Pteroylpolyglutamate hydrolase

Enzymes that hydrolyze the polyglutamate side chain of pteroylpolyglutamates are important in the bioavailability of dietary pteroylpolyglutamates. This assay uses the pentaglutamate derivative as substrate, and all intermediates on the pathway to pteroylmonoglutamate are detected. 

The pteroylglutamate derivatives were separated on a /xBondapak Ci8 column (2.9 mm X 300 mm, 10 /Am). The column was equilibrated with solvent A (0.1 M potassium phosphate buffer, pH 6.0). Following injection of sample, a linear gradient from 75% solvent A and 25% solvent B (solvent A containing 10% acetonitrile) to 15% solvent A and 85% solvent B was applied over a 20-minute time span. The effluent was monitored at 280 nm. 

The standard reaction mixture contained in a volume of 250 /aL 200 /aL of the enzyme source in 0.1 M acetate buffer (pH 4.5), 25 /aL of 0.1 M sodium acetate buffer, and 25 /aL of 1.0 M pteroylpentaglutamate. The reaction was started by adding substrate and the reaction was allowed to proceed at 37°C for 1 hour in the dark. The reaction was stopped by the addition of 250 /aL 

SURVEY OF ENZYMATIC ACTIVITIES ASSAYED BY THE HPLC METHOD 

The enzyme preparation was human serum that was equilibrated with 0.1 M sodium acetate buffer by chromatography on Sephadex G-25. 

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BHANDARI s D, GREGORY J F 3rd (1990) Inhibition by selected food components of human and porcine intestinal pteroylpolyglutamate hydrolase activity. ,4m J Clin Nutr. 51 87-94. 

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