Isolation of dipeptidyl peptidase IV (DP 4) isoforms from porcine kidney by preparative isoelectric focusing to improve crystallization

2011 ◽  
Vol 392 (7) ◽  
Author(s):  
Leona Wagner ◽  
Michael Wermann ◽  
Fred Rosche ◽  
Jens-Ulrich Rahfeld ◽  
Torsten Hoffmann ◽  
...  
Keyword(s):  
Isoelectric Focusing ◽  
Specific Activity ◽  
Dipeptidyl Peptidase ◽  
Dipeptidyl Peptidase Iv ◽  
Kidney Cortex ◽  
Porcine Kidney ◽  
Ph Gradients ◽  
Activity Staining ◽  
Ph Range ◽  
Gel Ph

AbstractIn the present studies we resolved the post-translational microheterogeneity of purified porcine dipeptidyl peptidase IV (DP 4) from kidney cortex. Applying SDS-homogeneous DP 4 onto an analytical agarose isoelectric focusing (IEF) gel, pH 4–6, activity staining resulted in at least 17 isoforms between pH 4.8–6.0. These could be separated into fractions with only two to six isoforms by means of preparative liquid-phase IEF, using a Rotofor cell. Starting off with three parallel Rotofor runs under the same conditions at pH 5–6, the fractions were pooled according to the specific activity of DP 4, pH and analytical IEF profile, and further refractionated without any additional ampholytes. Since excessive dilution of ampholytes and proteins was kept to the minimum, a second refractionation step could be introduced, resulting in pH gradients between 0.022 and 0.028 pH increments per fraction. By performing two consecutive refractionation steps, the high resolution necessary for the separation of DP 4 isoforms could be achieved. This represents an alternative method if isolation of isoforms with similar pI's results in precipitation and denaturation in presence of a narrow pH range. Furthermore, it demonstrates that preparative IEF is a powerful tool to resolve post-translational microheterogeneity of a purified protein required for crystallization processing.

Bestatin and bacitracin inhibit porcine kidney cortex dipeptidyl peptidase IV activity and reduce human melanoma MeWo cell viability

2020 ◽  
Vol 164 ◽  
pp. 2944-2952
Author(s):  
Laura Rivera Méndez ◽  
Yarini Arrebola ◽  
Mario E. Valdés-Tresanco ◽  
Lisset Díaz-Guevara ◽  
Gretchen Bergado ◽  
...  
Keyword(s):  
Cell Viability ◽  
Dipeptidyl Peptidase ◽  
Human Melanoma ◽  
Dipeptidyl Peptidase Iv ◽  
Kidney Cortex ◽  
Porcine Kidney ◽  
Mewo Cell

Effect of divalent cations on the porcine kidney cortex membrane-bound form of dipeptidyl peptidase IV

2011 ◽  
Vol 43 (3) ◽  
pp. 363-371 ◽  
Author(s):  
Isel Pascual ◽  
Hansel Gómez ◽  
Tirso Pons ◽  
Mae Chappé ◽  
Miguel Angel Vargas ◽  
...  
Keyword(s):  
Divalent Cations ◽  
Dipeptidyl Peptidase ◽  
Dipeptidyl Peptidase Iv ◽  
Kidney Cortex ◽  
Porcine Kidney ◽  
Membrane Bound

scholarly journals Comprehensive analysis of the isozyme composition of laccase derived from Japanese lacquer tree, Toxicodendron vernicifluum

2021 ◽  
Vol 67 (1) ◽  
Author(s):  
Mariko Takano ◽  
Masaya Nakamura ◽  
Masanobu Tabata
Keyword(s):  
Isoelectric Focusing ◽  
Laccase Activity ◽  
Maximum Activity ◽  
Acetone Powder ◽  
Blue Color ◽  
Buffer Solution ◽  
Activity Staining ◽  
Water Extracts ◽  
Ph Range ◽  
Toxicodendron Vernicifluum

