Characterisation of two probes for the localisation of enkephalinase in rat brain: [3H]thiorphan and a 125I-labeled monoclonal antibody.
We studied the binding of two radioactive probes, i.e. [3H]thiorphan and a 125I-labeled monoclonal antibody raised against the rabbit kidney enzyme, to enkephalinase (EC 3.4.24.11, membrane metalloendopeptidase) from rat cerebral membranes. [3H]Thiorphan binding at equilibrium to striatal membranes was monophasic with a KD (0.7 nM) and a pharmacology consistent with a selective labeling of the enzyme. The ratio of Vmax/Bmax was in the same range as the Kcat of the enzyme purified from peripheral tissues. The monoclonal antibody immunoprecipitated to a similar extent the solubilised enkephalinase activity and [3H]thiorphan binding sites from striatum. The regional distributions of binding sites for the two probes established either on isolated membranes or autoradiographic sections were highly heterogeneous and similar to that of enkephalinase activity. Hence the two probes appear to label membrane-bound enkephalinase in rat brain but, from a technical point of a view, the 125I-monoclonal antibody is a more sensitive and flexible tool.
D000603:Amino Acids, Sulfur; D000818:Animals; D000911:Antibodies, Monoclonal; D001345:Autoradiography; D001665:Binding Sites; D001921:Brain / Q000201:enzymology*; D010450:Endopeptidases / Q000378:metabolism*; D007457:Iodine Radioisotopes; D007700:Kinetics; D008297:Male; D015260:Neprilysin; D011869:Radioligand Assay; D051381:Rats; D011919:Rats, Inbred Strains; D015244:Thiorphan; D008625:Tiopronin / Q000031:analogs & derivatives; D014316:Tritium
D000603:Amino Acids, Sulfur; D000911:Antibodies, Monoclonal; D007457:Iodine Radioisotopes; D014316:Tritium; D015244:Thiorphan; D008625:Tiopronin; D010450:Endopeptidases; D015260:Neprilysin