VoL70,No,.1,1976

BIOCHEMICAL

INACTIVATION

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

OF ASPARTATE AMINOTRANSFERASE

PYRUVATE.

DURING TRANSAMINATION

OF CYSQgO IN CYTOSOLIC ISOENZYME

EVIDENCE AGAINST MODIFICATION Abdalla

WITH CHLORO-

Mohamed Osman* and Toshio

Yamano

Department of Biochemistry Osaka University Medical School Joancho, Kita-ku, Osaka 530 Japan Yoshimasa

Morino

Department of Biochemistry Kumamoto University Medical School Honjo, Kumamoto 860 Japan

Received

March

2,1976

SUMMARY: Both the cytosolic and mitochondrial isoenzyme of aspartate aminotransferase from pig heart were inactivated during transamination with chloropyruvate. Inactivation occurred with L-alanine as the amino group donor in the presence of potassium formate. When L-glutamate or L-aspartate was employed as the amino group donor in the transamination reaction with chloropyruvate, no inactivation occurred. This is in contrast to the case of inactivation by bromopyruvate ( Okamoto, M. & Morino, Y.(1973) J. Biol. Chem. 248, 82-90) where these natural dicarboxylic amino acid substrates were effective in the transamination reaction leading to syncatalytic inactivation (Birchmeier, W. & Christen,P.(1974) J. Biol. Chem. 249, 6311-6315). The Cys3go in the cytosolic isoenzyme which was modified in the syncatalytic inactivation was not modified under the present condition for inactivation with either chloropyruvate or bromopyruvate.

from

Both

the

cytosolic

pig

heart

were

8-chloro-L-alanine alanine is

residue residue

was tested than

inactivate

isoenzymes

for

the

the (3).

rather

reactive

With

than

the

to label

the active

modifier.

Indeed,

isoenzymes in

enzymes with

the

chloropyruvate

substrates

did

not

* On leave

from

lead

University

used are:

of aspartate

presence

in the to the

presence

inactivation

of Khartoum, Cys, cysteinyl

Copyright 0 1976 by Academic Press, Inc. All rights of reproduction in any form reserved.

site

than

of natural of both

of a

label to

transamination

incubation

dicarboxylic

of the amino

isoenzymes.

Sudan. residue; 153

to

bromopyruvate, was found

during whereas

the

(4).

as an affinity

chloropyruvate

formate

(5,6).

was claimed

label

reagent

aminotransferase

of potassium

isoenzymes

which

isoenzyme,

as an affinity

capability

residue

the modification

cytosolic

as an alkylating

with

by B-chloro-Llysyl

in both from

(EC 2.6.1.1)

reaction

and bromopyruvate

as a syncatalytic both

during

Inactivation

coenzyme

to result

to be Cys3go

modifier less

& 4).

of an essential

was shown

was identified

L-alanine

Abbreviations

with

aminotransferases

inactivated (2,3

modification

formation

in both

Chloropyruvate,

with

covalent

as a syncatalytic

rather

to be irreversibly

by bromopyruvate

cysteinyl act

from

aspartate

or bromopyruvate

in aldimine

Inactivation modified

shown (1)

resulted

involved

and mitochondrial

EDTA, ethylenediaminetetraacetate.

acid

VoL70,No.

