Altered subcellular and submitochondrial localization of CTP:phosphatidate cytidylyltransferase in the Morris 7777 hepatoma.

The subcellular and submitochondrial localization of CTP:phosphatidate cytidylyltransferase is altered in the Morris 7777 hepatoma. Mitochondria in this poorly differentiated tumor are the principal sites of CDP-diacylglycerol synthesis, in contrast to normal rat liver where the endoplasmic reticulum is most active. This enzyme activity was increased 17-fold in the outer mitochondrial membrane, and a 22% increase was noted in the inner mitochondrial membrane of the 7777 hepatoma as compared with the corresponding fractions from normal rat liver. Increased mitochondrial CTP:phosphatidate cytidylyltransferase was present in six other Morris hepatomas, but it was not found in fetal rat liver mitochondria, suggesting that rapid growth alone is not responsible for the difference. Evidence is presented which indicates that mitochondrial lipid degradation is similar in normal liver and the 7777 hepatoma, in vitro. The increased activity of CTP: phosphatidate cytidylytransferase is thought to be responsible in part for the moderately increased diphosphatidylglycerol content of 7777 hepatoma mitochondria.     

Phosphatidylserine biosynthesis in mitochondria from the Morris 7777 hepatoma.

Mitochondria from the 7777 hepatoma incorporate substantial amounts of l-[U-(14)C]serine into phospholipid by a Ca(2+)-dependent base-exchange reaction. This reaction is virtually absent in normal liver mitochondria. The finding cannot be attributed to microsomal contamination of the sucrose gradient-purified 7777 hepatoma mitochondria. The reaction is also absent in the rapid-growth controls, fetal rat liver and regenerating rat liver. [(14)C]Serine incorporation into 7777 hepatoma mitochondrial phospholipid by base-exchange requires Ca(2+) and is inhibited by EDTA. Ca(2+) cannot be replaced by Mg(2+), Mn(2+), or Co(2+). The reaction is inhibited by a sulfhydryl reagent and by detergents and is abolished by heating to 70 degrees C for 10 min. Product analysis indicates that phosphatidylserine and its decarboxylation product, phosphatidylethanolamine, are formed by 7777 hepatoma mitochondria, while phosphatidylserine is the sole product with microsomes. The conversion of phosphatidylserine into phosphatidylethanolamine in 7777 hepatoma mitochondria is inhibited by KCN. This study provides further evidence of abnormal mitochondrial biogenesis in the 7777 hepatoma. Our earlier study indicated a greatly increased mitochondrial activity of CTP:phosphatidate cytidylyltransferase in the 7777 hepatoma (Hostetler, Zenner, and Morris. 1978. J. Lipid Res. 19: 553-560). The presence in mitochondria of these two enzymes, which are primarily microsomal in normal liver, does not appear to be due to rapid growth alone, since their intracellular distribution was not altered in fetal or regenerating rat liver.-Hostetler, K. Y., B. D. Zenner, and H. P. Morris. Phosphatidylserine biosynthesis in mitochondria from the Morris 7777 hepatoma.     

The synthesis of sphingomyelin in the Morris hepatomas 7777 and 5123D is restricted to the plasma membrane

The site of sphingomyelin synthesis in the rapidly growing Morris hepatomas 7777 and 5123D was determined by incubating plasma membrane, mitochondrial and microsomal membrane fractions with vesicles of phosphatidyl[methyl-14C]choline in the presence of phosphatidylcholine transfer protein. In agreement with a previous study on rat liver (Voelker, D.R. and Kennedy, E.P., 1982, Biochemistry 21, 2753-2759) we have demonstrated that sphingomyelin synthesis in these hepatomas is restricted to the plasma membrane. The greatly elevated sphingomyelin content of mitochondria and microsomes (Hostetler, K.Y., Zenner, B.D. and Morris, H.P., 1979, Cancer Res. 39, 2978-2983) suggests that rapidly growing hepatomas, in contrast to liver, have an effective mechanism of intracellular sphingomyelin transfer.     

Phospholipid exchange between mitochondria and microsomes of rat hepatoma 27]

The phospholipid exchange in vitro between mitochondria and microsomes from rat liver and rat hepatoma 27 was investigated. On incubation with a postmicrosomal protein fraction the phospholipid exchange between subcellular fractions of the tumor was found to proceed much faster and less specific than between mitochondria and microsomes from normal liver. These results indicate that the earlier demonstrated lipid dedifferentiation of tumor cell membranes may be connected with an altered transmembrane phospholipid exchange in vivo.     

