The separation and characterization of two forms ofTorpedo electric organ calelectrin

122

Biochimica et Biophysica Acta, 957 (1988) 122-130

Elsevier BBA 33244

The separation and characterization of two forms of Torpedo electric organ caldectrin Ulrich Fritsche a, Annette von Kieckebusch a , , , Martin Potschka b, Victor P. Whittaker a and Veit W i t z e m a n n a " Abteilung Neurochemie and h Abteilung Bioci~¢mie, Max.Planck-h:stitut f ~ biophysikalische Chemie, G~ttingen (£R. G.)

(Received 19 May 1988)

Key words: Calelectrin; Electric organ; (T. marmorata)

Two methods for extracting calelectrin, a CaZ+-regulated membrane-binding protein from the electric organ of Torpedo marmorata, have been compared and the more promising one was modified to increase the yield to 7-8 mg. k g - t wet weight of tissue, that is 4-5-times greater than the original method. The calelectrin so obtained could be resolved into a minor component (designated L-calelectrin) eluted from an anion-exchange column at relatively low ionic strength (100 mM NaCi) and a major component (H-calelectrin) eluted at higher ionic strength (300 mM NAG). The two forms were also separated by chromatography on a hydrophobic resin. Electmphoresis on cellulose acetate indicated that L-calelectrin had a lower mean isoelectric point that the H-form and polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate showed that under reducing conditions (presence of 5%/3-mercaptoethanol) both forms migrated as single species, the L-form having a lower apparent relative molecular mass (Mr 32000) than the H-form (34000). Under non-reducing conditions, there was no change in the migration of L-calelectrin but the H-form was resolved into two components of Mr 34000 and 32000. The addition of 2 mM Caz+ had no effect on the migration of either form. Both forms were equally recognized by an anti-calelectrin antiserum and were microheterogeneous with respect to their isoelectric points (pH 4.3-5.5) in two-dimensional gel electrophoresis. Physical measurements were carried out on the major H-form. The Stokes radius was estimated to be 3 nm, c¢, responding to an apparent M r of 44000. It was unaffected by changes in ionic strength, pH or Caz+ concentration. Analytical ultracentrifngation gave a sedimentation constant of 2.9 S and an apparent Mr of 36000. Measurements of circular dichroism indicated that 78% of the molecule was in the a-belix configuration and 22% in random coil. Ca2+ had no significant effect on the conformation.

* Present address: Labor fOr Biochemie II, Eidgenbssische Technische Hochschule, ZUrich, Switzerland. Abbreviations: Bistris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyi)propane-l,3.diol; EGTA, ethylene glycol bis(/3aminoethyl ether)-N,N,N',N'-tetraacetic acid; FPLC, fast-performance liquid chromatography; SDS-PAGE, sodium dodecyl sulphate-polyacrylamide gel electrophoresis; TBS, Tris-buffered saline. Correspondence: V.P. Whittaker, Arbeitsgruppe Neurochemie, Max-Planck-lnstitut f'~ biophysikalische Chemie, Postfach 2841, D-3400 Gbttingen, F.R.G.

Introduction The electric organ of Torpedo marmorata has been extensively used as a model system for cholinergic neurotransmission and has facilitated the purification and characterization of synapseassociated components. Ca 2+ is essential for synaptic transmission and therefore attempts have been made to identify proteins in this specialized tissue that interact selectively with this ion. This resulted in the discovery [1] of a novel protein of

0167-4838/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)

