Identification of two somatomedin a active polypeptides and in vivo effects of a somatomedin a concentrate

Vol. 61, No. 3, 1974

IDENTIFICATION

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

OF TWO SOMATOMEDIN A ACTIVE POLYPEPTIDES AND IN VIVO EFFMCTS

OF A SOMATOMEDIN A CONCENTRATE Linda

Fryklund,

peptide

Knut Uthne

Laboratory,

Received

S-104

October

and Hans Sievertsson, 25 Stockholm,

AB Kabi,

The Recip

Poly-

Sweden.

16,1974

Summary The first chemical characterization of two polypeptides from human serum which stimulate the --in vitro incorporation of 35s sulfate into chick cartilage is described. These two polypeptides, designated Somatomedin Al and A2 have a molecular weight of approximately 7000. Although each peptide contains 1 cysteine residue and has asparagine as amino terminal residue, there are apparent differences in the amino acid composition. Administration of a Somatomedin A concentrate to hypophysectomized rats gave an increase in tibia1 width similar to that obtained with 20 ughuman growth hormone.

Introduction In their --in vitro sulphate

now classical

stimulatory into

dependent

several

anabolic

sulphation

the activity the

[6]

serum from

factor

at least

chemical

properties cartilage

e.g. [6].

of a somatomedin

A. At the

second cartilage

stimulating want

by the

factor.

the growth

Since

then

it

factor

to give

since

of labelled

somepreliminary

957

defined

that different

assomatomedin C, characterized

on the having

of

stimulatory

of hypophysectomized to report

[5]

assay

was demonstrated

a somatcmedin

we want uptake

In 1973 Uthne cartilage

to be due to a factor

of two polypeptides the

in 1972 on the bio-

was tentatively

segments

of

as a moregeneral

chicken

somatomedins,

A concentrate.

Copyright El 1974 b.v Academic Press, Inc. All rights of reproduction in any form reserved.

reported

same time

appeared

communication

characterization We also

sulphation

of a human somatomedin.

van Wyk et al[2]isolatec

In the present first

that

of labelled

was introduced [4]

measured

of cells

A. This in the

found

was due to an intermediary,

Hall

two different

types

B 151. Subsequently activity

[3].

activi~ty

somatomedin

somatomedin

incorporation

named the

the name somatomedin

and purification

on various

by its

they

[l]

have been ascribed to the --in vitro in serum or serum fractions [2]. As a result

somatomedin

contains

effects

of cartilage

activity

as the

and Daughaday

obtained

sulphation

defined

Salmon

of serum on the

which

effects effects

logical Hall

factor

factor

multiple

term for

effect

proteoglycans

hormone

these

study

mice. isolationandthe somatomedin

sulphate

into

data

on --in vivo

A

chicken acticn

Vol. 61, No. 3, 1974

Materials

BIOCHEMICAL

and Methods

The purification the

isolation

of the

procedure

an acid

ethanol

described extract

chromatographed

over This

material

further

being

and amino

time

of ectomy

from the diet

with

free

glycemia

access

with

NaCl per Sweden)

given

100 ml.

The rats Tibia1

were width

assay animal Results

ml

into

hormone

[6].

remaining

step.

as described weighing

groups

End

[7].

60 - 75 g at

defined

serum determined

was tested

at dosages

0.9 g

Stockholm,

last

same medium. injection. et al

of 15 U per rat

as the

inwere

plus

of Greenspan

at a dosage

arbitrarily

human reference

animals

in the the

to the

Subcutaneous

AB Kabi,

was dissolved 24 h after

hypo-

5 days prior

The control

to the method

ectomy,

to prevent

of 2.5 g human albumin

was studied

CrescormonR

week after

water

of 6 rats.

days.

and sacrificed

A is

the first

drinking

(CrescormonR,

according

A preparation

biological

[81. per

day.

activity

by the chick

embryo

of 20 and 40 ng per

and day. and Discussion The first shown

found

in fraction

activity

[5,7].

studies

in rats

gel

filtration

in Fig. A part while

in Figures treatment

on Sephadex

1. The major

1, while

The results shown

During

A preparation

daily

of a normal

the

at pH 1.5 and pH 5.0

performed

to the

5 consecutive

Human growth

of somatomedin

of Hall

was

the somatomedin

in rats,

was discontinued

divided

for

was determined

pattern

following

were

daily

somatomedin

The somatomedin One unit

added

The antibiotic

weighed

studies

(Sprague-Dawley)

