Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.4.21.4 (trypsin)
42,187 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Surface peptidase activities on the human monocytic lineage cell line U937 were characterized. Two diisopropyl phosphofluoridate (DFP)-inhibitable serine peptidases were identified by differences in their hydrolytic activities on chromogenic peptides: one removed tripeptides from the free NH2-terminal end of the synthetic peptide Ala-Ala-Phe-p-nitroanilide (pNA) and was not inhibited by inhibitors of metallo-, cysteic-, and aspartic-proteinases, or by those of elastase-, trypsin- and chymotrypsin-like enzymes, suggesting the presence of a hitherto unidentified serine tripeptidyl endopeptidase; the other peptidase catalyzed the release of Gly-Pro from Gly-Pro-pNA and was inhibited by DFP, phenylmethyl sulfonyl fluoride and diprotin A, thus resembling dipeptidyl peptidase IV (DPP IV) with respect to its substrate specificity and inhibitor profile. A group of N-exo-aminopeptidase activities specifically inhibited by bestatin, was also detected when Ala-, Leu-, Arg- and Lys-pNA were used a substrates. The activities were surface associated and not secreted as determined by extracellular location of product and enzymatic recovery in highly purified U937 cell membranes. Peripheral monocytes and macrophages were found to virtually exhibit identical levels of these two classes of peptidase activities when compared to those detected on U937 cells. The relative contributions of these hydrolytic enzymes to the cleavage of bioactive and radioiodinated cytokines including tumor necrosis factor-alpha (TNF-alpha), interleukin-1 alpha and interferon-gamma was next examined. The results indicated that N-aminopeptidases do not appear to participate in the catabolism of any tested cytokine. In contrast, the most interesting finding was that both serine peptidases participate in TNF-alpha degradation. Analysis of the final proteolytic digestion products demonstrated the disappearance of the native 17-kDa molecule TNF-alpha, and the concomitant release of biologically inactive fragments of less than or equal to 2 kDa. Together, these observations indicate new roles for both the DPP IV-like enzyme and the tripeptidyl endopeptidase located at the surface of human monocytic cells, including the regulation of the extracellular TNF-alpha concentration. Thus, the identification of functional ectopeptidases provides insight into their potential role in both normal and malignant monocytic function.
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PMID:Human U937 cell surface peptidase activities: characterization and degradative effect on tumor necrosis factor-alpha. 134 32

Clinical and veterinary uses of growth hormone-releasing factor [GRF(1- 29)NH2] require the design of analogs that are resistant to proteolysis by serum and liver degrading enzymes. This study investigated rat GRF(1-29)NH2 processing in serum and liver homogenate by means of high pressure liquid chromatography (HPLC). Synthetic rGRF(1-29)NH2 (30 microM) was incubated (0-120 min, 37 degrees C) in serum (49 +/- 8 mg prot./ml). The rGRF(1-29)NH2 (10 microM) was also incubated (0-120 min, 37 degrees C) with liver homogenate (200 +/- 6 micrograms prot./ml). Time course studies of rGRF(1-29)NH2 disappearance showed apparent half-lives of 18 +/- 4 min and 13 +/- 3 min in serum and liver homogenate, respectively. This was accompanied by the appearance of degradation products that were all less hydrophobic than the native peptide. In the serum, two major metabolites were detected and isolated by preparative HPLC. Combined results of amino acid analysis, sequencing, and chromatography with synthetic homologs revealed the presence of rGRF(1-20)OH and (3-20)OH. A small amount of rGRF(12-29)NH2, coeluting with rGRF(3-20)OH, was also found by sequencing. In the liver, rGRF(1-18)OH, (3-18)OH, and (1-10)OH were identified. The peptide bond Ala2-Asp3 (DPP IV cleavage site) was hydrolyzed in both serum and liver. Other tissue-specific cleavage sites were Arg11-Arg12 and Arg20-Lys21 (trypsin-like cleavage site) in the serum, and Tyr10-Arg11 and Tyr18-Ala19 (chymotrypsin-like cleavage site) in the liver.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Catabolism of rat growth hormone-releasing factor(1-29) amide in rat serum and liver. 143 11

