Characterization of dehydropeptidase I in the rat lung

Abstract

The activity of dehydropeptidase I in rat tissues decreases in the order of lung > kidney > liver-spleen > other tissues, while aminopeptidase activity is high in the kidney, and lower in the lung than in other tissues.Dehydropeptidase I was solubilized from the membrane fraction of rat lung by treatment with papain and purified by DEAE-cellulose column chromatography, affinity chromatography on concanavalin-A-Sepharose and highperformance liquid chromatography gel filtration. The purified preparation was found to be homogeneous on sodium dodecyl sulfate/polyacrylamide gel electrophoresis. The relative molecular mass was estimated to be 150000 by gel filtration, comprising a homodimer of two 80000-Mr subunits. The enzyme activity was inhibited by cilastatin, o-phenanthroline and ATP. This enzyme catalyzed the hydrolysis of S(substituent)-L-cysteinyl-glycine adducts such as L-cystinyLbis(g1ycine) and N-ethylmaleimide-S-L-cysteinyl-glycine, as well as the conversion of leukotriene D4 to F4. Furthermore it catalyzed a hydrolytic splitting of L-Leu-L-Leu, but not S-benzyl-L-cysteine p-nitroanilide, which is a good substrate for aminopeptidase. Our enzyme preparation was immunologically identical to the rat renal dehydropeptidase I. The physiological significance of the pulmonary dehydropeptidase I on the metabolism of glutathione and its adducts is discussed.The degradation of reduced and oxidized glutathione and of glutathione adducts is initiated by y-glutamyl transferase [l , 21, which removes the y-glutamyl moiety to form L-cysteinylglycine(L-Cys-Gly), L-cystinyl-bis(g1ycine) (~-m y s -G l y J and S (substituent)-L-Cys-Gly adducts, respectively. These dipeptides or dipeptide adducts are further hydrolyzed by dipeptidases or by aminopeptidase M [3,4].Campbell et al. purified the dipeptidase hydrolyzing glycyl-dehydrophenylalanine from porcine [S -71 and human [8] kidneys. Recently, it has been reported that this enzyme specifically hydrolyzes the dehydropeptide bond in carbapenem antibiotics, and it has been referred to as dehydrope tiddse I (DHP-I) [9, 101. We have previously shown that L-h ys ys-Gly, and S(substituent)-L-Cys-Gly adducts are mainly hydrolyzed by the renal DHP-I rather than by aminopeptidase M, and we suggested that DHP-I is an important enzyme in the metabolism of glutathione and glutathione adducts [Ill.In the present study, the tissue distribution of activities of DHP-I and aminopeptidase in rats was investigated, and DHP-I was purified from the rat lung, where its specific activity was found to be higher than in the kidney. The immunological identity between pulmonary and renal DHP-I was also compared using anti-(renal DHP-I) antibody.

Membrane Dipeptidase Is the Receptor for a Lung-targeting Peptide Identified by in Vivo Phage Display

“…MDP expression has also been observed on the endothelial cells of submucosal microvessels in the human trachea (26). The level of MDP activity was shown to be highest in the lung (27,28).…”

Leukotriene D 4 and cystinyl-bis-glycine metabolism in membrane-bound dipeptidase-deficient mice

“…However, unlike GGT-deficient mice, which show ϳ2,500-fold higher levels of GSH in the urine, MBD-deficient mice have only a 3-to 5-fold elevation of urinary cys-bis-gly (7.9 M in wild-type versus 33.8 M in MBD-deficient mice). MBD has been implicated as the enzyme responsible for cleavage of cys-bis-gly to cysteine and glycine (11,27). We adapted a reversed phase ion-exchange HPLC method used to separate GSH and GSSG (36) to examine cys-bis-gly cleavage to cysteine.…”

“…The degradation of reduced and oxidized glutathione (GSH and GSSG) is initiated by GGT to form cysteinyl-glycine (cys-gly) and cystinyl-bis-glycine (cysbis-gly) (11,12). It is generally believed that cys-bis-gly is further metabolized into its constituent amino acids by MBD (11,12,25,27). Because the majority of cys-gly in the renal tubules exists in the oxidized form, the recycling of GSH is postulated to be mediated via the consecutive actions of these two enzymes (12).…”

The Art of Fusion: From Penams and Cephems to Penems

“…This invalidates studies in these animal species. Furthermore, DHP-I is localised on the plasma membranes not only in the kidney but also in the lung, liver, spleen and small intestine of small rodents [62,63], so that most penems are degraded in these tissues.…”