Studies on heterogeneity in ferritin subunits

Biochimica et Biophysica A cta, 743 (1983) 98-105

98

Elsevier Biomedical Press BBA31487

STUDIES O N HETEROGENEITY IN FERRITIN S U B U N I T S NAOKI WATANABE * and JIM DRYSDALE **

Department of Biochemistry and Pharmacology, Tufts University School of Medicine, 136 Harrison Avenue, Boston. MA 02111 (U.S.A.) (Received August 9th, 1982) (Revised manuscript received November 12th, 1982)

Key words: Ferritin; Subunit heterogeneity

Subunits prepared by dissociating rat and human ferritins by acid/urea or S D S can be resolved by isoelectric focusing in urea/Triton gels into many discrete forms. Most of these are not true isosubunits but aggregation artefacts formed during electrofocusing. The distribution of H and L subunit classes in these aggregates indicates that HeLa and heart ferritins contain similar classes of H and L subunits but that one or both of these classes is different in liver and kidney ferritins. To avoid aggregation artefacts, we examined subunits synthesised in vitro from exogenous mRNA. Our results indicate that HeLa and rat liver cells synthesise only one class of L subunit but two classes of H subunit.

Introduction Most mammalian cells contain multiple forms of the iron-storage protein, ferritin. These isoferritins have characteristic tissue distributions and may have different functins in iron metabolism [1-4]. A total of about 20 isoferritins have been found in several species, including human, rat, mouse and horse. Apoferritin is a roughly spherical molecule consisting of 24 subunits. Present evidence indicates that most shells are heteropolymers fashioned from two subunit classes whose proportions vary progressively through the isoferritin spectrum [1,2,4]. The subunit classes, designated H and L, have molecular weights of about 21 000 and 19000, respectively. Although sharing extensive sequence homologies, they probably arise from distinct mRNAs rather than from post-translational modification [1,5].

* Present address: 4th Department of Internal Medicine, Sapporo Medical College, S-l, W-16, Sapporo, Japan. ** To whom correspondence should be addressed.

While the two subunit model is consistent with the immunological and physico-chemical properties of most cellular ferritins [6], other factors, such as heterogeneity within H and L subunit classes, may also contribute to ferritin phenotypes. For example, multiple species can be resolved from the subunits of both human and horse ferritins by isoelectric focusing in the presence of 8 M urea [1,7]. Further evidence for heterogeneity in H and L classes comes from analyses of rat heart and liver isoferritins, some of which have the same apparent p I but different proportions of H and L subunit classes [4]. It is not yet known whether these presumptive 'isosubunits' result from different mRNAs, post-translational modifications or experimental artifacts. This paper re-examines the question of subunit heterogeneity in rat and human ferritins. It provides further characterization of the multiple species obtained by electrofocusing ferritin subunits in urea gels. These analyses indicate that many of the multiple forms previously seen by electrofocusing subunits from dissociated shells are aggregation artifacts. To avoid such aggregation,

99 we analyzed free ferritin subunits synthesized from exogenous mRNA in wheat-germ lysates. These analyses indicate that there is only one form of the L subunit but two distinct forms of the H subunit in rat liver and HeLa cells. This heterogeneity in the H subunit class may account for compositional differences in tissue isoferritins. Materials and Methods

Reagents. Insofar as was possible, all reagents were analytical grade. Reagents for electrophoresis and isoelectric focusing in polyacrylamide gel were purchased from BioRad, CA. Highly purified SDS came from BDH, Poole, U.K.; urea from Schwarz-Mann and Triton X-100 from Sigma. The urea was prepared immediately before use and was treated with mixed-bed resin A G 501-X8 D (BioRad, CA) to remove isocyanate and other ionic decomposition products. Apparatus. Apparatus for gel electrophoresis and electrofocusing were obtained from MRA Corporation, FL. Ferritins. Human ferritins were obtained from fresh post-mortem samples of liver, kidney and heart or from HeLa cells grown in Eagle's minimum essential medium supplemented with 10 #g i r o n / m l as ferric ammonium citrate [5,8]. Rat liver ferritins were obtained from male Sprague-Dawley rats, 150-200 g, which had received an intraperitoneal injection of 400 #g iron as ferric ammonium citrate 18 h prior to killing to increase ferritin levels [9]. Tissue ferritins were prepared by standard procedures involving heat extraction, ammonium sulphate precipitation, gel filtration, ultracentrifugation and preparative gel electrophoresis [1 ]. The purity of the ferritins was confirmed by analytical gel electrophoresis and electrofocusing, showing coincidence of iron and protein staining and by subunit analyses in SDS gels [1].

