Proc. Natl. Acad. Sci. USAVol. 82, pp. 5332-5336, August 1985Biochemistry

The 90-kDa component of reticulocyte heme-regulated eIF-2a(initiation factor 2 a-subunit) kinase is derived from the(8 subunit of spectrin

(protein synthesis/translational control/phosphorylation/membrane skeleton/heme-controlied repressor)

WIESLAW KUDLICKI, SUSAN FULLILOVE, GISELA KRAMER, AND BOYD HARDESTY*Clayton Biochemical Institute, Department of Chemistry, The University of Texas at Austin, Austin, TX 78712

Communicated by Esmond E. Snell, May 6, 1985

ABSTRACT Antibodies from three different lines ofmonoclonal hybridomas crossreact with both the .3 subunit ofspectrin and the 90-kDa peptide present in highly purifiedpreparations of the heme-controlled eIF-2a (initiation factor 2a-subunit) kinase from rabbit reticulocytes. Antibodies fromtwo of the three lines enhance the enzymatic activity of thekinase preparation for phosphorylation of the a subunit ofeukaryotic translational initiation factor 2 (eIF-2) and forphosphorylation of the 100-kDa peptide thought to be a peptideof the kinase that is phosphorylated during its activation. Also,it is shown that both the .8 subunit of spectrin and the 90-kDapeptide can be phosphorylated by two protein kinases fromreticulocytes, the catalytic subunit of cAMP-dependent proteinkinase and a cAMP-independent protein kinase similar tocasein kinase II. Furthermore, a phosphorylated 90-kDa pep-tide can be derived from phosphorylated f3 subunit of spectrinby tryptic proteolysis. We conclude that the 90-kDa peptide isderived by proteolysis from the 13 subunit of spectrin, probablyfrom its carboxyl terminus, and suggest that the heme-sensitiveeIF-2a kinase, like the 56-kDa phosphatase [Woliny, E.,Watkins, K., Kramer, G. & Hardesty, B. (1984)J. Biol. Chem.259, 2484-2492], is associated with an element of the membraneskeleton in intact reticulocytes.

Protein synthesis in mammalian reticulocytes is controlled byheme through a system that has been called the heme-controlled repressor, HCR (1). Under conditions of hemedeficiency, eukaryotic translational initiation factor 2 (eIF-2)is phosphorylated in its smallest, or a, subunit (eIF-2a) andthereby inactivated with a concomitant inhibition of proteinsynthesis. The cAMP-independent protein kinase of the HCRsystem remains an enigma with respect to its isolation, itsphysical characteristics, and the mechanism by which it isactivated (for review, see ref. 2). Gross and Rabinovitz (3)ascribe a molecular size 300-400 kDa to the HCR. However,a molecular mass of 140 kDa was assigned to the purifiedkinase in one report (4). Wallis et al. (5) observed large formsofthe enzyme and Kramer and Hardesty (6) have emphasizedits chromatographic heterogeneity during its purificationfrom the postribosomal supernatant fraction of rabbitreticulocytes. Heme-sensitive eIF-2a kinase activity hasbeen associated with a 100-kDa phosphopeptide observed byautoradiography after NaDodSO4/PAGE of the activatedenzyme. The peptide appears to be phosphorylated duringthe activation process (7-9) and may be an integral compo-nent of the holoenzyme. Autophosphorylation has beensuggested (9), but the role of phosphorylation in the activa-tion process and the mechanism by which heme suppresseskinase activity are unclear. Wallis et al. (10) observed asecond peptide, of 90 kDa, in highly purified preparations of

the active kinase and suggested that it also is associated withthe enzyme.

