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PubMed1987

Release and functional characterization of the leukotriene D4-metabolizing enzyme (dipeptidase) from human polymorphonuclear leucocytes.

Polymorphonuclear leucocytes released LTD4-dipeptidase activity in a time-, calcium-, and cell number-dependent fashion. The LTD4-dipeptidase released from polymorphonuclear leucocytes (PMN) by incubation with calcium (0.91 mM) was detectable up to a cell concentration of 1 X 10(6)/ml and increased with higher concentrations. Maximal LTD4-dipeptidase activity within the extracellular environment was detected after 15 min of incubation (2 X 10(7)/ml) in the presence of 2-4.5 mM calcium or after 30 min, when stimulation was carried out with 0.91 mM calcium. The activity of the released LTD4-dipeptidase was modulated by various metal ions and other compounds. The addition of Mn2+, Co2+, and Zn2+ (final concentration 1 mM) enhanced the LTD4-dipeptidase activity, while Cu2+ led to a complete inhibition. In the absence of exogenous calcium EDTA inhibited LTD4-dipeptidase. Calcium up to a concentration of 5 and 10 mM decreased the dipeptidase activity. The LTD4-dipeptidase is not affected by bestatin, leupeptin, or N-ethyl-maleinimide (NEM). The Km of LTD4-dipeptidase for LTD4 was 0.95 +/- 0.2 microM and Vmax was 737.5 +/- 112.5 pmol/min X mg protein (n = 3 +/- SEM). The highest LTD4-dipeptidase activity was obtained at physiological pH values. LTD4-dipeptidase activity can also be released from other cell types, but the enzyme activity from human PMN exceeded that of other cells (e.g. human lymphocytes/monocytes and basophils (LMB) and human lung cell suspension).

Basophils↗

PubMed1987

Hydrolysis of the brain dipeptide N-acetyl-L-aspartyl-L-glutamate. Identification and characterization of a novel N-acetylated alpha-linked acidic dipeptidase activity from rat brain.

High performance liquid chromatography studies documented the presence of an enzyme activity, N-acetylated alpha-linked acidic dipeptidase (NAALA dipeptidase), in rat brain membranes that cleaves the endogenous brain dipeptide, N-acetyl-L-aspartyl-L-glutamate to N-acetyl-aspartate and glutamate. With ion exchange chromatography, which quantitatively separated [3,4-3H]glutamate from N-acetyl-L-aspartyl-L-[3,4-3H]glutamate, we found that NAALA dipeptidase activity was essentially restricted to nervous tissue and kidney. We characterized NAALA dipeptidase activity in lysed synaptosomal membranes obtained from rat forebrain. Membrane-bound NAALA dipeptidase activity was optimal between pH 6.0 and 7.4 at 37 degrees C. Eadie-Hofstee analysis of kinetic data revealed a rather high apparent affinity for N-acetyl-L-aspartyl-L-glutamate with a Km = 540 nM and a Vmax = 180 nM/mg of protein/min. While NAALA dipeptidase showed a requirement for monovalent anions such as Cl-, the polyvalent anions phosphate and sulfate inhibited enzyme activity 50% at 100 microM and 1 mM, respectively. The divalent metal ion chelators EGTA, EDTA, and o-phenanthroline completely abolished activity, which was partially restored by manganese. Treatment of membranes with 1 mM dithiothreitol abolished NAALA dipeptidase activity. NAALA dipeptidase activity was also sensitive to the aminopeptidase inhibitors bestatin and puromycin, although not to the selective aminopeptidase A inhibitor amastatin. Structure-activity relationships inferred from inhibitor studies suggest that this enzyme shows specificity for N-acetylated alpha-linked acidic dipeptides. NAALA dipeptidase was also potently inhibited by the excitatory amino acid agonist L-quisqualate. Comparison of the properties of NAALA dipeptidase to those of previously characterized enzymes suggests that this is a novel peptidase which may be involved in the synaptic degradation of N-acetyl-L-aspartyl-L-glutamate.

Animals↗

PubMed1992

Characterization of two dipeptidases purified from hepatic schistosome egg granulomas in mice. Leukotriene D4 hydrolases of granulomatous tissue.

