Comparative studies of CDP-diacylglycerol synthase in rat liver mitochondria and microsomes.

CDP-diacylglycerol for polyglycerophosphatide biogenesis can be synthesized within rat liver mitochondria. Contamination by microsomal membranes cannot account for the CDP-diacylglycerol synthesis found in the mitochondria. Phosphatidic acid from egg lecithin was the best substrate for the synthesis of CDP-diacylglycerol in both subcellular fractions. Concentration curves for CTP and Mg2+ differed for the two subcellular fractions. Microsomal CDP-diacylglycerol synthase was specifically stimulated by the nucleotide GTP; this stimulatory effect by GTP was not observed in the mitochondrial fraction. By comparison, the microsomal enzyme was more sensitive towards sulfhydryl inhibitors than the mitochondrial enzyme. The enzymes could be solubilized from the membrane fractions using 3-[(cholamidopropyl)dimethylammonio]-1-propanesulfonate, and the detergent-soluble activity could be partially restored by addition of phospholipids. Based on the differences in properties, it was concluded that there are two distinct enzyme localizations for CDP-diacylglycerol synthesis in mitochondria and microsomes from rat liver.

CDP-diacylglycerol synthesis in rat liver mitochondria.

CDP-diacylglycerol for polyglycerophosphatide biogenesis can be synthesized within rat liver mitochondria. This membrane-associated enzyme was predominantly located in the inner mitochondrial membrane. GTP had a significant effect in activating the microsomal CDP-diacylglycerol synthase, especially if the microsomes were preincubated with GTP in the presence of phosphatidic acid. This stimulatory effect of GTP on the microsomal enzyme was not detected in the mitochondrial fractions. The enzymes could be solubilized from the membrane fractions using CHAPS, and the detergent-soluble activity partially restored by addition of phospholipids. Mitochondrial and microsomal CDP-diacylglycerol synthase activity could be completely separated by anion-exchange column chromatography. The mitochondrial and microsomal CDP-diacylglycerol synthases appear to be two distinct enzymes with different localization and regulatory characteristics.

Biosynthesis of phosphatidic acid by glycerophosphate acyltransferases in rat liver mitochondria and microsomes.

The acyltransferases that catalyze the synthesis of phosphatidic acid from labelled sn-[14C]glycero-3-phosphate and fatty acyl carnitine or coenzyme A derivatives have been shown to be present in both isolated mitochondria and microsomes from rat liver. The major reaction product was phosphatidic acid in both subcellular fractions. A small quantity of lysophosphatidic acid and neutral lipids were produced as by-products. Divalent cations had significant effects on both mitochondrial and microsomal fractions in stimulating acylation using palmitoyl CoA, but not when palmitoyl carnitine was used as the acyl donor. Palmitoyl CoA and palmitoyl carnitine could be used for acylation by both mitochondria and microsomes. Mitochondria were more permeable to palmitoyl carnitine and readily used it as the substrate for acylation. On the other hand, microsomes yielded a better rate with palmitoyl CoA and the rate of acylation from palmitoyl carnitine in microsomes was correlated with the degree of mitochondrial contamination. The enzymes were partially purified from Triton X-100 extracts of subcellular fractions. Based on the differences of substrate utilization, products formed, divalent cation effects, molecular weights, and polarity, the mitochondrial and microsomal acyltransferases appeared to be different enzymes.

Examination of the potential role of the glycerophosphorylcholine (GPC) pathway in the biosynthesis of phosphatidylcholine by liver and lung.

The potential involvement of the glycerophosphorylcholine (GPC) pathway for the synthesis of phosphatidylcholine (PC) has been examined in rat liver and lung and in a human line, the A549 cell which possesses characteristics representative of mature alveolar type II epithelial cells. Although mitochondrial and microsomal fractions from the above sources readily incorporated radioactive glycerophosphate into lipids, the only incorporation observed with radioactive GPC was a small variable labelling with the mitochondrial and microsomal fractions from rat lung. Even with these fractions, no radioactivity from GPC was incorporated into PC or lysoPC. Attempts to increase the incorporation of GPC into lipids by manipulating the incubation conditions were unsuccessful. It was concluded that the occurrence of the GPC pathway in liver and lung is unlikely.

Cell-free synthesis of a putative precursor to the rat liver mitochondrial glycerol-3-phosphate dehydrogenase.

