- Email: [email protected]
5.1) is the only major protein extracted from HeLa nucleoli with 3M urea
Life Sciences, Vol. 51, pp. 915-920 Pranted an the USA
PROTEIN
Ben]amln
Pergamon Press
B 2 3 ( M . W . / p I = 37 k D / 5 . 1 ) IS T H E O N L Y M A J O R E X T R A C T E D F R O M H e L a N U C L E O L I W I T H 3M U R E A Yat-Mlng
Y u n g I*,
Erlc
Ka-Wa±
Hul I and
PROTEIN
Pul-Kwong
Chan 2
I D e p a r t m e n t of P h a r m a c o l o g y , Chang Gung Medlcal College, T a l w a n , R . O . C . a n d 2 D e p a r t m e n t of P h a r m a c o l o g y , B a y l o r C o l l e g e of M e d l c l n e , H o u s t o n , T e x a s 7 7 0 3 0 , U . S . A .
(Recelved an f~nal form July 8, 1992)
Summary H e L a n u c l e o l l w e r e i s o l a t e d u s l n g the N P - 4 0 m e t h o d a n d subsequently extracted w l t h 3M u r e a . The e x t r a c t w a s i n c u b a t e d at 6 0 ° C for 30 mln, a n d p r e c l p l t a t e d protelns were removed by c e n t r ± f u g a t l o n . The supernatant was analyzed by one- and two-dimensional SDS polyacrylamlde gel e l e c t r o p h o r e s l s (PAGE). P r o t e l n B23 w a s the o n l y m a 3 o r p r o t e i n e x t r a c t e d f r o m H e L a n u c l e o l ± by t h i s p r o cedure. Using thls procedure, 1 mg of p r o t e i n B 2 3 was obtained f r o m 2 g of H e L a c e l l s . T h e p u r ± t y of the ext r a c t e d p r o t e i n B23 w a s 98%, as m e a s u r e d by one- and two-dlmenslonl gel electrophoresls. The n u c l e o l u s 1s a h l g h l y s p e c l a l l z e d organelle, producing ribosomal R N A ( r R N A ) w h l c h is a s s e m b l e d into rlbosomal particles and eventually transported to the c y t o p l a s m (i). P r e v ± o u s s t u d l e s h a v e s h o w n t h a t the p h o s p h o p r o t e l n B 2 3 (37 k D / p I 5.1) is o n e of the m a 3 o r n u c l e o l a r p h o s p h o p r o t e l n ldentlfled in t u m o r c e l l s (24). P r o t e l n B23 is a s s o c l a t e d w l t h p r e - r R N P s , a n d m a y be i n v o l v e d in the m a t u r a t i o n of r l b o s o m e s (4). R e c e n t s t u d l e s s h o w t h a t p r o t e i n B23 t r a n s l o c a t e s f r o m the n u c l e o l u s to the n u c l e o p l a s m w h e n rRNA processlng is l n h l b l t e d by s e r u m s t a r v a t l o n or l n h l b l t o r s s u c h as a c t ± n o m y c l n D, l u z o p e p t l n s , t o y o c a m y c l n , or h l g h d o s e s of ~-amanltln (5-8). Proteln B23 was prevlously purlfled from Novlkoff hepatoma cell nucleoll uslng the method developed by M l c h a l l k et al (9). Thls method involved three ma3or steps, (i) 4M u r e a / 3 M L i C l extraction of n u c l e o l l , (11) d l a l y s l s of the e x t r a c t against 4M urea/20 mM Trls-Malate/pH 5.5, a n d (ili) D E A E - c e l l u l o s e chromatography (9). T h e p r o c e d u r e requlred 4 d a y s , a n d the y l e l d w a s about 2 mg/100 g tlssue. Thls communlcatlon r e p o r t s an i m p r o v e d new procedure f o r the isolatlon of p r o t e i n B 2 3 f r o m H e L a c e l l s w l t h h i g h e r y l e l d (i m g / 2 g c e l l s ) a n d s h o r t e r t l m e r e q u i r e m e n t .
* TO w h o m r e p r l n t r e q u e s t s s h o u l d be s e n t to: Dr. B e n 3 a m l n Y a t M l n g Y u n g , P h . D . , C h a n g G u n g M e d ± c a l C o l l e g e , D e p a r t m e n t of P h a r macology, 259 W e n - H w a ist Road, K w e 1 - S a n , Tao-Yuan 333, T a l w a n , R.O.C. 0024-3205/92 $5.00 + .00 Copyright © 1992 Pergamon Press Ltd All r~ghts reserved.
