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Purification of calmodulin from human brain
Life Sciences, Vol. 29, pp. 2781-2787 Printed in the U.S.A.
Pergamon Press
PURIFICATION OF CALMODULIN FROM HUMAN BRAIN N o b u h i s a Kobayashi, Y o s h i n o b u Nakao, M i c h i z o Kishihara, Yasuto Baba, Takuo Fujita, and Kyozo H a y a s h i * Third Division, Department of Medicine, Kobe University School of Medicine, Kusunoki-cho, Chuo-ku, Kobe 650, Japan *Department of Biological Chemistry, Faculty of Pharmaceutical Science, Kyoto University, Kyoto 606, Japan (Received in final form November 5, 1981) S UMMARY C a l m o d u l i n was p u r i f i e d from human brain by a m m o n i u m sulfate p r e c i p i t a t i o n , gel filtration, and anion e x c h a n g e chromatography. The p u r i f i e d c a l m o d u l i n was h o m o g e n o u s w h e n e v a l u a t e d by p o l y a c r y l a m i d e gel e l e c t r o phoresis. The b i o l o g i c a l and p h y s i c o c h e m i c a l p r o p e r t i e s of human brain c a l m o d u l i n such as the ability to activate c a l m o d u l i n - d e f i c i e n t bovine p h o s p h o d i e s t e r a s e , m o l e c u l a r weight, and amino acid c o m p o s i t i o n were almost the same as bovine brain calmodulin. C a l m o d u l i n is a heat stable, small acidic C a 2 + - b i n d i n g protein, and is known to m e d i a t e a number of Ca = - m e d i a t e d effects in various c e l l u l a r reactions (1-4). Recently, this protein has been i s o l a t e d from b o t h plants and animals. D e t a i l e d analyses of calmo d u l i n from bovine brain (5), bovine uterus (6), rat testis (7), rat islet cell tumor (8), murine m a c r o p h a g e cell lines (9), and human p l a c e n t a (i0) have shown little v a r i a t i o n in its structure. Nevertheless, there are no d e t a i l e d reports on b i o l o g i c a l and physi c o c h e m i c a l p r o p e r t i e s of human brain c a l m o d u l i n in the h o m o g e n o u s state as yet. Human c a l m o d u l i n may help to e l u c i d a t e the mechan i s m of the m a n y C a Z + - r e g u l a t e d b i o l o g i c a l process in man and the i d e n t i f i c a t i o n of f u n c t i o n a l l y i m p o r t a n t amino acid residues in c o m p a r i s o n with other species of calmodulins seems challenging. As such, we made an attempt to isolate pure human brain c a l m o d u l i n by a m m o n i u m sulfate p r e c i p i t a t i o n , gel filtration, and anion e x c h a n g e chromatography. In this article, we deal w i t h the i s o l a t i o n and some p h y s i c o c h e m i c a l p r o p e r t i e s of c a l m o d u l i n from human brain, and d i r e c t l y c o m p a r e d it to w e l l - c h a r a c t e r i z e d bovine brain calmodulin.
*This study was s u p p o r t e d in part by the Research Grant for the I n t r a c t a b l e Disease from the M i n i s t r y of H e a l t h and W e l f a r e of Japan (1980), the F o u n d a t i o n of Research for M e t a b o l i c Disease and Life Science of Japan (1980). **Requests for reprints should be a d d r e s s e d to N. Kobayashi.
0024-3205/81/262781-07502.00/0 Copyright (c) 1981 Pergamon Press Ltd.
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MATERIALS
AND
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METHODS
Materials: H u m a n b r a i n was o b t a i n e d from the c a d a v e r of a y o u n g japanese woman, two hours a f t e r she d i e d in an accident. Bovine b r a i n c a l m o d u l i n was the g e n e r o u s gift of Dr. S h i r o K a k i u c h i , Osaka University. B o v i n e h e a r t p h o s p h o d i e s t e r a s e and snake v e n o m (Crotalus atrox ) w e r e p u r c h a s e d from S i g m a C h e m i c a l Co. St. Louis, M.O. B i o - R a d A G I - X 2 (Chloride form, 2 0 0 - 4 0 0 3 m e s h ) was o b t a i n e d from B i o - R a d l a b o r a t o r i e s , Richmond, C.A. [ H ] c y c l i c AMP and A Q U A S O L - 2 w e r e o b t a i n e d from New E n g l a n d Nuclear, Boston, M.A. A s s a y of P h o s p h o d i e s t e r a s e A c t i v a t o r A c t i v i t y : Phosphodiesterase a c t i v i t y and the a b i l i t y of c a l m o d u l i n to s t i m u l a t e the a c t i v i t y