Distribution,properties, and functions of midgut carboxypeptidases and dipeptidases from Musca domestica larvae

Dipeptidase and carboxypeptidase A activities were determined in cells and luminal contents of the fore-, mid-, and hind-midgut of Musca domestica larvae. Dipeptidase activity was found mainly in hind-midgut cells, whereas carboxy-peptidase activity was recovered in major amounts in both cells and in luminal contents of hind-midguts. The subcellular distribution of dipeptidase and part of the carboxypeptidase A activities is similar to that of a plasma membrane enzyme marker (aminopeptidase), suggesting that these activities are bound to the microvillar membranes. Soluble carboxypeptidase A seems to occur both bound to secretory vesicles and trapped in the cell glycocalyx. Based on density-gradient ultracentrifugation and thermal inactivation, there seems to be only one molecular species of each of the following enzymes (soluble in water or solubilized in Triton X-100): membrane-bound dipeptidase (pH optimum 8.0; Km 3.7 mM GlyLeu, M_r 111,000), soluble carboxypeptidase (pH optimum 8.0; Km 1.22 mM N-carbobenzoxy-glycyl-L-phenylalanine (ZGlyPhe), M_r45,000) and membrane-bound carboxypeptidase (pH optimum 7.5, Km 2.3 mM ZGlyPhe, M_r58,000). The results suggest that protein digestion is accomplished sequentially by luminal trypsin and luminal carboxypeptidase, by membrane-bound carboxypeptidase and aminopeptidase, and finally by membrane-bound dipeptidase.     

Cleavage of di- and tripeptides by Prevotella ruminicola

The final step in the conversion of protein to amino acids by the common Gram-negative rumen bacterium, Prevotella (formerly Bacteroides) ruminicola , is the cleavage of di- and tripeptides. Dipeptidase and tripeptidase activities were predominantly cytoplasmic, and toluene treatment increased the rate of Ala2 and Ala3 hydrolysis by whole cells, suggesting that transport limited the rate of hydrolysis of extracellular di- and tripeptides. The hydrolysis of Ala2 and Ala3 by whole cells was not affected by protonophores, ionophores or dicyclohexylcarbodiimide, but Ala2 hydrolysis by EDTA-treated cells was inhibited by the Ca2+/H+ ionophore, tetronasin. Ala3 hydrolysis was not affected by protonophores or ionophores in EDTA-treated cells. The dipeptidase of strain M384 was inhibited > 99% by 1,10-phenanthroline and 39% by EDTA but not other protease inhibitors, consistent with the enzyme being a metalloprotease. Tripeptidase was insensitive to protease inhibitors, except for a 33% inhibition by EDTA. Cleavage of tripeptides occurred at the bond adjacent to the N-terminal amino acid. Distinct di-, tri- and oligopeptidase peaks were obtained by anion-exchange liquid chromatography of disrupted cells. Banding patterns on native PAGE using activity staining also indicated that P. ruminicola M384 had separate single dipeptidase and tripeptidase enzymes which hydrolysed a range of peptides. The dipeptidase of strain M384 was different from other strains of P. ruminicola: strains GA33 and B(1)4 had activities which ran at the same R(f); strain GA33 had another band of lower activity; strain 23 had two bands different from those of the other strains. The tripeptidases ran at the same R(f) for the different strains. Dipeptidase activity of all strains was inhibited by 1,10-phenanthroline on gels. Gel permeation chromatography indicated that the M(r) of the dipeptidases from strains M384 and B(1)4 were 115,000 and 114,500 respectively, and 112,500 and 121,500 for the corresponding tripeptidases. Thus the metabolism of small peptides by P. ruminicola involves separate permeases and intracellular peptidases for di- and tripeptides.     

Membrane peptidases in the pig choroid plexus and on other cell surfaces in contact with the cerebrospinal fluid.