916
P r o t e l n B23 and U r e a E x t r a c t i o n
Vol.
51, No.
12,
1992
Methods Cells H e L a $3 c e l l s w e r e g r o w n in m o n o l a y e r wlth Eagle's mlnlmum essentlal medium (MEM) supplemented wlth 10% f e t a l c a l f serum, glutamlne and antlbot±cs in a 5% C O 2 - h u m l d ± f l e d incubator at 37°C. Preparation of Nucleoll Nucleoll were isolated u s i n g the NP-40 method prevlously described (5,10). Cells were suspended in 20 v o l of R S B b u f f e r (0.01M Trls HCI/0.01M NaCl/l.5 mM NaCl/pH 7.2) f o r 30 m ± n a n d t h e n c e n t r l f u g e d at 630 g f o r 8 mln. S w o l l e n cells were resuspended in 20 v o l of R S B b u f f e r containing 0.5% NP-40. The cells were homogenized wlth a Dounce homogenizer (10 up-and-down strokes), a n d the c r u d e n u c l e i w e r e c o l l e c t e d by c e n trifugatlon at 630 g f o r 8 mln. T h e n u c l e i w e r e r e s u s p e n d e d in 10 v o l of 0 . 2 5 M s u c r o s e / 1 0 mM MgCI2, and underlayered w i t h an e q u a l vol of 0.88 M sucrose/0.05 mM MgCI2. After centrlfugatlon at 1700 g f o r i0 mln, the n u c l e i were resuspended in i0 v o l o f 0.34 M sucrose/0.05 mM MgCI2 . The suspension was sonlcated f o r 1 mln, underlayered w l t h an e q u a l v o l o f 0 . 8 8 M s u c r o s e / 0 . 0 5 mM MgCI 2 , and centrifuged at 3 0 0 0 g f o r 18 mln. T h e p e l l e t (nucleoll) was then collected. Purlflcat±on of Protein B23 Nucleoll (0.i g) f r o m H e L a cells were suspended in i0 vol (i ml) of 3M urea/l n~ phenylmethyl sulfonyl fluoride (PMSF)/I mM leupeptln/l mM pchloromercurlphenyl sulfon±c ac±d (pCMPS), and were homogenized with a Dounce homogenizer (20 u p - a n d - d o w n strokes) on ice. The extract was then incubated at 6 0 ° C f o r 30 m l n a n d c e n t r i f u g e d at 2 7 , 0 0 0 g f o r 20 m ± n at 5°C. T h e s u p e r n a t a n t , which contalned pur l f ± e d p r o t e l n B23, w a s c o l l e c t e d . Polyacrylamlde Gel Electrophores±s One-dlmenslonal SDSPAGE was performed accordlng to the m e t h o d o f L a e m m l l (Ii). T w o d±menslonal PAGE was done by isoelectrlc focuslng (12) a n d S D S PAGE. G e l s w e r e e l t h e r s t a ± n e d w i t h 0 . 1 % C o o m a s s l e Brlll±ant-Blue R (S±gma) or sllver-stalned (13). Proteln Assay B±o-Rad prote±n assay
Prote±n (14).
concentration
was
determined
by
the
Results The improved isolat±on procedure for p r o t e l n B 2 3 is s h o w n in FIG. i. H e L a c e l l n u c l e o l l t h a t w e r e p u r l f l e d b y the N P - 4 0 m e t h o d (5) w e r e u s e d as the s t a r t l n g materlal. The nucleoll were extracted wlth 3M u r e a a n d s u b s e q u e n t l y incubated at 6 0 ° C f o r 30 mln. Conslderable amounts of insoluble protelns (90% of the startlng protelns) were obta±ned, whlch were subsequently removed by centrlfugatlon at 2 7 , 0 0 0 g f o r 20 mln. FIG. 2 s h o w s the o n e dlmens±onal SDS-PAGE patterns of the p r o t e l n s from varlous fract±ons of the purlflcatlon procedure. There are approxlmately 45 Coomassle blue-sta±n±ng protelns ±n t h e g e l o f the t o t a l e x t r a c t of H e L a n u c l e o l l (FIG. 2B). A 37 k D b a n d w h l c h c o r r e s p o n d s to t h e pos±tlon of protein B23 was observed. By desltometrlc scannlng, thls band accounted for about 10.4+1.6% of the total amount of protemns (FIG. i). FIG. 2C s h o w s the i n s o l u b l e proteln aggregate f r o m the 3M u r e a e x t r a c t of nucleoll after mncubatlon at 6 0 ° C . Thls preclpltated fractlon contalned m o s t o f the same proteln
Vol.
