scholarly journals Purification, characterization and specificity of chondroitin lyases and glycuronidase from Flavobacterium heparinum

1995 ◽  
Vol 312 (2) ◽  
pp. 569-577 ◽  
Author(s):  
K Gu ◽  
R J Linhardt ◽  
M Laliberté ◽  
K Gu ◽  
J Zimmermann
Keyword(s):  
Catalytic Activity ◽  
Chondroitin Sulphate ◽  
Kinetic Properties ◽  
Apparent Homogeneity ◽  
Sds Page ◽  
Isoelectric Points ◽  
Chondroitin Ac Lyase ◽  
Flavobacterium Heparinum ◽  
Oligosaccharide Mapping

The chondroitin lyases from Flavobacterium heparinum (Cytophaga heparinia) have been widely used in depolymerization of glycosaminoglycan and proteoglycan chondroitin sulphates. Oligosaccharide products derived from chondroitin sulphate can be further degraded by glycuronidases and sulphatases obtained from the same organism. There has been no reported purification of these enzymes to homogeneity nor is there any information on their physical and kinetic characteristics. The absence of pure enzymes has resulted in a lack of understanding of the optimal conditions for their catalytic activity and their substrate specificity. This has limited the use of these enzymes as reagents for preparation of oligosaccharides for structure and activity studies. Reproducible schemes to purify a chondroitin AC lyase, a glycuronidase and chondroitin B lyase from Flavobacterium heparinum to apparent homogeneity are described. Chondroitin AC lyase (chondroitinase AC, EC 4.2.2.5), glycuronidase [chondro-(1-->3)-glycuronidase, no EC number] and chondroitin B lyase (chondroitinase B, no EC number) have M(r) values (assessed by SDS/PAGE) of 74,000, 41,800 and 55,200 respectively, and isoelectric points (determined by isoelectric focusing) of 8.85, 9.28 and 9.05 respectively. Chondroitin lyase AC and B contain pyroglutamic acid at their N-termini precluding their analysis by Edman degradation. Deblocking with pyroglutamate aminopeptidase facilitated the determination of their N-terminal sequences. The kinetic properties of these enzymes have been determined as well as the optimum conditions for their catalytic activity. The specificity of the glycouronidase, determined using 17 different disaccharide substrates, shows that it only acts on unsulphated or 6-O-sulphated 1-->3 linkages. The chondroitin lyases are both endolytic enzymes, and oligosaccharide mapping shows their expected specificity towards the chondroitin and dermatan sulphate polymers.

scholarly journals Role of arginine 292 in the catalytic activity of chondroitin AC lyase from Flavobacterium heparinum

2002 ◽  
Vol 1597 (2) ◽  
pp. 260-270 ◽  
Author(s):  
Ishan Capila ◽  
Yi Wu ◽  
David W Rethwisch ◽  
Allan Matte ◽  
Miroslaw Cygler ◽  
...  
Keyword(s):  
Catalytic Activity ◽  
Chondroitin Ac Lyase ◽  
Flavobacterium Heparinum

scholarly journals Purification and characterization of phosphatidylinositol 4-phosphate 5-kinases

1992 ◽  
Vol 288 (2) ◽  
pp. 637-642 ◽  
Author(s):  
N Divecha ◽  
C E L Brooksbank ◽  
R F Irvine
Keyword(s):  
Bovine Brain ◽  
Kinetic Properties ◽  
Type Ii ◽  
Purification And Characterization ◽  
Kinase C ◽  
Apparent Homogeneity ◽  
Sds Page ◽  
Single Polypeptide ◽  
Phosphatidylinositol 4

We detail the purification and characterization of three distinct isoforms of PtdIns4P 5-kinase present in bovine brain. One of these, PtdIns4P 5-kinase C, was purified to apparent homogeneity, and SDS/PAGE analysis demonstrated a single polypeptide and molecular mass 53 KDa. These three isoforms were shown to differ in their kinetic properties, and immunological characterization with an antibody raised to PtdIns4P 5-kinase C demonstrated that this isoform was unrelated to the other two. Furthermore, PtdIns4P 5-kinase C was shown to be the bovine brain homologue of the Type II PtdIns4P 5-kinase previously purified from human erythrocytes [Bazenet, Ruano, Brockman & Anderson (1990) J. Biol. Chem. 265, 18012-18022].

