Characterization of the expression products of recombinant human ...

THEJOURNAL OF BIOLOGICAL CHEMISTRY 0 1987 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 262, No. 29, Issue of October 15, pp. 14204-14212,1987 Printed in U.S.A.

Characterization of the Expression Products of Recombinant Human Choriogonadotropin and Subunits* (Received for publication, June 19, 1987)

Joyce LustbaderSB, Steven BirkenS, Susan PollakS, LeslieLevinsonll, Edward BernstinelJ, Nancy Hsiungll , and RobertCanfield* From the $Departmentof Medicine, College of Physicians and Surgeons and VHoward Hughes Medical Institute, Protein Chemistry Core Laboratory. Columbia Universitv. ” _New York, New York 10032, and lllntegrated Genetics, Znc., FramingLm, Massachusetts 01 701

Human choriogonadotropin (hCG) is a placental gly- subunit. When a is combined with 8, the local structures coprotein hormonecomposed of a 92-amino acida sub- around thea glycosylation sites are apparently altered unit noncovalently linked to a 145-amino acid j3 sub- so as to make the synthesis of triantennary chainsless unit. We report here the expression of biologically favorable. active hCG in mouse C127 cellstransfectedwith expression vectors containingthe DNA coding for both subunits. In addition, the same cell line was used to Human choriogonadotropin (hCG)’ is a glycoprotein horexpress thea subunit alone. The expression products were purified by affinity mone, whose primary function early in pregnancy is thought chromatography using specific monoclonal antibodies to be stimulation of the corpus luteum to maintain an endoto hCG or its subunits. The system secreting biologi- metrial environment that is favorable for the implanted fercally active hCG also produced a 10-fold or greater tilized ovum. The hCG molecule is composed of two nonimolar excess of free j3 subunit. The dimeric hormone, dentical subunits, a and p, each of which is first synthesized as well as the excess j3 subunit, resembles the standard as a larger precursor molecule containing a signal peptide (1, urinary hCG and j3 subunit by chemicaland biological 2), which is removed by proteolytic cleavage prior to selection. criteria. In contrast,when the vector encoding for the Only dimeric hCG, not the single subunits, possesses biologa subunit was expressed alone, the a subunit had a ical activity (3). The 92-amino acid (Y chain is glycosylated at higher molecular weight than both standarda and the asparagines 52 and 78, while the 145-amino acid p chain is a found in the expressed dimeric hormone. glycosylated at asparagines 13 and30 and also at four serine The molecular weight difference between expressed residues located near the COOH terminus of the polypeptide a subunit and standarda was found to reside in thea peptide consisting of residues 52-91 which contained chain (4, 5). The hormone contains approximately 30%carbohydrate by all of the carbohydrate of the a subunit. The N-asparweight (3), and proper glycosylation is thought to be required agine-linked carbohydrate moieties in the recombinant for biological function. HF-treated or enzymatically deglycoa were found to be triantennary in contrast to biantennary in urinary a, and this hyperglycosylation was sylated hCG is significantly impaired in its capability to responsible for the higher molecular weight of the a stimulate CAMP formationand steroidogenesis under in vitro subunit when it was expressed alone. We found no assay conditions despite a high binding affinity of the deglyevidence of 0-threonine glycosylation at position aS8 cosylated hormone for its receptor (6-9). This loss of biologreported to be present in free formsof the a subunit; ical activity may reflect a role for carbohydrate in hormone however, the companion paper (Corless, C. L., Bielin- action, or it could be due to perturbations in the tertiary ska, M., Ramabhadran, T. V., Daniels-McQueen, S . structure caused by the chemical treatment (8) or due to Otani, T., Reitz, B. A., Tiemeier, D. C., and Boime, I. contaminatingprotease activity when glycosidase enzymes (1987)J. Biol Chern. 262, 14197-14203) findsa small are used (10). The carbohydrate structure also contributes to quantity of 0-glycosylation. a prolonged circulating half-life of hCG in plasma (11). Since the excess j3 subunit appears to be of normal Knowledge of the chemistry and immunochemistry of this size and contains the expected complement of sugars, glycoprotein hormone permits it to be used as a model to only free a subunit seems to be a potential substrate study the regulation and synthesis of complex dimeric glycofor additionof extra sugarmoieties. No large j3 subunit proteins. Inthis report we describe the construction and forms havebeen found by others, while large a subunits expression of two vectors: one containing both subunits of have been described both clinically and in tissue cul- hCG and the other containing only the a subunit. The dimeric ture systems. These observations imply that the con- hCG expression product appearsto be chemically, biologically, formation of the free a subunit, in the regions of the easier access for and immunologically identical to the hCG that is isolated glycosylation recognition sites, allows glycosyltransferases than thosesame sites in the 8 from the urine of pregnant women. By contrast recombinant (Y subunit, when expressed alone, had a significantly higher * This work was supported by National Institutes of Health Grant PO-HD-15454. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. To whom correspondence should be addressed Dept. of Medicine, College of Physicians and Surgeons of Columbia University, 630 West 168th St., New York, NY 10032.

