Micellar high-performance liquid chromatographic determination of folylpolyglutamate hydrolase activity.
A rapid and sensitive method for the quantitative determination of folylpolyglutamate hydrolase activity in crude tissue extracts was developed. The procedure is based on high-performance liquid chromatographic separation of folate analogue mono- and polyglutamates on a reversed-phase column using sodium dodecyl sulfate in water as the mobile phase. Interfering substances in tissue extracts were removed by gel filtration on centrifugally-eluted mini-columns of Sephadex G-25 prior to incubation of polyglutamate substrate with tissue extract hydrolase. Reactions were terminated by denaturation of the enzyme in sodium dodecyl sulfate, which subsequently served as the micellar solvent system for chromatographic separation of substrate from reaction products.
D000818:Animals; D002851:Chromatography, High Pressure Liquid; D007668:Kidney / Q000201:enzymology; D008727:Methotrexate / Q000032:analysis; D051379:Mice; D008808:Mice, Inbred CBA; D008823:Micelles; D010453:Peptide Synthases / Q000032:analysis*; D011799:Quinazolines / Q000032:analysis; D012967:Sodium Dodecyl Sulfate; D013379:Substrate Specificity
D008823:Micelles; D011799:Quinazolines; D012967:Sodium Dodecyl Sulfate; C038838:5,8-dideazaisopteroyl triglutamate; D010453:Peptide Synthases; C023103:folylpolyglutamate synthetase; D008727:Methotrexate