Characterization of Glycoprotein Fraction from Carp Pituitaries Isolated Using Concanavalin A as the Affinity Ligand

1998 ◽  
Vol 63 (3) ◽  
pp. 434-440 ◽  
Author(s):  
Irena Hulová ◽  
Jana Barthová ◽  
Helena Ryšlavá ◽  
Václav Kašička
Keyword(s):  
Concanavalin A ◽  
Reversed Phase ◽  
Amino Acid Sequences ◽  
Gel Chromatography ◽  
Affinity Ligand ◽  
Sds Page ◽  
Terminal Amino ◽  
Α And Β Subunits

Glycoproteins that have affinity to Concanavalin A were isolated from the acetone-dried pituitaries of common carp (Cyprinus carpio L.). Two fractions of glycoproteins were separated using gel chromatography on Superdex 75HR. The fraction with lower molecular weight (30 000) corresponding to the carp gonadotropin cGtH II was composed of two subunits as determined using SDS-PAGE. This protein fraction was further divided into four components using reversed-phase HPLC. Two fractions were pure α and β subunits of cGtH II as follows from immunodetection and from determination of N-terminal amino acid sequences. The other two were a mixture of α and β subunits as was also revealed by N-terminal analysis. Capillary electrophoresis was also used for characterization of isolated glycoproteins.

scholarly journals Gating Competence of Constitutively Open CLC-0 Mutants Revealed by the Interaction with a Small Organic Inhibitor

2003 ◽  
Vol 122 (3) ◽  
pp. 295-306 ◽  
Author(s):  
Sonia Traverso ◽  
Laura Elia ◽  
Michael Pusch
Keyword(s):  
Chloride Channels ◽  
Bound State ◽  
Side Chain ◽  
Pore Channel ◽  
Kinetic Properties ◽  
Wild Type ◽  
High Affinity ◽  
Critical Residue ◽  
Fast Gating

Opening of CLC chloride channels is coupled to the translocation of the permeant anion. From the recent structure determination of bacterial CLC proteins in the closed and open configuration, a glutamate residue was hypothesized to form part of the Cl−-sensitive gate. The negatively charged side-chain of the glutamate was suggested to occlude the permeation pathway in the closed state, while opening of a single protopore of the double-pore channel would reflect mainly a movement of this side-chain toward the extracellular pore vestibule, with little rearrangement of the rest of the channel. Here we show that mutating this critical residue (Glu166) in the prototype Torpedo CLC-0 to alanine, serine, or lysine leads to constitutively open channels, whereas a mutation to aspartate strongly slowed down opening. Furthermore, we investigated the interaction of the small organic channel blocker p-chlorophenoxy-acetic acid (CPA) with the mutants E166A and E166S. Both mutants were strongly inhibited by CPA at negative voltages with a >200-fold larger affinity than for wild-type CLC-0 (apparent KD at −140 mV ∼4 μM). A three-state linear model with an open state, a low-affinity and a high-affinity CPA-bound state can quantitatively describe steady-state and kinetic properties of the CPA block. The parameters of the model and additional mutagenesis suggest that the high-affinity CPA-bound state is similar to the closed configuration of the protopore gate of wild-type CLC-0. In the E166A mutant the glutamate side chain that occludes the permeation pathway is absent. Thus, if gating consists only in movement of this side-chain the mutant E166A should not be able to assume a closed conformation. It may thus be that fast gating in CLC-0 is more complex than anticipated from the bacterial structures.

Determination of surface composition of alloy nanoparticles and relationships with catalytic activity in Pd–Cu/SiO2 cogelled xerogel catalysts

2004 ◽  
Vol 270 (1-2) ◽  
pp. 201-208 ◽  
Author(s):  
Stéphanie Lambert ◽  
Benoı̂t Heinrichs ◽  
Alain Brasseur ◽  
André Rulmont ◽  
Jean-Paul Pirard
Keyword(s):  
Catalytic Activity ◽  
Surface Composition ◽  
Alloy Nanoparticles

Electrochemical determination of (S)-7-hydroxywarfarin for analysis of CYP2C9 catalytic activity

2021 ◽  
pp. 115937
Author(s):  
Alexey V. Kuzikov ◽  
Tatiana A. Filippova ◽  
Rami A. Masamrekh ◽  
Victoria V. Shumyantseva
Keyword(s):  
Catalytic Activity ◽  
Electrochemical Determination

Selective oxidation of propene to acrolein on FeMoTeO catalysts: determination of active phase and enhancement of catalytic activity and stability

2017 ◽  
Vol 7 (20) ◽  
pp. 4629-4639 ◽  
Author(s):  
M. Tonelli ◽  
M. Aouine ◽  
L. Massin ◽  
V. Belliere Baca ◽  
J. M. M. Millet
Keyword(s):  
Catalytic Activity ◽  
Selective Oxidation ◽  
Active Phase ◽  
Propene Oxidation

Multicomponent FeMoTeO catalysts have been synthesized and studied for mild propene oxidation to acrolein.

