Acylphosphatase and calcium transport across erythrocyte membrane.

ADVANCES

IN

EXPERIl\/lENTAL

MEDICINE

AND

BIOLOGY

i"Tff.

ACYLPHOSPHAÎASE AND CALCIUM ÎRANSPORT ACROSS ERYTHROCYTE MEMBRANE '-Í:t

'

_{Chiara Nediani,}

Gianfranco

Liguri, Niccolò

Taddei'

'j::"

:j:i. Elena Marchetti, Gianpietro Ramponi- and Paolo Nassi

Dipartimento

di

Sclenze Biochimiche

Universj.tà

di

Firenze,

Firenze,

ltaly

INTRODUCTION

Acylphosphatase

is a

widespread

cytosolic

enzyme

that

catalyzes

the hydrolysis of

carboxylphosphate

bond

compounds. Among natural

acylphosphates hydrolyzed by

the

enzyme

are

3-phosphoglyceroylphosphate (1)

carbanoylphosphate

(2),

succinoylphosphate (3).

In

mammaLi-an

tissues

acylphosphatase

exists in trro

isoenzymatic

forms: one j.s

prevalent

in skeletal

muscle

(4-7),

the other

in

red blood

cells

(RBCs)

(B).

The

tuo

isoenzymes are

very

sma11 basj-c protej.ns wj-th the

sarne

nunber

of

aminoacids

but differing significantly in

primary

structure.

They

exhibit simj.lar substrate specificity and

kinetic

properties, except that erythrocyte

isoenzyme appears

to

have a higher

catalytic

pob/er.

The

physiologj-ca1

functj.on

of

acylphosphatase

is sti11

debated.

It

has

been

postulated

that, by

hydrolyzlng 3-phosphoglyceroylphosphate'

acylphosphatase may

hasten glycolysis

at

the

expence

of

ATP formation, when

the rate of this

pathway

j.s limited

by

low

concentrations

of

inorganic

phosphate and ATP.

In a recent

research project

we investigated acylphosphatase content

and activity during the

human

erythrocyte lifespan (9). In this

study

erythrocytes

were

age-fractj.oned

by isopicnic

centrifugation

in

Percoll

density gradients.

Acylphosphatase

content

wa6

deterrnined

by a

non-competitive enzyme-linked inmunoadaorbent

assay

(ELISA)

caried out

wlth

policlonal

antierythrocyte

acylphosphatase

antlbodies

(10).

Acylphosphatase

actlvity

waÉ measured by

a

continuous

optical test with

benzoylphosphate as

substrate

based

on the difference in

absorbance

at 283

run between

benzoylphosphate and benzoate

(11).

Acylphosphataae content

and activity

rose

with

ageing

of

red blood

ce1ls.

Maximum

values occuned j.n

mature

erythrocytes

and

these

values

decreased

slightly in

the older

cells

(Fig.

1).

This

singular

behavi.our may

be attributed to

a

de-novo syntheeis

of

Rcd Btd CcMging, Editcd by M. M4!z!, rrld A. De Flora- Plenum Prcss. New Yott. l9l r, r;i&_r1: - -€**

F

Ji*," s:lir%, .ìEl :--{Ì--_'i' :4f:,: ..:

i:s*

.;$:. ,Jl, qF! -ra

ffi

#

#

#h

&

rffi-#

_ffi

',**{ mtF

.ffi

#

"1<ì &., 207

'5ìi 'i.+h-.

.;t::

,*ff

_{AcyLpHospHATASE AND} cALcruM îRANSPoRT AcRoss ERYTHROCYTE MEMBRANE

.1:..

Chiara

Nediani-,

Gianfranco

Liguri, Niccolò

Taddei'

ìiii'ElenaMarchetti,GianpietroRamponiandPaoloNassi

Dipartimento

di

Sci-enze Biochimiche

Università

di

Firenze,

Firenze'

ltaly

INTRODUCTION

AcyJ-phosphatase

is a

widespread

cytosolic

enzyme

that

catalyzes

the hydrolysis of

carboxylphosphate

bond

compounds. Among natural

acylphosphates hydrolyzed by

the

enzyme

are

3-phosphoglyceroylphosphate (1)

carbamoylphosphate

(2),

succinoylphosphate

(3)'

In

manmalian

tissues

acylphosphatase

exists j,n trro

isoenzynatic

forns: one is

prevalent

in skeletal

muscle

(4-7),

the other

in

red blood

cells

(RBCs)

(B).

The two isoenzynes are

very

sma1l baslc

proteins

wj.th the

sane nunber

of

aminoacids

but differing significantly Ln

primary

structure.