Ethanol metabolism by a transplantable hepatocellular carcinoma. Role of microsomes and mitochondria.

1. Ethanol metabolism in slices or homogenates of transplantable hepatocellular carcinoma HC-252 (HC-252) was 50 to 60% of the rate found in host liver slices or homogenates when they were expressed per gram of tissue wet weight and 70 to 80% of the liver when the rates were expressed per milligram of tissue protein. At 10 mM ethanol, the activities of alcohol dehydrogenase in tumor and liver supernatants were comparable. 2. Tumor microsomes did not oxidize ethanol in the presence of a NADPH-generating system, indicating the absence of the microsomal ethanol-oxidizing system and catalase-mediated peroxidation of ethanol. The HC-252 microsomes were contaminated with catalase, and acetaldehyde production occurred in the presence of a H2O2-generating system (xanthine oxidase). The virtual absence of ethanol oxidation and drug metabolism (aminopyrine demethylase and aniline hydroxylase) in HC-252 microsomes may be due to the low activities of NADPH-cytochrome c reductase, NADPH oxidase, and NADPH-dependent oxygen uptake. 3. Microsomal oxidation of ethanol was present in Morris hepatoma 5123C, a well-differentiated tumor of intermediate growth rate, while activity was negligible in microsomes from Morris hepatoma 7288CTC, a less differentiated tumor. Microsomal NADPH oxidase was present in the well differentiated tumor 5123C but was lacking in the less differentiated tumor 7288CTC. Several microsomal, mitochondrial, and cytosolic properties of HC-252 are similar to those of Morris hepatoma 7288CTC but differ from those of the more differentiated 5123C tumor and normal liver. 4. The content of mitochondrial protein in HC-252 was only 25% that of liver, and oxygen consumption per gram of tumor was only 28% that of the liver. When corrected for the mitochondrial protein content, oxygen uptake in tumor HC-252 and liver homogenates was comparable. Isolated tumor and liver mitochondria displayed comparable State 4 and 3 rates of oxygen consumption with succinate and glutamate as substrates. The activities of the reconstituted malate-aspartate and alpha-glycerophosphate shuttles were only slightly lower in isolated HC-252 mitochondria compared to liver mitochondria, when shuttles were reconstituted with purified enzymes. 5. Antimycin inhibited alcohol metabolism,and pyruvate stimulated alcohol metabolism, much less in tumor slices than in liver slices, suggesting the presence of an augmented mitochondria-independent, cytosolic mechanism for oxidizing reducing equivalents in the tumor. These factors suggest that oxidation of NADH is the limiting factor in ethanol metabolism. Whereas, in the liver mitochondrial reoxidation is predominant, in HC-252, cytosolic reoxidation of NADH also plays a major role.     

Adenine nucleotide translocase activity and sensitivity to inhibitors in hepatomas. Comparison of the ADP/ATP carrier in mitochondria and in a purified reconstituted liposome system

Adenine nucleotide uptake was found to be lower in mitochondria from hepatoma 7777, 7800, and 9618A than in the host livers. Moreover, in the fast-growing hepatoma 7777 the sensitivity of the adenine nucleotide translocase to inhibition by carboxyatractylate and bongkrekic acid was considerably decreased. Purification of the ADP/ATP carrier from hepatoma 7777 mitochondria and its reconstitution into an artificial liposome system reversed the abnormal kinetics in that the adenine nucleotide uptake and response to inhibitors were identical in proteoliposome preparations from host liver and tumor mitochondria. Analysis of the lipids of the hepatoma inner mitochondrial membrane indicated considerable differences from normal in the levels of phospholipids and cholesterol. Most striking was the increase in cholesterol and sphingomyelin of the hepatoma 7777 inner membrane. An artificial liposome system containing cholesterol in addition to the standard phospholipids could produce alterations in kinetics of the purified ADP/ATP carrier from heart mitochondria similar to those seen in the hepatoma 7777. In general, these results support the suggestion that alterations in the lipid environment of the inner mitochondrial membrane rather than intrinsic changes in the carrier protein itself produce the aberrant observations of adenine nucleotide translocase activity in hepatoma mitochondria.     

