Structural characterization of the cytoplasmic pole of band 3 from bovine erythrocyte membranes

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 243, No. 1, November 15, pp. 228-237,1985

Structural Characterization of the Cytoplasmic Pole of Band 3 from Bovine Erythrocyte Membranes’ RYUICHI

MORIYAMA,

TAKASHI KITAHARA, SHIO MAKIN02

TAKUJI

SASAKI,

AND

Department

of Food Science and Technology, Faculty of Agriculture, Chihxsa-ku, Nagoya, Aichi 464, Japan Received

April

10,1985,

and in revised

form

August

Nagoya

University,

2,1985

In an earlier study, we found that chymotryptic digestion of band 3 isolated from bovine erythrocyte membranes produces a 38,000-Da fragment in nonaethyleneglycoln-dodecylether solution or a 50,000-Da fragment in deoxycholate solution as a primary fragment [Makino et al (1984) J. B&hem 95,1019]. In the present study, these fragments were purified in an aqueous medium without detergent and their structural properties were examined. Several lines of evidence showed that the 50,000-Da fragment constitutes the entire cytoplasmic pole of bovine band 3 and that the 38,000-Da fragment is a subfragment of the 50,000-Da fragment. The large fragment was suggested to be divided into two distinct regions, the 12,000- and 38,000-Da portions, differing in their conformational thermal stability. However, attempts to identify the 12,000-Da portion as an isolable segment were without success. The cytoplasmic pole was characterized as a dimer which adopts an elongated gross conformation with helix of -35%. Treatment of the fragments with dimethylmaleic anhydride dissociated the dimers into the monomers, accompanied by a significant conformational change of the 38,000-Da portion. Comparative studies suggested that the cytoplasmic domain of bovine band 3 has structurally different region(s) from that of human band 3, though their gross conformation shows extensive Similarity. o 1%~ Academic press. IX

Band 3 from the human erythrocyte membrane is a major intrinsic protein of apparent molecular weight - 95,000 and exists as oligomers in the membrane (l-3). The protein is easily cleaved at probably a single site of the cytoplasmic surface of the membrane by mild chymotryptic or tryptic treatment, yielding a water-soluble fragment of molecular weight - 40,000 and a membrane-associated fragment of molecular weight - 55,000 (4-6). The water-soluble fragment contains the amino-terminal end of band 3 (5, 7) and supplies binding sites for several membrane’s cytoskeletal 1 This research was supported in part by Grant-inAid for Scientific Research 58380034 from the Ministry of Education, Science and Culture of Japan. 2 To whom correspondence should be addressed. 0003-9861/85 Copyright All rights

$3.00

8 1985 by Academic Press. Inc. of reproduction in any form reserved.

and cytoplasmic proteins (8-12). The membrane-bound fragment, the carboxylterminal portion of band 3, is involved in the transport of inorganic anions (13-15) and spans the membrane several times (4, 5, 16, 17). In bovine band 3, its molecular disposition in the membrane appears to be similar to that of human band 3 (18-21). Chymotryptic digestion of inside-out membrane vesicles produces two fragments from bovine band 3, a membrane-associated 58,000Da fragment and a water-soluble 38,000Da fragment which is the cytoplasmic pole origin (21). Their molecular weights, however, do not sum to that of intact band 3, 107,000. We further observed that chymotryptic treatment of isolated bovine band 3 generates a 50,000-Da piece in deoxycho228

CHARACTERIZATION

OF

HYDROPHILIC

late solution or a 38,000-Da piece in nonaethyleneglycol-n-dodecylether (C12E9)3 solution, and that the 50,000-Da segment was cleaved to yield the 38,000-Da segment by second chymotryptic digestion in deoxycholate-free media (21). These observations suggest that the cytoplasmic pole of bovine band 3 consists of the 50,000-Da segment, of which an -lO,OOO-Da portion is excised during proteolysis in aqueous media. However, direct evidence for this suggestion was not provided in that study. This paper reports the isolation of the 50,000- and 38,000-Da fragments from bovine band 3, the identification of the fragments as the cytoplasmic pole origin, and the structural characterization of the fragments. The results were discussed in comparison with available structural properties of the cytoplasmic domain of human band 3. MATERIALS

