INHIBITION OF MITOCHONDRIAL ENERGY TRANSFER REACTIONS BY 5, 5'-DITHIOBIS(2-NITROBENZOIC ACID), ELLMANS REAGENT

The role of -SH groups in mitochondrial energy transfer was studied by using 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB)1 1 Abbreviation: DTNB, 5,5′-dithiobis(2-nitrobenzoic acid); ATP, adenosine 5′-triphosphate; ADP, adenosine 5′-diphosphate; Pi, inorganic phosphate; α-KG, α-ketoglutarate; β-OH, β-hydroxybutyrate; succ, sodium succinate; ascorb, ascorbic acid; TMPD, N,N,N′,N′-tetramethyl-ρ{variant}-phenylenediamine; EDTA, disodium ethylenediamine tetraacetate; Tris-HCl, tris(hydroxymethyl) aminomethane-HCl; DNP, 2,4-dinitrophenol; PCMB, ρ{variant}-chloromercuribenzoate., the specific reagent for the oxidation of -SH groups in biological materials. Observations on rat liver mitochondria have revealed that DTNB inhibits ADP-stimulated and arsenate-stimulated respiration without giving any effects on State-4 respiration, the ADP/O ratio, and the DNP-stimulated respiration. The activity of DNP-stimulated ATPase is arrested by DTNB with a slight enhance of the activity of Mg2+-stimulated ATPase, but the ATP-Pi exchange reaction is suppressed only partially. Ca2+-stimulated respiration is also inhibited by DTNB. The results indicate that in intact mitochondria -SH groups to be attacked by DTNB will not be essential for the segment of the energy transfer pathway concerned with the ATP-Pi exchange but essential for that concerned with the DNP-stimulated ATPase. Thus, it is suggested that the effecting site of DTNB is located between the sites to be stimulated by DNP and sensitive to oligomycin in the pathway of oxidative phosphorylation. © 1969.