The abbreviations used are: hCG, human choriogonadotropin; bp, base pair(s); kb, kilobase(s); FPLC, fast protein liquid chromatography; BPV, bovine papillomavirus; HPLC, high performance liquid chromatography; SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis; endo F, endoglycosidase F; RCM, reduced S-carboxymethylated; PTH, phenylthiohydantoin; TFA, trifluoroacetic acid; THF, tetrahydrofuran.

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Characterization of Expressed hCG and Subunits

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molecular weight than did the cy subunit derived from urinary hCG. It has been noted previously that a free a subunit can be secreted with a larger molecular size than the a subunit isolated from urinary hCG (12-17). One reported reason for this increased cy subunit size was the addition of an 0-linked oligosaccharide at residue Thr-39 found in the free cy subunit purified from bovine pituitaries (12) and from cell cultures (Refs. 13-17 and 40). Blithe and Nisula (18) also reported that a free cy subunit in pregnancy urine exhibited different bindingcharacteristics with lectins and mayhave had an CY altered carbohydrate structureas compared to the standard subunit derived from the dimerichormone. We report here that, when cy subunit is expressed without its subunit complement, the N-linked carbohydrate moieties of the secreted product contain additional sugar residues in contrast to the normal situation where cy and p subunits are expressed together in the same cell. These findings suggest that theseveral glycosyltransferases involvedin the construction of the oligosaccharide chains on glycoproteins are affected by the conformation of the glycosylation site. These results have been obtained by structural analyses of purified expressionproducts. An accompanyingcommunication by Corless et al. (19) showsa similar finding using in uiuo radiolabeling techniques.