1, 1976

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

EXPERIMENTAL

PROCEDURES

Materials: Both the cytosolic and mitochondrial aspartate aminotransferases were purified from pig herat by a modification of the procedure described for beef liver isoenzymes (7). Chloropyruvic acid was synthesized b the method described 1 4Clacetate (57 mCi per (8) and recrystallized from hot chloroform. Sodium iodo[lmmol) was obtained from the Radiochemical Center, Amersham. Methods: Assay for the normal transamination between L-aspartate and a-ketoglutarate was performed as described previously (3). Progress of inactivation was monitored by determination of transaminase activity on aliquots withdrawn at various time intervals from the reaction mixtures prepared for control and inactivation as described for the individual experiments. For the analysis of sulfhydryl groups, the cytosolic isoenzyme (50 mg) was incubated at 37" C with 100 mM chloropyruvate in the presence of 0.3 M L-alanine, 3 M potassium formate, 1 nM dithiothreitol and 0.2 M sodium cacodylate buffer (pH 6.5) in a final volume of 3 ml. As a control experiment, the reaction mixture identical to that described above except for the omission of L-alanine was prepared and incubated at 37°C. After incubation for 3 hours the remaining activities were 4 % and 94 % in the presence and absence of L-alanine, respectively. Then both preparations were dialyzed for 24 hours against 2 liters of 0.1 M sodium acetate buffer (pH 6.0). The enzyme activity was checked and found to be the same as described above. The samples were reduced by NaBH and dialyzed against 10 mM sodium acetate The dry samples were dissolved in buffer (pH 5.5) for 6 hours and 4 yophilized. 2 ml 6 M guanidine-HCl containing 0.1 M Tris-HCl buffer (pH 8.0), 1 mM EDTA and 1 mM dithiothreitol and incubated for an hour at 37°C. To these solutions was added a sufficient amount of sodium iodo[l-14Clacetate (2.2 x 105 dpm per ,,mol) to make a final concentration of 10 mM and the mixtures were incubated for 10 minutes at 37°C. The samples were dialyzed against distilled water and heavy precipitates formed were collected by centrifugation. Precipitates were dissolved in 3 ml of 70 % formic acid and 50 molar excess of cyanogen bromide was added. The mixtures were kept at room temperature (15 to 2O'C) for 24 hours and then diluted 10 times with distilled water and lyophilized. Dried samples were dissolved in 5 ml of 6 M urea in 5 % formic acid and applied to a Sephadex G-75 column (3 x 130 cm) equilibrated with the same solvent. Peptides were eluted with 6 M urea in 5 % formic acid. Fractions of 5 ml were collected at a flow rate of 15 ml per hour. The addition of urea gave better resolution and abolished the anomalous elution of the peptide with 75 residues before the one with 212 residues (4,g). Bray's solution (10) was used as the scintillation medium. Radioactivity was determined in a Packard scintillation spectrometer model 3385. Absorption spectra were obtained with a Hitachi recording spectrophotometer model 124 and circular dichroism spectra with a Jouan Dichrograph II. RESULTS Inactivation

of aspartate

Incubation

of both

inactivation

of these

inactivation

with

amino the

acid

effective vation

isoenzymes

enzymes.

chloropyruvate

cosubstrates.

natural

presence

aminotransferase

amino (Fig.

1).

increased

latory action aminotransferase

with

substrate,

was found L-glutamate

When L-alanine was required the

chloropyruvate

alone

did

not

lead

to the

In contrast to the case with bromopyruvate (3,4), was strongly dependent on the nature of the

L_Alanine

acid

of formate

with

by chloropyruvate

increase

to be the most or L-aspartate,

was used as an amino for

efficient in the

inactivation.

concentration

acid

effective

was much less substrate,

the

The rate

of inacti-

of formate.

of formate is analogous to that found in the reaction with L-alanine (1) or B-chloro-L-alanine (1,ll).

154

whereas

This

stimu-

of aspartate

VoL70,No,

1,1976

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

0 0

60

I20

180

0

Time

in

60

120

160

min.

