The inactivation of chick tyrosine aminotransferase by acid and alkaline phosphatases

Vol. 43, No. 5, 1971

BIOCHEMICAL

THE INACTIVATION

AND BIOPHYSICAL

OF CHICK TYROSINE AMINOTRANSFERASE

BY ACID AND ALKALINE Beverly Laboratory

Received

of Physiology,

April

22,

RESEARCH COMMUNICATIONS

National

PHOSPHATASES

Peterkofsky

Cancer

Institute,

Bethesda,

Maryland

20014

1971

SUMMARY

Chick tyrosine aminotransferase is inactivated by incubation in the presence of acid or alkaline phosphatases from various sources. The inactivating activity as well as the phosphatase activity of these preparations was inhibited by NaF in the case of acid phosphatase and stimulated by MgClp in the case of intestinal alkaline phosphatase. The mechanism of inactivation appears to be initial dephosphorylation of loosely bound pyridoxal phosphate followed by denaturation of the labile apoenzyme. The enzyme rat

liver

catalyze

(1) its

of interest

tyrosine

aminotransferase

and hepatoma degradation

when we observed of chick

extracts

or DEAE cellulose

A similar

could

embryo

that

liver

preparation

(4).

This

ase by acid

and alkaline

describes

enized

from

in a buffered

5'-phosphate

cellulose

at 105,000 by a method

obtained:

(I)

embryos,

medium

as described

centrifugation

were

19-day-old

containing

previously x

which unadsorbed

g for will

from

rapidly

enzymes

It

in

which

was therefore in crude

the enzyme activity stable

in heated

when stored

at

alanine-phenylglyoxal

and in that

inactivation various

very

activity

enzyme with

the

instance

the

inactivation

an alkaLine

phosphatase

of tyrosine

aminotransfer-

sources.

AND METHODS lo-day-old

chicks

or 80 g rats

1OmM o-ketoglutarate (5).

1 hour

solution

was fractionated

(II)

1171

elsewhere protein

were

homog-

and 0.08 n-M pyridoxal-

The supernatant

be described protein,

found.

the enzyme

the purified

over

specific

was quite

spheroides

phosphatases

but

while

extracts

MATERIALS Livers

lost

had been made for

report

(2,3)

aminotransferase

was rapidly

by treating

turns

have not been

tyrosine

of Rhodopseudomonas

be duplicated

cells

fractionated

observation

aminotransferase

culture

or inactivation

extracts

4'.

tissue

(EC 2.6.1.5)

obtained

batchwise (6).

eluted

with

Three with

DEAE

fractions

O.lM

KC1 and

after

Vol. 43, No. 5, 1971

(III)

protein

activity

BIOCHEMICAL

eluted

with

and tyrosine

while

fraction

III

AND BIOPHYSKAL

0.3 M KCL.

Fractions

aminotransferase contained

I and II

inactivating

tyrosine

RESEARCH COMMUNICATIONS

contained

activity

aminotransferase

acid

measured

purified

phosphatase at pH 4.1,

approximately

thirtyfold. Partially E. & from

purified

alkaline Worthington

purchased

from

Tyrosine

and pig

Biochemical

Corp.

Sigma Chemical

p-nitrophenyl

phosphate

that

a total min.

and alkaline in the

Acid

described

by Jenkins

Inactivation

contained

O.lN buffer,

intestine phatase have

all

solutions

alkaline

a sample wards.

were

(pH,

removed

into pyridoxal

reaction.

dialyzed

before

phosphate

Samples

the formation Phosphatase

and time

points

at pH 4.1

using

of p-

activities

against

by Lowry were

(7)

taken

at 0

O.lN acetate

aminotransferase

was measured Reaction

milliunits

or aspartic were

buffer buffer

was measured

before

mixtures

as

in no-enzyme

use against

mixtures

to be assayed

for

were aspartic

1172

aminochicken

containing

controls.

Results

tubes.

phospresented

The aminotransferase buffer, were

pH 8,

of the

reaction

at 37'

and at 45 and 90 min after-

aminotransferase

equilibrated

concentration)

tyrosine

O.OlM triethanolamine

the reaction tyrosine

or rat

O.OlM MgCln(if

in control

The components

for

out a preliminary

the preincubation

an enzyme fraction

observed

initiating

for

per ml of chick

used)and

losses

by carrying

aminotransferase,

buffer

to be assayed reaction

was

(8).

200-300

for

purchased

O.lN 2-amino-2-methyl-propanediol

Aspartate

2mM a-ketoglutarate.

