Int. J. Peptide Protein Res. 8, 1976, 4 3 5 4 Published by Munksward Copenhagen, Denmark No part may be reprodudd by any proeess without written permission from the author6)

PRIMARY STRUCTURE OF EQUINE GROWTH HORMONE M. M. ZAKIN,E. POSKUS, A. A. LANGTON, P. FERRARA, J. A. SANTOMB, J. M. DELLACHA and A. C. PALADINI Departamento de Qufmica Bioldgica, Facultad de Farmacia y Bioquimica, Universidad de Buenos Aires and Centro para el Estudio de 14s Hormonas Hipojsarias, Buenos Aires, Argentina

Received 15 July 1975 The study of the structure of the peptides arising from native, oxidized, reduced and alkylated or citraconylated equine growth hormone on incubation with trypsin and chymotrypsin is reported. The data obtained, combined with results already published, support the assembly of a unique sequence of amino acids for the polypeptide chain of the protein.

Equine growth hormone (EGH) was isolated by Saxena & Henneman (1) and Hartree et al. (2) who determined its amino acid composition and N- and C-terminal residues. In 1973 Conde et al. (3) described a new procedure for obtaining EGH of high purity, and basically confirmed the structural data obtained by the mentioned authors. Oliver & Hartree in 1968, reported the structure around both disulphide bridges present in the molecule (4). Further studies in our laboratory, using the Conde et al. preparation of EGH, established the structure of a 62 amino acid fragment which included the two cystines in the molecule (5). These results did not confirm the findings of Oliver & Hartree on one of the cystine bridges. In connection with immunological studies we have recently isolated and characterized the cyanogen bromide fragments obtainable from this hormone (Poskus et al., unpublished results) and in the present paper we describe the amino acid composition and sequence of the tryptic and chyrnotryptic peptides. This last information, together with that already published (9, leads to the unambiguous establishment of the primary structure of EGH molecule. A preliminary report of this investigation has been published (6).

EXPERIMENTAL PROCEDURES

Materials EGH was prepared by the method of Conde et al. (3). This protein was homogeneous as judged by disc electrophoresis, ultracentrifugation, gel electrofocusing, gel filtration and end group analyses. Its growth promoting activity was 1.6 USP unitslmg. TPCK-treated trypsin and a-chymotrypsin were from Worthington Biochemical Corp., thermolysin was from Calbiochem, carboxypeptidase A and B were from SchwarzIMann. All other reagents were of AR grade. Methods Chemical modifcations of EGH. Performic acid oxidation, and reduction and aminoethylation of the protein were done as previously described (5, 7). Reduction and carboxymethylation was performed as indicated by Crestfield et al. (8). Citraconylation of the protein was carried out by the method described by Gibbons & Perham (9). Enzymic digestions. The hormone, either in the native state, oxidized or reduced and aminoethylated, was digested with trypsin as indicated 435

M. M. ZAKIN ET AL.

by Seavey et al. (10). The ratio enzyme:protein was 1 to 66 w/w. For chymotrypsin digestions the conditions were as follows: 200 mg of reduced and carboxymethylated hormone were suspended in 66 ml of water and adjusted to pH 8.0with0.3 M potassium hydroxide. The pH was maintained constant with the use of a pH-stat. Other parameters were: temperature 35”C, initial ratio enzyme:protein of 1 to 50 w/w, further additions of enzyme after 4 and 15 h of incubation brought this ratio successively to 1 to 25 and 1 to 17. Total digestion time was 20 h. At the end of the incubation period the pH was lowered to 3.0 with concentrated acetic acid and the reaction mixture was lyophilized. Chromatographic solvents. The solvents used for paper chromatography were: solvent 1, butan-1 01-acetic acid-water (4: 1 :5 , by vol.); solvent 2, butan-1-ol-acetic acid-water-pyridine (15: 3: 12: 10, by vol.); solvent 3, butan-1-01-concentrated ammonia-water-ethanol (50:2 :18 :15, by vol.); solvent 4, butan-1-ol-formic acid-water (15:3 :2, by vol.) and solvent 5 , butan-1-ol-formic acidwater (9 :9 :2, by vol.). Tryptic and chymotryptic maps. Peptide mapping of native or modified EGH was made according to Zakin et al. ( 5 ) using 5 mg of digested protein; solvent 1 was used for the descending chromatography run. The paper band containing the neutral peptides was cut out, stitched to a new sheet of Whatmann 3MM paper and submitted to descending chromatography at right angles to the original direction. Solvent used was number 2. The peptides, located with 0.02% ninhydrin solution in acetone, were eluted with 50% formic acid or water and these solutions were used for amino acid analyses or sequence studies. Fractionation and purification of chymotryptic peptides. The lyophilized chymotryptic digest was dissolved in 0.2 M pyridine-acetate buffer, pH 3.1 and the solution was chromatographed at 40°C on a column (0.9 cm x 180 cm) of the sulphonic resin Aminex AG 50W-X2 (120 to 325 mesh) equilibrated with the same buffer. The elution was started by a linear gradient obtained with 800 ml of the buffer of pH 3.1 and an equal volume of 2 M pyridine-acetate buffer of pH 5. Fractions of 2.2 ml were collected at a flow rate of 11 ml/h.

