The primary structure af sheep pituitary growth hormone

ARCHIVES

OF

BIOCHEMISTRY

The Primary

AND

Structure

CHOH HA0 The Hornlone

166, &%-~08

BIOPHYSICS

af Sheep

LI, DANIEL

Research Laboratory,

Pituitary

GORDON,

AND

of California,

University Received

(1973)

January

Growth

Hormone

JEAN KNORR San EIrancisco,

California

19, 1973

The complete amino acid sequence of the sheep growth hormone molecule been elucidated. The proposed st’ructure is shown. It consists of 191 amino residues with two disulfide bridges formed by residues 53-164 and 181-189. Isolation and partial characterization of growth hormone (SGH)’ from sheep pituitary glands were first reported by Papkoff and Li (1) and subsequently by other investigators (2-5). SGH, which has an isoelectric point at pH 6.3 (1, 5), is a monomer of 22,000 daltons in solutions of pH’s 3.6 and 11.5 and a dimer at pH 8.2 (5, 6). Circular dichroism studies of the hormone show that the a-helix content (45-50%) does not change with pH (6, 7). This paper reports the complete amino acid sequence of the hormone. A preliminary account of this investigation has appeared (8). EXPERIMENTAL The method of isolation of SGH has been previously described (I); the product was further purified (6) by gel filtration on Sephadex G-100 in 0.01 M NH,HCO, buffer of pH 8.4. Materials employed in this investigation were the same as previously described (9,lO). The methods used for determination of the amino acid sequence were as previously described (9, 10). RESULTS

Table I gives the amino acid composition of SGH computed on the basis of a molecular weight of 22,000 (6). These values are in agreement with those reported by earlier investigations (5, 11). As reported earlier (1, 5), SGH has two NHz-terminal residues 1 Abbreviations: SGH, sheep pituitary growth hormone; BGH, bovine pituitary growth hormone; HGH, human pituitary growth hormone; 0-, performic acid derivative; M-, maleylat,ed derivative. 493 Copyright All rights

@ 1973 by Academic Press, of reproduction in any form

Inc. reserved.

94122

has acid

(phenylalanine and alanine) in approximately equal amount and one COOH-terminal amino acid (phenylalanine). The NHzterminal amino acid sequences, as determined by the dansyl-Edman Method (12), were found to be: H-Ala-Phe-Pro-Ala-MetSer-Leu- and H-Phe-Pro-Ala-Met-Ser-Leu-. The carboxypeptidase A digest of SGH gave -Ala-Phe-OH as the COOH-terminal sequence. The react’ion product of SGH with cyanogen bromide was first submitted to gel filtration on Sephadex G-25 (superfine) in 0.1 x acetic acid as shown in Fig. 1. nlaterials in peak 2 and 3 were rechromatographed and recovered by lyophilization. Peak 3 containing CBl was further purified by high voltage electrophoresis at pH 2.1 to give two fractions (CBlA and CBlB). Peak 2 contains CB5 without further purification. The material in peak 1 was further fractionated on Sephadex G-100 in 20 % formic acid to yield the elution profile shown in Fig. 2; without additional purification, the material in Peak 4 contained CB3 fragment and that in Peak 2 CB (2+4). Oxidation of CB (2+4) with performic acid gave two fragments (OCBZ and OCB4) after gel filtration on Sephadex G-50 (fine) in 0.1 N NH,OH (Fig. 3). From 500 mg SGH, yields of CXBr fragments were: CBl, 10 mg; CB2, 130 mg; CB3, 50 mg; CB4, 16 mg; CB5, 19 mg. Table II presents the amino acid composition and terminal amino acids of the five CXBr fragments. It may be noted that these fragments account for the total protein composition. From the terminal amino acid analysis, it is

494

LI,

GORDON,

evident that CBl is the NHz-terminal fragment whereas CB5 is at the COOH terminus. Table III gives the source and steps of purification of peptide fragments derived from SGH. The amino acid composition of each purified peptide is given in Table IV. Each of these peptides was submitted to complete or partial sequence determination. The steps employed with each peptide are summarized in Table V; a number above an amino acid residue gives its residue position number in the SGH molecule. DISCUSSION