0.5 ml of a medium consisting

and the

171. Thus,

from human plasma

G-50 yielding

was used as a final

were

water.

were

The animals were

treated

of one

to drinking

and infections.

jections

et al

out

in the --in vivo study. The rats were purchased Company, Chicago, Ill. and kept on a standard rat chow

and terramycin

experiment.

analysis rats

by Fryklund

electrophoresis

(fine)

and

used

Hormone Assay

5 % glucose

G-50

female

were

in the

by zone

acid

Hypophysectomized the

paper

in rats

have been carried

IV obtained

used

purified

studies

peptides

G-15 and Sephadex

on Sephadex

analysis

A active

of Cohn fraction

was partially

Gel filtration

used in the

in the companion

Sephadex

A concentrate.

group

concentrate

of the two somatomedin

by the

[7].

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

part

fraction

2 contained

of the material the remaining

of the

effects

2 and 3. There with

the

G-50 gave the

of the

on total

G-50-12-1

was further

was

A preparation

was used

B in the

purified.

body weight

was no significant

somatomedin

A activity

most of the somatomedin

of fraction part

chromatographic

somatomedin

and tibia1

effect

widht

on total

whereas

are

bodyweight

20 1~g of human

Vol. 61, No. 3, 1974

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

280 nm 2.000

1.500

1.000

0.500

Figure

1

Gel chromatography on Sephadex G-50 medium (2.5 Flow rate 25 ml/h, fraction size 6 ml. Fraction the SM-A active material [6].

x 100 cm) in 1 % formic acid. G-50-12-l contained most of

0 ControL 0 15U SM-A 0 2opg HGH

I

w

15U

SM-A

cl

HGH 4opg HGH 2Opg

P g 20 .c s 0 days

Figure

a



2

Effect on body weight of hypophysectomized somatomedin A concentrate (G-50-12-1). Figure

P

L

:rats

treated

with

HGH and the

3

Effect on the tibia1 width of hypophysectomized the somatomedin A concentrate (G-50-12-1).

959

rats

treated

with

HGH and

Vol. 61, No. 3, 1974

A230

“m

BIOCHEMICAL

VO

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

VT I

ln5ulin

A 230

nm

r

lflSUlill

v,

1.4.

1.2-

l.O-

II)-

D.B-

Da-

0.6-

06-

04-

Q4"

I

lo

20

30

1

40

.

1

SO

60

70

lo

I

20

I

30

I

1

40

60

60

Fraction Figure

Fmction

4

Gel chromatography of somatomedin A, on Sephadex G-50, fine, 0.02 N HCl. Flow rate 10 ml/h, fraction size 2.3 ml. Fraction sulphate uptake in vitro in chick cartilage [6]. Figure

70

(1.5 x 88 cm) in C increased

5

Gel chromatography of somatomedin A2. Experimental conditions see Figure Fraction C increased sulphate uptake in vitro in chick cartilage [6].

growth

hormone

significantly

caused

a normal

response

affected

by the

administration

a 30 % increase human growth

being hormone

contains

the major

tography

on Sephadex

observed. (Fig.

portion

3).

This

corresponds

the

to a combined

effect

of several

A activity.

This

somatomedin

G-50,

of highly When the (G-50-12-1,

factors, question

purified

somatomedin

somatomedin Fig. l),

A active

somatomedin

to the

effect

A activity

preparation

of peptides of similar the stimulatory effect

tibia1

A preparation

somatomedin

However,

However,

of the

consists of a mixture not be excluded that

amounts

2).

The somatomedin

of the

G-50.

(Fig.

width

of 20 i.lg of

following

be answered

region

was subjected

960

from

rats

chromaand

can therefore be attributed

some of them not necessarily A becomes

in the

homogeneous

molecular size. It on the tibia might

can only

was

A concentrate,

used

is not

5.

carrying

when sufficient

available. the

filtration

to electrophoresis

on Sephadex at pH 7.5 most

of

Vol. 61, No. 3, 1974

Table

I.