Fatty acid composition of phospholipids in mitochondria and microsomes during diethylnitrosamine carcinogenesis in rat liver

Changes in lipid composition and function of subcellular organelles have been described in transplanted and primary tumours. We examine here the fatty acid composition of individual phospholipids (PL) in hyperplastic nodules and primary hepatoma induced by diethylnitrosamine (DEN), compared to that of normal liver and of transplantable Yoshida AH-130 hepatoma. Phosphatidylcholine and phosphatidylethanolamine fatty acid composition in mitochondria and microsomes from primary hepatoma were markedly different from normal liver; C18:0/C18:1 ratio was lower and the ratio between monosaturated and polyunsaturated fatty acids was higher. Linoleic acid content of mitochondrial cardiolipin, usually very high in normal rat liver, was notably lower in primary hepatoma. Cholesterol/phospholipid ratio in both microsomes and mitochondria from DEN-induced hepatoma was higher than in normal liver. Hyperplastic nodules showed no changes in cholesterol content whereas modifications in fatty acid composition were already observable. These modifications of membrane structure may be related to the functional changes found in nodular cells. Changes in fatty acid composition of membrane phospholipids, occurring in both primary hepatoma and preneoplastic nodules, might be one of the causes for decreased rate of lipid peroxidation peculiar to these tissues.     

Phospholipid-transfer proteins in human liver and primary liver carcinoma

Investigations have been carried out on phospholipid-transfer activity of the cytosol and the phospholipid composition of subcellular membranes from human liver and primary liver carcinoma. In both human liver and primary liver carcinoma cytosolic fractions, the transfer activity for phosphatidylcholine (PC), phosphatidylethanolamine (PE) and sphingomyelin has been observed for the first time. The transfer rate of PC and PE in normal human liver was almost equal, whereas sphingomyelin-transfer activity was much slower. In carcinoma cells, the transfer activity for PE and PC was significantly enhanced, while sphingomyelin transfer remained unchanged. Comparative investigations with HepG2 cultured cells have revealed a high PE-transfer activity in this cell line. Parallel with the phospholipid-transfer activity modifications in neoplasic cells, changes in the phospholipid composition of microsomes and mitochondria have been observed. The content of PC and PE in hepatocarcinoma cells was decreased in microsomes, while in the mitochondria it was increased. The possible role of the phospholipid-transfer proteins in the maintenance of membrane composition and structure is discussed.     

Investigation of lipid-exchange lipoproteins from rat liver and hepatoma 27]

A phospholipid exchange lipoprotein from the postmicrosomal supernatant of rat hepatoma 27, which stimulated in vitro the exchange of sphingomyelin between mitochondria and microsomes, was found. Sphingomyelin is incorporated into the mitochondria under incubation of this complex with rat liver mitochondria (in which sphingomyelin is absent) an microsomes. Under the same conditions the phospholipid exchange lipoproteins of rat liver do not transfer sphingomyelin form microsomes to mitochrondria.     

Effect of thyroid dysfunction upon phospholipid composition and CDP-choline incorporation in mitochondria and microsomal fraction isolated from liver and brain of suckling rats

The phospholipid composition and the in vitro incorporation of radioactive CDP-choline into phosphatidylcholine was studied in mitochondria and microsomal fraction obtained from liver and brain of 20 day old hyperthyroid or hypothyroid rats. The chemical composition of the subcellular membranes isolated from brain differed markedly in both conditions. In hyperthyroidism the microsomal fraction was slightly affected while the mitochondria were also affected, but not as severely as in hypothyroidism, in which the microsomal fraction showed no alterations.The incorporation of the radioactive precursor into brain mitochondria isolated from hyperthyroid rats was markedly decreased, while no changes were observed in microsomes. However, incorporation into brain microsomal fraction obtained from hypothyroid rats was increased, while no changes were observed in mitochondria. Similar results were obtained in the studies performed with liver subcellular membranes from hyperthyroid animals while no changes were found in those from hypothyroid rats.Our results indicate that both experimental conditions affect in a different way the structure and function of brain mitochondria and microsomal fractions. They also give further support to our hypothesis that mitochondria have a certain degree of autonomy for the synthesis of phosphatidylcholine.Abbreviations used PS+PI phosphatidylserine+phosphatidylinositol - Sph sphingomyelin - PC phosphatidylcholine - PE phosphatidylethanolamine     

Lipids of dystrophic and normal mouse muscle: whole tissue and particulate fractions