2). Northern blot analyses demonstrated that the sizes of the major transcripts for both cy and p are about 1.2 kilobases, which is consistent with the size of a transcript using the regulatory signals of the metallothionein gene (results not shown). Biological Activity of Expressed Materials-In uitro bioassays showed that therecombinant hCG possessed a bioactivity very similar to the urinary hCG standard, including the finding of parallel slopes in the competitive receptor bindingassay (Fig. 3). An in vivobioassay using ascorbic acid depletion also indicated that the expressed hCG had similarbiological activity to thatof standard hCG (Fig. 4). Since the concentration of recombinant hCG was determined by immunoassay,a precise unitage comparison between standard hCG and the recombinant form is not presented in this report. Purification of Expressed Materials-Immunoaffinity purification of sufficient quantities of expressed hCG products for structuralanalyses was accomplished by the use of high capacity IgG-Sepharose columns constructed with 30-59 mg (3-8 mg/ml gel) of purified specific monoclonal IgGs (26). The crude medium was absorbed batchwise. Similarly, washing of the gel with water and elution with 2 M acetic acid was also accomplished batchwise. We elected to elutewith acetic acid because of its volatility. A final fast protein liquid chromatography gel filtration step served to remove any contamMATERIALS AND METHODS A N D RESULTS~ inant IgG leached from the immunoabsorbant gel as well as toseparate expressed products with different molecular DISCUSSION weights. Indeed, expressed cy could be segregated into a preHuman choriogonadotropin is the first two-subunit glyco- dominantly high molecular weight cy and a small component protein hormone to be expressed by recombinant technology, (5%) of normal standard sized cy (Fig. 5). andthishas been found toresultin a biologically active Characterization of Purified Expressed Products-Each of protein very similar to the product isolated from pregnancy the purifiedexpressed proteins wasexamined on SDS-gel. urine(41). In thisreport we demonstratetheunexpected electrophoresis. It was immediately apparent that the subfinding that the carbohydratemoieties, when the cy subunit is units of the expressed hCG, as well as theexpressed excess p expressed alone, differ from those in the cy subunit when it is subunit, all migrated exactlyas did the standard urinaryhCG expressed in the presence of the p subunit. Since the stateof proteins (Fig. 7). In the system expressing dimeric hormone, glycosylation appears tobe important to properfolding of the a 10-foldor greaterproduction of free /3 subunit was observed polypeptide chain as it is synthesized (42,43) and also to the which had a sizesimilar to standard urinary p subunit. Strucin uitro and in uiuo biological activity of the hormone (Refs. tural analyses of the dimeric hormone and the excess free p 6-9 and 441, factors effecting changes in carbohydratebiosyn- subunit, produced by the samesystem, were indistinguishable thesis are important. from urinary standard equivalents (Tables VI and VII). Construction of Expression Vectors-We have describedthe In contrast, the cy subunit, when expressed in the absence construction of expression vectors containing the intact bo- of itscomplementary p subunit, migrated at a molecular vinepapillomavirusgenome, theentire mouse metallothionein gene, and either the cy or both cy and @’ hCG DNA sequencesin Fig. 1. When the cy and p genes, on separate expression vectors, were cotransferred into mouse recipient C127 cells, bovine papillomavirus transformants were identified and analyzedfor production of hCG (Table I). The highest producing clones expressed hCG at a level of about lo-’’ mIU/ce11/24 h (Table I). The bovine papillomavirus transformants producing and secreting hCG were further characterized. The structures of the DNA of the higherproducing lines werebasically not rearranged. Clone CMaplh, our best producing line, based on expression levels and growth properties, contains25-50 copies of both the cy and /3 subunit DNA. The ratio of a/P DNA present within each of the transformants varied widely (Fig.

* Portions of this paper (including “Materials and Methods,” “Results,” Figs. 1-6 and 10-14,and Tables I-IV and VI-IX) are presented in miniprint at the end of this paper. Miniprint is easily read with the aia of a standard magnifying glass. Full size photocopies are availahle from the Journal of Biological Chemistry, 9650 Rockville Pike, Rethesda, MD 20814. Request Document No. 87M-2077, cite the authors, and include a check or money order for $10.80 per set of photocopies. Full size photocopies are also included in the microfilm edition of the Journal that is available from Waverly Press.

1 2 3 4 5 6 7 8 9 1 0

FIG.7. SDS-polyacrylamide gel electrophoresis analysis of purifiedurinary

and recombinant hCG and subunits. The purified proteins were treated with sample buffer containing 5% 6mercaptoethanol, boiled for 3 min, and applied to a 15% Laemmli gel. Lanes: I, 4, 7,and 10, molecular weight markers; 2, urinary a;3, recombinant a; 5, urinary 8; 6, recombinant 8; 8; urinary hCG; 9, recombinant hCG. The mobilities of the molecular weight standards are indicated to the left of the gel.

Characterization of Expressed hCG Subunits and

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weight approximately 4000-5000 greater than the standard urinary a subunit (Fig. 7). This higher molecular weight was consistent with its elution volume from the Superose 12 gel filtration column. The possible causes of the higher molecular weight of expressed a were individually explored and are enumerated as follows: 1) extraNHz-terminal or COOHterminal aminoacids; 2) 0-glycosylation at Thr-39 as reported by Parsons et al. (12) and Cole et al. (40) for excess pituitary a or a expressed in tissue culture;or 3) extra N-glycosylation, i.e. tri- or tetraantennary oligosaccharide chains. The question of whether extra amino acids were present within expressed a was readily addressed by NHz-terminal sequence analyses and carboxypeptidase digestions. Automated sequence analysis indicated that expressed a contained the expected NHz-terminal structurewith none of the heterogeneity that hasbeen observed for the a subunit polypeptide sequence in protein isolated from pregnancy urine (see Table 111).Carboxypeptidase digestions showed that theexpected a COOH terminus was intact (Table IV). Amino acid analyses of both the expressed and the urinary a were very similar (Table 11). Therefore, we assumed that carbohydrate differences were responsible for the higher molecular weight of the individually expressed a! subunit. Carbohydrate analysis of the expressed a subunit did, indeed, confirm the presence of carbohydrate differences in comparison to standard urinary a (Table V).

