Vol. 68, No. 4, 1976

BIOCHEMICAL

THE CALCIUM-BINDING MENTS. G. Sandri, Biologica

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

GLYCOPROTEIN AND MITOCHONDRIAL CALCIUM MOVE-

E. Panfili - Universita

and G.L. Sottocasa - Istituto di Trieste - Trieste - Italy.

di

Chimica

Received December15, 1975 ABSTRACT The calcium-binding glvcoprotein isolated from mitochondria can be shown to move from one mitochondrial compartment to another as a function of calcium and magnesium presence as well as calcium transport. The movement is reversible in vitro and the possibility is therefore considered that the gGc=ein may behave as a mobile calcium-carrier. In the presence of acetate and phosphate, calcium-pre-loaded mitochondria release the cat? ion upon addition of uncoupling concentrations of pentachlorophenol. The rate of calcium efflux can be modulated either by changing pentachlorophenol or phosphate concentrations. Simultaneously a release of calcium-binding glycoprotein can be detected and a negative linear relation has been found between amount of glycoprotein released and rate of calcium passive efflux. The data are interpreted to indicate that calcium efflux occurs only when the glycoprotein is bound to the mitochondrial membranes. INTRODUCTION The problem

of

energy-driven

calcium-transport

in mitochondria

is currently a much debated problem (I-3). Attempts to isolate the calcium-specific carrier from mitochondria (4-9) have led to the isolation of a glycoprotein with high affinity in a lanthanum- ruthenium to the finding dria capable mitochondria ed a possible

red-sensitive manner. This feature, that such a glycoprotein is present

in addition in mitochon-

of active calcium transport and absent from those which genetically lack this ability, have suggestrole of it in the calcium-transport system. Inte-

restingly, the addition of glycoprotein to an artificial lipid bilayer decreases notably the electrical resistence only in the presence of calcium and in a ruthenium red-sensitive manner(l0). Studies of the intramitochondrial location of the calcium-binding glycoprotein have revealed, on the other hand, a rather homogeneous distribution of the compound throughout the mitochondrial compartments (except fortie matrix space) (9), a finding PCP = pentachlorophenol; Abbreviations: cylate; EDTA = ethylendiaminotetracetic co1 bis (aminoethyl) tetracetic acid.

Copyright All rights

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

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LIS = lithium diiodosaliacid: EGTA = ethylengly-

Vol. 68, No. 4, 1976

which

is

BIOCHEMICAL

somewhat inconsistent

function for the glycoprotein. tible with a mobile-carrier

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

with an membrane-bound carrier Such a location is however compahypothesis

in which

the position

of

the glycoprotein depended upon the presence or absence of calcium and calcium-transport in the system. We have investigated the problem more in detail by measuring the release of the glycoprotein into the medium by osmotic treatment (intermembrane various conditions. Data are here reported showing

space) under that the amount

of glycoprotein bound to the membranes is dependent upon the existence of calcium in the medium and is increased by calcium-transport. It will be also tions of the uncoupler causes the organelles

shown that the addition of high concentraPCP to mitochondria pre-loaded with calcium to become unable to release calcium in the

medium. Simultaneously the calcium-binding glycoprotein is detached from the mitochondrion. The two phenomena show a linear negative correlation. MATERIALS AND METHODS Rat liver mitochondria were isolated in 0.2M mannitol as described by Johnson and Lardy (11). Glycoprotein released was measured after polyacrylamide gel electrophoresis and toluidine blue staining by integration of the densitometric peak (Hewlett Packard integrator mod. 3770 E). For the evaluation of the membrane-bound glycoprotein, the particulate fraction after swelling and centrifugation was extracted with LIS (0.2 M) according to Marchesi and Andrews (12). Details are given in the tables and figures. Calcium uptake and efflux was followed either by the murexide spectrophotometric method of Mela and Chance (13) or by the dynamic dialysis technique described by Colowick and Womack (14).Protein content of the suspension was determined according to Gornall et al. (15). For further technical details see legends. to tables and figures.