Fig. 1. Effect of amino acid substrates and formate ion on the inactivation of aspartate aminotransferase isoenzymes by chloropyruvate. Each reaction mixture contained, in a total volume of 1.0 ml, 2.5 mg mitochondrial isoenzyme (A) or 1.5 mg cytosolic isoenzyme (B), 150 mM (A) or 100 mM (B) chloropyruvate, and 0.2 M Tris-HCl buffer (pH 7.5). Incubations were at 25°C. Curve 1, in the absence of any amino acid substrate. Following additions were made in the experiments represented by curves 2-5. Curve 2, 100 mM L-glutamate; curve 3, 40 mM L-cysteine sulfinate; curve 4, 0.3 M L-alanine and 3 M potassium formate; curve 5, 100 mM L-aspartate. Activity for normal transamination was determined on aliquots withdrawn from each incubation mixture at the indicated times. In both A and B, addition of 0.3 M L-alanine alone gave results essentially identical to those indicated by curves 1. Specific activities of the cytosolic and mitochondrial isoenzymes were 210 and 190 pmoles per min per mg protein at 25"C, respectively, and these values were taken as 100 %. The rather substrates

puzzling

could

ineffectiveness

be attributed

produced

from

the

corresponding

competes

with

chloropyruvate

can be seen from Table protection

against

chloropyruvate explains the

isoenzymes

those

profound

that

acid

during

presence

the

a-ketoglutarate

acid

to the amino

active acids

site were

prior

inactivation.

substrates This

AS

exhibited

finding

strong

suggests

to inactivation.

ineffective

acid

transamination

enzyme against

of keto

amino or oxalacetate

enzymatic

by chloropyruvate.

that

Moreover,

as cosubstrates

of chloropyruvate-inactivated

considerable

gross

change

treated with chloropyruvate species (Fig. 2, bottom,

of the change

Either

the

analysis

revealed

preparations the inactivated

fact

amino

dicarboxylic

it

during

reaction.

The spectral

reagent

bind

why the natural

inactivation

from

I,

natural

to the

and protects

inactivation

must

of the

control

of chloropyruvate

and inactivate

in the active

site

or bromopyruvate

the enzyme

in comparison Circular

through

155

distinctly

that

upon

chromophore could

alkylation

with dichroic

2) were

suggesting

preparations,

had occurred

alone. curve

preparations

react

of both the control spectra

of

different inactivation

or its simply

of nucleophilic

a

vicinity. as an alkylating side

chains,

Vol.70,No.1,1976

BIOCHEMICAL

AND BIOPHYSICAL

Table

RESEARCH COMMUNICATIONS

I

Protection of aspartate aminotransferase isoenzymes against chloropyruvatemediated inactivation by competitive keto acid substrates. In a final volume of 1.0 ml each, 1.5 mg either isoenzyme were incubated with 50 mM chloropyruvate in the presence of 3 M potassium formate and 0.1 M potassium cacodylate buffer (pH 6.5) at 25°C. When indicated, concentrations of L-alanine, a-ketoglutarate and pyruvate were at 0.3 M, 50 mM and 50 mM, respectively. Specific activities of the cytosolic and mitochondrial isoenzymes were 210 and 190 Pmoles per min. per mg protein at 25"C, respectively, and these values were taken as 100 %. Remaining

Isoenzyme

Incubation Time (minutes)

Activity

(per

Chloropyruvate t

Chloropyruvate t

Chloropyruvate

L-Alanine

L-Alanine t

L-Alanine t

2

73 %

120 Lii

in addition

to acting

aminotransferase substrate

as a keto

is

pair

(9)

bromopyruvate-mediated cytosolic easily

isoenzyme be labeled

9"; 88

87

95

97

97

36 53

68 78

82 87

i:

Cys 3go of the

cytosolic

consequently peptide whether

is

identified

syncatalytically

known

(4).

(12,

13),

Since

the primary

each of the

carboxymethylation

with

by chromatographic

in the

as the modified five

separation

showed

that

each of three

approximately fractions

Cyslgl.