436

Aliquots from every third tube until fraction 450 were analysed by the Rosen reaction (11). Appropriate fractions were pooled and stored at 4°C until used. All pooled fractions were submitted to high voltage electrophoresis at pH 6.5 for 60 min at 60V/cm. If necessary, further purification was achieved by paper chromatography, using solvents 2 (descending chromatography), 3 or 4 (ascending chromatography). Fragmentation of peptides. Thermolysin digestion was carried out on some large peptides. The experimental conditions were: 2 h at 40°C in a solution 20 mM in ammonium acetate and 0.5 mM in calcium chloride, pH 8.6, with a ratio enzyme:substrate of 1 to 20 w/w. Partial acid hydrolyses of peptides were performed in 12 M hydrochloric acid as described in ( 5 ) or in 0.03 M hydrochloric acid as indicated by Tsung & Fraenkel-Conrat (I 2). Amino acid analysis and sequence determinations. Amino acid analyses were performed as previously described (7). Tryptophan was determined by the method of Matsubara & Sasaki (13). Edman degradations were carried out by the procedure described by Elzinga (14) or by the ‘dayfsyl’ Edman method (1 5 ) . The ‘dansy1’-amino acids were identified by two dimensional thin byer chromatography (16). Carboxypeptidase digestions ere performed as described by Santomd et al. (17). Hydrazinolysis was carried out by the procedure indicated by Braun & Schroeder (18). Amide residues were assigned on the basis of electrophoretic mobilities of peptides at pH 6.5 (19). In some cases this method was applied to the Edman degradation products. Nomenclature The peptides obtained from the tryptic, chymotryptic and peptic hydrolysates bear the prefix T, C and P,respectively, and are numbered according to their position in the sequence.

RESULTS

Tryptic peptides A summary of the isolation procedure and

analysis of the tryptic peptides isolated from

4

w P

1.0

52

C

N

N.D.~ + I

0.9

1.7

1.0

0.9

0.6 1.0

3.4

0.7

2.5

2.0

1.5

1.1

N

N.D.

0.7 1.8

2.3 1 .o

2.8

2.2

1.1

I .o

N

-1

0.8

1.1

2.0

1 .o

N

N.D.

1.0

1.1 2.0

1.8 0.8 0.8

2.6

4.4

1.o

0.8

52

N

53 54

A

A

0.7

N.D.

o

0.8

0.7

0.8

1.5

1.2

1.6

1.0 4.0

2. I

0.9

1.0

+I

-1

1.0 0.7 1.1

1.1

1.1 2.4 2.1 1.0 1.3 0.8 2.7

1.0

N

-1

1.1

1.1

3.1

0.7 1.0

C

-1

1.2

2.1

0.9

N

-2

1.1

1.0

0.9 0.8 1.2 1.1

1.3

52

N

*

0.7

1.7

1.1

o

+

1.1

52

N

o

1.o

0.8

1.7 0.8

1.2

N.D.

0.9

1.3

0.8 5.8

1.0

0.9 0.9

2.5 2.0

1.8 I .9 1.8 1 .o 2.1 1 .o 1,0

I .o 1.1

0.8

0.8

0.9

N

N

N

N

+ I -2 -2 + I

1.0 0.8 0.9

0.9 0.8 2.0

0.9

1.0 1.0 1.2

1 .o

3.2

0.8 1.1

52

N

0

1.2

1.1

1.2

1 .o

1.0

1.2

2.6 2.0

0.9

1.1

1.1

N

52

O

0

1.0 0.8

-1

2.2

1.1

1.1

A

+I

+

0.9 0.9

2.1 2.1

1.1

0.8 1.1

2.2 0.8 0.7

A

+I

1.0

1.0

1.1

0

+1

1.5

1.1

1.o

2.2

1.1

I .o

0.8

N

4-1

1.0 0.8

1.1

0.8

52

N

0

0.7

1.o

1.0

0.6

1.1

1.1

N

+I

1 .o

0.7

I .o

O

0

1.0

1.0

0

+I

1.6

1.0

0

-2

1.7

0.8

0.9

1.0

1.7

1.1

52

N

0

1 .o

1.7

I .8 0.8

0.9

1.0

1.7

*

'