They

exhibit similar substrate specificity and

kinetic

properties, except that erythrocyte

isoenzyme appears

to

have a higher

catalytic

pou/er.

The

physi-otogical

function of

acylphosphatase

is sti11

debated.

It

has

been

postulated

that, by

hydrolyzing 3-phosphoglyceroylphosphate'

acylphosphatase may

hasten glycolysis

at

the

expence

of

ATP formation, when

the rate of this

pathway

is limÍted

by

low

concentrations

of

inorganic

phosphate and ATP.

In a recent

research project

we investigated acylphosphatase content

and activity during the

human

erythrocyte lifespan (9). In

thì.s study

erythrocytes

were

age-fractioned

by isopicnic

centrifugation

in

Percoll

density gradients.

Acylphosphatase

content

waa

determined

by a

non-competitive enzyme-linked imnunoadaorbent

assay

(ELISA)

caried out

with

policlonal

antÍerythrocyte acylphoaphata6e

antlbodies

(10).

Acylphosphatase

actlvity

v/as measured by

a

continuous

optical test with

benzoylphoephate as

substrate

based

on the difference in

absorbance

at 283

run between

benzoylphosphate and benzoate

(11).

Acylphosphataae content

and activity

rose

with

ageing

of

red blood

ce11s.

Maximum

valuea

occumed

in

mature

erythrocytes

and

these val-ues decreased

slightly in

the older

cells

(Fig.

1).

This

singular

behaviour rnay

be attributed to

a

de-novo synthesis

of

Rcd Btd Cctl Aging, Editcd by M. M4D.ru arl

A. De Ftora- Plcnun hcss. Ncw Yort. l9l

_ _-:-it!* ' .#l*ò +'*F, +; ':-",..î 4*. -., ,# . :íts-r $

:

- .{-îilÈ!!

iffi

#

w

&

r#k

iffi

#

'fiF

*qs! ';d#-r, ,$#.

#h

:.t*,

#

20't

the

enzyrne during

reticul0cyte

_{stage and}

_its

_storage,

_virtuarly

_unaffected,

in

rnature

erythrocytes. As grycorysis

_is

_the

_{only energJr source}

_of

the

red cerì'

'

the

increase

of

acylphosphatase during erythrocyte _{maturation and}

_its

persistence

_at

_high

_{levels in}

_{o1d cel1s could}

contribute

to

a

progressive

reduction

_{of the}

_energeti-c

_{yield of}

_glycolysi.s

due

to

an uncoupr-ing

effect

of thls

enzyme produced _by

_{the hydrolysis}

_of

_the

carboxylphosphate bond

of

3-phosphoglyceroylphosphate _. 40 .;

,

o€

-oO IE >\: o o c o a î rc o oo c\

ù^

_{

a l.a 0.5 234 Fract ion n.

Fig. 1.

Acylphosphatase (Acyl-Pase)

_levels

j.n

_six

_{c.Lasses o1- increasing age}

erythrocytes.

Each

point

represents

_the

_mean

_{t S.D. of}

_six

deterrninations. (A) acylphosphatase

_activi-ty

_{(units,/g hemoglobin).}

(B) acyrphosphatase _content

_(yc/e

_henoglobin).

_îhe

changes observed

in

fracti.ons 2

_{vs. 1, 3 vs. 2 and 4 vs. 3 were statistically}

significant

(p

< O.05).

Acylphosphatase, however,

_{might affect in other}

_was

_the

_energy

::!i0"1::t

of erythrocytes.

Hunan

red blood celt

nembrane

_has

_a

L;a

_-Aîpase system

that

pumps _ca2*

_{ions out}

_of

_the

_ce'l

_coupring

calcìum

transport

_to

ATp

hydrolysis,

the latter

_{proceeding through}

_{a series of}

reactions

_that

_invoLve

_the

_ca

.-

_{dependent forrnation}

of

an acyrphosphorylaied

intermediate.

_{ft is well}

_known

_that

_human

RBC Carr_Or""".

_is

_activated

_by

calmoduli.n and

_{that this effect is}

_associated

_wlth

an

increased

_{rate in}

phosphorylated _{intermediate formation}

(12;. 6rr.r,

RBCs, however, seems to

contain

_{other soluble}

_{(r-3) and}

_{particulate factors (14) that can}

rnodulate

!/e

supposed

that

RBc acylphosphatase,

on

the

basis

of

a

potential-hydrolytlc effect

on

the

acylphosphorylated intermediate, might represent an

additional

modulator

of

ca2*-ATpase

that

would

effect both

ATp

hydrolysis

and*calcium

transport.In

other words,

the

efficiency of

erythrocyte rnembrane

Ca

pump. The studj-es

that

we conducted

to

examine

this

hypothesis are here

reported.

