A radiochemical assay for lysosomal carboxypeptidase A in human B- and T-lymphocytes

The purpose of the present investigation was to develop a sensitive radiochemical assay for lysosomal carboxypeptidase A, which would not be subject to interference from cellular constituents in crude extracts on B- or T-cell enriched preparations. N-Benzyloxycarbonyl-L-glutamyl-L-tyrosine is a substrate of high affinity for mammalian carboxypeptidase and the aromatic group of the tyrosyl residue can be radiolabelled with [125I]iodine. Thus, separation of radiolabelled product of proteolysis, 125I-tyrosine, from the radiolabelled dipeptide could be the basis for assay. Cell extracts from normal subjects and patients were used, and enzyme activity was measured using quantitative paper chromatography to detect amino acids released from amino-protected dipeptides by crude cell extracts. Radioactive compounds were located in the developed chromatograms by exposure to autoradiographic film. The results were compared with those of a spectrophotometric determination. The sensitivity of the new method is better. (Auth.)