Protein phosphatases in crude extracts from rabbitreticulocytes exhibit rather similar heterogeneity with re-spect to size and chromatographic properties. Fullilove et al.,(11) identified a 230-kDa protein and smaller peptides derivedfrom it that appear to be involved in the regulation ofa 56-kDareticulocyte phosphatase. The protein, named regulin, seemsto be associated with spectrin in the reticulocyte membraneskeleton. Spectrin, the most abundant protein of theerythroid membrane skeleton, is a flexible rod-shaped mol-ecule about 100 nm long (12) that is composed of oligomersof a (240-kDa) and , (220-kDa) subunits to which otherproteins are known to bind (reviewed in ref. 13). Spectrin canbe phosphorylated in vitro (14) or in intact cells (15) by oneor more protein kinases. Phosphorylation occurs at four sites,all of which are located within a 20-kDa fragment that can begenerated from the carboxyl-terminal end of the ,B subunit byreaction with cyanogen bromide (15). Both spectrin andregulin appear to be exquisitely sensitive to endogenousproteases in reticulocytes. We have used monoclonal anti-bodies to identify smaller peptides in the postribosomalsupernatant fraction of reticulocytes that are immunological-ly crossreactive with spectrin or regulin.Here we present evidence that the 90-kDa peptide reported

previously to be associated with the heme-regulated eIF-2akinase is derived from the (3 subunit of spectrin, apparentlyby proteolysis. We show that the activity of the kinase isstimulated by several but not all types of monoclonal anti-bodies that also recognize the P3 subunit of spectrin. Theresults favor the hypothesis that the heme-sensitive eIF-2akinase, like the 56-kDa protein phosphatase, is associatedwith the membrane skeleton in reticulocytes but can bereleased into the soluble fraction by one or more endogenousproteases.

EXPERIMENTAL PROCEDURESMaterials. Polylysine (Mr 90,000) was purchased from

Sigma and was linked to CNBr-activated Sepharose (Sigma)according to instructions provided by Pharmacia (16). HRP-substrate for peroxidase used in the ELISA was fromBio-Rad. [y-32P]ATP was from New England Nuclear. Allother materials and chemicals were ofreagent grade and fromsources given previously (17).

Isolation of the eIF-2a Kinase. Fractionation of the rabbitreticulocyte postribosomal supernatant to give the PC100fraction has been described (17). The details of furtherpurification of the eIF-2a kinase will be reported elsewhere.Briefly, the PC100 fraction was chromatographed on Sepha-

Abbreviations: HCR, heme-controlled repressor; eIF-2, eukaryotictranslational initiation factor 2; eIF-2a, a subunit of eIF-2.*To whom reprint requests should be addressed.

5332

The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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Proc. Natl. Acad. Sci. USA 82 (1985) 5333

dex G-150 and then on a polylysine-Sepharose column.Subsequently, the eIF-2a kinase fraction was subjected topreparative gel electrophoresis under nondenaturing condi-tions, using a BRL apparatus (Bethesda Research Labora-tories). Enzyme activity was followed by inhibition ofproteinsynthesis in the standard lysate assay system derived fromrabbit reticulocytes (18). One unit of eIF-2a kinase is definedas the amount ofprotein that causes 50o inhibition of proteinsynthesis in this 50-,ul assay system containing 10 1.d of lysate.

Preparation of Spectrin. Spectrin was extracted from thereticulocyte membrane fraction by the procedure of Litmanet al. (19) that was developed for the isolation of spectrin fromerythrocyte ghosts. Spectrin was further purified and sepa-rated from regulin by chromatography on DEAE-cellulose in20 mM Tris HCl, pH 7.5/20 mM KCl with batch-elution by400 mM KCl at pH 8.3 after intermediate washes of thecolumn matrix with 100 and 200 mM KCl. The details of theisolation procedure will be described elsewhere.

