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PubMed1991

Purification and characterization of dipeptidyl peptidase IV from Streptococcus salivarius HHT.

Dipeptidyl peptidase IV (DAP IV) was purified from Streptococcus salivarius HHT by anion-exchange chromatography, gel filtration and affinity chromatography after lysis of cell walls with N-acetylmuramidase. DAP IV was purified 114-fold with a yield of 16.6% from total activity of the crude extract. The purified enzyme was shown to be homogeneous by disc gel electrophoresis. The molecular weight of the enzyme was estimated to be about 109,000 by gel filtration and 47,000 by sodium dodecylsulphate SDS-polyacrylamide gel electrophoresis, suggesting that the native enzyme is a dimeric form. The optimum pH for the reaction was 8.7 in Gly-NaOH buffer, and the isoelectric point of the enzyme was pH 4.2. The enzyme hydrolysed specifically N-terminal X-Pro from X-Pro-p-nitroanilides. The enzyme activity was hardly affected by various cations, sulphydryl-blocking reagents and metal chelators. The enzyme activity was markedly inhibited by 1 mM diisopropylfluoride, and the desialysed enzyme was attacked by proteinases.

Chromatography, Affinity↗

PubMed1991

Evidence that thymocyte-activating molecule is mouse CD26 (dipeptidyl peptidase IV).

We previously described a developmentally regulated, Mr 115,000 (reduced) and 110,000/128,000 (nonreduced) mouse T cell-activating molecule (THAM) also expressed on a variety of epithelial cell surfaces, and associated with neutral exoaminopeptidase activity. In the present study, we show that THAM is the mouse counterpart of the human T cell-activating ectoenzyme CD26 (dipeptidyl peptidase IV, DPP IV) and that highly purified THAM lacks neutral exoaminopeptidase activity. This conclusion is based on the following: 1) the N-terminal segments of the THAM Mr 110,000 and 128,000 components shared the same amino acid sequence with the rat DPP IV. These N-termini comprised a short intracytoplasmic tail of six residues followed by a downstream hydrophobic transmembrane segment. 2) THAM-specific mAb H194-112-Affi-Gel immunoadsorbent was capable of removing DPP IV enzymatic activity from mouse thymoma cell detergent extracts. 3) H194-112 reactivity pattern on developing thymocytes was found to parallel that previously reported for membrane-bound DPP IV enzymatic activity. The extent of THAM N-glycosylation, as measured by N-glycanase treatment of H194-112 immunoprecipitates, was found to be similar to that of human and rat DPP IV (i.e., approximately 20 kDa). Cross-linking experiments indicated that THAM was expressed at the cell surface as a dimer of approximately 220 kDa. Its two subunits were found to be structurally related but not identical as shown by their different Mr under nonreducing conditions and by their slightly distinct peptide profiles after proteolytic cleavage. We conclude from these data that DPP IV, in addition to its extracellular matrix receptor and ectoenzymatic functions, is a T cell-activating structure in both human and mouse species.

Amino Acid Sequence↗

PubMed1990

Evidence for tripeptide-proton symport in renal brush border membrane vesicles. Studies in a novel rat strain with a genetic absence of dipeptidyl peptidase IV.

We have investigated the transport characteristics of L-phenylalanyl-L-prolyl-L-alanine in renal brush-border membrane vesicles isolated from Japan Fisher 344 rats. This particular rat strain genetically lacks dipeptidyl peptidase IV. Owing to the absence of this enzyme, the tripeptide was found to be completely resistant to hydrolysis by the renal brush-border membrane vesicles. Uptake of the tripeptide into these membrane vesicles in the presence of an inwardly directed Na+ gradient was slightly greater than in the presence of a K+ gradient, but there was no evidence for active transport. On the contrary, uptake was very rapid in the presence of an inside-alkaline transmembrane pH gradient, and accumulation of the tripeptide inside the vesicles against a concentration gradient could be demonstrated under these conditions. The uptake was drastically reduced by dissipation of the pH gradient. The uptake was stimulated by an inside-negative membrane potential and inhibited by an inside-positive membrane potential. Moreover, the uptake was greater in voltage-clamped membrane vesicles than in control vesicles. Many di- and tripeptides inhibited this pH gradient-stimulated uptake of Phe-Pro-Ala. The apparent dissociation constant for the tripeptide was 48 microM. High performance liquid chromatography analysis of the intravesicular content at the peak of the overshoot revealed that the tripeptide was transported across the membrane almost entirely in the intact form. These data provide the first direct evidence for the presence of an electrogenic tripeptide-proton symport in renal brush-border membranes.

