The action of interstitial cell-stimulating hormone upon tyrosinase activity in the weaver bird (Steganura paradisaea )

Vol. 20, No. 6,1965

BIOCHEMCAL AND BlOPHYSICAL RESEARCH COMMUNICATIONS

The Action

of

Interstitial

upon Tyrosinase

Cell-Stimulating Activity

(Steganura Kimihiko Department

of

cause

The black

of ICSH

is

to

in the

hormone feathers

resulting

1960).

It

biosynthesis

(Lerner,

of melanin

of melanin

to weaver

tyrosinase

in

so-called

right

imported al.,

days later rosinase

(dopa)

the

from 1965).

from

and left

Africa

and cared

The bird

(Witschi,

regenerating rate-limiting

conversion

of tyrosine

by the

demonstrates

enzyme that

ICSH

the activity --in vivo increases which responding feathers arise

Weaver

tract

the

catalysed

report

ventral

The ventral

one feather activity.

birds

skin

and Materials

This

the

feather

(Steqanura

for

as described tracts

was removed was then

of (the

tracts).

birds

feather

shown

administration

in the that

is

has been

birds

exogenous

is probable

1953).

(ICSH)+

of weaver

from

deposition

3,4-dihydroxyphenylalanine

Methods

of Pittsburgh,

Pennsylvania

of the

due to the

administered

et

13,

color

(Rawles,

tyrosinase

Bird

F. Hall

University

Eell-stimulating

1940).

step

and Peter

Physiology,

pigmentation

feathers

Weaver

12, 1965

Interstitial to

the

paradisaea)"

Okazaki

Pittsburgh

ReceivedJuly

in

Hormone

paradisaea) elsewhere

were plucked for

injected

measurement with

ICSH

were (Hall and 7 of ty(SO0

*These investigations were supported by the National Science Foundation Research Grant GB1923. +Abbreviationa used - FSH: follicle-stimulating hormone. ICSH: interstitial cell-stimulating hormone (also called luteinizing hozmone). MSH: melanocyte-stimulating hormone.

667

Voi.20,No.6,1965

BJOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

pg dissolved later

the

saline,

bird

activity way

in

was sacrificed

performed

each

bird

remaining

as its

tracts

in

centrifuged

at 700 x g for

potassium

(approximately

tract)

was then

incubated

lowing

additions:

flask),

DL-dopa

celite

volume by passing

column

Pomerantz

exchange

between

incubation

with

value

presence equivalent per

per

subtracted

from

with

the

fol-

urnale

per

the

tritium

as described

of

Non-enzymatic was measured

boiled

for

content

(SD) of

by

as mumoles

by

10 minutes

of water

The non-enzymatic

* 0.01

was re-

a charcoal-

is expressed

had been

buffer

water

per mq protein.

enzyme.

0.5 mumole

feather

through

and water

which

of untreated to

hour

per

Tritiated

measured

and

The super-

and phosphate

medium

activity

were

pH 6.8)

(5 ~c:O.l

flask)

tyrosine-3,5-3H enzyme

0'.

at 37'

-3,5-3H per

Pomerantz

(O.lM

at

1 hour

content

Enzyme

hydroxylated

buffer

incubation

and tritium

(1964).

In this

200 mg each)

2 ml at pH 6.8.

the

tyrosine

the

for

umole

of

of

2 mg protein

L-tyrosine (0.01

hours

tyrosinase

tract.

method

10 minutes

fraction

covered

by the

phosphate

natant

to a final

feather

(approximately

homogenized

of

four

own control.

was measured

Feather

Twenty

and measurement

on the

acted

Tyrosinase (1964).

intramuscularly).

in

exchange

tyrosine-3H

per

and the was

hour

flask. In

tyrosine

order -3,5-3H

dopa-3H

demonstrate

that

and water

measured

measurements

tyrosinase, performed

to

in

the

presence

(Pomerantz,

5-3H exchanges

with

the

in these

of dopa-3H of

exchange

Half

water

and half

1964).