Production and Purification of Monoclonal AntibodiesAgainst Spectrin. The strategy described previously for theproduction of monoclonal anti-regulin antibodies was fol-lowed (11). The antibodies were assayed in an ELISA withperoxidase linked to the second antibody as reported (11),except that HRP-substrate (Bio-Rad) was used. Ref. 11 alsogives the details of the purification of the monoclonal anti-bodies of the IgG2a or IgG2b subclass. Classification ofmonoclonal antibodies was carried out by use of a procedureand materials from Zymed Laboratories (Burlingame, CA).Antibodies of the IgM class were partially purified bychromatography on Sephacryl S-300 in 20 mM Tris HCl, pH7.5/100mM KCl (solution A). In some cases these antibodieswere further purified by affinity chromatography on aspectrin-Sepharose column. Antibodies adsorbed in solutionA were eluted by the addition of 3 M KSCN, then concen-trated in an Amicon concentrator equipped with a YM10membrane, and dialyzed against solution A. Antibodies werestored at -80°C in small aliquots.

Phosphorylation Assay. Protein kinase activity with eIF-2as substrate was determined as described (10). Routinely, anautoradiogram was prepared from the dried gel afterNaDodSO4/PAGE. Then the stained band corresponding toeIF-2a was cut from the gel and its radioactivity wasdetermined. Preparation of eIF-2 was described by Odom etal. (20). When the effect of antibodies on the phosphorylationof eIF-2 was measured, kinase in amounts given in the figurelegends was preincubated with the antibodies for 45 min onice before the phosphorylation assay was carried out.

Protein Determination. Protein concentrations were deter-mined routinely by absorbance at 280 and 260 nm (21).HPLC. This was carried out as detailed previously (17),

except that a SynChropak GPC 300 column (Synchrom,Linden, IN) was used.

RESULTSFor the experiments described below, eIF-2a kinase activitywas purified from the reticulocyte postribosomal supernatantas outlined under Experimental Procedures. The preparationobtained after polylysine-Sepharose chromatography wassubjected to size-exclusion chromatography. The materialloaded on the column had a specific activity of 11,100units/mg of protein as determined in the reticulocyte lysatesystem. The elution profile (Fig. 1) indicates the distributionof protein measured by A20. Inhibitory activity determinedin the lysate system was associated with the first proteinpeak, which eluted near the void volume at a position nearthat of ferritin (480 kDa). The peptide composition of afraction from peak 1 as well as phosphopeptides formed inthis fraction are shown in Fig. 1 Inset. A 100-kDa peptide and

E*10.¢01 A\**3 I||W-lOOkDa-4*o~~~10 43~~~9OkDa-

Cl

X~~~~ b

M.0 .0

< ~~~~~~~~~~~ab

4 8 12 16Fraction

FIG. 1. Molecular size determination of the eIF-2a' kinase prep-aration. The eIF-2a kinase fraction after polylysine-Sepharose chro-matography was subjected to chromatography on a SynChropakGPC 300 column used in conjunction with a Beckman HPLC system.The column was equilibrated in 20 mM Tris HCl, pH 7.5/100 mMKCI/0.5 mM dithioerythritol. About 300 ,&g of protein was applied.The column was developed at a flow rate of 0.5 ml/min; the eluatewas monitored at 280 nm and scanned at a chart speed of0.5 cm/min.This scan is shown by the continuous line. Fractions of 0.3 ml werecollected. The void volume (V0) is indicated, as are the positions ofmolecular size markers (o; 1, ferritin; 2, /3-amylase; 3, bovine serumalbumin; 4, ovalbumin; 5, cytochrome c) used to generate thecalibration curve shown. A 3-pl aliquot of each fraction was addedto a reticulocyte lysate system and incorporation of [14C]leucine (40Ci/mol; 1 Ci = 37 GBq) was determined (o). (Inset) Aliquots offraction 6 were incubated with [(-32P]ATP in the absence or presenceof eIF-2, then analyzed by NaDodSO4/PAGE plus autoradiography.Tracks: a, 10 Al of fraction 6, stained with Coomassie blue afterelectrophoresis; b, autoradiogram of track a; track c, autoradiogramafter electrophoresis of 3 Al of fraction 6 incubated with [y-32P]ATPand about 2.6 gg of eIF-2. 8, f3 subunit of spectrin.

