the interaction of calmodulin with human and avian spectrin

Vol. 122, No. 3, 1984 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

August 16, 1984 Pages 1194-1200

THE INTERACTION OF CALHODULIN WITH HUMAN AND AVIAN SPECTRIN

Athar Husain, Geoffrey J. Howlett and William H. Sawyer

The Russell Grimwade School of Biochemistry, University of Melbourne, Parkville, Victoria 3052, Australia

Received July 3, 1984

An air-driven ultraE?t$trifuge was used to investigate the calcium- dependent interaction of I-calmodulin with human and avian spectrins. The equilibrium Constants (Ka) for the interaction between calmodulin and human

~~e~~~~ ?O",-' nd tetramer un er on-denaturing conditions were estimated to

and 7.3 x IO' M-' respectively. The denaturatitn 01 human spectrin by urea (5 M) increased th;! K, for calmodulin to 4.6 x 10 M- . The Value of K,.for the interaction of calm,$duljn with avian spectrin dimer under non-denaturing conditions was 5.1 x 10 M . A bifunctional reagent crosslinked both avian spectrin and human spectrin to calmodulin in a calcium-dependent manner.

Intracellular calcium has been implicated in the control of erythrocyte

shape and deformability and it has been suggested that the effect might be

mediated by the interaction of calmodulin with cytoskeletal proteins (1). An

interaction between spectrin, the major cytoskeletal protein of human

erythrocytes, and calmodulin in the presence of urea has been demonstrated

(2). However, subsequent experiments performed using a gel overlay procedure

(3) or affinity chromatography (4) have failed to detect such an interaction

under non-denaturing conditions. In this communication we have employed the

method of sedimentation equilibrium in an air-driven ultracentrifuge to

provide a more quantitative analysis of these interactions (5).

EXPERIMENTAL

Materials: Ovalbumin and Oextra25 T40 were purchased from Sigma and Pharmacia Fine Chemicals, rf$gectlvely. I-Bolton and Hunter reagent (N-succinimidyl 3-(4-hydroxy, 5-C 11 iodophenyl) propionate) was from New England Nuclear. Phenothiazine-Sepharose, Bio-Gel A15m, AG501-X8(0),

and a mixed bed ion exchange resin, were from Bio-Rad Laboratories. Oithio-bis-N-hydroxy-

succinimidylpropionaie (Lomant's reagent) was obtained from Pierce and fluphenazine - HCl was a gift from Squibb, Australia. DE-52 was from Whatman.

Methods: Bovine brain calmodulin was purified according to the procedure of Sharma and Wang (6). Where necessary, final purification was carried out by


affinity chromatography on a column of phenothiazine-Sepharose (7) or by ion exchange chromatography on DE-52 in the presence of calcium (8). The final product migrated as single band on 12% SDS-polyacrylamide gels run in a discontinuous buffer system (9). RadioiodinTa;eiol; s carried out according to

procedures describef25 previously (10). I-calmodulin contained on average 1.4 mole of I per mole of calmodulin.

Human spectrin dimers and tetramers were prepared by the method of Cohen and Foley (11) which minimizes contamination of the preparation with trace amounts of other cytoskeletal proteins, particularly actin. Avian spectrin was extracted from avian erythrocyte ghosts prepared as described by Beam et al. (12). Spectrin was dissociated by incubation of membranes (30 min at 37oc) in low ionic strength medium (0.1 mM Tris-HCl, 0.1 mM EDTA, pH 7.5). The supernatant obtained after centrifugation (30,000 xg, 25 min) was extensively dialysed against 25 mM Tris-HCl, 100 mM NaCl, 0.1 mM EDTA, pti 7.5. After a second ultracentrifugation step (150,000 xg, 35 min) the clear supernatant was chromatographed on a Bio-Gel A15m column. Human and avian spectrins were homogeneous as judged by SDS-polyacrylamide

electrophoresis. gel

Sedimentation equilibrium experiments were performed in a Beckman Airfuge as previously described (13). The experimental data in the form of radioactivity (C) s aliquot number were converted to plots of C/C distance (r), where C t e radia1 is the initial radioactivity before ten rifugation. The apparent weight-avgrage molecular weight (M ) derived from the slope of the data in the linear region (r = 1.1 to 1.3 cm)wis defined as:

d In (C/C,) 2RT Rw =

dr2 . (1 - b), 2

where J is the partial specific volume, p the solution density, w the angular velocity of the rotor, and T the absolute temperature.

RESULTS AND DISCUSSION

Fig. 1 shows the stimulation of human erythrocyte membrane Ca2+-Mg2+-

ATPase activity by native calmodulin and by calmodulin radiolabeled with 1251-

Bolton-Hunter reagent. The results show that 1251-labeled calmodulin

activates the Ca2+ -Mg2+ATPase to the same extent as unmodified calmodulin.

