Amino acid composition of bovine ‘proinsulin’

Vol. 29, No. 3, 1967

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

AMINO ACID COMPOSITION

ReceivedSeptember

29, 1967

The structure and the

pancreas tence

of insulin

the

same embryonic

the

works (1965)

establish

existence

this

hy,pothesis

using

rabbit

materials.

the

existence

in

a separate

incubation rat in this

islets. crystallized purified

protein

pancreas

on fish

islets

calf

islet

insulin

this

existence amino

as the show

in details

et al

tissues

to

years,

period

'proinsulin'

adenoma

failed

laboratory

be reported

of

the

have

.past three

Steiner

and the

and by Wang

pancreas

during

and will

communication,

are

the

Recently,

of human

e_t a.&, 1965).

In this

during

formation

an attractive

extract

so obtained

on the

bovine

exis-

and fetal

communication.

In this

made the

have

pancreatic

such a protein

in vitro

type

(Givol

(1965)

pancreas

196733) have reported

exocrine

of such a protein.

Results of

and the

investigators

on bovine

of two poly-peptide

endocrine

endocrine

has been explored

ligated

source

of

by Humbel

and Carpenter the

cell

in the

to a number

composed

of the

of a 'proinsulin'

However,

being

differentiation

from

hypothesis

'PROINSULIN‘

C.C. Yip and B.J. Lin and Best Department of Medical Research Best Institute, University of Toronto Toronto, Ontario, Canada.

Banting C.H.

chains

OF BOVINE

(1967a, from

the

and isolated of a 'proinsulin'

acid

composition

from

Dr.

of

presented.

METHODS ---AND MATERIALS Crystallized

bovine

insulin

was a gift

382

A.M.

Fisher

BIOCHEMICAL

Vol. 29, No. 3, 1967

of Connaught

Medical

chromatography

Research

was performed

(2.5

x 90 cm) equilibrated

(1.0

x 50 cm) was used

with

1M acetic

Cary-15 by the

method

1M acetic

(G-50,

acid.

analytical

The eluate

column was done

275 w

activity

(1964). acid

smaller

at

Insulin

120 amino

column

Elution

was monitored

and Berson

column

fine)

A

purpose.

spectrophotometer.

in a Model

Preparative

on a Sephadex

for

of Yarlow

was performed

Laboratories,

to

acid.

recording

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Amino

a

was assayed

acid

analyzer

with

analysis

(Spa&man

et_

al. - (1958).

RESULTS Analytical

column

recrystallized seen that

bovine

insulin

this

material

the

heavier

of U.V.

for

of

is

amount in

the

1 hr at

shown in Figure material

pooled

in

insulin

elution

volume

product

and of the

assays

of this

l/200,000 (Figure

dilution,

the

showed that

insulin

ahead of

clearly

the and

trypsinization in

the

formation

was similar

to

Immune-electrophoresis

1~).

original

they

maybe

position

resulted

one of which

(Figure

It

emerged

Limited

protein

of protein,

material

1B showing

material.

37O) of this

1A.

Re-chromatography

acid.

species

its

Figure

of protein

of two smaller in

of 2 mgm of a 4 times

shown in

1M acetic

is

absorbing

homogeneity

(*g/ml

insulin

a significant

of monomeric

absence

chromatography

material,

both

were strongly

at

immunoreactive

2). Results

of amino

protein

after

hydrolysis

bwine

insulin.

acids are

analysis

of the

shown in

Table

383

purified I in

heavier

comparison

to

Vol. 29, No. 3, 1967

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

A

0.44 0.3.

INSULIN 0.2 .

*

.02-

k .OlY iB

70

60

50

ELUTION

Figure

1 - Column

bovine

is proinsulin'.

&

after

same Sephadex

The area

insulin.

column.

trypsin

The fractions

marked

insulin

on Sephadex

Re-chromatography

limited

for

ML

chromatography

of crystalline

the

VOLUME

40

C.

treatment by the

Chromatography

stripes

immunoactivity.

