Ligandin heterogeneity: Evidence that the two non-identical subunits are the monomers of two distinct proteins

Biochimica et Biophysica Acta, 492 (1977) 163-175

© Elsevier/North-Holland Biomedical Press BBA 37656 LIGANDIN HETEROGENEITY: EVIDENCE THAT THE TWO NON-IDENTICAL SUBUNI'IS ARE THE MONOMERS OF TWO DISTINCT PROTEINS

N. M. BASS, R. E. K1RSCH, S. A. TUFF, I. MARKS and S. J. SAUNDERS UCT-MRC Liver Research Group, Department of Medicine, University of Cape Town (Republic of South Africa}

(Received November 19th, 1976)

SUMMARY Purified ligandin (Y-protein) a 46 000-dalton protein, has been shown to consist of two subunit species (tool. wts. 22 000 and 24 000) on discontinuous polyacrylamide gel electrophoresis in sodium dodecyl sulphate. This technique was used to define further the nature of these subunits. The Y sulphobromophthalein-binding fraction of rat hepatic cytosol was shown to contain three major subunit bands designated subunit Ya, subunit Yb and subunit Yc in ascending order of size. Purified ligandin was found to comprise Ya and Yc subunit species, and also gave two bands on isoelectric focusing. The two subunit species in purified ligandin were partially separated by an additional purification step. Antiserum to ligandin reacted monospecifically with the purified protein, as well as hepatic, renal and small intestinal mucosa cytosol, but gave lines of identity and partial identity with cytosol from testis, ovary and adrenal gland. The Y fraction of testis was found to contain only Yb and Yc species, while all three major bands were found in liver, kidney and small intestinal mucosa. Phenobarbital treatment increased the concentration of Ya and Yb in the liver, but had little effect on Y¢. These findings suggest that the Y, and Y~ ligandin subunits are the monomers of two proteins: YaY~ and YcY~.

INTRODUCTION Subsequent to the discovery of 3 organic anion-binding protein fractions, X, Y and Z in rat liver cytosol [1], the protein responsible for anion-binding in the Y fraction was isolated and named ligandin [2]. This protein is found in abundance in the cytosol fraction of mammalian liver, proximal renal tubules and small intestinal mucosa [3]; and has been shown to bind non-covalently a large variety of organic anions including bilirubin and sulphobromophthalein [1, 4], anionic metabolites of steroid hormones [4--6] thyroid hormones [7], penicillin [8], and radiographic contrast media [9]; and also to bind azocarcinogen dyes covalently [6]. Ligandin is thought to influence uptake of organic anions by the liver [10] and kidney [8] and Abbreviations used: SDS, sodium dodecyl sulphate; "fEAE-cellulose, triethylaminoethyl cellulose.

164 may be involved in the intracellular transport of haem [11]. In addition, ligandin is identical with glutathione-S-transferase B (EC 2.5.1.18) [12], one of a group of cytoplasmic enzymes catalysing glutathione conjugation with a large range of substrates [13]. Ligandin is a basic protein (pI = 9.0) of 46 000 daltons and has been thought to exist as a dimer composed of two identical 23 000 dalton monomers. Recently, purified ligandin has been shown to exhibit two non-identical subunit species on discontinuous polyacrylamide gel electrophoresis in SDS [14]. Whether these subunits represent components of a single protein or of two separate and distinct proteins, has not been resolved. This paper deals with the partial separation of these components, further studies on their distribution in different tissues and their unequal response to phenobarbital administration. MATERIALS AND METHODS

Chemicals All reagents used were of analytical grade. TEAE-cellulose, myoglobin, cytochrome c, carbonic anhydrase (EC 4.2.1.1), lysozyme (EC 3.2.1.17) and Coomassie Brilliant Blue R-250 were obtained from Sigma Chemical Co. and ovalbumin and aldolase (EC 4.1.2.7) from Boehringer. Bovine serum albumin and 2-mercaptoethanol were purchased from Miles Serevac, sodium sulphobromophthalein from Hynson, Westcott and Dunning Inc. and Sephadex gels from Pharmacia Fine Chemicals. Ampholines were products of LKB instruments while complete Freund's Adjuvant and agar noble were obtained from Difco laboratories. Human gamma globulin and human serum albumin were kind gifts from Dr. John de la Harpe. All other reagents were supplied by BDH Chemicals. Acrylamide and N,N'-methylenebisacrylamide were recrystallized from chloroform and acetone, respectively, before use [15].