Antibodies to purified glycerol-3-phosphate dehydrogenase were raised in rabbits and purified from serum by affinity chromatography on enzyme-bound Sepharose columns. RNA from membrane-free polyribosomes, or poly(A)+ RNA (total cellular RNA) of rat liver, was translated in a rabbit reticulocyte protein-synthesizing system in the presence of [35S]methionine, and the glycerol-3-phosphate dehydrogenase synthesized was isolated by immunoprecipitation using the antibody. The in vitro product moved on sodium dodecyl sulfate-polyacrylamide gels as a polypeptide that was about 5,000 daltons larger than the subunit of the mature enzyme (74,000 daltons). Digestion of both the mature and the in vitro newly synthesized forms of the enzyme yielded respective sets of peptide fragments which had similar patterns upon sodium dodecyl sulfate-gel electrophoresis. When the presumptive precursor that had been synthesized in vitro was incubated with isolated intact rat liver mitochondria, it was converted to "mature" subunits that were no longer susceptible to externally added proteases. Import of the presumptive precursor is dependent upon an electrochemical potential across the inner mitochondrial membranes. The mature form of the protein is assembled in its native location (the outer surface of the inner mitochondrial membrane).

Characterization of CDPdiacylglycerol hydrolase in mitochondrial and microsomal fractions from rat lung.

CDPdiacylglycerol pyrophosphatase (E.C. 3.6.1.26) activity has been examined in rat lung mitochondrial and microsomal fractions. While the mitochondrial hydrolase exhibited a broad pH optimum from pH 6-8, the microsomal activity decreased rapidly above pH 6.5. Apparent Km values of 36.2 and 23.6 microM and Vmax values of 311 and 197 pmol.min-1.mg protein-1 were observed for the mitochondrial and microsomal preparations, respectively. Addition of parachloromercuriphenylsulphonic acid led to a marked inhibition of the microsomal fraction but slightly stimulated the mitochondrial activity at low concentrations. Mercuric ions were inhibitory with both fractions. Although biosynthetic reactions utilizing CDPdiacylglycerol require divalent cations, addition of Mg2+, Mn2+, Ca2+, Zn2+, Co2+, and Cu2+ all inhibited the catabolic CDPdiacylglycerol hydrolase activity in both fractions. EDTA and EGTA also produced an inhibitory effect, especially with the mitochondrial fraction. Although addition of either adenine or cytidine nucleotides led to a decrease in activity with both fractions, the marked susceptibility to AMP previously reported for this enzyme in Escherichia coli membranes, guinea pig brain lysosomes, and pig liver mitochondria was not observed. These results indicate that rat lung mitochondria and microsomes contain specific CDPdiacylglycerol hydrolase activities, which could influence the rate of formation of phosphatidylinositol and phosphatidylglycerol for pulmonary surfactant.

Phosphatidylinositol 4,5-bisphosphate phosphodiesterase in higher plants.

A phospholipase C which hydrolyses phosphatidylinositol 4,5-bisphosphate to release inositol trisphosphate was detected in a sedimentable fraction from celery and from some other higher plants. The particulate enzyme also hydrolyses phosphatidylinositol, whereas the soluble phosphatidylinositol phosphodiesterase described previously [Irvine, Letcher & Dawson (1980) Biochem. J. 192, 279-283] acts only on phosphatidylinositol, and we were unable to detect activity of this soluble activity on phosphatidylinositol 4,5-bisphosphate. Activity of the particulate enzyme is markedly enhanced in the presence of deoxycholate, but not of other detergents; the particulate enzyme can also be solubilized by extraction with deoxycholate.

Origins of the phospholipids in animal mitochondria.

As is the case for the assembly of protein components of the membranes in animal mitochondria, the bilayer phospholipids arise from a complicated interplay of intra- and extra-mitochondrial reactions. Our early studies indicated that the bulk of mitochondrial phospholipids (typified by phosphatidylcholine) had their origin in the endoplasmic reticulum and were transported to the mitochondria as complexes with phospholipid-exchange proteins. The polyglycerophosphatides (typified by diphosphatidylglycerol) were apparently synthesized in situ by intramitochondrial membrane-bound enzymes using CDP-diglycerides as intermediates. The case for the precursors in the latter pathway is less clear, although evidence has been presented for dual localization of enzymes for glycerophosphate acylation and CTP:phosphatidate cytidylyl transfer in both mitochondria and microsomes. Phosphatidylethanolamine also shows evidence for two sites of origin: by translocation from its site of synthesis in the endoplasmic reticulum and by translocation of phosphatidylserine followed by decarboxylation within the mitochondria. In the latter case mitochondrial phosphatidylserine decarboxylase may play an important role in the regulation of phospholipid metabolism throughout the cell.

