Stable ornithine decarboxylase in promastigotes of Leishmaniamexicana

Vol. 161, No. 2, 1989

BlOCHEMlCALANDBlOPHYSlCALRESEARCHCOMMUNlCATlONS Pages

June 15,1989

STABLE ORNITHINE

DECARBOXYLASE IN PROMASTIGOTES C.P.

Instituto

Sbnchez,

de Investigaciones

Ciencias

Exactas

April

24,

Gonza'lez

and I.D.

Bioquimicas

y Naturales,

A. Machado Received

N.S.

OF LEISHMANIA

(1405)

Campomar",

Facultad

Aires

and CONICET,

de Buenos

Buenos

MEXICANA MEXICANA

Algranati

"Fundacion

Universidad

151,

754-761

Aires,

de

Argentina

1989

Studies on the decarboxylation of ornithine in Leishmania mexicana have shown that this activity corresponds to a true ornithine decarboxylase rather than to an oxidative decarboxylation or aminotransferase reaction, both of which also give rise to the release of C02. The stoichiometric relationship between substrate and products has indicated that extracts of L. mexicana were able to catalyse the formation of an unknown compound besidesputrescine and CO?. The addition of cycloheximide to cultures of L. mexicana allowed us to demonstrate that ornithine decarboxylase degradationTn was extremely slow in this --vivo parasite. This remarkable stability of theenzyme is only comparable to that found in Trypanosoma brucei and contrasts with the high turnover rate of ornithine decarboxylasesotfferent mammalian cells. 0 1989 AcademicPESS, 1°C.

Results tant the

from

role

in

many laboratories

cell

enzymes

down

proliferation

ornithine-

rate-limiting

in

the

Uifluoromethylornithine

(DFMO),

decarboxylase,

can block

the

brucei

In

in

relative

mice to

brucei

(9).

its

The

mexicana,

here a

demonstrate putrescine

the in

a highly

several

cell

mammalian studies

causing parasite.

active

from -L. mexicana shows to the culture.

to

ODC but

cure

seem to cell

conversion The cell-free only

a remarkable

traces stability

added

0006-291X/89 $1.50 Copyright 0 1989 by Academic Press, Inc. AN rights of reproduction in any form reserved.

the

754

is

highly

diseases.

ornithine of viva“,

parasi-

Trypanosoma

on the slow

visceral

arginine "in

with

pathways

of

slow (4,5).

several

metabolic

extracts of

infecions

which

to

of ornithine

and

inhibitor

and

used

inhibitor cells

of the

enzyme

been

of

catalyse

trials

be due to

on polyamine cutaneous

inhibitors

clinical

acute

effects

host

intracellular

this

irreversible

differential

to the

parasite

known

an impor-

which

have

of mammalian

able

Several

polyamines, and

a well

play

decarboxylases,

models

proliferation

DFMO is

mammalian

ODC compared

We present

tain

addition

animal

of

polyamines (l-3).

methionine

biosynthesis

in

(6-8).

that

and differentiation

growth

tes

indicated

and S-adenosyl

steps

tumor

have

and Leishmania

parasite

turnover

of -T. (10).

unstable in

Leishmania Our

results

arginine mexicana

decarboxylase

(AK).

even when

putrescine

into conODC is

Vol. 161, No. 2, 1989

BIOCHEMICAL

MATERIALS

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

AND METHODS

Chemicals: Brain-heart infusion and tryptose were from Difco laboratories, Detroit,Michigan. Streptomycin, penicillin, cycloheximide, sulfosalicylic acid, and HEPES buffer were obtained from Sigma. pyridoxal 5-phosphate L-[U-14C]ornithine L-[U-14C]arginine (323 Ci/mol), (266 Ci/mol), L-Cl-14C]ornithine (55.9 Ci/mol) and L- 35S]methionine (1129 Ci/mmol were from M.A. DFM5 was a generous gift of d errell Dow New England Nuclear, Boston, Pharmaceutical Co.

Parasite cultures: Promastigotes of Leishmania mexicana mexicana originally isolated by Dr. Rodrigo Zeledon, University of Costa Rica, were obtained from stocks of the Instituto Fatala Chaben (Buenos Aires, Argentina) and grown with shaking at 26“C in karren's modified liquid medium (11) with the addition of 20 mg of haemin.HCl per liter, 20% fetal calf serum and antibiotics (100 ,ug/ml streptomycin and 100 U/ml penicillin). Leishmania growth was followed by cell counting.

