Studies on aldolase isozymes during amphibian development I. Evidence for the presence of two types of aldolase in eggs from Rana, pipiens

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Vol. 30, No. 4, 1968

STUDIES ON ALDOLASE ISOZYMES DURING AMPHIBIAN I.

EVIDENCE FOR THE PRESENCE OF TWO TiPES EGGS FROM Rana pipiens* Kozaburo

ReceivedJanuary

which

of investigation

with

and development.

ly, fetal

1967a)

are

aldolase major

tissues

reassociation

+

regard

to possible

changes

this

of animal classical

Five

and electrophoresis of mixtures

were 1959;

53706

represent during

(Penhoet

primarily

with

Cahn et al.,

1962).

Recent-

forms

of aldolase after

of each two-membered

of tissues

(Penhoet

and catalytic

aldolase

et al.,

either

and rat

et al.,

1966)

B from or from

1967)

(Rensing

in et al.,

properties,

liver, several

These and of the

and of the rat

have been demonstrated --in vitro set

dissociation

of parental

1966).

These

Supported by grants from the U.S. Atomic Energy Commission Contract No. AT(ll-l)-1631) and from the National Institutes (Grant No. AM-00922), U.S. Public Health Service. The abbreviations used are: FDP, fructose-1,6-diphosphate; fructose-l-phosphate.

343

present

have been demonstrated,

et al.,

(Herskovitz

differentiation

aldolase

of molecular

FDP aldolase

an interesting

concerned

--et al. 9 1967)

musclexenzyme,

brain

1967a).

field

and MBller,

(Herskovitz

of the chicken

in extracts

in

On the basis

rabbit

et al.,

chromatography

*

A, the

C from

(Rensing

--in vivo

types

forms

of fructose-1,6-diphosphate'

of the fowl

distinct

in multiple

(Markert

have appeared.

aldolase

and H. J. Sallach

exist

studies

on the types

tissues

three

Early

dehydrogenase

reports

Chen,

11, 1968

The many enzymes

lactate

Lee-Jing

OF ALDOLASE IN

From the Department of Physiological Chemistry, of Wisconsin Medical School, Madison, Wisconsin

University

area

Adachi,

DEVELOPMENT

by and

aldolases results

or are

(Research of Health FlP,

BIOCHEMICAL

Vol. 30, No. 4, 1968

explained three

most

readily

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

on the basis

of a molecule

four

rather

than

subunits. Studies

stage

on the relationship

of development

tory.

In this

of aldolase found

in the

in adult

killed

0.001

(eggs

the eggs before

shaken

gently

HCl buffer, times

these

from

of a pituitary stripped

during

homogenized

Gravid

liver

ovary) case,

gravid

suspension

labora-

to those

homogenization

with

four

pH 7.5,

for

this

treatment.

in an equal

of 0.125

40 minutes.

for

a column before

of Blostein

D. J.,

enzymatic

assay.

and Rutter

(1963)

J.

was removed which

G-25,

except

L.,

344

that

personal

a slight

in 0.005

was changed

were

supematant

eggs were containing centri-

solutions

equilibrated

0.008

was assayed M

M Tris-

three

procedure,

homogenates

activity

from

Eggs were

pH 7.5,

previously

Aldolase

is

buffer,

The resulting

of Sephadex

and Hedrick,

All

by in-

of Rugh (1961).

and Hedrick'.

M Tris-HCl

of

(eggs,

to ovulate

procedure

the dejellying

of 0.05

pH

of nonin-

ovulation

coat

The buffer

Following

volume

the ovary

M 2-mercaptoethanol

20 minutes.

buffer,

The two types

to the method

by Gusseck

The fresh

M Tris-HCl

induced

The jelly

were

removed.

after

were

following

described

about

were

obtained

later.

by the

volumes

(Rana pipiens)

of 0.05

females

at 100,OOOg

Gusseck,

this

of two types

compared

eggs from

according

24 to 48 hours

of the method

(w/v>

and those

fuged

method

are

grassfrogs

were

M 2-mercaptoethanol.

same buffer,

the presence

and muscle

studies

M EDTA and 0.01

through

and the in

M 2-mercaptoethanol.

0.001

passed

for

female

M EDTA and 0.01

In the latter

modification

of aldolase

investigation

and these

in two volumes

used in

females

Eggs were

under

is presented

and brain,

homogenized

egg preparations

jection

are

eggs of Rana pipiens

and Methods.

were

spawned).

amphibian

the types

tissues.

containing

duced

between

evidence

by decapitation

tissues 7.5,

in the

report,

Materials

1

with

were

with

by the

FDP was used.

communication.

the

Zone

BIOCHEMICAL

Vol. 30, No. 4, 1968

electrophoresl:

was performed

which

contained

0.001

strips

(Gelman

acetate two hours

at 4'C.