Amino Acid Sequence↗

PubMed1989

Primary structure of rat liver dipeptidyl peptidase IV deduced from its cDNA and identification of the NH2-terminal signal sequence as the membrane-anchoring domain.

Two forms of dipeptidyl peptidase IV (DPP) were purified from rat liver plasma membranes: a membrane form (mDPP) extracted with Triton X-100 and a soluble form (sDPP) prepared by treatment with papain. Apparent molecular masses of mDPP and sDPP were 109 and 105 kDa, respectively, when determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The NH2-terminal sequences of the two forms were found to be completely different from each other. For further information on the molecular structure, we constructed a lambda gt11 liver cDNA library and isolated two cDNA clones for DPP, lambda cDP37 and lambda cD5. The 3.5-kilobase cDNA insert of lambda cDP37 contains an open reading frame that encodes a 767-residue polypeptide with a calculated size of 88,107 Da, which is in reasonable agreement with that of DPP (87 kDa) immunoprecipitated from cell-free translation products. Eight potential N-linked glycosylation sites were found in the molecule, accounting for the difference in mass between the precursor and mature forms. Of particular interest is that the deduced NH2-terminal sequence with a characteristic signal peptide is completely identical to that determined for mDPP. In addition, the NH2-terminal sequence of sDPP is identified in the predicted sequence starting at the 35th position from the NH2 terminus. These results indicate that the signal peptide of DPP is not cleaved off during biosynthesis but functions as the membrane-anchoring domain even in the mature form. It is also found that the primary structure thus predicted has striking homology to that of gp 110, a bile canaliculus domain-specific membrane glycoprotein (Hong, W., and Doyle, D. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 7962-7966).

Amino Acid Sequence↗

PubMed1985

[Dipeptidyl peptidase IV. Purification for use in peptide sequencing].

Isolation and purification of dipeptidyl peptidase IV from pig kidney are described. The specific activity of the enzyme is 24.9 U/mg protein. Chromatography on Gly-Pro-AH-Sepharose is the most important procedure for the separation from other enzyme activities. The enzyme preparation is free of aminopeptidase, dipeptidase and prolyl endopeptidase activity; it can be used for peptide-sequence analysis.

Amino Acid Sequence↗

PubMed1986

[Diagnostic value of the enzyme dipeptidyl peptidase IV (DP IV) in abdominal cancers].

Activity changes of the enzyme dipeptidyl peptidase IV (DP IV) were observed in connection with various diseases. The increase of serum activity in case of liver diseases and the decrease of activity in tissue and serum in case of malignant diseases are known. This investigation examined serum activity of DP IV at 168 patients with malignant diseases of the gastro-intestinal system. The 5%-, 50%- and 95%-percentiles of serum activity of DP IV were determined. Age and sex did not influence the activity of DP IV as less as attendant diseases like diabetes mellitus and hypertension. Malignant diseases of the gastro-intestinal system without liver metastases do have a significantly lower serum activity of DP IV. Malignant diseases of the pancreas and the bile duct and liver metastases lead to an increase of DP IV-serum activity.

Abdominal Neoplasms↗

PubMed1986

[Determination of dipeptidyl-peptidase IV in biological materials].

Dipeptidyl-peptidase IV (EC 3.4.14.5) can be assayed relatively specifically in crude biological material by determination of the initial rate of formation of 4-nitroaniline from aminoacyl-proline-4-nitroanilides. For clinical purposes tricine buffer, pH 7.4, is employed, because unspecific cleavage of the substrate is minimal under these conditions. Various types of chromogenic and fluorogenic leaving groups can be used to detect the enzyme on solid supports. During the enzymatic hydrolysis of the tripeptides Xaa-Pro-Yaa, there is an increase of absorbance at 228 nm, which is useful for monitoring the enzyme activity. Enzymic activity can also be measured titrimetrically in unbuffered solutions, whereby corrections must be made for the protonation of both substrate and products.

Buffers↗

PubMed1987

Alteration in dipeptidyl peptidase activities in cultured human carcinoma cells.

