Low density lipoproteins reconstituted with steroids containing the nitrobenzoxadiazole fluorophore.
Academic Article
Overview
abstract
A new cholesterol analog, N-(7-nitrobenz-2-oxa-1,3-diazole)-23,24-dinor-5-cholen-22-amine-3 beta-ol, with fluorescent properties similar to those of fluorescein, has been synthesized. The fluorescence lifetimes, quantum yields, and wavelength maxima of N-(7-nitrobenz-2-oxa-1,3-diazole)-23,24-dinor-5-cholen-22-amine-3 beta-ol and its linoleate ester were solvent-dependent. The cholesterol analog was a satisfactory substrate for lecithin:cholesterol acyltransferase. The fluorescent sterol, added in ethanol, gave half-maximal suppression of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity in cultured human fibroblasts at 2.5 microM and was twice as effective as cholesterol. The fluorescent steryl ester, incorporated into low density lipoprotein, was used to demonstrate high affinity cellular uptake and degradation of the reconstituted lipoproteins, intracellular accumulation of the free sterol and simultaneous suppression of 3-hydroxy-3-methylglutaryl coenzyme a reductase in fibroblasts. Half-maximal suppression was achieved at 10 micrograms mL-1 of low density lipoproteins reconstituted with the fluorescent steryl ester, compared to the same degree of suppression produced by 2 micrograms mL-1 of native low density lipoproteins. The interaction of low density lipoproteins reconstituted with N-(7-nitrobenz-2-oxa-1,3-diazole)-23,24-dinor-5-cholen-22-amine-3 beta-ol linoleate with cells was readily visualized by fluorescence microscopy and quantified by fluorimetry. These analogs will facilitate the studies of lipoprotein-cell interactions and phospholipid vesicle-cell interactions, the selection of cell mutants defective in lipoprotein metabolism, and the assessment of the immediate environment of the steroids in cellular membranes.
