132 Btochlml~a etBtophv~t¢a A~ta, 788 (1984) 132-137 ENevter

BBA 31927

A REAPPRAISAL OF THE SELF-ASSOCIATION OF HUMAN SPECTRIN

MICHAEL MORRIS and G B RALSTON *

Department of Biochemtstrv, Unwerslt~ o/Sydney, Sydney,, NSI4 2006 (Au~traha)

(Received October 20th, 1983) (Revised manuscript received March 19th, 1984)

Key words Spe~trm ohgomer. Self -asso~mtlon, Sedtmentatton equthbrmm

The self-association behaviour of human spectrin has been re-examined through a study of sedimentation equilibrium, sedimentation velocity and gel electrophoresis. In all cases we find evidence for oligomers of spectrin larger than the tetramer, even at low concentration. The data are not consistent with a simple dimer-tetramer model, but instead indicate an open, or indefinite, pattern of association. Although a good fit to the data can be achieved with the isodesmic reaction model, with an equilibrium constant in agreement with the value previously determined for the dimer-tetramer reaction, there is other evidence suggesting that the actual association scheme may be somewhat more complex.

Introduction

The red blood cell cytoskeleton, a two-dimen- sional protein network comprised of spectrm, actin and other components, hnes the cytoplasmic face of the red-cell membrane and plays an essential role in maintaining the shape and stability of the red cell [1]. Spectrln, the principal component, has been extracted at low lomc strength from erythro- cyte ghosts as a heterodlmer at 37°C, and pre- dominantly as a tetramer at low temperature [2]. It has been reported that spectrln ohgomers larger than the tetramer have not been detected and that the dlmer and tetramer are m simple thermody- namic equdtbrlum [3]. Recent evidence, however, has cast some doubt on this simple, generally accepted scheme Cychc hexamers of spectrln have been detected m electron microscope studies [4] and non-denaturing acrylamtde gradient electro° phores~s of spectrin at low concentrations has re- vealed a band consistent with a hexamer [5]. At very high spectrln concentranons a series of higher

* To whom correspondence should be addressed

polymers has been detected [6]. We have re-examined the self-association of

spectrin in vitro using sedimentation equlhbrium, sedimentation velocity and acrylamide gradient electrophoresls. In each case we find evidence for oligomers of spectrin larger than the tetramer, even at low concentrations. An lsodesmlc reaction model fits the sedimentation equlhbrlum data much better than a monomer-dimer model

Materials and Methods

Preparatton of spectrm Spectrm dlmer was extracted and purified from

human red blood cells as previously described [7] but with the inclusion of 0.3 mM sodium azide m all buffers After repeated chromatography on a column of Sepharose 4B (3 0 × 50 cm) m a buffer comprlsmg 0.1 M NaC1/0.01 M sodmm phos- phate (pH 7.5)/1 mM EDTA/0 .1 mM dlthloth- reitol, the dimer fraction was treated with 0.3 mM phenylmethylsulfonyl fluoride and was centrifuged at 30000 × g for 40 mm to remove possible insolu- ble material. The purified spectrm was used ira-

0167-4838/84/$03 00 "' 1984 El,,evler Science Pubhshep, B V

mediately for experimentation to minimize the proteolytic damage that may occur during storage.

Sedlmentatton equthbrtum expertments Spectrln samples at three different initial load-

ing concentrations were centrifuged for 24 h at 9000 rev . /min in a Beckman-Spinco analytical ultracentrifuge fitted with an RTIC unit. In order to ensure chemical eqmhbration, experiments were performed near 30°C. An An-D rotor and Yphan- us 12 mm six-channel centrep~ece were used. At 24 h the Rayle~gh interference pattern was photo- graphically recorded and the plates measured on a Nlkon comparator at 10 × magnificatton. Baseline corrections were made and the molecular weight distribution was calculated [8]. The effective monomer molecular weight was taken as 480000 [7] and a concentration conversion factor of 4.04 fringes per 1 g/1 was used [9].

Apparent weight average molecular weights were determined from the concentration distribu- tion by means of a seven-point shdmg linear re- gression fit of log (concentration) on the square of the radial distance Apparent number average molecular weights, used in the methods of Adams et al. [10] to test reaction models, were determined by trapezoidal integration of the apparent weight average molecular weight distributions [10].