For the investigation of the possibility of its being a marker enzyme for tumor cells, the activity of dipeptidyl peptidase (DPP) IV (EC 3.4.14.5), a membrane-bound enzyme, in cultured human carcinoma cells was examined. The homogenates of three carcinoma cell lines (HeLa, KB, and K-44) contained lower glycylprolyl methylcoumarinamide (Gly-Pro-MCA) hydrolase activities at pH 8.7 (assumed to be DPP IV) and higher activities of alkaline phosphatase and gamma-glutamyl transpeptidase, which are also membrane-bound enzymes, than those of normal human fibroblasts (HF). Examination of carcinoma cells for the subcellular localization and pH optimum of Gly-Pro-MCA hydrolase activity revealed that the activity of a lysosomal enzyme that hydrolyzes Gly-Pro-MCA at pH 6.4 was markedly increased in carcinoma cells, but not in normal cells. The separation and characterization of Gly-Pro-MCA hydrolases by gel filtration, affinity chromatography, and substrate specificity demonstrated that HF have three peaks indicating DPP IV, DPP II, and an unknown enzyme, whereas the three carcinoma cell lines gave a prominent peak indicating DPP II and a trace of DPP IV. The DPP II activity was 6- to 24-fold higher in carcinoma cell lines than in HF, and it also was 2.85- to 4.13-fold higher than the DPP IV activity in carcinoma cell lines but was 10-fold lower in HF. These clear enzymatic differences between carcinoma cells and normal HF may be useful as a marker of malignancy.

Alkaline Phosphatase↗

PubMed1987

Dipeptidyl peptidase IV activity in cells of T-lymphoid origin is decreased in cultures with 12-0-tetradecanoylphorbol-13-acetate (TPA).

Dipeptidyl peptidase IV (DPP IV) is a specific enzyme for cells of T-lymphocytic lineage. It has been attempted to induce DPP IV activity in DPP IV negative T-lymphoid leukaemias by 12-o-tetradecanoylphrobol-13-acetate (TPA). Isolated cells from peripheral blood of 6 healthy blood donors and 22 patients with various types of leukaemia were cultivated in RPMI-1640 medium with 20% fetal calf serum alone (control) or supplemented with 20% human placenta conditioned medium (HPCM) or with 16 nmol/l TPA for 3 days. The percentage of DPP IV positive lymphocytes from blood donors remained unchanged in control and HPCM cultures, but decreased significantly in TPA cultures. Leukemic cells from three DPP IV positive cases of acute T-lymphoblastic leukaemia (T-ALL) reacted to the TPA treatment in a similar manner. Leukaemic cells of two DPP IV negative T-ALL cases remained negative in all three types of cultures, thus no induction of DPP IV activity was found. Other normal blood cells as well as leukaemic cells of 7 null-ALL, 1 preB-ALL, 3 B-CLL and 6 AML patients were DPP IV negative before and after cultivation in all types of culture. These findings showed that DPP IV is specifically expressed in cells of T-lymphocytic lineage even after short-term cultivation. HPCM was found to have no effect on DPP IV activity in T-lymphoid cells.

Blood Cells↗

PubMed1987

Identification of gp108, a pathogenic antigen of passive Heymann nephritis, as dipeptidyl peptidase IV.

Our previous paper showed that the glycoprotein of Mr 108,000 (gp108) is one of the major components of rat renal tubular brush border antigens and that an injection of anti-gp108 antiserum in rats induces passive Heymann nephritis with acute and severe proteinuria. In this study, gp108 was identified as a monomer of dipeptidyl peptidase IV (DPP IV). The anti-gp108 antibody was shown to immunoprecipitate DPP IV from Triton-extract of renal tubular membrane fractions. DPP IV was co-purified with gp108 from the Triton-extract by columns of DEAE-cellulose and Bio-gel A-1.5 m. The ratio of DPP IV activity and gp108 content was nearly constant throughout the purification steps. The final purified gp108 showed a high specific activity of DPP IV, comparable to that reported for purified rat DPP IV. These results indicate that gp108 is a monomer of DPP IV.

Animals↗

PubMed1988

Remodeling of a rat hepatocyte plasma membrane glycoprotein. De- and reglycosylation of dipeptidyl peptidase IV.

