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Substrate specificity of X-prolyl dipeptidyl-aminopeptidase (dipeptidyl aminopeptidase IV) was examined by using newly synthesized 8 chromogenic substrates, X-Y-p-nitroanilides. Homogeneous enzyme from human submaxillary gland hydrolyzed glycylproline p-nitroanilide almost specifically, except alanylalanine p-nitroanilide which had 11% activity.
Zinc is a nutritionally essential trace element required for many biological functions to be successfully carried out. The aim of the present work was to study the influence of zinc on the intestinal absorption of L-threonine and on the aminopeptidase N activity in rabbit jejunum, after in vitro addition and/or oral administration of ZnCl2 in drinking water. Results obtained show that zinc decreases L-threonine absorption in the jejunal tissue. This effect would appear to be owing to an action mainly located in active amino acid transport, because zinc does not seem to modify the amino acid diffusion across the intestinal epithelium, of the mucosal border of the intestinal epithelium. Zinc has also been shown to inhibit the (Na(+)-K+)-ATPase activity of the enterocyte, which might explain the inhibition of the L-threonine Na(+)-dependent transport. Nevertheless, a direct action of the zinc on carriers of active transport cannot be rejected. However, zinc did not significantly modify the aminopeptidase N activity in rabbit jejunum.
The aminopeptidase N (TH-4AP) of Streptomyces sp. TH-4 was purified from a culture supernatant. The purified enzyme had a molecular mass of 95 kDa. The gene encoding TH-4AP was cloned and sequenced. The primary structure of the protein possessed the PepN-conserved motif GxMEN and the zinc-binding motif HExxHx18E, and showed 88% identity with that of PepN from Streptomyces lividans strain 66. We succeeded in overproducing a His-tagged recombinant enzyme using Escherichia coli. The enzyme had a 1.5-fold higher activity in the presence of cobalt ions than in their absence. To evaluate the possible application of TH-4AP to decrease the content of bitter peptides, we investigated the ability of Streptomyces aminopeptidases to hydrolyze synthetic peptides by a coupling method using L-amino acid oxidase and peroxidase. The substrate specificity of TH-4AP toward synthetic peptides was significantly different from that toward aminoacyl-p-nitroanilide derivatives.
We have cloned the Aspergillus niger dapB gene. Analysis of its nucleotide sequence and the corresponding protein sequence indicates that the gene encodes a type IV dipeptidyl aminopeptidase (DPP IV). Based upon its deduced sequence we predict the presence of a transmembrane domain in the protein. Furthermore, dapB-overexpressing transformants display an increase in intracellular DPP IV activity. This is the first reported characterisation of a dipeptidyl aminopeptidase with a transmembrane domain from a filamentous fungus. Using the dapB sequence as a query, we were able to identify 14 DPP IV-encoding genes, and 12 additional DPPIV proteases in public genomic databases. Phylogenetic analysis reveals that in yeasts there are two clades of genes that encode DPP IV proteases with a transmembrane domain. In this study we demonstrate that, as in yeasts, two classes of DPP IV-encoding genes exist in filamentous fungi. However, only one of these codes for DPP IV proteases with a transmembrane domain. The second type present in filamentous fungi encodes extracellular DPP IV proteases. The dapB gene belongs to the first cluster. We propose that DapB plays a role in the proteolytic maturation of enzymes produced by A. niger.
A highly sensitive and specific method for the assay of dipeptidyl-aminopeptidase II (DAP II) in crude enzyme preparations such as serum and tissue homogenates has been established by using a newly synthesized fluorogenic substrate, 7-Lys-Ala-4-methylcoumarinamide. The enzymatically formed 7-amino-4-methylcoumarin was determined by high-performance liquid chromatography with fluorescence detection. The activities of other aminopeptidases in human serum and rat brain homogenates were completely inhibited by o-phenanthroline without any effect on DAP II activity to permit specific determination of DAP II. The limit of sensitivity for DAP II activity was about 300 fmol/30 min. DAP II activity was found to be increased in sera from cancer patients, in contrast to the decrease in serum DAP IV activity. DAP II activity was found to be unequally distributed in rat brain regions, and the highest activity was found in the hypothalamus.
