Interaction of D-beta-hydroxybutyrate apodehydrogenase with phospholipids.

The interaction of a soluble homogeneous preparation of D-beta-hydroxybutyrate apodehydrogenase with phospholipid was studied in terms of restoration of enzymic activity and complex formation. The purified apoenzyme, which is devoid of lipid, is inactive. It is reactivated specifically by the addition of lecithin or mixtures of phospholipids containing lecithin. Mitochondrial phospholipid, i.e. the mixture of phospholipids in mitochondria, reactivates with the highest specific activity (approximately 100 micromol of DPN reduced/min/mg at 37 degrees and with the greatest efficiency (2.5 to 4 mol of lecithin/mol of enzyme subunit). Each of the lecithins of varying chain length and unsaturation reactivated the enzyme, albeit to differing extents and efficiencies. In general, lecithins containing unsaturated fatty acid moieties reactivated better than those containing the comparable saturated lipid. Optimal reactivation can be obtained for the various lecithins when they are microdispersed together with phosphatidylethanolamine. When the lecithins are added microdispersed together with both phosphatidylethanolamine and cardiolipin, maximal efficiency is obtained. Also, PC6:0 and 8:0 reactivate as soluble molecules, so that a phospholipid bilayer is not necessary to reactivate the enzyme. Complex formation was studied using gel exclusion chromatography. It can be shown that each of the phospholipids which reactivate combines with the apoenzyme. Mitochondrial phospholipid, which reactivates the best, binds most effectively; PC8:0, which reactivates with poor efficiency, can be shown to bind with low affinity, and negligible binding occurs at concentrations which do not reactivate the enzyme. Since the apoenzyme is apparently homogeneous and devoid of phospholipid or detergents, it would appear that reactivation does not involve reversal of inhibition such as by removal of a regulatory subunit or detergent from the catalytic subunit. Rather, we conclude that phospholipid is a necessary and integral portion of this enzyme whose active form is a phospholipid-protein complex. The apoenzyme also forms a complex with phosphatidylethanolamine and/or cardiolipin, which do not reactivate enzymic activity. Salt dissociates such complexes in contrast with the lecithin-apoenzyme complex. Binding of phospholipid is a necessary but not sufficient requisite for enzymic activity. The same energies of activation are obtained from Arrhenius plots for the membrane-bound enzyme and for the purified soluble enzyme reactivated with mitochondrial phospholipid or different lecithins. This observation is compatible with the view that the purified enzyme has not been adversely modified in the isolation. Furthermore, essentially the same energies of activation were obtained for saturated lecithins below their transition temperatures and for unsaturated lecithins above their transition temperatures. Hence, there is no indication that a lipid phase transition occurs to influence the activity of this enzyme.     

Cyclopropane fatty acid synthase of Escherichia coli. Stabilization, purification, and interaction with phospholipid vesicles.

The cyclopropane fatty acid (CFA) synthase of Escherichia coli catalyzes the methylenation of the unsaturated moieties of phospholipids in a phospholipid bilayer. The methylene donor is S-adenosyl-L-methionine. The enzyme is loosely associated with the inner membrane of the bacterium and binds to and is stabilized by phospholipid vesicles. The enzyme has been purified over 500-fold by flotation with phospholipid vesicles and appears to be a monomeric protein having a molecular weight of about 90 000. The enzyme binds only to vesicles of phospholipids which contain either unsaturated or cyclopropane fatty acid moieties. CFA synthase is active on phosphatidylglycerol, phosphatidylethanolamine, and cardiolipin, the major phospholipids of E. coli, and also has some activity on phosphatidylcholine. The enzyme is equally active on phospholipid vesicles in the ordered or the disordered states of the lipid phase transition. Studies with a reagent that reacts only with the phosphatidylethanolamine molecules of the outer leaflet of a phospholipid bilayer indicate that CFA synthase reacts with phosphatidylethanolamine molecules of both the outer and the inner leaflets of phospholipid vesicles.     

