A dialyzable inducer for the glutamotransferase of chick embryo retina

Vol.

23, No.

1, 1966

BIOCHEMICAL

AND

A DIALYZABLE GLUTAMOTRANSFERASE

BIOPHYSICAL

INDUCER OF CH1C.K

RESEARCH

FOR THE EMBRYO

COMMUNICATIONS

RETINA”

:x :I: Liane

Rfeif

Department Harvard Medical

Received

February

In the is

not

and

enzyme

retina

is

Kirk,

removed

Amos,

which

the

high

can

be made

other

Kirk,

Supported by Grant E-348.

Much of Fellowship

the

and

the

Immunology Massachusetts

1965) of

obtained

enzyme, At

level

embryo Kirk

glutamotransferase

this

time

(Rudnick

to

and

the

appears

Waelsch,

much

1955).

earlier

cultured

in

Moscona,

(GTF)

enzyme

and

appear and

if

the

a standard

1963;

medium

Moscona

and

1965).

investigators

In

have

the

Amos,

appearance

we

Amos

Boston,

day.

a very

1963;

and

and

derepression.

1’7th

from

Reif

1965;

retina

the

and Hubby,

We

or

until

1965;

of Bacteriology School,

embryo

activity

(Moscona

that

chick

reaches

This

Harold

28, 1966

detectable rapidly

and

have the

this for

obtained

enzyme

vitro

report,

we

would

an

“inducer”,

Grant

here

and

evidence

--in

U. S. P. H. ;S.

work reported CA 16, 919.

(Moscona

is

39

carried

which

suggests

to

which

was

1965;

consistent

like

AI-00957

Kirk,

present

American

out

under

us

induction the

to

and to

with

appears

and

Reif

be

evidence a substance

Cancer

Society

U. S. P. H. S.

Vol.

23, No.

of

BIOCHEMICAL

1, 1966

relatively

low

Methods

and

molecular

were

development.

Each with

flasks

at

(70-72

RPM)

were

10%

37OC.

done

for on the

saline-washed Enzyme using

activity

calf The

flasks

the

culture

sample

the

at

in

in

incorporated

are

shaken

COMMUNICATIONS

by and

refer

to All as

14

a rotary days).

All

of

method

(pH

enzyme

activity,

the 7.4-7.

6).

(1955), of

incorporated of

analyses

disruption

of Waelsch

-1eucine

specific

shaker

method the

basal Erlenmeyer

buffer

by

values

ml

phosphate

the

of

Eagle’s

50

sonic

protein C

10 days

of

l-5

mild

of 0. 06M

2,

reported

on

(usually by

determined

5 ml

stoppered

period

protein.

in

tightly

were

2 ml

embryo

cultured

obtained

incubation

TCA-precipitable

was

serum

studies

3

RESEARCH

.

from

retina

was

a 2-hour

protein

removed

retina

Incorporation

of

1

weight

BIOPHYSICAL

Materials.

Retinas

medium

AND

Lowry

(1951).

into

cold

activity

and

counts

i. e. , per

unit

weight

.

Materials

were

dialyzed

for

24

or

48

hours

at

4OC

in

8 or

2Omm

1.

There is a possibility that we may also be dealing with a demonstrable repressor component. The main evidence for this is based on the fact that in many experiments the addition of components of fresh medium to OCM was appreciably less effective in inducing enzyme than changing the medium. Also supporting this idea is the finding that retinas are less responsive to inducer during the first 24 hours of culture than after 2 or 3 days.

2.

Product 37oc.

3.

formation

in

this

assay

is

linear

for

Units of enzyme activity are the Klett readings unit of optical density) divided by the pg protein day retinas assayed shortly after excision give and 0. 020. Therefore 0.015 has been subtracted presented here as a zero point.