Purification and characterization of glycerol-3-phosphate dehydrogenase (flavin-linked) from rat liver mitochondria.

We have purified the membrane-intrinsic glycerol-3-phosphate dehydrogenase from both normal and hyperthyroid rat liver mitochondria by extraction with Triton X-100, hydrophobic affinity chromatography, ion exchange chromatography, gel filtration, and FAD-linked Sepharose 4B affinity chromatography. The yields in both cases were over 20%, and purification ranged from 800- to 650-fold in mitochondria from hyperthyroid and normal rats, respectively. The final preparations appeared to be greater than 95% pure by polyacrylamide gel electrophoresis in the presence or absence of sodium dodecyl sulfate. The pure enzyme focused at pH 5.5 and produced a biphasic thermal inactivation plot at 50 degrees C. The holoenzyme was found to have a molecular mass of 250,000 daltons on gel filtration. The subunit molecular mass was found to be 74,000 daltons +/- 3,000 by sodium dodecyl sulfate-gel electrophoresis and high-performance liquid chromatography gel filtration in 0.1% sodium dodecyl sulfate. 1 mol of the holoenzyme preparation contains 1.1 mol of non-heme iron and 0.7-0.9 mol of noncovalently bound FAD. The absorption spectrum has a maximum at 375 nm and a shoulder at 450 nm which is bleached on treatment with sodium dithionite. The enzymatic reaction is competitively inhibited by glyceraldehyde 3-phosphate, dihydroxyacetone phosphate, phosphoenolpyruvate, and phosphoglycolic acid. The apparent Km for DL-alpha-glycerol 3-phosphate and noncovalently bound FAD were found to be 6 mM and 7 microM, respectively.

Triton solubilization of proteins from pig liver mitochondrial membranes.

Various aspects of membrane solubilization by the Triton X-series of nonionic detergents were examined in pig liver mitochondrial membranes. Binding of Triton X-100 to nonsolubilized membranes was saturable with increased concentrations of the detergent. Maximum binding occurred at concentrations exceeding 0.5% Triton X-100 (w/v). Solubilization of both protein and phospholipid increased with increasing Triton X-100 to a plateau which was dependent on the initial membrane protein concentration used. At low detergent concentrations (less than 0.087% Triton X-100, w/v), proteins were preferentially solubilized over phospholipids. At higher Triton X-100 concentrations the opposite was true. Using the well-defined Triton X-series of detergents, the optimal hydrophile-lipophile balance number (HLB) for solubilization of phosphatidylglycerophosphate synthase (EC 2.7.8.5) was 13.5, corresponding to Triton X-100. Activity was solubilized optimally at detergent concentrations between 0.1 and 0.2% (w/v). The optimal protein-to-detergent ratio for solubilization was 3 mg protein/mg Triton X-100. Solubilization of phosphatidylglycerophosphate synthase was generally better at low ionic strength, though total protein solubilization increased at high ionic strength. Solubilization was also dependent on pH. Significantly higher protein solubilization was observed at high pH (i.e., 8.5), as was phosphatidylglycerophosphate synthase solubilization. The manipulation of these variables in improving the recovery and specificity of membrane protein solubilization by detergents was examined.

CDP-diglyceride hydrolase from pig liver mitochondria.

A CDP-diglyceride hydrolase activity, measured by the release of [3H]CMP from labeled CDP-diglyceride, has been identified in pig liver mitochondria. A modified preparatory method for the synthesis of [3H]CDP-diglyceride of high specific activity and purity is also reported. Activity of the hydrolase is enriched 2.5-fold in mitochondrial membranes (over whole mitochondria) and can be solubilized by nonionic detergents such as Triton X-100 with further enrichment of activity (i.e., 7.9-fold). The CDP-diglyceride hydrolase has a Km of 12.8 microM for CDP-diglyceride and a broad pH range with optimum activity at approximately pH 6.2. Of the CDP-diglycerides tested, the hydrolytic rate is highest for dioleoyl CDP-diglyceride. Activity is inhibited by all divalent cations in whole mitochondria, except in the presence of phosphatidylglycerol in which CMP release is stimulated by Co2+ and Mn2+. The increase in CMP release in the presence of Co2+ or Mn2+ can be accounted for entirely by diphosphatidylglycerol synthase activity which requires either cation. This effect is not seen in Triton X-100 solubilized mitochondrial membranes which contain no diphosphatidylglycerol synthase. All preparations are inhibited by mixed phospholipids (Asolectin) and by Trixon X-100 which abolishes activity completely at concentrations greater than 0.5% (w/v). CDP-diglyceride hydrolase is also inhibited by AMP (46%) and by cytidine nucleotides (CTP greater than CDP greater than cytidine) except CMP. A role for this activity in the regulation of biosynthesis of mitochondrial polyglycerophosphatides is proposed.