51, No.
12,
1992
P r o t e i n B23 a n d U r e a E x t r a c t i o n
HeLa
Nucleoll,
2.
(no
Cells
10.5
3M U r e a
mg
(10.4%
B23)
L
Extraction
1. 6 0 ° C I n c u b a t i o n f o r 30 m l n Centr~fugatlon 2 7 , 0 0 0 g , 20 m~n.
/
",,,
pellet, 9 . 0 mg B23 d e t e c t a b l e )
Isolatlon
917
Scheme
supernatant, (98%
for
FIG.
1
HeLa
Nucleolar
1.12 B23)
Prote±n
mg
B23.
bands as the total extract (FIG. 2B), execpt protein B23. As s h o w n in FIG. 2E, p r o t e l n B 2 3 w a s f o u n d m a l n l y in the s u p e r n a t a n t f r a c t l o n w i t h a p u r l t y of o v e r 95%, d e t e r m l n e d by d e n s l t o m e t r l c scannlng. Immunoblot results conflrmed t h a t the p u r l f l e d p r o t e l n was p r o t e i n B 2 3 (FIG. 2F). W h e n the 60 °C l n c u b a t l o n step was omltted, the s u p e r n a t a n t contained not only protein B23, but also small quantltles of other protelns. FIG. 2D s h o w s t h a t t h e s e o t h e r p r o t e l n s a r e of hlgher molecular weight proteins. The purity of protein B23 was analyzed by 2D gel electrophoresls. A l a r g e d e n s e s p o t a n d a f e w m l n o r s p o t s (FIG. 3A) a r e l o c a t e d in the p o s i t i o n of B 2 3 (M.W. 37 k D / p I 5.1). T h i s r e s u l t a g r e e s w l t h p r e v l o u s s t u d l e s (5) w h i c h s h o w t h a t t h e r e a r e mult±ple forms of protlen B23. FIG. 3B shows the Western immunoblot of the purified protein B23 after 2D gel electrophoresls. The purlfled protein shows identlcal immunostalnlng and sllver-stalnlng patterns. DlSCUSSlOn T h l s s t u d y i n d l c a t e s t h a t p r o t e l n B23 is the o n l y m a ] o r p r o teln extracted from HeLa nucleol± w l t h 3M u r e a . T h e i n c u b a t i o n s t e p (at 6 0 ° C ) is n e c e s s a r y to e l l m l n a t e m l n o r c o n t a m l n a n t s (FIG. 2D) In the e x t r a c t . T h e f a c t t h a t p r o t e l n B23 is the o n l y m a ] o r p r o t e l n s o l u b l e in 3M u r e a s u g g e s t s t h a t the s t r u c t u r a l o r g a n l z a t l o n of p r o t e i n B23 in n u c l e o l l m a y be d i f f e r e n t f r o m t h a t of other nucleolar protelns. Compared to the m e t h o d developed by Mlchallk et al (9), t h i s n e w i s o l a t l o n procedure is s i m p l e a n d rapld. The new isolatlon p r o c e d u r e t a k e s o n l y 3 hrs, instead
918
P r o t e & n B23 a n d U r e a E x t r a c t & o n
Vol.
[
FIG.
51, No.
12, 1992
F
2
8% S D S - P A G E of n u c l e o l a r protelns at v a r l o u s s t e p s of purlflcatlon of p r o t e l n B23. The polyacrylamlde gel was stalned wlth Coomassle Blue. Lane A - purlfled proteln B23 from Novlkoff hepatoma cell nucleo]l (9). L a n e B - total HeLa nucleolar protelns (whole nucleoll dlss o l v e d in S D S s a m p l e b u f f e r ) . L a n e C - t h e p r o t e l n p r e clpltate ( p e l l e t ) o f t h e 3M u r e a e x t r a c t o f H e L a n u c l e o11. Lane D - supernatant of the 3M u r e a e x t r a c t of HeLa nucleoll (wlthout incubatlon at 6 0 ° C f o r 30 m l n ) . Lane E - supernatant of t h e 3M u r e a e x t r a c t o f H e L a nucleoll (with incubatlon at 6 0 0 C f o r 30 m ± n ) . L a n e F protelns in L a n e E w e r e t r a n s f e r r e d onto a nltrocellulose sheet and immunostalned wlth the ant±-B23 antlbody (5,7). An ± m m u n o b a n d correspondlng to p r o t e l n B23 was observed.
of 4 days, and HeLa cells).
it
has
a
hlgher
yield
(I
mg
proteln
B23/2
g
of
Vol.