A comprehensive survey of 11 peptidases, all of which are markers for renal microvillar membranes, has been made in membrane fractions prepared from pig choroid plexus. Two fractionation schemes were explored, both depending on a MgCl2-precipitation step, the preferred one having advantages in speed and yield of the activities. The specific activities of the peptidases in the choroid-plexus membranes were, with the exception of carboxypeptidase M, lower than in renal microvillar membranes: those of aminopeptidase N, peptidyl dipeptidase A ('angiotensin-converting enzyme') and gamma-glutamyltransferase were 3-5-fold lower, those of aminopeptidase A and endopeptidase-24.11 were 12-15 fold lower, and those of dipeptidyl peptidase IV and aminopeptidase W were 50-70-fold lower. Carboxypeptidase M had a similar activity in both membranes. Alkaline phosphatase and (Na+ + K+)-activated ATPase were more active in the choroid-plexus membranes. No activity for microsomal dipeptidase, aminopeptidase P and carboxypeptidase P could be detected. Six of the peptidases and (Na+ + K+)-activated ATPase were also studied by immunoperoxidase histochemistry at light- and electron-microscopic levels. Endopeptidase-24.11 and (Na+ + K+)-activated ATPase were uniquely located on the brush border, and the other two peptidases appeared to be much more abundant on the endothelial lining of microvessels. Dipeptidyl peptidase IV and aminopeptidase W were also detected in microvasculature. Pial membranes associated with the brain and spinal cord also stained positively for endopeptidase-24.11, aminopeptidase N and peptidyl dipeptidase A. The immunohistochemical studies indicated the subcellular fractionation did not discriminate between membranes derived from epithelial cells (i.e. microvilli) and those from endothelial cells. The possible significance of these studies in relation to neuropeptide metabolism and the control of cerebrospinal fluid production is discussed.     

Neurokinin B is hydrolysed by synaptic membranes and by endopeptidase-24.11 (enkephalinase) but not by angiotensin converting enzyme

The major site of hydrolysis was the Gly8-Leu9 bond. Angiotensin converting enzyme (peptidyl dipeptidase A, EC 3.4.15.1) from pig kidney hydrolysed substance P releasing the C-terminal tripeptide Gly-Leu-MetNH2 but failed to hydrolyse neurokinin B. Pig brain striatal synaptic membranes hydrolysed neurokinin B producing a similar pattern of products as did endopeptidase-24.11. Substantial inhibition of this activity was achieved with the selective inhibitor phosphoramidon. A combination of phosphoramidon and bestatin abolished the hydrolysis of neurokinin B by synaptic membranes. Thus, a bestatin-sensitive aminopeptidase may play a role in the synaptic metabolism of neurokinin B in addition to endopeptidase-24.11. This aminopeptidase appears to be distinct from aminopeptidase N (EC 3.4.11.2).     

The metabolism of neuropeptides. Phase separation of synaptic membrane preparations with Triton X-114 reveals the presence of aminopeptidase N.

The property of solutions of Triton X-114 to separate into detergent-rich and detergent-poor phases at 30 degrees C has been exploited to investigate the identities of the aminopeptidases in synaptic membrane preparations from pig striatum. When titrated with an antiserum to aminopeptidase N (EC 3.4.11.2), synaptic membranes solubilized with Triton X-100 revealed that this enzyme apparently comprises no more than 5% of the activity releasing tyrosine from [Leu]enkephalin. When assayed in the presence of puromycin, this proportion increased to 20%. Three integral membrane proteins were fractionated by phase separation in Triton X-114. Aminopeptidase activity, endopeptidase-24.11 and peptidyl dipeptidase A partitioned predominantly into the detergent-rich phase when kidney microvillar membranes were so treated. However, only 5.5% of synaptic membrane aminopeptidase activity partitioned into this phase, although the other peptidases behaved predictably. About half of the aminopeptidase activity in the detergent-rich phase could now be titrated with the antiserum, showing that aminopeptidase N is an integral membrane protein of this preparation. Three aminopeptidase inhibitors were investigated for their ability to discriminate between the different activities revealed by these experiments. Although amastatin was the most potent (IC50 = 5 X 10(-7) M) it failed to discriminate between pure kidney aminopeptidase N, the total activity of solubilized synaptic membranes and that in the Triton X-114-rich phase. Bestatin was slightly more potent for total activity (IC50 = 6.3 X 10(-6) M) than for the other two forms (IC50 = 1.6 X 10(-5) M). Puromycin was a weak inhibitor, but was more selective. The activity of solubilized membranes was more sensitive (IC50 = 1.6 X 10(-5) M) than that of the pure enzyme or the Triton X-114-rich phase (IC50 = 4 X 10(-4) M). We suggest that the puromycin-sensitive aminopeptidase activity that predominates in crude synaptic membrane preparations may be a cytosolic contaminant or peripheral membrane protein rather than an integral membrane component. Aminopeptidase N may contribute to the extracellular metabolism of enkephalin and other susceptible neuropeptides in the brain.     