Animals Male Wistar rats (200-300 g body weight) were used throughout this study except when female tissues were required. Phenobarbital treated rats received 8 mg/ 100 g body weight phenobarbital subcutaneously for 14 days. Control rats received equivalent volumes of saline for the same period of time.

Preparation of cytosol fractions Cytosol fractions were prepared at 4 °C from 20 different tissues which were removed from animals anaesthetized with ether. Where possible, the tissues were perfused with ice-cold saline. In each case a 25 ~ (w/v) homogenate was prepared in 0.01 M sodium phosphate buffer pH 7.4, 0.25 M sucrose using a Teflon-glass homogenizer. The homogenate was centrifuged at 100 000 x g in a Beckman L ultracentrifuge, and the supernatant fraction carefully removed without disturbing the floating lipid. All samples not used immediately were stored at --20 °C and used within 1 week.

Analytical methods Protein concentration in cytosol and pooled fractions was quantitated by the method of Lowry et al. [16] with bovine serum albumin as a standard. Protein in column fractions was determined at 280 nm in a Unicam SP 1700 spectrophotometer.

165 Sulphobromophthalein was determined spectrophotometrically at 580 nm after alkalinization. Ligandin concentration was measured by radioimmunoassay [17]. Densitometry of stained protein bands in gels was performed with a Joyce Loebl Chromoscan.

Purification of ligandin Ligandin was purified essentially by the method previously described [4, 8]. In brief, cytosol obtained from 1130g of rat liver was dialysed against 0.01 M Tris. HCI pH 8.8 (Buffer A) and chromatographed on a column of TEAE-cellulose in Buffer A. The single protein peak eluted was pooled, concentrated (Amicon cell UM-10 membrane) and after mixing with sulphobromophthalein, chromatographed on a column of Sephadex G-1130 in 0.01 M sodium phosphate buffer, pH 7.4, 0.1 M NaCi. Protein fractions exhibiting maximum sulphobromophthalein binding were pooled, concentrated, dialysed against Buffer A and chromatographed on a column of QAESephadex A-50 in Buffer A. The single protein peak eluted was taken to represent purified ligandin [4, 8] and was pooled and stored in sterile tubes at 4 °C. Rechromatography of the purified ligandin was also performed on QAESephadex using Buffer A with pH raised to 9.1.

Polyacrylamide gel electropk, oresis in SDS Polyacrylamide gel electrophoresis in 0.I ~ SDS was performed by two methods. Method 1 employed a discontinuous buffer system as described by Maizel [18] and comprised a 3 ~ stacking gel in 0.0625 M Tris/phosphate buffer pH 6.7 and a 10 or 12.5~ resolving gel in 0.375 M Tris.HC1 buffer pH 8.9. The electrode buffer was 0.005 M Tris/glycine pH 8.6. Gels were light-polymerized in the presence of riboflavin and electrophoresis performed in a 25 x 10 x 0.15 cm slab system at 10 mA for 15 h. Samples were prepared for electrophoresis by heating at 100 °C for 2 min with 1 ~ SDS, 1 ~ 2-mercaptoethanol, 10 ~ glycerol, and 0.002 ~ Bromphenol Blue in 0.0625 M Tris/phosphate buffer, pH 6.7. Method 2 employed 10 ~ gels in a continuous 0.1 M sodium phosphate buffer pH 7.2, as described by Weber and Osborn [19]. 5 4 0 #1 of each sample containing between 1 and 100/~g of protein were applied. After fixing in 12.5 ~ (w/v) trichloroacetic acid for 1-2 h, gels were stained for 2.5 h in 0.2~ Coomassie Brilliant Blue in methanol/water/acetic acid (50:50:7 v/v) and destained in a solution of 5 ~ methanol/7 ~ acetic acid.

Isoelectric focusing Isoelectric focusing in 10~ polyacrylamide gel was performed according to the method of Wrigley [20] in an MRA water-cooled apparatus at 4 °C, in both pH 3.5-10 and pH 7-10 ampholine ranges. Gels were stained with Coomassie Brilliant Blue as described by Ketterer et al. [21].

