A High Molecular Weight Nuclear Basic Protein from the Bivalve [PDF]

Jul 20, 1981 - From the Unidad de Quimica Macromolecular del Consejo Superior de Investigaciones Cientificas, Escuela Te

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THEJOURNALOF BIOLOGICAL CHEMISTRY Vol. 257, No. 6 , Issue of March 25, pp. 2802-2805, 1982 Printed in U S.A.

A High Molecular Weight NuclearBasic Proteinfrom the Bivalve Mollusc Spisula solidissima* (Received for publication, July 20, 1981)

Juan Ausioand Juan A. Subirana From the Unidad de Quimica Macromolecular del Consejo Superior deInvestigaciones Cientificas, Escuela Tecnica Superior deIngenieros Industriales, Diagonal999, Barcelona 28, Spain

A basic proteinhas been isolated from sperm of the protamine was recovered by one or two subsequent extractions with bivalve Spisula solidissima Its characteristics are rem-0.25 N HCl; most was recovered in the first extraction. In order to remove the histones which remained and to completely iniscent of both histoneH1 and of fish protamines. Itis unusual in several respects:it contains similar amounts purify the protamine, column chromatography was used. Both gel of lysine (24.8%)and arginine(23.1%), plus a residueof filtration and ion exchange were equally effective. In the fist case, Bio-Gel P-100 (Bio-Rad) was used and elution was carried out with tryptophan per molecule. Its size is very large, -297 0.02 N HCl containing 0.1%of 1,1,l-trichloro-2-methyl-2-propanol. amino acid residues.It shows a tendency toaggregate, Ion-exchange chromatography was done on CMC-25 Sephadex. The an unusual propertygiven the strongly charged nature sample was dissolved in 1 M NaCl, 50 mM sodium acetate, pH 6.7, and of this protein. was eluted with a linear gradient of NaCl from 1 to 2 M in the same

EXPERIMENTALPROCEDURES

Specimens of the surf clam S. solidissima were obtained at the Marine Biological Laboratory, Woods Hole, MA. The sperm was collected as previously described (Subirana et al., 1973).Observation at the optical microscope showed that more than 95% of the cells were motile gametes. The spermatozoa obtained were successively washed once with sterile sea water and twice with 0.15 M NaCl, 20 mM EDTA, pH 8. The latter solvent was used in order to inhibit protease digestion (Zubay and Doty, 1959). The protamine was separated from most of the histones by selective acid extraction as described elsewhere (Subirana et al., 1973). Histones were removed with 35% acetic acid. Besides the histones, this extract contained a large amount of other protein components. The partially purified

* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

RESULTS

The spermatozoa of S. solidissima contain a major protamine component of low electrophoretic mobility plus a small quantity of histones, as shown in Fig. 1. The electrophoretic pattern of the histones presentin ripe spermatozoa (Fig. 1A) is practically identical to that of calf thymus histones (not shown) and thefive histone components are apparently present in the preparations, although no attempt was made to fractionate them. The presence of residual histones is common in mollusc sperm nuclei (Subirana et al., 1973; Colom and Subirana, 1979, 1981). Digestion of nuclei with micrococcal nuclease did not show any nucleosome particles (Ausio, 1980), but only a broad distribution of DNA fragments of variable size. Theprotaminecomponent canbe partially purified by selective acid extraction (Fig. 1, B and C).Column chroma-



Ausio, J., and Subirana, J. A. (1982)Biochemistry, submitted for publication.

2802

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In the sperm of most animals, DNA is associated with basic proteins. These proteins vary significantly in each particular species. In some cases, they are very similar to somatic histones, as found, for example, in echinoderms (Subirana and Palau, 1968). On the other hand, in the well known case of fish protamines (Ando et al., 1973), these proteins are very small in size (30-35 amino acids) and arevery rich inarginine; lysine is usually absent. In the molluscs, a wide variety of sperm proteins is found (Subirana et al., 1973), with variable amounts of lysine and arginine and differentmolecular weights. In this paper we report the characterization of a mollusc sperm protein which is rather unique for its high molecular acid tryptophan. weight and also because it contains the amino As foundinmost bivalve molluscs (Subirana et al., 1973; Colom and Subirana, 1979), it contains about equal amounts of lysine and arginine. Throughout this work we will refer to this protein as a protamine, but it may also be related to histones as discussed at the endof this paper. The purpose of the present work is not only to achieve a better knowledge of the composition and conformation of theseunusual proteins, but also to contribute to a better understanding of their evolutionary significance.

buffer. In both cases the fractions were monitored by the absorbance at 230 nm. The protein was recovered by dialysis against 0.25 M HCl and precipitation with acetone. Polyacrylamide gel electrophoresis was carried out in either slab gels or tubes. A urea-acetic acid buffer was used as described by Panyim and Chalkley (1969). This gel system was also appropriate for preparative purposes. A gel system at pH9.2 with 12%acrylamide and 10%glycerol wasalso used. The buffer contained 12.5 mM glycine plus 25 mM Tris-HC1. In both methods, the gels were preelectrophoresed a t 100 V until the current was constant. The gels were stained for 5-6 hours in 0.1% Amido black 10B dissolved in 20% ethanol, 7% acetic acid. Amino acid analyses were carried out in a Beckman 119C analyzer. For the detection of tryptophan, the samples were hydrolyzed for 24 h at 110 “C in 3 N p-toluenesulfonic acid containing 0.2% tryptamine, as described byLiu (1972). This amino acid was also determined chemically with N-bromosuccinimide(Spande andWitkop, 1967).The fluorescence of the protein was studied in a Jovin-Yvon spectrofluorometer after excitation at 295 nm. Circultv dichroism was studied in a Cary 60 spectropolarimeter. The molecular weight of the protein was determined by sedimentation equilibrium at high speed (Yphantis, 1964),as described elsewhere.’ The partial specificvolume, taking into accountthe associated chloride ions, was calculated to be 0.702. Sedimentation coefficients were determined from the rate of movement of the boundary maximum with either schlieren or interference optics. In the latter case, the method of Van Holde and Weischet (1978)wasusedin the calculations.