The strategy employed to define the location of the carbohydrate difference, i.e. 0-versus N-glycosylation, was based on earlier data characterizing tryptic digestion of the native a subunit as described by Birken et al. (28). The expressed a subunits, as well as urinary a standards, were each digested with trypsin, reduced and S-carboxymethylated, and applied directly to reverse phase HPLC (Fig. 8). Allof the peptide peaks were isolated and analyzed by NHp-terminalsequence analysis (Table VIII; data for noncarbohydrate-containing peptides not shown), SDS-gel electrophoresis (Fig. 9), and carbohydrate analysis (Table V). It was observed that peptide a 3 6 4 2 from expressed a behaved on HPLC exactly the same as thispeptide from urinary a. Thus, we concluded there was no 0-glycosylation at Thr-39 (Fig. 8). In addition, both peptides were sequenced through position 39 with recoveries of expected quantities of Thr-39, indicating that the hydroxyl group was not significantly substituted with a carbohydrate. Carbohydrate analyses of both expressed and urinary ( ~ 3 6 - 4 2 also confirmed the apparentabsence of carbohydrate. By contrast, peptide a62-91 from expressed a appeared as a mixture with (Y46-91 and was shown to be responsible for the extra molecular weight of expressed a as determined by SDSgel electrophoretic analysis (Fig. 9). Sequence analysis indicated an identical primary structure of the recombinant carbohydrate-containing peptides when compared to the standard urinary peptide (Table VIII), but the carbohydrate con-

TABLE V Carbohydrate analysis A. Analysis of intact subunits and hCG Identification

Glucosamine"

Galactosamine"

Galactose"

Urinary a 0.2 (0) 4.0 (4) 7.2 (8)' 1.1 10.6 4.1 13.4 Recombinant a-ld 0.15 Recombinant ~ x - 2 ~ 7.9 9.9 4.0 (4) 8.3 (8) 7.9 (8) Urinary j3 8.9 7.9 4.7 Recombinant j3 16.0 (16) Urinary hCG 4.3 (4) 12.4 (12) Recombinant hCG 17.4 5.1 14.0 15.1 B. Analyses of RCMa carbohydrate-containing peptides Identification and figure location

Mannose"

Sialic acidb

6.0 (6) 6.0 6.0 6.0 (6) 6.0 12.0 (12) 12.0

2.8

Mannose" Galactose" Glucosamine"

4.2 Urinary a Fraction 49 (Fig. 8B, (Y62-9l)C 7.6 4.4 Urinary a Fraction 51-52 (Fig. 8B, ( ~ 6 2 - 9 ~ ) ~ 7.8 7.2 Recombinant a Fraction 51 (Fig. 8 A , a62.91). 10.2 5.9 Recombinant a Fraction 52 (Fig. 8 A , a62-91). 10.0 6.3 Recombinant a Fraction 53 (Fig. 8 A , a6g1). 9.6 C. Periodate oxidation studies of RCM recombinant a subunit and peptides Identification

10.1 9.6 13.4

Glucosamine"

GalactoseD

6.0 6.0 6.0 6.0 6.0 Mannose"