Elevation of glutathione in phenylalanine mustard-resistant murine L1210 leukemia cells

Murine L1210 leukemia cells resistant to the antineoplastic agent L-phenylalanine mustard have a 1.5-2.0-fold elevation in their cellular GSH and GSSG content as compared to drug-sensitive cells. Cellular uptake of L-[U-14C]cystine and its incorporation into GSH of the resistant tumor are correspondingly elevated. Synthesis of gamma-glutamylcysteine, GSH, and GSSG is elevated 1.5-2.0-fold in cell-free preparations of the resistant tumor. This increased synthesis of GSH is attributed to increased cellular content (1.6-fold) of gamma-glutamylcysteine synthetase. GSH synthetase activity is equivalent in both drug-sensitive and -resistant cells. Investigation into the hydrolysis of selected peptides by cell-free preparations of both sensitive and resistant tumors suggest that aminopeptidase M participates in the formation of L-cysteine from L-Cys-Gly. This is supported by the observation that these preparations readily degrade L-Leu-p-nitroanilide and L-Ala-L-Ala-L-Ala, known substrates for aminopeptidase M, but not dipeptidase. The failure of the tumors to degrade Gly-D-Ala, a dipeptidase substrate, and the marked inhibition of L-Ala-Gly, L-Cys-Gly, and L-Ala-L-Ala-L-Ala hydrolysis by Bestatin further support a role for aminopeptidase M in the generation of L-cysteine from L-Cys-Gly. These results suggest that the drug-resistant tumor cell has developed an efficient mechanism for maintenance of elevated GSH which involves both gamma-glutamyl transpeptidase-initiated catabolism of GSH to cysteine and its reutilization by gamma-glutamylcysteine synthetase.     

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.     

Dug1p Is a Cys-Gly Peptidase of the ��-Glutamyl Cycle of Saccharomyces cerevisiae and Represents a Novel Family of Cys-Gly Peptidases

