J. Biol. Chem., Vol. 256, Issue 17, 8963-8969, 09, 1981
Dicyclohexylcarbodiimide modification of bovine heart mitochondrial transhydrogenase
RM Pennington and RR Fisher
Dicyclohexylcarbodiimide (DCCD) inhibits the reduction of oxidized 3-
acetylpyridine adenine dinucleotide (AcPyAD+) by NADPH catalyzed by
purified and bovine heart submitochondrial particle transhydrogenase.
Kinetic studies demonstrate that the modification of 1 residue results in
complete inactivation. Both transhydrogenase preparations were labeled with
[14C]DCCD. Labeling of the purified enzyme was time- dependent and
paralleled the extent of inhibition. The incorporation of approximately 1
mol of [14C]DCCD/monomer resulted in complete inactivation of the enzyme.
At longer preincubation times or at higher DCCD concentrations, more than 1
mol of DCCD reacted and cross-linked dimers of transhydrogenase were
formed. The effect of substrates on DCCD inactivation was investigated.
AcPyAD+ provided no protection, and NADH gave partial protection in
submitochondrial particles, but not of the purified enzyme. NADPH and NADP+
stimulated inhibition. These results indicate that DCCD modification occurs
outside the active site. In experiments with transhydrogenase reconstituted
into K+-loaded phosphatidylcholine liposomes, DCCD inhibited the rate of H+
uptake into the vesicles to a significantly greater extent than
transhydrogenation. The incorporation of approximately 1 mol of DCCD/mol of
transhydrogenase monomer completely inhibited H+ translocation, whereas
complete inactivation of hydride ion transfer accompanied the incorporation
of approximately 2 mol of DCCD. These results indicate that DCCD modified
transhydrogenase outside the active site, possibly in a putative H+-binding
domain that functions to translocate protons across the membrane by a pump
rather than by a loop mechanism.
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