digitonin and Liver-Neoplasms

Previous studies have indicated that the early steps in the isoprenoid/cholesterol biosynthetic pathway occur in peroxisomes. However, the role of peroxisomes in cholesterol biosynthesis has recently been questioned in several reports concluding that three of the peroxisomal cholesterol biosynthetic enzymes, namely mevalonate kinase, phosphomevalonate kinase, and mevalonate diphosphate decarboxylase, do not localize to peroxisomes in human cells even though they contain consensus peroxisomal targeting signals. We re-investigated the subcellular localization of the cholesterol biosynthetic enzymes of the pre-squalene segment in human cells by using new stable isotopic techniques and data computations with isotopomer spectral analysis, in combination with immunofluorescence and cell permeabilization techniques. Our present findings clearly show and confirm previous studies that the pre-squalene segment of the cholesterol biosynthetic pathway is localized to peroxisomes. In addition, our data are consistent with the hypothesis that acetyl-CoA derived from peroxisomal beta-oxidation of very long-chain fatty acids and medium-chain dicarboxylic acids is preferentially channeled to cholesterol synthesis inside the peroxisomes without mixing with the cytosolic acetyl-CoA pool.

Topics: Acetyl-CoA C-Acetyltransferase; Animals; Carbon-Carbon Double Bond Isomerases; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Membrane Permeability; CHO Cells; Cholesterol; Cricetinae; Cricetulus; Digitonin; Fatty Acids; Fluorescent Antibody Technique, Indirect; Geranyltranstransferase; Hemiterpenes; Humans; Hydroxymethylglutaryl-CoA Synthase; Immunohistochemistry; Indicators and Reagents; Liver Neoplasms; Peroxisomes; Phosphotransferases (Alcohol Group Acceptor); Phosphotransferases (Phosphate Group Acceptor); Squalene; Terpenes

The number of proteins known to be associated with lipid droplets (LDs) is increasing. However, the reported distribution of a given protein in the LDs was, in some cases, found not reproduced by other groups. We report here that the choice of the fixation and permeabilization method is important in order to observe LD proteins using immunofluorescence microscopy. Formaldehyde fixation followed by treatment with Triton X-100, one of the most frequently used protocols for the immunolabeling of cultured cells, was not appropriate to label adipocyte differentiation-related protein (ADRP), TIP47, or Rab18 in LDs. Formaldehyde fixation followed by treatment with digitonin or saponin, allowed the visualization of all these proteins in LDs. When cells were fixed with glutaraldehyde, permeabilization by Triton X-100 could also be used for ADRP. These observations suggest that LD proteins are likely to be solubilized by some detergents, and strong cross-linkage to the surrounding protein matrix or mild permeabilization is necessary for their retention on the LD surface.

Topics: Adipose Tissue; Carcinoma, Hepatocellular; Cell Line, Tumor; Digitonin; DNA-Binding Proteins; Humans; Immunohistochemistry; Intracellular Signaling Peptides and Proteins; Lipid Metabolism; Liver Neoplasms; Membrane Proteins; Perilipin-2; Perilipin-3; Pregnancy Proteins; rab GTP-Binding Proteins; Saponins; Solubility; Tissue Fixation; Vesicular Transport Proteins

The question of whether and to what extent the in vivo cytochrome c oxidase (COX) capacity in mammalian cells exceeds that required to support respiration is still unresolved. In the present work, to address this question, a newly developed approach for measuring the rate of COX activity, either as an isolated step or as a respiratory chain-integrated step, has been applied to a variety of human cell types, including several tumor-derived semidifferentiated cell lines, as well as specialized cells removed from the organism. KCN titration assays, carried out on intact uncoupled cells, have clearly shown that the COX capacity is in low excess (16-40%) with respect to that required to support the endogenous respiration rate. Furthermore, measurements of O2 consumption rate supported by 0.4 mM tetramethyl-p-phenylenediamine in antimycin-inhibited uncoupled intact cells have given results that are fully consistent with those obtained in the KCN titration experiments. Similarly, KCN titration assays on digitonin-permeabilized cells have revealed a COX capacity that is nearly limiting (7-22% excess) for ADP + glutamate/malate-dependent respiration. The present observations, therefore, substantiate the conclusion that the in vivo control of respiration by COX is much tighter than has been generally assumed on the basis of experiments carried out on isolated mitochondria. This conclusion has important implications for understanding the role of physiological or pathological factors in affecting the COX threshold.