AND

METHODS

Materials. a-Chymotrypsin was obtained from Miles. Diisopropylfluorophosphate was a product of Fluka. Sepharose 4B and Sephadex G-100 superfine were products of Pharmacia. Anion exchanger, DE52, was a product of Whatman. Detergents used here were as follows: SDS from Nakarai Chemicals, &Es from Nikko Chemicals, and sodium deoxycholate from Yoneyama Kagaku. Preparation procedures of aminoethyl-Sepharose 4B and dimethylmaleic anhydride were described previously (18). All other reagents were purchased from Wako Pure Chemicals. Preparation of e-rythrocgte membranes. Unsealed ghosts from intact cells and chymotrypsin-treated cells, and their alkali-stripped membranes, were prepared according to the procedures described previously (21). Isolutia of band 3. Bovine and human band 3 were purified in 10 mM Tris-HCl, 1 mM NaN,, 0.1% C12E,, pH 8.0, at 4”C, by elution from an aminoethyl-sepharose 4B column with a linear NaCl gradient (18). Isolation of ~8,s,ooO- and sO,OMl-Llaw Isolated bovine band 3 was digested with 10 @g/ml chymotrypsin for 2 h at room temperature in a 5 mM N%HPO,-NaH2P0, buffer, pH 8.0, containing2% GE, (for the 38,000-Da fragment) or 2% deoxycholate (for the 50,000-Da fragment) (21). The hydrolysis was terminated by the addition of diisopropylfluorophosphate

’ Abbreviations used: C,,Eg, nonaethyleneglycol-ndodecylether; SDS, sodium dodecyl sulfate; R., Stokes radius.

DOMAIN

OF

BOVINE

BAND

3

229

(0.1 mM final). The solution was dialyzed for 24 h against 2 liters of a 5 mM Tris-HCl, 1 mM NaN3 buffer, pH 8.0, with one change of the external buffer. Then the solution was applied to a DE-52 column (2 X 20 cm) preequilibrated with the dialysis buffer, and proteins were eluted at pH 8.0 with 200 ml of a linear gradient of NaCl (the final concentration of NaCl was 0.4 M) containing 5 mM Tris-HCl and 1 mM NaNa. The 38,000- and 50,000-Da fragments were eluted at NaCl concentrations of 0.15-0.20 and 0.22-0.27 M, respectively. Each fragment was concentrated by Amicon PM-10 membrane and further purified on a Sephadex G-100 superfine column (2.2 X ‘75 cm) which had been equilibrated with a 10 mM Tris-HCl, 50 mM NaCl, 1 mM NaN, buffer, pH 8.0. These chromatography experiments were carried out at 4°C. Treatment of the fragments with dimethylmaleic anhydride. Ten milligrams of solid dimethylmaleic anhydride/mg polypeptide was gradually added to the isolated fragment solutions at 10°C for 1 h under stirring, maintaining the pH at 8.0 by the addition of 2 M NaOH. After the evolution of acid had ceased, the materials were dialyzed overnight against 10 mM TrisHCI, 50 mM NaCl, 1 mM NaN3 buffer, pH 8.0, at 4°C. Preparation of antiserum. Antiserum of the 38,000Da fragment of bovine band 3 was raised in a young male white rabbit. A mixture of the purified fragment (-1 mg) and complete Freund’s adjuvant was injected intramuscularly. Three weeks later another injection (-1 mg) was carried out, and the animal was bled 1 week later after the injection. Analytical procedures. Concentrations of the fragments were determined according to the modified Lowry method (18), using bovine serum albumin as a standard. Calibration was done by quantitative amino acid analysis. The polypeptide concentratioqs determined by the Lowry method were corrected for the 0.92- and 0.93-fold lower color yields for the 38,000and 50,000-Da fragments, respectively. Polyacrylamide gel electrophoresis in the presence of 0.1% SDS was performed at a gel concentration of 6% for cylindrical gels (0.6 X 8 cm) or 7.5% for slab gels (0.2 X 14 X 14 cm), using a 0.1 M NazHP04NaH2P0, buffer, pH 7.0, as an electrophoresis buffer. Usually, protein samples (30-60 pl), containing 25-50 pg protein, 5% SDS, and 0.1% 2-mercaptoethanol, were heated at 90°C for 5 min and then loaded on gels. Proteins were stained with Coomassie brilliant blue R. Phosphorylase b, bovine serum albumin, catalase, aldolase, chymotrypsinogen, and cytochrome c were used as molecular weight standards. Amino acid analysis was performed on a JASCO JLH-8AH machine. The fragments were hydrolyzed in 6 N HCI for 20, 48, and 72 h at 110°C. The stated abundances of Val, Ile, Leu, and Phe were from the 72-h analysis. The values for Thr, Ser, and Tyr were determined by extrapolation of the three analyses to zero hydrolysis time. Tryptophan was measured

