Evidence in vitro for an enzymatic synthesis of phosphocitrate

BIOCHEMICAL

Vol. 170, No. ‘I, 1990

AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 251-258

July 16, 1990

EVIDENCE: Luigi

in

vitro

Moro,

FOR AN ENZYMATIC

Nicola

Dipartimento

Stagni, Benedetto di

A.

Biochemistry

May 21,

34136

Department,

GPO Box Received

32,

252C

D.

e Trieste,

Tas

and

delle

di

Trieste,

Italy

University

Hobart

Sallis,

Chimica

Studi

degli

Valerio

OF PHOSPHOCITRATE

Luxich,*John Bernard

Biofisica

Universita'

Via *

Elena de

Biochimica,

Macromolecole,

SYNTHESIS

of

Tasmania,

7001,Australia

1990

SUMMPiRY: A biological synthesis of phosphocitrate is described from precursors, citrate and adenosinetriphosphate reacting in the presence of rat liver homogenate. Identity of the newly formed product was examined by enzymatic digestion of reactions mixtures, HPLC chromatography and 'H-NMR spectra. Authenticity of product was established by comparison to chemically synthesized phosphocitrate. Recognition of the existence of a biologically synthetic pathway adds credence to the known presence of phosphocitrate in mitochondria and a postulated role to control calcium phosphate deposition in that organelle. 01990 Academic Press, Inc. In recent over

years,

biomineralization

phosphocitrate

in

1980

to vitro

offers

be

phosphorylated

very

powerful (l-4).

to

control

events

The formation

of

and

magnesium

by

phosphate

granules

in kidney

influence

under

atherosclerotic

diet adhesion

to the is

PC

hydrogen

restricted

adhesion

(6).

aortic

an early is

Following

established

virtually

damaging

eliminated

of

its

of rats In the

also

maintained presence

In addition,

calcific ammonium

example,

can be

of

calcium

by PC (3).

on high

cholesterol

of PC however, compound

Its

Monocyte

impressive.

the

in of PC

the

deposition

can be prevented is

synthesis

of

for

is

formation

magnesium

in rats,

of

now

administration

i.e.

and

conditions

potential acid

some

stones

phosphate,

(7).

exciting

exogenous

"mixed"

influence

of hydroxyapatite

surrounding

mitochondria

event.

the

reports

that

Nephrocalcinosis

endothelium

of exerting

carboxylic

inhibitor

have

states.

phosphate

a

capable

has revealed

studies

potential

diseases

This

and in vivo

(2,5),

in compounds

processes

(PC).

recognized both

interest

monocyte is

known

0006-291W90 251

$1.50

Copyright 0 1990 by Academic Press, Inc. Ail rights of reproduction in any form reserved.

Vol.

BIOCHEMICAL

170, No. 1, 1990

to

restrict

muscle

uptake

cells

(81,

of

compound

calcium

another

important

providing

PC does

is

(10).

It

absence

of any form

occur

not

of cell

(7,111.

established.

Its

this

uncontrolled

role

transformation

of

hydroxyapatite,

PC probably

the

and growth

deposition The

(2,131.

stereochemistry

The

actual

established. must

presented

for

synthesizing

be

undoubtedly

PC in

the

is described

and thereby

size

PC

found

in

it

In the that

presence

through

by binding

to occur

time

the

phosphate

there

has never

mitochondria

been is

damaging

of

preventing

the

crystalline

interfering

ratio

to

effect

to

of growing

an

administered

is

In

is

crystal combined

with surfaces with

its

responsible.

involved.

the first

calcium

to

mitochondria

however,

the

the

(9).

tissue

(12).

sites

PC with

at least,

in

of

in certain

for

compound

against

charge

of

the

compound

on nucleating

negative

origin

in rats

precipitation

acts

For synthesis

reaction

product

high is

the

amorphous

soft

function

organelle

phosphate

target

in

smooth

development

such damage

that when

for

overall

into

such as can occur

found

endogenous

subcellular calcium

being

toxicity

A considered

protect

in the

against

surprising

true

lipoprotein

potential

role

naturally

is therefore

exogenously

another

a protective

in fact

phase

membranolysis

osteoarthritis

RESEARCH COMMUNICATIONS

and low density

Crystal-induced

atherogenesis. forms

of

AND BIOPHYSICAL

mitochondria

seems likely current

a rat

of citrate

liver

that

in vitro

to be

kinase

type

a study,

homogenate

and ATP.