Labeling using the iodogen procedure (14) destroyed calmodulin activity and

this procedure was therefore abandoned.

The interaction of 1251-calmodulin with purified spectrin dimer and

tetramer was investigated using the technique of sedimentation equilibrium in

an air-driven ultracentrifuge (13). Fig. 2(a) shows the distribution of

125 I-calmodulin with and without human spectrin tetramer. The slope of the

line drawn through the data obtained in the presence and absence of spectrin

tetramer yields values for Mw of 17,500 and 17,800, respectively. These

1195


- 0 0 , A io - -21-21 C%4OO%N ini? 2500 0 2 1.1 l-3 1.5 1.7 1.9 1.1 1.3 1.5 1.7 1.9

r' rz

Figure 1. Stimulation of Mg*+ dependent Ca*+ - stimulated ATPase of human

erythrocyte membranes by calmodulin and radioiodinated calmodulin. Assays were performed in 25 mM Hepes, pH 7.5. The procedures for iodination and for measuring ATPase activity have been described p?$%iously (10,20). The symbols are as follows. (0) Native calmo#Jin, (W) I-calmodulin labeled using Bolton and Hunter reagent, (A) I-calmodulin labeled using the iodogerl procedure (14).

Figure 2. Sedimentation equilibrium distribution of 1251-calmodulin in

kFe1q5 esence and absence of human spectrin tetramers. The concentrations I-calmodulin and spectrin were 0.41 uM and 0.68 uM, respectively.

Solutions in a total volume of 160 ul were centrifuged for 22 hr and at the conclusion of centrifuqation 10 ~1 fractions were sequentionallv withdrawn using a microtube fractionator (Beckman Instruments, Palo Alto,-Ca.). The temperature of the rotor at the end of centrifugation was 9+1"C. Oextran T-40 (4 mg/ml) was used to provide density stabilization and ovalbumin (200 ug/ml) was used as an inert pIr~;_iali;,,:;en ~1;;: ~,'~,;~,~tnr,i;,',; minimize non-specific adsorption of were (a) 25 mM Tris-HCl, pH 7.!jF50.3 mM CaC12 6-mercaptoethanol, 0.002% NaN3. I-calmodulin (O),

I5OI# KC~, 0.3 mM I-calmodulin f

same conditions as in (a) + 5 M deionized urea. I-calmodulin + spectrin tetramer (A). The rotor

values are in close agreement with the value of 16,790 reported for the

calmodulin monomer (15). In the presence of spectin tetramer, the depletion

of 1251-calmodulin over the radial distance 1.05 to 1.26 cm is attributed to

the sedimentation of 1251-calmodulin-spectrin complexes to the bottom of the

tube. The results summarized in Table 1 indicate that approximately 15% of

the calmodulin is bound to spectrin tetramer under these conditions. Similar

results were obtained for the binding of spectrin dimer to 1251-calmodulin.

The interaction between 1251-calmodulin and spectrin was specific in the sense

that excess unlabeled calmodulin considerably reduced the extent of binding.

Moreover fluphenazine and EGTA, antagonists of calmodulin action, completely

prevented the interaction. The interaction was favoured by increasing the

1196


TABLE 1: CALCIUM-DEPENDENT INTERACTION OF CALMODULIN WITH HUMAN AND

AVIAN SPECTRIN a

-

ADDITIONAL COMPONENTS b C/Co at meniscus

% Binding

----- Spectrin tetramer Spectrin dimer Spectrin dimer f EGTAC (0.3 mM) Spectrin tetramer + EGTA c (0.3 mM) Spectrin tetramer + fluphenazine (0.2 mM) Spectrin tetramer + excess unlabeled calmodulin Avian spectrin dimerd

0.51 --- 17,800 0.431 15.5 17,500 0.438 14.1 17,600 0.509 0.2 17,600 0.502 1.6 17,500 0.50 2.0 18,900 0.501 1.7 18,000

0.345 32.5 21,100

Apparent

plw

a The conditions for the experiments were the same as those described in Fig. 2(a). Tubes were incubated for two hours at 4°C before centrifugation at 37,500 rpm for 22 hr.

b Final concentrations of proteins were as follows:

I125 -calmodulin in each tube 0.41 PM; spectrin tetramer 0.68 uM; spectrin dimer 1.99 uM; unlabeled calmodulin 5.9 uM, avian spectrin 0.529 uM. Fluphenazine concentration was 0.2 mM.

' CaC12 absent.

d Final concentration of 1251-calmodulin in this tube was 0.17 uM.

ionic strength to loo-150 mM (data not shown). The interaction between

1251-calmodulin and avian spectrin dimer indicated that approximately 32% of

the calmodulin was bound under the conditions used (Table 1).

