Roderick A. Capaldi

Identification and extent of fluid bilayer regions in membranous cytochrome oxidase

Abstract Membranous cytochrome oxidase isolated from mitochondria provides a useful model membrane for studies of hydrophobic lipid association with a fairly well characterized functional protein complex. In order to examine the lipid environments, isotropic and oriented samples of cytochrome oxidase of varying phospholipid content were spin labeled with 5-doxylstearic acid (the 4′,4′-dimethyloxazolidine-N-oxyl derivative of 5-ketostearic acid), and examined by ESR spectroscopy. The isotropic samples exhibit two spectral components. Based on the relative intensity of the two components, the mobility, and the response to hydration, the two components are interpreted as arising from two lipid environments: (1) a layer of lipid bound to the hydrophobic protein surface (boundary lipid) and (2) fluid phospholipid bilayer regions. The extent of the bilayer regions is estimated from integration of the two components of the ESR spectra for samples with different phospholipid/protein ratios. The anisotropy of macroscopically oriented membranous cytochrome oxidase samples containing 0.33 and 0.49 mg phospholipid/mg protein confirms the presence of phospholipd bilayer regions. A spectral analysis reveals a remarkable similarity between the phospholipid bilayer regions in the cytochrome oxidase model membranes and bilayers formed by the isolated lipids, whereas the boundary lipid component has no counterpart in lipid bilayers.

The polypeptide composition of cytochrome oxidase from beef heart mitochondria

Cytochrome oxidase, the terminal member of the mitochondrial electron transfer chain, has been studied extensively but the subunit structure of the enzyme has not yet been clearly defined. In this paper we describe a preparation of the enzyme which is active, has a high heme content, is devoid of other cytochromes, and has only a small number of polypeptide components which are low in molecular weight.

Biological membrane structure. III. The lattice structure of membranous cytochrome oxidase

Abstract Membranous cytochrome oxidase forms a regular two-dimensional array, as visualized by negative staining and electron microscopy. The lattice formed by the spots in the micrographs, which are assumed to be the cytochrome oxidase protein complexes, belongs to the two-dimensional rectangular space group pg; the unit cell dimensions are 88 ± 4 × 127 ± 7 A , with two protein complexes per unit cell. By using the unit cell area in conjunction with the chemical composition of the membrane and the molecular weights and volumes of the constituents, it was possible to construct a geometrical model for the membrane which quantitatively accounts for the measurements in hand. The lipids are present as lamellar bilayer, and the protein complexes pass all the way through the bilayer.

Membrane proteins and membrane structure

Fig. 1. A schematic cross-sectional view of a hypothetical plasma membrane to show: i) The localization of intrinsic (I) and extrinsic (E) proteins. ii) The amphipathic nature of intrinsic proteins. The polar portion of the molecules (unshaded) is exposed to the aqueous medium while the nonpolar portion (shaded) is in contact with the hydrocarbon tails of the lipid. iii) The organization of the majority of proteins into complexes which span the membrane. The size of complexes isolated from most membranes is in the range of lOO,OOO-200,000 daltons. The subunit polypeptides generally have molecular weights between 10,000 and 50,000 daltons. Association of proteins into complexes allows the possibility of channels through which solutes may traverse the membrane. iv) The spanning of the membrane by glyoproteins (C) as single polypeptides.

DISCUSSION PAPER: A COMPARATIVE STUDY OF THE AMINO ACID COMPOSITIONS OF MEMBRANE PROTEINS AND OTHER PROTEINS*

Results of a comparative study of the amino acid compositions of membrane proteins versus nonmembrane proteins will be presented in this paper. We have concluded on the basis of this study that intrinsic membrane proteins, as defined earlier in this Conference by G. Vanderkooi, do indeed have amino acid compositions that statistically differ from those of nonmembrane proteins, as well as from the membrane-associated extrinsic proteins. The procedure followed is similar to that employed by other workers in the namely, the division of the amino acid residues into polar and nonpolar classes, and summing the percent polar. The principal reason that we have found a consistent difference between intrinsic membrane and nonmembrane proteins, in contrast to the lack of difference found by earlier workers,3 is the larger amount of composition data now available on purified membrane proteins.