Extracts prepared from tissue with granulomatous inflammation experimentally produced in liver of CBA-strain mice showed increased hydrolysis of leukotriene D4 (LTD4), Leu-Leu and Ala-Gly as compared with normal hepatic cells. Two dipeptidases, Leu-Leu dipeptidase and Ala-Gly dipeptidase, were purified from hepatic granulomas, and quantitative conversion of LTD4 into leukotriene E4 (LTE4) by both enzymes was demonstrated. M(r) values of the purified enzymes were 178,000 for Leu-Leu dipeptidase and 183,000 for Ala-Gly dipeptidase. The enzymes showed homogeneity, appearing as a single band on SDS/PAGE, and the M(r) values of the subunits were 56,000 and 57,000 for Leu-Leu and Ala-Gly dipeptidase respectively. The amino acid compositions of the two enzymes differed considerably from each other. The activity of Leu-Leu dipeptidase was inhibited by bestatin and captopril and stabilized with MnCl2. The Km for LTD4 was 25 microM with a V(max.) of 49.0 mumols/min per mg. In contrast, the activity of Ala-Gly dipeptidase was inhibited by cilastatin, cytinylglycine, EDTA and dithiothreitol, and also by captopril. The Km for LTD4 was 5.3 microM with a V(max.) of 50.4 mumols/min per mg. The findings indicate that the conversion of LTD4 into LTE4 by microsomal dipeptidases is elevated during granulomatous tissue reaction. This enzyme activity may become useful for biochemical quantification of the pathological tissue reaction that occurs in organized granulomas.

Amino Acid Sequence↗

PubMed1994

Activation of the glycosyl-phosphatidylinositol-anchored membrane dipeptidase upon release from pig kidney membranes by phospholipase C.

Incubation of pig kidney microvillar membranes with Bacillus thuringiensis or Staphylococcus aureus phosphatidylinositol-specific phospholipase C (PI-PLC) resulted in the release of a number of glycosyl-phosphatidylinositol (GPI)-anchored hydrolases, including alkaline phosphatase (EC 3.1.3.1), amino-peptidase P (EC 3.4.11.9), membrane dipeptidase (EC 3.4.13.19), 5'-nucleotidase (EC 3.1.3.5) and trehalase (EC 3.2.1.28). Of these five ectoenzymes only for membrane dipeptidase was there a significant (approx. 100%) increase in enzymic activity upon release from the membrane. Maximal activation occurred at a PI-PLC concentration 10-fold less than that required for maximal release. In contrast solubilization of the membranes with n-octyl beta-D-glucopyranoside had no effect on the enzymic activity of membrane dipeptidase. A competitive e.l.i.s.a. with a polyclonal antiserum to membrane dipeptidase indicated that the increase in enzymic activity was not due to an increase in the amount of membrane dipeptidase protein. Although PI-PLC cleaved the GPI anchor of the affinity-purified amphipathic form of pig membrane dipeptidase there was no concurrent increase in enzymic activity. In the absence of PI-PLC, membrane dipeptidase in the microvillar membranes hydrolysed Gly-D-Phe with a Km of 0.77 mM and a Vmax. of 602 nmol/min per mg of protein. However, in the presence of a concentration of PI-PLC which caused maximal release from the membrane and maximal activation of membrane dipeptidase the Km was decreased to 0.07 mM while the Vmax. remained essentially unchanged at 624 nmol/min per mg of protein. Overall these results suggest that cleavage by PI-PLC of the GPI anchor on membrane dipeptidase may relax conformational constraints on the active site of the enzyme which exist when it is anchored in the lipid bilayer, thus resulting in an increase in the affinity of the active site for substrate.

5'-Nucleotidase↗

PubMed1977

Histological localization of two dipeptidases in the pig small intestine and liver, using immunofluorescence.