has yet

is

Identification

evidence

is

capable

of of the

HPLC and 'H-NMR. MATERIALS

AND METHODS

Chemicals: Reagents obtained from commercial suppliers were all of analytical grade. PC standard was prepared by previously reported methods (2,5). Bovine semen acid phosphatase was a product from SIGMA, MO (USA). Rat Liver Homogenate: A 10% rat liver homogenate was prepared essentially according to Schnaitman and Greenawalt (141, in 0.225 M mannitol, 0.075 M sucrose and 1 mM HEPES. Assay Conditions: The typical microassay pl containing 150 ug of proteins, 1 mM ATP and 75 mM TEA buffer, pH 7.6. The incubation time reaction was stopped by adding 50 ,ul centrifugation of the mixture at 6000 x g for 252

system had a volume of 100 20 mM sodium citrate in a was 60 min at 37" C and the of 160 mM H PO . After 2 min, an aliqu30t4 (20 ~1)

Vol.

170,

No.

was

subjected

area

measurement,

to

Hydrolysis of the semen

ng= 37"

The

reaction Areas of

as

adding

in

of Separation

2 ,ul

of

was

phosphatase: lb) was

The diqested

stopped eluting

COMMUNICATIONS

evaluated

by

reaction with

peak

product bovine

the volume of a typical assay was aliquot constituted the control; of pH to 4.5 with 100 mM acetate

a solution

pH 4.5.

of

The

by adding at around

acid

phosphatase

incubation

time

23 ,ul of 3.6 min

lyophilized Digestion with

(

was

66

a solution of (10 experiments

and acid

dissolved phosphatase

in

1

h at

160

mM ) as

100 ,JJ~ was then

(a).

with 260

an nm

with

UV-visible ) permitted

HPLC ( C18,

was 5,um

10 mM KH2P04

),

a Beckman

system

detector. detection

performed equilibrated

( adjusted

with

sulphate. The elution H PO to pH 2 ) at a flow enab 3 4 ed full resolution of

( adjusted with acid conditions entirely

(a) One

buffer

Reverse-phase Ultrasphere ODS

solution containing tetrabutylammonium

GOLD

Monitoring of all

using

a 250 x 4.6 for 120 h

H3P04

to

pH

model

at due relevant mm i.d. with a

2 ) and

5

mM

solvent used was 10 mM KH PO rate of 1 ml/min. The st?ona assay mixtures as the molecules

protonated.

(b) 'H-NMR. S amples were H-spectra were obtained

mm lH-selective resonance of using a 36 suppressled were

Fiq.

RESEARCH

PC present

Phosphocitrate: was accomplished

equipped ( 220 and

wavelengths

1

acid in

lb, were collected, buffer pH 4.5.

described

Identification (a) HPLC. instrument compounds. column of

of

by

was peaks

BIOPHYSICAL

amount

profiled

mM acetate

in Fig. mM acetate

followed

were

by

50

(b)

H3P04* profiled of 100

( as

digested

) in

c.

The

phosphatase as follows: two aliquots of 50 ,ul. aliquot, after adjustement

was 1 nU

AND

phosphocitrate

mixture

acid into second

buffer,

HPCC.

of

assay

divided the

BIOCHEMICAL

1, 1990

probehead. water at 4.8

The ppm.

pulse with 8 by presaturation.

accumulated

achieved broadening

and

by of

lyophilized using a Bruker

s

and

chemical Spectra

shift were

repetition

scale collected

rate.

For transformed.

Fourier

dissolved in deuterated AM 300 WB spectrometer,

each

was

The

of

a 5

referenced to at room temperature

solvent

sample 160 free A resolution multiplication,

a Lorentz-Gauss exponential -0.75 and a Gaussian broadening

water. with

the

signal induction enhancement

was decays was

using

a line

0.1.

RESULTS As

revealed

wavelength and

in of

260

citrate

At

Addition

of

3.65:

shown

by

0.15

standard

prompted

an Fig.