123

this type, subsequently named calelectrin [2], that did not conform in its properties to any of the then known Ca2+-regulated proteins. Calelectrin is a protein of Mr 34000 that binds to membranes in the presence of Ca 2+. Immunochemically and biochemically similar proteins have been identified in and isolated from cells, membranes and organelles of diverse origin, including chromaffin granules [3-5], lymphocytes [6,7], other types of leucocytes [7], intestinal epithelial cells [8], smooth muscle membranes [9,10], liver [11,12], adrenal medulla [3,11,12], brain [11,12], kidney, intestine, pancreas [11] and mammary epithelial cells [13]. A structural relationship between these proteins was first suggested when an antiserum to the Torpedo electric organ calelectrin was found to cross-react with several of them [11]. Although the precise function and cellular localization of these proteins (for which the name annexin has been proposed [14]) remains to be elucidated, they are now generally agreed to represent members of a new Ca 2+- and phospholipiddependent group which is distinct from the calmodulin and calmodulin-related Ca2+-binding proteins [14,15]. Their ability to interact with Ca 2+ and phospholipids may be linked in some cases to a specific interaction with membrane-bound enzymes or substrates for such enzymes. Two members of the class, proteins p35 and p36, serve as substrates for retrovirally coded protein kinases [8,16,17], suggesting that they are involved in some way in intracellular chemical signalling. Torpedo electric organ calelectrin was first detected and partially purified by extracting cornminuted electric organ membranes with EGTA following extensive washing of the membranes with Ca2+-containing solutions [1]. Later, it was prepared by releasing it from membranes by an initial extraction with EGTA, and purified by a cycle of Ca2+-induced resorption to membranes and EGTA-induced release [18]. The biochemical and biophysical characterization of the protein and the raising of monospecific antibodies to it require its complete purification. In this paper we describe an improved purification procedure and some characteristics of the purified calelectrin. The work forms part of two recent theses [19,20].

Materials and Methods Extraction of calelectrin from electric organ Tissue. T. marmorata, supplied by the Station Biologique d'Arcachon, France, were kept in tanks of circulating artificial sea water at 15-18°C until required. The electric organs were removed from fish anaesthetized by immersion in sea water containing 0.05% Tricaine methanesulphonate and were stored frozen in liquid nitrogen. The extraction procedures for calelectrin were based on Refs. 1 and 18. Extraction method I. Frozen electric organs were crushed to a fine powder in the presence of liquid nitrogen. Tris-buffered saline iso-osmotic with Torpedo body fluids (0.4 M NaCl, 10 mM Tris-HCl (pH 7.4); TBS) containing 10 mM CaC! 2 was added to the tissue thawed to 0 ° C in a ratio of 2 : 1 (v/w). The resultant slurry was homogenized in a Waring blender for three 15-s periods at low speed and two 15-s periods at high speed. The homogenate was passed through four layers of cheese cloth and centrifuged at 2000 × g for 15 rain. The resulting supernatant was again centrifuged at 1 3 0 0 0 0 × g for 30 min. The pelleted membrane fraction was resuspended using a glass-Teflon homogenizer in TBS contairdng I mM CaCI 2 and pelleted again at 130000 × g for 30 min. The membranes were washed a further three times using the same buffer and centrifugation conditions before being extracted for 15-30 min with TBS containing 10 mM EGTA. The solubilized calelectrin was recovered in the supernatant obtained after removal of extracted membranes by centrifugation at 130000 x g for 60 min and was dialysed twice against 5 1 of 10 mM Tris-HCl (pH 7.4). After recentrifugation at the same speed to remove a small precipitate, the solubilized calelectrin was dialysed against 5 1 of 50 mM imidazole (pH 6.0) containing 5 mM fl-mercaptoethanol. Extraction method II. The crushed electric organ powder was suspended in TBS containing 1 mM EGTA instead of CaCI 2 to release all membranebound calelectrin. The supernatant remaining after centrifugation at 2000 x g for 15 min contained membrane fragments to which calelectrin bound when CaCl 2 was added to a final concentration of

124 10 mM. The membranes together with their bound calelectrin were sedimented at 100000 × g for 30 rain and washed three times with 0.05 M NaC1, 10 mM Tris-HC1 (pH 7.4) containing 3 mM CaCl2. The calelectrin was then resolubilized by resuspending the membranes in TBS adjusted to pH 9.2-9.4 and containing 10 mM EDTA. Dialysis was performed as in method I, but the final dialysis was against 20 mM Bistris buffer (Sigma, Deisenhofen, F.R.G.) (pH 6.0) and 5 mM flmercaptoethanol.