3.0 2.6 5.0 Recombinant aS2- Afg 3.0 3.0 5.1 Recombinant a s 2 4Bg 2.8 1.8 4.7 Recombinant a b 2 4 Bg,periodate treated 3.1 3.0 5.3 Recombinant (~76-9~ Ag 3.1 3.0 5.1 Recombinant a7691 Bg 1.9 2.5 4.9 Recombinant a7691 Bg,periodate treated 6.0 5.1 9.6 Recombinant a whole RCM" Recombinant a whole RCM", periodate treated 9.1 4.5 4.1 Analysis performed by F. Perini. Values are normalized to number of residues per 6 or 12 residues of mannose (or 3 for peptides containing only one carbohydrate moiety). * Fluorometric assay of sialic acid (32). Parentheses show expected values for the hormone isolated from pregnancy urine. Recombinant al-gel filtration fast protein liquid chromatography (Superose 12 column). Recombinant a2reverse phase HPLC subfractionation (Vydac C4,300A column). e Sequencing data for these peptides is shown in Table VIII. Note that the recombinant peptides also contain minor sequences. 'Preparation A of (Y62-63 contains a minor sequence of a non-carbohydrate-containinga peptide (see Table IX). See Table IX for details and sequencing data for preparations A and B. This RCM recombinant a is the parentof preparation B peptides. A clean sequence was obtained for 12 cycles (data notshown).


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IA

FIG. 9. SDS-polyacrylamide gel electrophoresis analysis of tryptic digests of urinary and recombinant a. 18% reduced Laemmli gel silver stained. Lune I , urinary n;lane 2, urinary n trypsin digest; lane 4, recombinant a;lane 5, recombinant a trypsin digest. Carbohydrate-containing RCM tryptic peptides are shown in lanes 7-12. Lane 7, urinary a tryptic peptides Fraction49 (Fig. 8R);lane 8, urinary a tryptic peptides Fraction 51/52 (Fig. 8B);lane IO, recombinant n tryptic peptides Fraction 51(Fig. 8A ); lone 11, recombinant a trypticpeptidesFraction52 (Fig. SA); lane 12, recombinant n tryptic peptides Fraction 53 (Fig. 8A ); lanes 3 and 6, molecular weight standards shown to the left of the gel. The tryptic digestions were performed a t 37 "Cfor 2 h, trypsin was added a t time zero and 1 h at a final enzyme:substrate concentration of 1:50.

02

be devoid of carbohydrate a t one of the glycosylation sites. This was investigated further by additional isolations of peptides from the recombinant a subunit and cleavage of the carbohydrate-containingpeptidesinto peptides containing FRACTION NUMBER only one carbohydrate moiety. Intact RCM recombinant CY FIG.8. Reversed phase HPLC (Vydac C4 300A column) of was also cleaved by trypsin to produce such peptides. Each of reduced S-carboxymethylated trypsin digests of recombinant the newly isolated peptides was sequenced (Table IX) and ( A )and urinary ( B )a. Mobile phase, 0.1% trifluoroacetic acid ( A ) ; analyzed for carbohydrate content. The composition results 0.1% trifluoroacetic acid in 60% acetonitrile ( B ) ;gradient: 10 min, moieties 100% A, 1 ml/min; 60 min to60% B ; 10 minto 100% B. 1-ml fractions presented in Table V indicate that both carbohydrate in the recombinanta subunit are similar and are likely to be were collected. Therecombinantandurinary profiles aresimilar triantennary. This was further supported by the results of except for the carbohydrate-containing peptides and The urinary a subunit profile always displays peptide al-nsasa major V). Complete loss of a periodateoxidationstudies(Table and minor peak (28). Each of the peaks was subjected to automated single mannose residue with only a small loss of galactose sequence analysis, and the identifications are shown on this figure. after periodate oxidation indicates the presence of triantenThe sequence data from the carbohydrate-containing peptides are nary chainswhich are heterogeneously sialylated. The higher shown in Table VI11 while the data from the other peptides are not sialic acid content of recombinant a (Table V) and the more shown to conserve space. acidicisoelectricprofile (Fig. 6) is also consistent witha triantennary structure rather than the biantennary branching tents were significantly different(Table V). Carbohydrateanalysesindicated amorecomplex sugar known to be present in urinary a.Furthermore, recombinant which contained a was resistantto endoglycosidase F cleavage, while the structure on the recombinant peptidea52-91, ahigher content of glucosamine and galactose (Table V). biantennary urinary a was readily digested (Fig. 10). Carbohydrate analyses of all of the other hCG expression Further evidence of the complexity of the recombinant a sugar moiety comes from the detailed evaluationof this pep- products were similar to the standard urinary preparations tide, which elutedas a triplet in Fig. 8. Amino-terminal (Table V). Since amino acid and structural analyses of all of the tripletbegan a t other expressed products showed them tobe identical to those sequencing indicated that the last peak of the urinary standards (Tables VI and VII), we concluded cy Thr-46 (TableVIII) instead of Asn-52 as expectedfrom the results of digestion of the standard urinary a. Perhaps the that the only difference in the recombinant materialsoccurs larger sugar structure, present on recombinant a, sterically when a subunit isexpressed alone. The sole structural change is the addition of extra sugars hinders the trypsin from cleaving as expected between resito both of the N-asparagine-linkedoligosaccharide chains (a dues Lys-51 and Asn-52. behaved as expected in the Asn-52 or a Asn-78). This is in concurrence with theaccomIt was also noted that a46.gl Sequencer in that residue 52 was not recovered as is usually panying paper (19). Blithe and Nisula (18)also found that a observed for N-asparagine-substitutedamino acids. However, preparation of free a subunit isolated from pregnancy urine from resembled the expressed free a described in this reportby its this was not the casefor recombinantpeptide as2-91 as as itsinability to combine which a significant quantity of asparagine was recovered a t altered carbohydrate content well the first Sequencer stepin contrast to theanalogous peptide with p subunit (Fig. 11). Cole et al. (40) reported that cultured JAr cells secreted from the urinary a subunit. A second preparation of recomIt is likely that hCG as well as free subunits but that only a subunit was binant cy yielded less of the peptide a52-91. there is heterogeneity in the synthesisof the carbohydrate in hyperglycosylated, while the /3 subunit was of normal size. In the recombinant preparation and that a small quantity may that case a was 0-glycosylated. There are numerous reports '