Peptides were obtained from fingerprints as indicated in Methods; they are presented in their correct sequence order. Fragmtnts originated by unspecific enzyme cleavage aTe indicated by an additional capital letter. New peptides originated by specific enzyme cleavage on aminethylated hormone are indicated by an additional lower-case letter. Estimated net charge at pH 6.5 of the peptides is indicated. a CySOsH, cysteic acid. AeCys, arninoethylcystcine 'Determined by the method of Matsubara and Sasaki(l4). N.D., not determined. N. native; 0, oxidized; A, arninocthylated; C. citracomylated hormone. S, chromatographic solvent; the numbers identify the composition as indicated in Methods. Isolated from citracomylated EGH by electrophoresis at pH 6.5 and paper chromatography using solvent 5 (see text).

TV Phe AeCysb LYs His Arg TrpC Net charge Derivative used for mappingC Further fractionation procedure'

Leu

GIY Ala HallCys Val Met Ile

Glu Ro

CyS03Ha ASP Thr Ser

M. M. ZAKlN ET AL.

TABU2 Sequence studies on tryptic peptides

Pcptide

Terminal residues numbers 1-16 13-16 17-29

30-33 17-33 34-41 42-52

Sequence Phe(Pro,Ala,Met,Pro,Leu,Ser,Ser,Leu,Phe,Ala,Asn,Ala,Val,Leu)Arg

+ + Ala-Glx-His(Leu,H is,Glx,Leu,Ala,Ala.Asx,Thr,Tyr)Lys +++ Glu(Phe.Glu)Arg + Ala(Va1,Leu)Arg

Ala-Glx-H is(Leu,His.Glx.Leu.Ala.Ala.Asx,Thr.Tyr)Lys-Glu(Phe,Glu)Arg Ala(Tyr.lle,Pro.Glu,Gly)Gln-Arg

+ c c +++++++ t t Phe-Ser-Glu~Thr-lle-Pro-Ala-Ro~(Thr,G ly-Lys.Asx.Glx, Ala,GIx.Glx)Arg + + +++ b AspGlu-Ala-Gln-Gln-Arg ++++ Ser-AspMet-Glu-Leu-Leu-Arg ++++

Tyr-Ser-Ile-Gln-Asn-Ala-GIn-Ala-Ala-Phe-AeCys I

53-63

+I++

64-69 70-76 70-74 77-94

Ser-Asp-Met-Glu-Leu Phe-Ser-Leu-Leu-Leu-lle(GIx,Ser,Trp,Leu,Gly,Pro,Val,Glx, Leu,Leu,Ser)Arg

95-I07

Val(Phe,Thr,Asx,Ser,Leu,Val.Phe,Gly,Thr,Ser.Asx)Arg

108-1 I I

112-113

++ + + + + + Val(Tyr,G lu)Lys + Leu-Arg

...

Th

114-124

I

Asp(Leu,Glu,Glu,Gly)lle-(GIn,Ala)(

+ + +

+

125-132

Glu-Leu(Glu,Asp.Gly,Ser.Pro)Arg

I334 38

Ala-Gly-Gln-lle-Leu-Lys

Leu,Met)Arg

4 4 44

'I

ELECTROPHORESIS

-

B 438

FIGURE I Tryptic map of native EGH (A)and chymotryptic map of reduced and carboxymethylated EGH (B). Neutral peptides in A were purified as indicated in Methods. Peptides selected for sequence studies in B are shaded.

PRIMARY STRIJCIWRE OF EQUMB GROWTH HORMONE

Peptide

Terminal residues numbers

muena

T15-16

139-149

T17

150-1 56

Ti 8

157-165

TIE.