RESULTS The distribution

in non-respiring

mitochondria

of calcium-bind-

ing glycoprotein between intermembrane space (osmotic supernatant) and membranes (sediment) as a function of calcium presence in the medium is reported in Table I. The data clearly show that the amount released

in the

absence of calcium

is

almost

twice

that

re-

leased in its presence. In addition, the experiment with EDTA shows that the chelator is a very potent extracting agent and can induce a considerably higher release of glycoprotein in the inter membrane space. It may be added that the total amount recovered

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Vol. 68, No. 4, 1976

BIOCHEMICAL

TABLE

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

I

INFLUENCE OF Ca2+ ON THE DEGREE OF ASSOCIATION

OF THE GLYCOPROTEIN

TO MEMBRANES Experimental conditions: mitochondria from one rat liver (140 mg) were taken up in seven ml IOmM triethanolamine-HCl buffer, pH 7.8 . . ;z?taining 10~s rotenone. Aliquots of 1 ml were added with O.lmM or O.lmM EDTA. After standing in ice for 10 min, 1 ml 0.9 M sucrose containing 2mM MgS04 and 2mM ATP was added to each sample. After centrifugation for 30 min at 105,000 x g, the supernatant was subjected to polyacrylamide gel electrophoresis using 10% gels Staining was performed with toluidine blue as already described (5). The sediments have been taken up in 2 ml 0.2 M LIS dissolved in 50mM Tris-HCl, pH 7.5 and extracted in this medium for 30 min under stirring at room temperature. After dialysis for 20 hours against the spacer gel buffer pH 6.7(17), the dialyzate was subjected to polyacrylamide gel electrophoresis and stained as described above. Densitometric traces were obtained as already described(5) and the signal integrated by means of a Hewlett Packard Integrator Mod. 3770 B.

Supernatant

Sediment

Conditions

Total peak area

Rotenone Rot. i- EDTA Rot.

+ Ca2+

(arbitrary

75.4 343.0 41.0

units)

85.3

160.7

46.8

389.8

104.4

145.4

in the presence of EDTA is almost three times higher than in its absence indicating that a considerable aliquot of the glycoprotein cannot be extracted by LIS unless divalent cations are chelated. The fact that such a result cannot be obtained using EGTA points to the importance of magnesiumizmaintaining the glycoprotein the results of an experiment bound to membranes. Table II collects in which the osmotic supernatant is recombined with the sediment

obtained in the presence of EDTA after removal of the chelator. It

appears that, under non-respiring conditions, the membranes can rebind a considerable portion of the glycoprotein in the presence indicate therefore that of calcium or magnesium. The data clearly the phenomenon is reversible and controlled by divalent cations.

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Vol. 68, No. 4, 1976

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

TABLE II

REVERSIBILITY OF ASSOCIATION OF THE GLYCOPROTEIN TO MEMBRANESAS A FUNCTION OF Ca2+ and Mq2+. Experimental conditions: mitochondria were swollen as described in the previous Table, but in presence of 1 mM EDTA. After contrac tion the supernatant was dialyzed through Hollow Fibers (Bio-fiber 50 beaker-Bio Rad Labs.) against 10 mM triethanolamine-HCl buffer, pH 7.8 at rate of 100 ml/min. After 35 min (3500 ml dialyzing solution) , the pellet was taken up in the dialyzed material and aliquots of the suspension were added with the different cations as indicated in the Table. After 10 min the samples were centrifuged and the supernatants further processed as described in Table I.

peak area (arbitrary units)

additions

Osmotic supernatant

Data

+

--

84.0

+ +

sediment sediment

+ 1.0 mM Mg

+ +

sediment sediment

+ 0.1 mP4Ca + 1.0 mM Mg"+

in Table

III

111.3 2+

show the

results

58.0

0.1

mM Ca2+ ',:I"0

of an experiment

the influence of calcium transport on the availability coprotein in the intermembrane space has been tested. when calcium is actively transported der these conditions promotes rather sport)

a net decrease

in which of the qly Clearly only

(lines 2 and 3; oliqomycin unthan inhibiting calcium tran-

in the amount of the qlycoprotein available reported space appears. The three experiments

in the intermembrane indicate that the glycoprotein

may

move

from the

intermembrane

ce into the membranes as a function of calcium presence and calcium transport in the mitochondrion. These findings are compatible with the view that the calciumbinding qlycoprotein behaves as a mobile carrier which becomes part of the membrane only when calcium is bound and is released when calcium is discharged, a finding reminiscent of early expe-