Molecular weight estimation The molecular weight of purified ligandin was estimated by chromatography on a 40 x 2.5 cm column of Sephadex G-75 in 0.01 M sodium phosphate buffer pH 7.4, 0.1 M NaC1 (flow rate 40 ml/h) with ovalbumin, bovine serum albumin, and carbonic anhydrase as standards. The molecular size of subunits was estimated by

166 discontinuous gel electrophoresis in SDS as described above, using gamma-globulin H and L chains, human serum albumin, cytochrome c, myoglobin, lysozyme, ovalbumin, aldolase and carbonic anhydrase as standards. Their molecular weights were taken to be 50 000, 23 500, 68 000, 11 700, 17 200, 14 300, 43 000, 40 000 and 29 000 respectively [19].

Immunology Immunization of male albino rabbits aged 3 months with purified ligandin in complete Freund's adjuvant, and collection of antisera was carried out as described previously [3, 8]. Double immunodiffusion in 1.2~o agar gels was performed as previously described [22]. Immunoelectrophoresis was performed in 1 ~o agar gels in 0.07 M veronal buffer, pH 8.7. Plates were run at 1.2 mA/cm for 2 h. Precipitates were stained with Amido Black. Y Fraction preparation Chromatography of 5-ml aliquots of cytosol fractions from various tissues was carried out at 4 °C on a 90 × 2.5 cm column of Sephadex G-100 in 0.01 M sodium phosphate buffer, pH 7.4, 0.1 M NaC1 at a flow rate of 30 ml/h, with collection of 3-ml fractions. The Y fraction of eluates obtained in this manner was defined as those fractions corresponding in elution volume to the Y sulphobromophthalein-binding peak obtained when hepatic cytosol with added sulphobromophthalein is separated by this method [1]. From each eluate, 15 ml of Y fraction (fractions 70-74) was pooled, mixed and a 5-ml aliquot concentrated 25 times in an Amicon B 15 concentration cell, prior to analysis. RESULTS Discontinuous slab polyacrylamide gel electrophoresis in SDS of liver cytosol and fractions from the various stages of purification of ligandin are shown in Fig. 1. The designations Ya, Yb and Yc refer to three major subunits species in unpurified cytosol in order of their relative mobilities on this system of analysis (Fig. 1A). This terminology has been introduced as these three species were found to correspond to the major components present in the Y peak of sulphobromophthalein-binding on Sephadex G-100 chromatograms of rat liver cytosol, when fractions from this peak were concentrated and examined by discontinuous gel electrophoresis in SDS (Fig. 2). As illustrated in Fig. 1, the method of purification used, results in a progressive enrichment of the subunit Ya and subunit Yc species. The Yb subunit band is no longer detected after the first ion exchange step, during the purification procedure (Fig. 1B) and thus probably represents a relatively acidic species in the Y protein fraction of hepatic cytosol. The finding of two non-identical subunit components on discontinuous gel electrophoresis in SDS in final purified ligandin preparations (Fig. 1D) is in keeping with the recent observations of other workers [14]. This heterogeneity is not revealed when the continuous buffer system [9] is used to examine the final protein product of purification. When this system is employed, a single protein band is evident even when the amount of protein loaded is varied over a range of 1-20/tg/gel. No decrease

167

Fig. 1. Discontinuous slab polyacrylamide gel electrophoresis in SDS (10~ resolving gel) of (A) hepatic cytosol (100 000 × g supematant), (B) pooled and concentrated protein peak from TEAEcellulose, (C) pooled and concentrated sulphobromophthalein-binding peak from Sephadex G-100 and (D) pooled protein peak from QAE-Sephadex. Ya-Yc represent the major subunit polypeptides found in the Y fraction of hepatic cytosol (see Fig. 2). Between 10 and 30/~g protein were loaded per sample after reduction (see text for details). The gel was stained with Coomassie Blue.

or increase in either subunit Ya or subunit Yo components was noted in purified ligandin preparations when stored at 4 °C for up to 6 months. When the purified ligandin fraction from the QAE-Sephadex step of purification was concentrated and rechromatographed on QAE-Sephadex at pH 9.1, the early peak fractions appeared to contain only subunit Ya species uncontaminated with subunit Yc (Fig. 3). Subunit Yc appeared in all subsequent fractions containing protein, and increased to an equal proportion to subunit Y, in the later fractions of