AbstractWe performed an analysis using isoelectric focusing to comprehensively clarify the isozyme composition of laccase derived from Japanese lacquer tree, Toxicodendron vernicifluum. When water extracts of acetone powder obtained from lacquer were subjected to isoelectric focusing, five bands within pI 7.35–9.30 and nine bands within pI 3.50–5.25 were detected using Coomassie staining. Similarly, laccase activity staining using guaiacol showed five bands within pI 7.35–9.30 and three bands within pI 3.50–4.25. However, laccase activity staining using gallic acid showed remarkable staining within pI 3.50–5.85, whereas staining was very weak within pI 7.35–9.30. When the water extracts of acetone powder were fractionated into the fractions containing bands within pI 7.35–9.30 and pI 3.50–5.85 by SP-Sepharose column chromatography, the former had a blue color and the latter a yellow color. The laccase activity was measured for each of the fractions in buffer solution in the pH range of 2.5–8.0. When syringaldazine, guaiacol, and 2,6-dimethoxyphenol were used as substrates, the yellow fraction showed considerably higher activity than the blue fraction for pH 5.5–7.5. When 3-methylcatechol and 4-methylcatechol were used as substrates, the yellow fraction showed higher activity for pH 4.5–6.5, and the blue fraction showed higher activity for pH 7.0–8.0. When 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) was used as the substrate, both fractions showed maximum activity at optimum pH of 3.0–4.0. Conventionally, in research on blue laccase derived from lacquer, the non-blue fraction corresponding to the yellow fraction lower than pI 6 has been removed during the purification process and thus has not been analyzed. Our results indicated that yellow laccase was present in the non-blue components of lacquer and that it may play a role in urushiol polymerization with previously reported blue laccase.

scholarly journals Influence of dipeptidyl peptidase IV on enzymatic properties of adenosine deaminase.

2006 ◽  
Vol 53 (3) ◽  
pp. 539-546 ◽  
Author(s):  
Svetlana Sharoyan ◽  
Alvard Antonyan ◽  
Sona Mardanyan ◽  
Giulio Lupidi ◽  
Gloria Cristalli
Keyword(s):  
Adenosine Deaminase ◽  
Immune Cell ◽  
Dipeptidyl Peptidase ◽  
Dipeptidyl Peptidase Iv ◽  
Soluble Form ◽  
Kidney Cortex ◽  
Peptidase Activity ◽  
Extracellular Medium ◽  
Ph Profile ◽  
Enzyme Preparations

The importance of ADA (adenosine deaminase) in the immune system and the role of its interaction with an ADA-binding cell membrane protein dipeptidyl peptidase IV (DPPIV), identical to the activated immune cell antigen, CD26, has attracted the interest of researchers for many years. To investigate the specific properties in the structure-function relationship of the ADA/DPPIV-CD26 complex, its soluble form, identical to large ADA (LADA), was isolated from human blood serum, human pleural fluid and bovine kidney cortex. The kinetic constants (Km and Vmax) of LADA and of small ADA (SADA), purified from bovine lung and spleen, were compared using adenosine (Ado) and 2'-deoxyadenosine (2'-dAdo) as substrates. The Michaelis constant, Km, evidences a higher affinity of both substrates (in particular of more toxic 2'-dAdo) for LADA and proves the modulation of toxic nucleoside neutralization in the extracellular medium due to complex formation between ADA and DPPIV-CD26. The values of Vmax are significantly higher for SADA, but the efficiency, Vmax/Km, in LADA-catalyzed 2'-dAdo deamination is higher than that in Ado deamination. The interaction of all enzyme preparations with derivatives of adenosine and erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA) was studied. 1-DeazaEHNA and 3-deazaEHNA demonstrate stronger inhibiting activity towards LADA, the DPPIV-CD26-bound form of ADA. The observed differences between the properties of the two ADA isoforms may be considered as a consequence of SADA binding with DPPIV-CD26. Both SADA and LADA indicated a similar pH-profile of adenosine deamination reaction with the optimum at pHs 6.5-7.5, while the pH-profile of dipeptidyl peptidase activity of the ADA/DPPIV-CD26 complex appeared in a more alkaline region.

