434 PRELIMINARY NOTES

Research Council, the Division of General Medical Science, U.S. Public Health Service(GM 12280-01) and the Agricultural Research Service, U.S. Department of Agri­culture (FG-Sw-I07).

Department of Biochemistry,University of Gothenburg,Goteborg (Sweden)andInstitute of Physics,University of Uppsala,Uppsala (Sweden)

Bo. G. MALMSTROM

ROLAND AASATORE VXNNGARD

I R. MOSBACII, Biochim. Biophys. Acta, 73 (1963) 204.2 L. BROMAN, B. G. MALMSTROM, R. AASA AND T. VANNGARD, J. Mol. Bioi., 5 (1962) 301.3 A. EIiRENBERG, B. G. MALMSTROM, L. BROMAN AND R. MaSBACli, j. Mol. Bioi., 5 (1962) 450.4 L. BROMAN, B. G. MALMSTROM, R. AASA AND T. VANNGARD, Biochim, Biophys. Acta, 75 (1963)

365·5 B. G. MALMSTROM AND T. VANNGARD, j. Mol. Biol., 2 (1960) u8.6 R. AASA AND rr. VANNGARD, Z. N'aturfcrsch., [9a ([964) 1425.7 W. E. BLUMBERG, J. E[SINGER, P. AlSEN, A. G. MORELL AND 1. H. SCIiEINBERG, ]. Bioi.

Chem., 238 (1963) 1675.8 W. E. BLUMBERG, W. G. LEVINE, S. Mh.RGOL1S AND J. PE1SACH, Biochem, Biopbys. Res.

Commun., IS (1964) 277·9 T. NAKAMURA, Biochim. Biophys. Acta, 30 (195 8) 44·

10 G. FAHRAEUS AND H. LJUNGGREN, Biocbim. Biophys. Acta, 46 (1961) 22.

Received August 30th, 1965

Biockim, Biophys. Acta, 110 (1965) 431-434

PN 61087H+/O ratio during Ca2+ uptake in rat-liver mitochondria

It was reported by CHANCEl that the amount of respiratory stimulation givenby I mole of Ca2+ is 2.2 times that given by I mole of ADP. CHAPPELL, COHN ANDGREVILLE 2 have reported for Mn2+ a value of about 6 Mn 2+10 with glutamate assubstrate. ROSSI AND LEHNtNGER 3 have reported that about 2 moles of Ca2+ areaccumulated per energy-conserving site. Simultaneously to the uptake of Ca2+ orMn2+, H+ is ejected. The Hr/cation ratio was either in the region of unit y 2,4- 6, ordecreased by the presence of an anion such as acetates.

We have obtained evidence that both the Ca2+10 and the H+/Ca2+ are depen­dent on the experimental conditions used. On the other hand the H+IO ratios seemto be constant and independent of the experimental conditions.

Rat-liver mitochondria were used in all the experiments. H+ ejection andoxygen uptake were recorded simultaneously. H+ was measured with a BeckmanpH meter and a Beckman glass electrode. O2 was measured polarographically.

In Table I are reported the effects of pH and ionic strength of the medium onthe H+/Ca 2+, Ca2+jO and 21-1+/0 ratios. By increasing the pH of the medium from6.5 to 7.7 or by replacing 250 roM sucrose with 240 roM NaCl the oxygen uptake was

Biocbim. Biophys. Aota, IID (1965) 434-436

PRELIMINARY NOTES 435

reduced to about half. (The inhibitory effect of high NaCl concentrations on the res­piratory stimulation caused by Ca2+ was reported to us by Rossr.) Due to this in­hibition of the respiratory stimulation, the Ca2+jO ratios calculated on the totaloxygen uptake increased from a value of 2 moles of CaN per energy-conserving siteto about II moles of Ca2+ per energy-conserving site. When the Ca2+jO ratio wascalculated on the L1 oxygen uptake the values increased from about 5 to 18 moles ofCa2+ per energy-conserving site. Parallel to the decrease of the oxygen uptake the

TABLE I

EFFECT OF pH AND IONIC STRENGTH OF THE MEDIUM ON H+ EJECTION AND OXYGEN UPTAKE

INDUCED BY Ca2+

The medium contained either sucrose or NaCl and 5 mM Tris buffer, 4 mM succinate, 111M rote­none. Amount of mitochondrial protein was 4 mg{mt. Final vol. '2 ml, Temperature: 25°. Amountof Ca2+ added was 250 I,M.

