Parvalbumins from coelacanth muscle I. General survey

263

Biochimica et Biophysica Acta, 5 3 6 ( 1 9 7 8 ) © Elsevier/North-Holland

Biomedical

263--268

Press

BBA 37980

PARVALBUMINS FROM COELACANTH MUSCLE I. G E N E R A L SURVEY *.**

JUAN

JAUREGUI-ADELL

and JEAN-FRAN7OIS

PECHERE

***

Centre de Recherches de Biochimie Macromoldculaire du CNRS. B.P. 5051, 34033 Montpellier Cedex (France) (Received

January

9th, 1978)

Summary Parvalbumins from coelacanth (Latimeria c h a l u m n a e ) myogen have been isolated by gel filtration on Sephadex G-75 and DEAE-cellulose chromatography. Disc electrophoresis and cellulose acetate electrophoresis showed the homogeneity of the three first major parvalbumin peaks .(pI = 5.44, pI = 4.95 and pI = 4.52). The fourth c o m p o n e n t was partially resolved into two more parvalbumins (pI = 3.78 and pI = 3.50) by preparative gel electrophoresis. Amino acid analyses and tryptic peptide maps separated the five components in two major categories. The two less acidic components differ only in the presence or absence of an N-terminal blocking group. The three more acidic components constitute the second category; in spite of this heterogeneity, they share the same amino acid sequence.

Introduction Previous work on muscular parvalbumins [1] has been mainly concerned with fish of amphibian proteins, which are easy to purify [2]. Their presence in muscles from higher vertebrates [3,4] has further increased the interest of this family of homologous proteins in molecular evolution studies [5]. The availability of a large sample of coelacanth muscle provided an exceptional opportunity to include this unique species into such studies. * C o n t r i b u t i o n N o . 1 5 5 f r o m t h e C e n t r e de R e c h e r c h e s . ** S u p p l e m e n t a r y d a t a t o t h i s article are d e p o s i t e d w i t h , a n d c a n be o b t a i n e d f r o m , Elsevier S c i e n t i f i c Publishing Company, BBA Data Deposition, P.O. Box 1345, 1000 BH Amsterdam, The Netherlands. Reference should be made to No. BBA/DD/081/37980/536 (1978) 263. The data include the p u r i f i c a t i o n t a b l e o f c o e l a c a n t h p a r v a l b u m i n s ; t h e u l t r a v i o l e t s p e c t r a o f c o e l a c a n t h p a r v a l b u m i n s ; the t r y p t i e p e p t i d e m a p s o f c o m p o n e n t s I a n d II, t h e t r y p t i e p e p t i d e m a p s of c o m p o n e n t s III, IV a n d V. * ** T o w h o m c o r r e s p o n d e n c e and reprint r e q u e s t s s h o u l d b e a d d r e s s e d .

264

Earlier reports on the protein components of coelacanth muscle have been published by Hamoir et al. [6]. The present study, based on a larger sample, includes a more detailed description of the parvalbumins of this muscle, and, in the following papers of this series, the amino sequences of the two major components. Preliminary reports of this work have been published in refs. 1 and 7. Material and Methods Coelacanth (Latimeria chalumnae) muscle was kindly provided by Prof. R. Acher, Laboratoire de Chimie Biologique, Universit~ de Paris VI. The animal (male) was frozen immediately after its capture, and thawed just before dissection. The parvalbumins were isolated and analyzed essentially as described before for other animal species [2,8], except that a m m o n i u m sulfate fractionation was omitted to minimize losses. Sephadex G-75 chromatography was carried out in 0.02 M Tris (HC1), pH 7.5, the low molecular weight protein peak lyophilized and dialyzed vs. the piperazine buffer before DEAE-cellulose chromatography. Preparative slab gel electrophoresis of some fractions obtained from DEAE-cellulose was carried out in an apparatus Ultraphor (Colora). Isoelectric points were determined from electrophoretic mobilities on cellulose acetate [8], with carp parvalbumin pI = 4.47 [2] as standard. Amino acid analyses were performed in a Beckman Multichrom instrument after 24 h and 72 h hydrolysis in 6 M HC1 at l l 0 ° C . N-terminus determination was carried out according to Sodek and Hofmann [9]. Peptide analyses were conducted as described previously [7] either on Chromobeads P in a pyridine gradient, or by double paper electrophoresis, first at pH 6.4 and then at pH 1.9. Tryptic digestion (pH 7.8, final E/S = 0.05, w/w) was performed on heat-denatured proteins [ 10], for 2 h. Results

The results * of the DEAE-cellulose chromatography of the low molecular weight protein peak from Sephadex G-75 are reported in Fig. 1. Spectral analyses of different fractions clearly identify most of them as parvalbumins [1]. Only the first peak (DV), as usual, and the fourth one (minor, SC) correspond to low molecular weight proteins of a different sort. The disc electrophoretic analysis at pH 9.4 [2] of these several peaks is shown in Fig. 2, it confirms the apparent homogeneity of the three first major parvalbumin peaks and the heterogeneity of the fourth one. Cellulose acetate electrophoresis at pH 5.5 [8] gives similar results. Preparative gel electrophoresis did not entirely resolve the components IV and V mixed into the fourth heterogeneous peak from DEAE-cellulose. For each of them, however, homogeneous fractions (as checked by analytical disc gel electrophoresis) were obtained; partial characterization was carried out on this small a m o u n t of pure protein. The amino acid composition of the five parvalbumins obtained is reported in Table I. From these data, the two less acidic components I and II (pI = 5.44 * Purification

and ultraviolet

s p e c t r a d a t a are d e p o s i t e d

with BBA Data Deposition.

