W.C. McMurray

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.

Mitochondrial biogenesis in cultured animal cells. I. Effect of chloramphenicol on morphology and mitochondrial respiratory enzymes.

The effects of chloramphenicol on the morphology and respiratory enzymes of BHK-21 cells in spinner culture have been examined with time. Cells treated with chloramphenicol double twice before growth ceases; these cells have increased size as measured by several techniques. Mitochondria are enlarged and appear to degenerate with prolonged treatment. Cytochrome c oxidase and succinate cytochrome c reductase activities are reduced while there is no decrease in the activities of monoamine oxidase, glutamate dehydrogenase or NADPH-cytochrome c reductase. Cytochromes aa3 and b disappear on treatment while cytochromes c + c1 appears to be unaffected. All these effects are reversible if chloramphenicol is removed within a limited period of time.

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.

Insulin and cortisol increase the response of rat hepatocytes in primary culture to 3,3′,5 triiodothyronine

Summary Primary cultures of adult rat hepatocytes respond to 3,3′,5 triiodothyronine added to the culture media. The specific activities of mitochondrial α-glycerophosphate dehydrogenase and cytosolic malic enzyme are increased. Although the addition of 3,3′,5 triiodothyronine causes a 30–70% increase in enzyme activity, the greatest response (150–200% increase) is obtained when both insulin and cortisol are present. Insulin and/or cortisol do not cause any marked increase in either enzyme in the absence of 3,3′,5 triiodothyronine.

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.

Mitochondrial biogenesis in cultured mammalian cells. III. Synthesis of mitochondrial phospholipids by subcellular fractions isolated from normal and chloramphenicol-treated BHK-21 cells.

The capacity of subcellular fractions isolated from chloramphenicol-treated BHK-21 cells to synthesize various mitochondrial phospholipids in vitro and examined. Both mitochondria and microsomes showed the capacity to acylate sn-glycerol 3-phosphate and dihydroxyacetone phosphate to lysophosphatidic acid and acyldihydroxyacetone phosphate and subsequently to phosphatidic acid. Both processes are inhibited in mitochondria from chloramphenicol-treated cells. The synthesis of CDPdiacylglycerol in mitochondria or microsomes, and the synthesis of phosphatidylinositol and of phosphatidylcholine in microsomes were stimulated in treated cells. A slight stimulation was also observed in the synthesis of phosphatidylglycerol and diphosphatidylglycerol when the labelled precursor was sn-glycerol 3-phosphate in treated cells, although the process was inhibited with labelled glycerol as the precursor. Conversion of phosphatidylglycerol phosphate to phosphatidylglycerol by mitochondria was rate limiting unless the post-microsomal supernatant fraction was added. These results are discussed in regard to the observed inhibition of phospholipid synthesis in BHK-21 cells in culture by chloramphenicol.

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.

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.

Mitochondrial biogenesis in cultured mammalian cells. II. Mitochondrial protein and phospholipid synthesis in chloramphenicol-treated BHK-21 cells.

The effect of growth of BHK-21 cells in chloramphenicol on the synthesis of cellular proteins and phospholipids has been examined. The incorporation of leucine into total cellular proteins, or into the proteins of specific subcellular fractions are not significantly reduced by cell culture in the presence of chloramphenicol. In cells treated with cycloheximide, a small amount of chloramphenicol-sensitive labelling of protein was detected within the first hour of exposure to the drug. Chloramphenicol inhibits the incorporation of delta-amino-levulinic acid into hemoproteins, only if it is present during both the 48-h culturing and 4-h labelling period. De novo synthesis of cellular lipids as measured by pulse labelling with 32Pi or [3H]glycerol, is decreased in chloramphenicol-treated cells. This decrease is observed in all sub-cellular fractions, although the mitochondrial fraction is most affected. All phospholipids are affected, with diphosphatidylglycerol labelling reduced to the greatest extent. Although fatty acid synthesis is inhibited, the labelling of diphosphatidylglycerol with fatty acids is stimulated on chloramphenicol treatment.