Evidence for Protein Penetration in Mitochondrial Membrane Structure

Lenaz, Giorgio; Bertoli, Enrico; Landi, Laura; Parenti-Castelli, G; Pasquali, P.; Sechi, A. M.

doi:10.1016/b978-0-08-017822-6.50032-0

Cited by 8 publications

(12 citation statements)

References 23 publications

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“…in pa-thological studies) might not be revealed if they occur at a step which is not rate-limiting in the enzymatic reaction. Moreover, CoQ, behaves as an inhibitor of Complex I, as has been pointed out previously [8]. In fact, NADH oxidation rates were the lowest when this homolog was used.…”

Section: Resultsmentioning

confidence: 67%

Assay conditions for the mitochondrial NADH:coenzyme Q oxidoreductase

et al. 1993

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The assay of Complex I activity requires the use of artiikial acceptors, such as short-chain coenzyme Q homologs and analogs, because the physiological quinones, such as CoQ,,, are too insoluble in water to be added as substrates to the assay media. The medical interest raised in the last years on the pathological changes of Complex I activity has focussed on the requirement of easy reliable assays for its analysis. We have undertaken a systematic examination of the assay conditions of Complex I in mitochondrial membranes, using a series of quinones as electron acceptors, particularly the coenzyme Q homologs CoQo, CoQl and CoQ,, and the analogs duroquinone and decylubiquinone. Our findings have pointed out that the most suitable electron acceptor for the NADH:CoQ reductase assay is the homolog CoQ1. The analog DB, commercially available, although yielding a high activity, nevertheless causes some problems for the standardization of the assay conditions.

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Section: Resultsmentioning

confidence: 67%

Assay conditions for the mitochondrial NADH:coenzyme Q oxidoreductase

et al. 1993

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“…The discontinuity in the Arrhenius plot seen with original submitochondrial particles containing very unsaturated endogenous lipids [44,45] and in the dioleoyl-phosphatidylglycerol-stimulated ATPase complex, are of difficult interpretation. It is possible that immobilization of phospholipid by proteins through polar and apolar interaction results in a change of the transition temperature [46] but the association of protein with lipids heterogeneously distributed within the membrane [47,36], the influence of upper and lower limits of transition [48], the cluster formation [37] or a change in the structure of water [49] near the ATPase are other possibilities which deserve consideration.…”

Section: Temperature-induced Changesmentioning

confidence: 99%

The role of phospholipid acyl chains in the activation of mitochondrial ATPase complex

Bruni

Dijck

Gier

1975

Biochimica et Biophysica Acta (BBA) - Biomembranes

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SUMMARY1. The role of length and unsaturation of phospholipid acy! chains in the activation of ATPase complex was studied with synthetic phosphatidylcholines and a phospholipid-dependent preparation obtained after cholate-extraction of submitochondrial particles (Kagawa, Y. and Racker, E. (1966) J. Biol. Chem. 241, 2467-2474.2. Micelle-forming, short-chain phosphatidylcholines produced activation only at critical micellar concentration. The reactivated complex was cold-stable but the oligomycin sensitivity was low.3. Bilayer-forming saturated phosphatidylcholines produced activation which was maximal at 9 carbon atoms in each chain but decreased sharply as the chainlength was increased and essentially disappeared at 14 carbon atoms. By contrast the oligomycin-sensitivity increased with the increase in chain length.4. Activation of ATPase complex reappeared when bilayers were formed with long-chain unsaturated phosphatidylcholines. The activity was oligomycin sensitive. Significant inhibition of activity was observed also after incorporation of cholesterol into the bilayers.5. By contrast the activation induced by negatively charged liposomes of diacylphosphatidyiglycerol was independent on acyi-chain composition and occurred at very low amounts of phospholipid.6. The discontinuity in the Arrhenius plot of activity of the ATPase complex reactivated with saturated phospholipids was found at temperatures close to the gelto-liquid crystalline transition of the lipid showing that the activity of ATPase complex was sensitive to the physical state of membrane phospholipids.7. It is concluded that (a) reactivation of ATPase complex by isoelectric phospholipids is an interracial activation, the minimum requirement for the lipid effect being micelle formation. (b) In order to gain the properties of the native complex a stable lamellar phase is needed. Both activity and oligomycin sensitivity are regulated by the chain length and degree of unsaturation of phospholipid acyl chains.

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“…Obviously the experiments reported here cannot alone clearly discriminate between the two possibilities, but a series of other findings appear to be in favor of the latter: (a) the bonds between the lipids and proteins appear mainly hydrophobic in nature (1)(2)(3)40); (b) phospholipase C attacks mitochondrial membranes and phospholipid vesicles to similar extents (48), so that protein is not a hindrance to lipolytic action; (c) all of the anionic groups of phospholipids in submitochondrial particles or mitochondrial membranes are available for ionic interaction with basic proteins (lysozyme and cytochrome c) (49).…”

Section: Discussionmentioning

confidence: 84%

Structural studies on the organization of proteins in mitochondrial membranes using proteolytic enzymes

et al. 1973

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Proteolytic enzymes, pronase and trypsin, digest protein in ETP and in SU-particles (devoid of the soluble ATPase) at similar rates and to the same extents for intact and lipid-depleted membranes, showing that lipids do not constitute a barrier to the action of the proteases. The rates and extents of hydrolysis are slightly depressed when membranes are reconstituted from lipid-depleted particles and phospholipids. The hydrolysis rates for the various particles are not greatly enhanced by detergent solubilization nor by other denaturing treatments, indicating that the rates measured in absence of treatments are maximal under the conditions used. The circular dichroism spectra of pronase treated ETP are noticeably altered showing modification of the original conformation. Moreover, enzymic activities of mitochondria and submitochondrial particles are progressively affected by proteases according to their localization at, or near to, a given surface of the membrane. The matrix enzyme, malate dehydrogenase, is not apparently reIeased from mitochondria during the initial incubation period. The results are tentatively discussed in terms of organization of lipids and proteins in the mitochondrial membrane.Previous investigations from our laboratory (1-3) have shown that the proteins of the inner mitochondrial membrane bind phospholipids hydrophobically in reconstitution experiments. Indirect reasons have suggested that the proteins which are bound hydrophobically in the intact membrane are those which may be considered "intrinsic" and are water-insoluble when separated from the lipids (4). Superficial "extrinsic" proteins (4) which are water-soluble when detached, may be bound by polar forces t o the membrane continuum (5).The type of binding alone may not be a sufficient tool t o evaluate the organization of proteins and lipids in the inner mitochondrial membrane. For example, hydrophobic binding between lipids and proteins is compatible with different membrane models, although the macromolecular organization is quite different in these models. Membrane proteins may be hydrophobically bound t o phospholipids according to any one of the following possibilities: (a) penetration of phospholipid fatty acyl chains from the lipid bilayer into superficial protein layers (6); (b) partial penetration of protein side residues between lipid molecules in the bilayer (7); (c) extensive penetration of protein molecules into interstices of the lipid bilayer, like in some mosaic models recently proposed (8-10);

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Evidence for Protein Penetration in Mitochondrial Membrane Structure

Cited by 8 publications

References 23 publications

Assay conditions for the mitochondrial NADH:coenzyme Q oxidoreductase

Assay conditions for the mitochondrial NADH:coenzyme Q oxidoreductase

The role of phospholipid acyl chains in the activation of mitochondrial ATPase complex

Structural studies on the organization of proteins in mitochondrial membranes using proteolytic enzymes

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