of b o v i n e h e a r t p h o s p h o d i e s t e r a s e w e r e a s s a y e d u s i n g a m o d i f i e d p r o c e d u r e of T h o m p s o n and A p p l e m a n (ii) and C h e u n g (12). A reaction m i x t u r e of 0.i ml c o n t a i n e d 40 m M T r i s - H C l (pH 8.0), ½0 m M MgCl2, 0.05 m M CaCl~, 0.i m M u n l a b e l l e d c y c l i c AMP, 1 ~ M [ H]cycllc AMP, and c a l ~ o d u l i n (i-i000 ng). The r e a c t i o n was i n i t i ated by the a d d i t i o n of c a l m ~ d u l i n - d e f i c i e n t b o v i n e h e a r t p h o s p h o diesterase. P r o d u c t i o n of [ H ] 5 ' - A M P was m e a s u r e d by i n c u b a t i o n w i t h snake v e n o m w h i c h was used as a source of 5 ' - n u c l e o t i d a s e , a f t e r w h i c h [ ~ H ] a d e n o c i n e was c o u n t e d in a i0 ml A Q U A S O L - 2 s c i n t i l l a t i o n fluid. P u r i f i c a t i o n of H u m a n B r a i n C a l m o d u l i n : The p r o c e d u r e a d o p t e d in this s t u d y c l o s e l y r e s e m b l e s that used by W a t t e r s o n (13). All o p e r a t i o n w e r e c a r r i e d out at 4°C. H u m a n b r a i n (280 g) was homogen i z e d in 2 v o l u m e s of 0.i M s o d i u m a c e t a t e (pH 7.2), 1 m M 2m e r c a p t o e t h a n o l , and 1 m M E D T A (Buffer A), and the h o m o g e n a t e w a s c e n t r i f u g e d at i0,000 x g for 60 min. The s u p e r n a t a n t fluid was m i x e d w i t h 600 ml of C M - S e p h a d e x C-50, a l l o w e d to stand for 30 min, and then f i l t e r e d t h r o u g h a B u c h n e r funnel. THe f i l t r a t e was a p p l i e d to a c o l u m n (2 x 50 cm) of D E A E - S e p h a d e x A-50 e q u i l i b r a t e d w i t h B u f f e r A. A d s o r b e d p r o t e i n s w e r e e l u t e d w i t h a linear salt g r a d i e n t f o r m e d w i t h 600 ml each of 0.05 M and 0.80 M NaCl in B u f f e T A as the s t a r t i n g and l i m i t i n g buffers. Five ml f r a c t i o n s w e r e c o l l e c t e d and m o n i t o r e d by r e a d i n g a b s o r b a n c e at 280 nm. A l i q u o t s of f r a c t i o n s h a v i n g a b s o r b a n c e peaks in the r e g i o n of 280 nm w e r e a n a l y z e d for p h o s p h o d i e s t e r a s e activity, and f r a c t i o n s c o n t a i n i n g c a l m o d u l i n w e r e pooled. S o l i d a m m o n i u m sulfate (313 g/ litter) was a d d e d to the p o o l e d f r a c t i o n s to b r i n g the s o l u t i o n to 50% s a t u r a t i o n . The m i x t u r e was s t i r r e d for 60 min, and c e n t r i fuged at 10,000 x g for 30 min. The s u p e r n a t a n t f r a c t i o n was a d j u s t e d to pH 4.0 w i t h s u l f u r i c acid. The m i x t u r e was s t i r r e d for 60 m i n and c e n t r i f u g e d at 10,000 x g for 30 min. The p e l l e t w a s d i s s o l v e d in 30 ml of 0.01 M a m m o n i u m b i c a r b o n a t e and d e s a l t e d on a c o l u m n (3 x 80 cm) of S e p h a d e x G-25 in 0.01 M a m m o n i u m b i c a r b o n a t e and l y o p h i l i z e d . The l y o p h i l i z e d p r o t e i n was d i s s o l v e d in 50 ml of 0.01 M T r i s - H C l (pH 7.5), 1 m M EDTA, 1 mM 2 - m e r c a p t o ethanol, and 0.2 M NaCI (Buffer B) and a p p l i e d to a c o l u m n (2 x 50 cm) of D E A E - S e p h a d e x A-50 e q u i l i b r a t e d w i t h B u f f e r B. THe c o l u m n was e l u t e d w i t h a linear salt g r a d i e n t c o n s i s t i n g of 600 ml each of B u f f e r B and 0.7 M NaCI in B u f f e r B as the s t a r t i n g and limiting buffers. The f r a c t i o n s c o n t a i n g c a l m o d u l i n w e r e p o o l e d and lyophilized. The l y o p h i l i z e d p r o t e i n was d i s s o l v e d in 20 ml of 0.5 M T r i s - H C l (pH 7.5), 1 m M EDTA, and 1 m M 2 - m e r c a p t o e t h a n o l (Buffer C) and a p p l i e d to a c o l u m n (4 x i00 cm) of S e p h a d e x G-100 in B u f f e r C. The p r o t e i n o b t a i n e d was h o m o g e n o u s w h e n e v a l u a t e d by s o d i u m d o d e c y l s u l f a t e - p o l y a c r y l a m i d e gel e l e c t r o p h o r e s i s in w h i c h no d e n a t u r a n t was present.
Vol. 29, No. 26, 1981
The y i e l d
of c a l m o d u l i n
Purification of Calmodulin
from the b r a i n was
2783
22 mg.
