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Glutathione-degrading enzymes of microvillus membranes
Abstract:
Microvillus membranes from rat kidney, jejunum, and epididymis have been purified by the Ca precipitation method. The membranes exhibit enrichment in specific activities of gamma-glutamyl transpeptidase, aminopeptidase M, and a dipeptidase. The latter has been characterized and shown to be the principal activity responsible for the hydrolysis of S derivatives of Cys-Gly (including cystinyl-bis-glycine (Cys-bis-Gly) and 5-hydroxy-6-S-cysteinylglycyl-1-7,9-trans-11,14-cis-eicosatetraenoic acid (leukotriene D4)). A method is described for the simultaneous purification of papain-solubilized forms of the three enzymes from renal microvilli. Dipeptidase (Mr = 105,000) appears to be a zinc metalloprotein composed of two Mr = 50,000 subunits. The enzyme is severalfold more effective in the hydrolysis of dipeptides than aminopeptidase M. Dipeptidase, in contrast to aminopeptidase M, is inhibited by thiol compounds; Cys-Gly, in particular, is a potent inhibitor (Ki = 20 microM). The inhibition of dipeptidase by thiols has been employed to probe the relative significance of dipeptidase and aminopeptidase M in the metabolism of glutathione and its derivatives at the membrane surface.
Insights
Researchers purified microvillus membranes and identified a key dipeptidase enzyme. This enzyme plays a crucial role in metabolizing glutathione derivatives, offering insights into membrane surface biochemistry.
Area of Science:
- Biochemistry
- Cell Biology
- Enzymology
Background:
- Microvillus membranes are crucial for nutrient absorption and transport.
- Specific enzymes like gamma-glutamyl transpeptidase and aminopeptidase M are abundant in these membranes.
- Understanding the function of associated peptidases is vital for comprehending membrane-associated metabolism.
Purpose of the Study:
- To purify and characterize microvillus membranes from rat kidney, jejunum, and epididymis.
- To identify and elucidate the properties of a specific dipeptidase involved in the hydrolysis of S-derivatives of Cys-Gly.
- To investigate the role of this dipeptidase in the metabolism of glutathione and its derivatives.
Main Methods:
- Calcium precipitation method for purifying microvillus membranes.
- Enzyme activity assays for gamma-glutamyl transpeptidase, aminopeptidase M, and dipeptidase.
- Papain solubilization for simultaneous enzyme purification from renal microvilli.
- Characterization of dipeptidase including molecular weight and inhibition kinetics.
Main Results:
- Purified microvillus membranes showed enrichment in gamma-glutamyl transpeptidase, aminopeptidase M, and a dipeptidase.
- The characterized dipeptidase is the primary enzyme responsible for hydrolyzing S-derivatives of Cys-Gly, including leukotriene D4.
- Dipeptidase, a zinc metalloprotein, is more effective than aminopeptidase M in dipeptide hydrolysis and is inhibited by thiol compounds like Cys-Gly.
Conclusions:
- A novel method allows for the simultaneous purification of key enzymes from renal microvilli.
- Dipeptidase's distinct properties and potent inhibition by thiols highlight its specific role in membrane-associated peptide metabolism.
- The inhibition of dipeptidase by thiols provides a tool to assess its significance in the metabolism of glutathione and related compounds.