Samples

directly

M MgCl,.

or appropriate

been corrected

containing

at pH 8.9 using

III)

were

phosphatase

as described

was measured

phosphatase

activity

(5).

essentially

or 8.9 at 37'.

(Fraction

alkaline

by measuring

previously

of aminotransferases

at pH 4.1

purified

aminotransferase

intestine

of 0.30 ml was used

and Sizer

incubation

transferase

Chicken

measured

phosphatase

of 0.01

chromatographically

aspartic

was assayed

were

phosphatase

presence

heart

as described

volume

phosphatase,

Corporation.

aminotransferase pyruvate

and 20-60

germ acid

phosphatase

hydroxyphenyl

except

wheat

activity

at 37'.

The conditions

sufficient

to stop

aminotransferase

the activity

mixed

were

at O",

placed

of the assay inactivation were

BIOCHEMICAL

Vol. 43, No. 5, 1971

added

to reaction

NADH was added was added

mixtures

containing

initiaLLy

all

to detect

to initiate

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

the

components

any NADH oxidase

the aminotransferase

except

activity

NADH and aspartate.

and then

aspartate

reaction.

RESULTS Initial

observations

responsible fraction

for

the

I were

that

phosphate

inhibited

In Table

I it

partial

inhibition

inactivation

of the cofactor was complete

even after was observed

1

time

of tyrosine

inactivation

after

concentration

90 min.

and that inhibition

activity

phosphate, there

was only

concentration however,

by wheat

was also

liver

was reversed.

At a higher

inactivation

be

pyridoxal-5'-

aminotransferase,

inhibited

inhibition

germ acid by

phosphate.

Enzyme Source Liver,

fraction

I

of pyridoxal-5'-phosphate (TAT) inactivation by acid

PH

PLP mN

TAT munits/ml

4.1

0

249

liver,

fraction

I

4.1

0

wheat

germ

4.1

0 0.04

on tyrosine phosphatase

TAT inactivated in 90 min. munits/ml

140

69.0

116.8 0

220

Percent Inhibition

96.0 29.8

1.0 3

by embryo

of aminotransferase,

and this

might

of pyridoxal

0.04 2

the

90 min.

In addition,

at pH 4.1

Table I. Effect aminotransferase

Expt.

at pH 4.1

but with

level

activity

aminotransferase

at a low concentration

saturating

and a lower

phosphatase

was optimal

inactivation,

of the

that

of tyrosine

may be seen that

mM, and a near

pyridoxal

suggested

inactivation

the

0.04

phosphatase

which

100

138.5 78.3

43.5

Preincubations to measure inactivation were carried out in a total volume of 0.30 ml as described under Methods and the protein concentration of the phosphatase-containing fractions used were: expt. 1, 6.7; expt. 2, 6.7 and expt. 3, 3.3 mglml.

1173

Vol. 43, No. 5, 1971

BIOCHEMICAL

The dephosphorylation sources

activity the

intestine

indirectly

substrate

by testing

by all

inhibited

the

and chicken

levels

competitive

alkaline

by phosphatases

embryo

(6.7

effect

of pyridoxal

of tyrosine

was inhibited alkaline

mM).

to some extent phosphatase

preparations

as shown

of acid

phosphatases

(12)

nitrophenyl

phosphate

The inhibition

appeared

phosphate

in Table

inhibited

and tyrosine

II.

both

was catalyzed Sodium

Enzyme Source

E.

coli

such

phosphate

on

of the but embryo

liver

be substantially (0.5

mM) and

to be of the

acid

fraction

germ

intestine

I

Phosphatase Activity units/ml

a known

phosphatase

aminotransferase

by the four

fluoride,

activity

inactivating

Table II. Relationship between phosphatase and tyrosine aminotransferase inactivating

chicken

could

of pyridoxal

aminotransferase

phatase

wheat

for

type.

Inactivation

liver,

checked

phosphatases

The dephosphorylation

low concentrations

of substrate

from various

germ acid

were

inhibitory

intestine

and wheat

phosphatases

of p-nitrophenylphosphate.

at relatively

saturating

Chick

of the phosphatases

phosphatase

phosphate

(9,10,11).

and E. &

dephosphorylation

acid

of pyridoxal

has been reported

and chicken

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

inhibitor

against activity

pof liver

activity activity

TAT inactivated in 90 min munits/ml

TAT inactivated phosphatase

PR

Additions

4.1

-

0.45

25.3

4.1

NaF

0.26

7.2

4.1

-

2.85

160.4

4.1

NaF

0.18

59.2

8.9

-

0.02

2.0

g.9

W 12

0.18

50.2

27.8

8.9

-

0.69

88.6

12.8

The volume of reaction the following concentrations wheat germ acid phosphatase, 6.67 pg/ml; E. & alkaline was used at a concentration out for 20 min. as described

phos-

x

5.6

5.6

mixtures in inactivation assays was 0.30 ml and of enzymes were used: embryo liver, 3.92 mg/ml; 3.33 mg/ml, chicken intestine alkaline phosphatase, phosphatase, 6.67 Kg/ml. Chick TAT (fraction III) of 180 munits/ml. Phosphatase assays were carried in the Methods section.