Medium pH H+ O~ total 0% extra H+{Ca~+ Caz+{O Ca~+{O 2 H+{O 2 H+jO(11M) (11M ) (11M) total extra total extra

250 mM sucrose 6.5 0 20g 2g.0 I9·4 0.84 4·3 6.4- 1.8 2.6250 mM sucrose 7.7 0 IIg 16·5 g.6 0.48 7.6 13.0 1.8 3.1240 mM NaCl 6·55 I47 18.6 g.8 0·59 6·7 12·7 2.0 3.8240 mM NaCl 7.7 6 45 5·5 3·5 0.18 22·7 35·7 2.0 3.2

H+ ejection also decreased to about half on increasing the pH of the medium or byreplacing sucrose with high NaCI concentrations. Due to the decrease of H+ ejectionthe H+/Ca2+ ratio decreased from about 0.9 to 0.2. On the other hand, a remarkableconstancy was observed in the H+/O ratios. Titration experiments at various pHvalues or NaCl concentrations indicated that the decreases of oxygen uptake and ofH+ ejection always proceeded parallel. The 2 H+jO ratios, when calculated on thebasis of the total oxygen uptake, were in the region of 2 for succinate under various

10

0--0--°

30

Protein concentration. mg/ml

Fig. I. The incubation medium contained 80 rnM NaCl, IO roM Tris buffer (pH 7.2), 3 mMsuccinate, 111M rotenone. Temperature 25°. 0-0, total O. uptake; _ •• extra O. uptake.

Biochim, Biophys. Acta, rIO (r965) 434-436

PRELIMINARY NOTES

experimental conditions (Table I). Values in the region of 3 were observed with glu­tamate. As shown in Table I, higher values are obtained when the H+/O ratios arecalculated on the basis of the L1 oxygen uptake.

The problem of the use of the extra oxygen uptake for calculations of energyrequirement has already been discussed by CHANCE AND WILLIAMS? and ERNSTERet ai», CARAFOLI et al. 9 have shown that the State 4 respiration can be used for Ca2+uptake. The experiment of CARAFOLI et al. agrees with previous findings of LEE,AZZONE AND ERNSTER10 and of HAASll where the uncoupled respiration of sub­mitochondrial particle was used to drive the transhydrogenase reaction. We suggestthe use of the total oxygen uptake on the basis of experiments where total and extra'Q.:'Cygen uptakes are measured at different protein and Ca2+ concentrations. As shownin Fig. I, while the total oxygen uptake remained constant at each Ca2+ concentration,the extra oxygen uptake decreased with increasing protein concentrations. The curvesfor the total and extra oxygen uptakes coincided when extrapolated to zero proteinconcentration. The extrapolation to zero oxygen uptake for each Ca2+ concentrationindicates the amount of protein required for the uptake of that amount of Ca2+ withoutrespiratory stimulation. The amount of Ca2+ taken up per mg protein without res­piratory stimulation was only slightly influenced by the Ca2+ concentration, the valueincreasing from 5.5 rruzmoles/mg protein at 50 flM Ca2+ to 7.3 mzcnolesjmg proteinat 190 flM Ca2+, under our experimental conditions. From the data of Fig. I it canbe easily calculated that the difference between extra and total oxygen uptake ateach protein concentration is accounted for by the amount of Ca2+ taken up per mgprotein without respiratory stimulation.

These results would seem to suggest that the basic stoichiometry for the res­piration-dependent cation uptake is the 2 H+/O ratio which is apparently I perenergy-conserving site.

The Unit "G. Vernoni" for the Study of Physiopathology,Institute of General Pathology,University of Padooa (Italy)

CRISTIANO ROSSIGIOVANNI FELICE AZZONE

1 B. CHANCE, Energy-linked Fumctions oj Mitochondria, Academic, New York, 1963, p. 253.2 J. B. CHAPPELL, M. COHN AND G. D. GREVILLE, Energy-linked Functions of Mitochondria,

Academic, New York, 1963, p. 2Ig.3 C. S. ROSSI AND A. L. LEHNINGER, J. Bioi. cu«. 239 (1964) 2971.4 N. E. SARIS, The Calcium. Pt'ffJP in Nfitoehondria, Dissertation, Helsinki-Helsingfors, Igb3·5 G. W. ENGSTROM AND H. F. DE LUCA, Biochemistry, 3 (19 63) 379.6 H. RASMUSSEN, B. CHANCE AND E. OGATA, Proc. Natl. Acad. Sci. U.S., 53 (1965) 1069 .7 B. CHANCE AND G. R. W. 'WILLIAMS, J. Biol. Chem., 217 (1955) 383.8 L. ERNSTER, G. F. AZZONE, L. DANIELSON AND E. C. WEINBACH, J. Bioi. Chem., 23 8 (1963)

1834.9 E, CARAFOLI, Z. DRAHm'A, C. S. ROSSI AND A. L. LEHNINGER, Fed Proc .• 24 (19 65) 425.

10 C. P. LEE, G. F. AZZONE AND L. ERNSTER, Nature, 201, (1964) 152.II D. W. HAAS, Bioehim. BioPhys. Acta, 89 (1964) 543.

Received September 6th, 1965

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