Bovine growth hormone: Evidence for two allelic forms

Vol. 43, No. 1, 1971

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

BOVINE GROWTH EVIDENCE FOR TWO

HORMONE: ALLELIC

FORMS

B. K. S E A V E Y , R. N. P. S I N G H and U. J. L E W I S Division of Endocrinology Scripps Clinic and R e s e a r c h Foundation L a Jolla, California 9Z037 Io Io G E S C H W I N D D e p a r t m e n t of A n i m a l Science, University of California Davis, California 95616

Received F e b r u a r y 2 2 , 1971 SUMMARY. Peptide m a p s of tryptic digests of bovine growth h o r m o n e isolated f r o m individual pituitary glands w e r e of three types. The h o r m o n e contained either an A or a B peptide or a 1:1 mixture of the two. A and B differed by a substitution of valine for leucine. The only difference noted between the ovine and bovine growth h o r m o n e s w a s that the ovine h o r m o n e had neither A nor B but instead a peptide that d~ffered f r o m A by a replacement of a glycine with valine. During a study of the peptide m a p

of bovine growth h o r m o n e ,

we

noted that the a m i n o acid compositions of two peptides w e r e identical except

for one

amino

forms

of the hormone

acid.

Since

this suggested

and that the forms

could

there

genization

hormone in water

was

isolated

to remove

of

isolated f r o m

T h e results are reported here.

MATERIALS Growth

be two

be characteristic

certain breeds of cattle, w e analyzed growth h o r m o n e individual pituitary glands.

might

AND from

blood

METHODS individual

proteins,

glands

extraction

by homoat pH

10,

c h r o m a t o g r a p h y first on Sephadex G - 1 5 0 and then on DEA]E-cellulose (i). In order to separate the two chains the L K B

A m p h o l i n e c o l u m n 8100

w a s used (2). The s a m p l e (50 rag) w a s electrofoeused for 7Z hours in a p H gradient (pH 7 to 9) containing 4 M urea.

189

Separation of the

Vol. 43, No~ 1, 1971

chains

was

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

confirmed

the two peaks

that were

by Ellis et al. (3). of I% NH4HCO for 2 hours

A 2-4 mg

0. i%

et al. (4).

Peptide

When

Similar

sample

at 37 ° with trypsin ketone.

of the NHz-terminal

separated.

3 containing

ehloromethyl Katz

by determination

was

dodecyl

sulfate,

been

of

reported

dissolved

in 0. Z ml

and hydrolyzed

with L-l-tosylamide-Z-phenylethyl-

mapping

the peptides

have

of hormone

sodium treated

results

residue

was

were

carried

out according

to

to be eluted for analysis,

the

paper w a s dipped into a dilute solution of ninhydrin (0.05% in ethanol) and as the spots appeared,

they w e r e cut f r o m the paper, w a s h e d with

acetone and the peptide eluted with 6 N HCI.

After hydrolysis for Z0 hours

at Ii0 ° the mixture w a s analyzed (5). T h e ratio of A and B peptides w a s determined by data f r o m a m i n o acid analysis.

RESULTS Bovine. found

By mapping

that two forms

pituitary

gland

a l:l mixture The

maps

or both.

The

that peptide marked

either

of the two. of three amino

present.

they contained

seen

amino

only when

B e c a u s e growth h o r m o n e

by itself or

A alone,

B alone,

(Table

acid composition,

the corresponding

individual

in Fig.

B were

of leucine

An

we

is shown

peptide

of A and

hormone

by cattle.

for the two forms

valine instead

A' and B' had the same

growth

one of the two modifications

Evidence types:

of bovine

are produced

acid compositions

B contained

A and B and were

digests

of the hormone

contained

were

tryptic

identical

except

I).

spots

The

respectively,

A or B peptide

i.

as

was

has been s h o w n (6) to lose a m m o n i a ,

and also because A and B w e r e m o r e

acidic electrophoretically, w e

believe that A' and B' are dearoidated f o r m s of A and B. The data on the growth h o r m o n e

of individual glands w e r e obtained

190

Vol. 43, No. 1, 1971

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Jr6 i

@@

@ ®

*

@ Q

~

ELECTROPHORESIS

(CATHODE)