Identification and characterization of human cardiolipin synthase

The mitochondrial phospholipid cardiolipin is synthesized from cytidinediphosphate-diacylglycerol and phosphatidylglycerol, a process catalyzed by the enzyme cardiolipin synthase. In this study, we identified a human candidate gene/cDNA for cardiolipin synthase, C20orf155. Expression of this candidate cDNA in the (cardiolipin synthase-deficient) crd1Delta yeast confirmed that it indeed encodes human cardiolipin synthase. Purified mitochondria of the crd1Delta expressing human cardiolipin synthase were used to characterize the enzyme. It has an alkaline pH optimum, requires divalent cations for activity and appears to have a different substrate preference for cytidinediphosphate-diacylglycerol species when compared to phosphatidylglycerol species. The possible implications for CL synthesis and remodeling are discussed.     

Cardiolipin is not essential for the growth of Saccharomyces cerevisiae on fermentable or non-fermentable carbon sources

Cardiolipin is a unique dimeric phospholipid, which is present throughout the eukaryotic kingdom and is specifically localized in mitochondrial membranes. It is widely believed that mitochondria possess an essential requirement for this phospholipid. To determine whether cardiolipin is essential for yeast growth, we generated a cardiolipin synthase null mutant by disrupting the CLS1 gene (open reading frame YDL142c on chromosome IV) of Saccharomyces cerevisiae . Biochemical analysis of the mutant indicated that it had no cardiolipin synthase activity and no cardiolipin in its membranes. The enzyme phosphatidylglycerolphosphate synthase, which catalyses the committed step of the cardiolipin pathway, remained unaffected in the null mutant. Haploid cells containing the null allele are viable in media containing glucose, galactose or glycerol/ethanol as the sole carbon source, although growth in galactose or glycerol/ethanol is somewhat reduced in the mutant compared with the wild type. These results indicate that cardiolipin is not essential for the growth of S . cerevisiae in fermentable or non-fermentable carbon sources.     

Effect of thyroxine on the activity of mitochondrial cardiolipin synthase in rat liver.

Cardiolipin is a major mitochondrial membrane lipid and plays a vital role in mitochondrial function. The effect of thyroxine on the activity of liver mitochondrial cardiolipin synthase was examined in this study. Treatment with thyroxine (250 micrograms/100 g) for 5 days increased cardiolipin synthase activity by 52%. Mitochondrial levels of cardiolipin appear to be regulated in part by the effect of thyroid hormone on the activity of cardiolipin synthase.     

Phospholipid accumulation during the cell cycle in synchronous cultures of the yeast, Saccharomyces cerevisiae

Phospholipid concentrations have been examined throughout successive cell cycles in synchronously growing cultures of the yeast, Saccharomyces cerevisiae. Total phospholipid phosphorus, as well as lecithin and phosphatidylethanolamine levels, exhibited stepwise increases during the cell cycle with step increments beginning just prior to new rounds of bud formation. Phosphatidylinositol and phosphatidylserine levels, on the other hand, showed what have been interpreted to be peak concentrations near the time of bud formation. Cardiolipin content varied considerably and was dependent upon the carbon source of the growth medium. Glucose-grown cells exhibited peak concentrations of cardiolipin near the time of bud formation, with marked decreases after this time. In contrast, galactose-grown synchronous cells exhibited stepwise increments in cardiolipin content, with step increases occurring near the time of new rounds of bud formation. Step or peak increases in cardiolipin, as well as all other phospholipids, were found to coincide with the time of stepwise increases in cytochrome c oxidase activity in these cells. No correlations were observed between the elaboration of mitochondrial membranes during the synchronous cell cycle and the observed patterns of phospholipid increase.     

Sigmoid kinetics of purified beef heart mitochondrial carnitine palmitoyltransferase. Effect of pH and malonyl-CoA