From the data summarized in Table V, the complete amino acid sequence of SGH can be formulated as shown in Fig. 10. The assignment for the two disulfide bridges is obvious as CB5 has two half-cystine residues (Table II). Thus, the disulfide bridges are formed by residues 53-164 and 181-189. Bellair (11) has reported the sequence of the 68 COOH-terminal amino acids. This is identical to the sequence herein described. While this manuscript was in preparation, Eernkdez et al. (13) published a proposed amino acid sequence of SGH partially based on homology with bovine growth hormone (14). The Argentina group reported that SGH contains 189 amino acids instead of 191. By the use of the peptide mapping technique, Seavey et al. (15) have pointed out that the only difference in the primary structures of SGH and BGH are located in a single tryptic peptide. This is the one listed as OT7 in Table V. Apparently, residue 130 is glycine in BGH (13, 14) and valine in SGH (see Fig. 10). If this is the only difference between SGH and BGH, it would be of interest to compare the proposed structure of SGH (Fig. 10) with the Argentine proposal for BGH (13, 14).2 There are two amino acids missing in the BGH structure; these are Leuso and Sersr. Other differences are : SGH:

Pro-Glu; 38 39

Gln; Gln-Ser; 69 84 85

Gly; Gin; Asp; Asp 88 91 99 168

2 The structure of BGH proposed earlier by Santomb et al. (14) is considerably different from that outlined by Fernhnder et al. (13). The comparison is made with the Fern&de2 structure.

AND BGH:

KNORR Gly-Pro; 38 39

Glu; Ser-Glu; 69 84 85

Glx; Glu; Asn; Asn 88 91 99 168

Whether or not these differences are real remains to be investigated. Nevertheless, the homology in the amino acid sequences of these two molecules is indeed very close. It is also of interest to compare the structure of SGH (Fig. 10) with that of HGH (16). The two sequences have been aligned as shown in Fig. 11; the alignment requires the presence of three nonhomologous gaps, two in SGH and one in HGH. It may be seen that a total of 123 positions are occupied by identical residues and an additional 46 positions contain either highly acceptable or acceptable replacements,3 giving a total of 169 homologous positions amounting to 88 %. This high degree of homology is consistent with the hypothesis that HGH and SGH have evolved from a common ancestor. ACKNOWLEDGMENTS The authors thank ful suggestions during tion. This work was from the American Foundation, and the

Dr. J. S. Dixon for his helpthe course of this investigasupported in part by grants Cancer Society, the Geffen Allen Foundation.

REFERENCES 1. PAPKOFF, H., AND LI, C. H. (1958) Biochim. Biophys. Acta 29, 145. 2. ELLIS, S., (1961) Endocrinology 69, 554. 3. WALLACE, A. L. C. (1961) Nature (London) 190, 535. 4. WALLACE, A. L. C., AND FERGUSON, K. A. (1963) J. Endocrinol. 26, 256. 5. DELLACHA, J. M., ENERO, M. A., SANTOM~, J. A., AND PALADINI, A. C. (1970) Eur. J. Biochem. 12, 289. 6. BEWLEY, T. A., AND LI, C. H. (1972) Biochemistry 11, 927. 7. EDELHOCH, H., AND LIPPOLDT, R. E. (1970) J. Biol. Chem. 946,4199. 8. LI, C. H., DIXON, J. S., GORDON, D., .IND 3 Dayhoff (17) has classified residue mutations according to their relative rates of acceptance into proteins. We have designated as “highly acceptable” those replacements Dayhoff lists as having acceptance rates equal to or greater than 40 times that predicted by chance, and those listed as “acceptable” are replacements with acceptance rates from 21 to 39. In our analysis, residue pairs representing replacement with acceptance rates from 0 to 20 were not considered sufficiently homologous.

PRIMARY

9. 10.

11. 12.

13.

14.

STRUCTURE

OF SHEEP

KNORR, J. (1972) Znt. J. Peptide Protein RPS. 4, 151. LI, C. H., DIXON, J. S., SND CHUNG, D. (1973) Arch. Biochem. Biophys. X6,95. LI, C. H., DIXON, J. S., Lo, T.-B., SCHMIDT, K. D., AND PARKOV, Y. A. (1970) Arch. Biochem. Biophys. 141, 705. BELL.~IR, J. T. (1972) Biochem. Biophys. Res. Comm. 46, 1128. GRAY, W. R., AND HARTLEY, B. S. (1963) Biothem. J. 89, 59P; WOOD, K. R., AND WANG, K. T. (1967) Biochim. Biophys. Acta 133, 396. FERN~NDEZ, H. N., Pi&-a, C., POSKUS, E., BISCOGLIO, M. J., PALADINI, A. C., DELLAcHA, J. M., AND SA4NTOM6, J. A. (1972) Fed. Eur. Biochem. Sot. Letters 26, 265. SANTOM~, J. A., r)ELL.ICH.I, J. M., PSLADINI, TABLE