668

studies

between

was due to

were made when incubation

ascorbate

1969).

of tritium

of the

to prevent tritium

appears

in

oxidation of

was of

tyPOSine-3,

dopa-3H

(Pomerantz,

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Vol. 20, No. 6, 1965

Because water,

it

the

compounds

was convenient

pare

dopa- l4C

from

the

bation

tritiated

for

to

use of tyrosine-u-14C

was isolated

and L-tyrosine-u-

Nuclear

Corporation. Study

LH-S-7

of the

and

from

The stoichiometry

comparison

of the

same assay

tritium on six

Expe.riment 1 2 3 4 5 6

Tyrosine

from from

of the

Endo(NIH-

from

with

was shown by

that

of dopa-3H

of enzyme

(Table

will

be seen that

Assay

on Feather

to

Tract

Dopa-3H relative 3H in water as 100

12,100 42,000 28,000 18,000 23,000 14,800

the

tritium

1).

1

86 98 104 95 110 93

The conditions of incubation are those reported by Pomerantz (1964). Each experiment was performed on one bird previously treated with ICSH (three daily injections, 100 bg each).

It

2.

Corporation.

assay

Dopa-3H (dpm)

14,100 43,000 27,200 19,000 21,000 16,000

New England

by CIBA.

batches

of Tyrosinase

3H in Water (dpm)

1964).

Nuclear

Biochemical

of water

different

Dopa-l')C

(Pomerantz,

decarboxylase

provided

content

incu-

of Health

of the.tyrosinase

Table The Stoichiometry

oxide

Institutes

Worthington

Apart

above.

No 69-16211-19)

National

a-MSH was generously

Results

mentioned

ICSH and FSH were gifts

NIH-FSH-2).

was obtained

Synthetic

in the

Section

and -S-8

faecalis

Ovine

to pre-

purity.

LO was purchased

14C (lot

with

106 cpm/flask)

on aluminum

(Batch

Chicago

mole;

same as those

by chromatography

order

radio-chemical

(1.0

were the

L-tyrosine-3,5-3H

crine

of

some exchange 14C in

use tyrosine-u-

determination

conditions

undergo

content

669

in

dopay3H

is

approx-

Vol. 20, No. 6, 1965

imately

equal

tritium

in

valid

measure

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

to that

the

in water,

water of

eluted

indicating

from

tyrosinase

the

Table

Purification

2 provides

Activity A

Sample

After addition of carrier 1st recrystallization 2nd V 3rd I, 4th I,

the

radiochemical

of homogenate

purity

of feather

(Pomerantz,

1964).

perimental

error,

the

908

596

914

608

916

activity

2) were obtained birds.

was provided

through

repeated from

Further

by treating

S. faecalis.

The specific

mole

of

and that

purification water of

by paper

l4 CO2 incurred

during

for

a sample

with

14C behaved

like It

is

the

the

of

the

therefore

Sample6

A and

prepared

identity

from

of dopa-14C decarboxylase

dopa-14C

was 3,182

from dpm/m

decarboxylation

after

and acid/

correcting

of dopa to compound

concluded

670

of ex-

of dopa-14C

on t-butanol/formic

conversion authentic

limits

tyrosine

14C following

dpm/m mole, the

within

preparations

chromatography

was 3,069

chromatography,

enzyme

incubation

and ascorbate

of two samples

evidence

dopamine-

70:15:15,

Dopamineof

the

after

crystallization.

activity

B

addition of authentic was added during reof dopa.

tyrosine-u-Cl4

specific

B (Table different

with

be seen that

constant

(dpm/mg)

912 914

61'1 603 606

will

remained

a

evidence

Sample

of dopa- 14C isolated

tract

It

is

of Dopa-l4C

Dopa-l4C was recrystallized from water after dopa (10 mg). A drop of N hydrochloric acid crystallization in order to prevent oxidation

for

column

of Dopa-l4C

Specific of

of

2

Recrystallization

Stage

measurement

charcoal-celite

activity. Table

that

that

in

the

for

dopamine. this the

system assays

loss

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Vol. 20, No. 6, 1965

4

3 Figure

The effect

1.

reported

here

Figure

fore

and 24 hours

(500

ug).

tract

It

protein). served

following

mones

and saline

saline

produced

Discussion Pomerantz enzyme

in

of tyrosinase of enzyme

be seen that

mumole

each

bird,

however,

demonstrable of

(1964)

is

skin.

