Development of a radiochemical assay for glycyl-leucine dipeptidase in human B- and T-lymphocytes.

Recent technical advances in the isolation of human lymphocytes have made it possible to obtain preparations enriched in Bor T-cells (Wybran, 1973; Pellegrino et al., 1976). This in turn facilitates studies on the comparative enzymology of Band T-lymphocytes, which may be relevant to some leukaemic states. Studies on lysosomal enzymes, including acid phosphatase, /3-glucuronidase (Meusers et al., 1976) and N-acetyl-8-glucosaminidase (Crockard et al., 1979) in Band T-cell-enriched fractions have already been carried out. Haschen & King (1966) found increased activities of the cytoplasmic enzyme N-glycyl-L-leucine dipeptidase (EC 3.4.13.1) in leucocytes from patients with chronic lymphatic leukaemia. The purpose of the present investigation was to measure the same activity in the fractionated lymphocytes and also to improve the assay by using a radioactive substrate, [2-3H] N-glycyl-L-leucine, since cellular constituents interfere with assays using ninhydrin. The substrate preparation was obtained as fallows: glycine, containing [2-)Hlglycine as tracer, at a final specific radioactivity of 2 mCi/mmol was converted into the N-benzyloxycarbony1 derivative and L-leucine to its benzyl ester. After condensation with l-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (Aldrich Chemicals Ltd. Gillingham, U.K.) both blocking groups were removed simultaneously from the conjugate (1.71 g) by hydrogenation with a Pd/charcoal catalyst (170mg). The yield in each of the first three steps was over 9096 and the final yield was over 57%. Cells (1 x 10’-2.5 x lo’) were homogenized in 0.25 M-sucrose (0.5ml) at OOC. Insoluble proteins were removed by centrifugation at 100OOOg. The cell extract was diluted with 0.25 M-sucrose. Assay mixtures contained diluted cell extract (1OOpl) and a solution (1OOpl) of dipeptide in 0.06 M-KH,PO,/ K,HPO, buffer, pH 8.1, containing 0.25 M-sucrose. Portions