230

MORIYAMA

spectrophotometrically in SDS solution (22). The total number of amino acid residues in the fragments was calculated assuming a molecular weight of 38,500 for the 38,000-Da fragment and of 50,000 for the 50,000Da fragment, respectively. Oxidative crosslinking of the fragments was performed with 1 mM o-phenanthroline, 0.2 mM CuSOl at 10°C for 1 h in a 10 mM Tris-HCl, 50 mM NaCI, 1 mM NaNa buffer, pH 8.0. The reaction was stopped by the addition of 0.4 mM EDTA. Ouchterlony double-diffusion analysis was performed by the method of Ouchterlony and Nilson (23). The plate was prepared with 1.2% (w/v) agarose, using a 5 mM NhHPO,-NaH2POI, 0.15 M NaCl buffer, pH 8.0, containing 0.5% ClzE9. Immunoblotting was performed according to the method of Towbin et al. (24), using the Immun-Blot assay kit of Bio-Rad. The proteins electrophoresed on 0.1% SDS, 7.5% polyacrylamide slab gels (0.1 X 6.5 X 8 cm) were transferred electrophoretically to a nitrocellulose paper, and the protein cross-reacted with the anti-38,000-Da fragment serum was visualized using horseradish peroxidase-conjugated goat antirabbit immunoglobulin G and I-chloro-1-naphthol. Stokes radii of the fragments were determined at 4°C using a Sephadex G-103 superfine column (2.2 X 75 cm) which had been equilibrated with 10 mM TrisHCl, 50 mM NaCl, 1 mM NaNs buffer, pH 8.0. The column was calibrated using standard proteins with known Stokes radii (ferritin, catalase, bovine serum albumin, hemoglobin, and chymotrypsinogen). The void and internal volumes were determined by eluting Blue Dextran and 2-mercaptoethanol, respectively. Sedimentation velocity measurements were made with a Hitachi UCA-01 analytical centrifuge equipped with Schlieren optics. The runs were done with a protein concentration of -1 mg/ml at 2O”C, in 10 mM Tris-HCl, 50 mM NaCl, 1 mM NaNa buffer, pH 8.0. Circular dichroic spectra were recorded on a JASCO J-40 spectropolarimeter, using a cell of 1 mm light path. The concentrations of proteins used were 0.20.3 mg/ml. Mean residue ellipticity was calculated using the mean residue weight of 111 for the 50,000Da fragment and 103 for the 38,000-Da fragment, based on the amino acid composition. The buffer used was 10 mM Tris-HCl, 50 mM NaCl, 1 mM NaNs, pH 8.0. The desired temperature of sample solution was maintained by circulating water from a thermoregulated bath through the jacket of the cell. RESULTS

Evidence showing that the isolated fragments are the c@plasmic pole Origin The isolated fragments were essentially free from other polypeptide components, as judged from the profiles of SDS-gel electrophoresis (Fig. 1).

ET

AL.

(-1

50K

-

38K

-

(+I

-

-

FIG. 1. SDS-polyacrylamide gel electrophoretic patterns of the 38,000and 50,000-Da fragments. Samples (-50 pg) which were purified by gel filtration were electrophoresed on 0.1% SDS, 6% polyacrylamide cylindrical gels.

To identify whether the isolated fragments are derived from the cytoplasmic pole of band 3, we tested the cross-reactivity of the anti-38,000-Da fragment serum against several fragments generated from bovine band 3 in the membrane-bound state after chymotryptic digestion. Bovine band 3 is split into two membrane-intercalating fragments of 67,000 and 41,000 daltons by extracellular chymotryptic attack, and the large fragment contains the cytoplasmic pole of band 3 (19, 21). Both fragments are not released from the membrane by alkali treatment. As shown in Fig. 2A (lanes 1 and 2), the 67,000-Da fragment primarily reacted with the antiserum. Subsequent chymotryptic digestion of leaky ghosts prepared from chymotrypsintreated cells generates membrane-traversing 17,000- and water-soluble 38,000-

CHARACTERIZATION

A

P- 9

Bond3-

OF

HYDROPHILIC

p ‘C 4

%’

DOMAIN

OF

BOVINE

BAND

3

231

B

d

67K-

8

38K-

1

2

3

4

FIG. 2. Immunoblotting and Ouchterlony double-diffusion analysis of band 3 fragments. Preimmune serum did not precipitate band 3 and its fragments. (A) Immunoblotting of band 3 fragments from bovine erythrocyte membranes digested with chymotrypsin. Five microliters (0.5-1.0 pg of proteins) of ghosts from chymotrypsin-treated cells (lane l), the same alkali-stripped membrane (lane Z), double-digested ghosts (lane 3) and the same alkali-stripped membrane (lane 4), were electrophoresed. Then polypeptides were transferred to a nitrocellulose paper and blotted with anti-38,000-Da fragment serum. (B) Ouchterlony double-diffusion analysis of the 50,000-Da fragment. Five micrograms (in 10 ~1) of samples was reacted with 10 ~1 of anti-38,000-Da fragment serum. Well 1, bovine band 3; well 2, the 50,000-Da fragment; well 3, the 38,000-Da fragment; well S, anti-38,000-Da fragment serum.