168

t-5

TT 00

/

I

Elution Volume [mll Fig. 2. Chromatography of hepatic cytosol and sulphobromophthalein on Sephadex G-100 showing X, Y and Z sulphobromophthalein-binding fractions. 5 ml of hepatic 100 000 × g supernatant was mixed with 5 mg sulphobromophthalein and separated on a column (90 x 2.5 cm) of Sephadex G-100 in 0.01 M sodium phosphate buffer pH 7.4, 0.1 M NaCI (flow rate 30 ml/h; fraction volume 3 ml). The fractions indicated by the horizontal bars were pooled (15 ml total volume), and a 5-ml aliquot concentrated to 0.2 ml of which 10 Itl was analysed by discontinuous slab gel electrophoresis in SDS (10~ resolving gel, shown in the insert). Ya-Yc represent the 3 major polypeptide subunits found in the Y fraction. The gel was stained with Coomassie Blue. - - ~ - - , absorbance at 280 nm; --A--, sulphobromophthalein absorbance at 580 nm. the peak. These findings suggest a less basic pI for subunit Yc relative to subunit Y, protein species.

Molecular weight estimation Molecular weight estimations of Ya and Yc subunits in purified ligandin preparations on discontinuous polyacrylamide gel electrophoresis in SDS by comparison with protein standards were 22 000 for subunit Ya and 24 000 for subunit Yc. If 2-mercaptoethanol was omitted from the ligandin sample preparation prior to electrophoresis, Ya and Yc subunits migrated in identical fashion to the bands from samples which had been reduced by heating with 2-mercaptoethanol. This suggests that these species are not disulphide-linked. Chromatography of purified ligandin preparations containing both subunit species on Sephadex G-75 and G-100 always resulted in elution of a single symmetrical protein peak which by comparison witlstandard proteins on Sephadex G-75, had a molecular weight of 46 000.

169

0.2 "T ¢.-

0.1

O OO

<

,

Elution Volume [ml] Fig. 3. Rechromatography of ligandin on QAE-Sephadex at pH 9.1. The protein peak eluted from QAE-Sephadex (38 × 2.5 cm column, flow rate 45 ml/h, fraction volume 3 ml) in 0.01 M Tris.HCl buffer pH 8.8 was pooled (fractions 25-35), concentrated to 5 ml, dialysed against 0.01 M Tris.HCl buffer pH 9.1 and chromatographed in this buffer on a new column of QAE-Sephadex with the same dimensions, flow rate and fraction volume as above. Aliquots of 25/~1 from fractions (a) 25, (b) 28, (c) 32 and (d) 35 were analysed by discontinuous slab gel electrophoresis in SDS (10 70 resolving gel) Y, and Yc represent ligandin subunit species. The gel was stained with Coomassie Blue.

Isoelectric focusing Isoelectric focusing of purified ligandin in the p H 3.5-10 range resulted in a single protein band focusing in the 8-9.5 p H range. When this procedure was performed in the p H 7-10 range, two bands were consistently found. The approximate p / f o r each band was estimated as p H 8.7 and p H 9.1.

Immunological studies Antiserum against purified rat ligandin containing both Y , and Yc subunits gave a single line o f complete identity on immunodiffusion against cytosol fractions from rat liver, kidney, small gut mucosa as well as purified ligandin (Fig. 4A). Besides cytosol fractions from liver, kidney and small gut mucosa, only supernatants from the testis, ovary and adrenal gave precipitin lines with the same ligandin antiserum (Fig. 4B). Cytosol fractions from these latter three organs, however, gave rise to 2 separate precipitin lines (Fig. 4B); a line of partial identity. (Ouchterlony type 3 reaction [22]) and a line of identity with purified ligandin. When cytosol from liver, kidney and small intestinal mucosa was used instead of purified ligandin in these immunodiffusion experiments, identical results were obtained. When 5-ml aliquots of cytosol fraction from liver (See Fig. 2), kidney, small intestinal mucosa and testis were eluted on Sephadex G-100, and the column fractions tested against ligandin

