RADIOLABELING OF THE LIPIDS OF CHINESE-HAMSTER OVARY CELLS WITH THE PROBE (TRIFLUOROMETHYL)-3(M-[(125)]IODOPHENYL)DIAZIRINE]

[I-125]TID [3-(trifluoromethyl)-3-(m-[I-125] iodophenyl)diazirine] is a commercially available, hydrophobic, photoactivatable, gamma-emittin g reagent mostly used to label protein hydrophobic domains. It has als o been used to radiolabel the phospholipids of lung surfactant (Gillia rd et al., Anal. Biochem. 193, 310-315, 1991). Since a nonspecific, hi ghly sensitive, lipid-labeling probe would be a very useful tool to in vestigate lipid-protein interactions in biological membranes, we chara cterized further the [I-125]TID-labeling products of lipids from cultu red Chinese hamster ovary cells (IR-CHO). After labeling of whole cell s, TLC analysis followed by autoradiography enabled detection of sphin gomyelin, phosphatidylcholine, phosphatidylinositol, phosphatidylserin e, phosphatidylethanolamine, cardiolipin, diglycerides, cholesterol an d its esters, and triglycerides. Analysis of the radioactivity associa ted with the saponification products of different lipids showed that [ I-125]TID was mostly (80%) extracted with the fatty acid moiety of the lipids whereas 20% remained associated with the hydrosoluble moiety. Similar radioactivity profiles were observed after labeling of whole c ells or extracted and liposome-reconstituted lipids; the [I-125]TID pr obe was able to diffuse in all intracellular organelles. Labeling was not equivalent between the different lipid classes, and it appeared th at the amount of associated radioactivity correlated well with the deg ree of lipid unsaturation. This was confirmed by studying [I-125]TID i ncorporation in phosphatidylcholines of different chain length and uns aturation. Taken together, our data demonstrate that [I-125]TID can be used as a radiolabel for lipids in cultured cells. It is rapidly inco rporated in the hydrophobic part of membranes, diffuses into all cellu lar compartments, and labels all lipid classes, including phospholipid s, cholesterol, and glycerides, with a sensitivity in the nanomolar ra nge. (C) 1997 Academic Press.