1174

10-l

Vol. 43, No. 5, 1971

fraction

BIOCHEMICAL

I and wheat

phosphatase

towards

germ acid both

activity

was compared

per unit

of phosphatase

phatase phate

was most

it

This

stimulated

of milliunits

activity,

of intestinal

alkaline

by MgC12.

When

of aminotransferase

may be seen that

intestinal

enzyme was also

most

inactivation

a specific

inactivated alkaline

sensitive

phos-

to pyridoxal

phos-

inhibition. Further

was shown

evidence by testing

crude

histone,

free)

were

that

the

several

crystalline

tested

phosphatase

other

at 0.1 mg/ml

at pH 4.1

nuclease

had approximately

showed

against

no inactivating p-nitrophenyl

reason

for

its

No inactivation could

be detected

on tyrosine

and purified

ribonuclease

to 3.33 mg/ml

of intestinal

20% of the

was detected

of rat .liver when the usual

is

assay

with

phosphatase

riboat pH 4.1.

activity solution

and the

unexplained.

aminotransferase

system

at pH 8.9.

phosphatase

in the ribonuclease

at present

tyrosine

phosphatase

of acid

protease

germ acid

was seen except

No acid

Lysozyme, (Sigma,

of wheat

alkaline

activity

at pH 8.9.

to inactivate

of phosphatase

the same conditions.

aminotransferase

activity

effect

under

and compared

phosphate

ability

proteins

and 6.7 +g/ml

effect

which

is

serum albumin

No significant

It

was greatly

on the basis

RESEARCH COMMUNICATIONS

The activity

phosphatase.

substrates

active.

AND BIOPHYSICAL

containing

by acid pyridoxal

phosphatase phosphate

was

Table III. Effect of acid and alkaline phosphatases on rat tyrosine aminotransferase

Enzyme Source

PLP in TAT assay

PH

Wheat germ

4.1

Wheat germ

4.1

Chicken

intestine

8.9

Chicken

intestine

8.9

TAT inactivated in 90 min. munits/ml 155.1

+

2.7 113.9

+

93.0

The initial concentration of rat tyrosine aminotransferase was 240 munits/ml; otherwise conditions were exactly the same as described in Table II. TAT was measured in the presence or absence of pyridoxal phosphate (PLP) as indicated.

1175

Vol. 43, No. 5, 1971

employed

(Table

an apparent

III).

loss

with

Purified

either

after

germ acid

and that

alkaline

phosphatase

remaining

was complicated

aminotransferase

holoenzyme

aminotransferase

activity

at pH 4.1

in

form

phosphatase

had at pH

nor

converted

into

of the enzyme with

Interpretation

of data

by the extreme

instability

phosphatase

however,

was stable

inactivated

phosphate

system,

inactivation.

and the presence

in the acid

acid

apoenzyme

resulted

of the pyridoxal

the assay

that

was neither

phosphatases.

phosphatase

from

indicating

the

aminotransferase

treatment

of the

was omitted

was observed,

alkaline

aspartic

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

the cofactor

the cofactor

Treatment

apoenzyme

If

of activity

dephosphorylated 4.1.

BIOCHEMICAL

obtained

with

wheat

of the aspartic

of endogenous

aspartic

preparation.

DISCUSSION Previous could

brief

inactivate

(13).

transferase

acid

report

observed

by an acid

phosphatase

that

E. &

in crude

in

suggest

by a mechanism

liver

aminomay be

and that,

'can carry

that

which

inactivation may be tentatively

E

+

of chick

tyrosine

outlined

+

in addition,

out such inactivation

supported

by the

apoenzyme

that

pyridoxal

observation which

phosphatase.