Characterization of subunits in tissue ferritins by isoelectric focusing. Ferritins were dissociated into subunits by treatment with SDS or by acid-denaturation at pH 2.5. For the former, ferritins at a concentration of 1 m g / m l were boiled for 10 rain in a solution containing 2% SDS and 0.5% (v/v) mercaptoethanol in 0.0625 M Tris-HC1, pH 6.8. This treatment completely dissociates ferritin into the H and L subunit classes [1]. Subunits so disso-

ciated were applied to the anodic surface of a 5% polyacrylamide gel containing 8 M urea, 2% (w/v) ampholytes, pH 3-10, and 2% (v/v) Triton X-100. Ferritin subunits were also prepared by exposure to 9 M urea at pH 2.5 at 20°C for 30 min [7]. These subunits were applied at the cathodic end of the gel surface. The anolyte and catholyte were papar wicks impregnated with 1 M phosphoric acid and 1 M NaOH, respectively. In both cases, the gel was cooled on a glass plate at 4°C for electrofocusing. Before sample application, gels were prerun at 100 V for 1 h to remove isocyanate that may have formed from the urea. After sample application, gels were run for 6-18 h at 300 V. In this system, the pH gradient formed after 1.5 h and did not change appreciably over 18 h. The ferritin samples and protein markers, including undissociated ferritin ( M r 480 000) formed stable banding patterns after 3 h. After electrofocusing, a strip of the gel was used to measure the pH gradient at 20°C by eluting the ampholytes from 0.5-cm sections with 1 ml boiled water. Protein patterns were detected l~y staining with Coomassie brilliant blue R250 [1]. Estimates of p I were made by superimposing the pH gradient on the stained pattern. These values were not corrected for differences due to temperature or urea. Two-dimensional analyses. Unstained strips (0.5 cm wide) from the initial isoelectric fractionation were used for further study in a second dimensional analyses by isoelectric focusing or by SDS gel electrophoresis. Tracks used for a second isoelectric focusing were treated in one of two ways (1) immersed for 30 min at 20°C in a 5% ampholyte solution, pH 5-7, in 6 M~area. The pH of this mixture remained near pH 6 during incubation, (2) immersed for 30 min at 20°C in 9 M urea maintained at pH 2.5 with HCl.~Tracks treated in either way were applied to the anodal end of a fresh 5% gel containing 2% ampholyte, pH 3-10, 8 M urea and 2% v / v Triton X-100. Focusing conditions for the second dimensiqn were those used for the first dimension. In other cases, the various components resolved by the first fractionation by isoelectric focusing were analysed by SDS gel electrophores'is. In one type of experiment, the focused track was stained with Coomassie blue to visualize the separated components. Individual components were sec-

100 tioned out. The gel slices were washed with 0.0625 M Tris-HCl, p H 6.8/2% S D S / 5 % mercaptoethanol for 30 min, then minced and boiled in 50 ~l of this solution for 10 min. These extracts were then loaded in 10% glycerol into the slots of a 15% SDS gel to determine their relative content of H and L subunits. [1]. After electrophoresis for 1 h at 80 V followed by 16 h at 150 V, the gel was stained with a sensitive silver stain [10] and scanned in a Laser Scanning Densitometer (Bio Med Instr., Chicago).

