Enkephalinases A and

Enkephalinase A (dipeptidyl carboxypeptidase) and enkephalinase B (dipep-tidyl aminopeptidase) are involved in the degradation of enkephalin, which has an opiate-like activity. Enkephalinase A cleaves methionine enkephalin (Tyr-Gly-Gly-Phe-Met) after the second glycine, whereas enkephalinase B cleaves after the first glycine. A postcolumn derivatization system is used to detect N-terminal tyrosine-containing peptides. 

Substrate and product peptides were separated on a reversed-phase column (200 mm x 4 mm) packed with TSK gel ODS-120T (5 /xm). The mobile phase was formed from solvents A and B formed from acetonitrile-sodium phosphate buffer (0.3 M, pH 2.3)-water mixed in ratios of 1 20 79 and 3 1 1 (v/v/v), respectively. For detection of N-terminal tyrosines (other tyrosines do not react), the eluate stream was mixed first with 50 /xm CoCl2 and 20 mM NH2OH, and then with 0.3 M borate (pH 11.4). The resulting mixture was passed through a reaction coil maintained at 75°C before monitoring at 435 nm (emission) and 335 nm (excitation). Calibration graphs for the products were linear up to at least 5 nmol per assay. 

The enzyme reaction ws conducted by adding 30 fiL of enzyme sample to a mixture containing 50 / L of 0.5 M Tris-HCl (pH 7.4), 50 /xL of 1 mM bestatin, 20 / L of 40 mM captopril, and 50 fiL of 100 fiM methionine enkephalin. Bestatin and captopril are included to inhibit aminopeptidase and angiotensin-coverting enzyme, respectively. Incubation at 37°C proceeded for 30 minutes, and then the reaction was terminated by heating at KXFC for 2 

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See also in sourсe #XX -- [ , ]

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