BOUNDARY LIPID AND FLUID BILAYER REGIONS IN CYTOCHROME OXIDASE MODEL MEMBRANES

Most current discussions of biological membranes stress a dynamic association of lipids and globular proteins in thin flexible structures forming closed surfaces.l-s One highly diagrammatic cross-sectional view is shown in FIGURE 1. This Figure does not represent any specific biological membrane, but instead indicates a few of the membrane proteins under investigation in various laboratories and summarizes some general ideas regarding membrane structure. On the left is the glycoprotein of the human red blood cell plasma membrane which has been isolated and partially characterized.6 The protein labeled C.O. is the cytochrome oxidase complex of the electron transport chain located in the inner mitochondria1 membrane. To the right in FIGURE 1 is cytochrome b, of the liver microsomal membrane fraction. The structure of the hydrophilic portion of this protein is now known to 2.8 A res~lu t ion .~ These intrinsic proteins penetrate into or through the phospholipid bilayer regions. Also shown in FIGURE 1 are extrinsic, peripheral or membrane-associated proteins 5 , such as cytochrome c that are easily released by mild aqueous treatments. The hydrophobic interior consists of phospholipid bilayer regions interrupted by hydrophobic protein complexes. Assuming this model to be substantially correct, there must exist a boundary between the fluid phospholipid bilayers and the hydrophobic protein components. Using membranous cytochrome oxidase as a model system, we have recently obtained spin-labeling data D* lo that are most easily accounted for in terms of a single boundary layer of immobilized lipid surrounding the protein complex, intercalated into lipid bilayer. At high phospholipid/protein ratios (> 0.2 mg phospholipid/mg protein) an additional component was observed and identified as fluid bilayer. The purpose of this paper is to introduce a second method of analyzing the data upon which the boundary lipid concept is based, and to summarize and discuss our previous results on membranous cytochrome oxidase.

On the subunit structure of oligomycin sensitive ATPase

Abstract The subunit structure of oligomycin sensitive ATPase has been determined. In addition to the components of F 1 , and the so-called oligomycin sensitivity conferring protein, there are four other polypeptides of molecular weights 55,000, 29,000, 20,000 and 10,000 which together form the intrinsic membrane portion of the enzymic complex.

A cross-linking study of the beef erythrocyte membrane: Extensive interaction of all the proteins of the membrane except for the glycoproteins

Abstract Treatment of beef red cell ghosts with 2% glutaraldehyde for 1 hour at 0° cross-links the bulk of the protein of the membrane. Less than 10% of the protein is soluble in SDS after this treatment and even less will penetrate 5% polyacrylamide gels. Yet the glycoprotein can be quantitatively extracted as monomers either in the SDS soluble fraction or by the chloroform, methanol, water extraction procedure. 1

Respiratory control in cytochrome oxidase

Abstract Vesicles of cytochrome oxidase, generated by dilution of the oxidase with a 15-fold excess of lipid by the Hinkle-Racker method, showed a respiratory control index of greater than 5 in presence of the combination of valinomycin and nigericin. Uncouplers were found to be ineffective in releasing respiratory control in the absence of valinomycin. Valinomycin titration in the presence of excess nigericin gave approximately a one to one stoichiometry with cytochrome oxidase. We propose that coupling of electron transfer to valinomycin K+ transport in cytochrome oxidase vesicles is a molecular event; the insensitivity of respiratory control to uncouplers is a consequence of the absence of the systems other than cytochrome oxidase which are required for the action of uncouplers.

Isolation of a major hydrophobic protein of the mitochondrial inner membrane.

Abstract A protein of molecular weight 29,000 has been isolated from the mitochondrial inner membrane. It is a major component of Rackers hydrophobic protein mixture and is also rather selectively released from the inner membrane by lysolecithin treatment. Data indicate that the 29,000 component may be as much as 10% of the total protein of the inner membrane.