Iodixanol (Optiprep), an improved density gradient medium for the iso-osmotic isolation of rat liver peroxisomes.
The suitability of Iodixanol {5,5'-[(2-hydroxy-1, 3-propanediyl)-bis(acetylamino)] bis-[N,N'-bis(2, 3-dihydroxypreopyl-2,4,6-triiodo-1,3-benzenecarboxamide)]}, a nonionic iodinated compound with a molecular weight of 1550, for the isolation of peroxisomes from rat liver was investigated. Centrifugation of light mitochondrial fractions in 20 to 40% (w/v) Iodixanol gradients, made iso-osmotic by the addition of sucrose, resulted in an excellent separation of peroxisomes from the remaining organelles, which were not able to enter the gradient. Peroxisomes banded around 30% (w/v) Iodixanol (d approximately 1.175) and, as revealed by marker enzyme analysis, were enriched 35- to 40-fold. Morphological examination of the peroxisomal fractions confirmed the near absence of other organelles and revealed structurally well-preserved peroxisomes. Free cores, also present in the starting fractions, migrated to higher densities and were trapped on a cushion. No interference of Iodixanol with marker enzyme determinations was observed, except for the UV-metric determination of urate oxidase and for the analysis of protein.
Van Veldhoven
P P
PP
Departement Moleculaire Celbiologie, Campus Gasthuisberg, Afdeling Farmakologie, Katholieke Universiteit Leuven, Belgium.
Baumgart
E
E
Mannaerts
G P
GP
D000818:Animals; D002469:Cell Separation / Q000379:methods*; D002499:Centrifugation, Density Gradient; D008099:Liver / Q000201:enzymology / Q000648:ultrastructure; D008297:Male; D008830:Microbodies / Q000201:enzymology; D051381:Rats; D014283:Triiodobenzoic Acids / Q000494:pharmacology*
D014283:Triiodobenzoic Acids; C044834:iodixanol