94] Assay of folylpolyglutamate hydrolase using pteroyl-labeled substrates and selective short-term bacterial uptake for product determination

Abstract

Publisher Summary This chapter describes an assay of folylpolyglutamate hydrolase using pteroyl-labeled substrates. The folate assay bacteria, Lactobacillus casei (ATCC 7469) and Streptococcus faecalis (ATCC 8043), exhibit specificity for the uptake as well as for the growth with folates of different numbers of glutamic acid residues. This specificity is exploited in short-term incubation, with bacterial cells in excess, to achieve quantitative incorporation of radioactive folates. Mono-, di-, and triglutamyl, but not heptaglutamyl, folate are taken up equally by L. casei cells, and monoglutamyl, but not di-, tri-, or higher conjugates are incorporated by S. faecalis cells. When the substrates for folylpolyglutamate hydrolases (conjugases) are labeled in the pteroyl moiety, short-term uptake of labeled folate products, by either bacteria, can be used to determine the enzyme activity. Combined use of L. casei and S. faecalis discriminates between the kinetics of a folylpolyglutamate peptidase. Three enzymes, with known differences in cleavage pattern, are used to illustrate the capacity of the assay described in the chapter. Enzyme concentrations are chosen to give complete hydrolysis after 40 min of incubation with respect to L. casei uptake. Whereas the time course of uptake by L. casei cells is very similar with all three enzymes, the corresponding S. faecalis uptake curves are found to be quite different.