The kinetics of purified beef heart mitochondrial carnitine palmitoyltransferase have been extensively investigated with a semiautomated system and the computer program TANKIN and shown to be sigmoidal with both acyl-CoA and L-carnitine. In contrast, Michaelis-Menten kinetics were found with carnitine octanoyltransferase. The catalytic activity of carnitine palmitoyltransferase is strongly pH dependent. The K0.5 and Vmax are both greater at lower pH. The K0.5 for palmitoyl-CoA is 1.9 and 24.2 microM at pH 8 and 6, respectively. The K0.5 for L-carnitine is 0.2 and 2.9 mM at pH 8 and 6, respectively. Malonyl-CoA (20-600 microM) had no effect on the kinetic parameters for palmitoyl-CoA at both saturating and subsaturating levels of L-carnitine. We conclude that malonyl-CoA is not a competitive inhibitor of carnitine palmitoyltransferase. The purified enzyme contained 18.9 mol of bound phospholipid/mol of enzyme which were identified as cardiolipin, phosphatidylethanolamine, and phosphatidylcholine by thin-layer chromatography. The data are consistent with the conclusion that native carnitine palmitoyltransferase exhibits different catalytic properties on either side of the inner membrane of mitochondria due to its non-Michaelis-Menten kinetic behavior, which can be affected by pH differences and differences in membrane environment.     

Cardiolipin activation of dnaA protein, the initiation protein of replication in Escherichia coli

ATP binding to dnaA protein is essential for its action in initiating the replication of plasmids that bear the unique origin of the Escherichia coli chromosome (oriC). ADP bound to that site renders dnaA protein inactive for replication. Diphosphatidylglycerol (cardiolipin), a diacidic membrane phospholipid, displaces the bound nucleotide, and in the presence of components that reconstitute replication, fully reactivates the inert ADP form of dnaA protein. The monacidic phosphatidylglycerol is one-tenth as active as cardiolipin, whereas the neutral phosphatidylethanolamine, the principal E. coli phospholipid, is inactive. Fluphenazine, a tranquilizer drug, blocks cardiolipin activation of dnaA protein, in keeping with the inhibitory action of such agents on phospholipid-dependent enzymes. With the use of this drug to terminate cardiolipin action, dependence of the activation on time, elevated temperature, and high levels of ATP was demonstrated. Cardiolipin binding of nucleotide-free dnaA protein prevents binding of ATP and initiation of oriC replication. Removal of a fatty acid from cardiolipin by phospholipase A reverses this inhibitory effect. The strong and specific interaction of cardiolipin, a cell membrane component, with an essential nucleotide-binding site of dnaA protein, the protein essential for the initiation of chromosome replication, may be an important element in regulating the cell cycle.     

Regulatory aspects of mitochondrial phospholipase A2 from rat liver: effects of proteins, phospholipids and calcium ions

This paper deals with the search for specific inhibitors or activators of the mitochondrial phospholipase A2. Convincing evidence for the existence of proteins in the mitochondrial or cytosolic fraction that function as specific regulators of this enzyme was not obtained. The enzymatic activity appeared to be inhibited at low substrate concentrations by lipocortin isolated from human monocytes. However, at higher substrate concentrations, the inhibition disappeared, suggesting either that lipocortin sequestered the phospholipid substrate or that the putative inactive complex of enzyme and lipocortin dissociated in the presence of excess phospholipids. The hydrolysis of the neutral phospholipid phosphatidylethanolamine was stimulated by the presence of cardiolipin and phosphatidylglycerol. It is unlikely that this is caused merely by the negative charge of these phospholipids, since other negatively charged phospholipids did not show this effect. Using a phospholipid extract from mitochondria as substrate, the enzymatic activity as a function of the Ca2+ concentration was determined. Only one enzyme activity plateau was observed. The calculated KCa2+ value of 0.05 mM suggests that the mitochondrial phospholipase A2 could be regulated strictly by the modulation of the free Ca2+ concentration in vivo. The two activity plateaus observed previously upon variation of the Ca2+ concentration using phosphatidylethanolamine as substrate could be explained by a Ca2+-induced transition of the phospholipid structure.     

Phosphatidyltransferase activity in Bacillus megaterium.

Phosphatidyl transfer between phosphatidylethanolamine, phosphatidylglycerol or phosphatidylserine as donors and primary hydroxyl acceptors including ethanolamine, glycerol, serine and Triton X-100 has been shown to be catalysed by membrane particles derived from Bacillus megaterium strains ATCC 13632 and ATCC 14581. The rate of cardiolipin synthesis from phosphatidylglycerol in the presence of ethanolamine was an order of magnitude greater than that of phosphatidylethanolamine formation. Cardiolipin synthesis from phosphatidylethanolamine in the presence of glycerol was also observed, and was 1.5-fold greater than the formation of phosphatidylglycerol. Similar heat lability, effects of pH and of Triton X-100 for phosphatidyl transfer and cardiolipin synthesis indicate that both reactions were catalysed by cardiolipin synthase.     