265

0.2

III ~'~

E c

=

o.,

DV

I

~pH

,v-v

II

i:

~__~

~ _

M NaCI x

-o.,

_

0

-0

5'0

100

150

2;0

Fr. nr

Fig. 1. D E A E - c e U u l o s e (4 X 42 c m ) c h r o m a t o g r a p h y of low m o l e c u l a r w e i g h t p r o t e i n s f r o m c o e l a c a n t h m u s c l e (85 g). T h e c o l u m n was e q u i l i b r a t e d w i t h 0 . 0 1 5 M c h l o r i d e a d j u s t e d t o p H 5.7 w i t h p i p e r a z i n e . E l u t i o n w a s s t a r t e d w i t h a g r a d i e n t of c h l o r i d e a d j u s t e d to t h e s a m e p H , at 50 m l / h ; t h e v o l u m e o f fract i o n s w a s 10.9 ml. U l t r a v i o l e t a b s o r p t i o n w a s r e c o r d e d at 2 5 4 n m w i t h t h e help of a L K B U v i c o r d II absorptiometer. The pH and the chloride content, the latter by conductivity m e a s u r e m e n t s , were determ i n e d i n t e r m i t t e n t l y . DV, d e a d v o l u m e ; SC, slow c o m p o n e n t ; I, II, I I I a n d I V - V , p a r v a l b u m i n c o m p o nents.

and pI = 4.95) appear to be identical; the major component III of intermediary acidity (pI = 4.52), is different from I and II, but very similar to the two most acidic acid components IV and V. Two major groups (I and II on one hand, IIIIV-V on the other hand) can thus be distinguished among coelacanth parvalbumins, on the basis of their amino acid composition and the ultraviolet spectra. As is the case with all parvalbumins isolated so far, no N-terminal amino acid

Fig. 2. Disc e l e c t r o p h o r e s i s o f c o e l a c a n t h m y o g e n a n d of the s u b s e q u e n t f r a c t i o n s leading t o t h e isolation of p u r e p a x v a l b u m i n s . D i a l y z e d e x t r a c t , 2 5 0 p g p r o t e i n ; S e p h a d e x G-75 e f f l u e n t , 1 4 0 ~g; C o m p o n e n t I, 25 pg; C o m p o n e n t I I , 25 pg; Slow c o m p o n e n t , 25 pg; C o m p o n e n t I I I , 50 pg; C o m p o n e n t I V - V , 50 g g .

I

16.19 5.83 3.18 14.25 1.03 10.95 10.10 0. 4.99 0.98 4.82 11.94 0.99 7.81 6.21 15.50 1.91 1.06 0.

111--112

16 6 3 14 1 11 10 0 5 1 5 12 1 8 6 15--16 2 1 0

16.27 6.01 3.12 14.38 1.02 11.04 10.13 0. 4.96 0.99 4.79 12.21 0.90 7.98 6.05 15.66 1.93 1.00 0. 111--112

16 6 3 14 1 11 10 0 5 1 5 12 1 8 6 15--16 2 1 0

MUSCLE

12.79 6.97 5.15 16.44 0. 7.70 13.98 0.70 5.06 O. 3.90 11.91 0. 8.61 7.06 12.59 1.Ol 1.00 0.

Exptl.

108--109

13 7 5 16 0 8 14 1 5 0 4 12 0 9 7 12--13 1 1 0

Assumed

Cpt lII (pl = 4.52)

COELACANTH

Assumed

Exptl.

Assumed

Exptl.

*

Cpt lI (pl = 4.95)

protein

FROM

Cpt I (pl = 5.44)

Residues/mol

OF PARVALBUMINS

* The values are derived from the analysis of duplicate samples of 24 h and 72 h amd hydrolysates.

To~l

Asx Thr Ser Glx Pro Gly Ala Cys Val Met He Leu Tyr Phe NH 3 Lys His Arg Try

Amino acid

AMINO ACID COMPOSITION

TABLE

12.81 6.81 4.84 15.63 0. 8.19 14.00 0.75 5.07 O. 4.05 11.95 0. 9.50 6.95 12.98 1.07 1.08 0.

Exptl.

109--110

13 7 5 16 0 8 14 1 5 0 4 12 0 9--10 7 13 1 1 0

Assumed

Cpt IV (pl = 3.78)

13.34 6, 7 9 5.15 17.37 0. 7.47 14.15 0.52 4.46 O. 3.68 11.96 0. 8.76 6.97 12.80 0.91 0.85 0.

Exptl.