Chromatography and electrophoresis Chromatography. This was performed with the Pharmacia (Freiburg, F.R.G.) LCC 500 FPLC system using stationary phases of appropriate composition. Anion-exchange chromatography was done on FPLC-MonoQ anion exchange resin. Calelectrin prepared by method I or II in the appropriate dialysis buffer was applied to columns previously equilibrated with the buffer and eluted in a 0-500 mM NaCI gradient generated by mixing, in continuously varying proportions, the appropriate dialysis buffer with the same buffer containing 1 M NaCI. Gel filtration was carried out on 10 x 300 mm FPLC-Superose 12TM and 6 TM columns with exclusion limits of 1000-300000 and 5000-5 000 000 respectively. The columns were calibrated with appropriate standards. Partition chromatography was carried out on FPLC-phenyI-Sepharose. The column was equilibrated with 20 mM Bistris buffer (pH 7.0) containing 2 mM Ca 2+, 0.5 mM dithiothrettol and 2 M (NH4)2SO4. Calelectrin was applied in the same solution and eluted in a 2-0 M (NH4)2SO4 gradient obtained by mixing the solution with (NH4)2SOefree dialysis buffer. Gel electrophoresis. Cellulose acetate electrophoresis was performed in 50 mM Tris-HCl (pH 7.0), 20 mM KCI and 0.1 mM EGTA at about 3-5 rnA. Calelectrin was rendered visible by staining with Amido-Schwarz or anti-calelectrin antibodies and horseradish peroxidase-conjugated second antibody. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) was carried out according to Laemmli [21]. Tw~-d~lensional electrophoresis was performed according to

O'FarreU [221.

Immunochemistry Two polyclonal antisera and a monoclonal antibody were used to investigate the immunochemical relationship between different forms of calelectrin. One polyclonal antiserum (I) was the one originally prepared by Dr J.H. Walker [1]; a second (II) was raised in a rabbit to purified native H-calelectrin by injecting 200 /tg of protein initially and three further amounts of 200 #g at 3, 4 and 5 weeks thereafter. The monoclonal antibody (designated B25) to H-calelectrin was prepared by conventional methods. Immunostaining was performed on blots of cellulose acetate electropherograms.

Physical methods Analytical ultracentrifugation. This was performed in a Beckman model E centrifuge, following standard procedures [23]. Sedimentation equilibrium experiments were done with 0.4~ (w/v) calelectrin in 40 mM potassium borate buffer (pH 7.0) containing 1 mM CaCI2 and 95 mM KCI to give an ionic strength of 100 mM. Spectroscopic measurements. UV absorption was measured in a Hitachi 220 spectrophotometer. The circular dichroism of calelectrin was determined in 20 mM cacodylate buffer (pH 8.0) to which either 2 mM Ca 2+ or 0.1 mM EGTA had been added, using a Jobin Yvon Dichrograph Mark V in ttle range 31-200 nm. The resulting spectra were recorded and analysed using the Jobin Yvon Dichro 7/80 program. Estimates of secondary structures were obtained using a program of Provencher and GlSckner [24]. Fluorescence measurements were performed in a MPF 4 Perkin-Elmer spectrofluorimeter. For all spectroscopic measurements calelectdn was submitted to gel filtration to ascertain its purity, concentration and buffer concentration. Protein concentrations. These were determined spectroscopically [25] using e2~Snm= 0.67 M -1. cm -1, or colorimetrically according to Lowry et al. [26] or Bradford [27]. Results

Isolation of two forms of calelectrin Extraction from tissue To optimize the initial extraction procedure a number of parameters were varied. Following the