Fig. I. Peptide m a p of tryptic digests of bovine and ovine growth h o r m o n e s m a d e by c h r o m a t o g r a p h y in butanol-acetic acid-water (4:1:5) and electrophoresis at p H 3. 7. T h e a m i n o acid compositions of the peptides are given in Table I. The h o r m o n e s gave identical m a p s except for peptides A, B, and C. A and B w e r e found in bovine growth h o r m o n e ; C only in the ovine h o r m o n e . G r o w t h h o r m o n e f r o m individual bovine pituitaries contained either A or B alone or a I:I mixture of the two. A' and B' are thought to be deamidated f o r m s of A and B. T h e peptides circled with dashes w e r e ninhydrin negative and w e r e located by detection sprays (IZ).

f r o m two different groups of pituitaries,

T h e first w a s a group of 1Z

glands r a n d o m l y selected f r o m a pool of about 50 pituitaries purchased from a commercial

supplier (Ersco, San Mateo,

California).

assurance of the b r e e d of the cattle could be given.

Eight of the 12 glands

had a i:i mixture of the A and B f o r m s of growth h o r m o n e . four had only the A form.

No

The other

The second group of individual pituitaries w a s

m a d e up of i0 glands f r o m three k n o w n breeds, Hereford,

Jersey, and

Holstein.

indicated two

Only females w e r e represented.

Herefords w e r e h o m o z y g o u s

Peptide m a p s

for the A f o r m and one the B form; a~l three

Jerseys had the B form; three Holsteins had a mixture of A and B and one

the A form. Bovine

growth

hormone

isolated

from

191

pooled

rather

than individual

Vol. 43, No. 1, 1971

Table I.

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Peptides f r o m M a p s

of Bovine and Ovine

Growth Hormones Peptide Number +

Amino

Acid Composition

asp thr glu Z pro gly leu arg] asp thr glu 2 pro gly val argj asp thr glu 2 pro val leu arg

A B C

Bovine Ovine

I* 2 3 4 5 6 7 8# 9 i0 ii IZ 13

See footnote asp2 thr 3 set 3 glu 3 pro gly Z ala Z cys 2 val ile 2 leu 3 tyr(2?) phe 2 lys arg asp ser glu leu 3 arg asP2 thr 2 ser 2 gly val Z leu phe arg aspz ser ala leu Z lys asp glu 2 gly m e t ile leu 4 lys arg thr Z glu leu tyr arg glu Z phe arg + peptide No. 15 lacking 1 arginine thr glu 3 pro gly ile tyr arg asP3 thr Z glu m e t tyr phe lys arg asp glu 4 ala lys a s p a l a Z v a l leu arg ] ;',-'* glu gly ala ile leu lysJ Poorly resolved

14 15 16 17 18 19 20 21 Z2

glu ser asp asp val leu arg lys asp

+ * # **

val glu thr leu met lys

tyr lys gly ala 2 cys Z phe arg 2 glu 2 ala 2 leu 2 phe lys his lys his lys

leu lys 2 his

C o r r e s p o n d s to n u m b e r given in Fig. i. Peptide i gave inconclusive analyses; this is being studied further. After reduction and carboxymethylation of the h o r m o n e , glu 2 phe arg remained but the cystine-peptide w a s found elsewhere. At times peptide 13 is seen without arginine-positive peptide 12. glands always had the two peptides A and B in a ratio of about Z:l (A:B). T w o such pools of Z4 glands each w e r e analyzed. hormone

obtained f r o m the Endocrinology

large pool of pituitaries,

Likewise,

Study Section,

growth

representing a

contained the two peptides in a Z:I ratio. The

relative a m o u n t s of the peptides found in pooled material would explain the Z:I ratio for leucine and valine found by Fellows and Rogol (7) in their

192

Vol. 43, No. 1, 1971

peptide

TpI ' 2 from

suggested were

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

fragment

except

are in complet It was

e agreement

possible

of the two

hormone

before

did not account

chains

hormone

indicated

data

in the

focusing

both were

to

the same

the two chains was

only peptide

residues.

a difference

After

allelism

having

as NH2-terminal

The

in the

of growth

indicated

A still had work

found

as noted

hormone

also by our alanlne

of Pe~a

for an allelic origin of the two chains

The

peptide

was

as peptide

the same

with valine

seen~

maps

hormone

had

I).

a peptide

and

et al. (8),

was

absent

found,

also

the composition

with these

by Dellacha

results

bovine except

and co-workers

of which

Also, growth

we

except

were

form

was

of peptide growth

not determined,

Our alkaline

did not observe

composition

that glycine

of bovine

hormone.

s

of these

I} whose

the ovine hormone

for the one very

which

(C in Fig.

that maps

hormone

of either

no deamidated

et al. (9) reported

from

growth

Instead

protein

Curiously,

not in the ovine hormone.

spot that was

and ovine

A and B.