GSH metabolism in yeast is carried out by the γ-glutamyl cycle as well as by the DUG complex. One of the last steps in the γ-glutamyl cycle is the cleavage of Cys-Gly by a peptidase to the constitutent amino acids. Saccharomyces cerevisiae extracts carry Cys-Gly dipeptidase activity, but the corresponding gene has not yet been identified. We describe the isolation and characterization of a novel Cys-Gly dipeptidase, encoded by the DUG1 gene. Dug1p had previously been identified as part of the Dug1p-Dug2p-Dug3p complex that operates as an alternate GSH degradation pathway and has also been suggested to function as a possible di- or tripeptidase based on genetic studies. We show here that Dug1p is a homodimer that can also function in a Dug2-Dug3-independent manner as a dipeptidase with high specificity for Cys-Gly and no activity toward tri- or tetrapeptides in vitro. This activity requires zinc or manganese ions. Yeast cells lacking Dug1p (dug1Δ) accumulate Cys-Gly. Unlike all other Cys-Gly peptidases, which are members of the metallopeptidase M17, M19, or M1 families, Dug1p is the first to belong to the M20A family. We also show that the Dug1p Schizosaccharomyces pombe orthologue functions as the exclusive Cys-Gly peptidase in this organism. The human orthologue CNDP2 also displays Cys-Gly peptidase activity, as seen by complementation of the dug1Δ mutant and by biochemical characterization, which revealed a high substrate specificity and affinity for Cys-Gly. The results indicate that the Dug1p family represents a novel class of Cys-Gly dipeptidases.GSH is a thiol-containing tripeptide (l-γ-glutamyl-l-cysteinyl-glycine) present in almost all eukaryotes (barring a few protozoa) and in a few prokaryotes (1). In the cell, glutathione exists in reduced (GSH) and oxidized (GSSG) forms. Its abundance (in the millimolar range), a relatively low redox potential (-240 mV), and a high stability conferred by the unusual peptidase-resistant γ-glutamyl bond are three of the properties endowing GSH with the attribute of an important cellular redox buffer. GSH also contributes to the scavenging of free radicals and peroxides, the chelation of heavy metals, such as cadmium, the detoxification of xenobiotics, the transport of amino acids, and the regulation of enzyme activities through glutathionylation and serves as a source of sulfur and nitrogen under starvation conditions (2, 3). GSH metabolism is carried out by the γ-glutamyl cycle, which coordinates its biosynthesis, transport, and degradation. The six-step cycle is schematically depicted in Fig. 1 (2).Open in a separate windowFIGURE 1.γ-Glutamyl cycle of glutathione metabolism. γ-Glutamylcysteine synthetase and GSH synthetase carry out the first two steps in glutathione biosynthesis. γ-glutamyltranspeptidase, γ-glutamylcyclotransferase, 5-oxoprolinase, and Cys-Gly dipeptidase are involved in glutathione catabolism. Activities responsible for γ-glutamylcyclotransferase and 5-oxoprolinase have not been detected in S. cerevisiae.In Saccharomyces cerevisiae, γ-glutamyl cyclotransferase and 5-oxoprolinase activities have not been detected, which has led to the suggestion of the presence of an incomplete, truncated form of the γ-glutamyl cycle (4) made of γ-glutamyl transpeptidase (γGT)⁴ and Cys-Gly dipeptidase and only serving a GSH catabolic function. Although γGT and Cys-Gly dipeptidase activities were detected in S. cerevisiae cell extracts, only the γGT gene (ECM38) has been identified so far. Cys-Gly dipeptidase activity has been identified in humans (5, 6), rats (7–10), pigs (11, 12), Escherichia coli (13, 14), and other organisms (15, 16), and most of them belong to the M17 or the M1 and M19 metallopeptidases gene families (17).S. cerevisiae has an alternative γGT-independent GSH degradation pathway (18) made of the Dug1p, Dug2p, and Dug3p proteins that function together as a complex. Dug1p also seem to carry nonspecific di- and tripeptidase activity, based on genetic studies (19).We show here that Dug1p is a highly specific Cys-Gly dipeptidase, as is its Schizosaccharomyces pombe homologue. We also show that the mammalian orthologue of DUG1, CNDP2, can complement the defective utilization of Cys-Gly as sulfur source of an S. cerevisiae strain lacking DUG1 (dug1Δ). Moreover, CNDP2 has Cys-Gly dipeptidase activity in vitro, with a strong preference for Cys-Gly over all other dipeptides tested. CNDP2 and its homologue CNDP1 are members of the metallopeptidases M20A family and have been known to carry carnosine (β-alanyl-histidine) and carnosine-like (homocarnosine and anserine) peptidase activity (20, 21). This study thus reveals that the metallopeptidase M20A family represents a novel Cys-Gly peptidase family, since only members of the M19, M1, and M17 family were known to carry this function.     

Cleavage of di- and tripeptides by Prevotella ruminicola

The final step in the conversion of protein to amino acids by the common Gram-negative rumen bacterium, Prevotella (formerly Bacteroides) ruminicola , is the cleavage of di- and tripeptides. Dipeptidase and tripeptidase activities were predominantly cytoplasmic, and toluene treatment increased the rate of Ala₂ and Ala₃ hydrolysis by whole cells, suggesting that transport limited the rate of hydrolysis of extracellular di- and tripeptides. The hydrolysis of Ala₂ and Ala₃ by whole cells was not affected by protonophores, ionophores or dicyclohexylcarbodiimide, but Ala₂ hydrolysis by EDTA-treated cells was inhibited by the Ca²⁺/H⁺ ionophore, tetronasin. Ala₃ hydrolysis was not affected by protonophores or ionophores in EDTA-treated cells. The dipeptidase of strain M384 was inhibited > 99% by 1,10-phenanthroline and 39% by EDTA but not other protease inhibitors, consistent with the enzyme being a metalloprotease. Tripeptidase was insensitive to protease inhibitors, except for a 33% inhibition by EDTA. Cleavage of tripeptides occurred at the bond adjacent to the N-terminal amino acid. Distinct di-, tri- and oligopeptidase peaks were obtained by anion-exchange liquid chromatography of disrupted cells. Banding patterns on native PAGE using activity staining also indicated that P. ruminicola M384 had separate single dipeptidase and tripeptidase enzymes which hydrolysed a range of peptides. The dipeptidase of strain M384 was different from other strains of P. ruminicola: strains GA33 and B₁4 had activities which ran at the same R_f; strain GA33 had another band of lower activity; strain 23 had two bands different from those of the other strains. The tripeptidases ran at the same R_f for the different strains. Dipeptidase activity of all strains was inhibited by 1,10-phenanthroline on gels. Gel permeation chromatography indicated that the M_r of the dipeptidases from strains M384 and B₁4 were 115 000 and 114 500 respectively, and 112 500 and 121 500 for the corresponding tripeptidases. Thus the metabolism of small peptides by P. ruminicola involves separate permeases and intracellular peptidases for di- and tripeptides.     