40

at

least

at 540 in the values from

3 hours

at

rnp (i. e. , a sample. Ten between 0. 010 all values

Vcl. 23, No. 1, 1966

dialysis for

BIOCHEMICAL

tubing 20

residue

of

and

in

distilled

water

be to

components

retinas

lyophilized

test

water

to dryness. or

Hanks

material

was

then

samples.

induced

at

will

no

inducer,

medium,

by

starting

a greater

supports 10%

serum. experiments,

test

or

by

1965)

the

lower

use of

latter

cultures

begun

in

fresh

protein

- leucine

, was

since null

about

very

1% or

3%

calf of

it

serum,

proved

time

consuming

to

be to

to by

the

is

that

evidenced

appears

presumably

OCM

situations serum

as

though

simply

media test

levels

well,

41

enzyme

baseline

Similar

to deteriorate, OCM,

is

14

to

cultures

concentrated 1).

these

tissue.

it

adding

in

Although

C

a uniform

cultures

tissue

us

contrast,

system,

(Fig.

tendency

of the

cultures

of

from

of

start

the

In

decreased

OCM.

excellent

Amos,

in

incorporation in

retinas led

to in

a maximum.

disadvantage

have

(= OCM)

activity

and

in

incubation

appeared

the

and

activity of

cultures

by

fresh

One

4 days

an

achieved

a disintegration

induction

The

of water

the

enzyme

proved

(Reif

by

of having

of

activity

medium

OCM

changing

medium.

medium

3 or

when

measured

could

be

follows:

was

reconstituted

reached

fresh

The

could

serum

This

that

enzyme

hours,

as

same

the

as

quantity

such

already

synthesis,

for

in

prepared

a small

the

after

No

had

activity

in

from

72-96

medium

in

(BSS).

constant

retinas. by

of

observation

collected

fresh

up

serum

early

remained

medium

the

) pi-e-boiled

Discussion.

An

by

taken

solution

place

Results

the

was

= 10/l)

was salt

in

even

fra’ction

(H20/serurn

balanced

or

Corp.

dialyzable

dialysate

used

Carbide

minutes. The

The

(Union

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

best

prepare.

have

virtue medium An

Vol.

23, No.

BIOCHEMICAL

1, 1966

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

Relative effects of different media on enzyme induction Figure 1. compared to effect on protein synthesis. Cultures in : A) 10% CS medium; B) OCM; C) changed to 0% medium after 1 day in OCM; D) changed to 10% CS medium after 1 day in OCM; E) 10% of fresh CS added after 1 day in OCM. All samples were harvested after 3 days in culture; each value is an average of three samples.

alternative Kirk

(1965)

with

fetal

found the as adult

that cultures in

test

system

that

no

calf

are

serum

(Fig.

appearance

of

an

the

permit

maintained

protein

an

suggested activity

(FCS)

does

OCM,

inducer,

enzyme

serum

FCS

was

inhibitory effect

an

of

as

long

early

addition

We

have

as

hours;

in

such

equal

to

that

96

about to

components

if medium, in

between or

of

since

of enzyme

distinguish

absence

and

cultured

level

and

42

Moscona

retinas

serum.

attempt

of

of

a low

usually

substance of

in

induction

is

report

appeared of adult

for

In

the

instead

synthesis 2).

by

the

depletion medium,

of

Vcl.

23,

No.

1, 1966

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

#,

COMMUNICATIONS

r

033

I I 0 20

, / 10.000

r; I I I; -2 0 ‘0 k

0 Pi ; 0 c.

0.15

7.500 E” \ E 8

)I z > ; =l t u” 0.10

E 6L si z -:

5,000

: .u ‘c : ::

.E ; 3 : 005

2.500

0’ ”

Figure 2. Comparison of enzyme induction and protein synthesis in fetal and adult serum. A) harvested after 1 day in 10% FCS medium; B) harvested after 3 days in 10% FCS medium; C) harvested after 1 day in 10% CS medium; D) harvested after 3 days in 10% CS medium.

individually

or

in

changing

medium.

enhanced

enzyme

dialysis

of

OCM

combinations, The

only

activity against

was component

was CS

calf or

restoring

the

inducer

activity

results).

In

contrast,

OCM

remained

ineffective.

(0%

medium)

compared whose serum

fresh to

OCM

dialyzed

10%

CS

(Rei against

the

effect

addition

(CS)

Moreover,

43

to

to

(Fig.

3).

medium and

0%

OCM Indeed,

resulted

Amos,

serum

of

in

unpublished free

medium,

medium

Vol.

23, No.

