Lipid radioactive precursors into

Incorporation of Radioactive Precursors into Lipids. The cultures of surface-adhering cells were exposed for 16 h to either 400 pCi of Hp/C- syo (final specific activity, 50 yCi/ ymol) or 10 pCi of / %/, galactose ( the rate of incorporation was linear during this period). After 16 hr. the radioactive medium was removed and the cultures were washed four times with 0.9 NaCl. The cells were removed from the surface with a rubber policeman and suspended in physiological saline. Lipids were extracted by Bligh and Dyer procedure (26) and analyzed for various lipids according to Neskovic, et al. (27)... 

A fundamental principle of membrane biosynthesis is that cells synthesize new membranes only by the expansion of existing membranes. Although some early steps in the synthesis of membrane lipids take place in the cytoplasm, the final steps are catalyzed by enzymes bound to preexisting cellular membranes, and the products are incorporated into the membranes as they are generated. Evidence for this phenomenon is seen when cells are briefly exposed to radioactive precursors... 

In hepatocytes cultured in the presence of oleic acid, incorporation of pH]-water into secreted lipids and TG was lower than incorporation into synthesized lipids with the 3-thia fatty acids. ° A similar phenomenon was observed with pH]-glycerol as the radioactive precursor. This suggests that some of the hypotriglyceridemic effects of 3-thia fatty acids may arise from a reduction in biosynthesis and/or secretion of TG. 

Fig. 5. "In vivo" incorporation of choline (%-methyl) in subcellular fractions of rat brain. (A) Incorporation into lipids of the different subcellular fractions was measured at various times after the intracranial injection of 2 pCi of the radioactive precursor. (B)...

Two hours after the intracranial injection of 2 /aCi of the radioactive precursor, a chase dose of 500 pg unlabelled choline was given intraperitoneally to each animal (arrow). Incorporation into lipids of the various subcellular fractions was measured at different times. Results in both cases are expressed as dpm/ijg of P and are the mean of six experiments. S.E.M. for each point was 10% or lower. 

Both, preparations of liposomal membranes and whole microsmal fractions of the moss protonema cells were incubated with radioactive oleoyl-CoA and glycerol-3-phosphate, in order to study incorporation into different lipid fractions. But so far practically no incorporation of the precursors into any lipid fraction could be demonstrated, even though the cells, used for the membrane preparations, were in a stage of high lipid... 

Since the specific radioactivity of ll- cl stearoyl CoA would be similar to the specific radioactivity of l- c oleyl CoA (Holloway and Holloway, 1974), the relative distribution of the l- C radioactivity in the stearic and oleic acid incorporated into lipids could account for the liver capacity to desaturated in vivo stearic acid. Nevertheless, the loss of oleic acid and its precursor, stearic acid, from the liver, excreted as lipoproteins... 

Fig. 7. "In vitro" incorporation of Na2 S04 into rat brain subcellu-lar fractions. Brain slices were incubated with the precursor for 15 min. and after two washings with fresh medium containing 0.1 mM-Na2S0 they were further incubated for different times. Isolation of subcellular fractions and extraction of lipids was performed as described under "Experimental Procedures". Radioactivity was measured in the total lipid extract. Results are expressed as dpm/brain and are the mean of three experiments. Symbols as in Fig. 5.

The distribution of radioactivity among retina lipids after 30 min incubation shows that in the toad retina the preferred flow of glycerol is towards PI, whereas arachidonate gets predominantly into PE (Table 3). By the contrary, the label from both precursors in cattle retina lipids is similarly distributed. 

On the other hand, Hendler 368, 369) has presented evidence that in hen oviduct tissue, both in vivo and in vitro, radioactive amino acids could be rapidly incorporated into a lipid fraction, which behaved as a precursor for the labeling of protein, in preference to free amino acids. The labeled amino acid, together with many others, could be recovered from the purified lipid fraction after acid hydrolysis. 

In these experiments rat brain slices were incubated with some phospholipid precursors (choline, arachidonic acid, etc,), and the effect of prostaglandins upon their incorporation and renewal in phospholipids was investigated. When tritiated ethanolamine was used, as the lipid precursor, radioactivity was found not only into ethanolamine phosphoglycerides (EPG) but also in smaller amounts... 

The potential of PET to inhibit cholesterol biosynthesis was determined by the inhibition of the incorporation of [2- H]acetate and R-[2- C]mevalonate into cholesterol (21). Human hepatoma cells (Hep G2) were cultured in DMEM supplemented with 10% fetal calf serum (PCS) in 12-well culture dishes (3x10 cell/well) at 37 C for 48 hours. After transferring to fresh medium, cells were treated with PET and labeled precursors ([2- H]acetate 2.5 pCi R-[2- CJmevalonate, 1.3 pCi). Lovastatin, in hydroxy acid form (5 and 10 pM), was used as a positive control. Incorporation experiment was carried out at 37 °C for 2 hours. After removal of medium and extensive washing, cells were harvested and crude total lipids were collected and saponified. Unlabeled cholesteiyl oleate was used as a carrier in saponification. Cholesterol was recovered by extraction with n-hexane. Radioactivity was measured by using a liquid scintillation counter. 

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