An evaluation of the role of a pyroglutamyl peptidase, a post-proline cleaving enzyme and a post-proline dipeptidyl amino peptidase, each purified from the soluble fraction of guinea-pig brain, in the degradation of thyroliberin in vitro

The degradation of thyroliberin (less than Glu-His-Pro-NH2) to its component amino acids by the soluble fraction of guinea pig brain is catalysed by four enzymes namely a pyroglutamate aminopeptidase, a post-proline cleaving enzyme, a post-proline dipeptidyl aminopeptidase and a proline dipeptidase. 1. The pyroglutamate aminopeptidase was purified to over 90% homogeneity with a purification factor of 2868-fold and a yield of 5.7%. In addition to catalysing the hydrolysis of thyroliberin, acid thyroliberin and pyroglutamate-7-amido-4-methylcoumarin the pyroglutamate aminopeptidase catalysed the hydrolysis of the peptide bond adjacent to the pyroglutamic acid residue in luliberin, neurotensin bombesin, bradykinin-potentiating peptide B, the anorexogenic peptide and the dipeptides pyroglutamyl alanine and pyroglutamyl valine. Pyroglutamyl proline and eledoisin were not hydrolysed. 2. The post-proline cleaving enzyme was purified to apparent electrophoretic homogeneity with a purification factor of 2298-fold and a yield of 10.6%. The post-proline cleaving enzyme catalysed the hydrolysis of thyroliberin and N-benzyloxycarbonyl-glycylproline-7-amido-4-methylcoumarin. It did not catalyse the hydrolysis of glycylproline-7-amido-4-methylcoumarin or His-Pro-NH2. 3. The post-proline dipeptidyl aminopeptidase was partially purified with a purification factor of 301-fold and a yield of 8.9%. The post-proline dipeptidyl aminopeptidase catalysed the hydrolysis of His-Pro-NH2 and glycylproline-7-amido-4-methylcoumarin but did not exhibit any post-proline cleaving endopeptidase activity against thyroliberin or N-benzyloxycarbonyl-glycylproline-7-amido-4-methylcoumarin. 4. Studies with various functional reagents indicated that the pyroglutamate aminopeptidase could be specifically inhibited by 2-iodoacetamide (100% inhibition at an inhibitor concentration of 5 microM), the post-proline cleaving enzyme by bacitracin (IC50 = 42 microM) and the post-proline dipeptidyl aminopeptidase by puromycin (IC50 = 46 microM). Because of their specific inhibitory effects these three reagents were key elements in the elucidation of the overall pathway for the metabolism of thyroliberin by guinea pig brain tissue enzymes.     

Purification and characterization of dipeptidase hydrolyzing L-cysteinylglycine from radish cotyledon

Dipeptidase activity was detected in the soluble fraction of radish (Raphanus sativus L.) cotyledon, and the purified enzyme had a specific activity of 7.32 nkat/mg protein for hydrolyzing L-cysteinylglycine. The dipeptidase was found to be a hexameric metalloenzyme, composed of homological 55 kDa-subunits. This is the first glutathione catabolism-related dipeptidase isolated from higher plants.     

