Evidence for the γ-glutamyl cycle in yeast

Vol. 68, No. 4, 1976

BIOCHEMICAL

AND BIOPHYSICAL

EVIDENCE FOR THE y-GLUTAMYL Elizabeth

of Chemistry, Brunswick,

Received

December

CYCLE IN YEAST

D. Mooz and Laura

Department

RESEARCH COMMUNICATIONS

Wigglesworth

Bowdoin

Maine

College

04011

19,1975 SUMMARY

Many previous studies have shown that yeast contains high concentrations of glutathione and enzymes needed for its synthesis. We report here that yeast also contains y-glutamyl transpeptidase, y-glutamyl cyclotransferase, dipeptidase, and 5-oxoprolinase activities, suggesting that the y-glutamyl cycle may be operative in yeast. The presence of the cycle enzymes in yeast offers a simple free-cell system which can probably be adapted to studies on the function of this cycle. It has long glutathione

been

(4-5

that

(1)

source

this

series

of six

and degradation has been

is

has been

the y-glutamyl

enzyme-catalyzed

of glutathione

suggested

that

concentrations

was first (2).

present

in yeast,

renewed

from

interest

(3,4)

and an apparently isolated

in glutathione

and there

reactions

functions

is

evidence in the

that

synthesis

of mammalian

tissues;

it

functions

in amino

acid

cycle

yeast

studies

enzyme has been

cycle,

y-glutamyl

of

isolated

Subsequent

of this

in a number

the

high

Glutathione

preparation

There in

contains

by Hopkins

purified

(5).

as the key reactant

kg).

synthetase

and highly

from this

yeast

and later

glutathione

homogeneous

that

mmoles per

by de Rey-Pailhade showed

known

transport

(637) . Although clearly

the

present

reactions whether

enzymes in yeast,

for

cycle.

this

microorganism

has the

it

does contain

y-glutamyl

that

the

that

of transpeptidases also

specificity

contains

of

the

synthesis

no information

of the y-glutamyl

and found

Yeast

needed

is

y-glutamyl

which

have been

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

about

sought

needed

for

transpeptidase

yeast

y-glutamyl

available

We therefore enzymes

of glutathione

cyclotransferase, 1066

the other

to determine glutathione

activity.

transpeptidase found

are

in various

is

utilization It

similar

mammalian

dipeptidase,

and

notable

is

to tissues.

Vol. 68, No. 4, 1976

BIOCHEMICAL

5-oxoprolinase

activities.

now make it the

possible

question

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

The finding

to examine

of these

enzymes

in a relatively

of the physiological

role

simple

in yeast

free-cell

of the y-glutamyl

should system

cycle

in this

organism. EXPERIMENTAL Materials Dr.

Alton

.

L-y-Glutamyl-L-a-aminobutyric

Meister.

L-y-Glutamyl-p-nitroanilide,

L-y-glutamyl-L-glutamic glycylglyine

acid,

were

obtained

or four

blender

to the

distilled

water

allowed

to stand

water

were

minutes.

yeast

consistency were

crumbled

yeast

at 37”.

and the mixture

The supernatant

Company. and dried

powder.

to one volume

10 hours

Three

of dried Then

over

three

yeast.

of

in a Waring

volumes

of prewarmed

The mixture

more volumes at 5,000

was stored

a period

briefly

was centrifuged

solution

and

was pulverized

of a coarse

added

for

added,

were

from

5-oxo-L-proline,

Sigma Chemical

The dried

days.

was obtained

L-y-glutamyl-a-naphthylamide,

from

Cakes of wet baker’s three

acid

was

of prewarmed

x g for

30

at 5’.

METHODS Glutathione synthesis

synthetase

of ophthalmic

activity acid

was determined

release

20 mM glycylglycine Tris-HCl magnesium

as given

transpeptidase

of p-nitroaniline

buffer

activity

previously

was determined

as follows.