Antibodies were raised in rabbits against glycylleucine dipeptidase (EC 3.4.13.2) and proline dipeptidase (EC 3.4.13.9) purified from the pig. IgG fractions were isolated and purified by affinity chromatography on Sepharose (dipeptidase) columns and then used in histological studies on the small intestine and the liver from pig, using the double layer immunofluorescence-staining technique. In the small intestine the dipeptidases were predominantly localized in the cytoplasm of the enterocytes. A reaction was also seen in the leukocyte cytoplasm in the stroma of the small intestine. The dipeptidases were present in the villi but not in the crypts. In the liver the dipeptidases were localized in the cytoplasm of the hepatocyte, but strong fluorescence was also obtained in the epithelium of the bile ducts. Glycylleucine dipeptidase and proline dipeptidase have been isolated from liver tissue and they were shown to be immunologically identical with the purified intestinal dipeptidases.

Animals↗

PubMed1995

N-acetylated alpha-linked acidic dipeptidase is expressed by non-myelinating Schwann cells in the peripheral nervous system.

N-acetylated alpha-linked acidic dipeptidase is a membrane-bound brain peptidase which cleaves the neuropeptide N-acetyl-aspartyl-glutamate to N-acetyl-aspartate and glutamate. In the present study, we have determined the localization of N-acetylated alpha-linked acidic dipeptidase in the peripheral nervous system. Using enzyme assays and immunoblotting, we demonstrate that sciatic nerve, phrenic nerve, cervical dorsal root ganglion and superior cervical ganglion contain N-acetylated alpha-linked acidic dipeptidase activity as well as an N-acetylated alpha-linked acidic dipeptidase-like protein. Furthermore, we show that N-acetylated alpha-linked acidic dipeptidase-like immunoreactivity is extensively co-localized in peripheral nerves with immunoreactivity for glial fibrillary acidic protein, a known marker for non-myelinating Schwann cells. Using electron microscopy, we demonstrate N-acetylated alpha-linked acidic dipeptidase-like immunoreactivity in cell membranes of non-myelinating Schwann cells in the superior cervical ganglion. These results show that N-acetylated alpha-linked acidic dipeptidase is expressed in the peripheral nervous system by non-myelinating Schwann cells. This cellular localization suggests that N-acetylated alpha-linked acidic dipeptidase may be involved in the signalling between axons and Schwann cells, for example during development or regeneration.

Animals↗

PubMed1984

A fluorometric method for dipeptidase activity measurement in urine, using L-alanyl-L-alanine as substrate.

A new method for the measurement of urinary dipeptidase activity is described. The action of dipeptidase on L-Ala-L-Ala results in production of an L-alanine, and this amino acid is simultaneously determined by an L-alanine dehydrogenase-diaphorase system. As urinary substances do not affect this reaction, the measurement can be accomplished without prior dialysis. The mean value +/- S.D. for normals was found to be 12.0 +/- 4.4 IU/g of creatinine. Elevated values were found in chronic nephritis (55.9 +/- 35.0 IU/g of creatinine, P less than 0.001 vs. normal), acute nephritis (46.6 +/- 29.9 IU/g of creatinine, P less than 0.001), and nephrotic syndrome (43.3 +/- 36.5 IU/g of creatinine, P less than 0.001). The dipeptidase activity thus measured showed a significant correlation with dipeptidase activity against L-Leu-L-Leu as substrate. On disc polyacrylamide gel electrophoresis, the urinary dipeptidase of a patient with chronic nephritis appeared as one band with similar mobility to human kidney dipeptidase F. Urinary dipeptidase in a patient with chronic nephritis was identical to human kidney dipeptidase on double immunodiffusion analysis.

Alanine Dehydrogenase↗

PubMed1990

Rat brain N-acetylated alpha-linked acidic dipeptidase activity. Purification and immunologic characterization.

N-Acetylated alpha-linked acidic dipeptidase (NAALA dipeptidase) is a membrane-bound metallopeptidase that cleaves glutamate from the endogenous neuropeptide N-acetyl-L-aspartyl-L-glutamate. In this report, we have solubilized NAALA dipeptidase activity from synaptosomal membranes with Triton X-100 and purified it to apparent homogeneity by sequential column chromatography on DEAE-Sepharose, CM-Sepharose, and lentil lectin-Sepharose. This procedure resulted in a 720-fold purification with 1.6% yield. The purified ezyme migrated as a single silver-stained band on a sodium dodecyl sulfate gel with an apparent molecular weight of 94 kDa. Using an enzymatic stain to visualize NAALA dipeptidase activity within a gel matrix, we have confirmed that the 94-kDa band is, indeed, NAALA dipeptidase. The purified enzyme was characterized and found to be pharmacologically similar to NAALA dipeptidase activity described previously in synaptosomal membrane extracts. Using the purified NAALA dipeptidase as antigen, we have raised specific and high titer polyclonal antibodies in guinea pig. Immunocytochemical studies show intense NAALA dipeptidase immunoreactivity in the cerebellar and renal cortices.