Regulation of phosphatidylcholine synthesis and the activity of CTP-cholinephosphate cytidylytransferase in myoblasts by 12-O-tetradecanoylphorbol-13-acetate.

The tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) increases rate of incorporation of 32P into phosphatidylcholine and phosphatidylethanolamine about twofold in a line of rat skeletal myoblasts (L6). This effect is specific for TPA and is not caused by the nontumor-promoting analogue 4 alpha-phorbol didecanoate (alpha-PDD). Cycloheximide and actinomycin D have no effect on the TPA-dependent increase in labelling. There is no significant difference in the rate of disappearance of [14C]glycerol from phospholipids in cells exposed to TPA. Several enzymes of phospholipid synthesis in myoblasts exposed to TPA are unaltered in activity, except CTP:cholinephosphate cytidylyltransferase (EC 2.7.7.15) whose activity is enhanced 1.5- to 2-fold compared with cells exposed to alpha-PDD. Exposure of cells for only 15 min to a concentration of 1 X 10(-6)M TPA is sufficient to cause maximum activation of the enzyme. The cytidylyltransferase activity is associated largely with the particulate fraction in the cell-free extracts of myoblasts. Following gel filtration on Bio-Gel A-1.5m, the enzyme activity appears in the void volume and smaller molecular weight forms are not discernible. The kinetic parameters of cytidylyltransferase in preparations from treated cells are unaltered except for an increased Vmax. The apparent Km value for CTP for the enzyme from treated and untreated cells is about 0.9mM. Both enzyme preparations are weakly inhibited by lysophosphatidylcholine, but are unaffected by several other phospholipids.

A sensitive, continuous spectrophotometric method for assaying alpha-glycerophosphate dehydrogenase: activation by menadione.

By use of a more sensitive method than standard assays, it is demonstrated that alpha-glycerophosphate dehydrogenase activity can be monitored continuously in rat liver mitochondria; maximum activity was obtained by sonication in the presence of Triton X-100. Vitamin K3 (menadione) seems to enhance the activity of the enzyme. The assay in the presence of menadione is linear over a much greater mitochondrial concentration (up to 250 micrograms of protein in the reaction mixture).

Effects of hormones on the maintenance and mitochondrial functions of rat hepatocytes cultured in serum-free medium.

The survival and morphology of rat hepatocytes were examined in primary cell cultures that were maintained in serum-free medium supplemented with different hormones. Insulin and dexamethasone improved survival and maintenance of normal epithelial-shaped cells, although triiodothyronine did not alter cell survival or morphology when added to the medium alone or with other hormones. The level of mitochondrial alpha-glycerophosphate dehydrogenase and cytochromes a(+a3), b and c, but not c1, were increased in cultured hepatocytes by triiodothyronine. Although induction of alpha-glycerophosphate dehydrogenase did not require serum or growth hormone, the triiodothyronine effect was potentiated by insulin plus dexamethasone. This permissive effect of dexamethasone parallels its known glucocorticoid action of increasing the activity of the gluconeogenic enzyme tyrosine aminotransferase in the cultured hepatocytes. Glucagon, which also elevated tyrosine aminotransferase activity, had no effect upon the induction of alpha-glycerophosphate dehydrogenase by triiodothyronine. Since the culture medium was completely defined and triiodothyronine did not alter survival or morphology of the hepatocytes, the effects upon mitochondrial functions are direct cellular actions of the thyroid hormone.

Concerning the coidentity of phosphatidic acid phosphohydrolase and phosphatidylglycerophosphate phosphohydrolase in rat lung lamellar bodies.