The equations for each reaction model used to fit the apparent weight average molecular weight distributions by non-hnear regression were as fol- lows: For the monomer-dimer model the weight average molecular weight, M,~, is given by [10]'

M w = 2M l ( ] +4/ t2() ' '2/[1 + (1 +4k,,.. )' ,2]

where k 2 is the equilibrium constant and c the total protein concentration in the g/1 scale. M 1 is the effective monomer molecular weight. The molar equilibrium constant, K 2, is given by [11]:

K2= Mik2/2

For the isodesmlc reaction model, M,~ is given by [111:

M.~ = MI (1 +4k,.tc) W2

where k,n t IS the i n t r i n s i c equdlbrlum constant.

133

The molar intrinsic eqmhbrmm constant, K,n t, IS

given by [11]:

K,n t = Mlkmt

Apparent weight-average molecular weight, M,~,dp p, is obtained by substituting the appropriate func- tion for M,, into the following equation [10]:

M~ .pp = M. , . / ( 1 + BM,~, )

where B is the second vmal coefficient. Non-hnear regression analysis was performed

on apparent weight-average molecular weight versus concentration data, with the use of a Gauss-Newton algonth, using first derivatives calculated by central differences. Convergence to a final set of parameters was achieved from initial estimates on either side of the final values, with a tolerance of less than one part in 106 .

The use of silicone layering oil was avoided in sedimentation equilibrium experiments, as it ap- peared to produce irreversible aggregation of the protein. A similar effect of layering oils has been found with other proteins [12].

Sedirnentatton velocity expertments Spectrm dimer samples were incubated for 6 h

at 29-37°C. After Incubation, the samples were chilled in ice and centrifuged at 6°C m a Beck- man-Spinco analytical ultracentrifuge using an An-D rotor and aluminium-filled Epon double- sector centrepieces. Sedimentation was followed with the aid of schheren optics for concentrations above 2 g/1 and a photoelectric scanner for con- centratlons below 2 g/ l .

Electrophorests Spectrm samples were examined by means of

acrylamide gel electrophoresis in the presence of sodium dodecyl sulfate according to the method of Fairbanks et al. [13]. Samples were found to be greater than 98% pure, even at the complenon of centnfugatlon. No trace of actm or band 4.1 could be detected in any of the samples (Fig. 1).

The distribution of ohgomers of spectrm was examined by means of acrylamide gradient elec- trophoresls. After rapid chilling of incubated spec- trln solutions, 10-50 #1 samples were applied to acrylamlde gradient gels (2-13%) m a buffer com-

134

Fig 1 SDS-acrylamlde gel electrophoresls of three different loading concentrations of spectrm after purification by means of repeated gel filtration on Sepharose 4B Although all three samples were heavily overloaded, no trace of actm or band 4 1 could be detected

prising 17 mM sodium phosphate (pH 7.5)/1 mM E D T A / 0 . 2 mM dlthlothrettol. Electrophoresls was

performed for 24 h at 50 V, at a temperature of 2 - 4 ° C

Results

Fig. 2 displays the apparent weight-average molecular weight of spectrin as a funct ion of total

concentra t ion for a sedimentat ion equi l ibr ium ex- per iment performed at 28.8°C. The plots for the

three lnitml loading concentra t ions of spectrln dr-

mer supenmpose, mdtcatmg that all species have chemically equil ibrated durmg the time-course of the experiment.

Ungewxckell and Gratzer [3] concluded that the self-association of spectrin was restricted to a dt- mer- tetramer equlhbrium. In view of this, the sedi- menta t ion equthbr lum results were tested w~th a monomer-d lmer reaction model (in which the ef-

fective monomer is the spectrln heterodimer) With this reaction model, plots of the appropriate func-

tion of the weight fraction of monomer agamst

concentra t ton [10] (Fig. 3a) showed distmct up- ward curvature, mdlcat lng that ohgomers larger

than the tetramer were formed. Furthermore, tt was not possible to obtain a

close functional fit to the molecular weight dtstrt-

but ton using non-hnear regression with both the equi l ibr ium constant and second virial coefficient as parameters (broken line, Fig. 2). The fit shown here is poor, wtth obvious non - r andom distribu-

t ion of residuals. In addit ion, the d lmer izauon

I o X

v

z~ z~ AU.,~ __~ a----~--O'-A'D" ~'a"-I#../£~-- OT--O'---O ..... I

;o t

7

o I I I I I I I I 0 0 2 0 4 0 6 0.8 10 12 14 16

Concn. (g/ l )