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spa-

Vol. 68, No. 4, 1976

BIOCHEMICAL.AND

TABLE

BIOPHYSICAL

RESEARCH COMMUNICATIONS

III

INFLUENCE OF Ca2+ TRANSPORT ON DEGREE OF ASSOCIATION PROTEIN TO MEMBRANES. Experimental tein/ml in amine-HCl Additions, CaCl 0.1 at 1 2'* for processed,

mitochondria were taken up at 16 mg proconditions: a medium containing 0.2 M mannitol, 25 mM triethanolbuffer pH 7.8, 8 mM MgSOq and 0.3 pg rotenone/mg protein. when indicated in the Table, were: 5 mM succinate, 1 mM mM PCP and 2pg oligomycin/mg protein.After incubation 10 min, the samples were spun down and the supernatant as indicated in Table I and II.

glycoprotein released in a hypotonic medium (integrator units)

Conditions

Mitochondria succinate II

riments

+

the

26,095

+

Ca2+

21,086

+ +

Ca2+ + oligomycin Ca2+ + PCP

20,036

by Gitler

expected that mitochondria on the

OF THE GLYCG

amount

and Montal

26,062

(16).

If

this

view

is

correct

it

was

the rate of passive calcium efflux from pre-loaded under uncoupled conditions would also be dependent of glycoprotein

amount of glycoprotein

present

in mitochondria.

bound to the particles

it

ry to cause an extensive swelling leading to rupturing er mitbchondrial membrane. This effect can be induced

TO change was necessathe outby high

concentrations of PCP and the extent of the effect can be modulated by variing the concentration of phosphate in the medium. Figure 1 shows the results of experiments in which the rate of calcium efflux was measured after addition of different concentrations of PCP. At 10OpM PCP the rate of calcium efflux was virtually zero. Figure 2 shows the effect of PCP on the rate of calcium efflux at high and low Pi concentrations. In both cases at low uncoupler concentrations calcium efflux increases till a maximum and then declines as PCP increases. The relation between the effectiveness of the uncoupler in inhibiting the rate of calcium

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Vol. 68, No. 4, 1976

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

5

mMSuccinate

).IM

10

50 100

RLM+ medium

PM ).IM

+ 50 PM murexide

Cai+ 500

Fig.

PCP

I

r-moles

1

min

I

1 - INFLUENCE OF PENTAFHLOROPHENOLCONCENTRATION ON THE PASSIVE EFFLUX OF Ca2 FROM RAT LIVER MITOCHONDRIA.

Experimental conditions: 3.5 mg rat liver mitochondria were ad7dedxa medium containing 0.1 M mannitol; 50 mM HCl; 25 mM TrisHCl buffer at pH 7.4; 8 mM MgS04; 15&M rotenone; 5 mM acetate buffer pH 7.4 and 5 mM phosphate; other additions as indicated in the figure. Dual Wavelength Phoenix Recording Spectrophotometer. The reaction was run at room temperature.

Fig.

-

0.66

t-4

6 rnM

mM

Pi

P,

2 - DEPENDENCEOF THE RATE OF Ca2+ EFFLUX ON PENTACHLOROPHENOL AND PHOSPHATE CONCENTRATIONS.

Experimental

conditions:

as in Fig.

1.

efflux and phosphate concentration is hyperbolic: the more concentrated phosphate in the medium, the more efficient PCP in inhibiting. By rapid centrifugation technique it has been possible to measure the amount of glycoprotein released upon addition of different concentrations of PCP or PCP plus phosphate. Figure 3 shows

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Vol. 68, No. 4, 1976

BIOCHEMICAL

Glycoprotein

Experimental conditions: red as described in Fig. chlorophenol the samples 3200 centrifuge and the staining and densitometric

of

released,

inteprator

units

2+ 3 - CORRELATION BETWEEN DECREASE OF RATE OF Ca EFFLUX AND GLYCOPROTEIN RELEASE.

Fig.

that

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

the

rate

of calcium

the glycoprotein

the rate of Ca2+ efflux has been measu1 and 2. Two min after addition of penta were centrifuged with an Eppendorf Mod. supernatant subjected to electrophoresis, scanning as described in the Tables.

efflux

is

inversely

related

to the

amount

released.