BIOCHEMICAL

1, 1966

AND

BiOPHYSlCAL

RESEARCH

COMMUNICATIONS

1

-

-. -

Figure 3. Effect of different media components on enzyme induction. A) harvested after 2 days in OCM; B) harvested after 4 days in OCM. (C through G were treated as indicated, after 2 days of culture in OCM, and were harvested 2 days after treatment. For additions, the amount of the specified substance added is equal to the amount of that substance initially added to make Eagle’s basal medium with 10% cs. ) C) add NaHC03; D) add amino acids; E) add vitamins; F) add CS; G) change to fresh 10% CS medium.

which

does

after

dialysis

isolating

not

or

BSS

dialysate fact

in

calf

inducer, (as

cases,

to

activity, serum

serum

described

proved most

enzyme

against

the

H20

induce

contain as

(Table

was under

acquires I).

As

dialyzed

against

Methods).

The

significant

prepared,

amounts it

44

was

essentially

this

capacity

a first

step

toward

10 volumes

of

concentrated of the

inducer; as

effective

in as

,&

OCM

FCS

10%

6

FCS

10%

0%

FCS

10%

5

2

1%cs

1

ENZYME

Mediuma

I.

Expt. No.

TABLE

I I

0.004

At timeb of treatment of parallel cultures

Untreated

INDUCTION

b

0. 019

Add

0. 244

(Specific

DIALYZABLE

At time of harvest of parallel culture 6

cultures

WITH

enzyme

CSc

’

EXTRACT

0.334

activitye)

10%

CALF

Change to CS medium

OF

0.301

0. 321

Add

Treated

SERUM.

CS

extract

cultures c,d

0. 327

0.302 h

Change to 0% medium vs. which CS has been dialyzed

Vol. 23, No. 1, 1966

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

46

Vol. 23, No. 1, 1966

whole

BIOCHEN\ICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

serum

when

the

(Table

retinas

reflects

what

tissue

in

was

the

control

and

repression

molecular

of the

(m-RNA)

have

reported

4

.

dialysates probably

poor

state

They becomes

the

m-RNA

for

these

findings

are

the

of the

that

currently

w:hen GTF

at

GTF

the

Kirk is

also

to

no

had

enzyme

had using

pursuing

also

not

OCM

as

the

for

indeed, serum

level

of messenger and

the

isolation

formation

of Actinomymade

in

made.

We

initial and

(1965)

freshly

the

effect

been

(and

Kirk in

that

been

to

induction

present

the

of

of the

reported

refractory

is

analogous

(1965) not

it

assume

1961);

effect

and

have

is

proposed

is

for

that to

Monod,

fraction)

m-RNA

retinas.

all

and

Moscona

the

conclude

reasonable

been

that

dialyzable

found

We

the

medium the

induction

(Jacob

Kirk

confirmed

is

have

indicates

gradually

medium,

1% CS

we

observed

bacteria

that

lo-day

cinD

or

of

regarding

It

which

synthesis.

enzyme

0%

dialyzable,

the

evidence

RNA

in

weight. for

in

experimental

of

is

mechanisms

presumably

effectiveness

mentioned

inducer

mechanism

excised

incubated

earlier

the low

the

reduced

media.

Since

that

The

were

such

relatively

I).

culture

FCS have 5

medium

identification

.

of the

inducer.

Acknowledgments. The

authors

wish

J.

Tersac

for

and

Mr.

4.

Dr. has

5.

A detailed

Sarah Ben-Or also confirmed account

to

thank

their

(Hebrew these of these

Mr.

technical

C.

W.

Christopher,

Miss

S.

assistance.

University Medical School, results (personal communication). experiments

47

will

be

published

Jerusalem)

shortly.

Hacke

Vol. 23, No. 1, 1966

5lOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

References. Jacob, Kirk, Kirk, Lowry,

F.

and Monod, J. J. Mol. Biol. 1, 318 (1961). L. and Moscona, A. A. Develop. Biol. S, 341 (1963). L. Proc. Nat. Acad. Sci. 4, 1345 (1965). 0. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. J. Biol. Chem. 193, 265 (1951). Moscona, A. A. and Hubby, J. L. Develop. Biol. 1, 192 (1963). Moscona, A. A. and Kirk, D. L. Science 148, 519 (1965). Reif, L. and Amos, H. Derepression of glutamotransferase in chick embryo retina cultures. Paper #46. Abstracts. Sixteenth Annual Meeting, Tissue Culture Association, May 1965. Rudnick, D. and Waelsch, H. J. Exp. Zool. 129, 309 (1955). Waelsch, H. in Methods in Enzymology, Colowick, S. P. and Kaplan, N. Vol. 117~. 269. Academic Press, New York, 1955. D. D.

48

0.