the a subunit ofexogenous eIF-2 were efficiently phosphoryl-ated when this fraction was incubated with ATP.The eIF-2a kinase fraction was purified further by prepar-

ative gel electrophoresis under nondenaturing conditions.The resulting enzyme preparation exhibited a specific activ-ity of about 100,000 units/mg of protein. Analysis of thishighly purified preparation after NaDodSO4/15% PAGE isgiven in Fig. 2A. Peptides visualized by staining in Coomassiebrilliant blue R are shown in track 1 and include a prominentspecies of apparent molecular mass 90 kDa. Tracks 2-4 showresults from electroblots and ELISAs of the same fraction.The ELISA (11) used monoclonal antibodies that recognizespectrin. The position of antigen was detected by reaction ofa chromogenic substrate with peroxidase linked to the secondantibody. Strong crossreaction of the 90-kDa peptide wasobserved with the three monoclonal antibodies used, each ofwhich recognizes both spectrin subunits as shown ih Fig. 2B.Similar crossreactivity between the two subunits by mnono-clonal antibodies has been reported (22). It should be em-phasized that the hybridorna cell lines used to produce themonoclonal antibodies were obtained from different cellfusions and were cloned twice. The antibodies recognize adifferent set of small spectrin peptides (Fig. 2B, tracks 2-4),indicating that they recognize different antigenic sites on thespectrin peptides.Data presented in Fig. 3 lead to the conclusion that the

90-kDa component of the eIF-2a kinase is derived from the,p subunit of spectrin. In Fig. 3B, phosphorylation of the90-kDa peptide by the catalytic subunit of cAMP-dependentprotein kinase and by the cAMP-independent casein kinase II

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5334 Biochemistry: Kudlicki et al.

A

90 kDa - !om~h~

_ -_m_

|-is,.

F

I1 i

FIG. 2. The 90-kDa peptide of the eIF-2a kinase is recognized byanti-spectrin monoclonal antibodies. The highly purified eIF-2akinase preparation after preparative gel electrophoresis (A) andspectrin (B) were subjected to NaDodSO4/PAGE, electrophoreticblot-transfer, and an ELISA (11). Tracks: 1, the separated peptidesstained with Coomassie blue; 2-4, peptides stained with BioradHRP-substrate after the ELISA. a and ,3, spectrin a and P3 subunits,respectively. Antibodies used in tracks 2 were from monoclonalhybridoma line C81, in tracks 3, line 4-3; and in tracks 4, line 1-5.

is shown. Both of the protein kinases were isolated fromreticulocytes as reported earlier (23, 24). Both kinases alsophosphorylate the ,3 but not the a subunit of spectrin (Fig.3A).A 90-kDa phosphopeptide also can be derived from the (3

subunit of spectrin by limited proteolysis with trypsin. Forthis experiment, spectrin subunit 3that previously had beenphosphorylated in the native molecule was isolated frompolyacrylamide gels by electrophoretic elution afterNaDodSO4/PAGE. The ,3-subunit peptide then was subject-ed to limited proteolysis as described in the legend to Fig. 3C.The results indicate that a phosphopeptide of 90 kDa is aprominent proteolytic-degradation product of the (3 subunitof spectrin. The derived phosphopeptide is recognized by thethree monoclonal antibody types that recognize the 90-kDapeptide of eIF-2a kinase preparation as shown in Fig. 2 (datanot given).The effect of anti-spectrin antibodies on the enzymatic

activity of the highly purified eIF-2a kinase was examined(Fig. 4). Preincubation of the enzyme with either oftwo of thethree monoclonal antibodies that recognize the 90-kDa pep-tide causes a concentration-dependent increase in eIF-2aphosphorylation. The effect is not produced by the othermonoclonal antibody (from line 1-5) or by regulin antibodies.The phosphorylation of the 100-kDa component of the kinaseincreased in parallel to the enhanced eIF-2a phosphorylation.Other experiments have shown that kinase activity can beseparated from the 90-kDa peptide, indicating that it is not thecatalytic subunit of the enzyme (10). We interpret these datato reflect an effect of the antibodies on a complex involvingthe kinase and the 90-kDa peptide that somehow causesenhanced enzymatic activity. Dissociation of the complex ora conformational change might be involved.The enzymatic activity for phosphorylation of the eIF-2a