20 untreated chronic periodontitis patients were given a full periodontal examination, including measurements of probing depth (PD), clinical attachment loss (CAL), gingival index (GI), bleeding index (BI) and plaque index (Pl.I.). At a second visit, gingival crevicular fluid (GCF) was collected on filter paper strips from the deepest accessible probing site of each tooth. GCF volumes were determined and the samples eluted into buffer. Protease activities in the resulting eluates were assayed with peptidyl derivatives of 7-amino-4-trifluoromethyl coumarin (AFC). Cathepsin B/L-like activity was determined with Bz-Val-Lys-Lys-Arg-AFC, elastase-like activity with MeOSuc-Ala-Ala-Pro-Val-AFC, tryptase-like activity with Z-Ala-Ala-Lys-AFC, trypsin-like activity with Z-Gly-Gly-Arg-AFC and dipeptidyl peptidase (DPP) IV-like activity with Ala-Pro-AFC. Total enzyme activities and enzyme concentrations both correlated positively with all clinical parameters in linear regression analysis. This was true on both a patient level, using mean patient values, and a site level, using either individual patient or pooled patient data. Most of these correlations were statistically significant, although the proportion was greater for total enzyme activity than concentration. With total activities, correlations with different enzymes and parameters generally followed the order: cathepsin B/L-greater than elastase- greater than DPP IV- greater than trypsin- greater than tryptase-like activity and PD greater than CAL greater than GI greater than BI greater than Pl.I respectively. Total enzyme activities had good diagnostic specificity and sensitivity as predictors of clinical parameters in this cross-sectional study, suggesting that GCF proteases might provide useful information on the periodontal condition.
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PMID:Cathepsin B/L-, elastase-, tryptase-, trypsin- and dipeptidyl peptidase IV-like activities in gingival crevicular fluid: correlation with clinical parameters in untreated chronic periodontitis patients. 153 11

Murine thymocytes are shown to possess at least three well-defined exo-N-aminopeptidase activities on their surface. One of them cleaves the prolyl bond in the synthetic dipeptide nitroanilide Gly-Pro-pNA (Km 0.95 mM and Vmax 8 nmol/h at pH 7.4 and 37 degrees C) and is specifically inhibited by phenylmethane sulfonyl fluoride, diprotin A, Gly-Pro-Ala and Gly-Pro-Gly-Gly. These data further support identification of this enzyme with a serine exopeptidase dipeptidyl peptidase IV (DPP IV), previously reported to be specific for collagen. The two other forms of N-exopeptidase activities are detected when Ala-pNA and Leu-pNA are used as substrates. Leu-aminopeptidase activity (Km 1.4 mM, Vmax 15 nmol/h) and Ala-aminopeptidase activity (Km 4.0 mM, Vmax 20 nmol/h) are inhibited by inhibitors for thiol- and trypsin-like proteinases, i.e. tosyl lysyl chloromethyl ketone, leupeptin and N-ethylmaleimide. Addition inhibition of Leu-aminopeptidase activity by peptstatin, a known inhibitor of carboxyl proteases, suggests that aminopeptidase activity detected with Leu-pNA is different in part from Ala-aminopeptidase activity. Among the various lymphoid cell populations tested, the three aminopeptidase activities are increased by three- to fourfold in the immature CD4-CD8- thymocyte subset as well as in the thymoma BW5147. In contrast, cortisone-resistant thymocytes, lymph node and spleen cells exhibit levels of activities almost similar to that of unfractionated thymocytes. During ontogeny, the levels of these activities are increased four- to sevenfold on fetal thymocytes (from days 14 to 16). Finally, when thymocytes or spleen cells are cultured with a mitogenic concentration of concanavalin A, their proliferative responses are correlated with an enhancement of the aminopeptidase activities (1.3- to 5-fold). From these results, a correlation between the presence of these peptidases on the cell surface of immature and mature lymphoid cells and biological responsiveness is suggested.
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PMID:Murine thymocytes possess specific cell surface-associated exoaminopeptidase activities: preferential expression by immature CD4-CD8- subpopulation. 210 42

Substrate impregnated paper discs were prepared using peptidyl derivatives of 7-amino-4-trifluoromethylcoumarin (AFC). After incubation with test solutions, the green, UV-induced fluorescence of AFC liberated by enzyme activity was distinguishable from the blue-violet fluorescence of the substrates. The AFC could then be coupled with p-dimethylaminocinnamaldehyde to form a colored Schiff base. Semi-quantiative assessments of disc fluorescence and color were made by comparison with AFC/substrate standards. Assays with discs impregnated with MeOSuc-Ala-Ala-Pro-Val-AFC, Z-Gly-Gly-Arg-AFC and Ala-Pro-AFC for elastase-, trypsin-, and dipeptidyl peptidase (DPP) IV-like activities respectively were evaluated using purified DPP IV and 100 eluates of crevicular fluid collected on filter paper strips from 10 gingivitis and periodontitis patients. The results showed that, within their working ranges, scores of disc fluorescence and color were reasonably accurate and reliable by comparison with enzyme activities measured in parallel quantitative fluorimetric assays with the same substrates. Using disc color, which was more sensitive than fluorescence, it was generally possible to measure all three enzyme activities in crevicular fluid samples from 5 periodontitis patients with varying degrees of gingival inflammation and pocketing. Disc color assays require no special apparatus and could be used for enzyme estimations in the clinical setting.
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PMID:A simple, combined fluorogenic and chromogenic method for the assay of proteases in gingival crevicular fluid. 214 76