E l e c t r o p h o r e t i c Method: Following treatment with sulfate (SDS) and 2 - m e r c a p t o e t h a n o l , the p r o t e i n c h r o m a t o g r a p h i c fractions were r o u t i n e l y a n a l y z e d sis on 12.5% p o l y a c r y l a m i d e gel, c o n t a i n i n g 0.1% by Laemmli (14). Gels were stained w i t h Coomasie (15).
sodium dodecyl c o m p o n e n t s of the by e l e c t r o p h o r e SDS, as d e s c r i b e d B r i l l i a n t Blue
A m i n o A c i d Analysis: Amino acid analysis was p e r f o r m e d on a Hitachi 835-50 single column s y s t e m a u t o m a t i c amino acid analyzer, a c c o r d i n g to the m e t h o d of S p a c k m a n et al. (16). The p r o t e i n fractions were h y d r o l y z e d w i t h 6 N HCI at II0°C for 24, 48, and 72 hours in the e v a c u a t e d tubes. T r y p t o p h a n was d e t e r m i n e d after h y d r o l y s i s w i t h 4 N m e t h a n s u l f o n i c acid at I10°C for 24 hours.
Fig.
1
E l e c t r o p h o r e t i c analysis of human b r a i n calmodulin: E l e c t r o p h o r e s i s was done in the presence of sodium dodecyl sulfate on a 12.5%(w/v) p o l y a c r y l a m i d e gel c o n t a i n i n g 2.5 mM EDTA. Samples were i n c u b a t e d at 100°C for 3 min before analysis. Lane A is 5 Dg of human b r a i n calmoduli~. Lane B c o n t a i n e d 5 D g of each m o l e c u l a r w e i g h t standard. From top to bottom, the standards are p o l y m e r a s e B p e ~ t a m e r (71,500), t e t r a m e r ( 5 7 , 2 0 0 ) , trimer(42,900), d i m e r ( 2 8 , 6 0 0 ) , and m o n o m e r ( 1 4 , 3 0 0 ) . Lane C is 5 ~g of bovine b r a i n calmodulin.
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Vol. 29, No. 26, 1981
RESULTS Pure human brain calmodulin was obtained by ammonium sulfate precipitation, gel filtration, and anion exchange chromatography. The purified protein migrated as a single band on SDS polyacrylamide gel electrophoresis and also comigrated with bovine brain calmodulin as shown in Fig. i. From these results, the molecular weight of this protein appeared to be similar to that of bovine brain calmodulin. The amino acid composition of this protein is shown in Table'l along with the composition by sequence of bovine brain calmodulin. The human brain calmodulin composition values h a v e been calculated by using estimated molecular weight (17,000) of bovine brain calmodulin (16,680) under the electrophoretic condition of Fig. i. One striking feature of the data is that one-third of the total amino acid is contributed from aspartic and glutamic acids following acid hydrolysis. Another feature is the absence of tryptophan and cystein. Both human and bovine brain calmodulin were tested in.triplicate for their ability to quantitatively stimulate 8 x 10 -4 units of activator-free bovine heart phosphodiesterase. Both brain calmodulins gave indistinguishable activation curves as shown in Fig. 2. Half-maximal stimulation of phosphodiesterase activity was obtained with 40 ng and 60 ng of human and bovine brain calmodulins respectively. Fully activated phosphodiesterase had an activity of 40 nmol of c-AMP hydrolyzed per mg phosphodiesterase.
• -
100
'///"
,---,human ----- bovine
/ JC
O_ .... 4 0 tO.
20
•~'!° ~ 50
100
~ 200 calmodul.in ( ng ) Fig.
1000
2
Comparison of human brain calmodulin ( - - ) and bovine brain calmodulin (---) by activation of activator-free phosphodiesterase. The closed ~ r c l e s refer to assay conducted in the presence of Ca- (50 ~M) and the open circles are values obtained in the presence of EGTA (i mM).
Vol. 29, No. 26, 1981
Purification of Calmodulin
TABLE
I
AMINO ACID COMPOSITION
Amino Acid
Aspartic
acid
Threonine Serine Glutamic
acid
H u m a n Brain a residues/ 17,000g
OF C A L M O D U L I N Bovine Brain b residues/ molecule
22 55
23
ii 28
12
5 40
4
29 34
27
Proline
2 82
2
Glycine
14 33
ii
Alanine
13 90
ii
Half-cystine
0 00
0
Valine
7.34
7
Methionine
5.58
9
Isoleucine
7.30
8
Leucine
9.30
9
Tyrosine
2.18
2
Phenylalanine
7.55
8
Lysine
6.92
7
Histidine
1.09
1
Arginine
5.89
6
Tryptophan
0.00
0
Trimethyllysine
N.D. c
1
a c a l c u l a t e d as d e s c r i b e d in the text f r o m c o m p o s i t i o n analysis u s i n g the e s t i m a t e d m o l e c u l a r w e i g h t of h u m a n b r a i n c a l m o d u l i n (17,000) from SDS p o l y a c r y l a m i d e gel analysis. b • C o m p o s l t l o n c a l c u l a t e d from amino acid s e q u e n c e (5). CNot d e t e r m i n e d .
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Vol. 29, No. 26, 1981
Fig. 2 shows also the r e q u i r e m e n t of Ca 2+ for the a c t i v a t i o n of p h o s p h o d i e s t e r a s e by b o t h h u m a n and b o v i n e b r a i n c a l m o d u l i n . Both c a l m o d u l i n s s t i m u l a t e d p h o s p h o d i e ~ e r a s e a c t i v i t y in an i d e n t i c a l m a n n e r in the p r e s e n c e of Ca ^° but did not a c t i v a t e p h o s p h o d i e s t e r a s e in the p r e s e n c e of C a Z + - c h e l a t o r , e t h y l e n e glycol-bis-(B-aminoethylether)-N,N'-tetraacetic acid (EGTA).
