Influence of Ca2+ on the isolation from rat brain mitochondria of a fraction enriched of boundary membrane contact sites.
Data have been obtained suggesting that the complex porin-hexokinase of brain mitochondria may be related to the contact sites between the outer and inner membrane. In the attempt to isolate from brain mitochondria the inner and outer membranes and the boundary membrane contacts, a procedure was developed based on swelling and shrinking of the organelles, followed by sonication and reverse discontinuous density gradient centrifugation. Three fractions were obtained by this technique, which were identified by measuring the relative specific activities of marker enzymes, namely succinate-cytochrome c reductase; NADH-cytochrome c reductase (rotenone insensitive); hexokinase and glutathione transferase, for the inner and outer membranes and contact sites, respectively. The fraction which contains the contact sites is characterized by the highest specific activity of hexokinase and glutathione transferase and by the highest calcium binding capacity; physiological concentrations of this cation produces a sharper separation of this fraction. Results indicate that both the porin-hexokinase gating system of the outer membrane and the calcium transporting complex of the inner membrane are present in the fraction which contains the contact sites.
Sandri
G
G
Dipartimento di Biochimica, Università degli Studi, Trieste, Italy.
Siagri
M
M
Panfili
E
E
D000818:Animals; D017920:Antiporters; D001665:Binding Sites / Q000187:drug effects; D001921:Brain / Q000648:ultrastructure*; D002118:Calcium / Q000378:metabolism / Q000494:pharmacology*; D002135:Calcium-Binding Proteins / Q000302:isolation & purification; D027682:Cation Transport Proteins; D002458:Cell Fractionation / Q000379:methods*; D006593:Hexokinase / Q000032:analysis / Q000302:isolation & purification / Q000378:metabolism; D007425:Intracellular Membranes / Q000032:analysis* / Q000187:drug effects / Q000201:enzymology / Q000648:ultrastructure; D008565:Membrane Proteins / Q000032:analysis / Q000302:isolation & purification* / Q000378:metabolism; D008928:Mitochondria / Q000032:analysis* / Q000187:drug effects / Q000201:enzymology / Q000648:ultrastructure; D018272:Porins; D051381:Rats; D050994:Voltage-Dependent Anion Channels
D017920:Antiporters; D002135:Calcium-Binding Proteins; D027682:Cation Transport Proteins; D008565:Membrane Proteins; D018272:Porins; D050994:Voltage-Dependent Anion Channels; C027614:calcium-hydrogen antiporters; D006593:Hexokinase; D002118:Calcium