PITUITARY

GROWTH

Trp LYS His Arg Asp Thr Ser Glu Pro

I

GUY Ala cyst Val Met Ile Leu Tyr Phe

This investigation __-~_-__ ExperiProposed mental 0.9 11.1 3.2 12.8 16.3 12.26 13.4b 24.4 5.8 9.4 14.8 4.2 7.1 3.9 6.7 27.0 6.1 12.7

495

A. C., WOLFENSTEIN, C. E. M., PI&~, C., POSKUS, E., D.INROT, S. T., BISCOGLIO, M.J., DE&X&Z. M.M., ANDDESANGUESA, A. V. F. (1971) Fed. Eur. Biochem. Sot. Letters 16, 198. 15. SEAVEY, B. K., SINGLE, It. N. P., LEWIS, U. J., AND GESCHWIND, I. I. (1971) Biochem. Biophys. Res. Commun. 43,189. 16. LI, C. H., LIU, W.-K., AND DIXON, J. S. (1966) J. Amer. Chem. Sot. 33,205O; LI, C. H., AND DIXON, J. S. (1971) Arch. Biochem. Biophys. 146, 233.; LI, C. H. (1972) Proc. Amer. Phil. Sot. 116, 365. 17. DAYHOFF, M. O., Atlas of Protein Sequence and Structure, Vol. 4, National Biomedical Research Foundation, Silver Spring, Maryland, 1969.

AMINO ACID COMPOSITIONS OF SGH Amino acid

HORMONE

1 11 3 13 16 12 13 24 6 9 15 4 7 4 7 27 6 13

Ref. 5

Ref. 11

1 12 3 12 15 11 11 23 6 10 14 4 6 4 7 24 6 12

1 11 3 12 16 12 12 23 8 11 15 4 7 4 6 24 6 12

a Residues per mole; the result represents the average of analyses carried out in duplicate on a single sample of hydrolyzate. b Corrected for destruction. c Cys determined as cysteic acid using performic-acid-oxidized samples.

1.5Or

1.50

r

1

TUBE NUMBER

TUBE NUMBER

FIG. 3. Gel filtration of oxidized CB (2 + 4) (28 mg) on Sephadex G-50 (fine) in 0.1 N NH,OH; 2 ml/tube/6 min. Column size, 2.3 X 102 cm.

FIG. 1. Gel filtration of CNBr-SGH reaction mixture (200 mg) on Sephadex G-25 (superfine) in 0.1 N HOAc. Column size, 1.9 X 60 cm; 3 ml/tube/8 min.

r

2 ‘.OO

TUBE NUMBER FIG. 2. Gel filtration of Peak 1 (Fig. ; 100 mg) on Sephadex Column size, 2.8 X 80 cm; 2 ml/tube/6 min.

11’1

a I 50

7

8 I 100

G-100 in 20% formic

9 10 II z I

I

150

200

VOLUME, ml

FIG. 4. Dowex

50 X 8 resin chromatography 496

of tryptic

digest

of 0-SGH

(100 mg).

acid.

PRIMARY

STRUCTURE

OF SHEEP

PITUITARY

TABLE

GROWTH

II

AMINO ACID COMPOSITION END TERMINAL AMINO ACID RESIDUES OF PURIFIED FRAGMENTS FROM SGH Amino acid

Fragment CBla

Trp LyS His Ax Asp Thr Ser Glu Pro Gly Ala Half-Cyst Val Meed Ile Leu Tyr Phe Total residues N-Terminal residue C-Terminal residued

CBlb

0 0 0 0 0 0 0 0

0 0 0 0 0 0 0 0

1.1 (1) 0 1.9 (2) 0 0 0.9 (1) 0 0 0 1.1 (1) 5 Ala

1.1 (1) 0 1.2 (1) 0 0 0.8 (1) 0 0 0 0.9 (1) 4 Phe

Met

Met

(1) (5) (2) (6) (8) (7) (10) (18) (4) (6) (9) (1) (5) (1) (6) (19) (3) (8) 119 Ser

0 2.3 (2) 0 2.2 (2) 3.9 (4) 2.7 (3) 0 4.2 (4) 1.1 (1) 1.1 (1) 1.1 (1) 0 1.1 (1) 0.4 (1) 1.0 (1) 2.1 (2) 0.8 (1) 1.0 (1) 25 Auz

Met

Met

OCB4

CBS

0

0 5.7 (6) 1.8 (2) 1.0 (1) 30 A%

0 1.0 (1) 0 2.0 (2) 0 0 1.0 (1) 1.2 (1) 0 1.1 (1) 2.1 (2) 1.5 (2) 0 0 0 0 0 2.0 (2) 12 LYS

Met

Phe

3.1 1.0 2.9 3.9 1.9 1.7 1.3

(3) (1) (3) (4) (2) (2) (1) 0

1.0 1.0 1.1 1.0 0.4

(1) (1) (1) (1) (1)

a Summation of amino acid content of each CB fragment except b Taken from Table I. 0 Half-cystine for OCB2 and OCB4 determined as cysteic acid. d Methionine determined as homoserine lactone.