Characterization of nascent ferritin subunits: preparation of ferritin mRNAs. Poly(A)mRNA enriched for ferritin mRNA was prepared from messenger ribonucleoprotein particles from rat liver and HeLa cells as previously described [5]. These preparations were translated in wheat germ lysates [ll], usually in a total reaction volume of 20 /~1 containing 5/tCi [3H]leucine. In some cases the nascent ferritin subunits were isolated immunologically using an excess of antibodies specific for rat and human ferritin and 3 #g rat or HeLa ferritin as carrier. Immune complexes were recovered by precipitation with protein A on insolubilized Staphylococcus aureus (IgSorb, New England Enzyme Center). After washing, the nascent ferritin chains were released from the complex by boiling in SDS and analyzed by electrofocusing in urea/Triton gels followed by fluorography as before [12,13]. In other cases, the labelled ferritin subunits were fractionated directly by a two-dimensional analysis combining electrofocusing and SDS gel electrophoresis. The translation products from the reaction mixture were precipitated with 5% trichloroacetic acid at 0°C for 1 h and pelleted by centrifugation at 12000 × g for 4 min. The pellets were washed twice with acetone to remove trichloroacetic acid and dissolved in l0 /~l of 8 M urea contining 2% (w/v) of a mixture of ampholytes, pH 3-10, 4-6 and 5-7. The solubilized proteins were fractionated in a 100-/xl gel cylinder by isoelectric focusing, then in a second dimension by SDS gel electrophoresis essentially as described by O'Farrell [14]. After staining, the radioactive peptides were displayed by fluorography after treatment with EN3HANCE ® (New England Nuclear). Approximately 0.5 • 1 0 6 cpm were applied to each gel and films were exposed for about 20 days at - 7 0 ° C .

Results When analyzed by SDS gel electrophoresis, most ferritin subunits resolve into two size classes of approximate M r 21 000 and 19000 depending on species [1]. However, when fractionated by surface charge, such as by electrofocusing, patterns considerably more complex are obtained. Because it is not clear whether these multiple forms are different subspecies of H and L classes [7] or aggregates formed during electrofocusing [1], we have re-examined this phenomenon. Fig. l shows patterns from acid-dissociated subunits of rat and human ferritins when analyzed by this procedure. Rat liver ferritin subunits (containing about 40% H, 60% L by SDS gel analysis) resolved into three major components of approximate p I 6.3, 6.2 and 6.0, together with other minor components. Only two major bands corresponding to the H and L classes were evident on SDS gel electrophoresis (not shown). Subunits from various human tissue ferritins also gave many more components on electrofocusing than the two size classes shown by SDS gel electrophoresis. About six distinct components were obtained from subunits of HeLa, heart, kidney and liver ferritins. These components had apparent p I values ranging from pH 5.9 to 5.0. In general, their distribution reflected the relative content of H and L subunit classes in the parent ferritins. The HeLa and heart ferritins both contained more than 70% H and both gave proportionately higher levels of the more acidic components than did the subunits from the L-rich

(9 --6.5 -6.0 pH -5.5 (~

-5.0 RL

HeLo

HH

HK

HL

Fig. 1. Gel electrofocusingin 8 M urea/l% Triton of 50 ~g of acid-dissociated subunits from rat liver (RL), HeLa cells and human heart (HH), kidney (HK) and liver (HL).

!01 kidney and liver ferritins (both greater than 70% L). In addition to these differences in distribution, there were also differences in the number of components obtained from the human ferritins. Of the six major components indicated in Fig. 1, four seemed to be common to all ferritins. H e L a and heart ferritin subunits both gave two components, 3 and 5, which were not apparent in the patterns from either the liver or kidney ferritin subunits. In order to eliminate the possibility that some of these components reflect aggregates from incomplete dissociation of the multimeric ferritins, we examined the patterns obtianed by dissociating ferritins by boiling in SDS rather than by acid dissociation. It is known that SDS is removed from SDS polypeptides during electrofocusing in the presence of urea and non-ionic detergents [15]. This approach is widely used to analyze proteins with low solubilities in aqueous media and complex protein mixtures [16]. Fig. 2 shows that, aside from minor differences in spacing due to differences in p H gradients, the patterns given by SDS dissociation were similar to those obtained by acid dissociation. Again there seemed to be qualitative as well as quantitative differences in banding patterns of subunits from H e L a and heart ferritins and those of kidney and liver ferritins. However, these and other analyses of SDS subunits typically gave considerable band distortion and trailing which complicated interpretation of banding patterns. In addition, the relative distribution of the bands varied considerably in different analysis. These effects are not usually evident with other proteins in this system [16].

