Labeling of cytochrome c oxidase with [35S]diazobenzenesulfonate. Orientation of this electron transfer complex in the inner mitochondrial membrane

Abstract

Isolated cytochrome c oxidase was fractionated by native-gel electrophoresis in Triton X-100, and a preparation of enzyme almost completely free of the usual impurities was recovered. This fraction was used to generate antibodies specific to cytochrome c oxidase. These antibodies inhibited cytochrome c oxidase activity rapidly and completely and immunoprecipitated an enzyme containing seven different subunits from detergent-solubilized mitochondria or submitochondrial particles. Reaction of detergent-solubilized cytochrome c oxidase with [35S]diazobenzenesulfonate labeled all seven subunits although I and VI were much less reactive than the other five components. When cytochrome c oxidase was immunoprecipitated from mitochondria which had been reacted with [35S]DABS, subunits II and III were the only components labeled. When the complex was immunoprecipitated from labeled submitochondrial particles, II, III, IV, V, and VII were all labeled. Polypeptides I and VI were not labeled from either side of the membrane. These results confirm earlier studies which showed that cytochrome c oxidase spans the mitochondrial inner membrane and is asymmetrically arranged across this permeability barrier.

The characterization of mitochondrial translation products in rat liver and rat hepatoma cells

“…21 500 and 9000 [4]. A [22] andNeurospora [23], and the Mr reported for similar result is also obtained with isolated hepatoma beef heart cytochrome b [24], we conclude that this cells labeled in the presence of cycloheximide ( fig.3, mitochondrially translated peptide is cytochrome b lanes 1,2). This observation is in partial variance with apoprotein.…”

“…This can be due to: (i) A low methionine content of subunit III; (ii) Unequal rates of formation and/or assembly of the subunits; (iii) Substoichiometric amounts of subunit III in the immunoadsorbed enzyme [24,25]; or (iv) The presence of antibodies directed specifically to subunit I and II resulting in the precipitation of these peptides even in the unassembled form. The latter is the least likely since the antiserum used can not interact with subunit I (not shown).…”

Deficiency in ubiquinone cytochrome c reductase in a patient with mitochondrial myopathy and lactic acidosis.

“…Protein samples were denatured in NaDodSO4, urea, and mercaptoethanol and after electrophoresis were stained for protein with Coomassie blue. Lane 3, 40 ,ug of purified beef heart cytochrome c oxidase; subunits are numbered on the left-hand side in order of decreasing molecular weights as described by Ludwig et al (15). Lane 4, 40 pg of purified beef heart complex m; subunits are labeled on the right-hand side according to their prosthetic groups and in order of decreasing molecular weight, according to Bell and Capaldi (16 Fig.…”

On the function of multiple subunits of cytochrome c oxidase from higher eukaryotes

“…Subunit Via (Buse) according to its app. M r of 14 000 [58] correlates with a band which we obtained only under certain conditions with the beef heart enzyme below subunit V and which probably corresponds to 'impurity a' of Capaldi's group [55]. This polypeptide has not yet been considered as a subunit by our group and would represent a 13th subunit.…”

“…Ludwig et al [55] studied beef heart cytochrome c oxidase by SDS-polyacrylamide gel electrophoresis and observed in addition to the 7 main components less intensive bands denoted a,b and c (between subunits 1V and VI) and further low M r peptides (below subunit VII). They suggested that these polypeptides represent impurities, since they could be removed or reduced by treatment of the enzyme with trypsin.…”