S1.20 Divalent metal binding to bovine heart F1 ATPase: An FT-ESEEM study

The divalent metal binding sites of beef heart mitochondria F1ATPase were studied by FT-ESEEM spectroscopy, using Mn(II) as a paramagnetic probe, which replaces the naturally occurring Mg(II) and maintains the enzyme catalytic activity. Purified F1ATPase still containing three endogenous tightly bound nucleotides, named MF1(1,2), was obtained under mild conditions, whereas a harsher treatment gave a fully nucleotide depleted enzyme, named MF1(0,0). When MF1(1,2) was loaded with Mn(II) in 1:0.8 ratio, the spectrum showed evidence of a nitrogen interacting with the metal, while this interaction was not present in the spectrum of MF1(0,0) loaded with Mn(II) in the same ratio. However, when MF1(0,0) was loaded with 2.4 Mn(II), the spectrum showed metal-nitrogen interaction resembling that of MF1(1,2) loaded with Mn(II) in 1:0.8 ratio. When MF1(1,2) was loaded with 2.4 Mn(II) the metal-nitrogen interaction signal remained and a phosphorous coordination to the metal was also evident, indicating a binding of Mn2+ to a site containing a tightly bound nucleotide but metal free. These results strongly support the role of the metal alone in structuring the catalytic sites of the enzyme while ESEEM technique appears to be a sensitive and suitable spectroscopic method for conformational studies of MF1 with the advantage of using proteins in frozen solution

The role of residual phospholipids and copurified proteins in the reconstitution of bovine heart mitochondrial ATPase complex.

The reactivation of mitochondrial ATPase by acidic and isoelectric phospholipids was studied comparatively with two purified enzyme preparations exhibiting different gel electrophoretic patterns: the preparation of Serrano et al. (1976, J. Biol. Chem. 251, 2453-2461) and the complex V of Galante et al. (1979, J. Biol. Chem. 254, 12372-12379). Isoelectric phosphatidylcholine liposomes showed marked differences in affinity for the two ATPase complexes and produced different maximal reactivations, whereas no significant differences were found with negatively charged liposomes. Analysis of residual phospholipids associated with the two ATPase preparations revealed a greater relative cardiolipin content in complex V. It is proposed that the different patterns of reactivation of the two ATPase preparations by isoelectric phospholipids result from different contents in residual cardiolipin and adenine nucleotide carrier

Mechanism of local anesthetic effect. Involvement of F0 in the inhibition of mitochondrial ATP synthase by phenothiazines.

The mechanism whereby tertiary amine local anesthetics affect the activity of membrane proteins was investigated by studying the interaction of phenothiazines with mitochondrial ATP synthase. These drugs caused inhibition of the activity of the membrane-bound enzyme at concentrations that do not perturb the phospholipid bilayer. The inhibitory effect appeared consequent to interaction with multiple sites located on both the F1 and the F0 components of the enzyme complex, since: (a) Dixon plots were parabolic; (b) the membrane-bound enzyme was more sensitive to the drug effect than the isolated F1 component; (c) conditions that decreased oligomycin sensitivity also decreased the sensitivity to phenothiazines; (d) irreversible binding of photochemically activated phenothiazines to the ATP synthase complex, followed by detachment of the F1 moiety and reconstitution with purified F1 resulted in an inhibited enzyme complex. These data are interpreted as indicating that tertiary amine local anesthetics affect the activity of membrane proteins by interacting with hydrophobic sites located on both their integral and peripheral domains

Quantification of muscle mitochondrial oxidative phosphorylation enzymes via histochemical staining of blue native polyacrylamide gels.

Blue native-polyacrylamide gel electrophoresis is a powerful technique that enables the separation of intact multi-subunit complexes. However, positive identification of particular enzymes generally requires further separation in a second dimension on a denaturing polyacrylamide gel. Histochemical staining is widely used to demonstrate enzyme activities in tissues, including oxidative phosphorylation enzymes. In this report, we demonstrate that the two techniques can be combined to quantify in situ mitochondrial enzymes, separated on nondenaturing polyacrylamide gels. The method gives quantitative results with human skeletal muscle as well as heart that contains higher mitochondrial numbers. Comparison of muscle from patients with oxidative phosphorylation enzyme deficiencies, such as those of two riboflavin-responsive patients, before and after vitamin treatment, gives results in agreement with those obtained by analyzing the activity of the mitochondrial enzymes in muscle homogenates

Regulation of phospholipid-ATPase complex interaction by the adenine nucleotide carrier.