"In viva" labeling with radioactive amino acids: Parasites at the log-phase of I)rowthre collec'te7[-6y centrltugafion, .?asXonce with PBS (0.14 M NaCl, 0.01 M Na phosphate, 3 mM KCl. pH 7.2) and resuspended in the same buffer or in arsinine-free Dulbecco's Modified Eaqle Medium at a concentration of 5-iom' cells per ml. Labeled ornithine br arginine (l-5 @i/ml) was added, and after 30 min incubation at 26°C oarasites were oelleted. washed with PBS and resuspended in 50-100 ~1 of 0.2 N HC104. Cell extracts were neutralized with KOH, centrifuged at 11,000 g for 10 min and the resulting supernatant fiuids were analysed by paper electrophoresis as described previously (12). Radioactive compounds were detected with a radiochromatogram scanner, and in some cases eluted and measured by scintillation counting. In order to follow protein biosynthesis in experiments designed to measure ODC turnover rate, 35S-methionine (10 Ci/ml, 0.1 PM final concentration was added to a portion of a L. mexicana cu r"ture at the log-phase of growth. 1 he labeied sample was divided into two aliquots, cycloheximide (50yg/ml) was added to one of them and samples were taken at different times to measure the incorporation tic acid-insoluble material. The of radioactive methionine into 10% trichloroace remaining non-labeled culture was used to follow the decay of ODC activity in the presence of cycloheximide.

Cell extracts and enzymatic assays: Parasites obtained from exponentially wing cultures-fter centrifui for 5 min at 10,000 g were washed once with PBS and then resuspended at a concentration of 1~10~ cells/ml in 50 mf4 Tris-HCl buffer, pH 7.2, containing 1 mM DTT, 0.5 mM EDTA and 0.1 mM pyridoxal 5-phosphate. Parasites were broken by 3 cycles of freezing and thawing followed by 3 periods of brief sonication (Branson Sonic Power, Co.) for 10 seconds each time. Homogenates were centrifuged for 20 minutes at 12,000 g and supernatant liquids were collected for enzyme assays. UUC activity was measured by the release of radioactive CO2 from labeled The characterization of the reacornithine as it will be described elsewhere. tion products as well as the stoichiometric relationship between them were with L[U-14C]ornithine by studied at the end of the incubation aper electrophoresis. The radioactive compounds were detected by scanning an a the Reaction mixtures to which HC104 was added at corresponding spots were counted. zero time served as blanks. In order to assay ADC a similar rocedure was followed using a reaction mixture containing 50 mM Tris-HCl buf Per, pH 7.5, 1 mM DTT, 0.5 mM EDTA, 0.08 mM pyridoxal 5-phosphate, 1 mM L-arginine and 0.2 jJCi LCU-I4Clarginine. 755

Vol. 161, No. 2, 1989

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

RESULTS AND DISCUSSION The

"in

studied

vivo'I

conversion

by incubation

Both

ornithine

other also

formed

from

latter

amino producing that

only

traces

In

order

this

was

to

organism.

CU2/h/my

of

further

the

protein)

and

have only

radioactive

shown).

could

through

the

possibilities

very

acids.

addition

to was

that

action

of

arginase

supported spp

the

by the

(13),

while

parasite.

biosynthetic

pathways

a high

low

been

ornithine

indicate

Leishmania

ODC and ADC in crude

indicated

in

are

in

has

amino

Labeled

result

reported

in this

we have measured

polyamines

putrescine

not

ornithine

been detected

results

these

and this

been

into

synthesize

OUC. These

ODC have

Our

with to

into

via

investigate

arginine

(results

arginine,

and

mexicana

able

converted

of ADC have

Leishmania

parasites

were

putrescine

arginase

and

characterized

radioactive

acid

ornithine

intact

arginine

not yet

before facts

of

and

compounds

of

levels

of

polyamines

extracts

activity

from

of ODC (60-100

of ADC (l-l.5

in

prepared

nmoles

nmoles

COz/h/mg

of

protein). The

assay

generally

radioactive

CO2

several

enzymes

catalyzing

aminotransferase

(15).

as a true

ornithine followed

measure

ODC activity

The

After

mixture

and

has

yet

not

two

investigation

been in

reaction

formation

subsequent

thin

layer

identified our

mixture. although

and

laboratory. In the

was completely

synthesized

by its

in

chromatography,

while

its

chemical

a somewhat

of the

whereas

the

lower

the

specific

amount.