VOLUME,

FIG. 5. Dowex

50 X 8 resin chromatography

CNBR

Suma CB3

OCB2

4.9 1.7 5.6 8.3 7.1 9.7 18.0 3.8 6.4 9.0 1.1 4.9 1.2 5.7 18.6 2.7 8.0

497

HORMONE

(1) 11 3 13 16 12 13 24 6 9 15 4 7 4 7 27 6 13 191

1 11 3 13 16 12 13 24 6 9 15 4 7 4 7 27 6 13 191 Phe, Ala Phe

CBlb.

ml

of the Peak 8 material

SGHb

of Fig. 4.

498

LI,

GORDON,

AND

KNORR

TABLE III SOURCE AND STEPS OF PURIFICATION OF PEPTIDE FRAGMENTS FROM SGH Peptide no. OT2 OT3 OT4 OT6 OT7 OT8 OT9 OTlO OT12 OT13 OT14 OT15 OT17 MT1 MT2 02Tl 02T2 02T3 02T4 02T5 02T6 02T7 02T8 02T9 M02Tl M02T2 M02T3 M02T4 M02T5 M02T6 3Tl 3T2 3T3 3-T4 3-T5 02Cl 02c2 02C3 02C4 02C5 02C6

Origin5 0-SGH 0-SGH 0-SGH 0-SGH 0-SGH 0-SGH 0-SGH 0-SGH 0-SGH 0-SGH 0-SGH 0-SGH 0-SGH M-SGH M-SGH O-CB2 O-CB2 O-CB2 O-CB2 O-CB2 O-CB2 O-CB2 O-CB2 O-CB2 MO-CB2 MO-CB2 MO-CB2 MO-CBS MOCB2 MO-CB2 CB3 CB3 CB3 CB3 CB3 O-CB2 O-CB2 O-CB2 O-CB2 O-CB2 O-CB2

Treatment* TD TD TD TD TD TD TD TD TD TD TD TD TD TD TD TD TD TD TD TD TD TD TD TD TD TD TD TD TD TD TD TD TD TD TD CD CD CD CD CD CD

Steps of purificationc Fig. 5, Fig. 5, Fig. 5, Fig. 5, Fig. 4, Fig. 4, Fig. 5, Fig. 4, Fig. 5, Fig. 5, Fig. 5, Fig. 4, Fig. 4, G-59N, G-59N, G-59N, Fig. 7, Fig. 6, G-59N, Fig. 6, Fig. 7, G-59N, Fig. 7, Fig. 7, Fig. 8, Fig. 8, Fig. 8, Fig. 8, Fig. 8, Fig. 8, G-25N, G-25N, G-25N, G-25N, G-25N, Fig. 9, Fig. 9, Fig. 9, Fig. 9, Fig. 9, Fig. 9,

Peak Peak Peak Peak Peak Peak Peak Peak Peak Peak Peak Peak Peak Peak Peak Peak Peak Peak Peak Peak Peak Peak Peak Peak Peak Peak Peak Peak Peak Peak Peak Peak Peak Peak Peak Peak Peak Peak Peak Peak Peak

9 3 2 6 6 2; Dowex 56, Peak 4; G-25N, Peak 1 1 2; Dowex 59, Peak 5 10 5 7 12 1; G-25N, Peak 1; G-59N, Peak 5 1; G-109N, Peak 1; G-56F, Peak 1 1; Dowex 50, Peak 6 3; Dowex 1, Peak 1 5 4; Dowex 59, Peak 2 2; Dowex 1, Peak 1 3 1 1 4 6 1; Dowex 50, Peak 3 4 5 2 1; Dowex 50, Peak 2 3 1; G-59N, Peak 4 3 2; G-59N, Peak 3; HVE, Band 5 2; G-59N, Peak 1 2; G-5ON, Peak 3; HVE, Band 3 2; G-15N, Peak 1; HVE, Band 2 10; G-15N, Peak 1 3 5; G-15N, Peak 2; HVE, Band 2 5; G-15N, Peak 2; HVE, Band 1 4; Dowex 59, Peak 2; G-25N, Peak 2

a See Tables I and II for the amino acid composition of SGH, O-CB2, CB-3, and O-CB4; 0-, performic acid oxidized sample; MO-, maleylated product of oxidized sample. * TD, Tryptic digests; CD chymotryptic digests. c High voltage electrophoresis (WE) was performed in pH 2.1 buffer; low voltage electrophoresis (LVE) was performed in pH 6.7 buffer; paper chromatography (PC) was performed in a solvent system consisting of n-butanol-acetic acid-water, in a ratio of 4:1:5 by volume; G followed by the grade number of the material is employed to designate separations by gel filtration on Sephadex with eluting solvents: N, 0.1 N NHIOH; F, 20% formic acid. Pooled fractions from gel filtration and ion-exchange ehromatography were numbered from left to right in the order in which they appeared on the chromatograph. Bands from electrophoresis were numbered from the anode. Bands from paper chromatography were numbered from the origin.

