Cardiolipins and mitochondrial proton-selective leakage.

The proton-selective leak (State 4 respiratory rate) but not delta psi, in mitochondria from thyroid-sensitive tissues, responds to in vivo stimuli in unique correlation with changes in cardiolipins, saturated and mono-unsaturated (extended) fatty acyl contents, cardiolipins/phospholipids ratios, and/or membrane outer-sidedness. Liver mitochondrial State 4 respiration, basal in fasted rats, contributes little to resting metabolic rate in fed rats, where State 3 depresses delta psi. In a proposed model, an essential inner-membrane outer-surface proton antenna collects protons and donates them, via a water-shuttle, to transmembrane porters: transient water-molecule-chains between extended phospholipid acyls; protonophores, and uncoupling proteins. Only cardiolipin microdomains can donate, from an anomalously-dissociating phosphate group in each headgroup; unadapted cardiolipins have few conducting water chains. Thyroid states regulate each cardiolipin property, and are permissive, via the proton antenna, for proton leaks, including those through adapted and possibly constitutive BAT and ectopic uncoupling proteins. Slow leakage in liposomes may reflect insufficient cardiolipin proton antennas.

Cardiolipins and biomembrane function.

Evidence is discussed for roles of cardiolipins in oxidative phosphorylation mechanisms that regulate State 4 respiration by returning ejected protons across and over bacterial and mitochondrial membrane phospholipids, and that regulate State 3 respiration through the relative contributions of proteins that transport protons, electrons and/or metabolites. The barrier properties of phospholipid bilayers support and regulate the slow proton leak that is the basis for State 4 respiration. Proton permeability is in the range 10(-3)-10(-4) cm s-1 in mitochondria and in protein-free membranes formed from extracted mitochondrial phospholipids or from stable synthetic phosphatidylcholines or phosphatidylethanolamines. The roles of cardiolipins in proton conductance in model phospholipid membrane systems need to be assessed in view of new findings by Hübner et al. [313]: saturated cardiolipins form bilayers whilst natural highly unsaturated cardiolipins form nonlamellar phases. Mitochondrial cardiolipins apparently participate in bilayers formed by phosphatidylcholines and phosphatidylethanolamines. It is not yet clear if cardiolipins themselves conduct protons back across the membrane according to their degree of fatty acyl saturation, and/or modulate proton conductance by phosphatidylcholines and phosphatidylethanolamines. Mitochondrial cardiolipins, especially those with high 18:2 acyl contents, strongly bind many carrier and enzyme proteins that are involved in oxidative phosphorylation, some of which contribute to regulation of State 3 respiration. The role of cardiolipins in biomembrane protein function has been examined by measuring retained phospholipids and phospholipid binding in purified proteins, and by reconstituting delipidated proteins. The reconstitution criterion for the significance of cardiolipin-protein interactions has been catalytical activity; proton-pumping and multiprotein interactions have yet to be correlated. Some proteins, e.g., cytochrome c oxidase are catalytically active when dimyristoylphosphatidylcholine replaces retained cardiolipins. Cardiolipin-protein interactions orient membrane proteins, matrix proteins, and on the outerface receptors, enzymes, and some leader peptides for import; activate enzymes or keep them inactive unless the inner membrane is disrupted; and modulate formation of nonbilayer HII-phases. The capacity of the proton-exchanging uncoupling protein to accelerate thermogenic respiration in brown adipose tissue mitochondria of cold-adapted animals is not apparently affected by the increased cardiolipin unsaturation; this protein seems to take over the protonophoric role of cardiolipins in other mitochondria. Many in vivo influences that affect proton leakage and carrier rates selectively alter cardiolipins in amount per mitochondrial phospholipids, in fatty acyl composition and perhaps in sidedness; other mitochondrial membrane phospholipids respond less or not at all.(ABSTRACT TRUNCATED AT 400 WORDS)

Thyroid control over biomembranes: VI. Lipids in liver mitochondria and microsomes of hypothyroid rats.

