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

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.

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Rapid Isolation of High Molecular Weight Urokinase from Native Human Urine

Thrombosis and Haemostasis ◽

10.1055/s-0038-1657167 ◽

1982 ◽

Vol 47 (03) ◽

pp. 197-202 ◽

Cited By ~ 23

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.

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

Biochemical Journal ◽

10.1042/bj2820711 ◽

1992 ◽

Vol 282 (3) ◽

pp. 711-714 ◽

Cited By ~ 49

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.

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Effects of tetrahydrofolate polyglutamates on the kinetic parameters of serine hydroxymethyltransferase and glycine decarboxylase from pea leaf mitochondria

Biochemical Journal ◽

10.1042/bj2920425 ◽

1993 ◽

Vol 292 (2) ◽

pp. 425-430 ◽

Cited By ~ 43

Author(s):

V Besson ◽

F Rebeille ◽

M Neuburger ◽

R Douce ◽

E A Cossins

Keyword(s):

Kinetic Parameters ◽

Serine Hydroxymethyltransferase ◽

Affinity Constant ◽

Plant Tissues ◽

Glycine Decarboxylase ◽

Glycine Cleavage System ◽

Matrix Space ◽

The Matrix ◽

Order Of Magnitude ◽

Glycine Cleavage

Plant tissues contain highly conjugated forms of folate. Despite this, the ability of plant folate-dependent enzymes to utilize tetrahydrofolate polyglutamates has not been examined in detail. In leaf mitochondria, the glycine-cleavage system and serine hydroxymethyltransferase, present in large amounts in the matrix space and involved in the photorespiratory cycle, necessitate the presence of tetrahydrofolate as a cofactor. The aim of the present work was to determine whether glutamate chain length (one to six glutamate residues) influenced the affinity constant for tetrahydrofolate and the maximal velocities displayed by these two enzymes. The results show that the affinity constant decreased by at least one order of magnitude when the tetrahydrofolate substrate contained three or more glutamate residues. In contrast, maximal velocities were not altered in the presence of these substrates. These results are consistent with analyses of mitochondrial folates which revealed a pool of polyglutamates dominated by tetra and pentaglutamates. The equilibrium constant of the serine hydroxymethyltransferase suggests that, during photorespiration, the reaction must be permanently pushed toward the formation of serine (the unfavourable direction) to allow the recycling of tetrahydrofolate necessary for the operation of the glycine decarboxylase T-protein.

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

Biochemical Journal ◽

10.1042/bj2430723 ◽

1987 ◽

Vol 243 (3) ◽

pp. 723-728 ◽

Cited By ~ 1

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.

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

BioMed Research International ◽

10.1155/2015/245649 ◽

2015 ◽

Vol 2015 ◽

pp. 1-8 ◽

Cited By ~ 13

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.

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First cloning and characterization of aspartate aminotransferase from river buffalo (Bubalus bubalis)

Biologia ◽

10.2478/s11756-011-0125-z ◽

2011 ◽

Vol 66 (6) ◽

Cited By ~ 1

Author(s):

Muhammad Shahid Nadeem ◽

Naeem Rashid ◽

Muzaffar Iqbal ◽

Qurra-tul-Ann Afza Gardner ◽

Muhammad Akhtar

Keyword(s):

Aspartate Aminotransferase ◽

Pyridoxal Phosphate ◽

Specific Activity ◽

Active Form ◽

Bubalus Bubalis ◽

Recombinant Enzyme ◽

Ionization Mass ◽

River Buffalo ◽

Apparent Homogeneity

AbstractAspartate aminotransferase catalyzes the transfer of an amino group from L-aspartate to α-oxoglutarate. We have purified cytosolic isozyme, to apparent homogeneity, from the heart of river buffalo. In order to clone the enzyme total RNA was isolated and the cDNA encoding complete polypeptide of 413 amino acids was amplified by reverse transcriptase polymerase chain reaction. The cDNA and the deduced amino acid sequence exhibited 98.2% and 99.5% identities, respectively, with that of cow. The cDNA was overexpressed in Escherichia coli and the gene product was obtained in enzymologically active form. The recombinant enzyme was about 40% of the total cell proteins. The purified recombinant enzyme displayed specific activity, Km, temperature and pH stability values similar to that of the native enzyme. Edman degradation and electrospray ionization mass spectra showed that the recombinant enzyme consisted of three species having N-terminal sequences of MAPPSIF, APPSIF and PPSIF, although the second one was the predominant species. Conditions are also described, which in the mass spectral analysis give either the three species corresponding to the apo- or the holo-enzyme; the latter retaining pyridoxal phosphate bound to the protein. To our knowledge this is the first report on sequence determination, cloning and characterization of aspartate aminotransferase from river buffalo.