These complex banding patterns led us to characterize the focused components from the SDS subunits. At first we examined their stability to refocusing under different conditions. Duplicate samples of SDS subunits from human kidney ferrin were subjected to gel electrofocusing in 8 M urea. One track was stained for protein as a control, another track was immersed in a solution of 8 M urea and 2% ( w / v ampholytes, p H 5-7, for 30 min. This track was then placed on top of a second slab gel containing 8 M containing 8 M urea, 2% ampholytes, p H 5-7, and the focused components reanalyzed by electrofocusing in the second dimension. The initial electrofocusing of the SDS subunits gave patterns similar to those previously obtained, with a major component near p H 5.9 and other more acidic minor components. On refocusing, a series of single spots on a diagonal were obtained (Fig. 3). This experiment shows that the components obtained by electrofocusing in the first dimension were stable and behaved as distinct entities on refocusing. However, when the focused components were exposed to urea at p H 2.5 before refocusing, a much more complicated

6.0

pH

5.0

-- 6.0

®

pH

--6.5 -

6.0

-

5 . 5 pH

- 5.0

(~

RL HeLo HH

HK

HL

Fig. 2. Gel electrofocusingin urea/Triton of 30 #g of SDS-dissociated subunits of ferritins in Fig. 1. RL, rat liver; HH, human heart; HK, human kidney.

--5.0

® Fig. 3. Two-dimensional analysis by gel electrofocusing of 50 Fg of SDS-disassociated subunits from human kidney ferritin. After focusing in urea/Triton the separated components in the horizontal plane were equilibrated at pH 6 in 8 M urea before refocusing in urea/Triton in the vertical plane. The horizontal pattern is a replicate track from the first dimension stained to show the distribution of components.

102

®

®® 6.0

pH

5.0

® 6.0

pH

5.0

6.0

pH

5.0

®

®

Fig. 4. Two-dimensional analysis by gel electrofocusing of 50 p,g of intact shells (left) and SDS-diassociated subunits (right) from human kidney ferritin. After focusing in urea/Triton in the horizontal plane, unstained tracks from both fractionations were treated at pH 2.5, 8 M urea before refocusing in urea/Triton in the vertical plane.

pattern was obtained (Fig. 4). More than 15 components were resolved on the diagonal. Running across the diagonal were two or three isoelectric series. Each series contained spots whose vertical alignment showed that initially they had the same isoelectric points. This result clearly shows that some of the components separated in the first dimension redistribute on focusing if first exposed to pH 2.5. This phenomenon could be due to a change in p I due to change in conformation, to chemical modification on exposure to acid/urea, or to aggregates. Since the additional components were on both sides of the diagonal, dissociation of aggregates seems the likely explanation for components which redistributed. Not all components redistribute on acid treatment. Possibly, conditions were insufficient to cause complete disaggregation. Alternatively, some of the stable components remaining on the diagonal represent charge isomers of H and L subunits. The left hand panel of Fig. 4 shows the pattern given by the parent ferritin sample when applied as intact shells in the first dimension before being dissociated by acid prior to the second dimension analysis. Most of the ferritin banded in about 4-5 components between pH 5.0 and 5.6, presumably as shells since they all contained iron (not shown) and had the same p I range as the native isoferritins. There were also minor components that correspond in p I to components from the focused subunits. These minor components probably arose

by partial dissociation of ferritin shells since they are not seen when ferritin is focused in the absence of urea Ill. However, when exposed to pH 2.5 before refocusing, each isoferritin gave rise to multiple components which banded in three or four isoelectric series. The p I range and relative amounts of the components derived from each isoferritin are similar to those obtained by acid treatment of kidney ferritin in solution (see Fig. l). They may, therefore, have similar compositions to the components in Fig. 1 and arise by similar mechanisms. The results confirm previous demonstrations by others of the remarkable stability of ferritin shells to urea at neutral pH and their ready dissociation into subunits at pH 2.5 [7,17]. The above experiments suggest that ferritin subunits, whether dissociated by acid/urea or by SDS, aggregate during electrofocusing even in the presence of 8 M urea and 1% Triton. In order to characterize these aggregates further we examined their relative content of H and L subunits classes by SDS gel electrophoresis. We found (Fig. 5) that many of the bands shown in Fig. 2 contained both H and L subunits classes, but in different proportions. The only apparent exceptions were bands 5

.~

H L---"

HeLa

L---"

HH

H ...,.,

HK

L~" I

2

3

4

5

6

Fig. 5. H and L subunit classes in components obtained by gel electrofocusing of SDS subunits from HeLa, human heart (HH) and human kidney (HK) ferritins. Stained bands from Fig. 2 were eluted with SDS and analysed for H and L by SDS gel electrophoresis.