157-163

Tim T20

164-165 157-166 167-1 70

Phc-Lys Asn-Tyr-Gly(Leu,Ltu.Ser)CySOtH-Phe-Lys-Lys AspLeu-His-Lys

Tz I

171-176

Ala-Glu-Thr(Tyr,Leu)Arg

Ti2

177-179

Val-Met-Lys

T.73 T23-24 Tzs

180-181 18&182 183-190

CyS03H-Arg CyS03H-Arg-Arg Phe(Val,Glu,Ser,Ser,CyS03H)Ala-Phe

T23--21

18&181 183-190

Tia-19

Gln-Thr(Tyr.Asx,Lys,Phe,Asx,Thr,Asx)Leu-Arg

++ c c ++++ Asn(Tyr,Gly,Leu,Leu,Scr,CySOJH,Phe)Lys + Asn-‘Llr-Gly(Leu,Leu,Ser)AeCys +++ Scr-AspAspAla-Leu-Leu-Lys

++ +-++ -+

+

f

I

1

Cys-Arg

t

Phe(Val,Glu,Ser,Cys)Ala-Phe

+,Residue found by “dansyl” Edman method or by the subtractive procedure; C, residue liberated on treat-

ment with carboxypeptidase A and/or B;*,residue determined by hydrazinolysis; Th, fragments obtained by further digestion of peptide Tl2 with thermolysin; points of selective rupture in peptides submitted to partial acid hydrolysis. The position of lysine, arginine or aminoethylcysteine in the peptides was assumed generally from the known specificity of trypsin. The sequences suggested for the regions in parentheses arc the correct ones found by other experiments in this work (see Table 4). maps of native, oxidized, reduced and aminoethylated and citraconylated EGH is given in Table 1 and their complete or partial sequences are presented in Table 2. In Fig. 1A is shown the tryptic peptide map of native EGH. Tryptic maps of oxidized, and reduced and aminoethylated protein have been published elsewhere (5). Peptides TI, T, and TI were obtained only from citraconylated-EGH by the following procedure : T1 was isolated from the peptide map applying the procedure for the purification of neutral tryptic peptides as indicated in Methods. TI is the peptide with the greatest Rr value; T7 was obtained directly from the peptide map of citraconylated hormone. Its electrophoretic and chromatographic mobilities resemble those of TI in the tryptic map of native EGH; peptide T, was

isolated by electrophoresis at pH 6.5 for 60 min at 60V/cm of the tryptic digest of citraconylated protein. The band in which the product was originally applied was cut out and stitched to a new sheet of Whatmann 3MM paper; submitted to ascending chromatography with solvent 5, it ran with a Rr near to 1. For sequence studies, some large tryptic peptides were submitted to further cleavage in order to obtain smaller units. Tlb (Table 2), after partial hydrolysis in concentrated acid, yielded a pentapeptide (Thr2,Pro, Ala, Ile) which was obtained pure by ascending bidimensional chromatography using solvents 3 and 4. Peptide T l z (Table 2) was digested with thermolysin and the fragments were purified by high voltage electrophoresis at pH 6.5.

