scholarly journals Purification and initial characterization of proline 4-hydroxylase from Streptomyces griseoviridus P8648: a 2-oxoacid, ferrous-dependent dioxygenase involved in etamycin biosynthesis

1996 ◽  
Vol 313 (1) ◽  
pp. 185-191 ◽  
Author(s):  
Christopher C. LAWRENCE ◽  
Wendy J. SOBEY ◽  
Robert A. FIELD ◽  
Jack E. BALDWIN ◽  
Christopher J. SCHOFIELD
Keyword(s):  
Active Site ◽  
Gel Filtration ◽  
Ferrous Ion ◽  
Typical Member ◽  
Apparent Homogeneity ◽  
Initial Characterization ◽  
Dependent Inhibition ◽  
Proline Hydroxylation ◽  
Time Dependent Inhibition

Proline 4-hydroxylase is a 2-oxoacid, ferrous-ion-dependent dioxygenase involved in the biosynthesis of the secondary metabolite etamycin. The purification, in low yield, of proline 4-hydroxylase from Streptomyces griseoviridus P8648 to near apparent homogeneity and its initial characterization are reported. In most respects proline 4-hydroxylase is a typical member of the 2-oxoacid-dependent dioxygenase family. It is monomeric (Mr approx. 38000) (by gel filtration on Superdex-G75) and has typically strict requirements for ferrous ion and 2-oxoglutarate. The enzyme was inhibited by aromatic analogues of 2-oxoglutarate. L-Proline-uncoupled turnover of 2-oxoglutarate to succinate and CO2 was observed. The addition of L-ascorbate did not stimulate L-proline-coupled turnover of 2-oxoglutarate, but did stimulate L-proline-uncoupled turnover. L-Ascorbate caused a time-dependent inhibition of L-proline hydroxylation. The enzyme was completely inactivated by preincubation with diethyl pyrocarbonate under histidine-modifying conditions. This inactivation could be partially prevented by the inclusion of L-proline and 2-oxoglutarate in the preincubation mixture, suggesting the presence of histidine residue(s) at the active site.

Rapid Isolation of High Molecular Weight Urokinase from Native Human Urine

1982 ◽  
Vol 47 (03) ◽  
pp. 197-202 ◽  
Author(s):  
Kurt Huber ◽  
Johannes Kirchheimer ◽  
Bernd R Binder
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Human Urine ◽  
Gel Filtration ◽  
Chain Structure ◽  
The Other ◽  
Single Chain ◽  
Apparent Homogeneity ◽  
Sequential Adsorption

SummaryUrokinase (UK) could be purified to apparent homogeneity starting from crude urine by sequential adsorption and elution of the enzyme to gelatine-Sepharose and agmatine-Sepharose followed by gel filtration on Sephadex G-150. The purified product exhibited characteristics of the high molecular weight urokinase (HMW-UK) but did contain two distinct entities, one of which exhibited a two chain structure as reported for the HMW-UK while the other one exhibited an apparent single chain structure. The purification described is rapid and simple and results in an enzyme with probably no major alterations. Yields are high enough to obtain purified enzymes for characterization of UK from individual donors.

Chromogenic Peptide Substrate Assays for Plasma Inhibitors of Plasma Kallikrein: Identification of the Major Inhibitors

1979 ◽  
Author(s):  
M.J. Gallimore ◽  
E. Amundsen ◽  
M. Larsbraaten ◽  
K. Lyngaas ◽  
E. Fareid
Keyword(s):  
Plasma Concentrations ◽  
Gel Filtration ◽  
Plasma Kallikrein ◽  
Peptide Substrate ◽  
Total Inhibition ◽  
Α2 Macroglobulin ◽  
Dependent Inhibition ◽  
Plus Fraction ◽  
Time Dependent Inhibition ◽  
Esterase Inhibitor

Plasma inhibitors of plasma kallikrein(KK) were studied using chromogenic peptide substrate assays. Both “immediate” and “time-dependent” inhibition was detected. Sephadex G-150 gel filtration revealed that fractions containing α2-macroglobulin (α2 M), C1 - esterase inhibitor (CIINH) and a low molecular weight component(KKI3) gave “immediate” inhibition. When fractions were tested for “total” inhibition (incubation of enzyme plus fraction for 300 seconds at 37°C) CIINH was found to be the major inhibitor. Both the α2M and KKI3-containing fractions exhibited more inhibition than in the “immediate” inhibition assay. Studies with purified preparations of CIINH and α2 M indicated that these are the two most important plasma inhibitors of KK. Preparations of α1-antitrypsin (α1AT), antithrombin III (ATIII) and α2-antiplasmin (α2AP) produced insignificant inhibition. When “total” KK inhibition in plasma samples from 20 healthy subjects was compared with plasma concentrations of CIINH, α2M and α1AT (immunochemical assays) a very good correlation (r=0.81) was found between percentage inhibition and CIINH concentration. Correlation values for the other antiproteases were α2M r=0.36 and α1AT r=0.19.