The present paper demonstrates the terminal de- and reglycosylation of a rat hepatocyte plasma membrane glycoprotein, dipeptidyl peptidase IV (DPP IV). Cultured hepatocytes were used in pulse-chase experiments with [3H]L-fucose and [14C]N-acetyl-D-mannosamine as markers for terminal carbohydrates, [3H]D-mannose as marker of a core-sugar, and [35S]L-methionine for labeling the protein backbone. Membrane DPP IV was immunoprecipitated with a polyclonal antibody which bound selectively at 4 degrees C to the cell-surface glycoprotein. The times of maximal labeling of hepatocyte plasma membrane DPP IV were 6-9 min for [3H]L-fucose, 20 min for [3H]D-mannose, and 25 min for [35S]L-methionine. When antibodies were bound to cell-surface DPP IV at 4 degrees C, the immune complex remained stable for more than 1 h after rewarming to 37 degrees C, despite ongoing metabolic and membrane transport processes. This was shown by pulse labeling with [35S]L-methionine at 37 degrees C, followed by cooling to 4 degrees C, and addition of antibody against plasma membrane DPP IV. During rewarming, the radioactivity in the complex remained constant. In a similar experiment with [3H]L-fucose, the radioactivity in the immune complex declined rapidly, indicating a defucosylation of the plasma membrane glycoprotein. Using the same experimental design with [3H]D-mannose, the radioactivity in the immune complex remained constant, showing that the core-sugar D-mannose is not cleaved from the membrane glycoprotein. Terminal reglycosylation (refucosylation and resialylation) was demonstrated as follows. Hepatocytes were maintained at 37 degrees C in a medium supplemented with tunicamycin in order to block the de novo synthesis of N-glycosidically bound carbohydrate chains. At 4 degrees C the antibody against DPP IV bound only to cell surface glycoprotein. During the rewarming period at 37 degrees C, radioactivity from [3H]L-fucose and [14C]N-acetyl-D-mannosamine became incorporated into the immune complex. This indicates a fucosylation and sialylation of the glycoprotein originally present at the cell surface. The mechanisms whereby terminal de- and reglycosylation of plasma membrane glycoproteins may occur during membrane recycling are discussed.

Animals↗

PubMed1988

Assay of dipeptidyl peptidase IV in serum by fluorometry of 4-methoxy-2-naphthylamine.

A new fluorometric assay for determining dipeptidyl peptidase IV (DPP IV; EC 3.4.14.5) was developed. The synthetic substrate glycyl-L-proline-4-methoxy-2-naphthylamide (20 mmol/L), Tris buffer (50 mmol/L, pH 8.3), and serum (20 microL) are mixed and incubated. The reaction is stopped with citrate (100 mmol/L, pH 4.0) and the released 4-methoxy-2-naphthylamine is measured fluorometrically. The mean value of DPP IV activity in serum for 64 healthy subjects was 58 (SD 16) mumol of 4-methoxy-2-naphthylamine released per liter of serum per minute. The proposed procedure is sensitive, rapid, and accurate and can easily be automated.

2-Naphthylamine↗

PubMed1988

Dipeptidyl peptidase IV--subcellular localization, activity and kinetics in lymphocytes from control subjects, immunodeficient patients and cord blood.

Dipeptidyl peptidase IV (DPIV), a T cell enzyme, has been implicated in the regulation of lymphocyte proliferation in response to lectins and allogeneic cells. A sensitive fluorimetric assay has been established for the enzyme and used to investigate DPIV activity, kinetics and the subcellular localization in lymphocytes from control subjects, cord blood and patients with common variable hypogammaglobulinaemia (CVH) and chronic lymphatic leukaemia (CLL). Using sucrose density gradient centrifugation and organelle marker enzyme assays, in conjunction with digitonin as a selective plasma membrane perturbant and diazotized sulphanilic acid as a non-permeant enzyme inhibitor, DPIV was shown to be a plasma membrane ecto-enzyme. A significant decrease in lymphocyte DPIV activity was observed in cord blood and in patients with CVH and CLL compared to controls. Kinetic analysis showed a marked decrease in the Vmax of lymphocyte DPIV from cord blood and patients with CVH and CLL compared to controls. The apparent Km for the substrate was unaffected in cord blood and patients with CLL. However, in patients with CVH the Km was significantly reduced. Various enzyme inhibitors showed no differences between control subjects and CVH lymphocyte activities. The decreased Km for DPIV provides further evidence for a stem cell defect rather than cell immaturity in CVH.

Agammaglobulinemia↗

PubMed1988

[Detection of dipeptidyl peptidase IV in follicular fluid--initial report].

Aspirates of ovarian follicles were obtained from patients subjected to gynaecologic surgeries. This is the first report of dipeptidyl-peptidase IV (DP IV) activity in human follicular fluid. This enzyme specifically cleaves regulatory important oligo- and polypeptides. Only from peptides with proline or alanine in position P1 of the N-terminal sequence a dipeptide is split off. DP IV activity was measured with glycyl-L-proline p-Nitroanilide as substrate. Levels in a range of 209.8 +/- 57.4 nmol l-1 s-1 were achieved.

Dipeptidyl Peptidase 4↗

PubMed1982

Enzymehistochemical staining of T mu lymphocytes for glycyl-proline-4-methoxy-beta-naphthylamide-peptidase (DAP IV).