PC at

3.6P0.12

Figure

mixture.

as

la, nm but

were

respectively.

lb)

Fig.

increase

and 220

citrate nm,

(mean

the

shows

a

min

a peak

eluted

PC

to

incubation

in

peak

retention

of

lb

the

show

10

lc. 253

absorbance

times

for

experiments)

typical

height

no

mixture of

of

absorbed

material

only ( Fig. eluting

a

standard

and

chromatogram which

at

6.47+0.4, the

reaction

at

'220

la at

PC

plus 3.60

nm. Fig. min

Vol.

170,

No.

1, 1990

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

6 d 0.030 r-

a

I

x

II

~bsodmnce

A

q

220nm

8=260nm (

;

(-)

Fig

1:

(a) (b) (c) (d) (e) (f)

Fig. citrate time

Id

depicts

was

omitted.

observed

in

observed shown). experiment

with No

the

when

elution

Only the

the peak

)

HPLC profiles of: standards of 2 mM PC (3.67 min) and of 5 mM citrate (6.47 min); enzymatic reaction mixture (see Methods); enzymatic reaction mixture with the addition of a standard 2 mM PC; enzymatic reaction mixture devoid of citrate; enzymatic reaction mixture devoid of rat liver homogenate; enzymatic reaction mixture, upon digestion with bovine semen acid phosphatase (see Methods).

profile

a very

previous control

was homogenate

small

peak

figures. assay

detectable was

for

a control

absen

(Fig. 254

3.60

from

was

detectable

at

An

identical

situation

containing around

mixture

only min

le).

homogenate in

a

further

The results

the

which elution was

( data control demonstrate

not

Vol.

BIOCHEMICAL

170, No. 1, 1990

that

eluting

citrate

material

Further min

at a retention

and the homogenate

lb can be considered

was

evidence gained

are

result

of the

regarding

the

from

treating

The minor

presence

nature the

ELUTION

(b:

of 3.65 min is formed

time

present.

Absorbance

Fig. (a)

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

of endogenous

of the

assay

TIME A = 220 “m I-)

2: HPLC profiles

peak

compound

incubation

appearance

8 = 260 (

eluting

mixture

“m )

of: eluates around 3.6 min from 10 experiments, lyophilized and dissolved in 10 mM KH2POq, adjusted to pH 2.0 with 160 mM H PO ; the same elua 2 es4subjected to acid phosphatase hydrolysis (see Methods). Peak eluting at 6.09 min corresponds to citrate.

255

when

in Fig.

compound.

1 minutesl ;

only

at with

3.65

an acid

Vol.

170, No. 1, 1990

BlOCHEMfCAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

,

I I I , I I

I , v,

3.35

,rTr

3.25

1T-

3.15

’

PPM

Fig.

3:

'H-NMR spectra of the enzymatic reaction mixture adjusted to pH 2.0. 0.3 ml of the sample was lyophilized and dissolved in 0.5 ml of deuterated water. Spectra were collected as described in Methods section. expanded plot in the range of 3.12-3.37 ppm. The A B2 doublet pattern of PC gave a J of 16.5 Hz; after addition of standard PC buffered tg e same sample at pH 2.0 (final concentration of the standard: 1 mM; the ordinate scales are different).

(a) (b)

phosphatase

from

corresponding obtained

bovine

to if

the

semen.

PC was

digestion

As

completely with

acid

shown

in

Fig.

abolished. phosphatase

eluting

around

3.65

mixture.

The peak

at 3.54

min was shown to disappear

6.16

corresponding

An of PC

examination in

confirmed

The typical citrate,

the

to citrate

by NMR provided enzymatic

an internal

AzBe pattern

of

was

recognizable

PC and

result

was

on the

peak

by re-chromatography whilst

2).

further

evidence

of the

standard (51,

The

of PC to the

shifted quantitation

shown). 256

peak

downfield was

of the peak

(Fig.

direct

peak

the

increased

mixture.

a

same

was noticeably

reaction

adding

by

followed

the

was performed

material

min

min

The

If,

presence

positions sample

were

(Fig.

relatively possible

at

(data

3). to not

Vol.

170,

No.