scholarly journals Occurrence of fatty acid epoxide hydrolases in soybean (Glycine max). Purification and characterization of the soluble form

1992 ◽  
Vol 282 (3) ◽  
pp. 711-714 ◽  
Author(s):  
E Blée ◽  
F Schuber
Keyword(s):  
Glycine Max ◽  
Gel Filtration ◽  
Soluble Form ◽  
Soluble Enzyme ◽  
Purification And Characterization ◽  
Epoxide Hydrolases ◽  
Major Activity ◽  
Apparent Homogeneity ◽  
A Minor

Epoxide hydrolases catalysing the hydration of cis-9,10-epoxystearate into threo-9,10-dihydroxystearate have been detected in soybean (Glycine max) seedlings. The major activity was found in the cytosol, a minor fraction being strongly associated with microsomes. The soluble enzyme, which was purified to apparent homogeneity by (NH4)2SO4 fractionation, hydrophobic, DEAE- and gel-filtration chromatographies, has a molecular mass of 64 kDa and a pI of 5.4.

scholarly journals Time-dependent inhibition of PHD2

2017 ◽  
Vol 37 (3) ◽  
Author(s):  
Isabelle Tcholakov ◽  
Charles E. Grimshaw ◽  
Lihong Shi ◽  
Andre Kiryanov ◽  
Sean T. Murphy ◽  
...  
Keyword(s):  
Active Site ◽  
Dissociation Rate ◽  
Time Dependent ◽  
Hypoxic Stress ◽  
Hypoxia Inducible Factors ◽  
Prolyl Hydroxylases ◽  
Enzyme Active Site ◽  
Dependent Inhibition ◽  
Time Dependent Inhibition ◽  
Slow Dissociation

Prolyl hydroxylases (PHDs) down-regulate the level of hypoxia-inducible factors (HIFs) by hydroxylating key proline residues that trigger the degradation of the protein and affect the cell and its ability to respond to hypoxic stress. Several small molecule PHD inhibitors are now in various preclinical and clinical stages for the treatment of anemia. The present study provides a detail kinetic analysis for some of these inhibitors. The data generated in the present study suggest that these compounds are reversible and compete directly with the co-substrate, 2-oxoglutarate (2-OG) for binding at the enzyme active site. Most of these compounds are pan PHD inhibitors and exhibit a time-dependent inhibition (TDI) mechanism due to an extremely slow dissociation rate constant, koff, and a long residence time.

scholarly journals Isolation and characterization of a novel haemoprotein b-559 from Bengal gram (Cicer arietinum)

1987 ◽  
Vol 243 (3) ◽  
pp. 723-728 ◽  
Author(s):  
C S Ramadoss ◽  
B C Shenoy ◽  
A Borthakur
Keyword(s):  
Gel Filtration ◽  
Molecular Size ◽  
Soret Band ◽  
Beta Band ◽  
Photosynthetic Membrane ◽  
Isolation And Characterization ◽  
Bengal Gram ◽  
Apparent Homogeneity ◽  
Reduced State

A haemoprotein was purified to apparent homogeneity from Bengal-gram seeds. The purified protein exhibited an absorption maximum at 412 nm (Soret band) that upon reduction with dithionite gave rise to a shift in the Soret band to 426 nm with concomitant appearance of an alpha-band at 559 nm and a beta-band at 530 nm. In the reduced state the Bengal-gram haemoprotein showed reactivity towards CO, nitrite and hydroxylamine. SDS/polyacrylamide-slab-gel electrophoresis showed that the haemoprotein has Mr 78,000. Gel-filtration and ultracentrifugal analyses suggest that the Bengal-gram haemoprotein is oligomeric in nature. Since it differs from photosynthetic membrane cytochrome b-559 in solubility in buffer, in reactivity towards CO and in molecular size, it appears to be a novel haemoprotein b-559.

scholarly journals Dynamic Compound-Dependent Acoustic Transfer to Investigate Inhibitor Reversibility

2016 ◽  
Vol 22 (5) ◽  
pp. 485-492
Author(s):  
Jennifer Nothstein ◽  
Elisabeth MacColl ◽  
Paul Zuck ◽  
Jason Cassaday ◽  
Victor N. Uebele ◽  
...  
Keyword(s):  
Dipeptidyl Peptidase 4 ◽  
Custom Made ◽  
Management Procedures ◽  
Dependent Inhibition ◽  
Labview Program ◽  
Time Dependent Inhibition ◽  
High Throughput Manner ◽  
Unique Compound ◽  
Specific Volumes