A Nuclear Basic Protein

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A B C D E F protamine aggregate

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H1 (H2,H3) S

2803

it will be shown below, the results given in Table I indicate that themolecule contains oneresidue of each of the aromatic amino acid residues (Trp, Tyr, and Phe). The electrophoreticanalysis of this protein always showed some material which did not penetrate the gel (Fig. 1). This band wasalso present in theprotamine which had been purified by column chromatography. When electrophoresis of the purifled protamine was carried outa t p H9.2, the amount of protein present at the origin increased significantly. All these results indicated that this protamine hasa tendency to aggregate, particularly at high pH. In order to c o n f i i this interpretation, the bandat theorigin was obtained by preparative electrophoresis and its amino acid analysiswas identical to thatshown in Table I. An additional testof homogeneity was carried out by analyzing sedimentation velocity experiments following the procedure of Van Holde and Weischet (1978). This method is

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TABLE I

H4

Amino acid composition of the purified protamine from S. solidissima Mole %

3.0 14.2 2.3 1.7 0.5 2.4 21.7 4.3 0.6 0.6 0.0 0.4 24.8 23.1 0.0 0.3 0.3 0.3

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FIG. 1. Gel electrophoresis. Gel electrophoresis of: A, whole sperm proteins; B , impure protamine recovered after a 4-h extraction of the sperm with 0.25 N HCI. The cells had been previously treated with 3 5 8 acetic acid in order to remove most of the histones; C, a second extraction for 24 h under the same conditions. The yield was -158 of the protein extracted in the previous step; D, histones recovered after column chromatography (fraction I in Fig. 2); E and F,purified protamine after column chromatography (fractions 2 and 3 in Fig. 2).

Amino acid

Glycine Alanine Valine Leucine Isoleucine Proline Serine Threonine Aspartic acid Glutamic acid Cysteine Methionine Lysine Arginine Histidine Phenylalanine Tyrosine TrvDtoDhan

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FIG. 2. Purification of protamine by ion-exchange chromatography in a salt gradient. Purification was carried out on a sample which had been previously extracted with acetic acid, so that the amount of histones shown in the figure is not representative of the total amount present in the sperm. The dimensions of the column were 2.2 X 50 cm. Fractions of 5 ml were collected a t a rate of 18.5 ml/h. The fractions indicated by I , 2, and 3 were pooled and the proteins recovered were subjected to gel electrophoresis, as shown in Fig. 1.

tography was used fora more thoroughpurification, as shown in Fig. 2. The proteins recovered are shown in Fig. 1, D to F. The aminoacid analysis of the purified protamine is given in previously Table I. The valuesshown are not identical to those reported (Subirana et al.,1973) because the protamine had not been thoroughly purified. The presence of tryptophan was c o n f i i e d by chemicaldetermination with bromosuccinimide. The fluorescence (Fig. 3) and absorption spectraa t basic and also c o n f i i t h epresence of this amino acidic pH (not shown) acid. Since this protein contains-297 amino acid residues, as

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xnm FIG. 3. Fluorescence spectra of N-acetyltryptophanamide -) and protamine (-) after excitation at 295 nm. The experiments were carried out in water solution. There is no shift in the position of the maximum given by the protein, when compared with the free amino acid, indicating that the chromophoric group of the protein is fully exposed to the solvent. (-

-

A Nuclear Basic Protein Sperm from Spisula the of

2804

based on determining the apparent sedimentation coefficient in different regions of the sedimenting boundary. The results obtained are shown in Fig. 4. Most of the points give similar sedimentation constants upon extrapolation. The curvature that is observed in the upper partof the inner diagram is due to the influence of concentration on the sedimentationcoefficient. The values of s increase slightly as 'they are taken across the boundary, anobservation which is consistent with a small amount of aggregation. The large aggregates detected by electrophoresis are probably not apparent because they will sediment very rapidly in the ultracentrifuge. Sedimentation equilibrium was also used to determine the molecular weight, taking into account the tendency of this protein to form aggregates. The influence of concentration on different values of the apparentmolecular weight is shown in Fig. 5. The plots are consistent with a moderate aggregation of the protein. This behavior is c o n f i i e d by representing the results by the methodof Roark and Yphantis (1969),as shown FIG. 7. Circular dichroism spectrum of the protamine disin Fig. 6. This methodwas developed for the studyof proteins

solved in either 0.15 or 1.5 M NaC1,O.OZ M NH,Cl, 0.02 M, pH 9.2.

coefficient.

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FIG. 4. Analysis of the boundary in a sedimentation velocity experiment according to the method of Van Holde and Weischet (1978). The inset shows the extrapolated sedimentationcoefficients in different regions of the boundary. The proteinwas dissolved in 0.25 M NaCl, 0.02 M NH,Cl, pH 9.2, at a concentration of 2.5 mg/ rnl. The experiment was carried out at 37.020 rprn at 20 "C.

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