The findings in

activity

in

3.

feather untreated

for

in

reported tracts

small

per mg

other

MSH, ACTH,

horFSH nor

activity.

described

by

amounts

of the

indicate

be

was ob-

using

tyrosinase

here

could

increase

tyrosinase

of measuring

feather

per hour

Neither

be-

of ICSH

untreated

Experiments

increase

capable

the

Table

assay

injection

no activity

considerable

tyrosin-

was measured

hydroxylated

of ICSH.

shown in

The method

in which

of the

some cases

tyrosine

injection are

activity

and in

activity.

6 experiments

of S -* paradisaea a single intramuscular

after

* 0.01 In

of

of tyrosinase

tracts

considerably

(ie

measure

results

feather

will

varies

measured

a valid

1 shows the in

6

of ICSH upon tyrosinase activity in feather The conditions of assay are given in bars represents a single determination of of the two feather tracts of one bird.

provide

ase activity

5

the

presence

of -* S paradisaea. The level birds is low and variable but

6’71

Vol. 20,No.6,

1965

considerable jection the

a black this

increase

of

fact

rosinase

ICSH

that bar

action

(Segal,

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

is observed

(Figure

a single

1).

1957). activity

MSH have been

of ICSH

has been

fish

(Chavin

shown to cause

Tyrosinase

Activity

of

Injection

Hormone

Dose

in keeping the

time

that

et

1963)

and that

of pelage

in the

before

and after

for

ACTH increases

Feather

Tracts

of Various

weasel

Hormones

Tyrosinase (mumoles/mg Treatment

Activity protein/hour) After

Treatment

a MSH

100 rg

0.1 so.01 *O.Ol

0.1
FSH

100 IJg

0.1 0.2 GO.01

x0.01 0.2
ACTH

2 units

co.01 co.01 co.01


Saline

0.1

0.1 *O.Ol co.01

0.2 *O.Ol co.01

ICSH

100 ug

0.1 ea.01

0.8 0.4

The effect of various hormones and of saline upon the tyrosinase activity of feather tracts of 2. paradi","","~ntlxx~~;~; (dissolved in saline) and saline were ln3ec e and tyrosinase assays were performed before and 24 hours after injection.

672

of

ICSH ty-

ACTH and

3

Before

ml

with

of injection;

to note al.,

in-

appearance

shown to be specific

darkening

Table

of a single

is

causes

at the

is of interest in

24 hours

observation

regenerating

hormone

It

This

injection

on feathers of the

within

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Vol. 20, No. 6, 1965

(Rust,

19651,

both

of these

effect

on tyrosinase

activity

not

cause

of these

the

appearance

birds

(Witschi,

hormones in

S. Daradisaea.

of black 1955;

were without

bars

Segal,

demonstrable MSH and ACTH do

on regenerating

feathers

1957).

ACKNOWLEDGMENT The authors Department able

are

grateful

of Biochemistry,

assistance

during

to

Dr.

University these

Seymour

H. Pomerantz,

of Maryland

for

valu-

studies.

REFERENCES Chavin, W., Kim, K. and Tchen, T.T., Annals New York Acad. Sci. 100, 678 (1963). Har P.F., Ralph? C.L. and Grinwich, D.L., General and Comparative Endocrinology, In Press (1965). Lerner, A.B., Advances in Enzymology, l4, 73 (1953). Pomerantz, S.H., Biochem. Biophys. Res. Comm., 16, 1886 (1964). Rawles, M.E.i in Biology and Comparative PhysioEgy of Birds, Ed. A.J. Marshall, Vol. 1, Page 190, Academic Press New York 1960. Rust, C.C., General and Comparative Endocrinology, 2, 222 (1965). Segal, S.J., Science 126, 1242 (1957). Witschi? E., Memoirs iifthe Society for Endocrinology No 4 Comparative Physiology of Reproduction, Edited by I. Chester Jones and P. Eckstein, Page 149, 1955. Witschi, E., Endocrinology, 2, 437 (1940).

673