DISCUSSION The a n a l y s e s r e p o r t e d here d i r e c t l y d e m o n s t r a t e that the s t r u c t u r a l and f u n c t i o n a l p r o p e r t i e s of h u m a n b r a i n c a l m o d u l i n have b e e n h i g h l y c o n s e r v e d d u r i n g v e r t e b r a t e evolution. This r e s u l t agrees w i t h that r e p o r t e d by D.M. W a t t e r s o n et al. (17) about a d e t a i l e d c o m p a r i s o n of c a l m o d u l i n s i s o l a t e d from several v e r t e b r a t e b r a i n s (pig, rabbit, chicken, and beef). The amino acid c o m p o s i t i o n g i v e n in Table 1 did not show any m a j o r d i f f e r e n c e s from that of the b o v i n e b r a i n c a l m o d u l i n . The c o m p o s i t i o n of this p r o t e i n is also c l o s e to the one r e p o r t for h u m a n b r a i n c a l m o d u l i n (18), b u t has the same c o n t e n t of a s p a r t i c acid as b o v i n e b r a i n c a l m o d u l i n . H u m a n b r a i n c a l m o d u l i n w a s also found to have the same activ i t y on p h o s p h o d i e s t e r a s e a c t i v a t i o n as those of the o t h e r v e r t e brate calmodulins. The a b i l i t y of p u r i f i e d h u m a n b r a i n c a l m o d u l i n to a c t i v a t e b o v i n e h e a r t p h o s p h o d i e s t e r a s e a g r e e d w i t h the r e s u l t s that r e p o r t e d by F. V a r g a s et al. (19). In the p h o s p h o d i e s t e r a s e a c t i v a t i o n , the p o t e n c y of h u m a n b r a i n c a l m o d u l i n was 1.5 times of that of b o v i n e b r a i n c a l m o d u l i n . This r e s u l t s u g g e s t s the p o s s i b i l i t y that h u m a n b r a i n c a l m o d u l i n also got a d i r e c t i o n a l c h a n g e d u r i n g the2+molecular e v o l u t i o n t o w a r d an increase' in the c a p a b i l ity in Ca - d e p e n d e n t enzyme a c t i v a t i o n as p r e v i o u s l y r e p o r t e d by S. K a k i u c h i et al. (20). As s h o w n above, we have found that c a l m o d u l i n p u r i f i e d from h u m a n b r a i n is v e r y s i m i l a r to b o v i n e b r a i n c a l m o d u l i n in m a n y r e g ~ e c t s such as f r a c t i o n a t i o n b e h a v i o r , e l e c t r o p h o r e t i c m o b i l i t y , Ca ~ - d e p e n d e n t p h o s p h o d i e s t e r a s e a c t i v a t o r activity, and amino acid c o m p o s i t i o n . The v a r i o u s f u n c t i o n a l roles of c a l m o d u l i n in man have not yet b e e n i n v e s t i g a t e d . However, the c h a r a c t e r i z a t i o n of h u m a n b r a i n c a l m o d u l i n r e p o r t e d h e r e p r o v i d e s a f i r m b a s i s for f u r t h e r b i o l o g i c a l study on the b i o l o g i c a l and b i o p h y s i c a l p r o p e r ties of h u m a n b r a i n c a l m o d u l i n .
ACKNOWLEDGEMENTS We thank Prof. Y. N i s h i z u k a and Dr. Y. Takai of D e p a r t m e n t of B i o c h e m i s t r y , Kobe U n i v e r s i t y School of M e d i c i n e , for e n c o u r a g e m e n t t h r o u g h o u t this w o r k and c r i t i c a l r e a d i n g of the m a n u s c r i p t .