Automated mechanism of action studies are introducing the need for tailored compound delivery, which can be challenging for standard compound management procedures. Jump dilution assays investigating inhibitor reversibility require compound delivery at specific volumes to assay specific concentrations of 10 × IC50 for each inhibitor. Creating custom-made source plates with unique compound concentrations to dispense a uniform single volume can be prohibitively slow. A broadly applicable tool that enables on-the fly dispensing of variable amounts of stock concentrations was developed using the Acoustic Transfer System (ATS). The Dynamic Transfer Modification Program (DTMP) is an integrated LabVIEW program used to automate customized volume transfers from each well based on compound identity within a given source plate. A jump dilution investigating the time-dependent inhibition of the enzyme dipeptidyl peptidase-4 (DPP4) with multiple inhibitors is described here to demonstrate the delivery of specific volumes of various compounds in a high-throughput manner. The ability to automate this process allows for the characterization of inhibitor reversibility earlier in the drug discovery process, resulting in better informed lead candidate selection.

scholarly journals Purification and Characterization of a Polyextremophilicα-Amylase from an Obligate HalophilicAspergillus penicillioidesIsolate and Its Potential for Souse with Detergents

2015 ◽  
Vol 2015 ◽  
pp. 1-8 ◽  
Author(s):  
Imran Ali ◽  
Ali Akbar ◽  
Mohammad Anwar ◽  
Sehanat Prasongsuk ◽  
Pongtharin Lotrakul ◽  
...  
Keyword(s):  
Specific Activity ◽  
Amylase Activity ◽  
Gel Filtration ◽  
Soluble Starch ◽  
Ammonium Sulfate Precipitation ◽  
Purification And Characterization ◽  
Apparent Homogeneity ◽  
Sds Page ◽  
Good Potential

An extracellularα-amylase from the obligate halophilicAspergillus penicillioidesTISTR3639 strain was produced and enriched to apparent homogeneity by ammonium sulfate precipitation and Sephadex G100 gel filtration column chromatography. The mass of the purified amylase was estimated to be 42 kDa by SDS-PAGE. With soluble starch as the substrate it had a specific activity of 118.42 U·mg−1andVmax⁡andKmvalues of 1.05 µmol·min−1·mg−1and 5.41 mg·mL−1, respectively. The enzyme was found to have certain polyextremophilic characteristics, with an optimum activity at pH 9, 80°C, and 300 g·L−1NaCl. The addition of CaCl2at 2 mM was found to slightly enhance the amylase activity, while ZnCl2, FeCl2, or EDTA at 2 mM was strongly or moderately inhibitory, respectively, suggesting the requirement for a (non-Fe2+or Zn2+) divalent cation. The enzyme retained more than 80% of its activity when incubated with three different laundry detergents and had a better performance compared to a commercial amylase and three detergents in the presence of increasing NaCl concentrations up to 300 g·L−1. Accordingly, it has a good potential for use as anα-amylase in a low water activity (high salt concentration) and at high pH and temperatures.

scholarly journals Resolution and characterization of the glycine-cleavage reaction in pea leaf mitochondria. Properties of the forward reaction catalysed by glycine decarboxylase and serine hydroxymethyltransferase

1988 ◽  
Vol 255 (1) ◽  
pp. 169-178 ◽  
Author(s):  
J Bourguignon ◽  
M Neuburger ◽  
R Douce
Keyword(s):  
Pyridoxal Phosphate ◽  
Gel Filtration ◽  
Serine Hydroxymethyltransferase ◽  
Molar Ratios ◽  
Apparent Homogeneity ◽  
The Matrix ◽  
Lipoamide Dehydrogenase ◽  
Glycine Cleavage ◽  
Low Ionic Strength

