Components of the mitochondrial inner membrane. 3. Characterization of a seventh different subunit of beef heart cytochrome c oxidase. Similarities between the beef heart enzyme and that from other species

Abstract

Beef heart cytochrome c oxidase has been resolved into seven subunits by electrophoresis in highly cross-linked gels containing urea and sodium dodecyl sulfate. The molecular weights of the polypeptides are estimated to be I, 35 400; II, 24 100; III, 21 000; IV, 16 800; V, 12 400; VI, 8200; and VII, 4400. It has been shown that subunits II and III can coelectrophorese on standard sodium dodecyl sulfate-polyacrylamide gels and appear as a single component with an apparent molecular weight of 22 500. This accounts for previous reports that the beef heart enzyme contains only six subunits. Amino acid analysis of the isolated subunits I, II, and III revealed that they have polarities of 35.5, 44.7, and 39.9%, respectively. All three subunits have an extremely high leucine content and a low percentage of basic amino acids relative to subunits IV-VII. The size, number, and properties of subunits in the beef heart cytochrome c oxidase complex suggest that it has essentially the same subunit structure as the complexes isolated from Saccharomyces cerevisiae and Neurospora crassa.

Immunological Studies on Cytochrome c Oxidase: Arrangements of Protein Subunits in the Solubilized and Membrane‐Bound Enzyme

“…The gels without urea were run at a constant amperage of 100mA, while the urea gels were run at 30 mA. Staining and de-staining were done as described in Weber and Osborn [23], with the exception that staining took place at 60 C for 2 h. In the case of the urea gels, the staining was preceded by the fixing step as recommended by Downer et al [25].…”

“…Subunits I1 and 111 not only co-migrate on dodecyl sulphate gcl clcctrophoresis (see below) but also appeared as a single subunit with identical molecular weight in gcl filtration chromatography in the presence of dodecyl sulphatc [I 51. Only when electrophoresed in the presence of urea [16] did subunit I11 migrate differently from subunit I1 [25]. The fraction that contained subunits IV to VII (routinely, the fractions were examined by gel electrophoresis for identification of the subunits) was used for isolation of individual subunits V. VI and VTT [15,25].…”

Mr‐values of mature subunits I and III of beef heart cytochrome c oxidase in relationship to nucleotide sequences of their genes

“…The Met content has been set out at 11.0 for the amino acid analysis since all of the Met residues are present in the N-terminus to residue 183. Subunit III, consisting of residues, 1-183, has M r ~ 21 000 which is the value obtained by SDS-polyacrylamide gel electrophoresis [6,7]. The amino acid composition calculated for this polypep- The fact that the Mr-values of subunits I and III are larger than indicated by SDS-polyacrylamide gels has important implications for the minimum M r of cytochrome c oxidase (i.e., the no.…”

“…After quenching the reaction with 1 mM 2-mercaptoethanol, the sample was electrophoresed on a 16% SDS urea polyacrylamide gel. The densitometric trace of a channel stained for protein [6] is shown (...) with the counts/ram gel slice of a n identical channel ( ). (B) Cleavage of subunit I with NTCB.…”

“…Samples (~100/ag) were electrophoresed on a 16% SDS-urea polyacrylamide gel with a stacking gel. This was stained for protein, sliced and prepared for counting as in [6]. The subunits are numbered according to [6] after identification on the basis of iodo[~4C]acetamide labelling [7].…”