Myofibrillar, mitochondrial, and microsomal fractions were prepared from normal and dystrophic mouse limb muscle by differential centrifugation and analyzed for phospholipids and cholesterol. Fatty acids and aldehydes of neutral lipids and of phospholipids from whole muscle and particulate fractions were also determined. Normal microsomes contained more lecithin and less total ethanolamine phospholipids and cardiolipin than mitochondria. The myofibrils had an intermediate phospholipid composition, but their cholesterol-phospholipid ratio was smaller than that of the other two fractions. Except for an increased percentage of phosphatidalethanolamine in the dystrophic mitochondria, only the composition of the dystrophic microsomes differed from normal by containing less lecithin but more total ethanolamine phospholipid, phosphatidalethanolamine, sphingomyelin, and cholesterol. No significant differences were found in the fatty acid composition of neutral lipid extracts from normal and dystrophic preparations, but there was a significant decrease in the percentage of 22:6 in phospholipids from both dystrophic whole muscle and microsomes (-25% and -37%, respectively), whereas the 20:4 content was unaltered. By contrast, the percentages of 18:0 and total fatty aldehyde increased significantly. Phospholipid extracts from all dystrophic samples showed a significant decrease in 16:0 and an increase in 18:1 as compared with the normal.     

Phospholipid composition and fatty acid patterns of isolated phospholipids from rabbit reticulocyte mitochondria

The phospholipid composition and fatty acid patterns of individual phospholipid classes were determined in mitochondria from rabbit reticulocytes. Compared to mitochondria from rat liver reticulocyte, mitochondria exhibit about twice the amount of phospholipids. The phospholipid pattern of reticulocyte mitochondria (phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol and cardiolipin) is comparable with other mitochondrial species. Mitochondrial fractions from reticulocytes are characterized, however, by an additional content of sphingomyelin. This sphingomyelin differs in its fatty acid composition from the sphingomyelin of the plasma membrane. The fatty acid patterns of all other phospholipids essentially correspond to those of mitochondria from other sources and to those of plasma membranes as well.     

Oxidative phosphorylation properties of mitochondria isolated from transplanted hepatoma.

Mitochondria were isolated from Morris hepatomas with rapid (types 3683, 7777, and 3924A) and intermediate (types 5123D and 7800) growth rates, using proteolytic digestion of minced tumor tissue to release the particles. Mitochondria isolated by the same procedure from rat liver were employed as controls. All the hepatoma mitochondria were capable of coupled respiration with normal phosphorylation yields (ADP/O) and respiratory control ratios ranging from 2 to considerably more than 10. Particles from hepatomas 7777 and 7800 exhibited properties closest to liver mitochondria, while those from hepatomas 3683 and 3924A showed the greatest difference. All the hepatoma mitochondria were capable of oxidizing succinate, 3-hydroxybutyrate and monoamines. However, the oxidation rates of the latter two substrates by mitochondria from hepatomas 3683 and 3924A were only a fraction of the control rates. These differences appeared to be due, at least in part, to the structural instability of the isolated hepatoma mitochondria. In contrast to the reports of others, all hepatoma mitochondria exhibited considerable stimulation of ATPase activity by uncouplers. Maximal stimulation of ATPase activity by representatives of three classes of uncouplers was in all instances comparable to the values obtained for rat liver mitochondria.     

Oxidative phosphorylation properties of mitochondria isolated from transplanted hepatoma

Mitochondria were isolated from Morris hepatomas with rapid (types 3683, 7777, and 3924A) and intermediate (types 5123D and 7800) growth rates, using proteolytic digestion of minced tumor tissue to release the particles. Mitochondria isolated by the same procedure from rat liver were employed as controls. All the hepatoma mitochondria were capable of coupled respiration with normal phosphorylation yields (ADP/O) and respiratory control ratios ranging from 2 to considerably more than 10. Particles from hepatomas 7777 and 7800 exhibited properties closest to liver mitochondria, while those from hepatomas 3683 and 3924A showed the greatest difference. All the hepatoma mitochondria were capable of oxidizing succinate, 3-hydroxybutyrate and monoamines. However, the oxidation rates of the latter two substrates by mitochondria from hepatomas 3683 and 3924A were only a fraction of the control rates. These differences appeared to be due, at least in part, to the structural instability of the isolated hepatoma mitochondria. In contrast to the reports of others, all hepatoma mitochondria exhibited considerable stimulation of ATPase activity by uncouplers. Maximal stimulation of ATPase activity by representatives of three classes of uncouplers was in all instances comparable to the values obtained for rat liver mitochondria.     