PROTEIN PROFILING OF DIFFERENT PLANT TISSUES FROM HERB PHYLLANTHUS NIRURI

2015 ◽  
Vol 77 (24) ◽  
Author(s):  
Ainul Mardhiah Mohd Nail ◽  
Noor Hasniza Md Zin
Keyword(s):  
Molecular Weight ◽  
Protein Band ◽  
Protein Profiling ◽  
Protein Extract ◽  
Phyllanthus Niruri ◽  
Two Dimensional Gel Electrophoresis ◽  
Plant Parts ◽  
Sds Page ◽  
Isoelectric Points

Herb Phyllanthus niruri (P. niruri) is known to have various pharmacological functions including anticancer, antibacterial, antioxidant, anti-hypertensive and also anti-diabetic properties.  In this research, the proteomic part of P. niruri was studied to determine the bioactive peptides that responsible for specific characteristics. Total soluble proteins from different plant parts of freshly collected P. niruri were extracted using TCA/acetone method and then quantified using Bradford assay. Fruits part was found to have a significantly higher amount of proteins (4.91µg/µl + 0.21) compared to leaves (4.18µg/µl + 0.15). To determine the quality of proteins in the crude extract, SDS-Page was carried out which separates proteins in the basis of molecular weight. Proteins extracted from leaves were widely distributed between the range of 3.5 kDa to 160 kDA. Meanwhile, proteins in fruits mainly distributed within the range of 15 kDa to 80 kDa. The most highly expressed protein band was found in fruit, located in between 30 to 40 kDa. The protein extracts were then further analyzed based on the molecular weight and isoelectric points using two-dimensional gel electrophoresis (2D-GE) approach. Based on the profile pattern obtained from 2D-GE analysis, protein extract from fruits seems to express more protein spots compared to protein extract from leaves. Protein spots from fruit are seen to be intensely resolved within pH 4 to 10 at molecular weight between 10 kDa to 80 kDa. On the other hand, protein spots from leaves were moderately resolved at pH 4 to 10 at molecular weight within 10 kDa to 50 kDa.

scholarly journals Sensitive Aptamer SERS and RRS Assays for Trace Oxytetracycline Based on the Catalytic Amplification of CuNCs

Nanomaterials ◽  
2021 ◽  
Vol 11 (10) ◽  
pp. 2501
Author(s):  
Shuxin Chen ◽  
Xiaowen Lv ◽  
Jifan Shen ◽  
Siqi Pan ◽  
Zhiliang Jiang ◽  
...  
Keyword(s):  
Catalytic Activity ◽  
Rayleigh Scattering ◽  
Molecular Probes ◽  
Resonance Rayleigh Scattering ◽  
Surface Enhanced ◽  
Surface Enhanced Raman ◽  
Enhanced Raman Scattering ◽  
Wet Chemical ◽  
Amplification Reaction

A new method for the determination of oxytetracycline (OTC) has been established by coupling the catalytic amplification reaction of copper nanoclusters (CuNCs) with the aptamer reaction. CuNCs prepared by a wet chemical method have the catalytic activity for the formation of gold nanoparticles (AuNPs) resulting from a HAuCl4-ethanol (En) reaction. The experimental results showed that OTC aptamer (Apt) can be adsorbed on the surface of CuNCs in a non-specific way, thus inhibiting its catalytic activity. When OTC was added to the solution, the OTC-Apt complex was generated by a specific reaction, which made the CuNCs desorb and restore their catalytic activity. With the increase of OTC, the recovery of the catalytic activity of CuNCs is strengthened, the reaction speed is accelerated, and the number of AuNPs is increased. The generated AuNPs exhibited surface enhanced Raman scattering (SERS) signals at 1615 cm−1 in the presence of Vitoria blue 4R (VB4R) molecular probes, and a resonance Rayleigh scattering (RRS) peak at 586 nm. There is a good linear relationship between the intensities of SERS, or RRS, and OTC concentration at the range of 37.5–300 ng/L or 37.5–225 ng/L, respectively. A new SERS and RRS assay for the determination of trace OTC based on the regulation of CuNCs catalysis was established.

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