170

Fig. 4. Immunodiffusion and Immunoelectrophoresis in agar gel, (A and B). The centre wells contain 10/tl rabbit antiserum to rat liver ligandin. The other wells contain in 10/~1: (A) 2, 4 and 6:1/~g purified ligandin; 1 : hepatic cytosol; 3 : kidney cytosol and 5 : small intestinal mucosa cytosol ; (B) 1, 3 and 5 : 1 #g purified ligandin; 2: testis cytosol; 4: ovary cytosol and 6: adrenal cytosol. No precipitin lines were seen with cytosol from heart, lung, brain, skeletal muscle, pancreas, spleen, bladder, gastric and colonic mucosa, uterus, thyroid, pituitary, salivary gland or seminal vesicles, when tested with the same antiserum. (C) Immunoelectrophoresis in 1% agar gel. The wells contain cytosol (10 /~1) from 1, liver; 2, testis; 4, small intestinal mucosa and 5, kidney with purified ligandin in well 3 (1/~g in 10/d). The troughs contain rabbit antiserum to rat liver ligandin. Electrophoresis was performed in 0.07 M veronal buffer, pH 8.7, at 12 mA for 2 h. The precipitates were stained with Amido Black. a n t i s e r u m b y i m m u n o d i f f u s i o n , i m m u n o r e a c t i v i t y was f o u n d to be exclusively c o n fined to t h e Y f r a c t i o n i n each case. O n i m m u n o e l e c t r o p h o r e s i s , c y t o s o l f r a c t i o n s o f liver, k i d n e y a n d s m a l l i n t e s t i n a l m u c o s a a n d p u r i f i e d l i g a n d i n gave rise to single

171 precipitin arcs with ligandin antiserum (Fig. 4C). Cytosol from testis gave rise to two distinct, closely migrating arcs (Fig. 4C). C o n t i n u a t i o n o f electrophoresis for a m i n i m u m period o f 2 h was f o u n d to be necessary in order to reveal this feature.

Comparison of Y fractions on discontinuous polyacrylamide gel electrophoresis in SDS Aliquots f r o m the p o o l e d and concentrated Y fraction f r o m cytosol o f liver,

Fig. 5. Discontinuous slab polyacrylamide gel electrophoresis in SDS of (A) purified ligandin, and the Y fractions of (B) liver (C) testis (D) small intestinal mucosa and (E) kidney (12.5 ~ resolving gel). A 10-/d aliquot of the pooled and concentrated Y fraction from Sephadex G-100 chromatography of each tissue was analysed by discontinuous slab gel electrophoresis in SDS (see text for details). Ya-Yc represent 3 major subunit species in the Y fraction. The gel was stained with Coomassie Blue.

172 kidney, small intestinal mucosa and testis were analysed comparatively on discontinuous slab gel electrophoresis in SDS. The results are shown in Fig. 5. The presence of 3 major subunit bands (Ya, Yb and Yc) were found with identical migration in the Y fraction of liver, kidney and small intestinal mucosa. The Y fraction of testis cytosol revealed major bands corresponding to subunit Yb and subunit Y¢, with a minor component migrating just ahead of subunit Yb- Significantly, subunit Y, was not seen. Thus Y,, if present in the testis must be present in quantities too small to be detected by this method.

Effect of phenobarbital admin&tration Immunoreactive ligandin concentration was significantly greater in phenobarbital-treated animals (mean 4- S.E. = 83.56 4- 1.9/~g/mg supernatant protein, n ~ 4) than controls (mean 4- S.E. = 44.07 4- 3.39, n -----4, P < 0.01). When whole hepatic cytosol from rats treated with phenobarbital was compared with that from saline treated controls on discontinuous gel electrophoresis in SDS, a marked effect was noted on the subunit Y, and subunit Yb bands. A typical example of this is illustrated in Fig. 6. A semi-quantitative estimate of the increase in Y polypeptides in the supernatant from phenobarbital treated rat liver relative to control supernatant derived from densitometry was: subunit Y~, 130~; subunit Yb, 240~o; and subunit Y~, 5%. DISCUSSION q-he presence of two non-identical subunits in purified rat and human ligandin on discontinuous gel electrophoresis in SDS, has been reported by Listowsky et al. [14]. Ligandin had previously been thought to consist of two identical 23 000-dalton subunits inferred from the finding of a single protein band on continuous SDS gel electrophoresis [2, 3, 8, 12, 14]. We have confirmed these findings, and have presented further data characterizing the nature of the ligandin subunits. A terminology has been introduced for these subunits, based on our finding that 3 major subunits are present in the Y sulphobromophthalein binding fraction of liver supernatant. We have termed these subunits Ya, Yb and Yc in ascending order of size on discontinuous polyacrylamide gel electrophoresis in SDS. If these subunits are tentatively assumed to represent the monomers of three dimeric proteins in the 40 1300-50 000-dalton class of liver cytosol proteins, then from their behaviour during the purification of ligandin as monitored by gel electrophoresis in SDS, a different order in terms of charge may be written for the dimers from most to least basic: Y~Ya > YcY~ ~ YbYb. In accordance with Listowsky et al. [14] we have found two subunit species in purified ligandin of molecular weight 22 000 and 24 000 and have further shown these to correspond to the Ya and Y¢ subunits of the unpurified Y fraction of hepatic cytosol. Analytical isoelectric focusing of ligandin reveals two protein bands in equal proportions, similar to findings previously reported [12, 14]. If it is assumed that ligandin comprises the two dimeric proteins YaY, and YcY¢, then the two charge species thus defined by isoelectric focusing might represent these two proteins separately, although charge heterogeneity in either one or both components might result in a similar pattern. Two components have been found to characterize ligandin during purification of this protein on CM-cellulose from the liver of the rat [23] and the