The plasma enzyme responsible for primary proteolytic cleavage of growth hormone-releasing hormone (GRH) at the 2-3 amino acid bond was characterized. Native GRH[GRH(1-44)-NH2 and GRH(1-40)-OH], and COOH-terminally shortened fragments [GRH(1-32)-NH2 and GRH(1-29)-NH2] were rapidly cleaved, while GRH(2-32)-NH2 was not degraded at this site. Moreover, degradation to GRH(3-44)-NH2 was unaffected by an aminopeptidase inhibitor, indicating that this metabolite was generated from a single step cleavage by a dipeptidylpeptidase (DPP) rather than sequential aminopeptidase cleavages. Conversion to GRH(3-44)-NH2 was blocked by diprotin A, a DPP type IV (DPP IV) competitive inhibitor. D-Amino acid substitution at either position 1 or 2 also prevented hydrolysis, characteristic of DPP IV. Analysis of endogenous plasma GRH immunoreactivity from a human GRH transgenic pig revealed that the major peak coeluted with GRH(3-44)-NH2. Native GRH exhibited trypsin-like degradation at the 11-12 position but cleavage at the 12-13 site occurred only with GRH(1-32)-NH2 and GRH(1-29)-NH2. Formation of these metabolites was independent of prior DPP IV hydrolysis but was greatly reduced by trypsin inhibitors. Evaluation of plasma stability of potential GRH super analogues, designed to resist degradation by these enzymes, confirmed that GRH degradation in plasma occurs primarily by DPP IV, and to a lesser extent by trypsin-like enzyme(s).
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PMID:Dipeptidylpeptidase IV and trypsin-like enzymatic degradation of human growth hormone-releasing hormone in plasma. 256 42

Crevicular fluid samples were collected from 20 gingivitis and periodontitis patients using filter paper strips; these were then eluted into buffer. Portions of each sample were combined and the activities of this pooled eluate against different peptidyl derivatives of 7-amino-4-trifluoromethyl coumarin (AFC) were examined with respect to their pH profiles and effector responses. Ca-thepsin B- and L-like activity was detected with Bz-Val-Lys-Lys-Arg-AFC; elastase-like activity with MeOSuc-Ala-Ala-Pro-Val-AFC; tryptase-like activity with Z-Ala-Ala-Lys-AFC; trypsin-like activity with Z-Gly-Gly-Arg-AFC; and dipeptidyl peptidase (DPP) IV-like activity with Ala-Pro-AFC. The selectivity and sensitivity of these assays were improved by choice of appropriate conditions. The cathepsin B- and L-, elastase-, tryptase-, and trypsin-like activities all had properties consistent with those from host sources, whilst partial inactivation of the DPP IV-like activity by heat treatment (60 degrees C for 30 min) suggested that it may have represented a mixture of human and Bacteroides gingivalis enzymes. Individual patient eluates showed wide variations in enzyme concentrations, but generally elastase-like activity was by far the highest. The sensitivity of the assays with AFC-linked substrates was such that it should prove possible to measure all five different types of activity in crevicular fluid samples from local periodontal disease sites.
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PMID:Detection of cathepsin B- and L-, elastase-, tryptase-, trypsin-, and dipeptidyl peptidase IV-like activities in crevicular fluid from gingivitis and periodontitis patients with peptidyl derivatives of 7-amino-4-trifluoromethyl coumarin. 257 34