1) The effect of phospholipids on a preparation containing the ATPase complex and the adenine nucleotide carrier is studied in the presence of ligands known to affect the conformation of these components of the mitochondrial inner membrane. (2) When ATPase activity is abolished by phospholipid depletion, the reactivation induced by phosphatidylcholine is prevented by the simultaneous addition of ATP. ADP partially reproduces the ATP effect. AMP, GTP, UTP, and Pi are ineffective. (3) The influence of ATP is associated with reduced phospholipid binding to the membrane fragments and is reversible. The ATP effect on reconstitution is not manifest when phosphatidylcholine is added together with negatively charged phospholipids. (4) Carboxyatractyloside does not modify the phospholipid-ATPase complex interaction but bongkrekic acid is as effective as ATP. In the presence of ADP, the influence of bongkrekic acid is considerably increased. (5) It is concluded that the binding of ATP to the adenine nucleotide carrier enables the complex to select between the charged and uncharged phospholipids. As a result of the carrier conformational change, the ATPase complex is induced to prefer a negatively charged phospholipid environment

Phospholipid-dependent assembly of mitochondrial ATPase complex.

1. Phosphatidylcholines of different acyl-chain composition and a preparation of ATPase complex depleted of phospholipids have been employed in order to evaluate the contribution of lipid bilayer to the assembly of this multi-subunit component of mitochondrial membrane. 2. At the minimal requirement for bilayer assembly (dinonanoylphosphatidylcholine, mixtures of lysophosphatidylcholine and phosphatidylcholine), fragments with oligomycin-insensitive ATPase activity are reconstituted. Conformational changes with dislocation of ATPase complex subunits may explain these results. 3. At increased strength of acyl-chain interaction (dilauroylphosphatidylcholine and higher homologues), the damage to the ATPase complex is prevented but this is not sufficient to achieve functional restoration. Bilayers with a tendency to coalesce and fuse aggregate in large amounts with the complex and yield low ATPase reactivation. Bilayers of high stability yield complexes with physiological content of phospholipids and efficient ATPase activity. Transition between these two possibilities is found at sixteen carbon acyl-chains. Only at this chain length does the cholate dialysis procedure of reconstitution become feasible. 4. It is concluded that a minimum of 16 carbon atoms in each chain are required to organize a bilayer structurable to maintain the ATPase complex conformation and to sustain the transmembrane position of the whole assembly

Molecular regulation of cholesterol metabolism: HDL-based intervention through drugs and diet

885-894The overloading of cholesterol in the arteries remains the principal cause of cardiovascular diseases. Since available anti-cholesterolemic drugs are not completely effective and have several severe adverse effects, the aim of this review is to analyze current research focused on the emerging, innovative therapeutic strategies based on both pharmacological and nutritional interventions to control cholesterol metabolism. Pharmacological interventions mainly involve the use of molecules capable of interfering with high-density lipoprotien (HDL) metabolism and the reverse cholesterol transport (RCT) through genetic control of apolipoprotein A-I (ApoA-I), agonism at liver X-receptor a (LXR<span style="font-family:Symbol;mso-ascii-font-family: " times="" new="" roman";mso-hansi-font-family:"times="" roman";mso-char-type:symbol;="" mso-symbol-font-family:symbol"="" lang="EN-GB">a), or inhibition of cholesteryl ester transport protein (CETP), scavenger receptor BI(SR-BI), and ecto F0F1ATPase/synthase. Nutritional interventions are based on the use of fibres, phytosterols, and probiotics acting through interference with absorption and re-absorption of cholesterol by enterocyte and hepatocyte specific transporters, thus influencing RCT final step. The search for new drugs is still at the very beginning and new molecules are not yet ready to enter clinical use. However, several promising findings coming from innovative biotechnological research are expected shortly to produce probiotics, fibres, and phytosterols to be used as therapeutic tools. Among the most important advantages of natural products in respect to traditional drugs are the lack of severe adverse effects and their low cost. </span

Membrane topology of ATP synthase from bovine heart mitochondria and Escherichia coli.

The polypeptides exposed to lipids in the membranous F0 sector of the mitochondrial and Escherichia coli ATP synthases were labelled with radioactive photoreactive lipids. Highly resolving gel electrophoretic conditions were used in order to separate all the eighteen components forming the bovine heart mitochondrial enzyme. The hydrophobic labelling was performed on fully active and inhibitor-sensitive ATP synthases. In the mitochondrial enzyme prepared according to Serrano et al. (1976) [J. Biol. Chem. 251, 2453-2461] seven polypeptides of Mr 30500; 11500; 10500; 10000; 9500; 8500 and 4500 were labelled. The major amount of radioactivity was associated with the 30500-Mr component, which is thought to be the adenine nucleotide carrier. In the preparation of Galante et al., (1979) which almost completely lacks this component [J. Biol. Chem. 254, 12372-12378] nine polypeptides of Mr 25000; 21000; 11500; 10500; 10000; 9500; 9200; 8500 and 4500 were labelled. In the ATPase synthase from E. coli the major amount of labelling was associated with subunit b and only a minor portion with subunit c