Radioactive lipids

The main problem in this approach is the very low permeability of mevalonic acid to membranes, resulting in very low incorporation. Positive results have been obtained by the use of cell-free systems incubated with [14C]-mevalonic acid,26,27 [14C]isopentenyl diphosphate,28 or [32P]orthophos-phate.29 Incubation of these radioactive lipids with glycosyl nucleotides labelled in the glycosyl group with a different isotope, followed by extraction and cochromatography in different solvent systems, may indicate that both compounds are present in the same molecule. When the lipid moiety becomes labelled from mevalonic acid or isopentenyl diphosphate, chromatography on DEAE-cellulose columns should be performed, in order to avoid confusion with steryl glycosides. 

A back-transfer of unlabeled and radiolabeled lipids from the acceptor to donor particles must be considered when calculating the lipid transfer rate. When transfer reactions beyond the initial rates are examined, it is necessary to account for the dilution of radioactive lipids in the donor particles by back-transfer of unlabeled lipid from the acceptor particles. Unless this is done, the apparent transfer rate will decrease with time. Equation (1) can be used to determine the transfer rate when back-transfer of unlabeled lipid is significant. 

Fig. 1. Lipid exchange between potato microsomes and mitochondria in vitro, (a) Incubation of radioactive lipid-labeled microsomes with unlabeled mitochondria, followed by reisolation of each organelle and determination of the specific radioactivity of its lipid, (b) Incubation of radioactive lipid-labeled mitochondria with unlabeled microsomes, and then as in (a). Mic, Microsomes Mit, mitochondria Sum, cytoplasmic supernatant , initial fraction of radioactive lipid (from Ben Abdelkader and Mazliak, 1970).

The quantity of lipid transferred from liposomes to membranes, per milligram of protein, increased with the quantity of radioactive lipid made available to the membranes in the initial mixture (Fig. 5). Also, the quantity of lipid transferred per milligram of protein increased when the quantity of membrane protein present in the initial mixture was decreased. Under optimal exchange conditions of low initial radioactivity and high protein concen-... 

Fig. 5. Transfer of radioactive lipids from liposomes to cauliflower mitochondria. Radioactively labeled liposomes were incubated with cauliflower mitochondria, and after various incubation periods the radioactivity in the mitochondria per milligram of protein was determined. Two experiments were carried out in the first (lower curve) 0.56 x 10 dpm in the liposomes was offered to 3.9 mg of mitochondrial protein, while in the second (upper curve) 3.6 X 10 dpm in the liposomes was offered to 2.1 mg of mitochondrial protein. The inset gives the plot of the percentage of the initial radioactivity recovered in the mitochondrion versus time, in the same two experiments (from Douady and Mazliak, 1975).

Similarly, it was recently found (Mazliak et al., 1977) that, in etiolated germinated sunflower cotyledons, newly synthesized radioactive lipids (labeled from [ C]acetate) accumulated first in the microsomes, second in the mitochondria, and much more slowly in the nuclei, which also suggests a lipid transfer from microsomes toward other cell organelles, such as mitochondria and nuclei. 

Scintillation solutions 1 and 2 are suitable for determining radioactive lipids in particular. The third cocktail resembles the well known Bray s solution and, like this, is used chiefly for measurements on aqueous solutions. Scintillation solution 4 contains methyl ceUosolve instead of dioxan as it is generally obtainable in a purer state. 

Fig. 5. Comparison of radioactive lipid in tectum following injection of 5yc of H-nievalonate into right eye in control goldfish and into goldfish whose optic nerve had been crushed 7 days prior to injection. Two groups of 3 fish each were averaged for each time point. Four groups v ere used for the l i-day point.

Our previous report (2) showed that the only metabolites (fatty acids) of oleate at sn-2 of 2-[ C]oleoyl-PC were ricinoleate and linoleate. Each radioactive lipid class shown in Fig. 1 (e.g. TG and free fatty acid, PA, PE, PC) was transmethylated and the esters separated by HPLC on a 5 cm, C g column. All lipid classes contained radioactive ricinoleate, linoleate and oleate. The percent radioactivity of fatty acids in each lipid class is shown in Table 1. 

Fig. 1. Time-course of UDPgalactose-independent MGDG synthesis. Spinach envelope membranes (150 ug protein) were sonicated with 520 nmol di[ C]oleoyl-PC and incubated with phospholipase C for various times at pH 7.2 (30 C). Indicated is the distribution of radioactive lipids.

After incubation, the radioactive lipidic material was separated in TLC with acid solvent as in Table 1 and the fluorogrs hic spots were scraped and counted in a liquid scintillator. The numbers are the average of two experiments.( ) Similar material from a parallel experiment was alkali-treated and the resulting product behaved as SX [Rf= 0.75]. 

Effect of Different Dehydrating Procedures on Retention of Radioactive Lipid in Adipose Tissue during Specimen Preparation for Electron Microscopy - ... 

In those experiments, neurons obtained from the spinal cord of embryonic rats were labeled by incubation with various radioactive lipid precursors, and exposed to bradykinin. Application of this peptide raised the levels of unesterified arachidonate, but had no effect on lysophospholipids, arguing against an involvement of PLA2. By contrast, appearance of the free fatty acid was preceded by a transient increase in 1,2 diacylglycerol content which took place within a few seconds of exposure to bradykinin. Because of its rapid time-course, this rise in 1,2 diacylglycerol may derive from the direct stimulation of a PLC activity. 

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