The association of metalloendopeptidase EC 3.4.24.15 at the extracellular surface of the AtT-20 cell plasma membrane

Endopeptidase EC 3.4.24.15 (EP24.15) is a soluble, neuropeptide-degrading m etalloenzyme, widely expressed in the brain, pituitary and gonads. For the physiological metabolism of neuropeptides, the enzyme should be located ext racellularly, either associated with the plasma membrane or in the extracel lular milieu, Western immunoblot analyses of crude cytosolic and post-nucle ar membrane fractions prepared by differential centrifugation revealed a sl ightly smaller molecular mass (similar to 2 kDa) for EP24.15 in the post-nu clear membrane fraction. This smaller EP24.15 species was also present in a n enriched fraction of plasma membrane prepared by Percoll gradient centrif ugation. To ascertain whether EP24.15 is associated with the extracellular surface of plasma membrane, two sets of experiments were carried out. First , Western immunoblot analysis of AtT-20 cells treated with the membrane-imp ermeable thiol-cleavable cross-linker, 3,3'-dithio-bis(sulpho-succinimidyl- propionate) (DTSSP), indicated an extracellular membrane association. After cross-linking and thiol-reduction, a distinct band corresponding to EP24.1 5 was significantly diminished under non-reducing conditions, Second, immun ocytochemical studies performed at 4 degrees C on non-permeabilized AtT-20 cells (i.e., non-fixed to prevent antibody internalization), indicated that EP24.15 was expressed on the surface of the AtT-20 cells. We furthermore d etermined that EP24.15 enzymatic activity is present on the extracellular s urface of the cell discernable from the secreted enzyme. These results sugg est that the EP24.15 is associated with the extracellular surface of the At T-20 cell plasma membrane and is enzymatically active. Taken together, the results are consistent with a putative role in the degradation of neuropept ides acting at the external cell surface. (C) 1999 Elsevier Science B.V. Al l rights reserved.