Cleavage by CD26/dipeptidyl peptidase IV converts the chemokine LD78β into a most efficient monocyte attractant and CCR1 agonist

Blood ◽  
2000 ◽  
Vol 96 (5) ◽  
pp. 1674-1680 ◽  
Author(s):  
Paul Proost ◽  
Patricia Menten ◽  
Sofie Struyf ◽  
Evemie Schutyser ◽  
Ingrid De Meester ◽  
...  
Keyword(s):  
Mononuclear Cells ◽  
Specific Activity ◽  
High Capacity ◽  
Dipeptidyl Peptidase ◽  
Dipeptidyl Peptidase Iv ◽  
Surface Glycoprotein ◽  
Peripheral Blood Mononuclear ◽  
Dpp Iv ◽  
Stromal Cell Derived Factor ◽  
Hiv 1

Abstract Chemokines are proinflammatory cytokines that play a role in leukocyte migration and activation. Recent reports showed that RANTES (regulated on activation normal T-cell expressed and secreted chemokine), eotaxin, macrophage-derived chemokine (MDC), and stromal cell–derived factor-1 (SDF-1) are NH2-terminally truncated by the lymphocyte surface glycoprotein and protease CD26/dipeptidyl peptidase IV (CD26/DPP IV). Removal of the NH2-terminal dipeptide resulted in impaired inflammatory properties of RANTES, eotaxin, MDC, and SDF-1. The potential CD26/DPP IV substrate macrophage inflammatory protein–1β (MIP-1β) and the related chemokine, LD78α (ie, one of the MIP-1α isoforms), were not affected by this protease. However, CD26/DPP IV cleaved LD78β, a most potent CCR5 binding chemokine and inhibitor of macrophage tropic human immunodeficiency virus–1 (HIV-1) infection, into LD78β(3-70). Naturally truncated LD78β(3-70), but not truncated MIP-1β, was recovered as an abundant chemokine form from peripheral blood mononuclear cells. In contrast to all other chemokines processed by CD26/DPP IV, LD78β(3-70) had increased chemotactic activity in comparison to intact LD78β. With a minimal effective concentration of 30 pmol/L, LD78β(3-70) became the most efficient monocyte chemoattractant. LD78β(3-70) retained its high capacity to induce an intracellular calcium increase in CCR5-transfected cells. Moreover, on CCR1 transfectants, truncated LD78β(3-70) was 30-fold more potent than intact LD78β. Thus, CD26/DPP IV can exert not only a negative but also a positive feedback during inflammation by increasing the specific activity of LD78β. CD26/DPP IV–cleaved LD78β(3-70) is the most potent CCR1 and CCR5 agonist that retains strong anti–HIV-1 activity, indicating the importance of the chemokine-protease interaction in normal and pathologic conditions.

scholarly journals Dipeptidyl peptidase IV, a kidney brush-border serine peptidase

1976 ◽  
Vol 157 (1) ◽  
pp. 169-182 ◽  
Author(s):  
A J Kenny ◽  
A G Booth ◽  
S G George ◽  
J Ingram ◽  
D Kershaw ◽  
...  
Keyword(s):  
Large Subunit ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Dipeptidyl Peptidase ◽  
Dipeptidyl Peptidase Iv ◽  
Sedimentation Equilibrium ◽  
Kidney Cortex ◽  
Serine Peptidase ◽  
Microvillus Membrane

Dipeptidyl peptidase IV, an enzyme that releases dipeptides from substrates with N-terminal sequences of the forms X-Pro-Y or X-Ala-Y, was purified 300-fold from pig kidney cortex. The kidney is the main source of the enzyme, where it is one of the major microvillus-membrane proteins. Several other tissues contained demonstrable activity against the usual assay substrate glycylproline 2-naphthylamide. In the small intestine this activity was greatly enriched in the microvillus fraction. In all tissues examined, the activity was extremely sensitive to inhibition by di-isopropyl phosphorofluoridate (Dip-F), but relatively resistant to inhibition by phenylmethylsulphonyl fluoride. It is a serine proteinase which may be covalently labelled with [32P]Dip-F, and is the only enzyme of this class in the microvillus membrane. The apparent subunit mol.wt. estimated by sodium dodecyl-sulphate/polyacrylamide-gel electrophoresis and by titration with [32P]Dip-F was 130 000. Gel-filtration and sedimentation-equilibrium methods gave values in the region of 280 000, which is consistent with a dimeric structure, a conclusion supported by electron micrographs of the purified enzyme. Among other well-characterized serine proteinases, this enzyme is unique in its membrane location and its large subunit size. Investigation of the mode of attack of the peptidase on oligopeptides revealed that it could hydrolyse certain N-blocked peptides, e.g. Z-Gly-Pro-Leu-Gly-Pro. In this respect it is acting as an endopeptidase and as such may merit reclassification and renaming as microvillus-membrane serine peptidase.

