Thimet oligopeptidase (THOP1), also known as endopeptidase EC 3.4.24.15 (EP24.15), is a zinc peptidase of the M3 family that also includes neurolysin/EC 3.4.24.16 and mitochondrial intermediate peptidase. Widely expressed by mammalian tissues and reported to be present in different subcellular locations, THOP1 is primarily a cytoplasmic enzyme capable of hydrolyzing a number of bioactive peptides and peptides released from by the proteosome, limiting antigenic presentation by MHC class I molecules. The amino acid sequence of human THOP1 is 90% and 86% identical to that of dog/mouse and rat. Thimet oligopeptidase (TOP) is a thiol-dependent metallopeptidase, which can cleave and thereby modulate the activity of many neuropeptides. The enzyme is active in many endocrine tissues, including testis, brain, and pituitary. In rat, the richest source of TOP is the testes, with a specific activity fivefold that of brain. The mechanism whereby rat TOP expression is regulated at the transcriptional level has been examined by reporter gene assay and electromobility shift assays after isolation of 1020 bp of upstream sequence. Computer analysis predicts a number of potential transcription factor-binding sites, which were examined by deletion analysis and DNA-binding studies. The promoter or its deletion fragments were fused to luciferase reporter gene vectors and introduced into GH3 pituitary, COS-1 kidney, MAT-Lu prostate, and GC-2spd(ts) spermatid cells. Two regions of the promoter have been identified: a positively acting region (-901/-219) and a strong negatively acting region (-219/-102). Concomitantly, potential transcription factors interacting with the cis-acting elements of the promoter were studied by gel electromobility shift assays. This work has identified a number of transcription factor-binding sites. However, no differences in the binding behavior in the various cell lines was observed. Applications: Western Blot: 0.1-0.2ug/ml with the appropriate secondary reagents to detect human THOP1. The detection limit for rhTHOP1 is approximately 5 ng/lane under non-reducing and reducing conditions. Immunoprecipitation: This antibody has been used to immunoprecipitate rhTHOP1 from conditioned media of transfected NS0 cells. Direct ELISA: This antibody can be used at 0.5-1.0ug/ml with the appropriate secondary reagents to detect human THOP1. The detection limit for rhTHOP1 is approximately 0.2 ng/well. Optimal dilutions should be determined by each laboratory for each application. Storage and Stability: Lyophilized and reconstituted products are stable for 12 months after receipt at -20C. Reconstitute with sterile PBS. Aliquot to avoid repeated freezing and thawing. Store at -20C. For maximum recovery of product, centrifuge the original vial after thawing and prior to removing the cap. Further dilutions can be made in assay buffer.