Da segments from the 67,000-Da fragment (21). In such double-digested ghosts, only the 38,000-Da polypeptide was immunoblotted (lane 3 of Fig. 2A). After the watersoluble 38,000-Da species was removed from the membrane by treatment with 0.1 M NaOH, the alkali-stripped, membranebound segments did not react with the antiserum (lane 4 of Fig. 2A). In a previous paper, we showed (21) that the 50,000-Da fragment is generated from the 67,000-Da fragment, and that chymotryptic cleavage of the 50,000-Da fragment in a deoxycholate-free medium produces the 38,000-Da fragment. The antiserum also reacted with the 50,000-Da fragment, as shown in Fig. 2B. These results strongly suggest that both fragments isolated in this experiment are released from the cytoplasmic pole of band 3, and that the small fragment is a subfragment of the large one. We could not detect a 12,000-Da piece from the 50,000-Da fragment as a stable and isolable segment in the following experiments. When the 50,000-Da fragment was digested with 10 pg/ml chymotrypsin for 1 h at room temperature in a deoxy-

cholate-free medium and the reaction mixture was analyzed by SDS-gel electrophoresis, only a 38,000-Da band was observed on the gel [Fig. 7 of Ref. (21)]. Similarly, in gel chromatography of the same reaction mixture, we were unable to detect any peak except the peak of the 38,000-Da fragment and a very broad one at the internal volume (not shown). Amino acid composition of the fragments. The amino acid composition of the fragments is shown in Table I, together with the discriminant function value which is an index of the polarity of protein (25). The discriminant function values of the fragments were within the range of an average value for many water-soluble proteins examined by Barrantes, 0.16 + 0.11 (25). By this criterion, either of the fragments can be classified as a water-soluble polypeptide. The hydrophilic nature of the fragments is more pronounced in the large fragment than in the small fragment. This would be a reflection of a distinct enrichment in negatively charged residues (include Asn and Gln) in the 12,000-Da part of the 50,000-Da fragment (mol% of Asx plus Glx

232

MORIYAMA TABLE

I

AWNO ACID COMPOSITION OF THE CYTOPLASMIC FRAGMENTS Residues/m01

Amino

acid

50,000-Da fragment

38,000-Da fragment

12,000-Da portion’

LYS His Arg Asx Glx Thr Ser Pro GUY Ala Cys (half) Val Met Ile Leu 5r Phe Trp

25.2 11.9 21.2 39.2 83.9 24.9 24.8 36.4 29.6 26.9 2.3 29.7 7.1 10.8 64.5 6.0 14.1 6.1

18.9 8.9 16.7 26.7 52.6 14.6 19.0 32.4 24.4 21.6 2.3 25.8 5.2 7.8 53.5 2.7 11.2 5.8

6.3 3.0 4.5 12.5 31.3 10.3 5.8 4.0 5.2 5.3 0 3.9 1.9 3.0 11.0 3.3 2.9 0.3

Sum Discriminant function valueb

464.6 0.15

350.1 0.27

114.5 -0.27

a The values were obtained by subtracting column 3 from column 2. * Diseriminant function (DF) is defined by Barrantes (25) as DF = -0.34544 (Lys + Arg + His + Asx + Glx)/ (Ile + Tyr + Phe + Leu + Val + Met) + 0.0006 (s.,), where &, is average hydrophobicity.

ET

AL.

in contrast to the previous observations that band 3 and its 67,000-Da fragment are resistant to complete denaturation in the medium because of the presence of the hydrophobic membrane-hound domain(s) (20). Figure 4 shows the effect of temperature on circular dichroic. intensity at 222 nm for the fragments. The 38,000-Da fragment showed a thermally irreversible transition at 65°C. On the other hand, the 50,000-Da fragment revealed a thermal transition at 50°C which is virtually reversible, in addition to the transition at 65°C. Treatment of the fragments with dimethylmaleic anhydride, a reagent which dissociates band 3 oligomers into the monomers (19), gave rise to a significant decrease in helical content of the 38,000-Da segment, and the transition at 65°C was not observed in the dimethylmaleic anhydride-treated fragments whereas the transition at 50°C was preserved. The results demonstrate that each of the 12,000- and 38,000-Da segments constitutes a structural domain in the 50,000-Da fragment. State of association of the fragments. Isolated bovine band 3 exists as oligomers composed of dimer and tetramer in ClzEg and deoxycholate solutions (19,27). Do the fragments isolated here participate in the formation of band 3 oligomers? Do multiple molecular species coexist in the fragments?