EXPERIMENTAL

Effect of

acYlPhosphatase on phosphoryfated intermediate from RBC membranes

RBC membranes were phosphorylated according

to

Luthra

et al.(15)

and

the

l-evel

_^of

phosphoenzyme

(np)

_{was taken as}

_{the difference}

_{between the}

amount

ol

3

2P

incorporated

into the

membrane

protein

in

a

medlum with

ca and in an identical

medi-um wÍthout

ca'* .

Labeled

membranes were

incubated

with

varyj-ng amounts

of

acylphosphatase from

2 to

10

units

per mg

menbrane protein.

Table 1 Effect of Dj.fferent Concentration of _{AcyLphosphatase on}

Phosphoryl-ated Intemedj.ate from RBC Membranes

Sanple Phosphate bound Phosphate rel-eased Labeled membranes Control-

for

spontaneous hydrolysi-s Acylphosphatase-treated labeled membrane 2U/ng 5U/ne 7OU/ng

pmol/rng protej.n pmol-/mg protein/min

1 . 81+O. 07

0. 3410.06

o. 43tO . 05

0.6110.09 0.8010.l-2

Labeled membranes (1 mg

protein)

_{were incubated}

_ìn

_{0.15O M Tris_HCl pH}

7.2

at

37oc wi.th varyi.ng amounts

_of

_{acylphosphatase from}

₂

_to

_{10 units}

per mg membrane

proteln (final

_{volume:1 m1). Results are means}

_t

_S.E.

of five

experiments

wlth different

menbrane preparations.

AlL

the

changes

in

32P

release

induced

by

acyLphosphatase

addition

were

statistically

signifì.cant

(p

< O.05).

( I I l I

l*"

209

As shown

i-n

Table

1,

the release

of

phosphate,

_{net of}

spontaneous

hydrolysis, rose significantly with the increase

inacyì.phosphatase

membrane

protein

_{ratio. This hydrolytic effect}

_{vras predictab.r.e (given}

the cataLytic properties of our

enzyme

and

the

acyrphosphate nature

of the intermediate) all the

more

so since

the

literature

has arready

reported simi-lar effect of

acylphosphatase

on

the

acylphosphorylated

intermediates

of

other transport

ATpase, such

as brain

(Na* _-K*)-ATpase

(16) and sarcoplasmic reticulum Ca2*-ATpase

(17).

_However,

_{it is}

_noter^/orthy

that

the

enzymatic

_{hydrotysis of Ep occu*ed to a}

_considerable

_extent

with

acylphosphatase amounts

_{that farl in the}

_{physiological}

_range.

rn

order to

kineti-carly

_{characterize the}

_{acyJ.phosphatase}

_effect

_rre

incubated

a

fixed

amount

_{of this}

enzyme

(5

_{units) with variable}

_amounts

_of

"?-labeled _{membranes and}

measured

_the

_{initial veiocity of}

_{dephosphorylation}

as

a

functi-on

of

the

phosphoenzyme _{concentration.}

o

₂₅

₅

pmof 32P

boundz ml

Fig' 2' rnitial

rate of

ca2"-ATpase

intermediate

dephosphoryration _by

acylphosphatase. ₅

_{unlts or}

_{acylphosphatase hrere incubated}

in

o.r-50

M Tris-HCI

(pH

_7.2

_at

_3Z"C)

_{with differing}

_amounts

of

label-ed

,i":b"T:":

EP concentration

in

the

medium

!/as

expressed

as

pmol

P bound/mr. phosphate _{rerease vras}

measured

_{at two}

_min

_{intervals for}

10 min and the initiaÌ rate of hydrolysis

was

estimated

by

calcurating the

first

derivative

value

at

zero

tine of the

curve

that

describes

the

phosphate

_{release, net}

_of

_{spontaneous hydrorysis,}

as

a functj-on

_of

time.

Each

point

represents

the

mean

vaLue of

four

determinations. The

inset

shows

the

double

reciprocal

p10t

of

the

data shown

_in

the

Figure.

c

:

c o 0.4 o o

E

oz 1 ,j l iì i i I lr t1

I

In these

conditions

(Fig. 2)

we observed

that

the

enzymatic dephosphorylation _rose

_with

_the

_j.ncrease

_in

Ep

along a hyperbolic curve whose _{paranete.rs were}

_K.

