Identification, purification, and subcellular localization of prostate-specific membrane antigen PSM' protein in the LNCaP prostatic carcinoma cell line.
An alternatively spliced variant of prostate-specific membrane antigen (PSMA) designated PSM' was originally described following identification of its mRNA in normal prostate. We have purified the PSM' protein from LNCaP cells using two immunoaffinity columns in tandem. The first column contained a monoclonal antibody (7E11) that was reactive with the NH2 terminus of PSMA, which specifically depleted the LNCaP lysate of full-length PSMA. The nonbinding fraction was then passed over a second column composed of a monoclonal antibody (PEQ226.5), the epitope of which was located within the 134-437 domain of PSMA and shared with PSM'. The protein eluted from the second immunoaffinity column produced a Mr 95,000 band on SDS-PAGE, which was slightly lower than the full-length PSMA at Mr 100,000. The band was NH2-terminally sequenced through 15 residues, and the assigned sequence coincided with the predicted sequence for PSM' protein minus the first two NH2 terminus amino acids. The PSM' protein, therefore, began with residue 60 of PSMA (alanine). LNCaP cells were fractionated, and PSM' was localized to the cytoplasm.
Grauer
L S
LS
Hybritech Incorporated, Beckman Coulter, Inc., San Diego, California 92196-9006, USA.
Lawler
K D
KD
Marignac
J L
JL
Kumar
A
A
Goel
A S
AS
Wolfert
R L
RL
D000818:Animals; D000951:Antigens, Neoplasm / Q000302:isolation & purification*; D000954:Antigens, Surface; D002268:Carboxypeptidases / Q000032:analysis / Q000235:genetics / Q000302:isolation & purification*; D002462:Cell Membrane / Q000737:chemistry; D003593:Cytoplasm / Q000737:chemistry; D043425:Glutamate Carboxypeptidase II; D006801:Humans; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D008970:Molecular Weight; D011471:Prostatic Neoplasms / Q000737:chemistry* / Q000648:ultrastructure; D012333:RNA, Messenger / Q000032:analysis; D014407:Tumor Cells, Cultured
D000951:Antigens, Neoplasm; D000954:Antigens, Surface; D012333:RNA, Messenger; D002268:Carboxypeptidases; C473848:FOLH1 protein, human; D043425:Glutamate Carboxypeptidase II