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i. S. KAKIUCHI, R. YAMAZAKI, and H. NAKAZIMA, Proc. Japan Acad. 46, 587-592, (1970). 2. W.Y. CHEUNG, Biochem. Biophys. Res. Commun. 38, 533-538, (1970). 3. W.Y. CHEUNG, Science 207, 19-37, (1980). 4. J.R. DEDMAN, M.I. WELSH, and A.R. MEANS, J. Biol. Chem. 253, 7515-7521, (1980). 5. D.M. WATTERSON, F. SCHARIEF, and T.C. VANAMAN, J. Biol. Chem. 255, 962-975, (1980). 6. R.J.A. GRAND, and S.V. PERRY, FEBS lett. 92, 137-142, (1978). 7. J.R. DEDMAN, R.L. JACKSON, M.E. SCREIBER, and A.R. MEANS, J. Biol. Chem. 253, 343-346, (1980). 8. J.C. HUTTON, E.F. PENN, P. JACKSON, and C.N. HALES, Biochem. 193, 875-885, (1980). 9. A. GORDON, JAMIESON, Jr., and T.C. VANAMAN, J. Immunol. 125, 1171-1177, (1980). 10. S.UMEI, S. NAGAO, and Y. NOZAWA, Biocim. Biophys. Acta, 674, 319-326, (1981). ii. W.J. THOMPSON, and M.M. APPLEMAN, B i o c h e m i s t r y i0, 311-316, (1971). 12. W.Y. CHEUNG, Anal. Biochem. 67, 130-138, (1975). 13. D.M. WATTERSON, W.G. HARRELSON, Jr., P.M. KELLER, F. SCHARIEF, and T.C. VANAMAN, J. Biol. Chem. 251, 4501-4513, (1976). 14. U.K. LAEMMLI, Nature 227, 680-685, (1970). 15. P.H. O'FARREL, J. Biol. Chem. 250, 4007-4021, (1975). 16. D.H. SPACKMAN, W.H. STEIN, and S. MOORE, Anal. Chem. 30, 1190-1206, (1958). 17. D.M. WATTERSON, P.A. MENDEL, and T.C. VANAMAN, B i o c h e m i s t r y 19, 2672-2676, (1980). 18. J. HAIECH, B. VALLET, R. AQUARON, and J.G. DEMAILLE, Anal. Biochem. 105, 18-23, (1980). 19. F. VARGAS, and A. GUIDOTTI, N~urochem. Res. 5, 673-681, (1980). 20. S. KAKIUCHI, K. SOBUE, R. YAMAZAKI, S. NAGAO, S. UMEKI, Y. NOZAWA, M. YAZAWA, and K. YAGI, J. Biol. Chem. 256, 19-22, (1981).
Pergamon Press
PURIFICATION OF CALMODULIN FROM HUMAN BRAIN N o b u h i s a Kobayashi, Y o s h i n o b u Nakao, M i c h i z o Kishihara, Yasuto Baba, Takuo Fujita, and Kyozo H a y a s h i * Third Division, Department of Medicine, Kobe University School of Medicine, Kusunoki-cho, Chuo-ku, Kobe 650, Japan *Department of Biological Chemistry, Faculty of Pharmaceutical Science, Kyoto University, Kyoto 606, Japan (Received in final form November 5, 1981) S UMMARY C a l m o d u l i n was p u r i f i e d from human brain by a m m o n i u m sulfate p r e c i p i t a t i o n , gel filtration, and anion e x c h a n g e chromatography. The p u r i f i e d c a l m o d u l i n was h o m o g e n o u s w h e n e v a l u a t e d by p o l y a c r y l a m i d e gel e l e c t r o phoresis. The b i o l o g i c a l and p h y s i c o c h e m i c a l p r o p e r t i e s of human brain c a l m o d u l i n such as the ability to activate c a l m o d u l i n - d e f i c i e n t bovine p h o s p h o d i e s t e r a s e , m o l e c u l a r weight, and amino acid c o m p o s i t i o n were almost the same as bovine brain calmodulin. C a l m o d u l i n is a heat stable, small acidic C a 2 + - b i n d i n g protein, and is known to m e d i a t e a number of Ca = - m e d i a t e d effects in various c e l l u l a r reactions (1-4). Recently, this protein has been i s o l a t e d from b o t h plants and animals. D e t a i l e d analyses of calmo d u l i n from bovine brain (5), bovine uterus (6), rat testis (7), rat islet cell tumor (8), murine m a c r o p h a g e cell lines (9), and human p l a c e n t a (i0) have shown little v a r i a t i o n in its structure. Nevertheless, there are no d e t a i l e d reports on b i o l o g i c a l and physi c o c h e m i c a l p r o p e r t i e s of human brain c a l m o d u l i n in the h o m o g e n o u s state as yet. Human c a l m o d u l i n may help to e l u c i d a t e the mechan i s m of the m a n y C a Z + - r e g u l a t e d b i o l o g i c a l process in man and the i d e n t i f i c a t i o n of f u n c t i o n a l l y i m p o r t a n t amino acid residues in c o m p a r i s o n with other species of calmodulins seems challenging. As such, we made an attempt to isolate pure human brain c a l m o d u l i n by a m m o n i u m sulfate p r e c i p i t a t i o n , gel filtration, and anion e x c h a n g e chromatography. In this article, we deal w i t h the i s o l a t i o n and some p h y s i c o c h e m i c a l p r o p e r t i e s of c a l m o d u l i n from human brain, and d i r e c t l y c o m p a r e d it to w e l l - c h a r a c t e r i z e d bovine brain calmodulin.
*This study was s u p p o r t e d in part by the Research Grant for the I n t r a c t a b l e Disease from the M i n i s t r y of H e a l t h and W e l f a r e of Japan (1980), the F o u n d a t i o n of Research for M e t a b o l i c Disease and Life Science of Japan (1980). **Requests for reprints should be a d d r e s s e d to N. Kobayashi.
0024-3205/81/262781-07502.00/0 Copyright (c) 1981 Pergamon Press Ltd.