439

0.7

S3 S4

0 E

0.8

1.0

0

E

E

1.0

0.9

0

0.9

I .a 1.0

I .o

+2

I .O 0.8

1.2

0.8

1.1

~~

E

3-1

0.8

1.0

1.0

E

0

0.6

1.2 0.9

1.6

-I E

1.3 0.8

1.6

1.0

1.1

1.1

0.9

1.2

1.0

-I E

1.1

1.7

1.0

1.0

0 E s3 s4

I .O 0.7

1.0

E

0

1.0

0.9

1 .O

1.0

E

0

1.0

1.0

1.0

2.0 0.8 1.3

2.1

0.8

1.0 1.2

1.0 3.5

2.1

sz

N.D. E

N.D. N.D. E

0.8

N . D . ~ 0.7

0.9

1.2 0.6 2.1

5.2 1.9 1.1 1.7

2.0

+

2.0 2. I

+I

1.0

1.o

0 E

2.3

1.0

0 E

+

0.8 1.1

1.3

1.0

E

0

2.9

0.9

1.2 0.9 1.0

+I

I .o

1.0

1.2

0.9

E

0

1.2

1.1

1.1

1.0 1.1

0.9

0.7

1 .o 3.0

1.2 1.1

3.8

0.9

E

0

E

-2

0.9 0.8

0.8

1.0

0.9

1.1 1.2 1.1

E

0

0.9

1.2

E

-1

1.5

2. I

0.7

0.9

0.7

2.3

3.1 0.8

I .O

0.9

+ I E

0.9

0.9

1.0

1.2

-2 E

0.6

1.0 0.6

3.4

0.7

4.6 1.1 1.3

0 E

0.8 0.8

1.2

s4

E

s3

-2

0.6

3.2

0.8

1.o

1.1

3.5

+I E

0.8

1.1

0.9

E

0

2.2

0.7

-I E

I .2

0.7

+

E

0

0.8

1.o

I .o

I .O

I .2

+-I E

0.6

1.1

I .O

1.3

-2 E

1.2

1 .O 1.2

I .9 1.4

+

Peptides are presented in their correct sequence order. The peptides indicated by a number with the addition of a prime are fragments of the larger peptide bearing the same number. The first step in the procedure used for the isolation of most peptides was ion exchange chromatography. Peptides C4, C l o and C,, were obtained from peptide map of reduced and carboxymethylated hormone. When further purification of peptides was necessary,electrophoresis at pH 6.5 or paper chromatography were used. Estimated net charge at pH 6.5 of the peptides is indicated. a CMCys, carhoxymethylcysteine. b N.D., not determined. C Determined by the method of Matsubara and Sasaki(l4). d E, high voltage electrophoresis at pH 6.5; S, and S. as indicated in note f, Table I .

His Arg TrpC Netcharge 0 Further E Fractionation S3 Procedured S4

LYS

1.0

LCU

0.9

0.9

2.0

1.5

~____

Tyr Phe

CMCysP ASP Thr Ser Glu Pro G ~ Y Ala Val Met Ile

LS

C20Cn c2:

-

0.4

C

L

c

P 0.3

FIGURE 2 Elution pattern of the chymotryptic peptides obtained from reduced and carboxymethylated EGH chromatographed through a column of Aminex AG 50 WX2, 120 to 325 mesh, 0 9 cm x 180cm, a t 40°C. The pyridine-acetate buffers used for elution are indicated in Methods; the rate was 11 ml/h. Fractions of 2.2 ml were collected and aliquots from every third tube were analysed by the Rosen reaction (11). Fractions pooled are indicated.

u c m Q,

n

8 0.2 u7

2 0.'

o -ioo

200

400

300 Fraction No

' 8-19 1C C ,s ,-r t

"7

8

1 w to 170 ll+tol= 190 L.u-Leu-krWI-Ru-CII-LE-Alsp-Leu-lllr-~I-Ala-Gl u-Thr-Tyr-Leu-Arg-Val - h f - L J I - C ~ - ~ P - ~ g - P k - V -Glu-Ser-kr-tys-Alr-PhrQI al +'ZO21.' k'ZZ---( 234 T25-

9 &23-24+

-C24-

PZ

p c 2 5 +

Pl

'

'23-25

'21

FIGURE 3 Amino acid sequence of EGH. T, C and P indicate tryptic, chymotryptic and peptic peptides respectively, obtained from native or modified hormone. 1indicates point of cyanogen bromide cleavage. The structure of the peptic peptides and cyanogen bromide fragments have already been published (5 and Poskus et al., unpubl. results). 44 1

M. M. ZAKIN ET AL.

TABLE 4 Sequence studies on chymtryptic jwpzides

Peptide

Terminal residues numbers 1-6 7-10 11-15

Sequence Phe-Pro-Ala-Met-Pro-Leu

+++++ Ser-Ser-Leu-Phe +++ Ala-Asn-Ala-Val-Leu ++++

14-15 16-20

Val-Leu Arg-Ala-Gln-His-Leu

16-18 2 1-28

Arg-Ala-Gln

22-28 24-28

Gln-Leu(Ala,Ala,Asp,Thr,Tyr) Ala-Ala-Asp-Thr-Tyr

29-3 1 32-35

Lys-Glu-Phe Glu-Arg-Ala-Tyr

36-42

Ile-Pro-Glu-Gly-Gln-Arg-Tyr

52-75 62-75

CMCys-Phe-Ser-Glu-Thr-Ile-Pro-Ala-Pro-Thr-Gly-LysAspGlu-Ala-Gln-Gln-Arg-Ser-Asp-Met-Glu-Leu-Leu Gly-Lys-Asp-Glu-Ala-Gln-Gln-Arg-Ser-Asp-Met-Glu-Leu-Leu

16-17

Arg-Phe

78-80 81-85

Str-Leu-Leu Leu-Ile-Gln-Ser-Trp

86-92

Leu-Gly-Pro-Val-Gln-Leu-Leu

93-96

Ser-Arg-Val-Phe

97-102

Thr-Asn-Ser-Leu-Val-Phe

-%

His-Gln-Leu(Ala,Ala.Asp,Thr.Tyr)

