Preparation of Alexa Fluor 350-conjugated nonradioactive or 3H-labeled GM1 ganglioside derivatives with different ceramides.
Alexa Fluor 350 hydrazide (AF) was coupled to the aldehyde group at C-6 of terminal galactose of oxidized GM1 gangliosides containing different fatty acid residues (GM1s). The AF-GM1 hydrazones obtained were reduced with NaBH(4) or [3H]NaBH(4) and purified by high-performance thin layer chromatography (HPTLC) and high-performance liquid chromatography (HPLC). Final yields of AF-GM1s exceeded 30%, purity was better than 97%, and radiochemical purity of 3H-labeled AF-GM1s was more than 94.5%. Structures of AF-GM1s were confirmed by electrospray ionization-mass spectrometry (ESI-MS). When added to HL-60 cell culture media, more than 81.6 or 78.9% of the AF-[3H]GM1s were taken up by cells in a bovine serum albumin- or trypsin-resistant manner, respectively. Approximately 70% of the AF-[3H]GM1s were recovered in HL-60 total plasma membrane fraction.
Panasiewicz
Mirosława
M
Department of Biochemistry and Molecular Biology, Medical Center of Postgraduate Education, Warszawa 01-813, Poland.
Domek
Hanna
H
Fedoryszak
Natalia
N
Pacuszka
Tadeusz
T
D000085:Acetates / Q000737:chemistry* / Q000493:pharmacokinetics; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism; D002518:Ceramides / Q000737:chemistry* / Q000493:pharmacokinetics; D002851:Chromatography, High Pressure Liquid; D002867:Chromones / Q000737:chemistry* / Q000493:pharmacokinetics; D005677:G(M1) Ganglioside / Q000737:chemistry* / Q000493:pharmacokinetics; D018922:HL-60 Cells; D006801:Humans; D021241:Spectrometry, Mass, Electrospray Ionization; D014018:Tissue Distribution; D014316:Tritium / Q000737:chemistry*
D000085:Acetates; C400304:Alexa 350; D002518:Ceramides; D002867:Chromones; D014316:Tritium; D005677:G(M1) Ganglioside