1, 1990

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

DISCUSSION The present biological

demonstrating

synthesis

existence

add

of PC in

incubation have

studies

soft

been able

a

phosphocitrate

molecule.

reaction

is

isolation

and purification

peak

little

profiles

reverse

synthesized

PC.

evident the

doubt

anticipated

appearance

as to

that

we

can

a

be 3-

kinase

must

type

await

the

of

the

understanding

chromatogram

peaks

chromatography

with

to

PC

signals

and

hydrolysis

known

to occur

with

chemically

and

citrate

the

simultaneous

with with

was

Further,

was added

confirmatory

on a

displayed

compounds.

peak

and

mixture

present

the respective

proved

time

reaction

produced

assigned

PC

by HPLC and NMR

Retention

products

phosphatase also

doublet

the

relevant

following

product

the

of

of

of the

of

one

Enrichment

and

source,

corresponding

mechanism

gain

the vitro

citrate

that

authenticity.

the

absence

strengths

its

to

The ' H-NMR patterns

field

the

likely

to

chromatography

of citrate

identity.

to

synthesized

identical

following

that

a

for in

of an energy

reaction

enzyme

biologically

indicated

characteristics

the

report

suitable

time

by

reaction.

following

phase

seems

of

of the

of the

leaves

presence

first

it

produced

documented

homogenate

details

of the

Identification

liver

be

Under

the

the

Whilst

involved,

characteristics

for

rat

can

to previous

include

to demonstrate by

PC

mitochondria.

which

phosphorylated

systems

credence

tissue

conditions

that

proof

of

the

typical

PC being

readily

demonstrable.

The significance should

lead

With

a blasic

be

possible

to

role

synthesis

of PC as

calcification mitochondria

determine

and ionic

of the a

of enzyme

and in

the

biologically

particular,

may be more easily

Studies

also

eventually,

physiological it

relating

promote

an

to determine

can now be contemplated. providing

basic discernable. 257

calcium

control phosphate

they

role

synthesized,

might

compound.

inhibitor

that

conditions

which

system

is

of the

cellular

species

natural

findings

understanding

of how PC is

to

distribution

present

a greater

concept

concentrations decreased

of the

over

of PC.

should

now

metabolite increased the

or

cellular Thus,

the

metastatic

precipitation

in

Vol.

170,

No.

1, 1990

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

ACKNOWLEDGMENTS Research supported by the Italian Ministery of University, Scientific Research and Technology and by the Italian National Research Council. Funding from the National Health and from Medical Research Council of Australia to support the technical assistance of MS Maree Anderson is acknowledged. We thank Doctors B.J. Kvam and P. Pollesello from the Laboratory of Biopolymers in Technology of Padriciano (Trieste) for the NMR measurements. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10.

11. 12.

13. 14.

Williams, G., and Sallis, J.D. (1979) Biochem. J. 184, 181-184. Tew, W.P., Mahle, C., Benavides, J., Howard, J.E., and Lehninger, A.L. (1980) Biochemistry 19, 1983-1988. C.D., Howard, J.E., and Lehninger, A.L. (1981) Proc. Tew, W.P., Malis, Natl. Acad. Sci. USA 9, 5528-5532. Shankar, R., Crowden, S., and Sallis, J.D. (1984) Atherosclerosis 52, 191-198. J.D. (1980) Anal. Biochem. 102, 365-373. Williams, G., and Sallis, Sallis, J.D.,Thomson, R., Rees, B., and Shankar, R. (1988) J. Urol. 140, 1063-1066. Shankar, R., Tuyethong, N., and Sallis, J.D. (1986) Atherosclerosis 62, 47-54. Ward, L.C.,Shankar, R., and Sallis, J.D. (1987) Atherosclerosis 65, 117-124. Sallis, J.D., Shankar, R., Rees, B., and Thomson, R.(l989) Biochem. Med. and Metab. Biol. 41, 56-63. J.D. (1987) In: Urolithiasis, Clinical and Williams, G., and Sallis, Basic Research (L.H. Smith, W.G. Robertson, and B. Finlayson Eds.) pp. 569-577. Plenum Press. N.Y. Tsao, J.W., Schoen, F.J., Shankar, R., Sallis, J.D., and Levy, R.J. (1988) Biomaterials 9, 393-397. Lehninger, A.L. (1983) In: Biomineralization and Biological Metal Accumulation ( P. Westbroek, and E.W. de Jong, Eds) pp. 107-121. 0. Reidel Publishing Co., Dordrecht, Netherlands. Williams, G., and Sallis, J.D. (1982) Calcif. Tissue Int. 34, 169-177. Schnaitman, C., and Greenawalt, I.W. (1968) J. Cell Biol. 38, 158-175.

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