The metabolism of the cell surface glycoprotein dipeptidyl peptidase IV(DPPIV) was studied in cultured rat hepatocytes. In pulse-chase labelling experiments using L-[35S]methionine a 100-kDa high-mannose precursor polypeptide is converted into the mature complex-type 110-kDa glycoprotein. Digestion with exo- and endoglycosidases and metabolic labelling with radioactive sugars demonstrate that the 110-kDa form contains about 6 complex-type oligosaccharides which are fucosylated and sialylated. About 25 min after the beginning of the pulse-labelled glycoprotein appears in the sinusoidal membrane. Physiologically only the 110-kDa form is found in the cell surface. If cell surface DPP IV was desialylated by sialidase at 4 degrees C, it is resialylated during incubation at 37 degrees C. This oligosaccharide reprocessing indicates that the surface glycoprotein has been recycled to the cell compartment containing terminal glycosyltransferases (presumably the trans Golgi system). Two different methods demonstrate internalization of cell surface DPP IV: 1) The complex cell surface DPPIV -anti-DPP IV-antibody -L-[35S]methionine-labelled secondary goat-anti-mouse-antibody formed at 4 degrees C becomes less accessible to trypsin during incubation at 37 degrees C. 2) Part of the complex plasma membrane DPP IV-anti-DPP IV-antibody formed in the cold cannot be recognized by the radioactive secondary antibody after rewarming. Internalization is not blocked by inhibition of protein synthesis with cycloheximide. During internalization of plasma membrane DPP IV its concentration in the membrane remains constant.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Oligosaccharide reprocessing and recycling of a cell surface glycoprotein in cultured rat hepatocytes. 810 Oct 88

A number of dipeptide diphenyl phosphonate esters were studied as inhibitors of dipeptidyl peptidase IV, focusing on the role of the P2 residue in the inactivation process. The active compounds were slow irreversible inhibitors of the catalytic activity of the enzyme. With proline (or alanine) in the P1 position, the rate constants of inactivation correlated with the acylation rate constants reported for homologous dipeptide derived substrates. The kinetic data indicate that the mechanism of inhibition consists of the formation of a fairly weak initial complex, followed by a slow irreversible inactivation step. This indicates that, as in the case of trypsin-like proteinases, dipeptide diphenyl phosphonate esters form a covalent adduct with the catalytic site of DPP IV, even though this enzyme belongs to a completely distinct class of serine peptidases. Enantioselectivity and secondary specificity further support the evidence that diphenyl phosphonate esters are mechanism-based inhibitors. The dipeptide diphenyl phosphonate esters had a half-life of 3-10 h at 37 degrees C in Tris buffer. The inhibitors were degraded in human plasma, depending on the type of amino-terminal amino acid. The compound with proline in the P2 position was the most resistant to degradation in plasma. Due to their stability and the irreversible nature of the inhibition, the diphenyl phosphonate esters promise to be useful tools in the continuing investigation of the physiological function of dipeptidyl peptidase IV.
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PMID:Dipeptide-derived diphenyl phosphonate esters: mechanism-based inhibitors of dipeptidyl peptidase IV. 864 10

The T-cell activation antigen CD26, is a type II membrane glycoprotein with intrinsic dipeptidyl-peptidase IV (DPP IV) activity, characterized by its capacity to cleave off N-terminal dipeptides containing proline as the penultimate residue. Independent of its catalytic activity, CD26 has also been characterized as adenosine deaminase binding protein. By using CD26 negative human C8166 cells, here we describe the existence of another cell-surface protein which manifests CD26-like DPP IV activity. For convenience, this protein will be referred to as DPP IV-beta. Consistent with the cell-surface expression of DPP IV-beta, intact C8166 cells manifested a high level of DPP IV, whereas, they manifested poor activity against substrates of DPP II known to have an intracellular localization. A partially purified preparation of CD26 from human MOLT4 cells, and the DPP IV-beta expressed on intact cells were found to possess similar catalytic activity and pH optimum. In addition, cell-surface CD26 and DPP IV-beta on intact MOLT4 and C8166 cells, respectively, resisted digestion by proteolytic enzymes such as trypsin and proteinase K. However, adenosine deaminase activity was not detectable on the surface of C8166 cells in contrast to CD26 positive MOLT4 cells. In accord with this, 125I-labeled adenosine deaminase which binds CD26 was found not to bind DPP IV-beta. Gel-filtration experiments using 0.5% Triton X-100 extracts from C8166 and MOLT4 cells, revealed that the apparent molecular mass of DPP IV-beta is 82 kDa, whereas that of CD26 is 110 kDa as expected. Taken together, our results suggest that DPP IV-beta is a CD26-like protein which could be characterized by distinct properties.
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PMID:Dipeptidyl-peptidase IV-beta, a novel form of cell-surface-expressed protein with dipeptidyl-peptidase IV activity. 870 27


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