In the present study, the voltage-dependent mechanism of spermine transport in liver mitochondria [Toninello, A., Dalla Via, L., Siliprandi, D., and Garlid, K. D. (1992) J. Biol. Chem. 267, 18393−18397] was further characterized by determining the rate constants Jmax and Km as functions of membrane potential. An increase in mitochondrial membrane potential from 150 to 210 mV promoted spermine transport, as reflected by an approximate 4-fold increase in Jmax and 25% decrease in Km. The mechanism for the voltage dependence of transport was examined using the β value, i.e., the slope of ln(flux) vs FΔΨ/RT plots. Flux-voltage analyses performed at very high and very low spermine concentrations yielded β values of 0.125 and 0.25, for Jmax and Jmax/Km, respectively. The physical significance of these β values was analyzed by means of a theory relating the enzyme reaction rate to the free energy profiles [Yagisawa, S. (1985) Biochem. J. 303, 305−311]. Depending on the nature of Km, two possible models could be proposed to describe the location and shape of the barriers in the membrane. Analysis of previous data concerning spermine binding [Dalla Via, L., Di Noto, V., Siliprandi, D., and Toninello, A. (1996) Biochim. Biophys. Acta 1284, 247−252] by a new rationale provided evidence for an asymmetrical energy profile composed of two peaks with the binding site near the membrane surface followed by a rate-determining energy barrier for the movement of the bound spermine toward the internal region of the membrane.
Kinetics and free energy profiles of spermine transport in liver mitochondria
TONINELLO, ANTONIO;DALLA VIA, LISA;
2000
Abstract
In the present study, the voltage-dependent mechanism of spermine transport in liver mitochondria [Toninello, A., Dalla Via, L., Siliprandi, D., and Garlid, K. D. (1992) J. Biol. Chem. 267, 18393−18397] was further characterized by determining the rate constants Jmax and Km as functions of membrane potential. An increase in mitochondrial membrane potential from 150 to 210 mV promoted spermine transport, as reflected by an approximate 4-fold increase in Jmax and 25% decrease in Km. The mechanism for the voltage dependence of transport was examined using the β value, i.e., the slope of ln(flux) vs FΔΨ/RT plots. Flux-voltage analyses performed at very high and very low spermine concentrations yielded β values of 0.125 and 0.25, for Jmax and Jmax/Km, respectively. The physical significance of these β values was analyzed by means of a theory relating the enzyme reaction rate to the free energy profiles [Yagisawa, S. (1985) Biochem. J. 303, 305−311]. Depending on the nature of Km, two possible models could be proposed to describe the location and shape of the barriers in the membrane. Analysis of previous data concerning spermine binding [Dalla Via, L., Di Noto, V., Siliprandi, D., and Toninello, A. (1996) Biochim. Biophys. Acta 1284, 247−252] by a new rationale provided evidence for an asymmetrical energy profile composed of two peaks with the binding site near the membrane surface followed by a rate-determining energy barrier for the movement of the bound spermine toward the internal region of the membrane.
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