384

(1.0

indicated of

on the

G-50

x 50 cm)

by the

'proinsulin'from of

A on

'proinsulin'

same Sephadex

were combined

stripes

column.

and assayed

Vol. 29, No. 3, 1967

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Figure

2 -

Immunoelectrophoresis assay of insulin activity. The immuno-assay was performed by the method of Yalow and Berson (1964). The tracings represent the scanning of the elecrf~f~~~~~~~t~;f;;~ for A. Contained only 1131xnsulin (Porcin) and guinea pig anti-insulin (Porcin) antibody; B. Addition of l/200,000 diluted 'proinsulin' material shown in Figure 18; C. Addition of l/200,000 diluted trypsinized 'proinsulin' material shown in 1C. (F-free insulin, Bantibody-bound insulin).

DISTANCE

DISCUSSION It

may be concluded

than

monomeric

This

protein

of the

insulin.

be converted

ine

is

also it

likely

to a smaller

and it

is

that

medium

crystallized

larger

bovine

of insulin

and of its

molecule,

like

the

any amino that

385

size

amino

acid

acid

acid

residues

the

amino

insulin

and can

analysis

which shows

absent acid

com-

of mono-

of insulin,

Amino

insulin. in view

to nor a multiple

immunologically

apparent

a protein,

an aggregate

reactive.

contain

data in

identical

behaves

immunologically

insulin,

the

not

elution

neither

It

does not

from

is present

of the

which

meric

that

is most

acidity

position

is

insulin,

AND CONCLUSION

in bov-

composition

Vol. 29, No. 3, 1967

Table

Amino

I.

BIOCHEMICAL

Amino

acid

Acid

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

composition

males/100,000 'Proinsulin'

of bovine

4

'proinsulin'*

rotein Bovine

Insulin**

LYS

14.5

" 2.1

14.5

Hist

26.6

+ 1.2

34.5

-63

30.8

+ 1.8

16.3

Asp

50.4

+ 1.1

53.0

Thr

17.0

2 0.4

17.3

Ser

47.1

+ 1.2

53.0

Glu

151.8

+ 1.2

115.8

Pro

42.8

GUY

0

16.7

117.1

+ 0.3

70.5

Ala

68.0

4 2.0

44.8

Half-Cys

83.9

f

2.6

52.0

Val

86.9

f

1.6

71.0

11.6

2 1.6

10.0

Leu

116.5

2 0.1

101.0

'Wr

61.5

+ 0.2

66.0

Phe

51.3

+ 4.2

49.8

***Ileu

Met Try * ** ***

+

0 not

0

determined

0

22-hr hydrolysis (duplicate) Corfield, M.C. and Robson, A., Biochem. J. 84, 146 (19621, cited in the Proteins, Vol. 1, p-48. ed. Neurath, H., Academic Press, New York (1963). 70-hr hydrolysis

386

BIOCHEMICAL

Vol. 29, No. 3, 1967

of the

'proinsulin'

significance before mined.

The value interest

it

an excess

of glutamic,

that

'proinsulin'

is

about

the

that appears

that

be adequately

of the

'proinsulin'

from

Table

glycine 2 in

to meet

I that

protein of these

of isolated

rat

e_t a&,1967).

is

present

the

criteria

The discussed is

the

and proline

this

2 (Steiner a protein

of insulin.

cannot

incorporation

fraction

of approximately

which

weight

is apparent

of Leu/Phe

may be stated

insulin,

it

from

differences

molecular

to note the

a ratio

exact

However,

contains

into

is different

of these

the

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

deter-

'proinsulin'

residues.

and it

is

two amino islets

also

of acids showed

In summary,

in crystallized

bovine

of a 'proinsulin'.

REFERENCES Givol,

If., DeLorenzo, F., Goldbergar, R.F. and Anfinsen, C.B., Proc. N.A.S. 53, 676 (1965). 53, 853 (1965). Humbel, R.E., Proc. N.A.S. 30, 1190 Spa&man, D.H., Stein, W.H. and Moore, S., Anal. Chem. (19582 Steiner, D-F., Cunningham, D., Spigelman, L. and Aten, B., Science 157, 697 (196733). Steiner, D.F. and Oyer, P.E., Proc. N.A.S. 57, 473 (1967a). ?lang, S.S. and Carpenter, F-H., J. Biol. Chem., 240, 1619 (1965). Yalow, R.S. and Berson, S-A., Methods of Biochemical Analysis, Interscience Publishers, Inc., N.Y. (1964). Vol. XII, p.69,

387