Dipeptidyl aminopeptidase IV is a dipeptidylpeptide hydrolase (EC 3.4.14) which hydrolyzes bond at the carboxyl group of proline releasing X-Pro dipeptides from the amino-terminus of polypeptides. The enzyme was purified 440-fold in 37% yield from swine kidney by ammonium sulfate fractionation, DEAE-cellulose chromatography, gel filtration and affinity chromatography with dipeptide-substituted Sepharose 4B. The enzyme released X-Pro from all X-Pro-beta-naphthylamides and polypeptides tested. The released dipeptides were not further degraded, and were readily identified in digests. The enzyme is suitable for use in the dipeptidyl aminopeptidase method for sequence analysis of polypeptides.
The biochemical characterization of dipeptidyl aminopeptidase II activity was investigated in the supernatant of centrifuged homogenates of adult Schistosoma japonicum using a lysine-alanine oligopeptide derivative of 4-methoxy-2-naphthylamide as a substrate. It was observed that the pH optimum of the enzyme is in the acid range, with an optimum at pH 6.3. Time and enzyme concentration studies, along with temperature studies, support the premise that the reaction is enzymatic. The Km was 3.3 X 10(-3) M, at pH 5.5 and 37 C. Tris and diisopropyl phosphofluoridate, when incorporated into the assay system at final concentrations of 500 and 2 mM, respectively, significantly inhibited the reaction by 70.9 and 75%, respectively. Leupeptin (5 X 10(-4) mM) had no effect. The results indicate that the enzyme under study in the present investigation strongly resembles mammalian dipeptidyl aminopeptidase II due to its affinity for substrate, sensitivity to Tris and diisopropyl phosphofluoridate inhibition, and pH optimum. Its inhibition by diisopropyl phosphofluoridate indicates that it may belong to the serine class of proteases. Cytochemical studies revealed reaction product in the lipid-like globules in the gastrodermis, adding further credence that these globules are lysosomal.
1. The subcellular distribution of dipeptidyl aminopeptidase activities in guinea-pig brain was investigated. Our studies show that DAP I (Gly-Arg-NH-Mec hydrolase) type activity was found to have an acidic optimum and was associated with the nuclear pellet. 2. No DAP II (Lys-Ala-NH-Mec hydrolase) type activity could be detected. Apparent hydrolysis was mainly due to aminopeptidase activity. 3. DAP III (Arg-Arg-NH-Mec hydrolase) type activity is largely cytoplasmic, but there was evidence of a membrane form associated with the synaptosomes. 4. DAP IV (Gly-Pro-NH-Mec hydrolase) type activity is present on the synaptosomal membrane, and also enriched in the microsomes. A soluble form of Gly-Pro-NH-Mec hydrolase activity is also present in the cytoplasm. Whether this activity is a DAP II or IV type activity is still yet to be determined.
We have previously demonstrated the expression of aminopeptidase N (APN, CD13) on synovial T cells from patients with different forms of arthritis. T cells of peripheral blood and serous body fluids are CD13-negative but can be stimulated to express CD13 after activation, e.g., with Con A. In the present report, double-labelling and flow cytometry analyses were performed to characterize the phenotype of tumour-infiltrating lymphocytes (TIL). A large panel of antibodies specific for different activation-associated molecules on T cells was used. In contrast to TIL of lung cancer, TIL of renal cell carcinoma (RCC) consisted of significantly higher percentages of T cells expressing CD13, dipeptidylpeptidase N (DPIV, CD26) and HLA-DR, whereas T cells of lung cancer expressed more CD25, CD69 and CD54/ICAM1. No differences could be found in the expression of CD45RO, CD49a/VLA-1 and CD62L/L-selectin. Our results demonstrate that T cells in RCC and lung cancer differ in their phenotype, especially with respect to surface aminopeptidases. Investigations into the function of APN on T cells could be of help in gaining deeper insight into tumour defence as well as into general mechanisms of T cell functions.