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Purification of Calmodulin
MATERIALS
AND
Vol. 29, No. 26, 1981
METHODS
Materials: H u m a n b r a i n was o b t a i n e d from the c a d a v e r of a y o u n g japanese woman, two hours a f t e r she d i e d in an accident. Bovine b r a i n c a l m o d u l i n was the g e n e r o u s gift of Dr. S h i r o K a k i u c h i , Osaka University. B o v i n e h e a r t p h o s p h o d i e s t e r a s e and snake v e n o m (Crotalus atrox ) w e r e p u r c h a s e d from S i g m a C h e m i c a l Co. St. Louis, M.O. B i o - R a d A G I - X 2 (Chloride form, 2 0 0 - 4 0 0 3 m e s h ) was o b t a i n e d from B i o - R a d l a b o r a t o r i e s , Richmond, C.A. [ H ] c y c l i c AMP and A Q U A S O L - 2 w e r e o b t a i n e d from New E n g l a n d Nuclear, Boston, M.A. A s s a y of P h o s p h o d i e s t e r a s e A c t i v a t o r A c t i v i t y : Phosphodiesterase a c t i v i t y and the a b i l i t y of c a l m o d u l i n to s t i m u l a t e the a c t i v i t y of b o v i n e h e a r t p h o s p h o d i e s t e r a s e w e r e a s s a y e d u s i n g a m o d i f i e d p r o c e d u r e of T h o m p s o n and A p p l e m a n (ii) and C h e u n g (12). A reaction m i x t u r e of 0.i ml c o n t a i n e d 40 m M T r i s - H C l (pH 8.0), ½0 m M MgCl2, 0.05 m M CaCl~, 0.i m M u n l a b e l l e d c y c l i c AMP, 1 ~ M [ H]cycllc AMP, and c a l ~ o d u l i n (i-i000 ng). The r e a c t i o n was i n i t i ated by the a d d i t i o n of c a l m ~ d u l i n - d e f i c i e n t b o v i n e h e a r t p h o s p h o diesterase. P r o d u c t i o n of [ H ] 5 ' - A M P was m e a s u r e d by i n c u b a t i o n w i t h snake v e n o m w h i c h was used as a source of 5 ' - n u c l e o t i d a s e , a f t e r w h i c h [ ~ H ] a d e n o c i n e was c o u n t e d in a i0 ml A Q U A S O L - 2 s c i n t i l l a t i o n fluid. P u r i f i c a t i o n of H u m a n B r a i n C a l m o d u l i n : The p r o c e d u r e a d o p t e d in this s t u d y c l o s e l y r e s e m b l e s that used by W a t t e r s o n (13). All o p e r a t i o n w e r e c a r r i e d out at 4°C. H u m a n b r a i n (280 g) was homogen i z e d in 2 v o l u m e s of 0.i M s o d i u m a c e t a t e (pH 7.2), 1 m M 2m e r c a p t o e t h a n o l , and 1 m M E D T A (Buffer A), and the h o m o g e n a t e w a s c e n t r i f u g e d at i0,000 x g for 60 min. The s u p e r n a t a n t fluid was m i x e d w i t h 600 ml of C M - S e p h a d e x C-50, a l l o w e d to stand for 30 min, and then f i l t e r e d t h r o u g h a B u c h n e r funnel. THe f i l t r a t e was a p p l i e d to a c o l u m n (2 x 50 cm) of D E A E - S e p h a d e x A-50 e q u i l i b r a t e d w i t h B u f f e r A. A d s o r b e d p r o t e i n s w e r e e l u t e d w i t h a linear salt g r a d i e n t f o r m e d w i t h 600 ml each of 0.05 M and 0.80 M NaCl in B u f f e T A as the s t a r t i n g and l i m i t i n g buffers. Five ml f r a c t i o n s w e r e c o l l e c t e d and m o n i t o r e d by r e a d i n g a b s o r b a n c e at 280 nm. A l i q u o t s of f r a c t i o n s h a v i n g a b s o r b a n c e peaks in the r e g i o n of 280 nm w e r e a n a l y z e d for p h o s p h o d i e s t e r a s e activity, and f r a c t i o n s c o n t a i n i n g c a l m o d u l i n w e r e pooled. S o l i d a m m o n i u m sulfate (313 g/ litter) was a d d e d to the p o o l e d f r a c t i o n s to b r i n g the s o l u t i o n to 50% s a t u r a t i o n . The m i x t u r e was s t i r r e d for 60 min, and c e n t r i fuged at 10,000 x g for 30 min. The s u p e r n a t a n t f r a c t i o n was a d j u s t e d to pH 4.0 w i t h s u l f u r i c acid. The m i x t u r e was s t i r r e d for 60 m i n and c e n t r i f u g e d at 10,000 x g for 30 min. The p e l l e t w a s d i s s o l v e d in 30 ml of 0.01 M a m m o n i u m b i c a r b o n a t e and d e s a l t e d on a c o l u m n (3 x 80 cm) of S e p h a d e x G-25 in 0.01 M a m m o n i u m b i c a r b o n a t e and l y o p h i l i z e d . The l y o p h i l i z e d p r o t e i n was d i s s o l v e d in 50 ml of 0.01 M T r i s - H C l (pH 7.5), 1 m M EDTA, 1 mM 2 - m e r c a p t o ethanol, and 0.2 M NaCI (Buffer B) and a p p l i e d to a c o l u m n (2 x 50 cm) of D E A E - S e p h a d e x A-50 e q u i l i b r a t e d w i t h B u f f e r B. THe c o l u m n was e l u t e d w i t h a linear salt g r a d i e n t c o n s i s t i n g of 600 ml each of B u f f e r B and 0.7 M NaCI in B u f f e r B as the s t a r t i n g and limiting buffers. The f r a c t i o n s c o n t a i n g c a l m o d u l i n w e r e p o o l e d and lyophilized. The l y o p h i l i z e d p r o t e i n was d i s s o l v e d in 20 ml of 0.5 M T r i s - H C l (pH 7.5), 1 m M EDTA, and 1 m M 2 - m e r c a p t o e t h a n o l (Buffer C) and a p p l i e d to a c o l u m n (4 x i00 cm) of S e p h a d e x G-100 in B u f f e r C. The p r o t e i n o b t a i n e d was h o m o g e n o u s w h e n e v a l u a t e d by s o d i u m d o d e c y l s u l f a t e - p o l y a c r y l a m i d e gel e l e c t r o p h o r e s i s in w h i c h no d e n a t u r a n t was present.