Role of cardiolipin peroxidation and Ca in mitochondrial dysfunction and disease

Cardiolipin is a unique phospholipid which is almost exclusively located at the level of the inner mitochondrial membrane where it is biosynthesized. This phospholipid is known to be intimately involved in several mitochondrial bioenergetic processes. In addition, cardiolipin also has active roles in several of the mitochondrial-dependent steps of apoptosis and in mitochondrial membrane dynamics. Alterations in cardiolipin structure, content and acyl chains composition have been associated with mitochondrial dysfunction in multiple tissues in several physiopathological conditions, including ischemia/reperfusion, different thyroid states, diabetes, aging and heart failure. Cardiolipin is particularly susceptible to ROS attack due to its high content of unsaturated fatty acids. Oxidative damage to cardiolipin would negatively impact the biochemical function of the mitochondrial membranes altering membrane fluidity, ion permeability, structure and function of components of the mitochondrial electron transport chain, resulting in reduced mitochondrial oxidative phosphorylation efficiency and apoptosis. Diseases in which mitochondrial dysfunction has been linked to cardiolipin peroxidation are described. Ca²⁺, particularly at high concentrations, appears to have several negative effects on mitochondrial function, some of these effects being linked to CL peroxidation. Cardiolipin peroxidation has been shown to participate, together with Ca²⁺, in mitochondrial permeability transition. In this review, we provide an overview of the role of CL peroxidation and Ca²⁺ in mitochondrial dysfunction and disease.     

Biosynthesis of Cardiolipin in Plant Mitochondria

The properties of cardiolipin synthase were investigated in mitochondria and submitochondrial fractions from etiolated mung bean (Vigna radiata L.) seedlings. Direct evidence is presented that the enzyme utilizes CDP-diacylglycerol in addition to phosphatidylglycerol for the synthesis of cardiolipin. Cardiolipin synthase had an alkaline pH optimum of about 9 and required divalent cations for activity. Maximal activity was obtained in the presence of 16 mM MnCl2. The apparent Km values for CDP-diacylglycerol and phosphatidylglycerol were 0.8 and 50 [mu]M, respectively. Cardiolipin synthase was localized predominantly in the inner membrane of mung bean mitochondria and displayed a substrate species specificity. Highest activities were measured with the dioleoyl species of both CDP-diacylglycerol and phosphatidylglycerol, and somewhat lower activities were measured with mixed species of the two substrates containing a palmitoyl and an oleoyl group. On the other hand, the cardiolipin synthase hardly used the dipalmitoyl species and strongly discriminated against CDP-dipalmitoylglycerol from a mixture with CDP-dioleoylglycerol.     

Proton-pumping N,N'-dicyclohexylcarbodiimide-sensitive inorganic pyrophosphate synthase from Rhodospirillum rubrum: purification, characterization, and reconstitution.

A new method has been developed for the isolation of the proton-pumping N,N'-dicyclohexylcarbodiimide-sensitive PPi synthase (H(+)-PPi synthase) from chromatophores of Rhodospirillum rubrum. The H(+)-PPi synthase was purified by extraction of chromatophores with a mixture of nonanoyl-N-methylglucamide and cholate, by fractionation with poly(ethylene glycol) 4000, hydroxyapatite chromatography, and affinity chromatography. The purified enzyme is homogeneous and has a specific activity of 20.4 mumol of PPi hydrolyzed min-1 mg-1 at pH 7.5 and 20 degrees C. The hydrolytic activity of the enzyme was stimulated by addition of phospholipids and Triton X-100. Of the lipids tested, cardiolipin proved to have the maximal activating effect. Reconstitution of the H(+)-PPi synthase by the freeze-thaw technique yielded an uncoupler-stimulated and N,N'-dicyclohexylcarbodiimide-sensitive PPi hydrolytic activity. The subunit composition of the purified H(+)-PPi synthase was investigated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. One band was obtained after silver staining with an apparent molecular weight of 56,000. The oligomeric structure of the H(+)-PPi synthase is discussed.     

