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 Garlid, K. D. (1992) J. Biol. Chem. 267, 18393-18397] was further characterized by determining the rate constants J(max) and K(m) 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 J(max) and 25% decrease in K(m). The mechanism for the voltage dependence of transport was examined using the beta value, i. e., the slope of ln(flux) vs FDeltaPsi/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 reaction rate to the free energy profiles [Yagisawa, S. (1985) Biochem. J. 303, 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 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.
Toninello
A
A
Dipartimento di Chimica Biologica, Università di Padova, and Centro di Studio delle Biomembrane del C.N.R. di Padova, Viale G. Colombo 3, 35121 Padova, Italy. toninell@civ.bio.unipd.it
Dalla Via
L
L
Stevanato
R
R
Yagisawa
S
S
D000818:Animals; D001692:Biological Transport; D007700:Kinetics; D008564:Membrane Potentials; D008930:Mitochondria, Liver / Q000378:metabolism*; D051381:Rats; D013096:Spermine / Q000737:chemistry / Q000378:metabolism*; D013223:Statistics as Topic; D013816:Thermodynamics
D013096:Spermine