51, No.
12,
1992
Protein
B23 and U r e a E x t r a c t z o n
FIG.
919
3
(A) T w o - d l m e n s l o n a l SDS-PAGE of p u r l f l e d proteln B23. A b o u t 1 m g of p u r l f l e d p r o t e l n B23 w a s l o a d e d o n t o the gel. T h e g e l w a s s l l v e r - s t a l n e d (13). A m a 3 o r s p o t at M.W./pI = 37 k D / 5 . 1 was observed. A large patch of stalnlng located in the b o t t o m l e f t c o r n e r of the gel corresponds to A m p h o l l n e . (B) W e s t e r n i m m u n o s t a l n l n g of p u r l f l e d p r o t e l n B23. A b o u t 5 m g of p u r l f l e d p r o t e l n B23 w a s l o a d e d o n t o a t w o - d l m e n s l o n a l S D S - P A G E gel. A f t e r the p r o t e l n w a s t r a n s f e r r e d to n l t r o c e l l u l o s e paper, the p a p e r w a s i m m u n o s t a l n e d w i t h the p r o t e l n B23 monoclonal antlbody.
Acknowledgments W e t h a n k Drs. H a r r l s B u s c h a n d D e l o n W u for t h e l r a d v l c e a n d encouragement durlng these studles. These studles were supported by C h a n g Gung Research Grant (CMRP 275) and Natlonal Science Councll (R.O.C.) Grant (NSC80-0412-B-182-41 and NSC81-0412-B-1825). References i. 2. 3. 4. 5.
H. B U S C H a n d K.J. S M E T A N A , T h e N u c l e o l u s . A c a d e m l c P r e s s , N e w York (1970). M . O . J . O L S O N , L.R. O R R I C K , C. J O N E S and H. B U S C H , J. B i o l . C h e m . 249 2 8 2 3 - 2 8 2 7 ( 1 9 7 4 ) . A. W. P R E S T A Y K O , G. R. K L O M P , D. J. S C H M O L L and H. B U S C H , Blochemlstry 13 1 9 4 5 - 1 9 5 1 ( 1 9 7 4 ) . D.L. S P E C T O R , R.L. O C H S a n d H. B U S C H , Chromosoma 90 1 3 9 - 1 4 8 ( 1984 ). P.K. C H A N , M. A L D R I C H a n d H. B U S C H , Exp. C e l l Res. 161 i01ii0 ( 1 9 8 5 ) .
920
6. 7. 8. 9. i0. ii. 12. 13. 14.
Protean B23 and Urea Extraction
Vol. 51, No. 12, 1992
B . Y - M . Y U N G , R.K. B U S C H , H. B U S C H , A.B. M A U G E R a n d P.K. C H A N , Blochem. Pharmacol. 34 4 0 5 9 - 4 0 6 3 (1985). B . Y - M . Y U N G , H. B U S C H and P.K. C H A N , Blochlm. B1ophy. Acta 826 1 6 7 - 1 7 3 ( 1 9 8 5 ) . B . Y - M . Y U N G , H. B U S C H a n d P.K. C HA N , C a n c e r Res. 46 9 2 2 - 9 2 5 (1986). J. M I C H A L I K , L.C. Y E O M A N a n d H . B U S C H , L i f e Scl. 28 1 3 7 1 - 1 3 7 9 (1981). M. M U R A M A T S U , Y. H A Y A S H I , T. O N I S H I , M. S A K A I , K. T A K A I and T. K A S H I Y A M A , Exp. C e l l Res. 88 345 ( 1 9 7 4 ) . U.K. L A E M M L I , N a t u r e ( L o n d . ) 277 6 8 0 - 6 8 1 (1970). P.K. C H A N , A. F E Y E R A B E N D , R.K. B U S C H and H. B U S C H , Cancer Res. 40 3 1 9 4 - 3 2 0 1 (1980). B.R. O A K L E Y , D.R. K I R S C H a n d N.R. M O R R I S , Anal. B1ochem. 105 361-363 (1980). M.M. B R A D F O R D , Anal. B1ochem. 72 2 4 8 - 2 5 4 ( 1 9 7 6 ) .