0 3 4

-2

is 26.5% for the 50,000-Da fragment, 22.7% for the 38,000-Da fragment, and 38.0% for the 12,000-Da portion). Secondary structure and um&rmational thermal transition of the fragments. Figure 3 shows circular dichroic spectra in the faruv region of the fragments in an aqueous buffer. Helical contents estimated by the method of Cockle and Epand (26) were 33% for the small fragment and 36% for the large fragment, respectively. Both fragments in 6 M guanidine hydrochloride exhibited circular dichroic curves which are typical for randomly coiled polypeptides,

-4 -6 -8

1

-10 IiT -121 200

210 220 230 240 Wavelength (nm)

250

FIG. 3. Circular dichroic spectra in the far-uv region of the fragments in 10 mM Tris-HCl, 50 mM NaCl, 1 mre NaN3 buffer, pH 8.0 (curves 1 and 2), and in the same buffer containing 6 M guanidine hydrochloride (curves 3 and 4). Curves 1 and 3, the 38,000-Da fragment; curves 2 and 4, the 50,000-Da fragment.

CHARACTERIZATION

Tempemiure

OF

HYDROPHILIC

(‘C)

FIG. 4. Effect of temperature on circular dichroic intensity at 222 nm of the fragments in the same buffer as described in Fig. 3. Ellipticity was measured after the sample solution had been maintained for 10 min at the indicated temperature. Temperature was gradually raised up to 80°C (open symbols) and then was lowered (closed symbols). (A) (0, l ), the 50,000-Da fragment; (Cl, n ), the 38,000-Da fragment. (B) (0, l ), the dimethylmaleic anhydride-treated 50,000-Da fragment; (0, n ), the dimethylmaleic anhydridetreated 38,000-Da fragment.

These problems were examined by gel filtration and sedimentation velocity experiments and molecular weight determination. Molecular weight was calculated according to, (28)

M = -6aNq Rssiw,w, r 1 - v;o where N is the Avogadro’s number, q and p are the viscosity and the density of water at 20°C Vis the partial specific volume, R, is the Stokes radius, and s~,~ is the sedimentation coefficient. Stokes radii of the fragments were determined at 4°C by Sephadex G-100 superfine column chromatography of the preparations which had been subjected to DE-52 purification, according to the plot described by Ackers (29). The result is shown in Fig. 5A. In this chromatography, either of the fragments was eluted as a single peak with an R, value of 52 +- 2 A for the 50,000-Da fragment or 40 f 2 A for the 38,000-Da fragment. The gel-filtration

DOMAIN

OF

BOVINE

BAND

3

233

experiment was found to yield identical results in the presence and absence of dithiothreitol. Sedimentation patterns of the purified fragments showed a single boundary (Fig. 5B), and sedimentation coefficients obtained were 4.7 +- 0.1 S for the large fragment and 4.4 f 0.1 S for the small fragment, respectively, at the polypeptide concentration of -1 mg/ml. Assuming a partial specific volume of 0.74 cm3/g (calculated based on the amino acid composition), the molecular weights were 107,000 f 6000 for the large fragment and 77,000 + 5000 for the small fragment. On the other hand, the polypeptide molecular weights were estimated as 50,000 + 1000 for the large fragment and 38,500 + 1000 for the small fragment by SDS-gel electrophoresis. Taken together, both fragments must be isolated as a single molecular species, a dimer. If the fragments were a globular particle, the predicted Stokes radius, RFin, would have been -34 A for the large fragment and -30 A for the small fragment, according to the equation, RFi” = (3 M,d 47rN)“3. (Calculation was done using the monomer molecular weight estimated from SDS-gel electrophoresis and assuming a hydration of 0.2 g water/g of the fragment.) The frictional ratio, RJR,“‘“, which is a measure of deviation from a spherical particle (28, 30), was 1.55 for the 50,000-Da fragment and 1.31 for the 38,000-Da fragment. Even though ambiguities are bound to the values of this ratio, e.g., by inaccurate estimation of protein hydration, the obtained values exceed the range of l.l1.25 expected for most globular proteins (28, 30), suggesting that the fragments adopt an extended conformation. The marked molecular asymmetry observed for the large fragment must be a reflection that the fragment is a two domains-linked complex. As shown in Fig. 6, both fragments were oxidatively crosslinked by o-phenanthroline/Cu2+, unlike the isolated human cytoplasmic fragment (6). The crosslinking did not occur after treatment of the fragments with dimethylmaleic anhydride. The dimethylmaleic anhydride-treated 50,000-

234

MORIYAMA

0.4

0.6

0.8 1.0 1.2 d'(lK(j)

ET

1.4

AL.

