Overexpression of the inhibitor protein IF(1) in AS-30D hepatoma produces a higher association with mitochondrial F(1)F(0) ATP synthase compared to normal rat liver: functional and cross-linking studies.
According to functional studies, the higher IF(1) content reported in mitochondria of cancerous cells is supposed to induce a higher association with the F(1)F(0) complex than in normal cells and therefore a better inhibition of its ATPase activity. The first structural evidence supporting this prediction is here presented. Densitometric analyses of Western blotting experiments indicated a 2-fold increase in IF(1) content of AS-30D submitochondrial particles compared to normal rat liver controls. The ratio of IF(1)/F(1) alpha subunit increased similarly as judged by Westernblot analyses. This IF(1) overexpression correlated with a slower rate of IF(1) release (F(1)F(0)-ATPase activation) from the F(1)F(0) complex in AS-30D than in normal rat liver submitochondrial particles. The IF(1)-IF(1), gamma-IF(1), and alpha-IF(1) cross-linkages previously formed with dithiobis(succinimidylpropionate) in bovine F(1)F(0)I and IF(1) complexes were reproduced in the F(1)F(0)I-ATP synthase of hepatoma AS-30D cells. However, a much lower yield of IF(1) cross-linkages was found in normal rat liver particles which made them almost undetectable in SMP as well as in the immunoprecipitated F(1)F(0)I complex. Modeling in vivo IF(1) overexpression of cancerous cells by in vitro reconstitution of excess recombinant IF(1) with rat liver submitochondrial particles devoid of IF(1) reproduced the same IF(1) cross-linkages observed in AS-30D particles.
Bravo
Concepción
C
Departamento de Bioquímica, Instituto Nacional de Cardiología, "Ignacio Chávez," México, México.
Minauro-Sanmiguel
Fernando
F
Morales-Ríos
Edgar
E
Rodríguez-Zavala
José S
JS
García
José J
JJ
D000818:Animals; D015153:Blotting, Western; D003432:Cross-Linking Reagents / Q000378:metabolism*; D003720:Densitometry; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000378:metabolism*; D008114:Liver Neoplasms, Experimental / Q000378:metabolism*; D008928:Mitochondria / Q000378:metabolism*; D025261:Mitochondrial Proton-Translocating ATPases / Q000378:metabolism*; D009841:Oligonucleotides; D010957:Plasmids / Q000235:genetics; D051381:Rats
D003432:Cross-Linking Reagents; D004791:Enzyme Inhibitors; D009841:Oligonucleotides; C474451:F1F0-ATP synthase; D025261:Mitochondrial Proton-Translocating ATPases