PROTEIN
Ben]amln
Pergamon Press
B 2 3 ( M . W . / p I = 37 k D / 5 . 1 ) IS T H E O N L Y M A J O R E X T R A C T E D F R O M H e L a N U C L E O L I W I T H 3M U R E A Yat-Mlng
Y u n g I*,
Erlc
Ka-Wa±
Hul I and
PROTEIN
Pul-Kwong
Chan 2
I D e p a r t m e n t of P h a r m a c o l o g y , Chang Gung Medlcal College, T a l w a n , R . O . C . a n d 2 D e p a r t m e n t of P h a r m a c o l o g y , B a y l o r C o l l e g e of M e d l c l n e , H o u s t o n , T e x a s 7 7 0 3 0 , U . S . A .
(Recelved an f~nal form July 8, 1992)
Summary H e L a n u c l e o l l w e r e i s o l a t e d u s l n g the N P - 4 0 m e t h o d a n d subsequently extracted w l t h 3M u r e a . The e x t r a c t w a s i n c u b a t e d at 6 0 ° C for 30 mln, a n d p r e c l p l t a t e d protelns were removed by c e n t r ± f u g a t l o n . The supernatant was analyzed by one- and two-dimensional SDS polyacrylamlde gel e l e c t r o p h o r e s l s (PAGE). P r o t e l n B23 w a s the o n l y m a 3 o r p r o t e i n e x t r a c t e d f r o m H e L a n u c l e o l ± by t h i s p r o cedure. Using thls procedure, 1 mg of p r o t e i n B 2 3 was obtained f r o m 2 g of H e L a c e l l s . T h e p u r ± t y of the ext r a c t e d p r o t e i n B23 w a s 98%, as m e a s u r e d by one- and two-dlmenslonl gel electrophoresls. The n u c l e o l u s 1s a h l g h l y s p e c l a l l z e d organelle, producing ribosomal R N A ( r R N A ) w h l c h is a s s e m b l e d into rlbosomal particles and eventually transported to the c y t o p l a s m (i). P r e v ± o u s s t u d l e s h a v e s h o w n t h a t the p h o s p h o p r o t e l n B 2 3 (37 k D / p I 5.1) is o n e of the m a 3 o r n u c l e o l a r p h o s p h o p r o t e l n ldentlfled in t u m o r c e l l s (24). P r o t e l n B23 is a s s o c l a t e d w l t h p r e - r R N P s , a n d m a y be i n v o l v e d in the m a t u r a t i o n of r l b o s o m e s (4). R e c e n t s t u d l e s s h o w t h a t p r o t e i n B23 t r a n s l o c a t e s f r o m the n u c l e o l u s to the n u c l e o p l a s m w h e n rRNA processlng is l n h l b l t e d by s e r u m s t a r v a t l o n or l n h l b l t o r s s u c h as a c t ± n o m y c l n D, l u z o p e p t l n s , t o y o c a m y c l n , or h l g h d o s e s of ~-amanltln (5-8). Proteln B23 was prevlously purlfled from Novlkoff hepatoma cell nucleoll uslng the method developed by M l c h a l l k et al (9). Thls method involved three ma3or steps, (i) 4M u r e a / 3 M L i C l extraction of n u c l e o l l , (11) d l a l y s l s of the e x t r a c t against 4M urea/20 mM Trls-Malate/pH 5.5, a n d (ili) D E A E - c e l l u l o s e chromatography (9). T h e p r o c e d u r e requlred 4 d a y s , a n d the y l e l d w a s about 2 mg/100 g tlssue. Thls communlcatlon r e p o r t s an i m p r o v e d new procedure f o r the isolatlon of p r o t e i n B 2 3 f r o m H e L a c e l l s w l t h h i g h e r y l e l d (i m g / 2 g c e l l s ) a n d s h o r t e r t l m e r e q u i r e m e n t .
* TO w h o m r e p r l n t r e q u e s t s s h o u l d be s e n t to: Dr. B e n 3 a m l n Y a t M l n g Y u n g , P h . D . , C h a n g G u n g M e d ± c a l C o l l e g e , D e p a r t m e n t of P h a r macology, 259 W e n - H w a ist Road, K w e 1 - S a n , Tao-Yuan 333, T a l w a n , R.O.C. 0024-3205/92 $5.00 + .00 Copyright © 1992 Pergamon Press Ltd All r~ghts reserved.