Aminopeptidase N (EC 3.4.11.2) (APN) is an ectopeptidase expressed in lung at the apical surface of alveolar type II epithelial cells. Its expression is up-regulated during fetal lung development. Recently several ectopeptidases have been recognized as possible regulators of growth and cell differentiation through their role in hydrolysis of autocrine and paracrine peptides that influence these processes. The studies reported here describe effects of factors known to promote lung development and differentiation of the alveolar epithelium on expression of aminopeptidase N during fetal lung development in organ culture. Fetal rat lung was placed in organ culture at the 15th gestational day and cultured for 6 days in the presence or absence of the synthetic glucocorticoid hormone dexamethasone or epidermal growth factor (EGF). Steady-state levels of APN mRNA increased approximately 10-fold during the 6-day culture. During this time the lung alveolar epithelium developed to the point where immature alveolar type II cells were recognized by the presence of lamellar bodies. Dexamethasone or EGF increased the levels of APN mRNA in the fetal lung 2-to 3-fold over control cultures by the third day in culture and concurrently accelerated the morphological development of the alveolar epithelium. Differences in treated and control cultures diminished after 6 days in culture when the epithelium appeared more mature, suggesting that the immature epithelium was more responsive to the treatments.
The inhibitory effect of catecholamines, their metabolic intermediates and derivatives on the activity of rat brain dipeptidyl aminopeptidase was studied. The enzyme was extracted from a crude mitochondrial fraction containing lysosomes (and also synaptosomes) and purified by three-step column chromatography. The activity of the purified enzyme was inhibited by a low concentration (IC50 2 approximately 5 mM) of dopamine, norepinephrine and epinephrine. Dihydroxyphenylalanine or dihydroxyphenylacetic acid, each having a carboxyl group, showed only a weak inhibitory effect (IC50 greater than 10 mM). These results suggest the possibility that brain dipeptidyl aminopeptidase activity may be regulated by the changing of the concentration of catecholamines in the central nervous system.
Dipeptidyl aminopeptidase IV activity staining was performed in various thyroid tissues to evaluate this enzyme activity as a thyroid tumor marker. A total of 195 thyroid tissues were tested for their enzyme activity expression. All papillary and follicular carcinomas, 40 cases and 3 cases, respectively, showed enzyme activity, although two other carcinomas, one medullary and one anaplastic, were not stained. Follicular adenoma expressed enzyme activity in 4 of 26 cases. Fifty-two cases with adenomatous goiter, 54 with Graves' disease and 13 with chronic thyroiditis were judged to be negative. Five normal thyroids expressed no activity except for occasional positive staining of capillary endothelia. These data suggest that dipeptidyl aminopeptidase IV activity staining is very useful for pathological diagnosis of thyroid tumors.
Dipeptidyl aminopeptidase IV (EC 3.4.14.-) was solubilized from a particulate membrane fraction of rat intestinal mucosa with Triton X-100. The solubilized enzyme was purified to homogeneity following ammonium sulfate fractionation, chromatography on DEAE-Sepharose and hydroxyapatite, gel filtration and preparative polyacrylamide gel electrophoresis. The final enzyme preparation had a specific activity of 55 units/mg protein representing a 1373 fold purification over the starting material. Purity was judged by polyacrylamide gel electrophoresis and double immunodiffusion. The molecular weight of the native undenatured enzyme was estimated to be 230000 by gel filtration and polyacrylamide gel electrophoresis. Electrophoresis under denaturing conditions (sodium dodecyl sulfate) indicated that the protein consists of two identical 98 kDa subunits. Dipeptidyl aminopeptidase IV is a glycoprotein containing approx. 8% carbohydrate by weight. A detailed analysis of the individual sugar components demonstrated that fucose, galactose, glucose, mannose, sialic acid and hexosamine sugars were present. The nature of the constituent asparagine linked oligosaccharide side chains was further examined following cleavage from the peptide backbone by hydrazinolysis. Following high voltage paper electrophoresis approx. 80% of the isolated oligosaccharide was found with the neutral fraction while the remaining 20% consisted of a single acidic component. Gel filtration of the neutral oligosaccharide fraction indicated that it contains approx. 19 sugar residues.