103 and 6 from HeLa and heart ferritin subunits which appeared to contain only H subunit classes. In all series there was generally a progressive increase in H / L ratio with decreasing pI. For example, with the exception of band 2 in the HeLa spectrum, the H content increased from about 60% in band 1 to about 100% in bands 5 and 6. A similar pattern was seen in the focused components from heart ferritin, so that bands of similar pI in heart and HeLa ferritins had similar contents of H and L subunit classes. A somewhat analogous correlation of banding patterns with H and L content was seen in the focused components from liver and kidney ferritin subunits. However, the relative content of H and L subunits from bands of similar pI from liver and kidney ferritin subunits were substantially different from the apparently corresponding bands of similar p I from heart and HeLa ferritins. In nearly all bands from liver and kidney ferritin subunits the content of L class was more than twice that found in bands of similar p l from heart and HeLa ferritin subunits. The above analysis led to three conclusions: (1) that many of the components seen in these and other [1,7] analyses of ferritin subunits by electrofocusing in urea represent aggregates of the H and L subunit classes; (2) that the H and L subunit classes in HeLa are similar to those in heart tissue; (3) that one or both of these subunit classes in HeLa and heart ferritins differ in surface charge from those in liver and kidney cells. Further evidence for this last point comes from Fig. 5. Although it shows that most of the focused components from kidney, heart and HeLa ferritin subunits contain both H and L classes, some such as components 5 or 6 from HeLa seemed to contain only H subunit classes. Since the p I values of the presumed aggregates are higher than those of the native isoferritins (see Fig. 4), it is possible that components 5 and 6 represent different aggregate states of the H subunit. However, both components have similar mobilities when analyzed by electrophoresis under non-dissociated conditions (e.g., pH 7, 8 M urea) in 5% polyacrylamide gels (unpublished data). This suggests that they have similar dimensions but different surface changes. This indication of two forms of the H subunit led us to examine the p I distribution of nascent subunits synthesized directly from ferritin mRNAs.

In one experiment, rat liver m R N A and HeLa cell m R N A enriched for ferritin mRNA were translated separately in wheat-germ lysate. We have found that the labelled subunits do not aggregate into shells in this system but chromatograph on gel filtration in the positions expected for free subunits (Watanabe, N., North, T.G. and Drysdale, J., unpublished data). After translation, the labelled ferritin subunits were recovered by immunoprecipitation together with 3 #g rat liver or HeLa ferritin as carrier. Ferritin subunits were released from the immune complexes by boiling in SDS and subjected to electrofocusing in urea/Triton gels. After electrofocusing, the gels were stained then subjected to fluorography. Because of the presence of nonferritin proteins, e.g., IgG and material solubilized from S. aureus, the position of shell subunits could not be determined from the stained pattern. However, it is apparent from the fluorogram (Fig. 6) that the distribution of radioactivity from the nascent subunits was similar to the focusing pattern given by subunits from ferritin shells. In the case of the nascent rat ferritin subunits, three major radioactive bands were obtained. These corresponded in pI, but not in relative proportions, to bands 1, 2 and 3 from the shell subunits (Fig. 3). Analyses of these three components showed that all contained both H and L subunit classes in the range 20% H, 70% L-60% H, 40% L (not shown). The nascent HeLa subunits also gave a complex pattern. In this case, the newly synthesized subunits corresponded in p I to components 2, 3 and 4 in Fig. 2, which all contained both H and L subunit classes (Fig. 5). These results indicate that the subunits translated from mRNA in wheat-germ lysates have similar properties and are probably interchangeable with subunits in ferritin shells. The differences in distribution given by fluorography and staining may reflect differences in radioactivity in the H and L subunit classes in the immune complexes (the H subunit has 4 methionine residues, the L subunit 3 [1,7]) together with differences in the amount of ferritin subunits available for heteropolymer formation in each track. The stained patterns (Figs. 1 or 2) were derived from approximately 30 #g ferritin subunit protein per track. By contrast, the track with labelled ferritin subunits contained only 3 #g ferritin protein.