that

In order 3,

to establish

reaction

the

mixtures

the required components perchloric acid (blank electrophoresis titated

and

by scintillation

the

stoichiometry

containing

radioactive counting.

two

6U min Both

compounds At the 7.56

as by danproduct

(X) under to

putrescine

compound

was still

strongly

suggest

reactions

using

ODC. decarboxylase parasite

or treated

at

in --L. mexihomogenates and zero

mixtures

were

detected

by scanning

same time

one was

currently

independent

of ornithine for

other is

remaining

of DFMO addition

results

involving

from

1A):

ODC inhibitor

L[U-14C]ornithine,

were incubated of the reaction).

the

labeled

These

of

reaction

the

as well

effect

unknown

extracts are able to catalyse -L. mexicana ornithine as substrate, one of them apparently

to (Fig.

structure

16 shows

action

L[U-14C]ornithine,

detected mobility

presence

blocked

were

the

substrate,

of the

with

electrophoretic

Fig.

labeled

In addition

products

with

activity

determination

extracts

of same

ornithine-Z-oxoacid

uniformly

and

the

sequential

via

CO2 producing

by using

products

radioactive

or by the the

of -L. mexicana by paper electrophoresis.

as putrescine

sylation

out

the

measurement

However,

decarboxylation

glutamate

incubation

(ornithine),

characterized

of of

carried of

was analysed

substrate

(14)

formation

the

ornithine.

an oxidative

identification

ODC was

involves

carboxyl-labeled

the

by characterization

stoichiometry.

the

to from

14C02 through can also generate production of 4-aminobutanamide

substrate concomitant

the

used

released

the

analysed

conccanitant

time

with

by paper were release

quanof

Vol. 161, No. 2, 1989

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

6

0

12

2L

18

cm

from

30

origin

Fig. 1. Reaction products formed by incubation of L. mexicana extracts with -toactive ornithine. Parasite homogenates were incubated for 1 h at 37°C in a mixture containing SD mY Hepes buffer (pH 6.9), 0.2 mM DTT, 0.1 mM EDTA, 0.1 mM PyridoxaT s-phosphate and LLU-14C]ornithine (g,uCi/ml, 0.5 mM). At the end of the reaction followed by treatment with HC1U4 and neutralization, the mixture was analysed by paper electrophoresis. The corresponding scans are shown. A and B represent reactions carried out in the absence and presence of 1 mM DFMO, respectively. Shaded or black spots indicate the position of standard compounds (Arg, arginine; Orn, ornithine; Spd, spermidine; Put, putrescine).

14C02 was

measured.

Table

The

decrease

experiments. putrescine duct

formation "X".

In that

of

assumption

this Table

it

TI

pyridoxal extracts

will the

5-phosphate

1.

CO2

in

the

Stoichiometry

reaction

for UTT

-L.

were

of

nmoles

labeled

carbon

mexicana

ODC

omitted

mixture,

the

of the reactions L-[U-14C]ornithine Variation

A Orn

amount of

was the of

two

different

equivalent

to

unidentified "X"

atoms

pro-

formed

we

have

(justification

elsewhere).

requirements and/or

for

ornithine

the

number

ornithine

reported

calculated

of

plus the

the

data

amount

release)

all be

the

the

calculate

contains

gives

and

Table

to

summarizes in

(or

order

assumed

I

in

substrate (nmoles/h)

Put

maximal the

enzymatic

and

activity.

preparation

activity

catalysed by L. as substrateof

A

both

mexicana

When of

was

extracts

markedly

with

products

A CO2

AX

Experiment

1

- 0.16

+ 0.14

f 0.12

+ 0.03

Experiment

2

- 0.29

+ 0.21

f 0.20

+ 0.08

Reaction mixtures were as described in Fig. 1. The products of the reaction were analysed as detailed in Materials and Methods, and radioactive CO2 released was counted. The amount of each compound was calculated using zero time reactions as blanks. and (+) represent decrease and increase of the indicated compounds, respect.iiel)y. 757

cell

Vol.

161,

No.

BIOCHEMICALAND

2, 1989

Table

Reaction

II.

