RESUMO
Accumulation of isoprenoids was studied in two cell lines derived from acute T-cell leukemia: CEM-C7 cells, whose growth is inhibited by the glucocorticoid dexamethasone, and CEM-C1 cells, which are resistant to this steroid. Isoprenoids were measured by growing the cells in serum-free medium in the presence of lovastatin, which blocks synthesis of mevalonate, and then labeling with exogenous [3H]mevalonolactone. In both cell lines, isoprenoids associated with proteins were detected in cytoplasm, nucleus, and chromatin, and in the chromatin residue that remains after extraction of histone and nonhistone proteins. Differences in labeling were detected after treatment with dexamethasone in the CEM-C7 line, showing a decrease in the cytoplasmic fraction with a corresponding increase in both the nuclear and chromatin fractions as compared with untreated cells. No change was seen in the CEM-C1 line. In both cell lines, 25-30% of the incorporated label was released by treatment with acid or alkali. However, the majority of the label required treatment with methyl iodide for the release of organic-soluble tritiated products. After extraction with chloroform, the lipid fractions contained farnesol, geraniol, dolichols, and possibly nerolidol.
Assuntos
Dexametasona/farmacologia , Proteínas de Neoplasias/isolamento & purificação , Linfócitos T/efeitos dos fármacos , Terpenos/isolamento & purificação , Divisão Celular/efeitos dos fármacos , Cromatina/química , Depressão Química , Resistência a Medicamentos , Humanos , Leucemia-Linfoma de Células T do Adulto/metabolismo , Leucemia-Linfoma de Células T do Adulto/patologia , Ácido Mevalônico/metabolismo , Frações Subcelulares/química , Linfócitos T/química , Células Tumorais CultivadasRESUMO
Phosphatidylinositol synthetic and intermembrane transfer activities were studied in rat in the developing whole brain and isolated cerebellum. Specific activities of CTP:phosphatidate cytidylyltransferase and CDPdiacylglycerol:inositol phosphatidyltransferase were found to have similar developmental patterns. Levels of phosphatidyltransferase seen in fetal animals (whole brain only) and neonatal (whole brain and cerebellum) were maintained through approximately postnatal day 15, peaked at day 28, and then declined to somewhat higher than fetal levels at day 60. Cytidylyltransferase activity varied from the phosphatidylinositol synthesizing enzyme in that specific activity continued to increase up to day 60. Whole brain phosphatidylinositol transfer specific activity showed a sharp peak at postnatal day 9 after which activity was maintained at or above the fetal levels to day 60. Cerebellum phosphatidylinositol transfer specific activity had a similar peak which was delayed 7-10 days compared to the whole brain. Phosphatidylinositol transfer protein was also determined immunologically: whole brain levels increased dramatically from fetal day 16 to 18 and then remained relatively constant, while cerebellum levels (measured from postnatal day 7) displayed a variable profile between days 7 and 28. The developmental pattern of CTP:phosphatidate cytidylyltransferase in rat brain is reported here for the first time.
Assuntos
Encéfalo/metabolismo , Proteínas de Transporte/metabolismo , Proteínas de Membrana , Nucleotidiltransferases/metabolismo , Fosfotransferases/metabolismo , Proteínas de Saccharomyces cerevisiae , Transferases (Outros Grupos de Fosfato Substituídos) , Animais , Western Blotting , Encéfalo/enzimologia , Encéfalo/crescimento & desenvolvimento , CDP-Diacilglicerol-Inositol 3-Fosfatidiltransferase , Cerebelo/enzimologia , Cerebelo/crescimento & desenvolvimento , Cerebelo/metabolismo , Eletroforese em Gel de Poliacrilamida , Feminino , Feto/enzimologia , Feto/metabolismo , Proteínas de Transferência de Fosfolipídeos , Gravidez , Ratos , Ratos EndogâmicosRESUMO
Thiophosphoester analogs of dioctanoyl and didecanoyl phosphatidic acids were synthesized for use as substrates in spectrophotometric assays. These substrates are easily dispersable in aqueous media and release thiodiacylglycerols after phosphomonoesterase catalyzed hydrolysis. The free sulfhydryl of these thiodiacylglycerols reacts with the colorimetric reagent 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), allowing the reaction to be followed. These analogs were shown to be good substrates for calf intestine alkaline phosphatase (highest activity at alkaline pH) and phosphomonoesterases of partially purified beef brain cytosol (highest activity at physiologic pH). Cationic amphiphilic drugs inhibit the actions of alkaline phosphatase on the dioctanoyl analog, but did not inhibit enzymatic hydrolysis of p-nitrophenyl phosphate. In contrast, the beef brain cytosolic fraction p-nitrophenyl phosphate hydrolysis was mildly inhibited, and the phosphatidic acid analog hydrolysis was increased slightly. Tetramisole inhibited all enzyme activities with p-nitrophenyl phosphate, but was inhibitory only to the alkaline-phosphatase activity with the phosphatidic acid analog.