Regulation of membrane enzymes by lipids

Characterization of membrane proteins in detergent solutions.

Cytochrome c oxidase. Towards a clarification of its structure, interactions and mechanism.

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.

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

Structure of cytochrome c oxidase.

Cytochrome c oxidase in detergent dispersion has been cross-linked with two reversible cross-linking agents, dithiobissuccinimidylpropionate (DSP) and dimethyl-3,3'-dithiobispropionimidate (DTBP), and the cross-linked products formed have been analyzed by two-dimensional gel electrophoresis. Under mild reaction conditions, several subunit pairs were seen including II and V, V and VII, IV and VI. With higher levels of DSP, larger aggregates were seen until a cross-linked product with an apparent molecular weight of 140 000 was the predominant band on gels. This is the smallest molecular weight aggregate to contain all seven subunits of the enzyme and most likely represents the "unit" or two heme and two copper containing complex of cytochrome c oxidase.

Near-neighbor relationships of the subunits of cytochrome c oxidase.

Cross-linking studies on a cytochrome c-cytochrome c oxidase complex.

Parallel modulation of catecholamine activation of adenylate cyclase and formation of the high-affinity agonist.cntdot.receptor complex in turkey erythrocyte membranes by temperature and cis-vaccenic acid

The subunit structure of the cytochrome c oxidase complex has been obtained for three preparations each isolated by a different detergent procedure. Six polypeptides were present in all samples with the following molecular weights: subunits I, 36000; II, 22500, III, 17100; IV, 12500; V, 9700; and VI, 5300. These subunits have been purified by gel filtration in sodium dodecyl sulfate or in 6 M guanidine hydrochloride and their amino acid compositions have been determined. Subunit I is hydrophobic in character with a polarity of 35.7%. Subunits II through VI are more hydrophilic with polarities of 45.5, 48.6, 47.8, 49.7, and 53.7%, respectively.

The subunit structure of the cytochrome c oxidase complex.

Publisher Summary Much important data about the arrangement of proteins in mitochondrial electron transfer complexes and in F 1 ATPase from several sources have been obtained from cross-linking studies. These experiments have for the most part been done on solubilized enzyme complexes and we are now beginning to perform cross-linking experiments on the intact membrane in the expectation of confirming the various dimmers seen in the isolated complexes. Such studies may also provide information about the longer-range interactions among different complexes in the membrane. The chapter discusses bifunctional reagents that have been used to investigate the spatial arrangement of components in multisubunit enzymes in complex structures such as ribosomes, and in membrane systems. The noncovalent associations that stabilize such structures are normally destroyed when these multicomponent aggregates are solubilized in a denaturing solvent. However, covalent cross-linking prevents the dissociation of apposed proteins and gel filtration or electrophoresis procedures can then be used to analyze for near neighbors in a complex. Cleavable bifunctional reagents are used to determine the arrangement of polypeptides in both complex III and cytochrome C oxidase. The chapter reviews the technical considerations in conducting these experiments as well as some of the problems encountered in analyzing the results.

Margaret M. Briggs

Papers

Near-neighbor relationships of the subunits of cytochrome c oxidase.

Cross-linking studies on a cytochrome c-cytochrome c oxidase complex.

Parallel modulation of catecholamine activation of adenylate cyclase and formation of the high-affinity agonist.cntdot.receptor complex in turkey erythrocyte membranes by temperature and cis-vaccenic acid

The subunit structure of the cytochrome c oxidase complex.

[57] Cleavable bifunctional reagents for studying near neighbor relationships among mitochondrial inner membrane complexes