+++

++++

+++ ++++ +

+

++ ++

+++++ +

++-,ttt 103- 109

GIy-Thr-Ser-Asp-Arg-Val-Tyr

110-122

Glu-Lys-Leu-Arg~As~Leu-GIu-Glu-Gly-Ile-Gln-Ala-Leu

113-1 14 123-137

Arg-Asp

138-141

Lys-Gln-Thr-Tyr

142-155

Asp-Lys-Phe-Asp-Thr-Asn-Leu-Arg-Ser-AspAsp-Ala-Leu-Leu

142-144 145-1 55

Asp-Lys-Phe Asp-Thr-Asn-Leu-Arg-Ser-Asp-Asp-Na-Leu-Leu

+ + +

I

I

I I + + +

I

C

f

Met-Arg-Glu-Leu-GlufAsdGly-Ser-Pro-Arg-Ala-G ly-Gln-lle-Leu I

'++

+++

++++ 4

442

f

I

PRIMARY STRUCTURE OF EQUINE GROWTH HORMONE

~~

Peptide

Terminal residues numbers

Sequence ~

c z1 c 22 c 23

c 2 4

CZ5 cZ6

156158 159-161 162-164 170-1 74

Lys-Asn-Tyr Gly-Leu-Leu Ser-CMCys-Phe Lys-Ala-Glu-Thr-Tyr

175-178 184190

Leu-Arg-Val-Met Val-Glu-Ser-Ser-CMCys-Ala-Phe

++ + +

-%

+++

The sequence of residues deduced from tryptic peptides (Table 2) is included. Conventions used are identical to those of Table 2. Chymotryptic peptides

The majority of the chymotryptic peptides were isolated by ion exchange chromatography of a digest of reduced and carboxymethylated hormone. Fig. 2 shows the elution pattern obtained. Only three peptides were isolated by peptide mapping of the reduced and alkylated hormone (Fig. 1B). The amino acid composition of all the peptides together with details of the methods used to isolate and purify them are presented in Table 3. Sequence studies are summarized in Table 4. The fragments are numbered and presented in their correct sequence order.

by homology considerations (6) is known to be aspartic acid (Table 1, peptide Tzo). EGH has a polypeptide chain with 190 amino acid residues, a molecular weight of 21,752daltons and the following amino acid composition, expressed in molar ratios: Cys4Aspl lAsnsThrsSerl~G1ul #3lnl2Pro7 Glys Alal, Met4 Val, Iles Leuls Tyr, PheI2 LysloHisrArgl *Trp,. The two disulphide bridges are formed by residues 52-163 and 180-188 respectively, as previously reported (5, 6). The primary structure of EGH established in this work, gives further support to the existence of a close analogy between all mamalian growth hormones (20-29).

DISCUSSION

All the information collected in Tables 1,2,3 and 4 is organized in Fig. 3 to give a unique sequence of amino acids for EGH. The sequence shown incorporates also the data previously published by us on the structure of the disulphide bridges (5) and of the cyanogen bromide fragments (Poskus et al., unpublished results). The sequences 16-17, 137-138 and 156-157 where the overlap is minimal (Fig. 3) are tentatively accepted since they belong to regions of the molecule which are highly homologous with the corresponding ones in human (20, 21), bovine (22-25) and ovine (26, 27) growth hormones. The resulting structure accounts remarkably well for the amino acid composition of the protein (3) and of the cyanogen bromide fragments (Poskus et al. unpublished results). Position 167 reported previously as asparagine

ACKNOWLEDGEMENTS

We thank Miss Dora M. Beatti for excellent technical assistance. M. M. Zakin, J. A. Santome, J. M. Dellacha and A. C. Paladini are Career Investigators of the Consejo Nacional de Investigaciones Cientificas y Tecnicas de la Rep~blicaArgentina. This work has been supported in part, by grants from the same Institution, from the Universidad de Buenos A i m and from the Regional Program of Scientific and Technological Development of the O.A.S.

REFERENCES

1. SAXENA, B.B. & HENNEMAN, P.H. (1966) Endocrinology 78, 561-567. A. S., MILLS,J. B., WELCH, R. A. S. & 2. HARTREE, THOMAS, M. (1968) J. Reprod. Fertility 17, 291-

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