173

B

l Fig. 6. Discontinuous slab polyacrylamide gel electrophoresis in SDS of 50-pg of hepatic cytosol from (A) saline- and (B) phenobarbital-treated rats (12.5 ~ resolving gel). After the gel was stained with Coomassie Blue, strips containing the protein bands were cut out and densitometry performed with a Joyce Loebl Chromoscan. Ya-Yc indicate the peaks of the corresponding subunit bands in the gel (see text for details). mouse (basic proteins II and III) [24, 25] but these have not been distinguished as yet, in terms of size heterogeneity. Ligandin has been shown by immunodiffusion methods to be present in cytosol of liver kidney and small intestinal mucosa [3, 26] and small quantities have also been found in cytosol of testis and ovary [26]. We have confirmed these findings and have in addition shown immunoreactivity to be present in the adrenal gland with antiserum to ligandin. A definite antigenic structural similarity between the Ya and

174 Yc subunits of ligandin could be inferred from the fact that antiserum raised against ligandin reacts in a monospecific fashion against purified ligandin and supernatants from liver, kidney and small intestinal mucosa on immunodiffusion and immunoelectrophoresis. Immunoreactivity for ligandin is confined to the Y fractions from these tissues, which all demonstrate the presence of subunit Y, and subunit Yc of ligandin, as well as the Yb subunit. The reason for the heterogeneity evident in the antiserum in reactions against testis, ovary and adrenal is not clear, but the finding that of the two ligandin subunits only subunit Yc is unequivocally present in the testis, may be significant in this regard. Further purification and characterization of the Y fraction components of the testis is required to answer this question more fully. Three combinations of the non-identical subunits present in purified ligandin are theoretically possible: YaYa, YaYe and Y~Y~. Two main points from our data support the hypothesis that subunit Y, and subunit Yc represent in vivo the monomers of two separate dimeric proteins, YaYa and Y¢Yc, without formation of a YaY¢ combination. Firstly, the finding of subunit Y~ existing without Ya in the testis, and secondly the clearly unequal response of these two components in the liver to phenobarbital administration. These data suggest that a YaY¢ combination is unlikely to occur in vivo. Phenobarbital has been shown to induce ligandin in the liver [3, 10] and this may partially account for the increased uptake of organic anions by the liver in animals treated with this drug [10]. It is therefore interesting to note that subunit Ya appears to be increased by phenobarbital treatment, whereas an effect on subunit Y¢ is not detectable. This would suggest that the YaYa protein species represents the true functional ligandin component. Strange et al have reported the presence of binding proteins with a low and a high affinity for cholic acid in the hepatic Y fraction [27]. Phenobarbital treatment increased the amount of the low but not of the high affinity binder. These binding proteins may well relate to the subunit Ya and subunit Yc (and possibly subunit Yb) protein species defined in this study. The Yb subunit species appears to represent the monomer of a major dimeric protein YbY~ in the Y fractions of hepatic and other tissue cytosols and is also increased in liver cytosol by phenobarbital treatment. Thus, although subunit Y~ is not a component of purified ligandin, it shows interesting similarities to the Ya and Y¢ subunit components of ligandin, and further study is required to establish its function and possible relationship to ligandin. A close relationship appears to exist between the subunit Y~ and subunit Yc components of ligandin in terms of size, charge and antigenic properties. As ligandin has been shown to be a major organic anion-binding protein [1-4], a target protein for carcinogens [6] and to be identical with glutathione-S-transferase B [12], the question is raised as to the roles of the subunit Ya and subunit Y¢ components of ligandin in these various functions. The interesting possibility of a precursor-product relationship between these two protein species also merits further study. ACKNOWLEDGMENTS This work was supported by the South African Medical Research Council, University of Cape Town and the Cancer Research Trust.

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