TY - JOUR
T1 - The association of metalloendopeptidase EC 3.4.24.15 at the extracellular surface of the AtT-20 cell plasma membrane
AU - Crack, Peter J.
AU - Wu, T. J.
AU - Cummins, Philip M.
AU - Ferro, Emer S.
AU - Tullai, John W.
AU - Glucksman, Marc J.
AU - Roberts, James L.
N1 - Funding Information:
The authors wish to thank Dr. Ruth M.E. Chalmers-Redman for technical advice. This work was supported by a National Science Foundation grant IBN-9512113 (J.L.R and M.J.G.). Additional support was provided by PHS 2T32-DA017135-16 (P.M.C.) and NIH T32DK07645 (T.J.W. and J.W.T.). Support for P.J.C. was provided in part by the John Douglas French Foundation for Alzheimer's Research. Support for E.S.F. was provided in part by F.A.P.E.S.P.
Copyright:
Copyright 2007 Elsevier B.V., All rights reserved.
PY - 1999/7/24
Y1 - 1999/7/24
N2 - Endopeptidase EC 3.4.24.15 (EP24.15) is a soluble, neuropeptide- degrading metalloenzyme, widely expressed in the brain, pituitary and gonads. For the physiological metabolism of neuropeptides, the enzyme should be located extracellularly, either associated with the plasma membrane or in the extracellular milieu. Western immunoblot analyses of crude cytosolic and post-nuclear membrane fractions prepared by differential centrifugation revealed a slightly smaller molecular mass (~ 2 kDa) for EP24.15 in the post-nuclear membrane fraction. This smaller EP24.15 species was also present in an enriched fraction of plasma membrane prepared by Percoll gradient centrifugation. 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-impermeable, 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 EP24.15 was significantly diminished under non-reducing conditions. Second, immunocytochemical studies performed at 4°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 determined that EP24.15 enzymatic activity is present on the extracellular surface of the cell discernable from the secreted enzyme. These results suggest that the EP24.15 is associated with the extracellular surface of the AtT-20 cell plasma membrane and is enzymatically active. Taken together, the results are consistent with a putative role in the degradation of neuropeptides acting at the external cell surface.
AB - Endopeptidase EC 3.4.24.15 (EP24.15) is a soluble, neuropeptide- degrading metalloenzyme, widely expressed in the brain, pituitary and gonads. For the physiological metabolism of neuropeptides, the enzyme should be located extracellularly, either associated with the plasma membrane or in the extracellular milieu. Western immunoblot analyses of crude cytosolic and post-nuclear membrane fractions prepared by differential centrifugation revealed a slightly smaller molecular mass (~ 2 kDa) for EP24.15 in the post-nuclear membrane fraction. This smaller EP24.15 species was also present in an enriched fraction of plasma membrane prepared by Percoll gradient centrifugation. 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-impermeable, 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 EP24.15 was significantly diminished under non-reducing conditions. Second, immunocytochemical studies performed at 4°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 determined that EP24.15 enzymatic activity is present on the extracellular surface of the cell discernable from the secreted enzyme. These results suggest that the EP24.15 is associated with the extracellular surface of the AtT-20 cell plasma membrane and is enzymatically active. Taken together, the results are consistent with a putative role in the degradation of neuropeptides acting at the external cell surface.
KW - AtT-20 cell
KW - EP24.15
KW - Extracellular membrane association
KW - Metalloendopeptidase
UR - http://www.scopus.com/inward/record.url?scp=0033600514&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=0033600514&partnerID=8YFLogxK
U2 - 10.1016/S0006-8993(99)01494-8
DO - 10.1016/S0006-8993(99)01494-8
M3 - Article
C2 - 10415366
AN - SCOPUS:0033600514
VL - 835
SP - 113
EP - 124
JO - Brain Research
JF - Brain Research
SN - 0006-8993
IS - 2
ER -