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Folypolyglutamate synthetase from beef liver: assay, stabilization, and characterization.

Authors
Type
Published Article
Journal
Canadian journal of biochemistry and cell biology = Revue canadienne de biochimie et biologie cellulaire
Publication Date
Volume
62
Issue
6
Pages
495–506
Identifiers
PMID: 6205735
Source
Medline

Abstract

A reliable assay for folylpolyglutamate synthetase has been devised and tested. Conditions have been established for the complete separation of [3H]glutamate and the tritium-labelled products on columns of DEAE-cellulose. The availability of this assay has aided us in partially purifying and characterizing the synthetase from extracts of beef liver. Suitable conditions have been found for the stabilization of the activity of both crude and partially purified folylpolyglutamate synthetase. The apparent Km values for L-glutamate (0.82 mM), dl-L-tetrahydrofolate (9 microM), ATP (25 microM with 10 mM MgCl2), KCl (3 mM), and 2-mercaptoethanol (5 mM) have been estimated. Several oxidized pteridine substrates have been tested. Of the antifolates tested, aminopterin is the more active substrate. The chain lengths of folate polyglutamates have been measured by chromatography on columns of DEAE-cellulose, with elution by a gradient of sodium acetate. Conjugates as long as hexaglutamates have been detected. The identities of the polyglutamates of tetrahydrofolate have been verified by hydrolysis in the presence of conjugase and by double-labelling experiments.

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