Neuropeptide-like immunoreactivities and carboxypeptide H activity associated with bovine brain clathrin coated...

Chromatographically purified clathrin coated vesicles (CCV) from bovine brain gray matter were collected and analyzed for their adaptor protein profile via SDS-PAGE, for their carboxypeptidase H (CPH) activity via radioenzymatic assays and for their NP (NPY1-36, CCK-8 and SS-14) content by radioimmunoassays. The results reveal the expression of CPH and neuropeptide-like immunoreactivities (NP-Li: NPY-Li, CCK-Li and SS-Li) associated with CCV. CPH activity associated with CCV is stimulated by cobalt, inhibited by 5 microM GEMSA and has an acidic pH optimum (4.5-6.5). The Michaelis-Menten kinetics assay of CPH in CCV reveals a Km = 28.9 microM and Vmax = 1.88 nmol/min/mg at pH 4.5 using 3H.Bz-FAR as a substrate. The NP-Li and CPH were mainly associated with the ascending portion of the CCV peak suggesting that these molecules are found in the larger diameter CCV subpopulation associated with CCV of the trans Golgi network (TGN), endocytic or recycling CCV. Analysis of the adaptor protein profile of the material eluting from the Sephacryl S-1000 column demonstrates a wide distribution of the Golgi-associated AP-1 (HA-I) molecules (AP47 and AP19) in relation to the CCV peak, spanning both the ascending and descending portions of the CCV peak. This adaptor protein pattern is consistent with the inherent size microheterogeneity of TGN CCV, large diameter CCV of the regulated secretory pathway and smaller diameter CCV of the lysosomal pathway.