e n c e to th e p r o t e i n c o n c e n t r a t io n , w h i c h h a d p r e v i o u s l y b e d n s t a t e d w i t h s t a n d a r d pr ot ei ns .

RESULTS AND DISCUSION

H M G p r o t e i n s w e r e e x t r a c t e d f r o m th e n o rm a l a nd p a t h o lo g i c t i s s u e s of t he h am st er . T h e d e n s i t o m e t r i c p a t t e r n s of p o l y a c r y l a -m i d e g el f r a g m en t s c o n t a i n i n g H M G 1 a n d H M G 2 f r a c ti o n s h av e b ee n s h o w n o n Fig. 1. T h e d e n s i t o g r a m s o b t a i n e d f r o m t he p r o t e i n e x -t r ac -t s -t a k e n f r om c e r e b r a l h e m i s p h e r e s , h e a l t h y l i v e r , K i r km a n- - R o b bi n s h e p a t o m a as w e l l as t he li ve r of tu m or b e a r i n g a ni ma l w e r e b a s i c a l l y si milar.

HMG]

HMG]

HMG1

HMG2

Hf

d )

Fig. 1. Densitometrie patterns of the HMG proteins extracts obtained from di -fferent cells of the normal and pathological tissues

a) from the cerebral hemispheres, b) from the normal liver, c) from the Kirk- man-Robbins hepatoma, d) from the Syrian hamster testis

Rys. 1. Densytogram ekstraktu białek HMG otrzymanego z różnych komórek tkanek normalnych i patologicznych

a) z półkul mózgowych, b) z normalnej wątroby, c) z miąsaka Kirkmana-Robbinsa d) z jąder chomika syryjskiego

Th e re l at i ve a mo u nt of H MG 1 a nd H MG 2 p r o t ei n s as re v ea l ed b y e l e c tr o p h o r e s i s a n d d e n s i ^ m e t r i c t r a c in g of the ge ls w er e al m os t eq u a l in v ar i o u s ti ss u es (Tab. 1). A q u i t e d i f fe r e n t den- s i to m e t r i c p a t t e r n wa s o b t a i n e d for the e x tr a c t s f rom the Sy ri an h a m s te r testes. In th is c as e the a mo u nt of H M G 2 f r a ct i on e x -c e e d e d th at of H M G 1 fra -ctio n. (Fig. Id a nd Tab. 1).

T h e f ac t t ha t no i n c r ea s e of t he r el a ti v e a mo u nt of H MG 2 p r o t e i n w a s fo u nd in K i r k m a n - R o b b i n s h e p a t o m a s u g g es t e d tha t ev en

T a b l e 1

The relative amount of HMG 1 and HMG 2 fractions Względna zawartość frakcji HMG 1 1 HMG 2

Analysed tissue n ■ 10 HMG 1 X HMG 2 7. Cerebral hemispheres 47,8 * 7 52,2 t 7,5 Liver 56,0 ± 5,8 43,8 ± 5,8

Liver tumor bearing animals 48,0 ± 9 52,0 ± 8,5 Kirkman-Robbins hepatoma 49,7 ± 5,9 51,3 ± 3,9 Testis 39,0 ± 8,5 61,0 i 8,5

N o t e: The sum of these proteins were considered to be 10/)%. The expe-riments were repeated 10 times.

d e e p c h a n g e s in t he s u b f r a c t i o n su b se t of hi s t o n e H 1 d i d not i n -f l ue n c e in c h a n g e s of th e r e l a t i v e a mo u n t of H MG 1 an d H MG 2 p ro t ei ns . In K i r k m a n - R o b b i n s t um o r f ro m 20% to 30% of the tota l H 1 h i s t o n e h as b e e n r e s e r v e d f or th e n e w f ra c ti o n - H i "slow", c h a r a c t e r i s t i c of t ha t t i s s u e [4, 9]. O u r r e s u l t s s ee m n ot to s u p po r t t he h y p o t e s i s c o n c e r n i n g the c o n t r i b u t i o n o f H MG 2 p r o t e i n in th e c el l d i v i s i o n r e g u l at i o n p r oc e ss e s. K i r k m a n - R o b b i n s h e p a t o m a ha s go t h i g h f r eq u e n c y of c e l l d i v i s i o n bu t n e v e r t h e l e s s the ra t i o of H M G 2 to H M G 1 r e -m a i n s c on s ta n t, e v e n in c o m p a r i s o n w i t h the c e r e b r a l h e m i s p h e r e s w h i c h l os t t h ei r a b i l i t y to c e l l d iv i si on . T h e r e h a v e b e e n s e ve r a l r ep o r t s s u g g e s t i n g t hat th e ra t io of H M G 2 t o H M G 1 i n c r e as e s p a r a l l e l y w i t h p r o l i f e r a t i v e a c t i v i t y in m u s c l e [12, 3], t es t is [12] a nd s a li v a r y g la n d t i ss u es [11]. H o -w e v e r o t h e r a u to r s r e po r t s o n r e g e n e r a t i n g rat li ver [7] an d m o u s e n e u r o b l a s t o m a c e l l s [13] d o no t n ote an i n c r e a s e d H M G 2/ /H MG 1 r a t i o d u r i n g p ro l i f e ra t i o n . T h e i n c re a s e of t he a m o u n t o f H M G 2 in S y r i a n h a m s t e r t es ti s se e m s t o be of p a r t i c u l a r i nt e res t. An a d di t i o n a l f r a c t i o n of H 1 h i s t o n e a p p e a r s i n t hi s o r g a n as wel l. H ow ev er , t he i nc re a se of H M G 2 c a n n o t b e d i r e c t l y a s s o c i a t e d w i t h th e i n c r e as e of the a m o u n t o f H 1 " slo w" as t h er e h a s b e e n n o c o r r e l a t i o n s ta t ed b e t w e e n t he s e tw o fa c ts in c a s e K i r k m a n - R o b b i n s h e p a t o m a R e ce n t l y B u c c i et al [1] f r a c t i o n a t e d v a r i o u s c el l s f ro m ra t t est is a n d on t he bas'is of t he r e s ul t s f r a m e d a- n e w h y p o t e s i s s ay i ng th at t hi s e n o r m o u s i n c r e a s e of H M G 2 is a s s o c i a t e d w i t h m e io s i s b ut no t p ro l if e r a t io n .

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Institute of Physiology and Biochemistry Medical Academy of Łódź

Hanna Modrzejewska, Grażyna Gałązka, Ilona Kowalska

WZGLĘDNA ILOŚĆ BIAŁEK HMG 1 I HMG 2 W RÓŻNYCH TKANKACH CHOMIKA SYRYJSKIEGO

Przebadano względną zawartość ilościową białek HMG 1 i HMG 2 w rożnych tkankach chomika syryjskiego. Stwierdzono, że zawartość tych białek w

połku-lach mózgowych, • wątrobie prawidłowej, wątrobie nosiciela guza i w wątrobiaku Kirkmana-Robbins jest bardzo podobna i niezależna od występowania dodatkowej subfrakcjl H 1 - H 1 "slow".

Względna zawartość białek HMG 1 i HMG 2 różni się w jądrach samców cho-mika syryjskiego. Ilość HMG 2 przewyższa znacznie zawartość HMG 1.