Topics: Adenosine Diphosphate; Carcinoma, Hepatocellular; Cell Differentiation; Cell Line; Cell Membrane Permeability; Digitonin; Electron Transport Complex IV; Glutamic Acid; Humans; Kinetics; Liver Neoplasms; Lung Neoplasms; Malates; Mitochondria; Multiple Myeloma; Neuroblastoma; Osteosarcoma; Oxygen Consumption; Potassium Cyanide; Tetramethylphenylenediamine; Tumor Cells, Cultured

We examined the effect of fatty acids on phosphatidylcholine synthesis and cytidylyltransferase activity in Hep G2 cells. Treatment of Hep G2 cells with oleic acid caused an increase in the incorporation of [methyl-14C]choline into phosphatidylcholine and a corresponding decrease in radioactivity in choline phosphate using a pulse-chase procedure. This result is consistent with a fatty acid-induced increase in the cytidylyl-transferase step in the choline pathway. We measured cytidylyltransferase activity in membrane fractions and in cytosol (100,000 x g supernatant or soluble enzyme released by digitonin). The activity increased in both membrane and cytosol. Thus, an increase in total activity occurred. Cytidylyltransferase protein determined by Western blot immunoassay increased after oleic acid treatment. Immunotitration of cytidylyltransferase protein also indicated that an increase in enzyme protein resulted from oleic acid treatment. Cycloheximide did not prevent the oleic acid-induced increase in cytidylyltransferase activity. The increase in enzyme activity was apparent when we measured the activity in the presence or absence of lipid activators. Separation of cytosolic cytidylyltransferase into H- and L-forms showed that the increase in cytosolic activity was due to an increase in H-form. The amount of L-form did not change. We interpret these results to suggest that fatty acid treatment of Hep G2 cells promoted the formation of active cytidylyltransferase (H-form) from a preexisting inactive form. The increased activity was distributed between membranes and the lipoprotein form in cytosol (H-form).

Topics: Blotting, Western; Carcinoma, Hepatocellular; Choline-Phosphate Cytidylyltransferase; Cycloheximide; Digitonin; Liver Neoplasms; Nucleotidyltransferases; Oleic Acid; Oleic Acids; Phosphatidylcholines; Tumor Cells, Cultured

The subcellular forms of cytidylyltransferase (EC 2.7.7.15) in rat lung, rat liver, Hep G2 cells, A549 cells and alveolar Type II cells from adult rats were separated by glycerol density centrifugation. Cytosol prepared from lung, Hep G2 cells, A549 cells and alveolar Type II cells contained two forms of the enzyme. These species were identical to the L-Form and H-Form isolated previously from lung cytosol by gel filtration. Liver cytosol contained only the L-Form. Rapid treatment of Hep G2 cells with digitonin released all of the cytoplasmic cytidylyltransferase activity. The released activity was present in both H-Form and L-Form. The molecular weight of L-Form was determined from sedimentation coefficients and Stokes radius values to be 97,690 +/- 10,175. Thus, the L-Form appears to be a dimer of the Mr 45,000 catalytic subunit. The f/f degrees value of 1.5 indicated that the protein molecule has an axial ratio of 10, assuming a prolate ellipsoid shape. The estimated molecular weight of the H-Form was 284,000 +/- 25,000. The H-Form was dissociated into L-Form by incubation of cytosol at 37 degrees C. Triton X-100 (0.1%) and chlorpromazine (1.0 mM) also dissociated the H-Form into L-Form. Western blot analysis indicated that both forms contained the catalytic subunit. An increase in Mr 45,000 subunit coincided with the increase in cytidylyltransferase activity in L-Form, which resulted from the dissociated of H-Form. The L-Form was dependent on phospholipid for activity. The H-Form was active without lipid. Phosphatidylinositol was present in the H-Form isolated from Hep G2 cells. The phosphatidylinositol dispersed when the H-Form was dissociated into L-Form. Phosphatidylinositol and phosphatidylglycerol cause L-Form to aggregate into a form similar to H-Form. Phosphatidylcholine/oleic acid (1:1 molar ratio) and oleic acid also aggregated the L-Form. Phosphatidylcholine did not produce aggregation. We conclude that the H-Form is the active form of cytidylyltransferase in cytoplasm. The H-Form appears to be a lipoprotein consisting of an apoprotein (L-Form dimer of the Mr 45,000 subunit) complexed with lipids. A change in the relative distribution of H-Form and L-Form in cytosol would alter the cellular activity and thus may be important in the regulation of phosphatidylcholine synthesis.