Distribution of dipeptidase activity between lysosomes and soluble fraction of rat liver.

Dipeptidase activity toward Arg-Phe, Arg-Gly, and Trp-Leu exhibited bimodal distribution in the lysosomal and soluble fractions of rat liver. The majority (50-70 percent) of the dipeptidase activity was present in the soluble fraction. Some evidence for a plasma membrane dipeptidase, which hydrolyzes Trp-Leu but not Arg-Phe or Arg-Gly, also was found. The lysosomal dipeptidase activity had a pH optimum of 6.0-7.0, and was activated by sulfhydryl reagents. Lysosomal localization for some of the dipeptidase activity was established with Triton WR-1339 fractionation and latency experiments.     

Thermostable dipeptidase from Bacillus stearothermophilus: its purification, characterization, and comparison with aminoacylase

Dipeptidase (dipeptide hydrolase [EC 3.4.13.11]) has been purified to homogeneity and crystallized from the cell extract of Bacillus stearothermophilus IFO 12983. The enzyme has a molecular weight of about 86,000, and is composed of two subunits identical in molecular weight (43,000). The enzyme contains 2 g atoms of zinc per mol of protein. A variety of dipeptides consisting of glycine or only L-amino acids serve as substrates of the enzyme; Km and Vmax values for L-valyl-L-alanine are 0.5 mM and 68.0 units/mg protein, respectively. The enzyme is significantly stable not only at high temperatures but also on treatment with protein denaturants such as urea and guanidine hydrochloride. The enzyme also catalyzes hydrolysis of several N-acylamino acids with Vmax values 3-30% of those for the hydrolysis of dipeptides. The thermostable dipeptidase shares various properties with bacterial aminoacylase [EC 3.5.1.14]: their subunit molecular weight, metal content and requirement, amino acid composition, and amino acid sequence in the N-terminal region are very similar.     

Peptidase-deficient mutants of Escherichia coli.

Mutant derivatives of Escherichia coli K-12 deficient in several peptidases have been obtained. Mutants lacking a naphthylamidase, peptidase N, were isolated by screening for colonies unable to hydrolyze L-alanine beta-naphthylamide. Other mutants were isolated using positive selections for resistance to valine peptides. Mutants lacking peptidase A, a broad-specificity aminopeptidase, were obtained by selection for resistance to L-valyl-L-leucine amide. Mutants lacking a dipeptidase, peptidase D, were isolated from a pepN pepA strain by selection for resistance to L-valyl-glycine. Starting with a pepN pepA pepD strain, selection for resistance to L-valyl-glycyl-glycine or several other valine peptides produced mutants deficient in another aminopeptidase, peptidase B. Mutants resistant to L-valyl-L-proline lack peptidase Q, an activity capable of rapid hydrolysis of X-proline dipeptides. Using these selection procedures, a strain (CM89) lacking five different peptidases has been isolated. Although still sensitive to valine, this strain is resistant to a variety of valine di- and tripeptides. The ability of this strain to use peptides as sources of amino acids is much more restricted than that of wild-type E. coli strains. Strains containing only one of the five peptidases missing in CM89 have been constructed by transduction. The peptide utilization profiles of these strains show that each of the five peptidases can function during growth in the catabolism of peptides.     

Ectoenzymes of the kidney microvillar membrane. Isolation and characterization of the amphipathic form of renal dipeptidase and hydrolysis of its glycosyl-phosphatidylinositol anchor by an activity in plasma.

Renal dipeptidase (EC 3.4.13.11) has been solubilized from pig kidney microvillar membranes with n-octyl-beta-D-glucopyranoside and then purified by affinity chromatography on cilastatin-Sepharose. The enzyme exists as a disulphide-linked dimer of two identical subunits of Mr 45,000 each. The purified dipeptidase partitioned into the detergent-rich phase upon phase separation in Triton X-114 and reconstituted into liposomes consistent with the presence of the glycosyl-phosphatidylinositol membrane anchor. The N-terminal amino acid sequence of the amphipathic, detergent-solubilized, form of renal dipeptidase was identical with that of the hydrophilic, phospholipase-solubilized, form, locating the membrane anchor at the C-terminus of the protein. The glycosyl-phosphatidylinositol anchor of both purified and microvillar membrane renal dipeptidase was a substrate for an activity in pig plasma which displayed properties similar to those of a previously described phospholipase D. The cross-reacting determinant of the glycosyl-phosphatidylinositol anchor was generated by incubation of purified renal dipeptidase with bacterial phosphatidylinositol-specific phospholipase c, whereas the anchor-degrading activity in plasma failed to generate this determinant.     