104

Rat pH

Rat+ Hela Hela

®

6.5 6.25--

5.45--

® Fig. 6. Fluorogram of gel electrofocusing pattern from HeLa and rat liver ferritin subunits synthesized from exogenous mRNA in wheat-germ lysates. After separate translation in the presence of [3H]leucine, the nascent ferritin subunits were isolated immunologicallyby co-precipitation with 3 #g cartier ferritin. The radioactive subunits were exracted with SDS and fractionated by gel electrofocusingin urea/Triton on the same gels as in Fig. 2.

In order to avoid such aggregate formation, we characterized the p I distribution of nascent subunits separated from a complex mixture of other proteins without immunoprecipitation or the addition of carrier ferritin. Fig. 7 shows two-dimensional analyses of nascent ferritin subunits separated directly from the translation products. Because both m R N A preparations are enriched for ferritin m R N A s [12], ferritin subunits can be visualized as discrete spots in the fluorogram. Their identity as ferritin has been established by demonstrating their specific removal by antiferritin antibodies prior to gel electrofocusing and by correlating their levels with those of functional ferritin m R N A (our unpublished data). In these experiments the likelihood of subunit aggregation is greatly reduced because of the very low levels of nascent subunits, and the high levels of other unlabelled proteins in the wheat-germ lysate. This analysis shows only three components, two H and

Fig. 7. Fluorogram of two-dimensional analyses of rat (top) and HeLa ferritin subunits (tight) synthesized from rat liver and HeLa cell ferritin mRNAs in wheat-germlysates.

one L, from both rat liver and H e L a cells. The two spots corresponding to the H subunits appeared to be discrete species, with no evidence of aggregate formation with L subunits. Discussion The preceding shows that during electrofocusing in urea gels ferritin subunits form aggregates whose pl, like the native isoferritins, varies with their relative content of H and L subunit classes. These aggregates are stable entities which do not redistribute on refocusing in urea gels, unless exposed to conditions which also dissociate the multimeric ferritin shells. This aggregation on electrofocusing shows both a pH- and concentration-dependency. It presumably occurs at the higher p H ranges encountered during electrofocusing since ferritin subunits behave as monomers during electrophoresis below p H 3 [18]. This conclusion seems at odds with circular dichroism studies which show

105

that acid-disassociated subunits do not renature in 8 M urea at neutral pH values [19]. However, there are large differences in protein concentration in the two systems. In the early stages of electrofocusing molecules can concentrate into very narrow zones of more than 100 mg protein/ml [20], which is very much higher than the levels in the circular dichroism studies. In considering the mechanism of subunit aggregation, it is interesting that heteropolymer formation seems to be independent of the prior history of the subunits. Molecules migrate to their isoelectric points as cations when dissociated with acid but as anions when dissociated by SDS. Presumably, subunit aggregation occurs before the H and L classes are resolved by surface charge since aggregates, once formed, seem to be stable in urea solutions near neutral pH. This propensity for aggregation in urea solutions is also seen with smaller fragments such as cyanogen bromide peptides [21] and probably accounts for the extensive heterogeneity seen in focusing such fragments [221. The molecular structure of the aggregates formed during electrofocusing is not known. However, the wide range in disribution of H and L subunit classes in the various aggregates suggests that they have large particle sizes. For example, a content of 5% H and 95% L requires that the complex contain at least 20 subunits, which is of the order of the complexity in the original ferritin shell. An alternative explanation that each band contains different unresolved classes of H and L subunits seems unlikely, but cannot be excluded from the present studies. Despite the pitfalls in electrofocusing ferritin subunits, our results do support the notion of some heterogeneity in the H and L subunit classes. Our findings that artifactual heteropolymers from Hela or heart ferritin subunits have higher H content than those of similar p I from liver or kidney is consistent with findings of Bomford et al. [4] from intact rat heart and liver ferritins. Secondly, our analyses of ferritin subunits synthesized in vitro provides direct evidence for two classes of H subunits in both rat liver and HeLa cells. Differences in expression of two H subunit classes may possibly account for such compositional differences in rat heart and liver ferritins.

Acknowledgements This work was supported by N I H Grant AM 17775-09. The authors thank Ms. Maureen Shields for expert technical assistance and Ms. Becky Haines for typing this manuscript.

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