Animals↗

PubMed1983

Association of renal dipeptidase with the Triton-insoluble fraction of kidney microvilli.

Renal dipeptidase, previously identified as a component of renal microvilli, has been investigated to determine its orientation within this organelle. Digestion of porcine renal microvilli with papain released essentially all of aminopeptidase M, an outer membrane marker enzyme from the microvilli within one hour; whereas less than 10% of renal dipeptidase was released under the same conditions. Antibody to purified renal dipeptidase produced 50% inhibition of the purified enzyme at an antibody/antigen molar ratio of 2:1. Inhibition by the renal dipeptidase-directed antibody was not observed when the enzyme was bound within the microvillar structure. Demembranation of the microvilli with Triton X-100 resulted in a distribution of 68% of renal dipeptidase in the insoluble pellet and 32% in the soluble supernatant. The same detergent treatment released 92% of animopeptidase M into the supernatant. These results indicate that renal dipeptidase is not located at the luminal surface of the microvillus membrane where it would be available for release of papain, inhibition by antibody, or solubilization by detergent. Fractionation of the Triton-insoluble pellet with 2 M NaCl resulted in the release of 64% of the peptidase into a pellicle fraction separated from insoluble pellet and soluble supernatant. Finally extraction of Triton-insoluble pellet with 0.05 mM ATP-0.10 mM MgCl2 X 6 H2O solubilized 57% of the renal dipeptidase.

Animals↗

PubMed1997

Identification of membrane dipeptidase as a major glycosyl-phosphatidylinositol-anchored protein of the pancreatic zymogen granule membrane, and evidence for its release by phospholipase A.

Membrane dipeptidase (EC 3.4.13.19) enzyme activity that is inhibited by cilastatin has been detected in pancreatic zymogen granule membranes of human, porcine and rat origin. Immunoelectrophoretic blot analysis of human and porcine pancreatic zymogen granule membranes with polyclonal antisera raised against the corresponding kidney membrane dipeptidase revealed that the enzyme is a disulphide-linked homodimer of subunit mass 61 kDa in the human and 45 kDa in the pig. Although membrane dipeptidase was, along with glycoprotein-2, one of the only two major components of carbonate high pH-washed membranes, no enzyme activity or immunoreactivity was detected in the zymogen granule contents. Digestion with bacterial phosphatidylinositol-specific phospholipase C (PI-PLC), and subsequent recognition by antibodies specific for the cross-reacting determinant, revealed that membrane dipeptidase in human and porcine pancreatic zymogen granule membranes is glycosyl-phosphatidylinositol-anchored. Membrane dipeptidase was released from the pancreatic zymogen granule membranes by an endogenous hydrolase, and the released form migrated as a disulphide-linked dimer on SDS/PAGE under non-reducing conditions. Under reducing conditions it migrated with the same apparent molecular mass as the membrane-bound form, and was still a substrate for bacterial PI-PLC. Treatment of kidney microvillar membranes with phospholipase A2 resulted in the release of membrane dipeptidase in a form that demonstrated electrophoretic and cilastatin-Sepharose binding properties identical to those of the endogenously released form of the enzyme from zymogen granule membranes. These results indicate that the glycosyl-phosphatidylinositol anchor on the pancreatic membrane dipeptidase is cleaved by an endogenous hydrolase, probably a phospholipase A, and that this cleavage may promote the release of the protein from the membrane.

Animals↗

PubMed1989

Inhibition by bestatin of a mouse ascites tumor dipeptidase. Reversal by certain substrates.