A of the bovine

(Table

Dellacha

of bovine

to peptides

had five spots,

that were

ment

by isoelectric

for the leucine-valine

the ovine

hormone

Our

to be the case.

That

two peptides,

C was

which

not found

electrofocusing.

only with respect

replaced

they

this view.

Ovine. differed

but this was

a 2:1 ratio,

no evidence

of two peptides

of valine for leucine.

A and B in about

finding that growth phenylalanine

could be a mixture

that the A and B peptides

separation

peptides

of this observation,

with this explanation.

(8) of the hormone

contain

an explanation

for a replacement

two chains

supports

As

that the tryptic peptide

identical

where

C.

contained

data are in agreepeptide

noted

in our bovine

map.

DISCUSSION Besides

the bovine

and ovine

growth 193

hormones,

we

one

have

determined

Vol. 43, No. 1, 1971

BIOCHEMICALAND BIOPHYSICAL RESEARCH COMMUNICATIONS

the a m i n o acid composition of peptides seen in m a p s these species, h u m a n

growth h o r m o n e

of the prolactins of

and rat growth hormone.

for the type of allelism seen with bovine growth h o r m o n e an allelism has been reported, however, immunoglobulin

kappa chains.

N o evidence

w a s noted.

Such

by T e r r y et al. (i0) for h u m a n

Seven individuals had valine at position

191 w h e r e a s three had both leucine and valine.

Recently M r o s s

et al. (II)

found a glycine-serine allelism in a fibrinopeptide f r o m individual gibbons. The n u m b e r

of individual glands and breeds that we e x a m i n e d w a s

too small to be able to m a k e

definite statements concerning the distribution

of the two forms of growth h o r m o n e

throughout the bovine population.

If

the two f o r m s w e r e expressed in simple Mendelian fashion, w e should find equal quantities of the A and B peptides in growth h o r m o n e

from

pooled glands, instead of the Z:I ratio that w a s noted.

individuals

become

As m o r e

available to us, w e shall be able to tell if s o m e breeds have

exclusively one form.

ACKNOWLEDGEMENT This work was made possible by PHS Grant AM09537 Cancer Society Grant P571. We thank the Endocrinology NIH, for the bovine and ovine growth hormones.

and American Study Section,

REFERENCES i. 2. 3.

4. 5. 6.

Lewis, U. J. and Cheerer, E. V., Endocrinology 8__!i, 1338 (1967). Svenson, H., Acta C h e m . Scand. I__55,3Z5 (1961); Haglund, H., Science Tools, 14, 17 (1967). Ellis, S., Grindeland, R. E., Callahan, P. X., Abstract No. 153, Fifty-second Endocrine Society Meeting, St. Louis, Missouri (1970), p. 113. Katz, A. M . , Dreyer, W. J., Anfinsen, C. B., J. Biol. C h e m . 234, 2897 (1959). S p a c k m a n , D. H., Stein, W. H., Moore, S., Anal. C h e m . 30__, 1190 (1958). Lewis, U. J., Cheerer, E. V., Hopkins, W. C., Biochim. Biophys. Acta ZI__~4, 498 (1970).

194

Vol. 43, No. 1, 1 9 7 1

7. 8. 9. i0. ii. iZ.

BIOCHEMICAL AND BIOPHYSICAL RESEARCHCOMMUNICATIONS

Fellows, R. E. Jr., and Rogol, A. D., J. Biol. Chem. 244, 1567 (1969). Pe~a, C., Paladini, A. C., Dellacha, J. M., Santom~, Ji A., Biochim. Biophys. Acta 194, 320 (1969); Wallis, M., F E B S letters 3, 118 (1969). Dellacha, J. M., Enero, M. A., Santomeq J. A., Paladi~l, A. C., Eur. J. Biochem. IZ___,289 (1970). Terry, W. D., Hood, L. E., Steinberg, A. G., Proc. Nat. Acad. Sei. (U.S.) 63__, 71 (1969). Mross, G. A., Doolittle, R. F., Roberts, B. F., Science 170, 468 (1970) Easley, C. W., Biochim. Biophys. Aeta I0___~7,386 (1965).

195