916
P r o t e l n B23 and U r e a E x t r a c t i o n
Vol.
51, No.
12,
1992
Methods Cells H e L a $3 c e l l s w e r e g r o w n in m o n o l a y e r wlth Eagle's mlnlmum essentlal medium (MEM) supplemented wlth 10% f e t a l c a l f serum, glutamlne and antlbot±cs in a 5% C O 2 - h u m l d ± f l e d incubator at 37°C. Preparation of Nucleoll Nucleoll were isolated u s i n g the NP-40 method prevlously described (5,10). Cells were suspended in 20 v o l of R S B b u f f e r (0.01M Trls HCI/0.01M NaCl/l.5 mM NaCl/pH 7.2) f o r 30 m ± n a n d t h e n c e n t r l f u g e d at 630 g f o r 8 mln. S w o l l e n cells were resuspended in 20 v o l of R S B b u f f e r containing 0.5% NP-40. The cells were homogenized wlth a Dounce homogenizer (10 up-and-down strokes), a n d the c r u d e n u c l e i w e r e c o l l e c t e d by c e n trifugatlon at 630 g f o r 8 mln. T h e n u c l e i w e r e r e s u s p e n d e d in 10 v o l of 0 . 2 5 M s u c r o s e / 1 0 mM MgCI2, and underlayered w i t h an e q u a l vol of 0.88 M sucrose/0.05 mM MgCI2. After centrlfugatlon at 1700 g f o r i0 mln, the n u c l e i were resuspended in i0 v o l o f 0.34 M sucrose/0.05 mM MgCI2 . The suspension was sonlcated f o r 1 mln, underlayered w l t h an e q u a l v o l o f 0 . 8 8 M s u c r o s e / 0 . 0 5 mM MgCI 2 , and centrifuged at 3 0 0 0 g f o r 18 mln. T h e p e l l e t (nucleoll) was then collected. Purlflcat±on of Protein B23 Nucleoll (0.i g) f r o m H e L a cells were suspended in i0 vol (i ml) of 3M urea/l n~ phenylmethyl sulfonyl fluoride (PMSF)/I mM leupeptln/l mM pchloromercurlphenyl sulfon±c ac±d (pCMPS), and were homogenized with a Dounce homogenizer (20 u p - a n d - d o w n strokes) on ice. The extract was then incubated at 6 0 ° C f o r 30 m l n a n d c e n t r i f u g e d at 2 7 , 0 0 0 g f o r 20 m ± n at 5°C. T h e s u p e r n a t a n t , which contalned pur l f ± e d p r o t e l n B23, w a s c o l l e c t e d . Polyacrylamlde Gel Electrophores±s One-dlmenslonal SDSPAGE was performed accordlng to the m e t h o d o f L a e m m l l (Ii). T w o d±menslonal PAGE was done by isoelectrlc focuslng (12) a n d S D S PAGE. G e l s w e r e e l t h e r s t a ± n e d w i t h 0 . 1 % C o o m a s s l e Brlll±ant-Blue R (S±gma) or sllver-stalned (13). Proteln Assay B±o-Rad prote±n assay
Prote±n (14).
concentration
was
determined
by
the
Results The improved isolat±on procedure for p r o t e l n B 2 3 is s h o w n in FIG. i. H e L a c e l l n u c l e o l l t h a t w e r e p u r l f l e d b y the N P - 4 0 m e t h o d (5) w e r e u s e d as the s t a r t l n g materlal. The nucleoll were extracted wlth 3M u r e a a n d s u b s e q u e n t l y incubated at 6 0 ° C f o r 30 mln. Conslderable amounts of insoluble protelns (90% of the startlng protelns) were obta±ned, whlch were subsequently removed by centrlfugatlon at 2 7 , 0 0 0 g f o r 20 mln. FIG. 2 s h o w s the o n e dlmens±onal SDS-PAGE patterns of the p r o t e l n s from varlous fract±ons of the purlflcatlon procedure. There are approxlmately 45 Coomassle blue-sta±n±ng protelns ±n t h e g e l o f the t o t a l e x t r a c t of H e L a n u c l e o l l (FIG. 2B). A 37 k D b a n d w h l c h c o r r e s p o n d s to t h e pos±tlon of protein B23 was observed. By desltometrlc scannlng, thls band accounted for about 10.4+1.6% of the total amount of protemns (FIG. i). FIG. 2C s h o w s the i n s o l u b l e proteln aggregate f r o m the 3M u r e a e x t r a c t of nucleoll after mncubatlon at 6 0 ° C . Thls preclpltated fractlon contalned m o s t o f the same proteln
Vol.