Bestatin, [(2S,3R)-3-amino-2-hydroxy-4-phenyl-butanoyl]-L-leucine, a known inhibitor of aminopeptidases, is shown to be a potent linear competitive inhibitor (KI,2.7 nM) of a dipeptidase purified from Ehrlich-Lettré hyperdiploid mouse ascites tumor cells. This inhibition can be classified as "slow binding" but not as "tight binding." Substrate protects the enzyme from bestatin inhibition when enzyme and inhibitor are in approximately equimolar concentrations. Addition of substrate (6 mM) partially (by about 20%) reverses dipeptidase inhibition by bestatin, but the time required for maximum recovery depends on the nature of the substrate. Substrates with lower Km (0.28-1.4 mM) values that exhibit substantial substrate inhibition require longer times (23-65 min) than those with higher Km values that show little substrate inhibition. Substrates with Km values higher than 1.5 mM do not reverse inhibition. The inhibition of the tumor dipeptidase by bestatin has been compared with inhibition by a variety of inhibitors of other Zn-metallo-proteolytic enzymes. These inhibitors were far less potent (KI, 0.063-10 mM), indicating a difference between the tumor dipeptidase and other enzymes of that class. Our results are discussed in terms of a postulated model of the bestatin molecule in the active site of the tumor dipeptidase, an enzyme which has not been studied by x-ray crystallographic means. The phenyl group of bestatin is placed in a hydrophobic pocket that is external but adjacent to the active site of the tumor dipeptidase. The shape of this pocket, as it appears from our results plus modeling, is such that only certain R groups of substrate can fit. The existence of such a pocket might explain the differential effect of substrates in the reversal of bestatin inhibition of the dipeptidase and also might explain substrate inhibition by misalignment of R groups into this pocket.

Animals↗

PubMed1983

Conversion of leukotriene D4 to leukotriene E4 by a dipeptidase released from the specific granule of human polymorphonuclear leucocytes.

Leukotriene D4 (LTD4), the most active spasmogenic leukotriene constituent of the slow reacting substance of anaphylaxis was converted by suspended human polymorphonuclear leucocytes (PMNs) to a single, less polar metabolite which was not further catabolized. This product was identified as leukotriene E4 (LTE4) by its retention time during reverse phase-high performance liquid chromatography (RP-HPLC) and subsequent bioassay on the guinea-pig ileum. LTD4 with a retention time of 21 +/- 1.6 min (mean +/- SD) and a contractile activity of 5.0 +/- 0.4 u./pmol (mean +/- SD) was quantitatively converted extracellularly by PMNs to LTE4 with a retention time of 26 +/- 1.8 min and a contractile activity of 1.2 +/- 0.3 u./pmol. Subcellular fractionations of PMNs revealed the recovered LTD4-to-LTE4 converting activity, termed LTD4 dipeptidase, to be localized only in he granule fraction. There was a time- and calcium-dependent extracellular release of LTD4 dipeptidase in association with lysozyme (r = 0.97, n = 16, P less than 0.001), a constituent of both specific and azurophilic granules, in the absence of release of cytoplasmic lactate dehydrogenase (LDH) and of beta-glucuronidase from the azurophilic granule. Phorbol myristate acetate (PMA), which selectively induces secretion of specific granules, released lysozyme and the LTD4 dipeptidase in a constant dose-dependent manner from PMNs (r = 0.96, n = 8, P less than 0.001). Calcium ionophore A23187 at concentrations less than 10(-7) M stimulated the parallel secretion of LTD4 dipeptidase and lysozyme (r = 0.91, n = 9, P less than 0.005), dipeptidase and lysozyme (r = 0.91, n = 9, P less than 0.005), whereas higher concentrations resulted in secretion of beta-glucuronidase and additional lysozyme without further release of dipeptidase. Thus, human PMNs can convert LTD4 to LTE4, a less vasoactive and spasmogenic leukotriene, via the secretion of a dipeptidase associated with the specific granules.

Cells, Cultured↗

PubMed1977

Intestinal dipeptidases and the dipeptide transport in the monkey and in man.