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in the literature, both concerning clinical situations as well as in vitro cell systems, describing production of free hCG subunits (12-17). In many cases a large a subunit is reported but never an abnormally large /3 subunit. It is known that the conformation of the a subunit is altered upon combination with p (45,46). Since the hCG subunits startcombining prior to leaving the rough endoplasmic reticulum (47-49), it is likely that some of the induced conformational changes alter the environment of the carbohydrate oligosaccharide core structures of the a subunit so that an additional N-acetylglucosamine residue cannot be added, thus blocking triantennary chain formation (50). When a subunit does not combine with its complementary p, the sugar residue can be added resulting in formation of additional antennae. Thep subunit does not appear to undergo such changes in its free state. These resultsand thatof the accompanying communication (19) show the importance of the conformation of a protein to the structure of its post-translation end product. The Nasparagine-linked oligosaccharide branches on the a subunit are biantennary when a combines with its /3 complement prior to completion of its biosynthesis. In contrast,we have shown that in the same cell system, a expressed without fi subunit contains triantennary instead of normal biantennary carbohydrate structures. While the presence of abnormal carbohydrate structures on the a subunit produced by in vitro or in vivo systems has been known for some time, this is the first demonstration that a is abnormally glycosylated when synthesized in the absence of /3 subunit. Acknowledgments-We thank Dr. M. A. Gawinowicz Kolks and S. Halpine from the Protein Chemistry Core Laboratory of Columbia University for amino acid analyses, Barbara Fleming of Integrated Genetics for skillful technical assistance, Dr. L.Cole for helpful discussions, and G. Mayewski and M. Zurack for typing of the manuscript. We are especially grateful to Dr. F. Perini of the University of Michigan who performed the carbohydrate analyses and periodate digestion. REFERENCES 1. Birken, S., Fetherston, J., Canfield, R., and Boime, I. (1981) J. Biol. Chem. 2 5 6 , 1816-1823 2. Fiddes, J. C., and Goodman, H. M. (1981) in Bioregulators of Reproduction (Jagiello, G., and Vogel, H. J., eds) pp. 279-304, Academic Press, Orlando, FL 3. Morgan, F. J., Canfield, R. E., Vaitukaitis, J. L., and Ross, G. T. (1974) Endocrinology 9 4 , 1601-1606 4. Morean. F. J.. Birken,. S.,. and Canfield, R. E. (1973) Mol. Cell. B&hem. 2,'97-99 5. Carlsen. R. B.. Bahl. 0. P.. and Swaminathan, N. (1973) J. Biol. Chem; 248,'6810-6827 ' 6. Manjunath, P., and Sairam, M.R. (1982) J. Biol. Chem. 2 5 7 , 7109-7115 7. Kalyan, N. K., Lippes, H. A., and Bahl, 0.P. (1982) J. Biol. Chem. 257,12624-12631 8. Keutman, H. T., McIlroy, P. J., Bergert, E. R., and Ryan, R. J. (1983) Biochemistry 22,3067-3072 9. Chen, H-C., Shimohigashi, Y., DuFau, M. L., and Catt, K. J. (1982) J. Biol. Chem. 2 5 7 , 14446-14452 10. Goverman, J. M., Parsons, T. F., and Pierce, J. G. (1982) J. Biol. Chem. 257,15059-15064 11. Pierce. J. G.. and Parsons, T. F. A. (1981) Annu. Rev. Biochem. 50,456-495 12. Parsons, T. F., Bloomfield, G. A. and Pierce, J. G. (1983) J. Biol. Chem. 2 5 8 , 2 4 4 13. Hussa, R. 0.(1980) Endocr. Rev. 1, 268-294 14. Ruddon, R. W., Hanson, C.A., Bryan, A. H., Putterman, G. J., White, E. L., Perini, F., Meade, K. S., and Aldenderfer, P. H. (1980) J. Biol. Chem. 255, 1000-1007 ~