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Purification and characterization of a low molecular weight xylanase from solid-state cultures of Aspergillus fumigatus Fresenius

Revista de Microbiologia ◽

10.1590/s0001-37141999000200005 ◽

1999 ◽

Vol 30 (2) ◽

pp. 114-119 ◽

Cited By ~ 22

Author(s):

Claudio Henrique Cerri e Silva ◽

Jurgen Puls ◽

Marcelo Valle de Sousa ◽

Edivaldo Ximenes Ferreira Filho

Keyword(s):

Molecular Weight ◽

Solid State ◽

Aspergillus Fumigatus ◽

Gel Filtration ◽

Low Molecular Weight ◽

Transferase Activity ◽

Purification And Characterization ◽

Apparent Homogeneity ◽

Sds Page

A xylan-degrading enzyme (xylanase II) was purified to apparent homogeneity from solid-state cultures of Aspergillus fumigatus Fresenius. The molecular weight of xylanase II was found to be 19 and 8.5 kDa, as estimated by SDS-PAGE and gel filtration on FPLC, respectively. The purified enzyme was most active at 55 °C and pH 5.5. It was specific to xylan. The apparent Km and Vmax values on soluble and insoluble xylans from oat spelt and birchwood showed that xylanase II was most active on soluble birchwood xylan. Studies on hydrolysis products of various xylans and xylooligomers by xylanase II on HPLC showed that the enzyme released a range of products from xylobiose to xylohexaose, with a small amount of xylose from xylooligomers, and presented transferase activity.

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Isolation and partial characterization of boar proacrosin

Collection of Czechoslovak Chemical Communications ◽

10.1135/cccc19900846 ◽

1990 ◽

Vol 55 (3) ◽

pp. 846-853 ◽

Cited By ~ 1

Author(s):

Věra Jonáková ◽

Brigita Vidimská ◽

Jana Urbanová ◽

Manfred Pavlík

Keyword(s):

Amino Acid ◽

High Performance ◽

Gel Filtration ◽

Reversed Phase ◽

Exchange Chromatography ◽

Relative Molecular Mass ◽

Reducing Conditions ◽

Apparent Homogeneity ◽

Step Procedure

Boar proacrosin was purified to apparent homogeneity by a three-step procedure: gel filtration on Sephadex G-50 medium, ion-exchange chromatography on CM-Cellulose 32, and reversed-phase high-performance liquid chromatography on a C4 column. The relative molecular mass (Mr) of the proacrosin estimated by gel filtration was about 70 000, whereas the results of an electrophoretic experiment on SDS-polyacrylamide gel with copolymerized casein under non-reducing conditions indicated an Mr of 55 000-60 000. The proacrosin reproducibly migrated on the gel as a double band. When purified, it remained stable at pH 8.0 for 30 min. The amino-acid composition of the homogeneous proacrosin was determined, the N-terminal amino-acid sequence being Arg-Asp-X-Ala-Thr-X-X-Gly-Pro-X-Gly-.

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Structural and Functional Characterization of the Alanine Racemase from Streptomyces coelicolor A3(2)

10.1101/089938 ◽

2016 ◽

Author(s):

Raffaella Tassoni ◽

L.T. van der Aart ◽

M. Ubbink ◽

G. P. Van Wezel ◽

N. S. Pannu

Keyword(s):

Pyridoxal Phosphate ◽

Streptomyces Coelicolor ◽

Functional Characterization ◽

Gel Filtration ◽

Structural Features ◽

Alanine Racemase ◽

Increased Sensitivity ◽

Bacterial Peptidoglycan ◽

Hinge Angle

AbstractThe conversion of L-alanine (L-Ala) into D-alanine (D-Ala) in bacteria is performed by pyridoxal phosphate-dependent enzymes called alanine racemases. D-Ala is an essential component of the bacterial peptidoglycan and hence required for survival. The Gram-positive bacterium Streptomyces coelicolor has at least one alanine racemase encoded by alr. Here, we describe an alr deletion mutant of S. coelicolor which depends on D-Ala for growth and shows increased sensitivity to the antibiotic D-cycloserine (DCS). The crystal structure of the alanine racemase (Alr) was solved with and without the inhibitors DCS or propionate, at 1.64 Å and 1.51 Å resolution, respectively. The crystal structures revealed that Alr is a homodimer with residues from both monomers contributing to the active site. The dimeric state of the enzyme in solution was confirmed by gel filtration chromatography, with and without L-Ala or D-cycloserine. Specificity of the enzyme was 66 +/− 3 U mg−1 for the racemization of L-to D-Ala, and 104 +/− 7 U mg−1 for the opposite direction. Comparison of Alr from S. coelicolor with orthologous enzymes from other bacteria, including the closely related D-cycloserine-resistant Alr from S. lavendulae, strongly suggests that structural features such as the hinge angle or the surface area between the monomers do not contribute to D-cycloserine resistance, and the molecular basis for resistance therefore remains elusive.

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

Biochemical Journal ◽

10.1042/bj3130185 ◽

1996 ◽

Vol 313 (1) ◽

pp. 185-191 ◽

Cited By ~ 62

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.

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