Enzymatic cleavage prior to antibody incubation as a method for neuropeptide immunocytochemistry.
When deplasticized Epon sections were treated with endo- and/or exopeptidases prior to incubation with antibodies, the neuropeptide immuno-reactivity of secretory nerves was often altered in a predictable way. Cleavage of neurosecretory material in octopus nerves by trypsin and carboxypeptidase-B enhanced enkephalin-like immunoreactivity, while Molluscan neuropeptide-like immunoreactivity was prevented by tryptic cleavage. The enzyme effects indicated the occurrence of a heptapeptide (Tyr-Gly-Gly-Phe-Met/Leu-Arg-Phe) that contains both the enkephalin and the Molluscan neuropeptide sequence. Vasopressin terminals of the rat neurohypophysis, which presumably contain enkephalin precursor sequences, exhibited enkephalin-like immunostaining after tryptic cleavage. ACTH/beta-endorphin cells of the rat intermediate pituitary, which synthesize the enkephalin sequence at the N-terminus of Beta-endorphin, exhibited enkephalin=like immunoreactivity when sections were treated with alpha-chymotrypsin or trypsin, but not after incubation with leucine-aminopeptidase or carboxypeptidase-B. Enkephalin-like immunostaining could not be induced in any way in ACTH/beta-endorphin cells of the anterior pituitary. Enzymatic cleavage may give additional information in immunocytochemical localization studies on neuropeptide sequences in secretory nerves and hormonal granules.
D000324:Adrenocorticotropic Hormone / Q000378:metabolism; D000818:Animals; D002268:Carboxypeptidases / Q000494:pharmacology; D004723:Endorphins / Q000032:analysis* / Q000378:metabolism; D004745:Enkephalins / Q000032:analysis*; D007124:Immunoenzyme Techniques; D009489:Neurosecretion; D049831:Octopodiformes; D010902:Pituitary Gland / Q000032:analysis; D010904:Pituitary Gland, Posterior / Q000032:analysis; D051381:Rats; D014357:Trypsin / Q000494:pharmacology; D014667:Vasopressins / Q000378:metabolism; D001615:beta-Endorphin
D004723:Endorphins; D004745:Enkephalins; D014667:Vasopressins; D001615:beta-Endorphin; D000324:Adrenocorticotropic Hormone; D002268:Carboxypeptidases; D014357:Trypsin