and 100-kDa peptide of the highly purified kinase used for theexperiments of Figs. 3 and 4 is suppressed by heme at 10 ,uM(Fig. 5). Heme at this concentration prevents activation ofHCR in reticulocyte lysates. Phosphorylation of the P subunitof eIF-2 is not affected by heme under these conditions. The(3 subunit is phosphorylated by traces of the casein kinase IItype enzyme (see Fig. 3A) that were present in the eIF-2preparation used for this experiment. These data indicate that

A

40.=

.. 2F 3 4

B

90 kDa-_w

1 3

C

/3'-

*-90 kDa

1 2 3 4 5

FIG. 3. Relationship between the 90(-kDa peptide and the 13subunit of spectrin. (A and B) The eIF-2a kinase (2 ,ug of protein) (A)or spectrin (2 ,g of protein) (B) were incubated in parallel with[.y-32P]ATP alone (tracks 2), or plus 0.1 ,ug of the casein kinase II(tracks 3), or plus 0.2 ,ug of the catalytic subunit of the cAMP-dependent protein kinase (tracks 4) in a phosphorylation assay asoutlined in Experimental Procedures. Phosphorylated peptides wereanalyzed by NaDodSO4/PAGE and autoradiography. Autoradi-ograms are shown in tracks 2-4; tracks 1: Coomassie blue-stainedpeptides in the spectrin (A) and eIF-2a kinase (B) preparations,respectively. (C) Spectrin was incubated with [y-32P]ATP and thenelectrophoresed in a NaDodSO4/polyacrylamide gel to separate thea and ,B subunits. The (3-subunit band (unstained) was cut out fromthe gel and the protein was eluted electrophoretically. Subsequentlythis peptide was subjected to limited proteolysis with variousamounts of trypsin (0-400 ng) for about 3-4 ,g of 8 subunit. After3 min incubation at 35°C, a 10-fold excess of trypsin, inhibitor overtrypsin was added. Resulting spectrin (8-subunit peptides wereanalyzed by NaDodSO4/PAGE and autoradiography. Tracks 1-5: 0,50, 100, 200, and 400 ng of trypsin, respectively.

the effects we observed are related to the heme-controlledkinase of the HCR system and show that sensitivity to hemneis retained even in highly purified preparations in which the90-kDa peptide is present.

DISCUSSIONThe three lines of evidence presented above justify theconclusion that the 90-kDa peptide associated with theheme-regulated eIF-2a kinase is derived from the (3 subunitof spectrin, probably from its carboxyl-terminal end, sincethe smaller peptide appears to contain the spectrin (3-subunitphosphorylation sites, all of which are located in this region(15). The spectrin (3 subunit itself is a prominent component

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Proc. Natl. Acad. Sci. USA 82 (1985) 5335

8

6-xE

44- X 1

Antibody. zg of protein

FIG. 4. Monoclonal anti-spectrin antibodies stimulate the activ-ity of the eIF-2a kinase. The enzyme (0.05 ,ug protein) was incubatedon ice for 45 mmn with various amounts of antibodies from the C81 line(0) or from the 1-5 line (a). Then eIF-2 and [y-32P]ATP were addedand the phosphorylation assay was carried out as described inExperimental Procedures. The samples were analyzed by gel elec-trophoresis and autoradiography; 32p in eIF-2a then was determinedby cutting this band out from the gels and measuring its radioactivityby liquid scintillation counting. (Insets) The eIF-2a bands on theautoradiogram, corresponding to the data points (a, o-o); b, *-*).a, a subunit of eIF-2.

