Abstract
Macromolecules as components of the physiological mitochondrial environment were substituted by dextrans of different molecular weight. The addition of 10% dextran (molecular weights varying between 20 and 500 kDa) affected neither basic mitochondrial parameters (state 4 and state 3 respiration) nor kinetic properties of soluble kinases. A significant increase by 10% dextran was however observed of the voltage sensitivity of isolated porin when reconstituted in planar bilayers. The pores adapted the low conducting state already at a voltage of 10 mV. This effect of the macromolecules may explain the higher diffusion resistance of adenine nucleotides across the outer membrane as observed in different experiments: (i) the Michaelis constant of adenylate kinase in the inter-membrane space increased, in contrast to the soluble enzyme, from 118 ± 10 μM to 193 ± 20 μM ADP, (ii) in the presence of competing external pyruvat kinase, the mitochondrial utilization of ADP, produced by adenylate kinase in the inter-membrane space, was improved 3-fold suggesting a reduced ADP diffusion out of the outer mitochondrial compartment. The influence of the various dextrans correlated with the increase in molecular weight of the dextrans. The effect on the kinetic constants was dependent on the dextran concentration in terms of weight and not of molarity. The oncotic pressure and viscosity of dextran solutions with different molecular weight showed a comparable dependence. In general, the data indicate that the outer membrane pore responds to an increased oncotic pressure by reducing adenine nucleotide permeability. This suggests the physiological existence of a third adenine nucleotide compartment between the two envelope membranes which may be important especially at high metabolic fluxes.
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