Arylamidases of rat liver and chemically induced hepatomas 1. Subcellular distribution of l-leucine 2. Naphthylamidase-active antigens

The subcellular distribution of arylamidase-active antigens in rat liver and in two chemically induced hepatomas (D23 and D33) was investigated. Soluble antigens or detergent-solubilized membrane antigens from isolated subcellular fractions were tested in fused rocket immunoelectrophoresis against antisera prepared against each of the fractions. The arylamidase active antigens were identified by means of a zymogram technique using l-leucine 2-naphthylamide as substrate.Two arylamidase-active antigens were shown to be shared between plasma membranes, microsomes, lysosomal membranes and lysosomal content of the hepatocytes. One of these occurred predominantly in the plasma membranes (the plasma membrane arylamidase) while the other was preferentially found in the lysosomal content (the lysosomal content arylamidase). Also a third arylamidase-active antigen was identified and was shown to be restricted to the microsomes and the lysosomal membranes (the microsomal/lysosomal arylamidase).The rat liver plasma membrane arylamidase-active antigen was also present in plasma membrane, microsomal an cell-sap fractions of both the hepatomas. However, in the hepatomas this antigen occurred predominantly in the microsomal fraction. The plasma membrane arylamidase was the only arylamidase-active antigen found in the hepatoma D33 while the plasma membrane and microsomal fractions of hepatoma D23 also contained another antigen with this activity. Neither the lysosomal content arylamidase nor the microsomal/lysosomal arylamidase could be detected in any of the hepatoma fractions.     

Arylamidases of rat liver and chemically induced hepatomas. 1. Subcellular distribution of L-leucine. 2. Naphthylamidase-active antigens.

The subcellular distribution of arylamidase-active antigens in rat liver and in two chemically induced hepatomas (D23 and D33) was investigated. Soluble antigens or detergent-solubilized membrane antigens from isolated subcellular fractions were tested in fused rocket immunoelectrophoresis against antisera prepared against each of the fractions. The arylamidase active antigens were identified by means of a zymogram technique using L-leucine 2-naphthylamide as substrate. Two arylamidase-active antigens were shown to be shared between plasma membranes, microsomes, lysosomal membranes and lysosomal content of the hepatocytes. One of these occurred predominantly in the plasma membranes (the plasma membrane arylamidase) while the other was preferentially found in the lysosomal content (the lysosomal content arylamidase). Also a third arylamidase-active antigen was identified and was shown to be restricted to the microsomes and the lysosomal membranes (the microsomal/lysosomal arylamidase). The rat liver plasma membrane arylamidase-active antigen was also present in plasma membrane, microsomal and cell-sap fractions of both the hepatomas. However, in the hepatomas this antigen occurred predominantly in the microsomal fraction. The plasma membrane arylamidase was the only arylamidase-active antigen found in the hepatoma D33 while the plasma membrane and microsomal fractions of hepatoma D23 also contained another antigen with this activity. Neither the lysosomal content arylamidase nor the microsomal/lysosomal arylamidase could be detected in any of the hepatoma fractions.     

Effect of Morris 7777 hepatoma on microsomal glucose-6-phosphatase latent activity

The change in the activity of several hepatic enzymes during hepatocarcinogenesis suggests a pattern of dedifferentiation. This category of enzymes includes glucose-6-phosphatase and gamma-glutamyltranspeptidase (GGT). A detailed kinetic analysis of microsomal glucose-6-phosphatase activity revealed that both the translocase and phosphohydrolase activities were markedly reduced in Morris 7777 hepatoma transplanted in male Buffalo rats. In addition, the activity of the translocase component increased 2.4-fold, while the phosphohydrolase activity decreased 1.6-fold in the liver of tumor-bearing animals. GGT activity in the host liver was not effected by the presence of the tumor. These results suggest differences in the effect of Morris 7777 hepatoma on: the phosphohydrolase and translocase activities of microsomal glucose-6-phosphatase and the sensitivity of glucose-6-phosphatase and GGT activities in the host liver.     

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.     

Changes in the phospholipid catabolism of mitochondria and microsomes during the development of rat liver.

The lipolytic activities of mitochondrial and microsomal fractions ('microsomes') isolated from foetal, suckling and adult rat liver were compared. The catabolism of endogenous phospholipids was followed by measuring the loss of phospholipids and the appearance of non-esterified fatty acids and lysophosphatides. The rate of mitochondrial phospholipid catabolism does not change significantly during development, but the rate of lipolysis of microsomal phospholipids increases 3-fold during development. Balance studies showed that, in mitochondria and microsomes of foetal, suckling and adult rat liver, fatty acid formation is greatly in excess of the fatty acids that can be accounted for by measuring phospholipid disappearance and lysophosphatide appearance. The hypothesis that this excess fatty acid formation resulted from the lipolysis of mitochondrial and microsomal triacylglycerols were tested and confirmed by preliminary experiments. Mitochondria and microsomes isolated from all developmental ages investigated had phospholipases with A1 and A2 activities. The degree of unsaturation of the fatty acids derived from the phospholipids of mitochondria did not vary significantly during development.