scholarly journals Protein heterogeneity of spinach pullulanase results from the coexistence of interconvertible isomeric forms of the monomeric enzyme

1998 ◽  
Vol 331 (3) ◽  
pp. 929-935 ◽  
Author(s):  
Anette HENKER ◽  
Ilka SCHINDLER ◽  
Andreas RENZ ◽  
Erwin BECK
Keyword(s):  
Isoelectric Focusing ◽  
Spinacia Oleracea ◽  
Specific Activity ◽  
Dissociation Constants ◽  
Protein Band ◽  
Enzyme Substrate ◽  
Spinacia Oleracea L ◽  
Starch Debranching Enzyme ◽  
Ph Range ◽  
The Individual

Purified pullulanase (starch-debranching enzyme, R-enzyme, EC 3.2.1.41) from spinach (Spinacia oleracea L.) chloroplasts separated into at least seven individual enzymically active proteins (isomers, numbered 1–7) on isoelectric focusing or column chromatofocusing. At their isoelectric points (between pH 4.7 and 5.2) these forms were rather stable. At slightly alkaline pH, each converted into the whole set of isomers. PAGE of the purified enzyme under denaturing or non-denaturing conditions resulted in one protein band. When substrate (amylopectin or pullulan) was included in the gel, the native enzyme as well as any of the individual isomers separated into two (sometimes three) bands (‘substrate-induced forms’, numbered I–III) with different specific activities, dissociation constants of the enzyme–substrate complexes and activation energies. Each substrate-induced form produced the whole set of seven isomers on isoelectric focusing. The specific activity of the total enzyme reflected the relative proportions of the substrate-induced forms. To some extent the relative proportions, as determined by crossed immunoelectrophoresis, could be shifted in favour of the more or the less active forms by reduction with dithiothreitol, and gentle oxidation respectively. Activation by dithiothreitol did not alter the mode of action of the enzyme but only increased the velocity of substrate degradation and extended its activity into the pH range of the chloroplast. As a consequence of isomer interconversion, microheterogeneity could serve to regulate pullulanase activity in a biochemical manner that shares some features with allosteric regulation.

Isoelectric focusing of milk-clotting enzymes

1977 ◽  
Vol 44 (1) ◽  
pp. 69-72 ◽  
Author(s):  
P. G. Righetti ◽  
Bruna M. Molinari ◽  
G. Molinari
Keyword(s):  
Thin Layer ◽  
Cellulose Acetate ◽  
Isoelectric Focusing ◽  
Polyacrylamide Gel ◽  
Acidic Ph ◽  
Ph Gradients ◽  
Milk Clotting ◽  
Cellulose Acetate Film ◽  
Ph Range ◽  
Highly Porous

SummaryIsoelectric focusing in thin-layer polyacrylamide gel has been applied successfully to the characterization and identification of calf rennet and its substitutes. The use of acidic pH gradients (pH range 2·5–6) allows the identification of calf and microbial rennets and bovine and pig pepsins.A new, very rapid and sensitive zymogram technique has been developed by overlaying the gel with a highly porous, transparent cellulose acetate film impregnated with casein.