PRIMARY

STRUCTURE

OF SHEEP PITUITARY TABLE

Pcptide no.

Origin” 0.CB2 0-CB2 (I-CB2 0-CB2

Cl) CD CD CD

02Cll 02c12 02C13 02c14 04Cl

0-CB2 O-CB2 O-CB2 O-CB2 0-CB4

Cl) Cl) CD CD Cl)

HORMONE

Steps of purificationc Fig. 9, Peak Fig. 9, Peak Fig. 9, Peak Fig. 9, Peak PC, Band Fig. 9, Peak Fig. 9, Peak Fig. 9, Peak Fig. 9, Peak LVR, Band

1 7 2; G-15N, 2; G-15N, 4 6 2; G-15N, 4; Dowex 2; B-15N, 3

Peak 1; HVE, Band 1; LVE, Band 5 Peak 1; HVE, Band 1; LVE, Band 3;

Peak 1; HVE, Band 1; LVE, Band 1 50, Peak 2; G-25N, Peak 1 Peak 1; HVE, Band 4

TUBE NUMBER

FIG.

6. Dowex

50 X 8 resin chromatography

of tryptic

digest, of 0-CB2

(56 mg).

TUBE NUMBER

FIG.

7. Dowex

499

III-Continued

Treatmenth

02C7 02C8 02c9 02C10

GROWTH

50 X 8 resin chromatography

of the Peak 5 material

of Fig. 6.

500

LI, GORDON,

AND KNORR

2.6

1.0

1.0

1.1

1.0

1.3 1.0

1.0

1.0

1.1 1.0

0.9

2.0

1.0

0.8 0.7

0.8

1.8

0.9

1.0

1.2

1.1 0.9

1.1 1.1 1.0

- -~

2.9

1.0 1.0

1.2 1.3 0.9 0.9 2.1 1.4

~.__

1.1

1.1 1.9

TABLE

1.1

0.9

1.1

1.0 1.2

1.2

1.7 1.0 2.0

IV--Continued

1.0

0.9 1.0

0.7

0.5

1.0

1.0 1.0 1.0 1.0 2.2 1.3 2.0 1.1 1.3 3.1 1.0 2.1

1.1 1.1 1.2

0.7

0.G 0.G

0.8 0.9 1.0

0.9

1.0 1.0

5 5 8 3 4 7 10 5 5 G 5 0 11 3 10

-~ .__ Q See Table III for the steps of purification employed to obtain the peptides; Cys determined as cysteic acid in oxidized samples; Met, determined as homoserine in CNBr fragments or methionine sulfone in oxidized samples; values for residues present in less than lo?-& of the l-residue figure are omitted. * Peptide MT1 contains one residue of tryptophan; 02T7 and O2C9 contain traces of tryptophan by color tests. OOTll, free lysine; OT16, free arginine.

02Cl 02c2 02C3 02C4 02C5 02CG 02C7 02C8 02c9 02ClO 02Cll 02c12 02C13 02C14 04c1

-

TABLE STRUCTURAL INVESTIGATIONS ___Peptides”’ b

V

ON PEPTIDES DERIVED StructureC

-~~

SGH

1 2 34567 Ala-Phe-Pro-Ala-Met-Ser.Leu-( -AA----

CBla

1 2 3 4 5 Ala-Phe-Pro-Ala-Met ---.--A---

MOST1

6 7 8 9 10 11 12 13 14 Ser-Leu-Ser-Gly-Leu-Phe-Ala-Asx-Ala(Val,Leu,Arg) -----_I ---

02Cl

12 13 14 15 16 Ala-Asn-Ala-Val-Leu --I---

02c2

OT2

M02T2

02C4

02T2

02C5

OT3

02C6

02T4

FROM SGH

)

17 18 19 20 21 Arg(Ala,Glx,His,Leu) 18 19 20 21 22 23 24 25 26 27 Ala-Gln-His-Leu-His-Gln-Leu-Ala-Ala-Asp-Thr-Phe-Lys ---I--------3--+4333334+ 18 Ala(Asx,Thr,Glx~,Ala2,Leu~,Phel,Lys,His2)Arg

