Cytochrome c oxidase subunits in contact with phospholipids. Hydrophobic photolabeling with azidophospholipids.

Self Cite

Beef heart cytochrome c oxidase has been reacted with [35S]diazobenzenesulfonate ([35S]DABS), [35S]-N-(4-azido-2-nitrophenyl)-2-aminoethylsulfonate ([35S]NAP-taurine), and two different radioactive arylazidophospholipids. The labeling of the seven different subunits of the enzyme with these protein modifying reagents has been examined. DABS, a water-soluble, lipid-insoluble reagent, reacted with subunits II, III, IV, V, and VII but labeled I or VI only poorly. The arylazidophospholipids, probes for the bilayer-intercalated portion of cytochrome c oxidase, labeled I, III, and VII heavily and II and IV lightly but did not react with V or VI. NAP-taurine labeled all of the subunits of cytochrome c oxidase. Evidence is presented that this latter reagent reacts with the enzyme from outside the bilayer, and the pattern of labeling with the different hydrophilic and hydrophobic labeling reagents is used to derive a model for the arrangement of subunits in cytochrome c oxidase.

Section: Resultsmentioning

confidence: 62%

Section: Methodsmentioning

confidence: 99%

Structure of the cytochrome c oxidase complex: labeling by hydrophilic and hydrophobic protein modifying reagents

Prochaska¹,

Bisson²,

Ra³

1980

Self Cite

“…To further exploit the potential of DAF as a photoactivatable reagent, we wanted to synthesize phospholipids incorporating DAFbased fatty acids. These phospholipids could be effectively used for studying reconstituted membrane-bound proteins as in the case of phospholipids derived from fatty acids containing an aryl azide (Bisson et al, 1979), phenyldiazirine (Takagaki et al, 1983), or [(trifluoromethyl)phenyl]diazirine (Brunner et al, 1983) group. In most of these cases the photoactivatable group is attached as a terminal group at the -carbon atom of the fatty acid via an ether or amide linkage.…”

Section: Discussionmentioning

confidence: 99%

Design, synthesis, and fluorescence studies of fluorenyl fatty acids as new depth-dependent fluorescent probes for membranes: getting over the looping-back problem

Lala

Dixit²,

Koppaka³

et al. 1988

Fluorescent fatty acids have proved very useful in studying the membrane hydrophobic core. They readily partition into membranes or can be converted to phospholipids, which form integral components of membranes. By attaching the fluorescent chromophore to different positions along the alkyl chain of fatty acids, e.g., an anthroyloxy group attached via an ester linkage to n-hydroxystearic acid, membranes have been probed at different depths. While this is an interesting approach and has been extensively used, relatively little attention has been paid to the molecular design of these probes in order to have minimal membrane perturbation. In the present study we have looked into the general problem of design of such depth-dependent membrane probes. We report here a series of fluorenyl fatty acids with varying fatty acid chain lengths, Le., (2-fluoreny1)acetic acid, -butyric acid, -hexanoic acid, and -octanoic acid, in order to obtain information at different depths in the membrane hydrophobic core. To see the effect of attachment of a hydrophobic tail on the orientation of such fatty acids in membranes, an n-butyl group was linked to the C-7 position of fluorene in (2-fluoreny1)butyric acid to get 4-(7-n-butylfluoren-2-yl)butyric acid. Further, to assess their ability to act as depth-dependent fluorescent probes, these fatty acids were incorporated in vesicles prepared from egg phosphatidylcholine, and their fluorescence quenching was studied with potassium iodide, Cu(II), 9,lO-dibromostearic acid, and 12-bromostearic acid. The Stern-Volmer plots so obtained clearly indicated the following depth order: 8-(2-fluorenyl)octanoic acid > 6-(2-fluorenyl)hexanoic acid > 4-(7-n-butylfluoren-2-yl)butyric acid > 4-(2-fluorenyl)butyric acid > (2-fluoreny1)acetic acid. These results indicate that the fluorenyl fatty acids reported here do probe the membrane at different depths, depending on their transverse location. In addition, the attachment of a hydrophobic tail helps in better aligning the probe in membranes and could thus overcome the problems associated with looping back of such fatty acids to the membrane-water interface. These results were also supported by fluorescence polarization data. The general design strategy utilized here can in principle be extended to other membrane probes like photoactivatable fatty acids.

“…In studies of cell membranes, hydrophobic probes of this sort have been used to identify hydrophobic regions of membrane proteins (Bayley & Knowles, 1980;Brunner, 1981;Pradhan & Lala, 1987), and hydrophilic probes have been used to label surface proteins (Staros et al, 1974;Dockter, 1979). Although photoactivable phospholipid analogues have been synthesized for use as amphipathic probes (Bisson et al, 1979;Ross et al, 1982;Burnett et al, 1985), they have been used chiefly to study the interactions of integral membrane proteins with phospholipids; none have been applied to peripheral membrane proteins, which lie in proximity to the membrane. A variety of investigations would profit from the availability of a photoactivable phospholipid probe which could specifically label and thus identify proteins lining the cytoplasmic side of the plasma membrane of intact cells.…”

mentioning

confidence: 99%

A photoactivable phospholipid analog that specifically labels membrane cytoskeletal proteins of intact erythrocytes

Pradhan¹,

Williamson²,

Schlegel³

1989

A radioactive photoactivable analogue of phosphatidylethanolamine, 2-(2-azido-4-nitro-benzoyl)-1-acyl-sn-glycero-3-phospho[14C]ethanolamine ([14C]AzPE), was synthesized. Upon incubation with erythrocytes in the dark, about 90% of [14C]AzPE spontaneously incorporated into the cells; of this fraction, about 90% associated with the membrane, all of it noncovalently. Upon photoactivation, 3-4% of the membrane-associated probe was incorporated into protein. Analysis of this fraction by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, as well as extraction of labeled membranes with alkali or detergent, showed that the probe preferentially labeled cytoskeletal proteins. [14C]AzPE appears to be a useful tool for the study of lipid-protein interactions at the cytoplasmic face of the plasma membrane of intact cells.