1.6

FIG. 5. (A) Determination of the Stokes radii of the 38,000- and 50,000-Da fragments by Sephadex G-100 superfine column chromatography at 4°C. Kd is distribution coefficient. Standard proteins are: 1, ferritin (R. = 64 A); 2, catalase (R, = 52 A); 3, bovine serum albumin (R, = 35 A); 4, hemoglobin (R. = 32 A); 5, chymotrypsinogen (R. = 22 A). (B) Sedimentation profiles of the 38,000-Da fragment (the upper picture) and the 50,000-Da fragment (the lower picture). Samples of 0.4 ml (a concentration of ~1 mg/ml) were centrifuged at 20°C in an RA rotor at a speed of 55,430 rpm. The profiles were recorded after centrifugation for 36 min.

(+) 1

-

-

-+.

2

3

4

FIG. 6. Profiles of SDS-polyacrylamide gel electrophoresis of the cytoplasmic fragments (gels 1 and 3) and their dimethylmaleic anhydride-treated materials (gels 2 and 4), which were treated with Cu”/o-phenanthroline. Samples without 2-mercaptoethanol were electrophoresed on 0.1% SDS, 6% polyacrylamide cylindrical gels.

Da fragment had a sedimentation coefficient of 2.9 S, which increased to 3.5 S by heating at 73°C for 5 min. The heat-treated (‘73”C, 5 min) 50,000-Da fragment sedimented as a 3.7 S component, sometimes accompanying with a 3.0 S minor component. Heating of the 50,000-Da fragment at 90°C for 5 min produced huge aggregates having sedimentation coefficients of more than 15 S. These findings suggest that heat denaturation of the cytoplasmic pole is accompanied by the disruption of the dimers into the monomers, which are followed by aggregations. The dimethylmaleic anhydride-treated or heat-denatured 50,000-Da fragment was sensitive to proteolysis, and no 38,000-Da fragment, or only a small amount, if produced, was generated from such a treated 50,000-Da fragment. The structural properties of the 38,000and 50,000-Da fragments are shown in Table II, together with those reported for the

CHARACTERIZATION

OF

HYDROPHILIC

DOMAIN

TABLE MOLECULAR

PROPERTIES

OFTHE

OF

BOVINE

BAND

3

235

II

CYTOPLASMIC

FRAGMENTSOF

BAND 3

Polypeptide Stokes radius (A)

Polypeptides 33,000-Da fragment bovine band 3

from

50,000-Da fragment bovine band 3

from

40,000-Da fragment human band 3’

from

Molecular

molecular

weight (x10-y

weight (x10-s)

Sedimentation coefficient(S)

Number

of subunits

Frictional ratio (R,/R,m’“)

Percent of helix

40

4.4

77

38.5

2

1.31

33

52

4.7

107

50.0

2

1.55

36

53

4.1

95

40.0

2

1.6

37

a Data taken from Appel and Low (6).

40,000-Da chymotryptic fragment of human band 3 (6). Studies on isolated human band 3. Isolated human band 3 was cleaved by chymotrypsin in 2% &Es and 2% deoxycholate solutions, and the results were compared with those of bovine band 3. As shown in Fig. 7A, under mild conditions, a primary chymotryptic product from human band 3 was a 40,000- to 45,000-Da polypeptide, which is assumed to be the cytoplasmic pole origin judging from the molecular weight value. The fragment obtained in the respective detergent solutions showed a different mobility on SDS-gel electrophoresis from that of bovine band 3. The fragment of bovine band 3 was found to have a greater resistance to proteolysis than that of human band 3 (lanes 4 and 6 of Fig. 7A). In addition, the antiserum raised against the bovine 38,000-Da fragment did not immunoprecipitate human band 3 (Fig. 7B). DISCUSSION

In an earlier study (21), we suggested by the molecular weight evaluation of several chymotryptic fragments that the only detectable water-soluble 38,000-Da fragment, which is released from bovine band 3 when proteolyzed at the intracellular surface of the membrane, does not account for the entire cytoplasmic pole of the band 3 polypeptide. The present experiments show unambiguously that the 50,000-Da frag-