(150 umoles,

chloride

and

pH 9),

(10 umoles),

(0.1-0.2

ml)

in a final

minutes,

2 ml of 1.5 M acetic

was read

against

acceptor

were

a blank separately

The reaction

volume

of 1.0 ml. is

at 410 mp. omitted

mixtures

added. Controls

were

employed.

1067

incubation

The absorbance in which

of

contained (4 umoles),

(20 pmoles) After

the

in presence

y-glutamyl-p-nitroanilide

glycylglycine

acid

(5).

by following

from L-y-glutamyl-p-nitroanilide (8)

the

(L-y-glutamyl-L-a-aminobutyrylglycine)

L-y-glutamyl-L-a-aminobutyrylhydroxamate y-Glutamyl

by measuring

and autolyzate at 37’

for

5

of the solution

substrate,

enzyme,

or

Vol. 68, No. 4, 1976

BIOCHEMICAL

y-Glutamylcyclotransferase L-a-aminobutyrate buffer

AND BIOPHYSICAL

activity

as substrate

(9).

RESEARCH COMMUNICATIONS

was assayed with L-y-glutamylThe reaction mixtures contained Tris-HCl

(50 umoles, pH 8), L-y-glutamyl-L-a-aminobutyrate

enzyme in a final

volume of 0.25 ml.

After

(4 umoles), and

incubation

at 37’ for 20 minutes,

the tubes were placed at 90° for 3 minutes to terminate the reaction. mixtures were put on was collected.

Dowex

50 columns (1x3

cm.)

The

and 2.5 ml of water effluent

The absorbance of the solutions containing

5-oxo-L-proline

was measured at 205 mu. Controls were run in which enzyme and substrate were omitted.

Similar results were obtained when L-y-glutamyl-L-glutamate

was used as a substrate. Studies on the utilization

of 5-0x0-L-[&

14C]-proline

glutamate was taken as a measure of 5-oxoprolinase activity

was determined qualitatively

(4 umoles), and enzyme in a final Samples of the reaction n-butanol:acetic

by incubating

to form L-[14C]

activity

(10).

L-a-aminobutyrylglycine

volume of 0.6 ml at 37’ for 60 minutes.

mixtures were chromatographed for 20 hours in

acid:water

(40:9:20)

together with appropriate

standards

and the chromatograms were developed with ninhydrin.

TABLE 1 EnzymeActivities

of the y-Glutamyl

Cycle

Specific

Enzyme

Activity*

y-Glutamyl

transpeptidase

8.4

y-Glutamyl

cyclotransferase

26.0 10.0

5-Oxoprol inase Glutathione

Dipeptidase

Synthetase

4.6

*Nanomoles of product formed per mg of autolyzate protein per minute under the conditions given in the text.

1068

BIOCHEMICAL

Vol. 68, No. 4, 1976

Protein

was determined

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

by the method

of Lowry,

--et al,

(11).

RESULTS Table cycle.

1 gives

the

The observed

not

accurately

the

enzyme increases

presence

reflect

II

acceptors

in the

activity

gives

by 60% after

for

y-glutamyl

activity partial

as the

transpeptidase

may

total

for

purification,

activity suggesting

the

in the autolyzate.

data

on the

of the y-glutamyl

autolyzate

the

absence

of added

in the presence

of enzymes of the y-glutamyl

value

the --in vivo

of an inhibitor

Table

yeast

enzyme activities

relative

of p-nitroaniline

acceptor

of various

amino

of y-glutamyl-p-nitroanilide.

moiety

release

activity

With

the

from y-glutamyl-p-nitroanilide

was about

of 20 mM glycylglycine.

acid

60% of the

activity

The autolysate

observed

contains

some free

TABLE II Activity

with

Amino

Acid

(20

Acceptor

of y-Glutamyl Various

Amino

n mol/min.

mw

Transpeptidase Acid

*

Acceptors Relative Activity to Glycylglycine (%I

Glycylglycine

6.28

(100)

L-Methionine

4.96

79

L-Alanine

4.39

70

L-Glutamine

4.50

71

Gfycine

4.71

75

L-Proline

4.58

73

L-Cysteine

5.15

82

3.77

60

L-Glutamic

Acid

*Enzyme activity was determined as described in the text. The formation product was demonstrated by paper of the y-glutamylglycylglycine chromatography in studies in which y-glutamyl p-nitroanilide and y-glutamyla-naphthylamide were the y-glutamyl donors.

