EFFECT OF MACROMOLECULES ON THE REGULATION OF THE MITOCHONDRIAL OUTER-MEMBRANE PORE AND THE ACTIVITY OF ADENYLATE KINASE IN THE INTER-MEMBRANE SPACE

Macromolecules as components of the physiological mitochondrial enviro nment 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 stat e 3 respiration) nor kinetic properties of soluble kinases. A signific ant increase by 10% dextran was however observed of the voltage sensit ivity of isolated porin when reconstituted in planar bilayers. The por es adapted the low conducting state already at a voltage of 10 mV. Thi s effect of the macromolecules may explain the higher diffusion resist ance of adenine nucleotides across the outer membrane as observed in d ifferent experiments: (i) the Michaelis constant of adenylate kinase i n the inter-membrane space increased, in contrast to the soluble enzym e, from 118 +/- 10 muM to 193 +/- 20 muM ADP, (ii) in the presence of competing external pyruvat kinase, the mitochondrial utilization of AD P, produced by adenylate kinase in the inter-membrane space, was impro ved 3-fold suggesting a reduced ADP diffusion out of the outer mitocho ndrial compartment. The influence of the various dextrans correlated w ith the increase in molecular weight of the dextrans. The effect on th e kinetic constants was dependent on the dextran concentration in term s 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 por e responds to an increased oncotic pressure by reducing adenine nucleo tide permeability. This suggests the physiological existence of a thir d adenine nucleotide compartment between the two envelope membranes wh ich may be important especially at high metabolic fluxes.