125

original extraction procedure [1] (here referred to as method I) the frozen electric organ was homogenized in a high ionic strength buffer in the presence of 10 mM Ca 2+, at pH 7.4. Coarse cell debris was removed by low-speed centrifugation and a membrane fraction containing calelectrin was obtained upon high-speed centrifugation. The resulting pellet was washed in the presence of 1 mM Ca 2+. Calelectrin bound to the pelleted membranes was solubilized by removing Ca 2+ with EGTA. Changing the pH of the homogenization buffer to 6.0 or 8.0 had little effect on the yield of calelectrin which was about 17 rag. kg -~ tissue (wet weight). Significantly higher yields were obtained by means of a later extraction procedure [18] which makes better use of the Ca2+-dependent binding of calelectrin to membranes. Calelectrin is solubilized during the first homogenization step by means of a buffer containing 1 mM EGTA. Call debris is removed and Ca 2+ is added to a final concentration of 10 mM to allow calelectrin to rebind to membranes. Sedimentation and washing of the resulting pellets in the presence of 3 mM Ca 2+ yields a membrane-bound calelectrin fraction, from which calelectrin is solubilized by suspending it in t...r ,.,,.,,fe.. containing 10 mM EGTA. The yield of calelectrin is 2-3-times greater (45 nag. kg -1 wet weight of tissue) than that of method I. We have now further improved the yield (method II) by changing the ionic strength and pH during the isolation procedure on the basis of an analysis of the interaction of calelectrins with lipids [30]; the EGTA-solubilized calelectrin is rebound to membranes at low ionic strength and eluted at high ionic strength and pH 9.2-9.4. This increases the yield of calelectrin still further, to 70-80 mg. kg-~ wet weight of tissue or 4-5-times greater than the original method [1,28]. If the protein content of electric organ is assumed to be 10 g. kg-~, calelectrin must constitute over 0.8% of the total protein.

Purification by ion-exchange chromatography Calelectrin prepared by method I and chromatographed in imidazole buffer emerged as a single peak at 300 mM NaCI (Fig. la) and behaved as a homogeneous polypeptide of the expected M r in SDS-PAGE (Fig. lb). One major and several minor impurities were removed by this

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Fig. 1. (a) Separation of calelectrin, prepared by method I, by FPLC on a column of MonoQ anion-exchange resin. A NaCI gradient (0-1.0 M; broken line) in imidazole buffer (50 raM, pH 6.0) containing 0.5 mM EGTA and 5 mM ,8mercaptoethanol was used for ehition. Calelectrin eluted as a sharp peak of UV absorbance at 280 nm (A28 o. continuous line) when the NaCI concentration (broken line) reached 300 mM (fractions 21 and 22). (b) Fractions 21 and 22, eluting at 300 mM NaCI, contain a polypeptide of Mr 34000 which migrates as a single band in SDS-PAGE in a 10% gel. The right-hand lane contains standard proteins of the Mr values ( × 10 -3) indicated.

procedure. However, when calelectrin prepared by method II was chromatographed in a Bistris buffer, the elution pattern was more complex (Fig. 2a) and in addition tu Ul~ mare peak eluting at ~0u mM NaC1, there was a smaller, but prominent peak at 100 mM together with other components, one of which was a shoulder on the high-ionicstrength side of the main peak at 300 mM NaCI. Only the prominent peaks at 300 and 100 mM NaC1 contained calelectrin as judged by their immunochemical reactions and ability to bind to lipids in a Ca 2+-dependent manner. The two main peaks, at 300 and 100 mM NaCI respectively, when submitted to further analysis by means of SDS-PAGE, proved to contain polyp e p t i d e s with a p p a r e n t M r values of 32000-34000; both components also responded to an anti-calelectrin antiserum. It is concluded that calelectrin, at least as prepared by method II, exists in two forms, which we refer to as the high-salt (H) and low-salt (L) forms respectively. Estimates based on the areas under the FPLC peaks indicated that the H-form accounts for about 9070 of the total extractable electric organ calelectrin. This was confirmed by the relative intensities of the immunochemically stained bands corresponding to the two forms of calelectrin after

126

electrophoretic sep~.ration on cellulose acetate of crude calelectrin pr~.pared by method I. Accordingly, this form v'as used for physical and biochemical characterization. For characterization, H-calelectrin was freed from buffer salts by gel filtration on FPLC-Superose 12TM. The resulting preparation contained no detectable contaminants in SDS-PAGE and had a defined, reproducible UV absorption spectrum (see below).