Among the powerful dipeptidases of the cytosol fraction the well-characterized 'master' dipeptidase of broad substrate specificity is an example of a 'true' dipeptidase. There are only a limited number of peptidases which together can hydrolyse the total range of the theoretically possible dipeptides. In the lumen, the dipeptides of dietary origin apparently enter the cell through a single transport system of broad specificity in the monkey and possibly also in man. Perfusion studies indicated impared absorption of glycine and Gly-Gly in Indian compared to English subjects, but the kinetic advantage of dipeptide uptake over that of the free amino acid was maintained. In patients with tropical sprue, apart from decreased peptide absorption, there was an increased 'backflow' of the constituent amino acids. The back-flow assumes nutritional significance since dipeptide uptake is competitively inhibited by amino acids of a certain specificity. Inhibition of brush border glycylglycine hydrolase (glycylglycine dipeptidase, EC 3.4.13.1) by L- leucine is non-competitive while that of cytosol enzyme is competitive. Certain other amino acids have no effect on glycylleucine hydrolase (glycylleucine dipeptidase, EC 3.4.13.2) from either fraction but inhibit Gly-Leu uptake. Thus, the inhibition of dipeptide uptake appears to be a consequence of interaction of amino acids directly with the dipeptide transport system and not with dipeptidases.

Amino Acids↗

PubMed1990

cDNA cloning and expression in Xenopus laevis oocytes of pig renal dipeptidase, a glycosyl-phosphatidylinositol-anchored ectoenzyme.

Clones expressing renal dipeptidase (EC 3.4.13.11) have been isolated from a pig kidney cortex cDNA library after employing the polymerase chain reaction technique to amplify a region of the dipeptidase cDNA. The complete primary sequence of the enzyme has been deduced from a full length cDNA clone. This predicts a protein of 409 amino acids, a cleavable N-terminal signal sequence of 16 residues and two N-linked glycosylation sites. At the C-terminus of the predicted sequence is a stretch of mainly hydrophobic amino acids which is presumed to direct the attachment of the glycosyl-phosphatidylinositol membrane anchor. Expression of the mRNA for pig renal dipeptidase in Xenopus laevis oocytes led to the production of a disulphide-linked dimeric protein of subunit Mr 48,600 which was recognized by a polyclonal antiserum raised to renal dipeptidase purified from pig kidney cortex. Bacterial phosphatidylinositol-specific phospholipase C released renal dipeptidase from the surface of the oocytes and converted the amphipathic detergent-solubilized form of the dipeptidase to a hydrophilic form, indicating that Xenopus laevis oocytes can process expressed proteins to their glycosyl-phosphatidylinositol anchored form.

Amino Acid Sequence↗

PubMed1997

Identification by site-directed mutagenesis of three essential histidine residues in membrane dipeptidase, a novel mammalian zinc peptidase.

Membrane dipeptidase (EC 3.4.13.19) is a plasma membrane zinc peptidase that is involved in the renal metabolism of glutathione and its conjugates, such as leukotriene D4. The enzyme lacks the classical signatures of other zinc-dependent hydrolases and shows no homology with any other mammalian protein. We have used site-directed mutagenesis to explore the roles of five histidine residues in pig membrane dipeptidase that are conserved among mammalian species. When expressed in COS-1 cells, the mutants H49K and H128L exhibited a specific activity and Km for the substrate Gly-D-Phe comparable with those of the wild-type enzyme. However, the mutants H20L, H152L and H198K were inactive, but were expressed at the cell surface at equivalent levels to the wild-type, as assessed by immunoblotting and immunofluorescence. These three mutants were compared with regard to their ability to bind to the competitive inhibitor cilastatin, which binds with equal efficacy to native and EDTA-treated pig kidney membrane dipeptidase. Expressed wild-type enzyme and mutants H20L and H198K were efficiently bound by cilastatin-Sepharose, but H152L failed to bind. Thus His-152 appears to be involved in the binding of substrate or inhibitor, whereas His-20 and His-198 appear to be involved in catalysis. Membrane dipeptidase shares some similarity with a dipeptidase recently cloned from Acinetobacter calcoaceticus. In particular, His-20 and His-198 of membrane dipeptidase are conserved in the bacterial enzyme, as are Glu-125 and His-219, previously shown to be required for catalytic activity.

Amino Acid Sequence↗

PubMed1983

Purification and properties of human pancreas dipeptidase.