Human T mu lymphocytes are detected in a histochemical reaction with glycyl-proline-4-methoxy-beta-naphthylamide-peptidase (DAP IV; EC 3.4.14.4). DAP IV occurs in 59.4 +/- 8.25 of human T lymphocytes in blood and marrow. Direct biochemical assays were in accord with the cytochemical findings. DAP IV-positive lymphocytes accounted for 89.5 +/- 0.6% of T-lymphocytes bearing Fc receptor for IgG contained 11.3 +/- 8.3% T-lymphocytes bearing Fc receptor for IgM.

Cell Separation↗

PubMed1984

Similarities of the substrate cleavage catalyzed by proline specific endopeptidase and dipeptidyl peptidase IV.

16 substrates of the types succinyl-alanyl-alanine-pX-anilide, and succinyl-alanyl-proline-pX anilide having different substituents in para-position of the aryl residue were synthesized and characterized. The influence of electronic as well as hydrophobic substituent constants, sigma and pi, on the hydrolysis of the substrates catalyzed by the enzyme Proline Specific Endopeptidase (PSE) has been investigated. In the Hansch approach, the catalytic constants lg kcat and lg (kcat/KM) of succinyl-alanyl-alanine-pX-anilides hydrolyzed by PSE correlate significantly with electronic substituent constants sigma, whereas no correlation in the case of succinyl-alanyl-proline-pX-anilides. The intercorrelation of the constants of the former substrates with corresponding data from Dipeptidyl Peptidase IV (DP IV) catalyzed hydrolysis of alanyl-alanine-pX-anilides suggest that both enzymes act by similar catalytic mechanism.

Chemical Phenomena↗

PubMed1994

Dipeptidyl peptidase IV: development, design, synthesis and biological evaluation of inhibitors.

Dipeptidyl peptidase IV (DPP IV, EC 3.4.14.5) is a highly specific serine protease which cleaves off N-terminal dipeptides from peptides with a penultimate proline or alanine. The enzyme is widely distributed in mammalian cells and tissues, but specific activities differ greatly. In the hematopoietic system it is found almost exclusively on T cells, where it is identified as CD26, a T cell activation molecule. DPP IV may be involved in the metabolism of peptides, intestinal assimilation and cell adhesion and it plays an integral role in T cell activation. DPP IV inhibitors may provide help during the further elucidation of the biological function(s) of this enzyme. Moreover, because of the integral role the enzyme plays in T cell activation, specific inhibition of DPP IV may constitute a new way of immune modulation. N-Peptidyl-O-acylhydroxylamines and boronic acid analogues of proline and alanine are two known DPP IV inhibitors. The major drawbacks for their therapeutic use are for the hydroxylamines, the toxicity and for the boronic acid derivatives, the chemical liability. A low toxicity, acceptable stability and a high specificity are essential criteria for the design of inhibitors that are suitable, not only for experimental, but also for therapeutic use. Therefore we proposed 5 types of potential DPP IV inhibitors: azapeptides, azetidines, Michael substrates, reduced peptides and phosphonic acids. All the synthesized compounds possess a substrate-like structure, which is a pre-requisite for recognition by the enzyme. We choose for a modified proline or alanine at the penultimate position, substituted with glycine, alanine, valine, isoleucine or phenylalanine at the N-terminus. The prepared compounds were screened biologically at a 5 mM concentration with a fluorometric method using Gly-Pro-4-Me-2NA as substrate.

Aza Compounds↗

PubMed1994

Components of biological variation in prolyl endopeptidase and dipeptidyl-peptidase IV activity in plasma of healthy subjects.

We investigated the components of biological variation in plasma prolyl endopeptidase (PEP; EC 3.4.21.26) and dipeptidyl-peptidase IV (DPP IV; EC 3.4..14.5) activity in healthy individuals. We took monthly blood samples from 26 healthy volunteers for determination of plasma PEP and DPP IV activity during one calendar year. The estimated CVs for PEP activity were: total (CVt) = 25.0%, interindividual (CVg) = 13.9%, and intraindividual (CVi) = 16.8%. There was a statistically significant (P < 0.0001) seasonal pattern in plasma PEP activity, with significantly higher values in the fall than in the other seasons. The peak-trough difference in the yearly variation in PEP activity, expressed as a percentage of the mean, was as high as 56.8%. The estimated CVs for DPP IV activity were: CVt = 17.1%, CVg = 14.5%, and CVi = 8.2%. DPP IV activity was significantly (P < 0.0001) higher in summer than in the other seasons but the amplitude of the yearly variation was small.

Adolescent↗