Determination of the amount of bound 1251-calmodulin allows calculation of

the equilibrium constants for the interactions as described previously (16).

Using values from Table 1 the results indicate a relatively weak interaction

between calmodulin and human spectrin dimer and tetramer characterized by the

equilibrium constants of 4.6 x lo4 M-l and 7.3 x lo4 M-l, respectively. On

the other hand the avian spectrin dimer interacts with Iz51-calmodulin with an

equilibrium constant of 5.1 x lo5 M-l, an order of magnitude higher than that

obtained for the interaction with human spectrin. The weak interaction of

125 I-calmodulin with human spectrin may explain the failure to detect an

interaction between 1251-calmodulin and human spectrin by the techniques of

gel overlay (3) and affinity chromatography (4).

The sedimentation equilibrium distributions of 1251-calmodulin in the

presence and absence of urea denatured human spectrin are shown in Fig. 2(b).

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TABLE 2: INTERACTION OF CALMODULIN WITH HUMAN SPECTRIN IN THE PRESENCE

OF UREA

ADDITIONAL COMPONENTSa C/C at men scus ?

% Binding Apparent flw

--- 0.601 --- 18,900 Spectrin dimer 0.519 13.7 19,500

Spectrin tetramer 0.510 15.1 18,200 Spectrin dimer + ureab Spectrin tetramer + ureab

0.223 62.9 0.208 65.4 1::

i?25 Final concentrations of proteins were as follows: I-calmodulin in each tube 79 nM; Spectrin dimer 1.8 uM;

Spectrin tetramer 0.545 PM. The experimental conditions were same as in Fig. 2(a) except that the total calcium concentration was 1.5 mM.

b Spectrin dimer and tetramer were dialysed in 6 M deionized urea for

1 hr at 4'C. Final concentration of urea in the centrifuge tube was 5 M.

The results indicate significant depletion of radioactivity near the meniscus

in the presence of spectrin. The values of C/Co for 1251-calmodulin and the

percent binding with urea denatured spectrin dimer and tetramer are given in

Table 2. Analysis of the interaction yielded an equilibrium constant of 4.6 x

lo5 M-l. After dialysis of the urea treated spectrin to remove urea, it was

found that the interaction of the spectrin with 1251-calmodulin was similar to

that observed with native spectrin. Recent results have shown that the

binding of calcium to calmodulin exposes a hydrophobic region in the protein

molecule which participates in protein-protein interactions (17,18). The

denaturation of human spectrin by urea could expose a region of hydrophobicity

thus enhancing its interaction with calmodulin.

The interaction between 1251-calmodulin and human and avian spectrins was

further analysed by covalent crosslinking with dithiobis-N-hydroxy-

succinimidylpropionate (Lomant's reagent) followed by SDS-polyacrylamide gel

electrophoresis and autoradiography (Fig. 3). 1251-calmodulin was found to

migrate as a single band of MW 17,000 both before and after treatment with

Lomant's reagent. Fig. 3 shows the results for mixtures of 1251-calmodulin

and human spectrin. When cross-linking was carried out in the presence of

calcium, high molecular weight material was observed near the origin (Lane 2).

Such material was not found when cross-linking was carried out in the absence

of calcium (Lane 1). For mixtures of 1251-calmodulin and avian spectrin

1198


Fiqure 3. Crosslinkinp250f 1251-calmodulin to human and avian spectrin. The concentration of I-calmodulin in each tube was 0.41 PM. Final

uman and avian spectrin were 1.17 PM and 0.24 uM, and spectrin incubated at room

addition of LomanTFreagent (0.35 mg/ml). Tubes were subsequently incubated for an additional hour at 4°C and the reaction was quenched with glycine (0.6 mM). SDS-polyacrylamide electrophoresis was carried out according to the Fairbanks procedIyf (18). The gels were dried and exposed to Kodak XRP-5 film for 20 hr. I-calmodulin f25human spectrin,

I-calmodulin + Lane I (no CaCl2) and Lane 2 (2.5 mM CaC12).

CaC12).

aVian SpeCtrin, Lane 3 (“0 CaCl2) and Lane 4 (2.5 mM

dimer, a crosslinked product in the presence of calcium (Lane 4) was observed

which migrated at a position only slightly above the position observed for

avian spectrin alone. This band was not observed when cross-linking was

carried out in the absence of calcium. The concentration of avian spectrin

used in these studies (0.11 mg/ml) was significantly lower than the value of

0.54 mg/ml for human spectrin (Lane 2). These results, therefore, support the

observations made using the air-driven ultracentrifuge that 1251-calmodulin

has a higher affinity for avian spectrin compared with human spectrin.

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the interaction of calmodulin with human and avian spectrin

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