ment isolated here constitutes the cytoplasmic pole of bovine band 3, indicating that the cytoplasmic pole of the protein has a larger molecular size than that of human band 3,40,000-43,000 Da (6, 31). As in the human case (6), the cytoplasmic pole of bovine band 3 was isolated as a dimer. The dimer was as susceptible to oxidative crosslinking as intact bovine band 3 in the membrane (19). The dimer was suggested to be dissociated to the monomer by heat denaturation, showing a 25% decrease in helical content, and the resulting monomer or its aggregates was very sensitive to proteolysis compared with the dimer. These demonstrate that the 50,000-Da fragment isolated here retains much of intact structure and that the structure of the cytoplasmic pole is stabilized by the formation of dimer. The cytoplasmic pole of human and bovine band 3 is quite similar in the gross conformation to each other, as shown in Table II. In the human case, Low et aZ. (32) reported that the cytoplasmic domain exists in a reversible, pH-dependent conformational equilibrium among three native states. This problem in the bovine case, however, has not been studied. A comparison of the thermal denaturation behavior between the 38,000- and 50,000-Da fragments demonstrates that the cytoplasmic pole of bovine band 3 consists of two domains, the 12,000- and 38,000Da portions, though the alignment of the two segments on the primary structure of

236

MORIYAMA

ET

AL.

-i 50K38K-

FIG. 7. (A) SDS-polyacrylamide gel electrophoretic patterns of isolated human and bovine band 3 treated with chymotrypsin in 2% CiaEe or 2% deoxycholate solution. Human band 3 (gel 1); its products digested with 2 pg/ml of ehymotrypsin at room temperature for 1 h in 2% ClzE9 (gel 2) or in 2% deoxycholate (gel 3); and its products digested with 10 &ml chymotrypsin at room temperature for 1 h in 2% CnE9 (gel 4). Bovine band 3 (gel 5), and its products digested with 10 fig/ml chymotrypsin for 1 h at room temperature in 2% ClzE9 (gel 6) or in 2% deoxycholate (gel 7). (B) Immunological cross-reactivity of human and bovine band 3. Five micrograms (in 10 pl) of isolated human band 3 (well 1) and isolated bovine band 3 (well 2) were reacted with 10 pl of anti-bovine 38,000-Da fragment serum (well S).

the band 3 polypeptide remains to be clarified. It is of interest to note that the secondary structure of the 12,000-Da segment is not affected by the dissociation of the dimer, and that its thermal transition is virtually reversible. These features exhibit a striking contrast to the structural properties of the 38,000-Da fragment. This may suggest that the 12,000-Da portion does not relate to the dimer formation of the cytoplasmic pole. The absence of molecular associates other than the dimer in the isolated fragments rules out the possibility that the cytoplasmic pole participates in further association of bovine band 3, so as to result in the tetramer in the solubilized state and probably in the membrane (19). It must be emphasized again that, under our conditions, we failed to identify the 12,000-Da domain as an isolable segment. In nondenaturing detergents which we used for solubilization of band 3, isolated bovine band 3 was cleaved to yield the 50,000-Da fragment in the medium containing deoxycholate and cholate. On the other hand, the 38,000-Da fragment was

obtained in nonionic detergent solutions such as CIZEg, Triton X-100, octyl glucoside, and dodecyldimethylamine oxide. Thus, the 12,000-Da piece appears to be very sensitive to proteolysis in the medium without bile salts, though the mechanism of such a protective effect by bile salt anions is obscure. With respect to the species specificity of the structure and function of band 3, Jay (33) found that the cytoplasmic domain of chicken band 3 differs in its structure from that of human band 3 and that chicken band 3 lacks the ability to bind glyceraldehyde-&phosphate dehydrogenase, a function associated with the amino terminus of human band 3 (12,34). In view of the fact that the sera raised against human band 3 in both rabbit (35) and sheep (7) have been directed against the amino-terminal region, Jay (33) further noted that “This may suggest that there is a large diversity between species in this part of the molecule while the membrane-associated domain may be conserved”. In accordance with this indication, the present results suggest that the cytoplasmic pole of bovine