108--110

13 7 5 17 0 7--8 14 1 4--5 0 4 12 0 9 7 13 1 1 0

Assumed

Cpt V (pl = 3.50)

to 03 O~

267 could be detected with dansyl chloride [9] reacting with 30 nmol of four of the five coelacanth parvalbumins. However, c o m p o n e n t I clearly shows a free N-terminal threonine. Peptide mapping was used in order to compare further the different components of a same group. The ion-exchange profiles of tryptic digests * of components I and II differ only in that the very first peak generated from c o m p o n e n t II is not present in the digest of c o m p o n e n t I, which shows in contrast a new peak retarded on the resin. It was subsequently confirmed (see accompanying paper II) that the different peptide was the N-terminal peptide, free (Cpt I) or N-acetylated (Cpt II). Similarly, a careful inspection of the chromatoelectrograms at pH 6.5 and at pH 3.5 * shows that exactly the same tryptic peptides are generated in comparable amounts ~rom Cpts III, IV and V. From these data, the peptide chain of these three components appears to be identical. Discussion

In the present study the very large amount of parvalbumin present in the muscles of Latimeria chalumnae was found to be distributed essentially in 5 components, one of which is predominant (component III, pI = 4.52). The earlier results obtained by Hamoir et al. [6] led only to the characterization of the more acidic components (III and IV-V). However, the t w o DEAEcellulose profiles of the Sephadex G-75 low molecular weight fraction are actually remarkably similar in both studies, the present one being more accurate because of a larger a m o u n t of starting material. On the other hand, the lack of resolution of the two more acidic ones (IV, V) could correspond to the real state in that sample. The present data, indeed, suggest that the heterogeneity of components III, IV and V might be due to the strong adsorption of variable amounts of an extraneous, ultraviolet-absorbing and acidic low molecular weight molecule, but does n o t reflect an heterogeneity of the peptide chain. Cpts I and II (pI values = 5.44 and 4.95) appear to differ only in the presence of an N-terminal blocking group in Cpt II, which accounts for their differing pI and their separation on the anion exchanger. By contrast, we have as of now no indication of the nature of the acidic ultraviolet chromogen responsible for the heterogeneity of Cpts III, IV and V, which share the same polypeptide chain. It has not been indeed possible to separate this chromogen from the peptide chain under various conditions of pH and ionic strength, or after denaturing treatment. On the other hand, establishment of the amino acid sequence (see paper III of this series) showed no 'abnormal' amino acid or peptide, which would suggest a covalent bond between the chromogen and the peptide moiety. Acknowledgements The authors are grateful to Professor R. Acher who generously provided the coelacanth muscle, to Dr. Jean Paul Capony and Conception Ferraz for amino * T r y p t i c p e p t i d e m a p s o f C p t s I t o V are d e p o s i t e d w i t h B B A D a t a D e p o s i t i o n .

268

acid analyses and helpful discussions and to Dr. Nguyen Van Thoai for his interest and constant support. This work was funded in part by the CNRS and the D~l~gation G~n~rale ~ la Recherche Scientifique et Technique. References 1 2 3 4 5 6 7 8 9 10

P e c h ~ r e , J . - F . , C a p o n y , J . P . a n d D e m a i l l e , J. ( 1 9 7 3 ) Sys¢. Z o o l . 2 2 , 5 3 3 - - 5 4 8 P e c h ~ r e , J . - F . , D e m a i l l e , J. a n d C a p o n y , J . P . ( 1 9 7 1 ) B l o e h i m . B i o p h y s . A e t a 2 3 6 , 3 9 1 - - 4 0 8 C a p o n y , J.P., Pina, C. a n d P e e h ~ r e , J . - F . ( 1 9 7 6 ) E u r . J. B i o c h e m . 7 0 , 1 2 3 - - 1 3 5 B l u m n , H . E . , L e h k y , P., K o h l e r , L., S t e i n , E . H . a n d F i s c h e r , E . H . ( 1 9 7 7 ) J . Biol. C h e m . 2 5 2 , 2 8 2 4 - 2838 G o o d m a n , M. a n d P e e h ~ r e , J . - F . ( 1 9 7 7 ) J. Mol. Evol. 9, 1 3 1 - - 1 5 8 H a m o i r , G, P i r o n t , A., G e r d a y , C h . a n d D a n d e , P . R . ( 1 9 7 3 ) J. Max. Biol. Ass. U . K . , 5 3 , 7 6 3 - - 7 8 4 T h a t c h e r , D . R . a n d P e c h 4 r e , J . - F . ( 1 9 7 3 ) E u r . J. B i o c h e m . 7 5 , 1 2 1 - - 1 3 2 P e c h e r e , J.-F., C a p o n y , J . P . a n d R y d ~ n , L. ( 1 9 7 1 ) E u r . J . B i o c h e m . 23, 4 2 1 - - 4 2 8 S o d e k , J. a n d H o f f m a n n , T. ( 1 9 7 0 ) C a n . J. B i o c h e m . 4 8 , 4 2 5 - - 4 3 1 C a p o n y , J.P., D e m a i l l e , J., P i n a , C. a n d P e c h d r e , J . - F . ( 1 9 7 5 ) E u r . J. B i o c h e m . 5 6 , 2 1 5 - - 2 2 7