Cloning and characterization of a cDNA encoding human cardiolipin synthase (hCLS1)

Cardiolipin (CL) is a phospholipid localized to the mitochondria, and its biosynthesis is essential for mitochondrial structure and function. We report here the identification and characterization of a cDNA encoding the first mammalian cardiolipin synthase (CLS1) in humans and mice. This cDNA exhibits sequence homology with members of a CLS gene family that share similar domain structure and chemical properties. Expression of the human CLS (hCLS1) cDNA in reticulocyte lysates or insect cells led to a marked increase in CLS activity. The enzyme is specific for CL synthesis, because no significant increase in phosphatidylglycerol phosphate synthase activity was observed. In addition, CL pool size was increased in hCLS1-overexpressing cells compared with controls. Furthermore, the hCLS1 gene was highly expressed in tissues such as heart, skeletal muscle, and liver, which have been shown to have high CLS activities. These results demonstrate that hCLS1 encodes an enzyme that synthesizes CL.     

Biochemical characterization and regulation of cardiolipin synthase in Saccharomyces cerevisiae

Cardiolipin (CL) synthase activity was characterized in mitochondrial extracts of the yeast Saccharomyces cerevisiae and was shown for the first time to utilize CDP-diacylglycerol as a substrate. CL synthase exhibited a pH optimum of 9.0. Maximal activity was obtained in the presence of 20 mM magnesium with a Triton X-100: phospholipid ratio of 1:1. The apparent Km values for phosphatidylglycerol and CDP-diacylglycerol were 1 mM and 36 microM, respectively. CL synthase activity was maximal at 45 degrees C and heat inactivation studies showed that the enzyme retained greater than 75% of its activity at temperatures up to 55 degrees C. To study the regulation of CL synthase, the enzyme was assayed in cells grown under conditions known to affect general phospholipid synthesis. Unlike many phospholipid biosynthetic enzymes including PGP synthase, which catalyzes the initial step in CL biosynthesis, CL synthase was not repressed in cells grown in the presence of the phospholipid precursor inositol. Detailed procedures for the enzymatic synthesis of 32P-labelled substrates are described.     

Studies on phospholipase C from Pseudomonas aureofaciens. I. Purification and some properties of phospholipase C.

Phospholipase C (phosphatidylcholine cholinephosphohydrolase, EC 3.1.4.3) from Pseudomonas aureofaciens was purified 3600-fold from the culture filtrate with a recovery of 1.6%. Purification was performed with the useof (NH4)2SO4 precipitation, Sephadex G-100 gel filtration and by ion-exchange chromatography on DEAE-Sephadex A-50 and CM-Sephadex C-50. The purified enzyme appeared to be homogeneous as revealed by polyacrylamide disc gel electrophoresis at pH 9.3. The molecular weight was estimated to be 35 000 by gel filtration on Sephadex G-75. Under our experimental conditions, phosphatidylethanolamine was more rapidly hydrolysed than phosphatidylcholine. Lyso forms of these two phosphatides were poor substrates. Phosphatidylserine, phosphatidylglycerol, phosphatidylinositol, cardiolipin and sphingomyelin were not hydrolysed. The enzyme activity with phosphatidylcholine as substrate was slightly stimulated by Ca2+, Mg2+, and Mn2+. However, these cations inhibited the activity with phosphatidylethanolamine as substrate. An anionic detergent, sodium deoxycholate, slightly enhanced the activity when phosphatidylcholine and phosphatidylethanolamine were used as substrates. A cationic detergent, cetyltrimethylammonium bromide, inhibited enzyme activity. EDTA and o-henanthroline inhibited the activity of the enzyme to a marked degree.     

In yeast,cardiolipin unsaturation level plays a key role in mitochondrial function and inner membrane integrity

Cardiolipin is the signature phospholipid of the mitochondrial inner membrane. It participates in shaping the inner membrane as well as in modulating the activity of many membrane-bound proteins. The acyl chain composition of cardiolipin is finely tuned post-biosynthesis depending on the surrounding phospholipids to produce mature or unsaturated cardiolipin. However, experimental evidence showing that immature and mature cardiolipin are functionally equivalents for mitochondria poses doubts on the relevance of cardiolipin remodeling. In this work, we studied the role of cardiolipin acyl chain composition in mitochondrial bioenergetics, including a detailed bioenergetic profile of yeast mitochondria. Cardiolipin acyl chains were modified by genetic and nutritional manipulation. We found that both the bioenergetic efficiency and osmotic stability of mitochondria are dependent on the unsaturation level of cardiolipin acyl chains. It is proposed that cardiolipin remodeling and, consequently, mature cardiolipins play an important role in mitochondrial inner membrane integrity and functionality.     

