A unique computer centered instrument for simultaneous absorbance and fluorescence measurements

“…The absorbance-corrected fluorescence intensity of dehydroergosterol excitation 324 nm, emission 380 nm) and diphenylhexatriene (excitation 362 nm, emission 424 nm) (1:1000 molar ratio to membrane lipid) was determined for unlabeled and trinitrophenylated plasma membranes (50 /zg protein/ml phosphate-buffered saline, pH 7.4) using a computer-centered spectrofluorimeter [38] as described [29]. The fluorescence lifetime of dehydroergusterol in the plasma membranes was determined against a reference of 1,4-bis[2-(4-methyl-5-phenyloxazolyl)] benzene (dimethyl POPOP) in absolute ethanol [39] using a T format SLM 4800 spectrofluorimeter (SLM Instru-ments, Champaign-Urbana, IL) as described [29].…”

Section: Fluorescence Measurements and Transbilayer Sterol Distributionmentioning

confidence: 99%

Polyunsaturated fatty acids alter sterol transbilayer domains in LM fibroblast plasma membrane

Sweet

Schroeder

1988

FEBS Letters

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Sterols are asymmetrically distributed between the leaflets of animal cell plasma membranes. Although transbilayer migration of sterols is extremely rapid, s to rain, previous experimental manipulations have not altered their transmembrane steady-state distribution. However, the effect of polyunsaturated fatty acids has not been reported. When cultured in a lipid-free, chemically defined culture medium, LM fibroblasts do not synthesize polyunsaturated fatty acids but will incorporate polyunsaturated fatty acids into their plasma membranes if supplied in the medium. Sterol transbilayer distribution in LM plasma membranes was determined from quenching of fluorescence of dehydroergosterol by trinitrophenyl groups selectively attached to the exofacial leaflet. When cells are cultured in lipid-free media, 28.1% of the plasma membrane sterol is located in the exofacial (outside) leaflet. In contrast, when cells are cultured with linoleate-or linolenatesupplemented medium, 71 ;8% and 75.5% of the plasma membrane sterol is exofacial, respectively.

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“…The 1,6-diphenyl-1,3,5-hexatriene (0.5/~g/ml) was incorporated into the VLDL (50/.tg lipid/m1) resuspended in phosphate-buffered saline, (pH 7.4) as in [ 11 ]. The computer-centered spectrofluorimeter in [ 12,13] was usedto measure absorbance, absorbance corrected fluorescence (corrected for the inner filter effect), relative fluorescence efficiency, corrected fluorescence emission, and light scattering corrections as in [14,15]. Excitation for ~-parinaric acid and 1,6-diphenylhexatriene was measured at 313 nm and 362 nm, respectively, while emission was measured at 420 nm and 424 nm, respectively.…”

Section: Methodsmentioning

confidence: 99%