51, No.
12,
1992
P r o t e i n B23 a n d U r e a E x t r a c t i o n
HeLa
Nucleoll,
2.
(no
Cells
10.5
3M U r e a
mg
(10.4%
B23)
L
Extraction
1. 6 0 ° C I n c u b a t i o n f o r 30 m l n Centr~fugatlon 2 7 , 0 0 0 g , 20 m~n.
/
",,,
pellet, 9 . 0 mg B23 d e t e c t a b l e )
Isolatlon
917
Scheme
supernatant, (98%
for
FIG.
1
HeLa
Nucleolar
1.12 B23)
Prote±n
mg
B23.
bands as the total extract (FIG. 2B), execpt protein B23. As s h o w n in FIG. 2E, p r o t e l n B 2 3 w a s f o u n d m a l n l y in the s u p e r n a t a n t f r a c t l o n w i t h a p u r l t y of o v e r 95%, d e t e r m l n e d by d e n s l t o m e t r l c scannlng. Immunoblot results conflrmed t h a t the p u r l f l e d p r o t e l n was p r o t e i n B 2 3 (FIG. 2F). W h e n the 60 °C l n c u b a t l o n step was omltted, the s u p e r n a t a n t contained not only protein B23, but also small quantltles of other protelns. FIG. 2D s h o w s t h a t t h e s e o t h e r p r o t e l n s a r e of hlgher molecular weight proteins. The purity of protein B23 was analyzed by 2D gel electrophoresls. A l a r g e d e n s e s p o t a n d a f e w m l n o r s p o t s (FIG. 3A) a r e l o c a t e d in the p o s i t i o n of B 2 3 (M.W. 37 k D / p I 5.1). T h i s r e s u l t a g r e e s w l t h p r e v l o u s s t u d l e s (5) w h i c h s h o w t h a t t h e r e a r e mult±ple forms of protlen B23. FIG. 3B shows the Western immunoblot of the purified protein B23 after 2D gel electrophoresls. The purlfled protein shows identlcal immunostalnlng and sllver-stalnlng patterns. DlSCUSSlOn T h l s s t u d y i n d l c a t e s t h a t p r o t e l n B23 is the o n l y m a ] o r p r o teln extracted from HeLa nucleol± w l t h 3M u r e a . T h e i n c u b a t i o n s t e p (at 6 0 ° C ) is n e c e s s a r y to e l l m l n a t e m l n o r c o n t a m l n a n t s (FIG. 2D) In the e x t r a c t . T h e f a c t t h a t p r o t e l n B23 is the o n l y m a ] o r p r o t e l n s o l u b l e in 3M u r e a s u g g e s t s t h a t the s t r u c t u r a l o r g a n l z a t l o n of p r o t e i n B23 in n u c l e o l l m a y be d i f f e r e n t f r o m t h a t of other nucleolar protelns. Compared to the m e t h o d developed by Mlchallk et al (9), t h i s n e w i s o l a t l o n procedure is s i m p l e a n d rapld. The new isolatlon p r o c e d u r e t a k e s o n l y 3 hrs, instead
918
P r o t e & n B23 a n d U r e a E x t r a c t & o n
Vol.
[
FIG.
51, No.
12, 1992
F
2
8% S D S - P A G E of n u c l e o l a r protelns at v a r l o u s s t e p s of purlflcatlon of p r o t e l n B23. The polyacrylamlde gel was stalned wlth Coomassle Blue. Lane A - purlfled proteln B23 from Novlkoff hepatoma cell nucleo]l (9). L a n e B - total HeLa nucleolar protelns (whole nucleoll dlss o l v e d in S D S s a m p l e b u f f e r ) . L a n e C - t h e p r o t e l n p r e clpltate ( p e l l e t ) o f t h e 3M u r e a e x t r a c t o f H e L a n u c l e o11. Lane D - supernatant of the 3M u r e a e x t r a c t of HeLa nucleoll (wlthout incubatlon at 6 0 ° C f o r 30 m l n ) . Lane E - supernatant of t h e 3M u r e a e x t r a c t o f H e L a nucleoll (with incubatlon at 6 0 0 C f o r 30 m ± n ) . L a n e F protelns in L a n e E w e r e t r a n s f e r r e d onto a nltrocellulose sheet and immunostalned wlth the ant±-B23 antlbody (5,7). An ± m m u n o b a n d correspondlng to p r o t e l n B23 was observed.
of 4 days, and HeLa cells).
it
has
a
hlgher
yield
(I
mg
proteln
B23/2
g
of
Vol.