Of these

blocked

by chloropyruvate.

derived

from

peptide

which

peptide

fragment.

the

three Cyslgl

absorbed

equal

(Fig.

sulfhydryls,

3). the

amount Fraction fully

peptide. in Fraction

cysteinyl

156

of the

residues

can

iodoacetate

and

bromide-cleaved to examine Such

was distributed

I should

Cy~4~.

contain

results

CysB2

Cys4S and CysB2 were

in Fraction

Fraction

a

during of

was employed chloropyruvate.

exposed

III

structure

of cyanogen

at 325 nm at pH 7.0 and hence

Radioactivity

presence

of radioactivity

Hence the radioactivity -containing

aspartate

residue

radioactive

fragments on a Sephadex column. This procedure or not Cys3go is involved in inactivation with

analysis into

substrate.

had been implicated

through

%

% ;: 97

acid

inactivation

a-Ketoglutarate

i':

known to be modified and it

Alone

85 % 69 60

Cytosolic 120

Chloropyruvate

t

Pyruvate

Mitochondrial

cent)

II

I represents

contained

should from

contain the

that

pyridoxyl the

CYSTIC-

Cys3g0-containing

Vol.70,No.1,1976

BIOCHEMICAL

I 300

350

400

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

1 450 WAVELENGTH,

350

400

450

nm

Fig. 2. Absorption and circular dichroic spectra of aspartate aminotransferases treated with chloropyruvate in the presence and absence of L-alanine. A. mitochondrial enzyme; B. cytosolic enzyme. Upper, absorption spectra; lower, circular dichroism spectra. Each reaction mixture contained, in a total volume of 1.0 ml, 3.0 mg mitochondrial isoenzyme or 2.8 mg cytosolic isoenzyme, 150 mM chloropyruvate, 3 M potassium for-mate and 0.1 M Tris-HCl buffer (pH 7.5). Curves 1, in the absence of L-alanine; curves 2, in the presence of 0.3 M Lalanine. Incubations were at 33°C for 4 hours. These spectra were taken after dialysis overnight against 0.1 M potassium pyrophosphate buffer (pH 7.5). Spectra were corrected for dilution due to dialysis. Remaining activities checked after dialysis were 97 % (curve 1) and 28 % (curve 2) in A., 96 % (curve 1) and 4 % in B.

$

0.3 III

E 2 a

0.2

FRACTION

NUMBER

Fig. 3. Fractionation of cyanogen bromide-cleaved peptides on a Sephadex G-75 column in 6 M urea containing 5 % formic acid. Experimental details were described under Methods. A. inactivated cytosolic enzyme (remaining activity, 4 X); B. control preparation (remaining activity, 94 %). 157

Vol.70,No.1,1976

peptide.

BIOCHEMICAL

Thus all

five

cysteinyl

identified by examining the of cyanogen bromide-peptides 14C-iodoacetate. pattern

residues

of radioactivity

from

and that

L-alanine

and potassium

formate.

was not modified

either

inactivation

resulted

in the

a preparation Fig.

cytosolic

which

between

This

the

finding

from modification

can be

chromatogram

has been treated

with

in the distribution

preparation

treated

by chloropyruvate

in the

indicated

in the presence

enzyme

on the

3, no difference

inactivated

residue

RESEARCH COMMUNICATIONS

of radioactivity

was observed

alone

present

distribution

As can be seen from

chloropyruvate

the

AND BIOPHYSICAL

that

or absence

with presence

any cysteinyl of L-alanine

of residue(s)

of

other

and hence

than

cysteinyl

residues.

DISCUSSION When the data

for

curves not yield

the resulting

plot

did

from

that

during

the fact

tective the

action

against

acid

substrates,

keto

reaction

mixture

Furthermore,

which acts

condition,

i.e.,

in an indirect methylcysteine

cyanogen

bromide-cleaved

the presence

Fig. result

is

inactivation in accord label)

of the amino

in the (4))

in

the

cysteinyl

the data

cosubstrate

from

that

Thus

by bromothat

of the control as with

modified

in the

isoenzyme, conditions.

the

preparation (a condition

in Fraction there

the

that,

on the

(as a syncatalytic

158

was performed

revealed

indicates

radioactivity

and reaction

formate.