Dipeptidase [EC 3.4.13] was purified from human pancreas; the activity was followed with L-Leu-L-Leu as a substrate. Polyacrylamide gel electrophoresis showed that the final preparation was homogeneous. The molecular weight of the dipeptidase was estimated to be 135,000 by gel filtration. From the result of SDS-polyacrylamide gel electrophoresis, it was found that the enzyme consisted of two subunits with equal molecular weights of 68,000. By atomic absorption analysis, the dipeptidase was shown to be a zinc metalloenzyme containing one atom of zinc for each subunit. Cu2+ and Hg2+ (1 mM) inhibited the enzyme by 50%. o-Phenanthroline strongly inhibited the enzyme. The dipeptidase hydrolyzed dipeptides such as L-Ala-L-Ala, L-Met-L-Met, L-Ala-L-Leu, L-Leu-Gly, and L-Leu-L-Leu but did not hydrolyze tripeptides, Bz-amino acids, CBz-amino acids, or L-amino acid beta-naphthylamides. The dipeptidase from human pancreas was immunologically distinct from human liver dipeptidase.

Cations, Divalent↗

PubMed2004

Characteristics of a new enantioselective thermostable dipeptidase from Brevibacillus borstelensis BCS-1 and its application to synthesis of a D-amino-acid-containing dipeptide.

A new thermostable dipeptidase gene was cloned from the thermophile Brevibacillus borstelensis BCS-1 by genetic complementation of the D-Glu auxotroph Escherichia coli WM335 on a plate containing D-Ala-D-Glu. Nucleotide sequence analysis revealed that the gene included an open reading frame coding for a 307-amino-acid sequence with an M(r) of 35,000. The deduced amino acid sequence of the dipeptidase exhibited 52% similarity with the dipeptidase from Listeria monocytogenes. The enzyme was purified to homogeneity from recombinant E. coli WM335 harboring the dipeptidase gene from B. borstelensis BCS-1. Investigation of the enantioselectivity (E) to the P(1) and P(1)' site of Ala-Ala revealed that the ratio of the specificity constant (k(cat)/K(m)) for L-enantioselectivity to the P(1) site of Ala-Ala was 23.4 +/- 2.2 [E = (k(cat)/K(m))(L,D)/(k(cat)/K(m))(D,D)], while the D-enantioselectivity to the P(1)' site of Ala-Ala was 16.4 +/- 0.5 [E = (k(cat)/K(m))(L,D)/(k(cat)/K(m))(L,L)] at 55 degrees C. The enzyme was stable up to 55 degrees C, and the optimal pH and temperature were 8.5 and 65 degrees C, respectively. The enzyme was able to hydrolyze L-Asp-D-Ala, L-Asp-D-AlaOMe, Z-D-Ala-D-AlaOBzl, and Z-L-Asp-D-AlaOBzl, yet it could not hydrolyze D-Ala-L-Asp, D-Ala-L-Ala, D-AlaNH(2), and L-AlaNH(2.) The enzyme also exhibited beta-lactamase activity similar to that of a human renal dipeptidase. The dipeptidase successfully synthesized the precursor of the dipeptide sweetener Z-L-Asp-D-AlaOBzl.

Amino Acid Sequence↗

PubMed1999

Behaviour of urinary dipeptidase in patients with chronic renal failure.

Renal dipeptidase (EC 3.4.13.19) activity in serum and urine from healthy volunteers (n = 20), patients with diabetes (n = 18) and patients with chronic renal failure (n = 5) was measured using glycyl-D-alanine as substrate. The assay was highly specific for the enzyme and was not affected by the various aminopeptidases present in serum and urine. No difference in serum renal dipeptidase activity was observed between the groups. The enzyme activity (U/L) in urine was higher than that in serum, irrespective of the group, suggesting the urine concentration was not affected by the serum concentration. The mean renal dipeptidase activities in urine were 2.56, 2.46 and 0.78 U/mol creatinine for healthy subjects, patients with diabetes and patients with chronic renal failure, respectively. The renal dipeptidase activity was significantly lower in the chronic renal failure group. The urinary excretion of dipeptidase (U/mmol creatinine) showed significant inverse correlations with that of beta 2-microglobulin, albumin and alpha 1-microglobulin, and with serum concentrations of creatinine, beta 2-microglobulin and alpha 1-microglobulin. We suggest that urine dipeptidase may be a useful marker of renal diseases.

Adult↗