1069

Vol. 68, No. 4, 1976

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

amino acids, as evidenced by paper chromatography. y-glutamyl

transpeptidase

and selective

enzyme (obtained by ammoniumsulfate

heat denaturation)

decreased in relation

the control

to the activity

in Table III

show that L-cystine

nitroanilide

as the substrate.

(12) *

These results

purified

fractionation

value without added acceptor

observed with added acceptor.

amino acid acceptors were methionine, glycine,

acids.

With partially

and cysteine.

is a very active

The best

The data given

acceptor for y-glutamyl-p-

L-Cystine was more active

than other amino

are similar with those found with a rat kidney preparation

In addition qualitative

studies (carried

out as stated under methods) show

the presence of dipeptidase activity. DISCUSSION Evidence presented here suggests that y-glutamyl in yeast.

cycle is operative

There is ample evidence that yeast has the enzymes required for

the synthesis of glutathione. enzymes of the y-glutamyl on y-glutamyl

The present studies show that the other

cycle are also present.

Results shown in Table II

transpeptidase with various amino acid acceptors suggest

that the enzyme has a broad acceptor specificity.

These results are similar

TABLE III Activities

of Various Amino Acids as Acceptors of the y-Glutamyl

Amino Acid Acceptor L-Cystine Glycylglycine

Group

nmoles/min * 5.40

(0.45 mM) (0.50 mM)

5.21

L-Cysteine

(0.50 mM)

4.90

L-Glutamine

(0.50 nM)

4.58

* Enzyme activity

was determined as described in the text.

1070

Vol. 68, No. 4, 1976

BIOCHEMICAL .AND BIOPHYSICAL RESEARCH COMMUNICATIONS

in somerespects to those shownpreviously kidney system (8). activity,

Our data show that glycylglycine

followed next by the sulfur-containing

cystine and methionine. activity

by Tate and Meister in the rat

One noticeable

has the highest

amino acids, cysteine,

difference

is that the acceptor

of glutamine in the yeast system is considerably

reported with the rat kidney system. y-glutamyl

of the purified

enzyme will

Studies on the activity concentrations

of y-glutamyl

as glycylglycine

those given previously

studies on the

transpeptidase with low suggests that L-cystine

by Thompsonand Meister (12) using a rat kidney

while intra-cellular

those of cystine;

at (0.45 mM)

at (0.50 mu). These results agree with

Blood plasma contains higher concentrations

preparation.

of yeast

be published subsequently.

of acceptors (Table III)

is about as active

cysteine,

Work on the purification

transpeptidase is now in progress; detailed

specificity

less than that

concentrationsof

cysteine are higher than

it has been suggested that an active

system within the cell may account for this finding yeast contains glutathione

of cystine than

glutathione

(12).

reductase

It is known that

reductase which uses NADPHat about one-hundred

times the rate with NADH. This enzyme exhibits

a high degree of specificity

for glutathione

We have also carried

disulfide

as a substrate (13).

studies which show that whole cells suspensions of yeast exhibit transpeptidase activity; conditions

no detectable cell disruption

y-glutamyl

occurs under the

employed. This suggests that the enzyme may be located on the

surface of the. cell wall,

as it is in many mammaliancells.

The yeast is

usually grown in a mediumcontaining added amino acids (14). that cell membranebound y-glutamyl transport

out

transpeptidase may function

It is possible in the

of amino acids into the cell. ACKNOWLEDGMENTS

This investigation was aided by a Faculty Research Grant from Bowdoin College. Wewish to thank Dr. OwenGriffith, Department of Biochemistry, Cornell University Medical College for the assays of S-oxoprolinase. We are indebted to Peter Pressman, in our laboratory, for the studies with dipeptidase.

1071

BIOCHEMICAL

Vol. 68, No. 4, 1976

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

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