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Fig. 3. (a) Chromatography on phenyl-sepharose e~ a preparation of calelectrin extracted from electric organ by method IL Continuous line, absorbanco at 280 nm (A2,o); broken line, (NH4)2SO 4 gradient. For other experimental details, see text. (b, c) Separation, on MonoQ anion exchange resin, of (b) protein not retained by phenyl-Sepharose, (c) protein removed as the concentrations of (NH4)2SO+ and Ca 2+ in the eluent foil to zero. Note that the main protein peak in (b) emerges at a NaCI concentration of 0-100 mM allowing the accompanying calelectrin to be identified as the L-form, while that in (c) emerges at 300 mM and is identified as consisting of H-calelectrin.

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FRACTION No. Fig. 2. Separation similar to that of Fig. 1 except that calelectrin was prepared by method IL (a) Separation by FPLC resolved calelcctrin, as extracted, into three or four components; only the two largest of these, oluting at 100 and 300 mM

NaC! respectively, had the immunochemical (Fig. 4) and Ca2+-r©gulated lipid-binding properties (Fig. 1, Ref. 30) characteristio of calolectrin. Analysis by SDS-PAGE (b, c) showed that both these peaks contained a single pcptide of apparent Mr in the expected range for calelectrin of 32000-34000.

and those containing protein eluted as the (NH4)2SO4 concentration fell towards zero (Fig. 3a) were separately pooled and submitted to anion-exchange chromatography on a MonoQ column (Figs. 3b, c). Fractionation in this way showed that L-calelectrin had not been retained by the column, but that the H-form had been and was only recovered from the hydrophobic matrix when the (NH4)2SO4 concentration fell to zero. L-calelectrin forms only a small part of the initial, incompletely resolved peaks seen in Fig. 3b since it is accompanied by other components that also have little or no affinity for the column. Fig. 3a shows that there is little separation of the material retained by the column, elution occurring only when the (NH4)2SO 4 (and Ca 2+) concentrations fall to zero. Thus a selective fractionation due to

127

differences in Ca2+-dependent hydrophobicities of the bound proteins appears unlikely. However, the fact that H-calelectrin was retained by the column under the conditions under which it was applied, whereas L-calelectrin was not indicates that the overall charge and hydrophobicity of these two related peptides differ significantly.

Electrophoretic properties H- and L.calelectrin obtained by separating a method-II extract on MonoQ were compared by electrophoresis on cellulose acetate (Fig. 4a, b), SDS-PAGE (Fig. 4c) and two-dimensional electrophoresis (Fig. 4d). Electrophoresis on cellulose acetate (Fig. 4a) showed that L-calelectrin had a lower mean isoelectric point than ferritin, i.e. less than 4.5, whereas the H-form had a higher one than ferritin, i.e. greater than 4.5. Both forms were equally immunoreactive to an anti-calelectrin antiserum (Fig. 4b). In SDS-PAGE (Fig. 4c) under reducing conditions (presence of 5~ fl-mercaptoethanol, lanes 1 and 5) both forms migrated as single species, the L-form having a lower apparent M r (32000) than the H-form (34000). Under non-reducing conditions (lanes 2, 4, 6, 8) there was no change in the migration of L-calelectrin (compare lane 6 with 5 and 8 with 7) but the H-form was resolved into two components of M r 34000 and 32000 (compare lane 2 with 1 and 4 with 3). The addition of Ca 2÷ (2 raM) had no effect on mobility under either condition. In two-dimensional electrophoresis, both calelectrin forms showed microheterogeneity in respect of their isoelectric points which ranged from pH 4.3 to 5.5. The mean value for H-calelectrin was, as in cellulose acetate electrophoresis, higher than that for the L-form.