An enkephalin-degrading aminopeptidase using Leu-enkephalin as a substrate was purified about 4100-fold from guinea pig serum. The purified preparation was apparently homogenous, showing one band on polyacrylamide gel electrophoresis. The molecular weight of the enzyme was approx. 92 000. The aminopeptidase had a pH optimum of 7.0 with Km values of 0.12 mM and 0.18 mM for Leu- and Met-enkephalin, respectively. The enzyme hydrolyzed neutral, basic and aromatic amino acid beta-naphthylamides, but did not the acidic one. The enzyme was inhibited strongly by metal-chelating agents, bestatin and amastatin and weakly by puromycin. Among several biologically active peptides, angiotensin III and substance P strongly inhibited the enzyme.
Five-hundred-and-six fresh isolates of rumen bacteria were tested for their ability to hydrolyse the synthetic substrate for dipeptidyl aminopeptidase type I, GlyArg-4-methoxy-2-naphthylamide (GlyArg-MNA), using a gel overlay technique. Twelve positive isolates were small Gram-negative rods which resembled Bacteroides ruminicola in their biochemical and morphological properties. SDS-PAGE of whole cell extracts indicated that two were similar to B. ruminicola strain B14, six resembled B. ruminicola strain M384, and four were similar to B. ruminicola GA33. All hydrolysed GlyArg-MNA, Ala2 and Ala5, and showed no activity against Leu-MNA. Ala3 and Ala2, but no Ala4, was produced from Ala5. The different groups had different, distinctive activity profiles. The two remaining positive isolates were Lactobacillus spp. with an exceptionally high Leu-MNA activity. It was concluded that, although different strains may only be distantly related, B. ruminicola forms the most important group of bacteria in the rumen to possess a dipeptidyl aminopeptidase type I activity.
Lactobacillus (Lb.) helveticus CNRZ32 possesses a 97-kDa metalloenzyme with aminopeptidase activity (PepN; EC 3.4.11.2). A 3.8-kb fragment encoding PepN was cloned into pIL253 and designated pSUW34. Transformation of Lactococcus (Lc.) lactis LM0230 with pSUW34 resulted in > 180-fold increase in general aminopeptidase (AP) activity using L-lysine-p-nitroanilide. Southern hybridization was conducted to determine the distribution of homology to the CNRZ32 pepN gene among lactic-acid bacteria (LAB). Hybridization was observed with strains of lactobacilli, pediococci, leuconostoc, streptococci and lactococci. The pepN gene was sequenced and found to encode a protein containing 844 amino acid (aa) residues. A comparison of Lb. helveticus CNRZ32 pepN to Lb. delbrueckii ssp. lactis DSM7290 pepN indicated 69.5% nucleotide (nt) identity and 71.8% aa identity, while comparison to pepN from Lc. lactis ssp. cremoris MG1363 indicated 61.1% nt identity and 49.2% aa identity. Alignment of peptidase aa sequences of LAB, Escherichia coli, yeast and mammalian origin display homology in the zinc-binding domain, as well as a conserved region upstream from the putative active site.
By means of immunohistochemical techniques we have investigated the presence of dipeptidyl aminopeptidase IV immunoreactivity in brain material derived from human fetuses, newborns and aged persons. It was revealed that the enzyme protein is abundantly present in the immature human CNS. On the contrary the adult human brain contains much less dipeptidyl aminopeptidase immunoreactivity. It is speculated that the enzyme might play an important role in neuronal proliferation and/or differentiation especially with regard to its possible action on certain neuronotrophic peptides (IGF II, growth hormone).
Proteolytic enzyme-mediated degradation of the extracellular matrix (ECM) is crucial for the formation of both tumor metastasis and angiogenesis. Recently, several reports have suggested that aminopeptidases are involved in this process, but precisely how is largely unknown. We found here that aminopeptidase N (APN/CD13) was selectively expressed in vascular endothelial cells including human umbilical vein endothelial cells (HUVEC) and human aortic endothelial cells (HAEC), and was not detectable in a majority of normal cells and tumor cell lines we examined. RNA interference (RNAi) of APN resulted in the inhibition of capillary tube formation of HUVEC on Matrigel. APN siRNA suppressed the migration of HUVEC through a fibronectin-coated Transwell membrane, and reduced the cellular adhesion to Matrigel and various adhesion molecules including type IV collagen, type I collagen and fibronectin. These findings suggest that APN is a multifunctional protein with important roles in vascular endothelial morphogenesis during angiogenesis.