_{= 3.41 1}

&d

V

_,r,

₌

1.19

_{t o.2o7 (s.r. )}

_prol

32

_p

reÌeàsed,/min.

vaLue

that

we found

for

acylphosphate

_hydrolis

_{suggests a}

210

lnitial

rate

of

concentration

1.16

(S.8.

₎

nM

The

very

1ow Km

high

affinity in

l-"#

thirs, enzÍme

for

EP, which seems

Ca

-AîPerse

units,

these

latter

about 0.I%

of total

protej.n (12)

to

be consistent

with

the

small number

of

onl,y accounting

in

human RBC membranes

_for

Effect of

acyLphosphatase _{on erythrocyte membrane Ca2*_Aîpase}

To

see

i.f

the

above

action

on

the

acylphosphorylated intermediate

resui"ted

_{in modified functional properties in}

_Ca2 * _{_ATpase,}

,e

investigated the effect of

acylphosphatase

addition

on

the

rate of

Ca2*

dependent RBc membrane ATp

hydrolysis. The

_mean

_{value of}

_{basar- ca2'}

-AÎPase

activity in

our membrane preparations

_ranged

_frorn

_1o9

_to

_{15o nmol}

of

ATP

split,/h per

mg

mernbrane

protein,

a var-ue which

is

close

to

that

obtained

by

Thakar

(rg).

when added

to

the

assay medium

in

_{concentrations}

that

ranged from

0.5

to 10 units per

mg membrane

protein,

_{acyJ-phosphatase}

si.gnificantly

increased

_{the rate of}

_{RBC membrare}

_{ATp hydrolysis.}

_The

i-ncrease depended

on the

amount

of

acyrphosphatase added

and

maximal

stimulation

(about two

fold

over basa]-

val-ue) was

observed

_at

₂

_units/mg

membrane

protein

(Tab.

_2).

_Îhe

_{effect of}

_{acylphosphatase was cumul"ative}

with

that of

carmodulin

since

ca 2

t

_-ATpase

_activity

_observed

_with

_optimar

calmodulin concentration

v,ras

further

enhanced

_{by the addition}

_of

acylphosphatase (Tab.

_{3). The stirnulatory effect of}

_{acyJ,phosphatase}

in

the

presence

_of

carmodulin was si-mi-lar

_{to that}

_{observed when}

_thi.s

_enzvme

al-one was added

to

the

membranes.

Table 2. Effect of Exogenous _{Acylphospatase on}

ca 2*-ATpase Erythrocyte Membrane

AcyJ-phosphatase _{Ca2*-ATPase actlvity}

units/mg mernbrane protein

0 0.5 1.0 1È 2.O 5.0 10. o

nmol-/h per mg membrane protein

A 109+11 134116 145+15 1 57r18 2L3!23 208t22 201i19 AA

+25

+36

+48

+

lO4

+99

+92

'ìi I ii * |$ *f i!t I

ResuLts

are

means

_{t S.E. of six}

_{determinations}

_{performed on}

dlffering

menbrane

preparations.

_{A represents ca2r_ATpase wi.th}

acylphosphatase. aA

indicates the

change

in

ca2*-ATpase with

acylphosphatase compared

_to

basaL

activity.

changes

_in

ca2*_ATpase

actlvity

observed

_with

_{increasi.ng anounts}

_of

_{acylphosphatase}

were

statistically

_{signi.ficant (p <O.0L}

_by

_the

_{one_way analysis}

_of

variance).

I

if

t

Table

_{3. Effect of}

_the

_SlmuLtaneous

Calmodulin on CaZ*_ATpase

Additi-on

of

Acylphosphatase and

Acylphosphatase _ca2iATpase

activity

AA

units/mg nembrane protein

0 1.O 1E t^ 'R

nrnol,/h

per

mg membrane _protein

249Ì31 tol req 301+38 348t4I JJ /AJ9 +42 +52 +99 +88

Results are means

t

s.D.

of six

assays performed

_{with different}

_membrane

preparations. Ìn all

assays. calmodul,in _{was added}

_at

₄₀

_ng/nl

(optirnal-concentration

_{in our conditions). AA indlcates the}

changes

in

ca 2*-AÎPase

_{actlvlty with}

_{acyJ-phosphatase}

compared

_with

_the

_activity

without

acyJ'phosphatase. _The

_difference

_observed

with

increasing

concentration

_of

_{acylphosphatase was statisti-ca11y}

signlficarrt

(p

< 0.01 by

the

one-way analysis

_of

_variance).