formate

of L-glutamate

the cytosolic

chloro-

experimental

inactivated

Cys3gD was not

(4).

that

residue

Cys3gD vJas modified

reported

inactivation idea

residues as carboxy14 enzyme with C-iodoacetate

experiment

of bromopyruvate

of

cysteinyl

finding

that

the

presence

the present

isoenzyme

presence

revealed

inactivating

acid

of the

was indistinguishable

A parallel

to the inactivation.

in the

and potassium

and potassium

and hence with

under

an inactivated

by chloropyruvate,

results a pro-

Indeed,

syncatalytic

reagent

This

probably

against

enzymes

in the

fashion,

and exerts

when added

in favor

of cytosolic

order

chloropyruvate.

are

a modified

treating

bromopyruvate.

by bromopyruvate

the

of unmodified

peptides

modes of action

an affinity

for

observation).

3) was insignificant

different nature

with

This

the enzymes

of L-alanine

of L-alanine

(unpublished

case of inactivation

syncatalytic

findings

search

on the preparation

in

with

cosubstrate

these

from

line. accumulates

inactivate

way by identification

pyruvate

inactivated

not

site-directed

study,

in a first

and a-ketoglutarate,

in the presence

resulting

Such analysis

inactivation

All

straight

pyruvate

protected

did

as an active

In the present

preparation

pyruvate

inactivation,

(4).

plotted

by competing

was an efficient

bromopyruvate

pyruvate

a typical

inactivation

for

1 were

the reaction

chloropyruvate

L-glutamate with

4 in Fig.

in this

for

III case.

( This

seems to be two modifier depending

or as upon the

Vol. 70, No. 1, 1976

The present by chloropyruvate modification of the

result does

has shown not

result

of some residue(s)

structure

is currently

BIOCHEMICAL

of the modified under

way with

the

that

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

inactivation

of the

cytosolic

from modification

of the

Cys390

within

the

site

in the

active

site.

cytosolic

use of radioactive

Direct

isoenzyme but

from

identification

and mitochondrial

isoenzyme

chloropyruvate.

REFERENCES Morino,

Y. (1972) Biochem. Biophys. Res. Commun. 47-, 498-504. Y. and Okamoto, M. (1970) Biochem. Biophys. Res. Commun. 40, 600-605. Okamoto, M. and Morino, Y. (1973) J. Biol. Chem. 248, 82-90. i: Birchmeier, W. and Christen, P. (1974) J. Biol. Chem. 249, 6311-6315. 5. Morino, Y. and Okamoto, M. (1973) Biochem. Biophys. Res. Commun. 50, 1061-1067. 6. Morino, Y. (1973) Seikagaku 9, 993-1014. 7. Morino, Y., Itoh, H. and Wada, H. (1963) Biochem. Biophys. Res. Commun. 13, 348-352. 8. Cragoe, E. J. and Charles, M. R. (1960) Org. Synth. 40, 54-60. 9. Birchmeier, W., Wilson, K. J. and Christen, P. (1973rJ. Biol. Chem. 248, 1751-1759. 10. Bray, G. A. (1960) Anal. Biochem. 1, 279-285. 11. Morino, Y., Abdalla, M. 0. and Okamoto, M. (1974) 3. Biol. Chem. 249, 6684-6692. 12. Ovchinnikov, Yu. A., Egorov, Ts. A., Aldanova, N. A., Feigina, M. Yu., Lipkin, V. M., Abdulaev, N. G., Grishin, E. V., Kiselev, A. P., Modyanov, N. N., Braunstein, A. E., Polyanovsky, 0. L., and Nosikov, V. V. (1973) FEBS Lett. 29, 31-34. 13. Doonan, S., Doonan, A. J., Hanford, R., Vernon, C. A., Walker, J. M'., Airoldi, iL. P. S., Bossa, F., Barra, D., Carloni, M., Fasella, P. and Riva, F. (1975) Biochem. J. 149, 497-506.

:: Morino,

159