of the kinase preparation until a late stage of purification (seeFig. 1). Avian erythrocytes appear to contain a pool of freespectrin subunits (25), the counterpart of which may providea source of spectrin p3 subunit in the reticulocyte post-ribosomal supern~atant. In addition to the 90-kDa fragment,partially purified kinase preparations contain several lessprominent, immunologically crossreactive phosphopeptideswhich also appear to be derived from the /3 subunit of

x.:

, \ ~ ~ ~~~-

10 " 0 30aHemin.gM

FIG. 5. Heme inhibits the purified eIF-2a kinase. About 0.1ag ofeIF-2a kinase and 2.6 gpigof eIF-2 were incubated with [y32P]ATP inthe absence or presence of various concentrations of heme. Phos-phorylation of eIF-2a was analyzed as described in the legend to Fig.4. The graph shows the 32P incorporated into eIF-2a under thedifferent experimental conditions. (Inset) The correspondingautoradiogram. a and 83, the a and /3 subunits of eIF-2.

spectrin. Furthermore, there appears to be no simple stoi-chiometry between the hypothetical catalytic subunit of theeIF-2a kinase and spectrin 3-subunit peptides. These factors,together with the tendency of spectrin 8 subunit and itspeptides to aggregate, presumably account for the heteroge-neity of the kinase that have made it extremely difficult toisolate and characterize by the common techniques of clas-sical enzymology.Although the results presented here do not demonstrate

that the kinase is associated with the membrane skeleton inintact reticulocytes, this appears to be very likely. Associa-tion of both the kinase and phosphatase (11) with large,protease-sensitive peptides that undergo reversible aggrega-tion into the cytoskeleton immediately suggests mechanismsby which both of these enzymes might be physiologicallyregulated. For example, the data of Fig. 5 show that heme hasa concomitant effect in reducing phosphorylation of the100-kDa peptide and on the enzymatic activity of the kinasefor phosphorylation of eIF-2a, even in highly purified prep-arations that contain the 90-kDa peptide. We have noted withconsiderable interest a report (26) indicating that heme affectsthe aggregation state of spectrin and the conformation of themembrane skeleton at a concentration similar to that whichlimits the activity of the eIF-2a kinase. Although specialcaution is warranted in interpreting the effect of heme on suchsystems, it appears possible that a common mechanism mightbe involved.A number of recent reports on diverse topics describe

peptides of about 90 kDa that appear to have many similarcharacteristics in addition to a common size. These includea heat shock protein [hsp9O (27)], a peptide of the chickenoviduct progesterone receptor (28), a component of the srckinase (pp6src) complex (29), and a prominent peptideidentified as a heat shock protein that is phosphorylated in thepresence of double-stranded DNA in extracts from a numberof cell types (30). The latter report is particularly intriguing inthat it lists a family of phosphopeptides that appear to beremarkably similar in size to those derived from the 8 subunitof spectrin (see Fig. 3). It appears likely that at least some ofthese peptides are derived from the p subunit of spectrin ora closely related counterpart in nonerythroid cells (31).

We thank M. Hardesty, M. Rodgers, and S. Demkowicz for theirexcellent technical assistance; M. E. Powers for preparation of thetypescript; and F. Hoffman for photography and art work. Wegratefully acknowledge some recent discussions and exchange ofantibodies with Dr. David Toft (Department of Cell Biology, MayoClinic, Rochester, MN). This work was supported by NationalInstitutes of Health Grant CA16608 to B.H.

1. Gross, M. & Rabinovitz, M. (1972) Proc. Natl. Acad. Sci.USA 69, 1565-1568.

2. Kramer, G. & Hardesty, B. (1980) in Cell Biology, A Compre-hensive Treatise, eds. Prescott, D. & Goldstein, L. (Academic,New York), Vol. 4, pp. 69-105.

