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J. Biochem, 1977, Vol. 81, No. 4 911-921
© 1977 Japanese Biochemical Society

research-article

Analysis of Reverse Acceptor Control in Mitochondria1

Takeshi TSUJIMOTO

Department of Physiology Wakayama Medical College 9-bancho, Wakayama, Wakayama 640

The reduction of NAD(P) by ADP, a reversal of the normal change, in rat kidney mitochondria incubated in isotonic media containing NaCl and EDTA was studied by dual-wavelength spectrophotometry. Dual-wavelength spectrophotometry and respiratory studies showed that with increasing concentrations of NaCl and EDTA, rapid respiratory rate and marked oxidation of NAD(P) became evident in state 4, respiratory stimulation by ADP was reduced considerably, and the oxidation-reduction pattern of NAD(P) by ADP was converted to a diminished initial oxidation and subsequent reductive-reoxidative changes. Spectroscopic and enzymatic assays confirmed that the NAD(P) reductive change by ADP observed in dual-wavelength spectrophotometry appeared to coincide with a remarkable swelling-shrinkage reaction larger in magnitude than the diminished oxidation of NAD by ADP. Under experimental conditions which might involve a significant swelling-shrinkage reaction, the redox kinetics observed by dual-wavelength spectrophotometry do no show the true NAD+: NADH change. The marked swelling-shrinkage was ascribed to an increase of ionic permeability due to the chelation by EDTA of membrane-bound magnesium with loss of potassium and of the integrity of mitochondria. ADP-induced inhibition of respiration and reduction of NAD, which will be designated here as "true reverse acceptor control," was not observed. Instead, the rapid respiratory rate in state 4, the decline of respiratory control, oxidized NAD in state 4, inhibition of further oxidation of NAD in state 3, and the small phosphorylation deficiency observed in the NaCl-EDTA media, suggested that a leaky or looseiy coupling state rather than uncoupling led to the apparent reduction.

1The purchase of a Hitachi 356 spectrophotometer was supported in part by a "grant for public school research equipment" in 1969 from the Ministry of Education, Science and Culture of Japan.


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