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.     

Purification and Characterization of Dipeptidase Hydrolyzing L-Cysteinylglycine from Radish Cotyledon

Dipeptidase activity was detected in the soluble fraction of radish (Raphanus sativus L.) cotyledon, and the purified enzyme had a specific activity of 7.32 nkat/mg protein for hydrolyzing L-cysteinylglycine. The dipeptidase was found to be a hexameric metalloenzyme, composed of homological 55 kDa-subunits. This is the first glutathione catabolism-related dipeptidase isolated from higher plants.     

Identification of aminopeptidase M as an enkephalin-inactivating enzyme in rat cerebral membranes

Two membrane-bound enkephalin-hydrolyzing aminopeptidase activities were partially purified from rat brain membranes. The first, which represents 90% of the total activity, was highly sensitive to both puromycin (Ki = 1 microM) and bestatin (Ki = 0.5 microM). The second was inhibited much more by bestatin (Ki = 4 microM) than by puromycin (Ki = 100 microM). The latter puromycin-insensitive aminopeptidase was found to resemble aminopeptidase M purified from rat kidney brush border membranes. Both displayed the same purification pattern and the same kinetic constants of substrates and inhibitors, and both were similarly inactivated by metal chelating agents. Moreover, antibodies raised in rabbits against rat kidney aminopeptidase M inhibited the aminopeptidase activities of both kidney and brain puromycin-insensitive enzymes at similar dilutions, while the brain puromycin-sensitive aminopeptidase activity was not affected. Thus, aminopeptidase M (EC 3.4.11.2) was found to occur in brain, and the role of this enzyme in inactivating endogenous enkephalins released from their neuronal stores is suggested.     

The metabolism of neuropeptides. Neurokinin A (substance K) is a substrate for endopeptidase-24.11 but not for peptidyl dipeptidase A (angiotensin-converting enzyme).

Both endopeptidase-24.11 and peptidyl dipeptidase A have previously been shown to hydrolyse the neuropeptide substance P. The structurally related peptide neurokinin A is also shown to be hydrolysed by pig kidney endopeptidase-24.11. The identified products indicated hydrolysis at two sites, Ser5-Phe6 and Gly8-Leu9, consistent with the known specificity of the enzyme. The pattern of hydrolysis of neurokinin A by synaptic membranes prepared from pig striatum was similar to that observed with purified endopeptidase-24.11, and hydrolysis was substantially abolished by the selective inhibitor phosphoramidon. Peptidyl dipeptidase A purified from pig kidney was shown to hydrolyse substance P but not neurokinin A. It is concluded that endopeptidase-24.11 has the general capacity to hydrolyse and inactivate the family of tachykinin peptides, including substance P and neurokinin A.     

Ectoenzymes of the kidney microvillar membrane. Affinity purification, characterization and localization of the phospholipase C-solubilized form of renal dipeptidase.

Renal dipeptidase (EC 3.4.13.11) was solubilized from pig kidney microvillar membranes with bacterial phosphatidylinositol-specific phospholipase C and then purified by affinity chromatography on cilastatin-Sepharose. The enzyme was apparently homogeneous on SDS/polyacrylamide-gel electrophoresis with an Mr of 47,000. Immunohistochemical analysis of the distribution of the dipeptidase showed it to be concentrated in the brush-border region of the proximal tubules in close association with endopeptidase-24.11) (EC 3.4.24.11). The purified dipeptidase was shown to contain 1 mol of inositol/mol and to possess the cross-reacting determinant characteristic of the glycosyl-phosphatidylinositol membrane-anchoring domain. The glycoprotein nature of renal dipeptidase was confirmed by chemical and enzymic deglycosylation. These results establish renal dipeptidase as a glycosyl-phosphatidylinositol-anchored ectoenzyme of the microvillar membrane.