Effects of phorbol ester and teleocidin on Ca2+-induced fusion of liposomes

The effects of different types of lipid membrane defects on Ca2+-induced fusion of liposomes containing phosphatidylserine (PS) were investigated using fluorescent probes. Teleocidin enhanced the fusion of phospholipid vesicles in an assay system using terbium/dipicolinic acid during mixing of internal aqueous phases of vesicles upon fusion. 12-O-Tetradecanoylphorbol-13-acetate (TPA) suppressed the fusion. This latter phenomenon was also observed by measuring the excitation energy transfer. The promotion of membrane fusion by teleocidin was ascribed to dehydration of the membrane surface, the suppressive effect of TPA to desorption of Ca2+ from the membrane surface. Thus, Ca2+-induced fusion of PS vesicles was shown to be sensitive to defects of the membrane surface, but insensitive to defects of the hydrophobic core of the lipid membrane.     

Complementation study of peroxisome-deficient disorders by immunofluorescence staining and characterization of fused cells

Summary Genetic heterogeneity in peroxisome-deficient disorders, including Zellweger's cerebrohepatorenal syndrome, neonatal adrenoleukodystrophy and infantile Refsum disease, was investigated. Fibroblasts from 17 patients were fused using polyethylene glycol, cultivated on cover slips, and the formation of peroxisomes in the fused cells was visualized by immunofluorescence staining, using anti-human catalase IgG. Two distinct staining patterns were observed: (1) peroxisomes appeared in the majority of multinucleated cells, and (2) practically no peroxisomes were identified. Single step 12-(1-pyrene) dodecanoic acid/ultraviolet (P12/UV)-selection confirmed that the former groups were resistant to this selection, most of the surviving cells contained abundant peroxisomes, and the latter cells died. In the complementary matching, [1^-14C]lignoceric acid oxidation and the biosynthesis of peroxisomal proteins were also normalized. Five complementation groups were identified. Group A: Zellweger syndrome and infantile Refsum disease; Groups B, C and D: Zellweger syndrome; Group E: Zellweger syndrome, neonatal adrenoleukodystrophy and infantile Refsum disease. We compared these groupings with those of Roscher and identified eight complementation groups. There was no obvious relation between complementation groups and clinical phenotypes. These results indicate that the transport, intracellular processing and function of peroxisomal proteins were normalized in the complementary matching and that at least eight different genes are involved in the formation of normal peroxisomes and in the transport of peroxisomal enzymes.     

Inhibition by gossypol of tumor promoter-induced arachidonic acid metabolism in rat peritoneal macrophages

Rat peritoneal macrophages were prelabeled with [3H]arachidonic acid. The release of radioactivity into the medium was increased by treatment with TPA-type tumor promoters, such as TPA, teleocidin and aplysiatoxin, and the non-TPA-type tumor promoter, thapsigargin. Gossypol, at concentrations of 3 and 10 microM, inhibited the release of radioactivity stimulated by both types of tumor promoter, although the mechanism of stimulation of arachidonic acid metabolism is different in the two types of tumor promoter. Stimulation of prostaglandin E2 production by these tumor promoters was also inhibited by treatment with gossypol. Calcium ionophore A23187-stimulated release of radioactivity and prostaglandin E2 production were also inhibited by gossypol treatment. The mechanism of inhibition by gossypol of prostaglandin E2 production is discussed.     

Involvement of sulfatide in activation of protein kinase C by tumor promoters

Sulfatide (cerebroside sulfate) activated protein kinase C to the same extent as phosphatidylserine did with the tumor promoters, 12-O-tetradecanoylphorbol-13-acetate (TPA), teleocidin and debromoaplysiatoxin. Sulfatide and phosphatidylserine both induced specific binding of [3H]TPA to protein kinase C, although the ratios of specific to non-specific [3H]TPA binding to protein kinase C with the two were not the same. It is concluded that sulfatide is involved in activation of protein kinase C by tumor promoters in a slightly different way from phosphatidylserine.     

Partial disassembly of peroxisomes

Rat liver peroxisomes were subjected to a variety of procedures intended to partially disassemble or damage them; the effects were analyzed by recentrifugation into sucrose gradients, enzyme analyses, electron microscopy, and SDS PAGE. Freezing and thawing or mild sonication released some matrix proteins and produced apparently intact peroxisomal "ghosts" with crystalloid cores and some fuzzy fibrillar content. Vigorous sonication broke open the peroxisomes but the membranes remained associated with cores and fibrillar and amorphous matrix material. The density of both ghosts and more severely damaged peroxisomes was approximately 1.23. Pyrophosphate (pH 9) treatment solubilized the fibrillar content, yielding ghosts that were empty except for cores. Some matrix proteins such as catalase and thiolase readily leak from peroxisomes. Other proteins were identified that remain in mechanically damaged peroxisomes but are neither core nor membrane proteins because they can be released by pyrophosphate treatment. These constitute a class of poorly soluble matrix proteins that appear to correspond to the fibrillar material observed morphologically. All of the peroxisomal beta-oxidation enzymes are located in the matrix, but they vary greatly in how easily they leak out. Palmitoyl coenzyme A synthetase is in the membrane, based on its co-distribution with the 22-kilodalton integral membrane polypeptide.     

Post-translational import of fatty acyl-CoA oxidase and catalase into peroxisomes of rat liver in vitro

Total polysomal RNA of rat liver was translated in vitro in a rabbit reticulocyte lysate system. The translation products were mixed with a postnuclear supernatant fraction of rat liver and incubated post-translationally at 26 degrees C for 15-60 min. The import assay mixture was separated into a particulate fraction and supernatant by centrifugation, both of which were analyzed by immunoprecipitation with a goat antibody against rat liver peroxisomal proteins, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and fluorography. One peroxisomal translation product (Mr 72,000) appeared in the particulate fraction, was partly proteinase K-resistant, and addition of detergents prior to proteolysis abolished this resistance. In isopycnic centrifugation of the uptake assay mixture, the protease-resistant 35S-polypeptide of Mr 72,000 cosedimented with the peroxisomes. This translation product was identified immunochemically as fatty acyl-CoA oxidase; both before and after import it was indistinguishable in size from subunit A of the purified enzyme by prolonged sodium dodecyl sulfate-polyacrylamide gel electrophoresis. When the cell-free translation products were incubated with highly purified peroxisomes, 35S-catalase entered peroxisomes (by the criterion of protease resistance), and its entry was stimulated by the addition of a high speed supernatant (cytosolic) fraction of rat liver. These results demonstrate the post-translational import into peroxisomes in vitro of at least two cell-free translation products.     

Effect of TPA on the mutagenicity of caffeine in the soybean mutation test

12-O-Tetradecanoylphorbol-13-acetate (TPA) is a well-known tumor promoter in mouse-skin carcinogenesis. Its effects on mutagenesis in a soybean test system were examined, and the effects were judged from the appearance of spots of various colors on the leaves. When soybean seeds were treated with TPA plus 0.03% caffeine, the frequency of spots per leaf decreased significantly and in proportion to the concentration of TPA. TPA alone at concentrations of 1–20 μg/ml did not induce any mutations. Mutations induced by γ-rays were not affected by administration of TPA either before or after exposure to γ-rays. The mechanism of suppression by TPA of mutations induced by caffeine is discussed.