Faculty Bibliography

Cardiolipin is a specific and functionally important phospholipid of mitochondria, and its biosynthesis is considered to be crucial for the assembly of this organelle. However, little information is available about the enzyme cardiolipin synthase, largely because it has not yet been isolated. We solubilized cardiolipin synthase from rat liver mitochondrial membranes with Zwittergent 3-14 and purified it by Mono Q anion exchange chromatography, Superose 12 gel filtration, and Mono P chromatofocusing. Cardiolipin synthase is one of the most acidic mitochondrial proteins (isoelectric point, pH 4-5) and appears as a 50-kilodalton band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified enzyme requires CO2+ for activity, has an alkaline pH optimum (pH 8-9), and exhibits Km values of 45 and 1.6 microM for phosphatidylglycerol and CDP-diacylglycerol, respectively. Cardiolipin synthase loses activity during purification, and the activity can be partially reconstituted by the addition of phospholipids. The most effective phospholipid is phosphatidylethanolamine which reactivates in a cooperative manner. Cardiolipin reactivates hyperbolically at low concentrations but inhibits the enzyme at higher concentrations. In addition, cardiolipin shifts the sigmoidal reactivation curve of phosphatidylethanolamine toward lower concentrations. It is suggested that cardiolipin synthase requires interaction with several molecules of phosphatidylethanolamine and at least one molecule of cardiolipin for full enzymatic activity

We recently synthesized phospholipid analogs with antiviral nucleosides in the polar headgroup and demonstrated their antiretroviral activity in vitro in human immunodeficiency virus-infected cells (Hostetler, K.Y., Stuhmiller, L.M., Lenting, H.B.M., van den Bosch, H. and Richman, D.D. (1990) J. Biol. Chem. 265, 6112-6117). Dideoxynucleoside analogs of cytidine diphosphate diglyceride (CDP-DG) represent one class of such phospholipid prodrugs from which the antiviral active principle may be released through established pathways of cellular phospholipid metabolism. We now demonstrate that the liponucleotides of dideoxycytidine, 3'-deoxythymidine and 3'-azido-3'-deoxythymidine (AZT, Zidovudine) can substitute to varying extents for CDP-DG in the biosynthesis of phosphatidylinositol, phosphatidylglycerol or diphosphatidylglycerol by rat liver subcellular fractions. In all three biosynthetic pathways dideoxycytidine diphosphate diglyceride was the most active donor of the phosphatidyl unit. The nearly stoichiometric formation of dideoxycytidine-5'-monophosphate during phosphatidylinositol biosynthesis supports the rationale that the antiretroviral liponucleotides may provide cells with a depot form from which the antiviral drug can be released in 5'-monophosphorylated form, thus bypassing the initial phosphorylation of free dideoxynucleosides

Molecular species in the three major mitochondrial lipids cardiolipin, phosphatidylcholine and phosphatidylethanolamine were analysed in bovine heart and Saccharomyces cerevisiae. In both organisms cardiolipin contains mainly diacylglycerol moieties with two unsaturated chains and a significant higher proportion of C18-C18 species than phosphatidylcholine and phosphatidylethanolamine. To study whether the specific acyl composition of cardiolipin has a functional significance in lipid-protein interaction, experiments were made with the isolated ADP/ATP carrier of bovine heart mitochondria since this dimeric protein is known to be tightly associated with six molecules of cardiolipin [Beyer, K. and Klingenberg, M. (1985) Biochemistry 24, 3821-3826]. This association seems to be very strong as protein-bound cardiolipin does not exchange with soluble cardiolipin on a time scale of hours. Analysis of the species composition suggests that one carriers dimer is associated with four molecules of tetralinoleoyl cardiolipin and two molecules of trilinoleoyl-monolinolenoyl cardiolipin. Catalytic hydrogenation of the acyl chains of carrier-bound cardiolipin does not result in release of cardiolipin as judged by 31P-NMR spectroscopy. The ADP/ATP carrier was reconstituted with saturated phosphatidylcholines and spin-labelled cardiolipin whose double bonds were subsequently saturated by catalytic hydrogenation. ESR spectroscopy shows that saturation of spin-labelled cardiolipin has no significant impact on its association with the ADP/ATP carrier. However, precipitation of the detergent-solubilized ADP/ATP carrier can only be induced by addition of unsaturated but not by saturated cardiolipin. It is concluded that the specific acyl composition of cardiolipin is not a prerequisite of its high affinity for the ADP/ATP carrier, at least when the protein is reconstituted in a saturated phosphatidylcholine environment