Acylphosphat""" (untt.r-g IOV

protein)

'10

Fig' 3 ' Effect of

acylphosphatase on ca2*

_{transport into}

_{RBC membrane}

inside-out

vesicl-es. Ca2* uptake

into

fOVs was measured

_at

₄

_min

intervals for

_{16 min by}

_{a rapid}

_firtration

technir

pore

sartorius

nenbrane

rirter.

_Each

_point

""o"""i1i""il:"r:i

i'

s'E. of four

deterrninations.

_crranges

_;a";;;;'

with differing

amounts

_of

_{acylphosphatase}

I

o.or. by one way analys1s .1'

"J:;""statistically

significant

(p< .a o o a 9 o E o o .g E

)

oa = E tr lr ir lr t) l; li ii li ii li 212

7

_*,

/

Effect of

acylphosphatase on ca2*

transport

into

RBC membrane inside-out

vesicles

(IOVs)

Acylphosphatase

also

affected ca2n

transport

across erythrocyte

membranes,

but thi.s

effect

was

of

_{opposite type} compareé

with that

on the

rate of

ATP

hydrolysi.s. rn fact

comparable amounts

of

acylphosphatase

decreased

the

rate

Ca'* transport

into

inside-out

RBC vesj.cles and nore

markedly

so with

hi-gher

enzyme amounts. Maximal

lnhibition of

ca2*

transport

was obtained

with

10 units/mg rnembrane

protein

and resul"ted

in

a

decrease

of

about 3O%, 4.O9

I

0.15

(S.8.)

nmol/min per mg fOV

protein,

with

respect

to

basal

value,

5,98

!

O,27

(S.E.)

nmof/min

per

mg fOV proteln

(rle.

3).

CONCLUSION

From

the

resul-ts here reported

acylphosphatase appears

to

have an

uncoupling

effect-

on

the

erythrocyte

Calt

pump. This

was

confirmed by

fillEili"r,"a

ca2'

_I

ATp

ratio tnàt we found in the

presence

of

acylphosphatase when we measured

in

the

sane

the

rate of

Ca2* uptake and

of

ATP

hydrolysis

by roVs. Thi-s value decreased

_{from 2.oL to 0,91 in}

_the

presence

of

acylphosphatase

at

a

concentration

of

10 units/mg rov protein.

As regards

the

mechanism underlying acylphosphatase

action, it is

general.J-y

accepted

that the operation of

RBC membrane ca-

-

pump proceeds through

the

sequence

of

elementary reactions reported

in

the

Scheme (12)

Ca'?' t AT P ì--=* Ca E, ATp ,1

I,

,1 2 ADP CaE, P x L

,'#

caEzp Ca 2'+ P;

Ca2*-ATPas.

exists in

two conformers,

E,

and Er.

Ieads

to

the

_{Ca'*-E -ATp complex. phosphorylation}

conversion

of

ca2*-Er-p

to ca2*-rr-r. After

the

dephosphoryl,ation E ₁

is

converted

into E,

and the

ide

propose

that

acylphosphatase-induced

_{hydrolysis of}

_the

phosphoenzyme _{intermediate occurs before}

_ca'*

_transport

_{has taken pLace,}

thereby

short

circuiting

the

system and

giving

rise to

an acceLerated ATp

hydrolysis

with

concomitant

j,nhibition

_of

_{ca2*pumpLng. The}

_effects

_above

described suggest

an addi,tionaL mechanism

of

energy wasting, which

-given the

hi.gher

acylphosphatase

levels

observed

in

mature RBCs with

respect

to

younger erythrocytes

_-

mi.ght i.ncrease

with

ageing

contributing

to

the

age-dependent decline

of

the

energetic

potential of

these ceLLs.

2.

Ca and ATP bindi.ng to E,

by ATP results j-n the

release of Ca 2 * and

cycle closes.

ii i

3.

ll

I

I

I f I I I I REFERENCES

G. Ramponi, C.

Treves

and A. Gueritore,

Hydrolytic

muscLe _{acylphosphatase on 3-phosphoglyceroylphosphate,}

23:1019 (1967).

u

1.

ac tivi. tl' ::

Experi e:.:::

G. Ramponi'

_F.

_Melani and

A. Gue*itore,

Azione

_{dellracil,fosfatasl ::}

muscol,o su carbarnilfosfato, _{G. Biochim. 1O:1g9 (1961).}

A.

Berti,

M.

Stefani,

G.

Li.guri,

_{G. Camici, G. Manao and}

_c.

_Rampc:.i:.

Acylphosphatase

action

on dicarboxyric

acyJ-phosphates,

_{rtar-. -.}

Biochem. 26:377 (1972).

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