Isolation and characterization of highly purified mitochondrial outer membranes of the yeast Saccharomyces cerevisiae (Method)

Mitochondrial outer membrane vesicles (OMV) from the yeast Saccharomyces cerevisiae were prepared by osmotic swelling and mechanical disruption of mitochondria that had been isolated at pH 6.0 and purified by density gradient centrifugation. The OMV were obtained in a yield of 1% (protein/protein) with respect to the mitochondria. The OMV were shown to be essentially free of mitochondrial inner membrane protein markers, while contamination with endoplasmic reticulum was around 5% (protein-based). The very low phosphatidylserine synthase activity present in the OMV preparation indicated that contamination with mitochondria-associated membranes (MAM) was negligible. The resistance of the outer membrane protein Tom40 to digestion by trypsin demonstrated the sealed nature and right-side out orientation of the OMV. Analysis of the phospholipid composition revealed that the contents of phosphatidylcholine and phosphatidylinositol are higher and the content of phosphatidylethanolamine is lower in the mitochondrial outer membrane as compared to whole mitochondria. Cardiolipin is largely depleted in the OMV.     

Purification and characterization of CDP-diacylglycerol synthase from Saccharomyces cerevisiae

The membrane-associated phospholipid biosynthetic enzyme CDP-diacylglycerol synthase (CTP:phosphatidate cytidylyltransferase, EC 2.7.7.41) was purified 2,300-fold from Saccharomyces cerevisiae. The purification procedure included Triton X-100 solubilization of mitochondrial membranes, CDP-diacylglycerol-Sepharose affinity chromatography, and hydroxylapatite chromatography. The procedure resulted in a nearly homogeneous enzyme preparation as determined by native and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Radiation inactivation of mitochondrial associated and purified CDP-diacylglycerol synthase suggested that the molecular weight of the native enzyme was 114,000. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified enzyme preparation yielded two subunits with molecular weights of 56,000 and 54,000. Antibodies prepared against the purified enzyme immunoprecipitated CDP-diacylglycerol synthase activity and subunits. CDP-diacylglycerol synthase activity was dependent on magnesium ions and Triton X-100 at pH 6.5. Thio-reactive agents inhibited activity. The activation energy for the reaction was 9 kcal/mol, and the enzyme was thermally labile above 30 degrees C. The Km values for CTP and phosphatidate were 1 and 0.5 mM, respectively, and the Vmax was 4,700 nmol/min/mg. Results of kinetic and isotopic exchange reactions suggested that the enzyme catalyzes a sequential Bi Bi reaction mechanism.     

Isolation and characterization of highly purified mitochondrial outer membranes of the yeast Saccharomyces cerevisiae (method).

Mitochondrial outer membrane vesicles (OMV) from the yeast Saccharomyces cerevisiae were prepared by osmotic swelling and mechanical disruption of mitochondria that had been isolated at pH 6.0 and purified by density gradient centrifugation. The OMV were obtained in a yield of 1% (protein/protein) with respect to the mitochondria. The OMV were shown to be essentially free of mitochondrial inner membrane protein markers, while contamination with endoplasmic reticulum was around 5% (protein-based). The very low phosphatidylserine synthase activity present in the OMV preparation indicated that contamination with mitochondria-associated membranes (MAM) was negligible. The resistance of the outer membrane protein Tom40 to digestion by trypsin demonstrated the sealed nature and right-side out orientation of the OMV. Analysis of the phospholipid composition revealed that the contents of phosphatidylcholine and phosphatidylinositol are higher and the content of phosphatidylethanolamine is lower in the mitochondrial outer membrane as compared to whole mitochondria. Cardiolipin is largely depleted in the OMV.