Cardiolipin (CL, 1,3-bisphosphatidyl-sn-glycerol) is a four-acyl-chain phospholipid whose molecular species composition cannot be analyzed by standard procedures. Here we report a method to resolve the molecular species of CL by high-performance liquid chromatography of its derivative 1,3-bisphosphatidyl-2-benzoyl-sn-glycerol dimethyl ester. The CL derivative was characterized by 1H nuclear magnetic resonance spectroscopy, ultraviolet (uv) spectroscopy, thin-layer chromatography, and fatty acid analysis. The derivatization procedure did not change the fatty acid profile and provided a virtually complete conversion to the highly apolar, uv-visible product. In HPLC separations, recorded by 228 nm absorbance, a linear correlation was found between the area of individual peaks and their amount of lipid phosphorus. Bovine heart CL was resolved into 11 molecular species of which 6 (together accounting for 97 mol%) could be identified. The molecular species of bovine heart CL feature a linear relationship between their logarithmic retention time and their double bond number

Liver mitochondrial cardiolipin (CL) is distinguished from other phospholipids by the presence of linoleoyl in almost all molecular species, and the biosynthesis of these species is not yet understood. The present study was carried out in order to test the hypothesis that the linoleoyl proportion of CL may be specifically enriched by a deacylation-reacylation cycle. Incorporation of [14C]glycerol 3-phosphate into the metabolites of the CL pathway was accompanied by formation of 14C-labelled monolyso- and dilyso-CL. Labelling of dilyso-CL was increased or decreased by stimulation or inhibition respectively of mitochondrial phospholipase A2. These data suggest a rapid deacylation of newly formed [14C]CL by phospholipase A2, whereas endogenous mitochondrial CL was very resistant to hydrolytic degradation. Unlike dilyso-CL, monolyso-CL could be reacylated by [14C]linoleoyl residues. [14C]Linoleoyl incorporation into CL was also observed when exogenous CL was added instead of monolyso-CL, thus indicating the concerted action of de- and re-acylation. Although 1-palmitoyl-2-[14C]linoleoyl-phosphatidylcholine was a suitable acyl donor under experimental conditions, the reaction was not a transacylation but required splitting of [14C]linoleic acid from phosphatidylcholine and formation of [14C]linoleoyl-CoA as an intermediate. The [14C]linoleoyl was mainly bound to the sn-2(2') position of CL, and a small portion (about 20%) to the sn-1(1') position. It is concluded that a cycle, comprising CL deacylation and monolyso-CL reacylation by linoleoyl-CoA, provides a potential mechanism for the remodelling of molecular species of newly formed CL

(1) Isolated rat liver mitochondria were subjected to catalytic hydrogenation using a water-soluble Pd complex and molecular H2. This treatment resulted in a reduction of double bonds on phospholipid acyl chains as judged by gas chromatography of fatty acid methyl esters and HPLC of dinitrobenzoyldiacylglycerols. (2) After hydrogenation, mitochondria lost their ability to hydrolyze endogenous phospholipids in alkaline, Ca2+ containing medium, while phospholipase A2 retained full activity against exogenous substrates, regardless of whether those substrates were hydrogenated or not. (3) Inhibition by hydrogenation of endogenous phospholipid hydrolysis correlated with the loss of polyunsaturated fatty acyls, rather than with changes of the bulk membrane fluidity as measured by ESR and fluorescence studies. (4) These data suggest that the unsaturation of mitochondrial membrane lipids might be important for regulation of phospholipid breakdown by endogenous phospholipases. In particular, polyunsaturated molecular species seem to be involved in making phospholipids accessible to phospholipase A-mediated hydrolysis