CHARACTERIZATION

OF

HYDROPHILIC

band 3 has a different structure compared to that of human band 3 as evidenced by a different molecular size, a different sensitivity to proteolysis, a different reactivity to o-phenanthroline/Cu2+, and the absence of cross-reactivity of human band 3 with antiserum raised against the bovine 38,000Da segment. However, their gross conformation showed extensive similarity and, furthermore, we have observed that the 50,000-Da fragment binds glyceraldehyde3-phosphate dehydrogenases from human and bovine erythrocyte membranes and inhibits the dehydrogenase activity in a similar fashion to the human case (unpublished result). Taken together, these observations may suggest that the cytoplasmic domain of human and bovine band 3 contains mutually homologous and heterogeneous region(s) in their structure, though only limited data are available at present. REFERENCES 1. STECK, T. L. (1978) J. Supramol. Struct. 8,311-324. 2. NIGG, E., AND CHERRY, R. J. (1979) Biochemistry l&3457-3465. 3. WEINSTEIN, R. S., KHODAD, J. K., AND STECK, T. L. (1980) in Membrane Transport in Erythrocytes, The Alfred Benzon Symposia (Lason, U. V., Ussing, H. H., and Weith, J. O., eds.), Vol. 14, pp. 35-50, Munksgaard, Copenhagen. 4. STECK, T. L., RAMOS, B., AND STRAPAZON, E. (1976) Biochemistry 15,1154-1161. 5. STECK, T. L., KOZIARZ, J. J., SINGH, M. K., REDDY, G., AND K~HLER, H. (1978) Biochemistry 17, 1216-1222. 6. APPEL, K. C., AND Low, P. S. (1981) J. BioL Chem 256,11104-11111. 7. FUKUDA, M., ESHDAT, Y., TARONE, G., AND MARCHESI, V. T. (1978) J. Bid Chem 253,2419-2428. 8. HARGREAVES, W. R., GIEDD, K. N., VERKLEIJ, A., AND BRANTON, D. (1980) J. BioL Chem 255, 11965-11972. 9. RICHARDS, S., HIGASHI, T., AND UYEDA, K. (1979) Fed Proc 38,798. 10. SALHANY, J. M., CORDES, K. A., AND GAINES, E. D. (1980) Biochemistry 19,1447-1454.

DOMAIN

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BOVINE

BAND

3

237

11. MURTHY, S. N. P., LIU, T., KAUL, R. K., K~HLER, H., AND STECK, T. L. (1981) J. BioL Chem. 256, 11203-11208. 12. TSAI, I.-H., MURTHY, S. N. P., AND STECK, T. L. (1982) J. BioL Chem. 257.1438-1442. 13. GRINSTEIN, S., SHIP, S., AND ROTHSTEIN, A. (1978) B&him Biophys. Acta 507,294-304. 14. CABANTCHIK, Z. I., KNAUF, P. A., AND ROTHSTEIN, A. (1978) Biochim Biophys. Ada 515,239-302. 15. DUPRE, A. M., AND ROTHSTEIN, A. (1981) B&him. Biophys. Acta 646,471-478. 16. DRICKAMER, L. K. (1977) J. Biol. C&m. 252,69096917. 17. RAMJEESINGH, M., GARRAN, A., AND ROTHSTEIN, A. (1983) Biochim. Biophys. Acta 729,150-160. 18. NAKASHIMA, H., AND MAKINO, S. (1980) J. Biochem 87,899-910. 19. NAKASHIMA, H., AND MAKINO, S. (1986) J. Bioehem. 88,933-947. 20. MAKINO, S., NAKASHIMA, H., AND SHIBAGAKI, K. (1981) J. Bioehem. 89,651-658. 21. MAKINO, S., MORIYAMA, R., KITAHARA, T., AND KOGA, S. (1984) J B&hem 95,1019-1029. 22. KOZIARZ, J. J., KHMER, H., AND STECK, T. L. (1978) AnaL Biochwn. 86,78-89. 23. OUCHTERLONY, O., AND NILSSON, L. A. (1973) in Handbook of Experimental Immunology (Weir, D. M., ed.), pp. 19.1-19.39, Blackwell, Oxford. 24. TOWBIN, H., STAEHELIN, T., AND GORDON, J. (1972) Proc. NatL Acad Sci USA 76,4350-4354. 25. BARRANTES, F. J. (1975) B&hem Biophys. Res. Ccnnmun 62,407-414. 26. COCKLE, S. A., AND EPAND, R. M. (1978) J. BioL Chem 253,8019-8026. 27. NAKASHIMA, H., NAKAGAWA, Y., AND MAKINO, S. (1981) Biochim Biophys. Ada 643,509-518. 28. TANFORD, C., NOZAKI, Y., REYNOLDS, J. A., AND MAKINO, S. (1974) Biochemistry 13,2369-2376. 29. ACKERS, G. K. (1967) J. BioL Chem 242,3237-3238. 30. TANFORD, C. (1961) Physical Chemistry of Macromolecules, Ch. 6, Wiley, New York/London. 31. JENNINGS, M. L. (1984) J. Mewzb. BioL 80,105-117. 32. Low, P. S., WESTFALL, M. A., ALLEN, D. P., AND APPEL, K. C. (1984) J. BioL Chem 2.59, 1307013076. 33. JAY, D. G. (1983) J. BioL Chem. 258,9431-9436. 34. KAUL, R. K., MURTHY, S. N. P., REDDY, A. G., STECK, T. L., AND K~HLER, H. (1983) J. BioL Chem. 258,7981-‘7990. 35. ENGLAND, B. J., GCJNN, R. B., AND STECK, T. L. (1980) B&him Biophys. Acta 623,171-182.