Amino

BIOCHEMICAL

acid

composition

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

of two peptides Somatomedin

Aspartic

acid

with

.A1 -

somatomedin

A activity

Somatomedin

A2

3.95

(4)

4.75

(5)

Threonine

4.08

(4)

1.85

(2)

Serine

7.46

(7-8)

3.50

(3-4)

4.84

(4)

4.65

(5)

Proline

Glutamic

acid

7.79

(8)

7.30

(7)

Glycine

9.72

(10)

7.10

(7)

Alanine

4.63

(5)

7.00

(7)

* 1.00

(I)

** 1.00

(I)

Valine

3.81

(4)

4.90

(5)

Methionine

1.00

(1)

1.00

(1)

Cysteine

Isoleucine

1.28

(1)

0.81

(1)

Leucine

2.00

(2)

3.30

(3)

Tyrosine

0.53

(1)

0.97

(1)

Phenylalanine

1.04

(1)

2.20

(2)

Histidine

1.38

(1)

3.75

(4)

Lysine

2.65

(2-3)

2.50

(2-3)

Arginine

3.82

(4)

4.65

(5)

NH2-terminal

Asn

* **

the

determined

as S-carboxymethyl

determined

as Cysteic

activity

further rated further

was found

resolved peaks gel

(peak

acid

in the neutral

on a small

Peak 2 gave the pattern

in somatomedin glycine

analysis A1

stimulated column

shown in Fig.

A 1. Fig. 5 shows the corresponding to somatomedin

as well

region.

This

at pH 5.0,

2 and 3) which

tomedin

End group

cysteine

on electrophoresis

filtered

Asn

Sephadex A2.

yielded

giving

s,ulphate

of Sephadex 4, fraction

asparagine

uptake.

was but

sepa-

Each peak was

G-50 (fine)

in 0.02

C corresponding

N HCl.

to soma-

of peak

3, fraction

as the amino

terminal

residue

A 2. However,

in SM-Al

traces

of threonine

961

material

two adjacent

G-50 purification

as in somatomedin

(< 5 %) and in SM-A2 traces

neutral

(< 10 %) were

also

identi-

C

of

Vol. 61, No. 3, 1974

fied.

The amino

entirely with

acid

analysis

homogeneous the

elution

peptides

is

of labelled dose response as a standard peptides

are

residue

volume

histidine

from

alterations

7000.

curve

into parallel

was not obvious

Both

substances

The chemical

we cannot

converted

the

small

into

the

the

the

between

the two

one cysteine

are the

serine

separated

possibility

in the amino

acid

considered

a

serum used

and the two

that

due to enzymatic

We have also from

However,

of only

chemically

other

incorporation

reference

presence

exclude

together of the two

[cl.

differences

differences

impurities. may derive

weight

similarities

is the

are not

composition

increased

we have apparently

peptides,

acid

of the arbitrary

The most apparent

Although

the two peptides

of chickencartilage

to that

obtained.

have been

two peptides

that amino

G-50 the molecular

and most striking

due to peptide

the

Sephadex

proteoglycans

and that

partially

indicates

Based on the

sulphate

A active

might

1).

approximately

contents.

peptide

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

also

(Table

in each peptide.

somatomedin

that

BIOCHEMICAL

one

or chemical

compositions

are

the possibility

a common "pro-somatomedin"

yet to be

discovered. Acknowledgement: Hospital, to thank cal

Stockholm, Mrs.

The valuable during

M. Hammarlund,

this

support

work

fromDr.

is gratefully

M. Liw&ng

and T. Uthne

K. Hall

at the

acknowledged. for

their

Karolinska

We also skilful

want

techni-

assistance.

Literature 1. 2. 3. 4. 5. 6. 7. 8.

Salmon W.D. jr and Daughaday W.H. (1957) --J. Lab. Clin. 49, 825-836. van Wyk J.J., Underwood L.E., Lister R.C. and Marshall R.N. (1973) Am. J. Dis. Child. 126, 705-711. ----Daughaday W.H., Hall K., Raben M.S., Salmon W.D. jr, van de n Brande J.L. and van Wyk J.J. (1972) Nature 235, 107. Hall K. (1972) Acta Endox Suppl 163, l-52. Uthne K. (1973) Acta Endocrinol Suppl 175, l-35. Hall K. (1970) AzEndocrinol 63, 338-350. Fryklund L., Uthne K., Sievertsson H. and Westermark B. (19 74) Biochem. Biophys. Res. Conunun (Companion paper). Greenspan F.S.I.H., Simpson M.E. and Evans H.M. (1949) , Endocrinology 65, 455-463.

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