Fig 2 Apparent weight-average molecular weight versus total protein concentration determined for a memscus depleuon sedlmenta- Uon equlhbnum experiment for three different mlhal loading concentrations of spectrm The dashed hne represents the best fit for the monomer-dlmer reacUon model and the sohd hne represents the best fit for the lsodesmlc reaction model, using non-hnear regression For the ~sodesnuc model the computed molar intrinsic equlhbnum constant, K,n t = (1 00_+0.03) 106 M - ! and the non-ldeahty constant, B= (499+0 11) 10 -7 l.mol.g -2 Symbols 3 7 g/1 (O), 1 8 g/l (rn). 09 g/1 (zx)

I ,.c

_ ,~J, ,o t °, a

b 3

2 , . S

, , .C

I o o

v 1 ?, o o

o % % u o

o o o

m . - ~ I

0 2 0 1 4

% o

o n ~

o n

n o o ~

o o o o o o

o u

0~6 OJ8 1'0 '1'2 1~ 16 ~8 Conen ( g / I )

Fig 3 Graphical tests for the self-assocmtlon model of spec- trm (a) Monomer-dlmer model Only the 0 9 g/l loading concentration has been graphed. The plots for the other con- centrattons showed similar curvature (b) Isodesmm model Symbols 37 g/l (O), 18 g/1 (13), 09 g/l (zx) For both models, a good fit would be m&cated by a straight hne of slope equal to the eqmhbrlum constant and passing through the origin [10]

c o n s t a n t ( K 2 = 4 . 6 1 . 1 0 6 M l) is u n a c c e p t a b l y

large c o m p a r e d wi th pub l i shed values [3], a n d the second v m a l coeff ic ient ( B = 4 . 6 . 1 0 ~ 1. m o l . g 2) is unrea l i s t i ca l ly smal l for a molecule the size a n d shape of spect r in . W h e n the d l m e n z a t l o n con- s t an t was c o n s t r a i n e d to the p u b l i s h e d value of 106

M 1 [3], the fit was still poor a n d a negat ive (and thus phys ica l ly mean ing less ) va lue of B was re- qu i r ed

The l sodesmm reac t ion mode l ( in which each effect ive m o n o m e r adds to the o l lgomer with the s ame change in free energy) gave a m u c h bet te r fit to the d i s t r i b u t i o n (solid line, Fig. 2). The fit was

ref ined us ing n o n - h n e a r regress ion a n d a va lue of 1 . 0 0 . 1 0 6 M - ~ was o b t a i n e d for the in t r ins ic equl-

135

h b r t u m cons tan t . Plots of the app rop r i a t e f u n c t i o n

of the weight f rac t ion of m o n o m e r agains t con- cen t r a t t on [10] (Fig. 3b) showed on ly sl ight d o w n - ward cu rva tu re wi th this model .

Fig. 4a shows the a b s o r b a n c e scan for a sedi- m e n t a t i o n veloci ty expe r imen t pe r fo rm ed on a

spec t rm sample of low ini t ia l c o n c e n t r a t i o n (1.3

g / l ) i n c u b a t e d at 37°C. The p ro t e in c o n c e n t r a t i o n

does no t reach a p l a t eau be tween the t e t r amer

b o u n d a r y a n d the base of the cell as wou ld be

, 429 ;>

06

04

02

" S D\

J,

Fig 4 Sedimentation velocity results for (a) spectrm (1 3 g/l) incubated for 6 h at 37°C and (b) spectrm (7 g/l) ,ncubated for 6 h at 29°C. After incubation, the samples were chilled ,n ice and centrifuged at 6°C. The scanner absorption profile in (a) was recorded at 40 mm at a wavelength of 292 nm The angular velocity was 40000 rev/mm The arrows indicate the ap- proximate posmons of the dlmer (D) and tetramer (T) boundaries The schheren photograph in (b) was recorded at 53 mm using a bar angle of 60 ° The angular velocity was 48000 rev/mln

136

expected if only the dlmer and tetramer species were present. The continuous rise in absorbance to the base of the cell indicates that ohgomers larger than the tetramer are present. In similar sedimen- tation velocity experiments at higher concentra- tion, schheren optics revealed a substantial amount of material sedimenting ahead of the main boundary and probably comprised of several ohgomerlc species of spectrln (Fig. 4b). After fur- ther sedimentation the main boundary resolved into two components which sedimented at the rate expected for the dimer and tetramer of spectrin. By the time the dlmer and tetramer boundaries had been resolved, however, the faster-moving material had sedimented and spread, so that it was no longer clearly distinguishable from the basehne. Ungewickell and Gratzer [3] performed similar sedimentation-velocity experiments on spectrin, but used single-sector cells and recorded the schlieren pattern only after the dimer and tetramer species had been resolved. The faster-sedimentlng material thus presumably escaped detection by Ungewlckell and Gratzer.

