Study of endogenous Tyr-Gly-Gly, a putative enkephalin metabolite, in mouse brain: validation of a radioimmunoassay, localisation and effects of peptidase inhibitors.

The tripeptide Tyr-Gly-Gly, a hydrolysis product of enkephalins and related opioid peptides obtained with 'enkephalinase', was identified and quantified in various regions of mouse brain by means of HPLC and a sensitive and specific radioimmunoassay. Similar levels i.e. about 8 pmol/brain were found after the animals were killed by various procedures, including microwave irradiation, suggesting its pre-mortem formation. The distribution of Tyr-Gly-Gly immunoreactivity among brain regions was highly heterogeneous and paralleled to a certain extent the [Met5]enkephalin distribution, molar levels of Tyr-Gly-Gly representing 10-30% of those of the enkephalin. Following gentle homogeneisation of striata in 0.32 M sucrose and centrifugation, 73% of Tyr-Gly-Gly immunoreactivity was recovered in the supernatant, a result consistent with its extracellular localisation in vivo. Administration of enkephalinase inhibitors rapidly elicited marked decrease in Tyr-Gly-Gly immunoreactivity whereas bestatin, an aminopeptidase inhibitor, elicited 100% increase and captopril, an ACE inhibitor, was without significant effect. These data indicate that the tripeptide is in a dynamic state in the brain and that its levels might reflect the release of endogenous enkephalins or related opioid peptides and their subsequent metabolism by enkephalinase.