Purification and Characterization of Human Microsomal Dipeptidase

Abstract

Human microsomal dipeptidase (MDP, formerly referred to as dehydropeptidase-I or renal dipeptidase) [EC 3.4.13.11] was solubilized from the membrane fraction of kidney by treatment with octyl-beta-D-glucoside and purified by a procedure including ion exchange chromatography and affinity chromatography on cilastatin-immobilized Sepharose. The purified human MDP was found to be homogeneous on sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. The apparent molecular weight (Mr) was estimated by SDS-polyacrylamide gel electrophoresis under non-reducing conditions to be 130 kDa, comprising a homodimer of two subunits. After treatment with endoglycosidase F, human MDP showed a single band with an apparent Mr of 42 kDa on SDS-polyacrylamide gel electrophoresis. Human MDP was found to bind to Con A-Sepharose and the activity was eluted with methyl-alpha-D-mannopyranoside, suggesting that human MDP is a glycoprotein. We also examined the substrate specificity of human MDP and found that human MDP catalyzed the hydrolysis of S(substituent)-L-cysteinyl-glycine adducts such as L-cystinyl-bis(glycine) and S-N-ethylmaleimide-L-cysteinyl-glycine, as well as the conversion of leukotriene D4 to leukotriene E4. These results suggest that MDP might play an important role in the metabolism of glutathione and leukotriene.

Dug1p Is a Cys-Gly Peptidase of the γ-Glutamyl Cycle of Saccharomyces cerevisiae and Represents a Novel Family of Cys-Gly Peptidases

“…Although ␥GT and Cys-Gly dipeptidase activities were detected in S. cerevisiae cell extracts, only the ␥GT gene (ECM38) has been identified so far. Cys-Gly dipeptidase activity has been identified in humans (5,6), rats (7)(8)(9)(10), pigs (11,12), Escherichia coli (13,14), and other organisms (15,16), and most of them belong to the M17 or the M1 and M19 metallopeptidases gene families (17).…”

Hydrolysis of S-aryl-cysteinylglycine conjugates catalyzed by porcine kidney cortex membrane dipeptidase

“…MDP has been implicated in the metabolism of Cys-Gly and the X-CG, leukotriene D 4 (LTD 4 ) (Adachi et al, 1989). The enzyme is found in the renal brush border microvillar membrane and also in the lung (Brewis et al, 1994;Campbell et al, 1990;Curthoys et al, 1980;Welch and Campbell, 1980).…”

“…In this study, we have purified MDP to homogeneity by PI-PLC-mediated cleavage of the GPI anchor, followed by cilastatin affinity chromatography; this strategy was based on literature precedents of renal MDP purification (Adachi et al, 1989;Hooper et al, 1987;Kraus et al, 2000) (Figure 4). We also synthesized several aromatic Cys-Gly conjugates and developed high performance liquid chromatography (HPLC) separation methods for their quantitation.…”

“…The procedure for purification of MDP was based on previous methods (Adachi et al, 1989;Hooper et al, 1987;Kraus et al, 2000). Sow kidneys were obtained from the Meat Science Laboratory, University of Guelph and stored at -70°C.…”

“…The enzyme purification protocol (Table 3) was based on those of previous publications (Adachi et al, 1989;Hooper et al, 1987;Kraus et al, 2000). Following PI-PLC incubation of the CHAPS-solubilized microsomal fraction and centrifugation, the supernatants and resuspended pellets were assayed for peptidase activity with DNP-CG substrate (500 µM).…”