51, No.
12,
1992
Protein
B23 and U r e a E x t r a c t z o n
FIG.
919
3
(A) T w o - d l m e n s l o n a l SDS-PAGE of p u r l f l e d proteln B23. A b o u t 1 m g of p u r l f l e d p r o t e l n B23 w a s l o a d e d o n t o the gel. T h e g e l w a s s l l v e r - s t a l n e d (13). A m a 3 o r s p o t at M.W./pI = 37 k D / 5 . 1 was observed. A large patch of stalnlng located in the b o t t o m l e f t c o r n e r of the gel corresponds to A m p h o l l n e . (B) W e s t e r n i m m u n o s t a l n l n g of p u r l f l e d p r o t e l n B23. A b o u t 5 m g of p u r l f l e d p r o t e l n B23 w a s l o a d e d o n t o a t w o - d l m e n s l o n a l S D S - P A G E gel. A f t e r the p r o t e l n w a s t r a n s f e r r e d to n l t r o c e l l u l o s e paper, the p a p e r w a s i m m u n o s t a l n e d w i t h the p r o t e l n B23 monoclonal antlbody.
Acknowledgments W e t h a n k Drs. H a r r l s B u s c h a n d D e l o n W u for t h e l r a d v l c e a n d encouragement durlng these studles. These studles were supported by C h a n g Gung Research Grant (CMRP 275) and Natlonal Science Councll (R.O.C.) Grant (NSC80-0412-B-182-41 and NSC81-0412-B-1825). References i. 2. 3. 4. 5.
H. B U S C H a n d K.J. S M E T A N A , T h e N u c l e o l u s . A c a d e m l c P r e s s , N e w York (1970). M . O . J . O L S O N , L.R. O R R I C K , C. J O N E S and H. B U S C H , J. B i o l . C h e m . 249 2 8 2 3 - 2 8 2 7 ( 1 9 7 4 ) . A. W. P R E S T A Y K O , G. R. K L O M P , D. J. S C H M O L L and H. B U S C H , Blochemlstry 13 1 9 4 5 - 1 9 5 1 ( 1 9 7 4 ) . D.L. S P E C T O R , R.L. O C H S a n d H. B U S C H , Chromosoma 90 1 3 9 - 1 4 8 ( 1984 ). P.K. C H A N , M. A L D R I C H a n d H. B U S C H , Exp. C e l l Res. 161 i01ii0 ( 1 9 8 5 ) .
920
6. 7. 8. 9. i0. ii. 12. 13. 14.
Protean B23 and Urea Extraction
Vol. 51, No. 12, 1992
B . Y - M . Y U N G , R.K. B U S C H , H. B U S C H , A.B. M A U G E R a n d P.K. C H A N , Blochem. Pharmacol. 34 4 0 5 9 - 4 0 6 3 (1985). B . Y - M . Y U N G , H. B U S C H and P.K. C H A N , Blochlm. B1ophy. Acta 826 1 6 7 - 1 7 3 ( 1 9 8 5 ) . B . Y - M . Y U N G , H. B U S C H a n d P.K. C HA N , C a n c e r Res. 46 9 2 2 - 9 2 5 (1986). J. M I C H A L I K , L.C. Y E O M A N a n d H . B U S C H , L i f e Scl. 28 1 3 7 1 - 1 3 7 9 (1981). M. M U R A M A T S U , Y. H A Y A S H I , T. O N I S H I , M. S A K A I , K. T A K A I and T. K A S H I Y A M A , Exp. C e l l Res. 88 345 ( 1 9 7 4 ) . U.K. L A E M M L I , N a t u r e ( L o n d . ) 277 6 8 0 - 6 8 1 (1970). P.K. C H A N , A. F E Y E R A B E N D , R.K. B U S C H and H. B U S C H , Cancer Res. 40 3 1 9 4 - 3 2 0 1 (1980). B.R. O A K L E Y , D.R. K I R S C H a n d N.R. M O R R I S , Anal. B1ochem. 105 361-363 (1980). M.M. B R A D F O R D , Anal. B1ochem. 72 2 4 8 - 2 5 4 ( 1 9 7 6 ) .