High-molecular-mass proteins from pea (Pisum sativum) mitochondrial matrix retained on an XM-300 Diaflo membrane (‘matrix extract’) exhibited high rates of glycine oxidation in the presence of NAD+ and tetrahydropteroyl-L-glutamic acid (H4 folate) as long as the medium exhibited a low ionic strength. Serine hydroxymethyltransferase (SHMT) (4 x 53 kDa) and the four proteins of the glycine-cleavage system, including a pyridoxal phosphate-containing enzyme (‘P-protein’ 2 x 97 kDa), a carrier protein containing covalently bound lipoic acid (‘H-protein’ 15.5 kDa), a protein exhibiting lipoamide dehydrogenase activity (‘L-protein’; 2 x 61 kDa) and an H4 folate-dependent enzyme (‘T-protein’ 45 kDa) have been purified to apparent homogeneity from the matrix extract by using gel filtration, ion-exchange and phenyl-Superose fast protein liquid chromatography. Gel filtration on Sephacryl S-300 in the presence of 50 mM-KCl proved to be the key step in disrupting this complex. During the course of glycine oxidation catalysed by the matrix extract a steady-state equilibrium in the production and utilization of 5,10-methylene-H4 folate was reached, suggesting that glycine cleavage and SHMT are linked together via a soluble pool of H4 folate. The rate of glycine oxidation catalysed by the matrix extract was sensitive to the NADH/NAD+ molar ratios, because NADH competitively inhibited the reaction catalysed by lipoamide dehydrogenase.

scholarly journals Purification and properties of a monoacylglycerol lipase in human erythrocytes

1995 ◽  
Vol 312 (2) ◽  
pp. 519-525 ◽  
Author(s):  
C Somma-Delpéro ◽  
A Valette ◽  
J Lepetit-Thévenin ◽  
O Nobili ◽  
J Boyer ◽  
...  
Keyword(s):  
Gel Filtration ◽  
Human Erythrocytes ◽  
Lipolytic Enzyme ◽  
Monoacylglycerol Lipase ◽  
Serine Hydrolase ◽  
Apparent Homogeneity ◽  
Apolipoprotein C ◽  
Purification And Properties ◽  
Nitrophenyl Phosphate ◽  
Dependent Inhibition

A membrane-bound monoacylglycerol lipase (MAGL) activity, previously demonstrated in intact human erythrocytes [Boyer, Somma, Vérine, L'Hôte, Finidori, Merger and Arnaud (1981) J. Clin. Endocrinol. Metab. 53, 143-148], has now been purified to apparent homogeneity by a five-step procedure involving solubilization in CHAPS and sequential chromatographies on Sephacryl S-400, DEAE-Trisacryl, Zn(2+)-chelating Sepharose and Superose 12 columns. The purified protein has a molecular mass of 68 +/- 2 kDa, as determined by SDS/PAGE and gel filtration, suggesting that the enzyme behaves as a monomer. The concentration-dependence of MAGL activity with monooleoylglycerol, the preferred substrate showed kinetics typical of an interfacial lipolytic enzyme displaying optimal activity on emulsified substrate particles; apparent Km values were 0.27 mM and 0.49 mM for the sn-1(3)- and sn-2-isomers respectively. MAGL had no, or negligible, activity towards tri-oleoylglycerol, di-oleoylglycerol, oleoylcholesterol, oleoyl-CoA and phosphatidylcholine; it was inhibited by di-isopropylfluorophosphate, PMSF and diethyl p-nitrophenyl phosphate, suggesting that MAGL is a serine hydrolase. MAGL activity was not modified by bile salt or apolipoprotein C-II, whereas a dose-dependent inhibition was observed with apolipoprotein A-I.

scholarly journals PROPERDIN FACTOR D: CHARACTERIZATION OF ITS ACTIVE SITE AND ISOLATION OF THE PRECURSOR FORM

1974 ◽  
Vol 139 (2) ◽  
pp. 355-366 ◽  
Author(s):  
Douglas T. Fearon ◽  
K. Frank Austen ◽  
Shaun Ruddy
Keyword(s):  
Active Site ◽  
Gel Filtration ◽  
Serine Esterase ◽  
Factor B ◽  
Alternate Pathway ◽  
Serine Esterases ◽  
Definition Of ◽  
Chromatographic Isolation

The activity of properdin factor D was measured by the generation of the hemolytically active cellular intermediate, EAC43B(D), bearing the C3b-dependent alternate pathway C3 convertase. Treatment of factor D with DFP prevented formation of EAC43B(D); thus, a serine esterase is essential for the generation of the alternate pathway C3 convertase, a situation analogous to the role of C1 in the formation of the classical C3 convertase, C42. The definition of factor D as a serine esterase prompted a search for its proenzyme form, and resulted in the chromatographic isolation from plasma of a single peak of trypsin-inducible factor D activity, distinct from activated factor D. Analytical gel filtration indicated an apparent mol wt of 25,000. This protein from which trypsin elaborated factor D activity, as assessed by the formation of EAC43B(D), the generation of the CoVF-dependent C3 convertase, and the cleavage of factor B in the presence of C3b, was designated "precursor factor D." The DFP resistance of precursor factor D, and the susceptibility of its trypsin-activated form to inactivation by DFP is analogous to the behavior of other plasma serine esterases, including C1.

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