Physical parameters of H-calelectrin The results of measurements of the Stokes radius, sedimentation coefficient relative to molecule mass, molar absorption coefficient and ahelical content of H-calelectdn are summarized in Figs. 5 and 6 and Tables I and II. Fig. 5 shows determinations of Stokes radius by a comparison of the elution volume of H-calelectrin with those of standard proteins after gel filtration through FPLC-Superose 6 TM. It will be

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seen that the Stokes radius is independent of the buffer used (borate-KF or cacodylate), the ionic strength (5-100 mM) and the pH (7 or 8) over the ranges tested. Similar results (not shown) were

128 TABLE i SUMMARY OF HYDRODYNAMIC PROPERTIES OF H-CALELECTRIN Method

Parameter lneasured

Value obtained

Unit

Mr estimated

Gel filtration Ultracentrifugation SDS-PAGE Amino-acid analysis

Pokes radius Sedimentation coefficient Amino-acid composition of hydrolysate

3.1 + 0.3 (13) 2.9 -

nm S -

44000 36000 34000

-

-

32 000

obtained in experiments with Sepharose 12TM and in which ionic strengths up to 700 mM and pH values down to 6 were tested. Ca :+ (2 raM) had no effect and did not produce self-aggregation. The Mr for a spherical protein of this Stokes radius is 44000 (Table I).

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REV Fig, 5, The Stokes radii (rstok,) Of four reference proteins plotted as ordinates against their relative elution volumes (REV) as abscissae when subjected to gel filtration through FPLC-Sepbarose 6 TM under varying conditions. Triangles and dotted line, cacodylate buffer (pH 7.0) of ionic strength 20 mM; squares and solid line, borate-KF buffer (pH 8.0), ionic strength 100 raM; circles and broken line, same buffer at ionic strength $ raM. The fiUed symbols indicate the relative elution volumes of H,calelectrin under the three conditions, projected onto the calibration lines. The point with the bar is the mean of six determinations at an ionic strength of 20 m M + S.E. The mean of all values for the Stokes radius of caielectdn+ S.E, is 3,1+0,3 (13), Duplicate runs with and without 2 mM Ca 2+ showed no effect of this ion on the Stokes radius. The departure of the points at 5 mM ionic strength from linearity indicates some degree of non-specific interaction with the column matrix at this low ionic strength. The magnitude of the Stokes radius is not, however, affected.

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Fig. 6. (a) UV absorption, (b) fluorescence emission spectra of H-calelectdn. in (b), almost identical spectra were obtained in (1) the absence, (2, 3) the presence of Ca 2+ at (2) 5 mM, (3) 10 mM concentration. (c) Circular dichroism of H-calelectrin in cacodylate buffer (pH 8.0) of ionic strength 20 mM, in the presence of 0.1 mM EGTA (broken line) or 2 mM Ca 2+ (continuous line). Ordinates: molar ellipticity at 222 nm (0222) in deg.cm2.dmol - I x l 0 -3. In neither (b) nor (c) does the presence of Ca 2+ have any significant effect.

129 TABLE I!

Discussion

MOLAR ELLIPTICITY A N D SECONDARY STRUCTURE O F H-CALELECTRIN Measurements were made at pH 7.0 and ionic strength 20 mM.

Ca2+ 0222 (mM) (deg.cm2.dmol-t) 0 2.0

-19275 -19280

Structure(~) a-hefix

B-fold sheet

random coil

79 77

0 0

21 23

Velocity and equilibrium sedimentation in an analytical ultracentrifuge indicated a sedimentation coefficient of 2.9 S and apparent Mr of 36000. Ca 2+ had no effect on the sedimentation properties of H-calelectrin and no evidence for Ca 2+-induced self-aggregation was obtained. Spectroscopic measurements are shown in Fig. 6. The UV absorption (Fig. 6a) shows a maximum at 278 mm; the molar absorption coefficient is 2.28- 104 M- ~- cm- ~assuming a molecular weight of 34000. In Fig. 6b, the fluorescence emission spectrum of H-calelectrin is shown. Tyrosine and tryptophan residues are responsible for the emission maximum at 303 nm and the additional signals at 328 and 348 nm. Ca 2+ at 5 or 10 mM concentrations caused no significant change in fluorescence. Circular dichroism in the far UV has been measured to determine whether Ca 2+ could induce conformational changes that could affect the biological properties of calelectrin. The measurements (Fig. 6c and Table II) were made in cacodylate buffer, whose excellent optical properties reduced the noise to signal ratio which is increasingly troublesome at wavelengths below 210 nm. However, similar results were obtained in Tris buffer, in which Ca2+-regulated binding to phosphatidylserine has been demonstrated [30] despite the less favourable signal to noise ratio. In spite of these precautions, some variation (less than 10%) is observed between different preparations. Measurements with and without 2 mM Ca 2+ revealed no significant changes in the proportion of a-helix and random coil configurations in H-calelectrin. Calelectrin is characterized by a high content of a-helix and little, if any B-configuration (Table II).

