Open AccessIsoforms of human cytochrome‐ c oxidase
Author(s) -
KUILENBURG André B. P.,
DEKKER Henk L.,
BOGERT Coby,
NIEBOER Popko,
GELDER Bob F.,
MUIJSERS Anton O.
Publication year - 1991
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1991.tb16162.x
Subject(s) - cytochrome c oxidase , cytochrome c , oxidase test , electron transport complex iv , cytochrome , coenzyme q – cytochrome c reductase , biochemistry , chemistry , cytochrome p450 reductase , cytochrome c peroxidase , protein subunit , hemeprotein , microbiology and biotechnology , heme , biology , mitochondrion , enzyme , gene
The subunit pattern and the steady‐state kinetics of cytochrome‐ c oxidase from human heart, muscle, kidney and liver were investigated. Polyacrylamide gel electrophoresis of immunopurified cytochrome‐ c oxidase preparations suggest that isoforms of subunit VIa exist, which show differences in staining intensity and electrophoretic mobility. No differences in subunit pattern were observed between the other nucleus‐encoded subunits of the various cytochrome‐ c oxidase preparations. Tissue homogenates, in which cytochrome‐ c oxidase was solubilised with laurylmaltoside, were directly used in the assays to study the cytochrome‐ c oxidase steady‐state kinetics. Cytochrome‐ c oxidase concentrations were determined by immunopurification followed by separation and densitometric analysis of subunit IV. When studied in a medium of low ionic strength, the biphasic kinetics of the steady‐state reaction between human ferrocytochrome c and the four human cytochrome‐ c oxidase preparations revealed large differences for the low‐affinity TN max (maximal turnover number) value, ranging from 77 s −1 for kidney to 273 s −1 for liver cytochrome‐ c oxidase at pH 7.4, I = 18 mM. It is proposed that the low‐affinity kinetic phase reflects an internal electron‐transfer step. For the steady‐state reaction of human heart cytochrome‐ c oxidase with human cytochrome c , K m and TN max values of 9 μM and 114 s −1 were found, respectively, at high ionic strength ( I = 200 mM, pH 7.4). Only minor differences were observed in the steady‐state activity of the various human cytochrome‐ c oxidases. The interaction between human cytochrome‐ c oxidase and human cytochrome‐ c proved to be highly specific. At high ionic strength, a large decrease in steady‐state activity was observed when reduced horse, rat or bovine cytochrome c was used as substrate. Both the steady‐state TN max and K m parameters were strongly affected by the type of cytochrome c used. Our findings emphasize the importance of using human cytochrome c in kinetic assays performed with tissues from patients with a suspected cytochrome‐ c oxidase deficiency.