The properties of the phosphatidylglycerophosphate phosphohydrolase activity in lamellar bodies from rat lung have been compared with the properties of the activities responsible for the degradation of aqueously-dispersed phosphatidic acid (PAaq) and membrane-bound phosphatidic acid (PAmb). Subcellular fractionation studies revealed that the phosphatidylglycerophosphate phosphohydrolase activity and the PAaq-dependent phosphatidic acid phosphohydrolase activity were predominantly associated with the mitochondrial and microsomal fractions, while the PAmb-dependent phosphohydrolase activity was associated with the cytosol. Although the lamellar body fraction contained less than 1% of the total activity, the phosphatidic acid phosphohydrolase activities associated with this fraction could not be explained by contamination with microsomes or cytosol. The three activities exhibited similar heat inactivation profiles at 55 degree C. However, differences in the responses of these activities to the presence of iodoacetate, p-chloromercuriphenyl sulphonate, mercaptoethanol, mercaptoethanol plus MgCl2, and Triton X-100 indicated that the enzymes responsible for these activities may be distinct. Furthermore, addition of up to a ten-fold greater amount of PAaq did not seriously affect the hydrolysis of phosphatidylglycerophosphate. These results indicate that the phosphatidic acid phosphohydrolase and phosphatidylglycerophosphate phosphohydrolase activities in rat lung lamellar bodies are not necessarily catalyzed by the same protein.

Regulation of malic enzyme and mitochondrial alpha-glycerophosphate dehydrogenase by thyroid hormones, insulin, and glucocorticoids in cultured hepatocytes.

Hepatocytes isolated from normal adult rats were cultured under serum-free conditions. Induction of mitochondrial alpha-glycerophosphate dehydrogenase (glycerol 3-phosphate dehydrogenase) (EC 1.1.99.5; sn-glycerol-3-phosphate: (acceptor) oxidoreductase) and cytosolic malic enzyme (EC 1.1.1.40; L-malate-NADP+ oxidoreductase (decarboxylating)) by 3,3'-5-triiodo-L-thyronine (triiodothyronine) in the culture medium follows the same time course as the in vivo response to thyroid hormones. The addition of 1 microM cycloheximide blocks the triiodothyronine response. Thyroxine is also capable of stimulating the activities of both enzymes. Although increases in alpha-glycerophosphate dehydrogenase and malic enzyme activities are observed when triiodothyronine is added to the culture medium for 3 days (62% and 36%, respectively), in the presence of insulin and cortisol the response is significantly greater. Dexamethasone is more potent than cortisol in increasing triiodothyronine action. In the presence of bovine serum albumin, to prevent metabolism of triiodothyronine, hepatocytes show increased enzyme activity at concentrations as low as 10(-10) M triiodothyronine.

Partial purification of diphosphatidylglycerol synthetase from liver mitochondrial membranes.

The enzyme responsible for the conversion of phosphatidylglycerol to diphosphatidylglycerol (cardiolipin) in the presence of cytidine diphosphate diacylglycerol is firmly associated with mitochondrial membranes and is not extracted with hypotonic or hypertonic media or with nonionic detergents. Some solubilization was obtained with bile salt solutions, but the zwitter-ionic detergent. Miranol H2M, was most effective in extracting the enzyme. The Miranol extracts were fractionated by column chromatography on Bio-Gel A-1.5 m. The solubilized enzyme is considerably more active in converting unsaturated than saturated phosphatidyl-glycerols, but shows little preference for the cytidine diphosphate diacylglycerols with different fatty acyl substituents. There is an absolute dependence upon divalent cations with the order of effectiveness: Co2+ much greater than Mn2+ greater than Mg2+. In the presence of optimal levels of Co2+ other divalent cations are inhibitory with the order of inhibition: Cd2+ greater than Zn2+ greater than Ca2+ greater than Ba2+ greater than Cu2+ greater than Hg2+ greater than Ni2+. The solubilized enzyme exhibited no requirement for added phospholipids and several phospholipids inhibited the reaction in the order: diphosphatidylglycerol greater than phosphatidylethanolamine greater than phosphatidylserine greater than phosphatidylinositol.

Partial purification and properties of mammalian phosphatidylglycerophosphatase.

The phosphatidylglycerophosphatase (EC 3.1.3.27) activity of rat liver mitochondria was investigated by assaying the conversion of 14C-labelled phosphatidylglycerophosphate to phosphatidylglycerol. The activity was associated with a mitochondrial membrane fraction and could not be released into solution employing techniques applicable to a peripheral membrane protein. The enzyme was partially purified by sonication, pH 5.0 precipitation, and gel filtration. Various ionic and nonionic detergents as well as numerous divalent cations inhibited the phosphatase. The enzyme displayed a high affinity for phosphatidylglycerophosphate.