Sidney Fleischer

The role of lipids in mitochondrial electron transfer and oxidative phosphorylation

Abstract 1. 1. The mitochondrion has been fragmented into a fraction containing the elementary particles (the seat of the electron-transfer chain) and a fraction containing the structural protein. On the basis of high-resolution electron microscopy and detailed chemical studies, the mitochondrion is pictured as a structural protein-phospholipid matrix to which are affixed many thousands of elementary particles. 2. 2. The essentiality of lipid for electron transfer can be demonstrated by extracting mitochondria with aqueous acetone to remove lipid. Restoration of activity is achieved by adding back both coenzyme Q (Q) and phospholipid. A phospholipid requirement has been demonstrated in three segments of the electron-transfer chain; succinate → Q; QH2 → cytochrome c; reduced cytochrome c → O2. 3. 3. Phospholipids can be oriented in water to form water-clear micelles. The micelle is the key to an understanding of the role of phospholipid and of the interaction of phospholipid with mitochondrial proteins. Two types of interaction have been characterized. The first is ionic, and occurs between acidic phospholipids and basic proteins like cytochrome c. The second, mainly hydrophobic in nature, involves the interaction of structural protein with all phospholipids tested, acidic as well as basic. 4. 4. The role of phospholipid in electron transport, oxidative phosphorylation and the energy-linked transport of ions across membranes is discussed and a generalized formulation of the molecular structure and properties of membranes is given.

Studies of the electron-transfer system XLIX. Sites of phospholipid involvement in the electron-transfer chain

Abstract The reactivation of succinoxidase activity in acetone-extracted heart mitochondria requires the addition of CoQ and purified phospholipids only. The restored activity is directly proportional to the amount of phospholipid taken up by the mitochondrion. The three successive reactions constituting collectively the oxidation of succinate, (a) the reduction of CoQ by succinate, (b) the reduction of cytochrome c , are all reduced CoQ, and (c) the reduction of oxygen by reduced cytochrome c , are all increased by the addition of phospholipid to extracted mitochondria, and in each case the reactivation is proportional to the amount of phospholipid bound by the mitochondrion. Studies with the purified submitochondrial enzyme complexes, succinate-CoQ reductase and CoQH 2 - cytochrome c reductase, indicate that these complexes, depleted of phospholipid by extraction with aqueous acetone, require the addition of phospholipid for maximal activity. In a companion communication it has been demonstrated that the activity of purified cytochrome oxidase is directly proportional to its phospholipid content. The present studies show that phospholipid is required for maximal activity in at least three segments of the electron-transfer chain.

Interaction of the protein moiety of bovine serum α-lipoprotein with phospholipid micelles: I. The use of lipid-requiring enzymes as assay systems

Abstract The relationship of lipid deficient enzymes of beef-heart mitochondria which require phospholipid for enzymic activity to soluble lipoproteins has been studied. α- and β-lipoproteins were isolated from bovine serum by flotation in high-density media. Although these soluble lipoproteins contain lecithin, this lipid is not available to reactivate mitochondrial β-hydroxybutyric apodehydrogenase, a lecithin-requiring enzyme. When freed from the protein and dispersed in aqueous media, however, the lipids from these soluble lipoproteins reactivate the enzyme. The protein moiety of α-lipoprotein (α-protein) was freed of its lipid by treatment with 95% ethanol. α-Protein inhibits the reactivation of β-hydroxybutyric dehydrogenase by lecithin. Inhibition of mitochondrial electron transport is also observed when α-protein is added to a mixture of cardiolipin and lipid-deficient beef-heart mitochondria. α-Protein is unique in this regard as compared with a number of proteins which do not normally exist as lipoproteins. Little or no inhibition occurs if the enzymes are allowed to interact with phospholipid before the addition of α-protein. These results indicate that α-protein is capable of binding both lecithin and cardiolipin in micellar form, and that the binding of protein to lipid may be similar in soluble lipoproteins and in membrane-bound enzymes. It is concluded that lipid-protein complexes (lipoproteins) exist as such because the protein moiety has an affinity to bind lipid.

Interaction of the protein moiety of bovine serum α-lipoprotein with phospholipid micelles: II. Isolation and characterization of the complexes

Abstract Complex formation between the protein moiety of bovine serum α-lipoprotein (α-protein) and micellar dispersions of phospholipids has been demonstrated directly by three methods; (1) chromatography on Sephadex G-200; (2) ultracentrifugation in salt solutions of high density; and (3) electrophoresis in starch gel. The binding of phospholipid to α-protein is of the same magnitude as that in the original α-lipoprotein. The saturating ratio of lipid to protein was found to be different for lecithin and cardiolipin, being 2.5 and 0.6 mg lipid per mg protein respectively. In the native lipoprotein the ratio is 1.7 mg total lipid per mg protein of which 0.7 mg per mg protein is phospholipid. When the lipids of α-lipoprotein are fractionated into a phospholipid and neutral lipid fraction, α-protein binds appreciably to the phospholipid and only slightly to the neutral lipids.