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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.
Insights
Human microsomal dipeptidase (MDP) was purified and found to be a glycoprotein. This enzyme catalyzes the hydrolysis of S-cysteinyl-glycine adducts and leukotriene D4, suggesting a role in glutathione and leukotriene metabolism.
Area of Science:
- Biochemistry
- Enzymology
- Molecular Biology
Background:
- Human microsomal dipeptidase (MDP), also known as dehydropeptidase-I, is an enzyme found in kidney membranes.
- Its precise physiological role, particularly in the metabolism of specific substrates, requires further elucidation.
Purpose of the Study:
- To purify and characterize human microsomal dipeptidase (MDP).
- To investigate the substrate specificity and potential metabolic roles of human MDP.
Main Methods:
- Solubilization of MDP from kidney membrane fractions using octyl-beta-D-glucoside.
- Purification via ion exchange and affinity chromatography on cilastatin-immobilized Sepharose.
- Characterization using SDS-polyacrylamide gel electrophoresis, endoglycosidase F treatment, and Con A-Sepharose binding.
Main Results:
- Purified human MDP was homogeneous, with an estimated molecular weight of 130 kDa (homodimer) that reduced to 42 kDa after endoglycosidase F treatment, indicating it is a glycoprotein.
- MDP demonstrated catalytic activity in hydrolyzing S-cysteinyl-glycine adducts and converting leukotriene D4 to leukotriene E4.
- Binding to Con A-Sepharose confirmed the glycoprotein nature of human MDP.
Conclusions:
- Human MDP is a purified glycoprotein with a molecular weight of 42 kDa (monomer).
- MDP's substrate specificity suggests a significant role in the metabolism of glutathione and leukotrienes.
- Further research into MDP's function in vivo is warranted.