BIOPHYSICALRESEARCH

Requirements

for

optimal

LIDC activity

DDC activity CO2/h/mg of

mixture (nmol

Complete

system

57.7

(100%)

111.3

(31.7%)

- UTT

44.1

(76.4%)

+ 4 mM Mg++

56.4

(97.8%)

- Pyridoxal

Reaction described

mixture in Fig.

in

5-phosphate

as

absence

of ELITA or and excess

the

case

rates

were

optimum

ptl and

kinetic

apparent

Km for

saturation the

affinity

DFMO is enzyme lated

from

inactivation

(the ODC as

corresponding the

of

alter are

enzyme

and Wilson

by the

product

inhibitor

at

the by

putrescine

reaction) using (2.1

was

a Dixon mM) was

pti

III.

-L. mexicana

optimum

Km pyridoxal

plot

ODC properties

mM

0.94

PM

>8 hrs.

Ki

0.20

Ki

DFMO DFMO at putrescine

determinations

mM

3 min

30°C

were

carried

758

2.10

mM

out

in

The

III. 5-phosphate

shows with

the

DFMO was calcutime-dependent

half-life

(results

three-fold

0.70

The

Table

occurs of

a competitive

Half-life

tIj2

All

5-phosphate

as it

The

6.9

Km ornithine

of protein.

of

inac-

3 min.

substrate.

Table

activity.

5-phosphate

studies

30°C.

the

reaction.

The Ki for

of DFMD was about

of

in

as

contrast,

pyridoxal

ON,

from

were

enzyme

pyridoxal

of the

(18)

the

of

Km for

co-factor

In

shown

conditions

of -L. mexicana or from --T. brucei (6,17).

Kitz

(16).

up to 15Opg

enzyme

of the

the

not

1 h with

the

value

sources

did

under

for

concentration

for

least of

low

enzyme

determined

Ki

at

measured

cells

method the

almost

protein)

the complete system duplicate assays.

inhibitor

mammalian of

Putrescine mexicana

for

to of

average

many other

parameters

mM. The

at infinite

the

of Mg'+

linear

of the

the

are

ODC of

an irreversible

with

tivation

for

ornithine

was 0.70

high

corresponding

and conditions 1. All values

reduced Reaction

COMMUNICATIONS

duplicate.

higher

inhibitor not than

of -L. shown). The the

Km for

Vol. 161, No. 2, 1989

The "in cific

vivo"

half-life

activity

mide more

BIOCHEMICALAND

of

higher

than

of the

incubation 8 hr. the

mammalian

cells

have

stimulate

th'eir

degradation

cana

presented

compound

from

substance

probably

observed

when

ZB),

increase

a very

short

half-life

its

the

enzyme

to the

has

cell

turnover.

was inhibited over a long a half-life

culture

In contrast,

(lower

than

30 min)

detect

ODC in extracts

seems to OK

from

and polyamines

(19). paper

allowed

with

radioactive does

that

of 10 mM putrescine than

together

incubations

indicating

rather

in this

promastigotes

these conditions protein synthesis ODC activity changed only slightly

The addition

stabilize

Data

Under while

(Fig.

enzyme

COMMUNICATIONS

of -L. mexicana ODC was estimated by measuring the speenzyme obtained from parasites incubated with cyclohexi-

for different times. than 95% (Fig. 2A)

period

BIOPHYSICALRESEARCH

us to

another

activity

ornithine. not

The

involve were

producing

formation

decarboxylation,

carried

of --L. mexian unknown labeled

of since

out

in the

presence

of -L. mexicana OUC for of the enzyme were also

optimal

activity

this

the

unidentified

same compound

was

of L[1-14C]ornithine

as substrate. The

requirements

kinetic

properties

sible

inhibitor

as determined

for

the

enzymes

lime 0

I

0

I

2

4

6 Time

as well

DFMO was a potent

of other

eucaryotic

cells

as several irrever(20).

(mid

30

I

0

studied.

60

I

u

a

24 (hr)

incorporation into 2. Stability of C, mexicana WC. A, [35S]methionine insoluble material In L. mexlcana promastigotes incubated in the absence (0) 6, ODC specific activity (nmoles and presence (0) of 50 ug/ml cycloheximide. for different times cu /h/mg of protein) obtained from L. mexicana incubated with 50,pg/ml cycloheximide (0) or 10mM putresclne (A). ;;a1

BIOCHEMICALAND

Vol. 161, No. 2, 1989

The unusual that

"in

reported

culate

that

turnover

rich

which

are

The

known

because, depletion

their

to provoke

a rapid

the

obtained

from

amino

glutamic

different

acid

serine

case

by irreversible

comparable

tempting have do

this enzyme leishmaniasis

a very not

slow

contain

(PEST

further

to

to spe-

regions)

of proteins

ODC deserves

inhibition

is

threonine

degradation

-L. mexicana of --T. brucei (lo), against strategies

only

It

probably

and

intracellular

COMMUNICATIONS

parasites

sequences

acid,

of

therapeutic

is

enzyme

proline,

stability

as in for

of ODC from -L. mexicana of --T. brucei (10).

the

decarboxylases

because in

remarkable

target

for

ornithine rate,

regions

stability

vivo"

recently

BIOPHYSICALRESEARCH

(21).

investigation

might provide based on

a useful polyamine

of DDC.