3. Gross, M. & Rabinovitz, M. (1973) Biochem. Biophys. Res.Commun. 50, 832-838.

4. Ranu, R. S. & London, I. (1976) Proc. Natl. Acad. Sci. USA73, 4349-4353.

5. Wallis, M. H., Kramer, G., Pinphanichakarn, P. & Hardesty,B. (1978) Fed. Proc. Fed. Am. Soc. Exp. Biol. 37, 1625.

6. Kramer, G. & Hardesty, B. (1981) Curr. Top. Cell. Regul. 20,185-203.

7. Farrell, P. J., Balkow, K., Hunt, T., Jackson, R. & Trachsel,H. (1977) Cell 11, 187-200.

8. Gross, M. (1980) Mol. Cell. Biochem. 31, 25-36.9. Trachsel, H., Ranu, R. S. & London, I. (1979) Proc. Natl.

Acad. Sci. USA 75, 204-208.10. Wallis, M. H., Kramer, G. & Hardesty, B. (1980) Biochemis-

try 19, 798-804.11. Fullilove, S., Wollny, E., Stearns, G., Chen, S.-C., Kramer,

G. & Hardesty, B. (1984) J. Biol. Chem. 259, 2493-2500.

Biochemistry: Kudlicki et al.

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12. Speicher, D. W. & Marchesi, V. T. (1982) in Differentiationand Function of Hematopoetic Cell Surfaces, eds. Marchesi,V. T. & Gallo, R. C. (Liss, New York), pp. 129-142.

13. Lux, S. E. (1982) in Differentiation and Function ofHematopoetic Cell Surfaces, eds. Marchesi, V. T. & Gallo,R. C. (Liss, New York), pp. 197-206.

14. Harris, H. W., Levin, N. & Lux, S. E. (1980) J. Biol. Chem.255, 11521-11525.

15. Harris, H. W. & Lux, S. E. (1980) J. Biol. Chem. 255,1X512-11520.

16. Anonymous (1979) Affinity Chromatography, Principles andMethods (Pharmacia Fine Chemicals AB, Sweden).

17. Wollny, E., Watkins, K., Kramer, G. & Hardesty, B. (1984) J.Biol. Chem. 259, 2484-2492.

18. Kramer, G., Cimadevilla, J. M. & Hardesty, B. (1976) Proc.NatI, Acad. Sci. USA 73, 3078-3082.

19. Litman, D., Hsu, C. J. & Marchesi, V. T. (1980) J. Cell Sci.42, 1-22.

20. Odom, 0. W., Kramer, G., Henderson, A. B., Pinphanichakarn,P. & Hardesty, B. (1978) J. Biol. Chem. 253, 1807-1816.

Proc. Nati. Acad. Sci. USA 82 (1985)

21. Warburg, 0. & Christian, W. (1942) Biochem. Zeitschr. 310,384-421.

22. Kuppuswamy, K., Fleming, J. & Harrison, P. (1983) Exp. CellRes. 144, 241-247.

23. Grankowski, N., Kramer, G. & Hardesty, B. (1979) Arch.Biochem. Biophys. 197, 618-629.

24. DePaoli-Roach, A. A., Roach, P. J., Pham, K., Kramer, G. &Hardesty, B. (1981) J. Biol. Chem. 256, 8871-8874.

25. Blikstad, I., Nelson, W. J., Moon, R. T. & Lazarides, E.(1983) Cell 32, 1081-1091.

26. Liu, S.-C. & Palek, J. (1985) J. Cell. Biochem. Suppl. 93, 25.27. Farrelly, F. W. & Finkelstein, D. B. (1984) J. Biol. Chem. 259,

5745-5751.28. Dougherty, J., Puri, R. K. & Toft, D. 0. (1984) J. Biol. Chem.

259, 8004-8009.29. Brugge, J., Yonemoto, W. & Darro, D. (1983) Mol. Cell. Biol.

3, 9-19.30. Walker, A. I., Hunt, T., Jackson, R. & Anderson, C. W.

(1985) EMBO J. 4, 139-145.31. Lazarides, E. & Nelson, W. J. (1982) Cell 31, 505-508.

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