DISCUSSION The data presented clearly indicate that mitochondria pre-loaded with calcium, under appropriate conditions, cannot release the ion into the medium despite the fact that a high gradient of calcium exists across the mitochondrial no energy is utilized to maintain such a gradient.

concentration membrane and 'The effect seen

is not artifactual (e.g. due to swelling interference in the spectrophotometric assav) because it can be measured as well by dynausing radioactive calcium, a method which is entiremic dialysis, ly independent of optical phenomena, and because the rate of swell ing measured in the absence of murexide cannot account for the drastic reached end of pitation

decrease in optical density. by back titration of calcium

The same conclusion has been in the medium by EDTA at the

the experiment. On the other hand, calcium phosphate preciinside the mitochondrion cannot be invoked to explain the

result because at low PCP concentrations calcium efflux occurs normally and a.precipitation of calcium phosphate promoted by PCP is not likely. On the basis of the above considerations the most rea-

1278

BIOCHEMICAL

Vol. 68, No. 4, 1976

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

sonable explanation for the phenomenon seems to be that dria have become impermeable to calcium. It is tempting de that the impermeability induced by high concentrations reflects volved

the absence of the carrier molecule which is in the calcium traffic across the mitochondrial

This idea is supported rate of calcium efflux ments the glycoprotein

mitochonto concluof PCP normally inmembrane.

by the linear relationship found between and glycoprotein released. In these experirelease was the only parameter measured and

it may be that the correlation is fortuitous. It would be necessary, however, to postulate the existence and simultaneous release of an unknown factor which would be the calcium carrier. We feel that this satisfies transport.

is unnecessary since the calcium-binding glycoprotein the pre-requisites for a role in mitochondrial calcium

ACKNOWLEDGEMENTS The authors are indebted to Mr. nical assistence. Research carried

Bruno Gazzin for out with financial

Consiglio

- Rome. (Contr.

Nazionale

delle

Ricerche

skilful techsupport of nr.

720089204).

REFERENCES

1)

Reynafarje

B. and Lehninger

A.L.

(1974)

J.Biol.Chem.249,

6067-

6073. (1975) Anal.Biochem.67, 44-53. 2) Reed K.C. and Bygrave F.L. Reed K.C. and Bygrave F.L. (1975) Eur.J.Biochem.55, 497-504. 3) Lehninger A.L. (1971) Biochem.Biophys.Res.Commun.42, 312-318. 4) Sottocasa G.L., Sandri G., Panfili E. and de Bernard B.(l971) 5) FEBS Lett. 17, 100-105. G.L., Sandri G., Panfili E., de Bernard B., Gazzotti 6) Sottocasa P Vasington F.D. and Carafoli E.(1972) Biochem.Biophys.Res. Col;unun. 4J, 808-813. E., Gazzotti P., Vasington F.D., Sottocasa G.L.,Sandri 7) Carafoli G., Panfili E. and de Bernard B. (1972) in "Biochemistry and Biophysics of Mitochondrial Membranes" (Azzone G.F., Carafoli E Lehninger A.L., Quagliariello E. and Siliprandi N.,Eds.) New York. PP: 623-640, Acad.Press, E., Sandri G. and Sottocasa G.L. (1974) Acta Vitamin. 8) Panfili Enzymol. 28, 323-330. Sandri G., Panfili E. and Sottocasa G.L. (1974) ibidem,317-322. Ii,’ Prestipino G., Ceccarelli D., Conti F. and Carafoli E. (1974) FEBS Lett. 45, 99-103. in "Methods in Enzymology" 11) Johnson D. and Lardy H.A. (1967) (Estabrook R.W. and Pullman M.E., Eds.) Vol.X, pp.94-96,Acad. Press, New York. Science 174, 1247-1248. 12) Marchesi V.T. and Andrews E.P. (1972) Mela L. and Chance B. (1968) Biochemistry I, 4059-4063. 13) Colowick S.P. and Womack F.C. (1969) J.Biol.Chem.244, 774-777. 14) Gornall A-G., Bardawill C.G. and David N.M. (1949) J.Biol.Chem. 15) 177,

16) Gitler Davis 17)

751-766.

C. and Mental M. (1972) FEBS Lett. B.J. (1964) Ann.N.Y.Acad.Sci. 121, 1279

28, 329-332. 404-427.