28

29

30

34

30 Lys(Glx,Phe) 2 31 32 33 34 Glu-Phe-Glu-Arg AA--f-+-+ 33 36 Glx(Arg,Thr)Tyr 35 36 37 38 39 40 41 Thr-Tyr-Ile-Pro-Glu-Gly-Gln-Arg 2-----14--+433+---f 37 Ile(Pro,Glx2,Gly,Arg)Tyr

42

43

43 44 45 46 47 48 49 50 51 52 Tyr-Ser-Ile-Gln-Asn-Thr-Gln-Val-Ala-Phe-Cys-Phe-( --Id~.-..k__)---2d-~ 34-a-++*3443

53

54

64 )Lys

a Peptides are listed in the order of their appearance in SGH, Met at the COOH termini determined as homoserine la&one; Cys determined as cyst,eic acid. b See Table IV for the amino acid composition. c -, dansyl-Edman procedure; -, phenylthiohydantoin amino acid derivative identified; -+, leucine aminopeptidsse digestion; t- carboxypeptidase A and/or B digestion. The number above each residue indicates its position in SGH. 50%

Peptide+

b

Structurec

R102T-l

02ci

53 5-l 55 56 57 58 59 60 Cys-Phe-Ser-C;lll-Thr-Ile-Pro-Ala-Pro-Thr _I-dd---------\2-

Gl

62

87

---f--)4-+.-+

()T4(02T5)

65 6G 67 68 69 70 Asn-C‘rh1-Ala-(:111-Gln-Lys -----+4+--f--+

02T(i”

71 72 73 74 75 76 Ser-Asp-Lell-(;lu-Leu-I,e~l-Arg -----2

77

02C8

77 78 79 80 81 Arg-He-Her-Leu-Leu ----A

MT1

78 79 80 81 82 t(3 84 85 86 Ile-Ser.Leu-Leu-Leu-He-Gin-Ser-Trp-Leu( i_I2---LA--

95 )Arg

--7

02ClO

87 88 89 90 91 Leu-Gly-Pro-Leu-(iln-Phe 2-----

O2Cll

93 94 95 90 97 Lell-Ser-Arg-Val-Phe ----i

M02T5

96 97 98 99 100 101 102 103 104 \‘~al-Phe-Thr-Asp-Ser-Lel~-~al-Phe-(;ly(Thr,Ser,Asp)Arg ---------

O2T8

02T9

O2C13

OT5

92

108

104 105 106 107 108 Gly-Thr-Her-Asp-Arg --22 --+ -3. -+ -+ 109 110 111 112 Val-Tyr-Glu-Lys -22 9 111 112 113 114 115 116 117 118 119 120121 (:lu-Lys-Leu-Lys-Asp-Le~~-~~l~~-(:lu-(;ly-Ile-Le~~ --_I-------113 114 115 116 117 118 119 120 121 122 I~e~~-Lys-Asp-Le~~-G1~~-C;l~~-Gly-Ile-I~eu-Ala(I~eu,Met)Arg -----------7-

d Glu and Asp were assigned

by low values

(
of NH3 in amino 503

125

acid analyses.

TABLE Peptide+

b

Structure”

OTG

122 123 124 125 Ala-Leu-Met-Arg ---

CB3

125 126 127 128 Arg-Glu-Leu-Glu( ----.A -f

3Tl

OT7d

3T2

3T3

3T4

3T5

OTS

CB4

OT9

04Cl

OTlO

MT2

V-Continued

149 )Met

7

125 Arg(Asx,Thr,Glxl,Pro,Val,Leu)Arg

133

126 127 128 129 130 131 132 133 Glu-Leu-Glu-Asp-Val-Thr-Pro-Arg ------7 7 134 135 136 137 138 139 Ala-Gly-Gln-Ile-Leu-Lys -A--44444 140 141 142 143 144 Gln-Thr-Tyr-Asp-Lys ---44444 140 141 142 143 144 145 Gin-Thr-Tyr-Asx-Lys-Phe(Asxz,Thr)Met 22----

149

145 146 147 148 149 Phe-Asp-Thr-Asn-Met ----44444 -T 140 141 Gln-Thr-(Thr,Asxa,Tyr,Phe,Lys)Met-Arg --

149 150

150 151 152 153 154 155 156 Arg-Ser-Asp-Asp-Ala-Leu-Leu( -->A---

178 179 )Val-Met tt

tt

151 152 153 154 155 156 157 Ser-Asp-Asp-Ala-Leu-Leu-Lys ----22 150 Arg(Asxa,Ser,Ala,Leu2,Lys)Tyr