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14209


14210

reported.

16.54 18.75 17.42 15.25 27.32 12.55 13.09 23.33 17.30

2.74 5.61 16.30 4.95

5.71 3.88 10.00

13.89

Aspartic Acid mraon&s

6 l o p a r t6.80 i 0 Acid6.99 Thm..bnin* ssrina 9 C l u t u8.87 i e Acid9.54 5.96 Proline1.49 c1yeins *1min*5.34 c,stsine V.1ir.B

7.48

8.49 9.75 7.14 5.85 5.52 6.07 7.09 7.18 2.85 3.02

n s t h i o n i0.89 na 1so1sveins lavcina3.97 2.95 Tyr0ain* p b s ~ l a l m i n4.09 s Hiatidina 6.00 LpilI* 2.83 Arginine

serine Glutamic Acid Pmlins 01reins

7.12

=

8.0 7.53

7.93 6.71

8 8

4.42

6.11 5.71 8.15 6.99

4 5 10

7.51 e

293 4

7 3

0.94

1.18

4.24 3.15 3.84 3.63 6.00 2.63

AIhniM cysteine Y8li.X nathlooias 1aDla"Cine 5 Laveins Tyroains Phewlalanine Hiatidins ymim 4 Arginine

7

2.97 6.W 2.63

1.23

1

4.86 2.53 3.80 2.74

4 4 4

3 6

3

18 170 21

0

264 75

5 199 28

252 76

42 239 57

----. . .

2 51 42

0

42

12.08 9.95 (2.35 9.11

24.10 8.27

7.m 7.91 12.68 .86 4.59 11.90 2.41 2.18 2.16 1.42 4.W 10.89 11.07

17

15.62 19.60 17.70 18.27 26.n 17.05 12.14 26.06 18.73

18 21 18 29 12 13 22 19 4

3.37

6

5.68 (6.24 5.26 5.60 4.14 10.00 13.88

16

7 6 4 10 15

11

10 13

9 22 8

8

12 12 1

12

3

12.69 10.25 13.76 9.54

25.55 8.22 7.34 10.23 12.77 .93 4.71 12.11 2.56

2 1

12

1.47 4.00


14211

A

1

2

3

4

5 . 8

7

e

9

8.

A YW

YW

"...

-2.w

,1001 -

.IO00 -

43.

43-

25.7

25.7-

18.4 14.3-

-

-

I 8.4

14.36.2-I)

6.2-

3-

1 2 3 4

I

2 3 4


14212

Y.W

A

0

.IO00

"w - 200

Y.W.

-

1 2 3 4 5

10.4 14.36.2-

90-

8070

60

12 3 4 5 6 7 8 9

so 40

30

~

~

-

" 10

-

-

24

28 FRACTION NUMBER

32

x 1000 -200

- 18.4