Kinetics and free energy profiles of spermine transport in liver mitochondria

In the present study, the voltage-dependent mechanism of spermine transport in liver mitochondria [Toninello, A., Dalla Via, L., Siliprandi, D., and G arlid, K. D. (1992) J. Biol. Chem. 267, 18393-18397] was further characteri zed by determining the rate constants J(max) and k(m) as functions of membr ane potential. An increase in mitochondrial membrane potential from 150 to 210 mV promoted spermine transport, as reflected by an approximate 4-fold i ncrease in J(max) and 25% decrease in K-m. The mechanism for the voltage de pendence of transport was examined using the beta value, i.e,, the slope of In(flux) vs F Delta Psi/RT plots. Flux-voltage analyses performed at very high and very low spermine concentrations yielded beta values of 0.125 and 0.25, for J(max) and J(max)/K-m, respectively. The physical significance of these beta values was analyzed by means of a theory relating the enzyme re action rate to the free energy profiles [Yagisawa, S. (1985) Biochem. J. 30 3, 305-311]. Depending on the nature of k(m), 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 Note, V., Siliprandi, D., and Toninello, A. (1996) Biochim. Biophys. Acta 1 284, 247-252] by a new rationale provided evidence for an asymmetrical ener gy profile composed of two peaks with the binding site near the membrane su rface followed by a rate-determining energy barrier for the movement of the bound spermine toward the internal region of the membrane.