Vol. 29, No. 26, 1981
The y i e l d
of c a l m o d u l i n
Purification of Calmodulin
from the b r a i n was
2783
22 mg.
E l e c t r o p h o r e t i c Method: Following treatment with sulfate (SDS) and 2 - m e r c a p t o e t h a n o l , the p r o t e i n c h r o m a t o g r a p h i c fractions were r o u t i n e l y a n a l y z e d sis on 12.5% p o l y a c r y l a m i d e gel, c o n t a i n i n g 0.1% by Laemmli (14). Gels were stained w i t h Coomasie (15).
sodium dodecyl c o m p o n e n t s of the by e l e c t r o p h o r e SDS, as d e s c r i b e d B r i l l i a n t Blue
A m i n o A c i d Analysis: Amino acid analysis was p e r f o r m e d on a Hitachi 835-50 single column s y s t e m a u t o m a t i c amino acid analyzer, a c c o r d i n g to the m e t h o d of S p a c k m a n et al. (16). The p r o t e i n fractions were h y d r o l y z e d w i t h 6 N HCI at II0°C for 24, 48, and 72 hours in the e v a c u a t e d tubes. T r y p t o p h a n was d e t e r m i n e d after h y d r o l y s i s w i t h 4 N m e t h a n s u l f o n i c acid at I10°C for 24 hours.
Fig.
1
E l e c t r o p h o r e t i c analysis of human b r a i n calmodulin: E l e c t r o p h o r e s i s was done in the presence of sodium dodecyl sulfate on a 12.5%(w/v) p o l y a c r y l a m i d e gel c o n t a i n i n g 2.5 mM EDTA. Samples were i n c u b a t e d at 100°C for 3 min before analysis. Lane A is 5 Dg of human b r a i n calmoduli~. Lane B c o n t a i n e d 5 D g of each m o l e c u l a r w e i g h t standard. From top to bottom, the standards are p o l y m e r a s e B p e ~ t a m e r (71,500), t e t r a m e r ( 5 7 , 2 0 0 ) , trimer(42,900), d i m e r ( 2 8 , 6 0 0 ) , and m o n o m e r ( 1 4 , 3 0 0 ) . Lane C is 5 ~g of bovine b r a i n calmodulin.
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Vol. 29, No. 26, 1981
RESULTS Pure human brain calmodulin was obtained by ammonium sulfate precipitation, gel filtration, and anion exchange chromatography. The purified protein migrated as a single band on SDS polyacrylamide gel electrophoresis and also comigrated with bovine brain calmodulin as shown in Fig. i. From these results, the molecular weight of this protein appeared to be similar to that of bovine brain calmodulin. The amino acid composition of this protein is shown in Table'l along with the composition by sequence of bovine brain calmodulin. The human brain calmodulin composition values h a v e been calculated by using estimated molecular weight (17,000) of bovine brain calmodulin (16,680) under the electrophoretic condition of Fig. i. One striking feature of the data is that one-third of the total amino acid is contributed from aspartic and glutamic acids following acid hydrolysis. Another feature is the absence of tryptophan and cystein. Both human and bovine brain calmodulin were tested in.triplicate for their ability to quantitatively stimulate 8 x 10 -4 units of activator-free bovine heart phosphodiesterase. Both brain calmodulins gave indistinguishable activation curves as shown in Fig. 2. Half-maximal stimulation of phosphodiesterase activity was obtained with 40 ng and 60 ng of human and bovine brain calmodulins respectively. Fully activated phosphodiesterase had an activity of 40 nmol of c-AMP hydrolyzed per mg phosphodiesterase.
• -
100
'///"
,---,human ----- bovine
/ JC
O_ .... 4 0 tO.
20
•~'!° ~ 50
100
~ 200 calmodul.in ( ng ) Fig.
1000
2
Comparison of human brain calmodulin ( - - ) and bovine brain calmodulin (---) by activation of activator-free phosphodiesterase. The closed ~ r c l e s refer to assay conducted in the presence of Ca- (50 ~M) and the open circles are values obtained in the presence of EGTA (i mM).