159

158 159 160 161 162 163 164 165 166 Asn-Tyr-Gly-Leu-Leu-Ser-Cys-Phe-Arg A------4443444 167 168 169 170 171 172 Lys-Asx-Leu-His-Lys-Thr(Thr,Glx,Leu,Tyr)Arg A>---504

177

TABLE

V-Col~tinu~d

Peptidesap b

StruclureC

OT12

168 169 170 171 Asp-Leu-His-Lys

OT13

172 173 174 175 176 177 Thr-Glu-Thr-Tyr-Leu-Arg 7

178 179 180 Val-Met-Lys -A -

OT14

180 181 182 183 184 185 186 187 188 189 190 191 Lys-Cys-Arg-Arg-Phe-Gly-Glu-Ala-Ser-Cys-Ala-Phe

OCB5

1., I

1.5

22 1.0 22 wz

!

VOLUME,

FIG. 8. Gel filtration of MO-CB2 3 min. Column size, 2.3 X 102 cm.

(35 mg)

ml

on Sephadex

(+-50 (fine) in 0.1 N NH&H.

1.2 ml/tithe/

-6

-5 I

-4

I I 5

-I3 500 VOLUME, FIG.

9. Dower

50 X 8 resin chromatography

ml

of chymotryptic 505

digest

of O-CB2

(73 mg).

MO2Tl I02Cl I-

Leu-Arg-Ile-Ser-Leu-Leu-Leu-Ile-Gln-Ser-Trp-Leu-Gly-Pro-Leu-Gln-Phe-Leu80 cCB2 MTl;l 02T6 -I MOZT4I I 02C8 I I-

02c10-

90

Ala-Phe-Cys-Phe-Ser-Glu-Thr-Ile-Pro-Ala-Pro-Thr-Gly-Lys-Asn-Glu-Ala-Gln-Gln-Lys-Ser-Asp-Leu-Glu-Leu60 CB2 OT4 02T5-I-----02T4i~M02T402C7 I I

Ala-Asp-Thr-Phe-Lys-Glu-Phe-Glu-Arg-Thr-Tyr-Ile-Pro-Glu-Gly-Gln-Arg-Tyr-Ser-Ile-Gln-Asn-Thr-Gln-Val30 40 CB2 I I - OT2 OT3 IOZTZ’ I -MOZTZ 02C6 I-02C402C5 II-

I

H-Ala-Phe-Pro-Ala-Met-Ser-Leu-Ser-Gly-Leu-Phe-Ala-Asn-Ala-Val-Leu-Arg-Ala-Gln-His-Leu-His-Gln-Leu-Ala10 I CBI CBZ I

I-

I I I

1

I

70

I

02T6-

02T4MO2T4-

MO2T2

OT2

02Cll

I I

MO2T5

Ser-Arg-Val-Phe-Thr-Asp-Ser-

I

02c2

20

-

100

50

MO2T5

1 -02T8 I I

02T9

-

1

I

02C13

3Tl

OT7 -

3T2

FIG. 10. Amino

Leu-Arg-Val-Met-Lys-Cys-Arg-Arg-Phe-Gly-Glu-Ala-Ser-Cys-Ala-Phe-OH 180 1 CB5 CB4 - OT13-’ - OT14 -1 MTZ-1 acid sequence

I

I

1

3T3-

of the SGH molecule.

190

I-

OT5

Ser-Asp-Asp-Ala-Leu-Leu-Lys-Asn-Tyr-Gly-Leu-Leu-Ser-Cys-Phe-Arg-Lys-Asp-Leu-His-lys-Thr-Glu-Thr-Tyr160 CB4; OTlO I I OT9 04Cl I

-

“YJ

Glu-Leu-Glu-Asp-Val-Thr-Pro-Arg-Ala-Gly-Gln-Ile-Leu-Lys-Gln-Thr-Tyr-Asp-Lys-Phe-Asp-Thr-Asn-Met-Arg140 130

-

“YG

Leu-Val-Phe-Gly-Thr-Ser-Asp-Arg-Val-Tyr-Glu-Lys-Leu-Lys-As~p-Leu-Glu-Glu-Gly-Ile-Leu-Ala-Leu-Met-Arg110

I-

1-

OTl2

OT8 3T5

-i MT2

170

120

I

-

1-0~6

OT13-

I 04Cl

150

1,3Tl-

I

I I

55

75

80

I

I

I

i

I

I

I

III

I

I

100

I

I

l

:I

I

I

i

I:

Ill

IIX:

I

IXXI

HGH:

SGH:

HGH:

SGH:

HGH:

-Lys-Phc-Asp-Thr-Asn-Ser

I IXXXl Cys-Arg-(

)-Ser-Val-Glu-Gly-Ser-Cys-Gly-Phe-OH

*I

Cys-Arg-Arg-Phe-Gly-Glu-&za-Ser-Cys-A!a-Phe-OH

185

I:

190

I

Tyr-Gly-Leu-Leu-Tyr-Cys-Phe-Arg-Lys-Asp-Met-AspLys-Val-Glu-Thr-P~e-Leu-Arg-

IllIxlrlrli~l’rr

170 160 lb5 Tyr-Gly-Leu-Leu-Ser-Cys-Phe-Arg-Lys-Asp-Leu-His-Lys-Thr-Glu-Thr-T~r-Leu-Arg-V~l-M~t-Lys-

I I Gln-lle-Phe-Lys-Gln-Thr-Tyr-Ser

*lrllxlllll~~~fllt~lI

I

IlX:

I

130

:xxt

I

85

175

b5

I

l

110

155

I

II:!’

I

I

135

*

90

I

180

ix

:I

Ile-Val-Gln-

I

I

)-Arg-Val-Tyr-Glu-Lys-

I

-Asn-Arg-Glu-Glu-Thr-

- His-AsrrAsp-Asp-Ala-Leu-Leu-Lys-Asn-

150 145 140 Gln~Ile-Leu-Lys~Gln-Thr-Tyr-Asp-Lys-Phe-Asp-Thr-Asn-Met-Arg-Ser-Asp-Asp-Ala-Leu-Leu-Lys-Asn-

I

SGH:

I

Leu~Lys-Asp-Leu-Glu-Glu-Gly-Ile-Gln-Thr-Leu-Met-Gly-Arg-Leu-Glu-Asp-Gly-Ser-Pro-Arg-Thr-Gly-

HGH:

1

105

II

-Val-Pl~c-Ala-Asn-Ser-Leu-Val-Tyr-Gly-Ala-Ser-Asn-Se~-Asp-Val-Ty~-A~pLe~-

125 115 120 Le~~Lys-Asp-Leu-Clu-Glu-Cly-Ilc-Leu-A~a-Leu-l~~et-Arg-Glu-Leu-Glu-Asp-Val-T~r-Pro-Arg-Afa-Gly-

(XX

SCH:

I

Gin-Phe-Leu-ArgSer

I

Gln-Phe-Leu-Ser-Arg-Val-Phc-T~r-A~p-Ser-Leu-Val-Phe-Gly-T~r-Ser-A~p-(

95

Gl~.Gl~-Lys-Ser-Asn-Leu-G~n-Leu-Lcu-Arg-Ile-Scr-Leu-Leu-Leu-~e-Gln-Ser-Trp-Leu-Glu-Pro-Val-

I

60

-Ilc-Pro-Thr-Pro-Ser

Gln-Gln-Lys-Ser-Asp-Leu-G~u-Leu-Leu-Arg-Ile-Ser-Lcu-Leu-Leu-Ile-Gln-Ser-Trp-Leu-Gly-Pro-Leu-

70

llI~ll:~i*~xl~

HGH:

SGH:

HGH:

SGH:

:Jlxl*:‘l Leu-G]n-As~-P~o-Gln-Thr-Scr-Leu-Cys-~he-Ser-Glu-Scr

HGH:

50

~Gln~As~-Th~~Gln-Val-A~a-~hc-Cys-~hc-Ser-Glu-Thr-Ile-Pro-Ala-Pro-Thr-Gly-Lys-Asn-Glu-~a-

Ilc

45

Gln-Leu-Ala-Ph~Asp-Thr-T~r-Gln-Glu-~he-Glu-Glu-Ala-Tyr-Ile-Pro-Lys-Glu-Gln-L~s-Tyr-Ser-Phe-

SGH:

HGH:

~IIIx:III**I:IIx

SGH:

IllXlI

25 30 35 GIn-Le~-~a-Ala-Asp-Thr-P~e-Lys-Glu-Phe-Glu-Arg-T~r-Tyr-Ile-Pro-Glu-Gly-Gln-Arg-Tyr-Ser-(

HGH: 40

1 5 10 15 20 H-Ala-Phe-Pro-A~-Met-Srr-Leu-Ser-Gly-Leu-Phe-Ala-Asn-ALa-V:al -Leu-Arg-Ala-Gln-His-Leu-Hisx I I . : * IlXll’ll I I I : i H-( )-Phc-Pro-ThrIle -Pr~Leu-Ser-ArgLcu-Ir’he-AsyAsn-Ala-~et-Leu-Arg-Ala-~is-ArgLeu-His-

SGH:

)-