The improved isolation procedure described here in which the extraction procedure of Siidhof et al. [18] has been optimized and followed by FPLC anion-exchange chromatography has resulted in a substantial improvement in the yield of calelectrin and its resolution into two forms, a major, H-form, eluting from the anion exchange resin and binding to a hydrophobic resin at relatively high ionic strengths and a minor, L-form, eluting from the anion exchange resin at low ionic strength and showing little affinity for the hydrophobic resin. It is estimated that 90~ of the calelectrin of the electric organ is in the H-form and that the two forms constitute about 1~ of the total protein of the organ. The two forms of calelectrin show similar immunoreactivity and behave similarly but not identically in gel electrophoresis and isoelectric focusing. As previously reported for total calelectrin, though with slightly different M r values [1,28], the H-form was resolved into two bands of apparent Mr 34000 and 32000 under non-reducing conditions but migrated as a single band of Mr 34000 in the presence of 5~ fl-mercaptoethanol. L-calelectrin migrated as a single band of Mr 32000 under both conditions. H-calelectrin had a lower isoelectric point than the L-form, but both showed charge microheterogeneity in gel isoelectric focusing. This may result from varying amounts of phosphorylation since other proteins of the calelectrin class are known to possess phosphorylation sites [29]. The UV absorption and fluorescence emission spectra of H-calelectdn were similar to those reported earlier [18] and were consistent with the presence of tryptophan and tyrosine residues in the polypeptide chain. A determination of the Stokes radius by gel filtration on Superose 6 TM and 12TM (not shown) gave a value of 3.1 nm which, for a typical globular protein of spherical shape implies a Mr of 44000. This is 22~ higher than the value obtained from measurements of the sedimentation coefficient and 23~ higher than that obtaine0 from SDS-PAGE, which were in good agreement with each other and with a value derived from amino-acid analysis [20~30]. Values fairly close to ours (Stokes radius 2.9 nm, sedi-

130

mentation coefficient 3.5 S) have recently been obtained [28] for calelectrin prepared according to the original method [1]. Measurements of circular dichroism revealed that a considerable portion of the molecule is in a random-coil configuration which would tend to increase hydrodynamic drag and might go some way to accounting for the discrepancies between the different estimates of the M r of H-calelectrin. An important, if negative finding is the lack of effect of C a 2+ o n any of the physical properties of calelectrin examined. The small effect (Table II) on the secondary structure is not considered to be significant. This is in marked contrast to the behaviour of calmodulin and other proteins of the 'EF-hand' group, and clearly distinguishes it from the latter. We were unable to confirm the previously reported self-aggregation of calelectrin [2,4] with our purified preparation; however, our findings are consistent with the previously reported lack of any effect of C a 2+ o n the ultraviolet and fluorescence spectra of calelectrin and failure to detect Ca2+-induced conformational changes with hydrophobic fluorescent probes [18]. Judged by the change in electrophoretic mobility induced by reducing conditions, the conformation of native H-calelectrin may be influenced by the redox state of internal disulphide bridges. The function of calelectrin in the electric organ is not known thought immunocytochemical evidence [31] points to a role in stabilizing filamentous structures in the electrocyte and the electromotor axons in their relation to membranes. It is interesting to speculate that H- and L-calelectrin may have cell-specific locations in the tissue, the more abundant H-form being associated with the electrocytes and the less abundant in the presynaptic electromotor nerve terminals or associated glial processes. Acknowledgements U.F. and A.v.K. were supported by stipends from the Max-Planck-Gesellschaft.

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