Purification and characterization of human microsomal dipeptidase.
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.
Adachi
H
H
Suntory Institute for Biomedical Research, Osaka.
Kubota
I
I
Okamura
N
N
Iwata
H
H
Tsujimoto
M
M
Nakazato
H
H
Nishihara
T
T
Noguchi
T
T
D000596:Amino Acids / Q000032:analysis; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D004150:Dipeptidases / Q000032:analysis / Q000276:immunology / Q000302:isolation & purification*; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007525:Isoelectric Focusing; D007668:Kidney / Q000201:enzymology; D008861:Microsomes / Q000201:enzymology*; D010455:Peptides / Q000032:analysis
D000596:Amino Acids; D010455:Peptides; D004150:Dipeptidases