ACKNOWLEOGMENTS We are cultures tially

indebted and

supported

Ueveloping Tropical

S.H.

Countries

Gonzalez

Dr.

B. from

(SAREC), Secretaria

Cientificas and I.D.

Franke

Goldemberg

by grants

Diseases,

Investigaciones N.S.

to

Dr.

the

Cazzulo

helpful

Swedish

and

L.

discussions. Agency

for

Programme

for

de Ciencia

y Tecnica

and the

are

(Argentina). career

C.P.

investigators

Sferco

for

This

work

Research

WHO Special y Tecnicas

Algranati

de

for

Research

Cooperation

of the

parwith

and Training

Consejo

Sa/ncher

parasite was

is

in

National

de

a fellow

and

latter

institu-

tion. REFERENCES 1. Jhnne, J., Pb‘ssb‘, H. and Raina, A. (1978) Biochim. Biophys. Acta, 473, 241-293. 2. Pegg, A.E. (1988) Cancer Res., g, 759-774. 3. Heby, 0. (1981) Uifferentiation, -I9-, l-20. 4. Sunkara, P.S., Baylin, S.B. and Luck, G.U. (1987) In: Inhibition of Polyamine Metabolism. Biological Significance and Basis for New Therapies (McCann, P.P., Pegg, A.E. and Sjoerdsma, A. eds.) Academic Press, Orlando, FL, pp. 121-140. 5. Talpaz, M., Player, J., Quesda, R., Benjamin, H., Kantarjian, H. and Gutter-man. J. (1986) Eur. J. Cancer Clin, Oncol.. 22. 685-689. 6. Bacchi, C;J., Nathan, H.D., Hutner, S.H. and McC%in, P.P. (1980) Science, ZJ, 332-334. 7. Hanson, W.L., Bradford, M.M., CHapman, W.L., Waits, V.B., McCann, P.P. and Sjoerdsma, A. (1982) Am. J. Vet. Res., 43, 1651-1653. 8. Assaraf, Y.G., Golenser, J., Spira, D.X and Bachrach, U. (1984) Biochem. J ., 222, 815-819. 9. Baccki, C.J. and McCann, P.P. (1987) In: Inhibition of Polyamine Metabolism. Biological Significance and Basis for New Therapies (McCann, P.P., Pegg, A.E. and Sjoerdsma, A. eds) Academic Press, Orlando, FL, pp. 317-344. 10. Phillips, M.A., Coffino, P. and Wang, C.C. (1987) J. Biol. Chem., 262, 8721-8727. Warren, L. (1960) J. Parasitol., 46, 529-538. 2: Cataldi, A.A. and Algranati, I.D.71989) J. Bacterial., in press. E.P., Coelho, J.A., Moraes, G. and Figuereido, E.N. (1978) Exp. 13. Camargo, Parasitol., 46, 141-144. 14. Smith, T.A. and Marshall, J.H.A. (1988) Phytochemistry, 27, 703-710. M.E. (1976) Biochem. J., 157, 33-39. 15. Murphy, B.J. and Brosnan, 760

Vol. 161, No. 2, 1989

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

16. Kim, B.G., Sobota, A., Bitonti, A.J., McCann, P.P. and Byers, T.J. (1987) J. Protozool., 34, 278-284. 17. Metcalf, B.W., Bey, P., Danzing, C., Jung, M.J., Casara, P. and Vevert, J.P. (1978) J. Am. Chem. Sot., 100, 2551-2553. 18. Kitz, R. and Wilson, 1.6. F62) J. Biol. Chem. 237, 3245-3249. 19. Kanamoto, R., Utsonomiya, K., Kameji, T. and Hayashi, S. (1986) Eur. J. Biochem., 154, 539-544. 2U. Pegg, A.E. m86) Biochem. J., 234, 249-262. 21. Rogers, S., Wells, R. and Rechsteiner, M. (1986) Science, 234, 364-368.

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