Rat prostatic acid phosphatase: androgenic control of isoelectric focusing patterns

1983 ◽  
Vol 61 (7) ◽  
pp. 744-749 ◽  
Author(s):  
J. Downey ◽  
D. Mahan ◽  
T. G. Flynn ◽  
C. E. Bird ◽  
A. F. Clark
Keyword(s):  
Enzyme Activity ◽  
Acid Phosphatase ◽  
Isoelectric Focusing ◽  
Specific Activity ◽  
Electrophoretic Pattern ◽  
Prostatic Acid Phosphatase ◽  
Staining Intensity ◽  
Adult Rats ◽  
Enzyme Specific Activity ◽  
Ph Range

To further characterize the androgen dependence of prostatic acid phosphatase (AP), the isoelectric focusing patterns of enzyme activity have been examined for normal and castrated adult rats and for rats receiving androgen injections. Isoelectric focusing was performed in polyacrylamide gels over the pH range 4–8. Naphthyl phosphate was used as substrate for staining. For normal rats there was a single lysosomal band (isoelectric point (pI) = 7.35 ± 0.04), four closely migrating secretory bands (pI = 5.96–5.63), and an androgen-dependent band (pI = 6.37 ± 0.05) which as yet has not been identified as either lysosomal or secretory. Following castration the secretory bands decreased significantly in staining intensity, the androgen-dependent band disappeared, and two new lysosomal bands (pI's = 7.13 ± 0.03 and 7.00 ± 0.03) appeared. With androgen replacement the latter two bands disappeared, the androgen-dependent band reappeared, and the secretory bands increased in staining intensity but with the most anodic of the four appearing before the others. This suggests that it could be a precursor to the others. The isoelectric focusing patterns of AP activity appear to be a better method of assessing the androgen status of the prostate than are the previously used parameters, namely, enzyme specific activity, degree of inhibition by tartrate, and polyacrylamide gel electrophoretic pattern.

Cleavage by CD26/dipeptidyl peptidase IV converts the chemokine LD78β into a most efficient monocyte attractant and CCR1 agonist

Blood ◽  
2000 ◽  
Vol 96 (5) ◽  
pp. 1674-1680 ◽  
Author(s):  
Paul Proost ◽  
Patricia Menten ◽  
Sofie Struyf ◽  
Evemie Schutyser ◽  
Ingrid De Meester ◽  
...  
Keyword(s):  
Mononuclear Cells ◽  
Specific Activity ◽  
High Capacity ◽  
Dipeptidyl Peptidase ◽  
Dipeptidyl Peptidase Iv ◽  
Surface Glycoprotein ◽  
Peripheral Blood Mononuclear ◽  
Dpp Iv ◽  
Stromal Cell Derived Factor ◽  
Hiv 1

Chemokines are proinflammatory cytokines that play a role in leukocyte migration and activation. Recent reports showed that RANTES (regulated on activation normal T-cell expressed and secreted chemokine), eotaxin, macrophage-derived chemokine (MDC), and stromal cell–derived factor-1 (SDF-1) are NH2-terminally truncated by the lymphocyte surface glycoprotein and protease CD26/dipeptidyl peptidase IV (CD26/DPP IV). Removal of the NH2-terminal dipeptide resulted in impaired inflammatory properties of RANTES, eotaxin, MDC, and SDF-1. The potential CD26/DPP IV substrate macrophage inflammatory protein–1β (MIP-1β) and the related chemokine, LD78α (ie, one of the MIP-1α isoforms), were not affected by this protease. However, CD26/DPP IV cleaved LD78β, a most potent CCR5 binding chemokine and inhibitor of macrophage tropic human immunodeficiency virus–1 (HIV-1) infection, into LD78β(3-70). Naturally truncated LD78β(3-70), but not truncated MIP-1β, was recovered as an abundant chemokine form from peripheral blood mononuclear cells. In contrast to all other chemokines processed by CD26/DPP IV, LD78β(3-70) had increased chemotactic activity in comparison to intact LD78β. With a minimal effective concentration of 30 pmol/L, LD78β(3-70) became the most efficient monocyte chemoattractant. LD78β(3-70) retained its high capacity to induce an intracellular calcium increase in CCR5-transfected cells. Moreover, on CCR1 transfectants, truncated LD78β(3-70) was 30-fold more potent than intact LD78β. Thus, CD26/DPP IV can exert not only a negative but also a positive feedback during inflammation by increasing the specific activity of LD78β. CD26/DPP IV–cleaved LD78β(3-70) is the most potent CCR1 and CCR5 agonist that retains strong anti–HIV-1 activity, indicating the importance of the chemokine-protease interaction in normal and pathologic conditions.

Sign in / Sign up

Export Citation Format

Share Document