Topics: Adenoma; Animals; Carcinoma, Hepatocellular; Centrifugation, Density Gradient; Chemical Phenomena; Chemistry, Physical; Choline-Phosphate Cytidylyltransferase; Cytosol; Digitonin; Humans; Liver; Liver Neoplasms; Lung; Lung Neoplasms; Male; Molecular Weight; Nucleotidyltransferases; Pulmonary Alveoli; Rats; Rats, Inbred Strains; Tumor Cells, Cultured

Rat and mouse liver mitochondria, when centrifuged in a sucrose density gradient (25--50%, w/w), showed the presence of heavy (H) and light (L) subfractions with buoyant densities 1.185 and 1.170--1.165 g/ml, respectively. Mild treatment with digitonin or EDTA (30 mM) shifted H-subfraction of mitochondria into the lighter zone of the gradient and as a result of this the mitochondria were distributed as a homogenous band with buoyant density 1.170--1.165 g/ml. Mitochondria isolated from both MD hepatoma and Zajdela rat hepatoma were characterized by a homogenous banding with buoyant density 1.160--1.165 g/ml. Regarding to this, the content and patterns of polyribosomes bound to outer membranes of mouse tumor mitochondria were studied. Analysis of polyribosomes as well as the results of RNA polyacrylamide gel electrophoresis indicate that the content of these polyribosomes in tumor mitochondria is less than that in normal liver ones. However, the decrease of cancer cell membrane-bound polyribosomes cannot account for the differences in buoyant densities of mitochondria from normal and tumor tissues.

Topics: Animals; Carcinoma, Hepatocellular; Centrifugation, Density Gradient; Digitonin; Edetic Acid; Liver Neoplasms; Male; Mice; Mitochondria; Mitochondria, Liver; Neoplasms, Experimental; Polyribosomes; Rats

Topics: Adenine Nucleotides; Adenosine Triphosphate; Animals; Carcinoma, Hepatocellular; Chromatography, Ion Exchange; Digitonin; Kinetics; Liver; Liver Neoplasms; Mitochondria; Mitochondria, Liver; Neoplasms, Experimental; Nucleotidyltransferases; Phosphoric Diester Hydrolases; Polynucleotides; Rats; Snakes; Solubility; Spectrophotometry, Ultraviolet; Structure-Activity Relationship; Tissue Extracts; Tritium; Venoms

Topics: Adenosine Triphosphatases; Animals; Carbon Isotopes; Carcinoma, Hepatocellular; Cell Line; Cytochrome c Group; Cytochrome Reductases; Dextrans; Digitonin; Electron Transport Complex IV; Liver Neoplasms; Membranes; Mitochondria, Liver; Mitochondrial Swelling; Neoplasms, Experimental; Permeability; Phospholipases; Phosphotransferases; Rats; Succinates; Trypsin

Topics: Animals; Carcinoma, Hepatocellular; Clone Cells; Coumarins; Digitonin; Edetic Acid; Female; Glucosamine; Glucuronates; Glucuronosyltransferase; Hexosyltransferases; Liver Neoplasms; Male; Microsomes, Liver; Nitrophenols; Nitrosamines; Orotic Acid; Phenols; Proteins; Rats; Stimulation, Chemical; Time Factors