JBC, Vol. 250, Issue 24, 9397-9403, Dec, 1975
Oxidation of NADPH by submitochondrial particles from beef heart in complete absence of transhydrogenase activity from NADPH to NAD
L. Djavadi-Ohaniance and H. Hatefi
Treatment of submitochondrial particles (ETP) with trypsin at 0 degrees
destroyed NADPH leads to NAD (or 3-acetylpyridine adenine dinucleotide,
AcPyAD) transhydrogenase activity. NADH oxidase activity was unaffected;
NADPH oxidase and NADH leads to AcPyAD transhydrogenase activities were
diminished by less than 10%. When ETP was incubated with trypsin at 30
degrees, NADPH leads to NAD transhydrogenase activity was rapidly lost,
NADPH oxidase activity was slowly destroyed, but NADH oxidase activity
remained intact. The reduction pattern by NADPH, NADPH + NAD, and NADH of
chromophores absorbing at 475 minus 510 nm (flavin and iron-sulfur centers)
in complex I (NADH-ubiquinone reductase) or ETP treated with trypsin at 0
degrees also indicated specific destruction of transhydrogenase activity.
The sensitivity of the NADPH leads to NAD transhydrogenase reaction to
trypsin suggested the involvement of susceptible arginyl residues in the
enzyme. Arginyl residues are considered to be positively charged binding
sites for anionic substrates and ligands in many enzymes. Treatment of ETP
with the specific arginine-binding reagent, butanedione, inhibited
transhydrogenation from NADPH leads to NAD (or AcPyAD). It had no effect on
NADH oxidation, and inhibited NADPH oxidation and NADH leads to AcPyAD
transhydrogenation by only 10 to 15% even after 30 to 60 min incubation of
ETP with butanedione. The inhibition of NADPH leads to NAD
transhydrogenation was diminished considerably when butanedione was added
to ETP in the presence of NAD or NADP. When both NAD and NADP were present,
the butanedione effect was completely abolished, thus suggesting the
possible presence of arginyl residues at the nucleotide binding site of the
NADPH leads to NAD transhydrogenase enzyme. Under conditions that
transhydrogenation from NADPH to NAD was completely inhibited by trypsin or
butanedione, NADPH oxidation rate was larger than or equal to 220 nmol
min-1 mg-1 ETP protein at pH 6.0 and 30 degrees. The above results
establish that in the respiratory chain of beef-heart mitochondria NADH
oxidation, NADPH oxidation, and NADPH leads to NAD transhydrogenation are
independent reactions.
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