The sedimentation velocity results agree quali- tatively with the non-denaturing acrylamide gradi- ent electrophoresis results for spectrin dimer in- cubated for 6-8 h at 28.8°C (results not shown). Several bands, each presumably representing a separate ollgomeric state of spectrln, appear in addition to the dimer and tetramer bands. The results are similar to the band patterns obtained by Marchesl and Morrow [6].

Discussion

The evidence presented here suggests that spec- trin undergoes an indefinite self-association reac- tion in vitro, in which oligomers larger than the tetramer form a significant proportion of the population even at moderately low concentrations, The close fit of the lsodesmlc model to the sedi- mentation equihbrlum results suggests that each ohgomerizatlon step beyond the tetramer occurs with a free-energy change of magnitude compara- ble with that for tetramer formation

Fig. 3b shows that an isodesmic model results in a reasonably constant value of the isodesmic equilibrium constant (the slope of Fig. 3b), al- though there is some evidence of a downward

trend as concentration increases. Th~s downward trend may be due to the overslmphflcatlon in the analysis inherent in the Adams-Fujlta approxima- tion for the second Vlrlal coefficient [10,14]. Alter- natively, it may indicate that the equilibrium con- stant for successive addmons of heterodlmer units becomes progressively smaller

Tyler et al. [4] suggested that the cyclic hexamer of spectrm seen occasionally in electron micro- graphs could be formed by the opening of one of the paired reciprocal head-to-head interactions in the tetramer and the addition of a further hetero- dimer unit. This process may be expected to con- tinue to allow progressive ohgomerlzation by the quasi-equivalent addmon of further heterodlmers [6]. A mechanism such as this relies heavily on the flexlblhty of spectrin to allow variable geometry, and it is likely that increasing sterlc hindrance to further additions may limit the size of polymers Thus, the binding of additional heterodlmers is likely to involve progressively smaller changes in free energy, consistent with the curvature m Fig. 3b.

Evidence from gradient gel electrophoresls has been presented elsewhere [15] to show that the equilibrium constant for spectrin association be- yond the tetramer may be smaller than that for tetramer formation. However, in experiments of this type, the possibility exists that the equilibrium may be perturbed during the rapid chilling, or during the separation of the oligomers on pro- tracted electrophoresls. In the present study, using sedimentation equilibrium analysis, it is possible to study the system at chemical equilibrium at every point in the cell. In addition, the satisfactory overlap of the curves for the three separate loading concentrations in Fig. 2 indicates that, m our experiments, spectrln self-association has reached chemical equilibrium, and no significant amounts of material are present that are incapable of par- ticipating in the reactions.

Our value for the equilibrium constant for the dimer-tetramer reaction is remarkably close to the value of 1,. 106 M 1 determined by Ungewlckell and Gratzer for similar solution conditions [3]. These authors were unable to detect h~gher ohgomers in their reaction mixtures, presumably because of the difficulty in resolving the large number of minor boundaries in single-sector oper-

ation. Additionally, Fig. 4b shows a series of sharp spikes below the tetramer boundary. These spikes may mdtcate convection, which has been observed m the sedimentation of other interacting macro- molecules (Nachol, L.W., personal communication). Any convection in the cell at this point would erode boundar tes corresponding to higher oligomers, making their detection even more dtf- ftcult. Ungewtckell and Gratzer, however, de- termined the equilibrium constant from absolute values of the concentrations of dimer and tetra- mer, which leads to a valid determination of the equilibrium constant for tetramer formation, even tn the presence of additional species in the equi- hbrium.

At the very high local concentrations of the spectrln found at the membrane surface [16], spec- trIn would be extensively assoctated. The implica- tions of this have been discussed by Marchesi and his colleagues [6,15]. Models that attempt to de- scribe the organtzation of the red-cell cytoskeleton must in future take into account the increased tendency of spectrin to self-associate.

Acknowledgements

We wish to thank Dr. Simon Easterbrook-Smith for allowing us to use his non-linear regression program. This work was supported by the Australian Research Grants Scheme.

137

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