The observation

tightly

bound

cofactor

tested

suggests

that

aminotransferase,

that is

free

phosphate rat

apparently that

(14),

phosphatase

PAL + Pi

E

form,

as follows:

1

inactive The conclusion

aminotransferase

PLP

1

tyrosine

embryo

extracts

sources

E. coli

of tyrosine

of chick

these

from various

and resolve

inactivation

extracts

present

(4)

phosphatase

degrees.

E - PLP

the

alkaline

aminotransferase has shown that

phosphatases

The results occurs

This

initially

and alkaline

to varying

have suggested

alanine-phenylglyoxal

tryptophanase

catalyzed

reports

rather which

tyrosine

than

affected bound

has a very

1176

the site

of phosphatase

aminotransferase,

stable

aspartic

is not

is

at pH 4.1,

by either cofactor labile

which

with contains

of the alkaline is attacked.

apoenzyme

is

was converted

by treatment

aminotransferase,

attack

form

Thus (5),

into acid a

phosphatases chick would

first

BIOCHEMICAL

Vol. 43, No. 5, 1971

be resolved

and its

These in the

observations

in this

process

aminotransferase

(17)

lysosomal

enzyme,

lysosomal

proteolysis.

oxygenase

to degradation

is

levels

removed

there

(3)

in rat has been

of tyrosine

from

rat

fluoride

in the

observed

that

decrease

affecting

may also

liver

tissue

the

be affecting

that (16)

to be inactivated

are more

susceptible

to the susceptibility

which

prevents

cells.

of the enzyme, through

its

to

(18).

the

decrease

occurs

when

it

was shown

Although

and this

a

of tryptophan

of tryptophan

normally

decrease

of

steroid that

was suggested

the possibility inhibitory

a

by crude

which

fluoride

play

and tyrosine

phosphatase,

in ATP levels

turnover

lysosomes

of acid

absence

culture

turnover

is

One

enzymes.

function

be analogous

aminotransferase

hepatoma

was a concomitant

as the factor

would

may be involved

dependent

been reported

be to form apoenzymes This

it

phosphate

A possible

at 37'.

phosphatases

aminotransferase

have

at pH 5. might

that

degradation

alanine

liver

RESEARCH COMMUNICATIONS

by incubation

possibility

protein

Both

of rat

preparations

inactivated

of pyridoxal

regarding (15).

lysosomal

induced

the

regulation theories

Recently

rapidly

suggest

intracellular

of the current role

apoenzyme

AND BIOPHYSICAL

exists action

that

on acid

phosphatase.

Acknowledgements. valuable

discussions

I wish

to thank

and Mrs.

Drs.

H. Bergman

T. R. Breitman for

excellent

and A. Peterkofsky technical

for

assistance.

REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13.

Kennay,F. T., Science 156, 525 (1967). Peterkofsky, B. and Tomkins, G. M., J. Mol. Biol. 30, 49 (1967). Hershko, A. and Tomkins, G. M., J. Biol. Chem. 246, 710 (1971). Turner, J. M., Biochim. Biophys. Acta, 77, 697 (1963). DeVivo, D. C. and Peterkofsky, B., J. Biol. Chem. 245, 2737 (1970). Peterkofsky, B., in preparation. Lowry, 0. H. in S. P. Colowick and N. 0. Kaplan (Editors), Methods in enzymology, Vol. IV, Academic Press, Inc., New York, 1957, p. 371. Jenkins, W. T. and Sizer, I. W., in S. P. Colowick and N. 0. Kaplan (Editors), Methods in enzymology, Vol. V, Academic Press, Inc., New York, 1955, p. 677. Andrews, M. J. and Turner, J. M., Nature, 210, 1159 (1966). Turner, J. M. and Happold, F. C., Biochem. J., 7S, 364 (1961). Pogell, B. M., J. Biol. Chem. E, 761 (1958). Shibko, S. and Tappel, A. L., Biochim. Biophys. Acta, 73, 76 (1963). Happold, F. C., Nature, 194, 580 (1962).

1177

Vol. 43, No. 5, 1971

14. 15. 16. 17. 18.

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

Scardi, V., Scotto, P., Iaccarino, M. and Scarano, E., Biochem. J. 88, 172 (1963). Coffey, J. W. and DeDuve, C., J. Biol. Chem., 243, 3255 (1968). Segal, l-l. L., Matsuzawa, T., Haider, M. and Abraham, G. J., Biochem. Biophys. Res. Communs. 2, 764 (1969). Auricchio, F. and Liguori, A., FEBS Letters, 12, 329 (1971). Schimke, R. T., Sweeney, E. W. and Berlin, C. M., Biochem. Biophys. Res. Communs. l5, 214 (1964).

1178