Lipid acyl double bonds in isolated mitochondrial membranes were gradually reduced by palladium-complex-catalysed hydrogenation, and the resulting saturation was monitored by fatty acid analysis of phosphatidylcholine, phosphatidylethanolamine and cardiolipin. The courses of hydrogenation of these phospholipids suggested that cardiolipin is in a membrane compartment which is less accessible to the applied catalyst. Native cardiolipin and its hydrogenation products were further characterized by analysis of their molecular diacylglycerol species. A decrease in the double bond content was accompanied by an increased amount of motionally restricted lipids at the hydrophobic interface of proteins as measured by two different spin-labelled lipids (C-14 positional isomers of spin-labelled stearic acid and phosphatidylcholine analogues). The protein-immobilized fraction of spin-labelled stearic acid increased in parallel with the hydrogenation of cardiolipin rather than of phosphatidylcholine or phosphatidylethanolamine. These data are interpreted in terms of a tight association of cardiolipin with membrane proteins, which becomes looser upon double bond reduction leading to the replacement of cardiolipin by spin-labelled stearic acid in the solvation shell. Thus the hydrophobic moiety of cardiolipin, characterized by double-unsaturated C18-C18 diacylglycerol species, seems to be an important structural requirement for the high protein affinity of this compound

Rat liver mitochondria were incubated with [3H]glycerol 3-phosphate, ATP, CTP and coenzyme A allowing acylatin of glycerophosphate with endogenous fatty acids and the further conversion of labelled phosphatidic acid (PA) to diacylglycerol (DG), CDP-diacylglycerol (CDP-DG) and phosphatidylglycerol (PG). In these glycerolipids, the distribution of label among the individual molecular species was found to be similar, with 16:0-18:1, 16:0-18:2 and 18:0-18:2/16:0-16:0 being the main species. It was concluded that mitochondrial enzymes involved in the de novo synthesis of these glycerolipids exhibited no acyl selectivity for their substrates. The pattern of molecular species of mitochondrial PA, DG and CDP-DG closely approached that of the same glycerolipids synthesized de novo in isolated rat liver microsomes

The mitochondrial fraction of adult rat lung contains choline phosphotransferase (EC 2.7.8.2) activity which can not be explained by microsomal contamination estimated on the basis of marker enzyme distribution. Mitochondrial (14C)glycerol-3-phosphate incorporation into PC (phosphatidylcholine) can be distinguished from the microsomal incorporation by different sensitivity to N-ethylmaleimide inhibition. The data indicate that rat lung mitochondria have the intrinsic capability to synthesize PC. Both synthesis of PC and PG (phosphatidylglycerol) are susceptible to isotonic tryptic attack against the cytoplasmic face of isolated rat lung mitochondria, suggesting the outer membrane location of crucial activities involved in the formation of these phospholipids. Rat liver mitochondria are different from rat lung mitochondria with respect to their capability to synthesize PC, their rate of (14C)glycerol-3-phosphate incorporation into PG as well as the submitochondrial site of PG formation

It is not yet completely understood how a cell is able to export specific phospholipids, like dipalmitoylphosphatidylcholine (dipalmitoyl-PC), which is secreted by pneumocytes type II, into pulmonary surfactant. The acyl species composition of [3H]PC which was synthesized in type II cells in the presence of [2-3H]glycerol resembled the species composition of PC localized in intracellular pneumocyte membranes. This species pattern was different from the pattern of PC of lamellar bodies, i.e., intracellularly stored surfactant, by a higher proportion of dipalmitoyl-PC mainly at expense of 1-palmitoyl-2-oleoyl-PC. Lamellar body PC in turn showed the same species distribution as surfactant PC. The data suggest that subcellular compartmentation and/or intracellular transfer of PC destined to storage in lamellar bodies, but not secretion of lamellar bodies, involves an enrichment of dipalmitoyl-PC and a depletion of 1-palmitoyl-2-oleoyl-PC. In contrast, the acyl species pattern of phosphatidylglycerol does not seem to undergo gross changes on the path from synthesis to secretion