The lipids of liver mitochondria prepared from normal rats and from rats made hypothyroid by thyroidectomy and injection with 131 INa contained similar amounts, per mg protein, of total lipids, phospholipids, neutral lipids and lipid phosphorus. Hypothyroidism caused a doubling of the relative amounts of mitochondrial cardiolipins (CL; to 20.5% of the phospholipid P) and an accompanying trend (although statistically not significant) toward decreased amounts of both phosphatidylcholines (PC) and phosphatidylserines (PS), with phosphatidylethanolamines (PE) remaining unchanged. The pattern of elevated 18:2 fatty acyl content and depleted 20:4 acyl groups of the mitochondrial phospholipids of hypothyroid preparations was reflected to varying degrees in the resolved phospholipids, with PC showing greater degrees of abnormality than PE, and CL showing none. Hypothyroidism produced the same abnormal pattern of fatty acyl distributions in liver microsomal total lipids as was found in the mitochondria. Hypothyroid rats, when killed 6 hr after injection of [1-14C] labeled linoleate, showed the following abnormalities: the liver incorporated less label into lipids, and converted 18:2 not exclusively to 20:4 (as normals do) but instead incorporated the label mainly into saturated fatty acids. These data, together with the known decrease in beta-oxidation, suggest that hypothyroidism involves possible defective step(s) in the conversion of 18:2 to 20:4.

A rapid controller of temperature for use in determining Arrhenius profiles in biomembrane systems.

To minimize artifacts in temperature-velocity (Arrhenius) profiles due to aging of preparations of biological membranes, a rapid controller of temperature was developed for spectrophotometric or polarographic (02 electrode) measurements. The reaction mixture is cooled or heated through contact with Peltier elements. One Pt temperature sensor in the cuvette or electrode holder controls current flow into the Peltier units, and another Pt temperature sensor in the reaction mixture is used to read out the sample temperature on a meter or recorder, and to provide feedback control. The sample temperature can be reproducibly set to within 0.1 degree C, with a noise level of 0.04 degrees C or less; a change of 4 degrees C takes 1 min.

Thyroid control over biomembranes. Liver-microsomal cytochrome b5 in hypothyroidism.

Hypothyroid rats were prepared by thyroidectomy and maintenance on a low-iodine diet (group A); Group B was additionally pretreated with 0.5 mCi of 131I as NaI, given intraperitoneally. Liver microsomes obtained from hypothyroid and normal rats were compared. After fasting and refeeding on 20% sucrose solution, high levels of microsomal fatty-acyl-CoA delta 9-desaturase (as measured spectrophotometrically by the rate constants for cytochrome b5 reoxidation) were induced in all the normal animals, half of the group A hypothyroid rats, and none of the group B hypothyroid rats. Hypothyroidism did not change desaturase Arrhenius profiles or V and Km for NADH-cytochrome c reductase, but increased content of cytochrome b5. The inability of adequately hypothyroid rats to induce the delta 9-desaturase seems to be specific, in that injection of methylcholanthrene successfully induced microsomal benzpyrene monooxygenase activity and increased cytochrome b5 contents in hypothyroid animals. The defects in overall fatty acyl desaturation reported in hypothyroid animals [Landriscina, C., Gnoni, G. V. & Quagliariello, E. (1976) Eur. J. Biochem. 71, 135-143] are suggested to be due to deficiencies in the specific desaturase(s).

Thyroid hormone action on mitochondria. IV. Redox states of intrinsic pyridine nucleotides in hypothyroidism, and influence of L-thyroxine.

Measurements of fluorescence at >420 nm and extracted NADPH in mitochondria obtained from the livers of hypothyroid rats show that the addition of Pi, ADP and glutamate rapidly reduces over 90% of the total reducible intrinsic pyridine nucleotides in State 3, compared with 20% in normals. The total fluorescence intensity change and reducible NADP+ is about twice normal in hypothyroid mitochondria. Adding 6-30 microM L-thyroxine to hypothyroid mitochondria in vitro decreases and delays the substrate-induced reduction of pyridine nucleotides, and excludes both NADP+ from such reduction and NADPH from oxidation by added ADP + Pi, without changing the high NADP(H) content. The correcting actions of the hormone are rapidly reversed by albumin, probably by binding free hormone. Changes in respiration do not appear to account for these observations. There is indirect evidence for decreased phosphorylation of added ADP in hypothyroid mitochondria, and a correction by added hormone. The hormonal actions on NADP(H) redox reactions are not reproduced by 1 to 6 microM dinitrophenol in vitro. L-Thyroxine appears to specifically block the participation of NADP(H) in redox reactions in mitochondria from hypothyroid rats, perhaps by effecting a sequestration of the nucleotide, by inhibiting the pyridine nucleotide transhydrogenase, or by activating an energy-linked process that competes with transhydrogenation.