The staining

solution

arsenate,

mg per

ml of rabbit

Sepraphore

contained

muscle

A strip

solution

electrophoresis, placed minutes

for

strips

color

were

Results extracts

(1963)

used

aldolase

activity

ratios

for

FlP obtained

demonstrated

tios

of brain. egg were

the

cent

fonnalin

their

I.

activity et&.,

for

egg preparations

activity

1.

ratios

however, here

--et al.,

345

10 to 20

the various and Rutter

as shown in Fig.

is not

saturated et al.

lowest (aldolase

reported

however,

(1967b),

that

Vm,,

of the reaction

for

was

and highest

A) was found previously

for

to this

The activity those

ra-

of muscle

from muscle, from

.

used in the

liver

1967a,b). between

1

by this

of the apparent

FlP of aldolase

of magnitude,

were

and staining

at the level

intermediate for

After

Blostein

by Rensing

were

than values

stain-

samples

for

studies,

muscle

Rensing

sample

The velocity

ratios

this

at 37OC for

on the basis

studies,

were

(Sigma),

fixing.

suggested

of Fig.

K m values

of the same order

both

activity

examined as

in

the

the dark

of FDP concentration

1963;

M

M NAD, 0.12

box.

containing

system,

tissues

ratio

0.00015

0.001

was soaked

In their

assay

the data

as follows:

pH 7.5,

M FlP,

cm for

dehydrogenase

staining,

The FDP/FlP

In these

The apparent

buffer, 0.03

per

activity

in a plastic

in

have been calculated

aldolases.

(Blostein for

After

in Table

The FDP/FlP

have a much lower tissue

strips

to be independent

system. brain

acetate

Therefore,

from

aldolase

paper

plate

in the

of FlP.

the

for

given

0.01 M FlP in

I,

acetate

and incubated

in 1 per

are

concentration

assay

strip

on cellulose

and 0.5 mg per ml of nitroblue

on a glass

and Discussion

tissue

and Table

methosulfate,

development.

soaked

for

0.1 M Tris-HCl

the cellulose

pH 8.5,

2.5 X 17 cm) at 25 volts

glyceraldehyde-3-phosphate

placed

on the staining

buffer,

M 2-mercaptoethanol,

stained

of cellulose

and then

barbital

M EDTA, 0.01 M FDP, or

24 pg per ml of phenazine

ing

M sodium

III,

were

Strip.5

0.001

tetrazolium.

in 0.06

M EDTA and 0.0025

The

sodium

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

liver

brain was

and and

Vol. 30, No. 4, 1968

BIOCHEMICAL

-100

0

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

loo

200 [FRUCTOSE- I -PHOSPHATE]-’

Fig. 1. Double reciprocal plots A, liver; x, brain; I, muscle;

of l/v with respect to l/[FlP]. o, eggs from ovary; l , eggs,

Table Aldolase extracts.

activity,

Organ

FDP/FlP

activity

Activity Units/mg

Muscle

Brain

5.5

Eggs from ovary

1.1

Eggs, *

of aldolase the aldol described

activity cleavage in the

FlP of frog

Km for

tissue

FlP

-2

5.7

1.1X10

1.3

0.41

"

1.1

"

8.6

0.95

"

7.9

1.4

"

12

spawned

One unit catalyzed conditions

and Km for

FDP/FlP activity ratio

52 2.7

spawned.

I

ratio,

for FDP tissue*

Liver

300

M

was defined as that amount of enzyme which of 1 micromole of FDP per minute under the assay procedure.

lower. The electrophoretic

patterns

of aldolase

346

from

various

organs

of the

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Vol. 30, No. 4, 1968

c

AB

Fig. frog A: B: C: D: E: F:

CD

are

patterns

shown in Fig.

activity,

which

two bands

of activity,

more anodic

results

are

et al.,

1966) than

Fig. 3. Electrophoretic patterns of aldolase staided for FlP and FDP. A: Muscle, stained for FlP. B: Liver, stained for FlP. C: Liver, stained for FDP. D: Muscle, stained for FDP.

of

The muscle

preparation

at the most

one coinciding band,

in contrast and rat

aldolase

and B was not

2.

was localized

second,

lity

EF

2. Electrophoretic tissue aldolases. Eggs from ovary. Spawned eggs. Brain. Spawned eggs. Liver. Muscle.

frog

Origin

A.

detected

which

found et al.,

The possible in

these

cathodic that

was specific

to those (Rensing

with

with 1967a),

presence

experiments.

347

showed

only

site,

whereas

of muscle

for

liver

aldolase which of hybrids

one band of liver

(aldolase

(aldolase B from

gave A) and a

B).

rabbit

These (Penhoet

had more cathodic between

The localization

mobi-

aldolase of aldolase

A

BIOCHEMICAL

Vol. 30, No. 4, 1968

activity

for

FlP

muscle

preparation

activity

in this

from

liver

that

the high

could

et al.,

aldolase

A in brain

that

ever,

this

both it

Brain

muscle

1966).

aldolases

additional

egg preparation

those

phoretical

in egg,

have

tissues

not

been

the predominance of the

with

the presence

rat

followed

development. of aldolase

How-

that

In order

of

to provide

of types

A and C,

extract

and then

bands

of egg aldolase

in an increased

must

during

later

that

in-

the aldolases

await

the results

of aldolase by a shift other

C, as well

to the

the

at earlier

stages,

--et al.

type)

as A, in embryonic

(1967a)

in most

organ-specific

Kaplan's

electro-

of development.