Vol. 29, No. 26, 1981
Purification of Calmodulin
TABLE
I
AMINO ACID COMPOSITION
Amino Acid
Aspartic
acid
Threonine Serine Glutamic
acid
H u m a n Brain a residues/ 17,000g
OF C A L M O D U L I N Bovine Brain b residues/ molecule
22 55
23
ii 28
12
5 40
4
29 34
27
Proline
2 82
2
Glycine
14 33
ii
Alanine
13 90
ii
Half-cystine
0 00
0
Valine
7.34
7
Methionine
5.58
9
Isoleucine
7.30
8
Leucine
9.30
9
Tyrosine
2.18
2
Phenylalanine
7.55
8
Lysine
6.92
7
Histidine
1.09
1
Arginine
5.89
6
Tryptophan
0.00
0
Trimethyllysine
N.D. c
1
a c a l c u l a t e d as d e s c r i b e d in the text f r o m c o m p o s i t i o n analysis u s i n g the e s t i m a t e d m o l e c u l a r w e i g h t of h u m a n b r a i n c a l m o d u l i n (17,000) from SDS p o l y a c r y l a m i d e gel analysis. b • C o m p o s l t l o n c a l c u l a t e d from amino acid s e q u e n c e (5). CNot d e t e r m i n e d .
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Vol. 29, No. 26, 1981
Fig. 2 shows also the r e q u i r e m e n t of Ca 2+ for the a c t i v a t i o n of p h o s p h o d i e s t e r a s e by b o t h h u m a n and b o v i n e b r a i n c a l m o d u l i n . Both c a l m o d u l i n s s t i m u l a t e d p h o s p h o d i e ~ e r a s e a c t i v i t y in an i d e n t i c a l m a n n e r in the p r e s e n c e of Ca ^° but did not a c t i v a t e p h o s p h o d i e s t e r a s e in the p r e s e n c e of C a Z + - c h e l a t o r , e t h y l e n e glycol-bis-(B-aminoethylether)-N,N'-tetraacetic acid (EGTA).
DISCUSSION The a n a l y s e s r e p o r t e d here d i r e c t l y d e m o n s t r a t e that the s t r u c t u r a l and f u n c t i o n a l p r o p e r t i e s of h u m a n b r a i n c a l m o d u l i n have b e e n h i g h l y c o n s e r v e d d u r i n g v e r t e b r a t e evolution. This r e s u l t agrees w i t h that r e p o r t e d by D.M. W a t t e r s o n et al. (17) about a d e t a i l e d c o m p a r i s o n of c a l m o d u l i n s i s o l a t e d from several v e r t e b r a t e b r a i n s (pig, rabbit, chicken, and beef). The amino acid c o m p o s i t i o n g i v e n in Table 1 did not show any m a j o r d i f f e r e n c e s from that of the b o v i n e b r a i n c a l m o d u l i n . The c o m p o s i t i o n of this p r o t e i n is also c l o s e to the one r e p o r t for h u m a n b r a i n c a l m o d u l i n (18), b u t has the same c o n t e n t of a s p a r t i c acid as b o v i n e b r a i n c a l m o d u l i n . H u m a n b r a i n c a l m o d u l i n w a s also found to have the same activ i t y on p h o s p h o d i e s t e r a s e a c t i v a t i o n as those of the o t h e r v e r t e brate calmodulins. The a b i l i t y of p u r i f i e d h u m a n b r a i n c a l m o d u l i n to a c t i v a t e b o v i n e h e a r t p h o s p h o d i e s t e r a s e a g r e e d w i t h the r e s u l t s that r e p o r t e d by F. V a r g a s et al. (19). In the p h o s p h o d i e s t e r a s e a c t i v a t i o n , the p o t e n c y of h u m a n b r a i n c a l m o d u l i n was 1.5 times of that of b o v i n e b r a i n c a l m o d u l i n . This r e s u l t s u g g e s t s the p o s s i b i l i t y that h u m a n b r a i n c a l m o d u l i n also got a d i r e c t i o n a l c h a n g e d u r i n g the2+molecular e v o l u t i o n t o w a r d an increase' in the c a p a b i l ity in Ca - d e p e n d e n t enzyme a c t i v a t i o n as p r e v i o u s l y r e p o r t e d by S. K a k i u c h i et al. (20). As s h o w n above, we have found that c a l m o d u l i n p u r i f i e d from h u m a n b r a i n is v e r y s i m i l a r to b o v i n e b r a i n c a l m o d u l i n in m a n y r e g ~ e c t s such as f r a c t i o n a t i o n b e h a v i o r , e l e c t r o p h o r e t i c m o b i l i t y , Ca ~ - d e p e n d e n t p h o s p h o d i e s t e r a s e a c t i v a t o r activity, and amino acid c o m p o s i t i o n . The v a r i o u s f u n c t i o n a l roles of c a l m o d u l i n in man have not yet b e e n i n v e s t i g a t e d . However, the c h a r a c t e r i z a t i o n of h u m a n b r a i n c a l m o d u l i n r e p o r t e d h e r e p r o v i d e s a f i r m b a s i s for f u r t h e r b i o l o g i c a l study on the b i o l o g i c a l and b i o p h y s i c a l p r o p e r ties of h u m a n b r a i n c a l m o d u l i n .
ACKNOWLEDGEMENTS We thank Prof. Y. N i s h i z u k a and Dr. Y. Takai of D e p a r t m e n t of B i o c h e m i s t r y , Kobe U n i v e r s i t y School of M e d i c i n e , for e n c o u r a g e m e n t t h r o u g h o u t this w o r k and c r i t i c a l r e a d i n g of the m a n u s c r i p t .
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Purification of Calmodulin
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