Rensing

A (muscle

hand,

in

stages

present

previously.

348

of brain

The electro-

the changes

of aldolase

On the

to that

are in progress.

isozymes

investigated

than

present.

suggests

concerning

the forms

or rabbit

concentration

or muscle

proof

results

between

and resulted

to

of aldo-

1967a)

C.

of all

Additional

reports

of aldolase

type

egg consists

brain

which

the

were

ratio.

observation

studies

several

to our knowledge,

have demonstrated

ported

and other

patterns

of aldolase

This

of the A and C types.

have been

either

and muscle

staining.

with

et al.,

over

B

1963;

was composed

forms

The distribution

of brain

of enzymatic

There

fetal

with

et al.,

egg was intermediate

the aldolases

was mixed

with

i.e.

A predominated from

aldolase

egg was similar

hybrid

of the low

has been proposed

in greater

from

from

coincided

However,

C was present

that

to electrophoresis.

of immunochemical

(Rensing

activity

subjected

of egg are

rat

as was the FDP/FlP

evidence

tensity

type

(Rutter

the frog

The

FlP may be related

in agreement

A and C and their

of aldolase

and of brain

is

Aldolase

that It

for

tissue

B.

because

suggests

liver

from

either

aldolase

FDP and FlP.

this

This

2).

in that

pattern

in

Aldolase

(Fig.

also

B from

aldolase

from

of liver

FlP activity

of both

hybrids.

tissue

differed

phoretical

cleavage

metabolism

A and C and their

for

observation

of aldolase

1964).

(Penhoet

the

the

of fructose

for

be stained

This

specificity

obtained

in

not

tissue.

et al.,

lases

3) was the same as that

catalyzes

the pathway Spolter

(Fig.

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

type

group

has re-

muscle

of the

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Vol. 30, No. 4, 1968

chick

(Herskovitz

the existence three

et al., of two parent

hybrids,

type)

1967).

However,

has been observed

blastocyst

stage

although

the early

muscle

types

type

(Auerbach

frog.

(Markert

in sea urchin

tion

et al.,

(Patton

upon fertilization

multiple

in unfertilized a singl.e

lactate

tissues

1967;

The number

eggs was shown to decrease

1967).

Possible

changes

and during

early

developmental

their

of malate eggs isozyme

the unfertilized

Rapola

for

with

forms

sea urchin

ovum to the

and Koskimies,

of mammals contain 1962).

evidence

dehydrogenase

in mouse ova from

and Ursprung,

ase isozymes

provide

A and C, together

Similarly,

and Brinster,

embryonic

studies

of aldolases,

have been demonstrated

et al.,

(heart

The present

in the egg of the

dehydrogenase (Patton

1967).

1967),

predominantly

the

of malate

dehydrogen-

following

fertiliza-

in aldolase stages

types are

in frog

under

eggs

investiga-

tion. REFERENCES Auerbach, S. and Brinster, R. L., Exp. Cell Res., 46, 89 (1967). Blostein, R. and Rutter, W. J., J. Biol. Chem., 238, 3280 (1963). Cahn, R. D., Kaplan, N. O., Levine, L., and Zwilling, E., Science, 136. 962 (1962). Herskovitz, J. J., Masters, C. J., Wasserman, P. M., and Kaplan, N. O., Biochem. Biophys. Res. Commun., 26, 24 (1967). Markert, C. L. and Mbller, F., Proc. Natl. Acad. Sci., 45, 753 (1959). Markert, C. L. and Ursprung, H., Develop. Biol., 2, 363 (19.62). Patton, G. W., Jr., Mets, L., and Villee, C. A., Science, 156, 400 (1967). Penhoet, E., Rajikumar, T. V., and Rutter, W. J., Proc. Natl. Adac. Sci. U. S ., If?, 1275 (1966). Rapola, J. and Koskimies, O., Science, 157, 1311 (1967). Rensing, U., Schmid, A., and Leuhardt, F., Hoppe-Seyler's Z. Physiol. Chem., 348, 921 (1967a). Rensing, U., S&aid, A., Christen, P., and Leuhardt, F., Hoppe-Seyler's Z. Physiol. Chem., 348, 1004 (1967b). Rugh, R., "Experimental Embryology: A Manual Techniques and Procedures", Burgess, Minneapolis, Minn., 1961, p. 102. Rutter, W. J., Blostein, R. E., Woodfin, B. M., and Weber, C. S., in Weber, G. (Editor), Advances in Enzyme Regulation, vol. 1, Pergamon Press Ltd., London, 1963, p. 39. Rutter, W. J., Federation Proc., 23, 1248 (1964). Spolter, P. D., Adelman, R. C., DiPietro, D. L., and Weinhouse, S., in Weber, G. (Editor), Advances in Enzyme Regulation, vol. 2, Pergamon Press Ltd., London, 1964, p. 79.

349