scholarly journals Identification, molecular cloning and functional characterization of a novel NADH kinase from Arabidopsis thaliana (thale cress)

2004 ◽  
Vol 385 (1) ◽  
pp. 217-223 ◽  
Author(s):  
William L. TURNER ◽  
Jeffrey C. WALLER ◽  
Wayne A. SNEDDEN
Keyword(s):  
Arabidopsis Thaliana ◽  
Affinity Purification ◽  
Functional Characterization ◽  
Gel Filtration ◽  
Cell Suspension Cultures ◽  
Redox Status ◽  
Ph Optimum ◽  
Yeast Saccharomyces Cerevisiae ◽  
Thale Cress ◽  
Nadh Kinase

NADH kinase (NADHK; ATP:NADH 2′-phosphotransferase; EC 2.7.1.86), an enzyme that preferentially utilizes NADH as the diphosphonicotinamide nucleotide donor, has been identified for the first time in plants. Low activity (0.4 nmol of NADPH produced/min per mg of protein) was observed in clarified protein extracts from Arabidopsis thaliana (thale cress) cell suspension cultures. However, unlike an NADHK from yeast (Saccharomyces cerevisiae) (POS5), the enzyme from Arabidopsis did not associate with the mitochondria. NADHK was cloned (gi:30699338) from Arabidopsis and studied as a recombinant protein following affinity purification from Escherichia coli. The enzyme had a pH optimum for activity of 7.9 and a subunit molecular mass of 35 kDa. Analytical gel filtration demonstrated that the recombinant enzyme exists as a dimer. Hyperbolic saturation kinetics were observed for the binding of NADH, ATP, free Mg2+ and NAD+, with respective Km values of 0.042, 0.062, 1.16, and 2.39 mM. While NADHK could phosphorylate NADH or NAD+, the specificity constant (Vmax/Km) for NADH was 100-fold greater than for NAD+. The enzyme could utilize UTP, GTP and CTP as alternative nucleotides, although ATP was the preferred substrate. PPi or poly-Pi could not substitute as phospho donors. PPi acted as a mixed inhibitor with respect to both NADH and ATP. NADHK was inactivated by thiol-modifying reagents, with inactivation being decreased in the presence of NADH or ATP, but not NAD+. This study suggests that, in Arabidopsis, NADP+/NADPH biosynthetic capacity could, under some circumstances, become uncoupled from the redox status of the diphosphonicotinamide nucleotide pool.

Enzymatic Studies on the Reversible Synthesis of Nicotinic Acid-N-glucoside in Heterotrophic Parsley Cell Suspension Cultures

1988 ◽  
Vol 43 (11-12) ◽  
pp. 835-842 ◽  
Author(s):  
Barbara Upmeier ◽  
Jürgen E. Thomzik ◽  
Wolfgang Barz
Keyword(s):  
Nicotinic Acid ◽  
Cell Suspension ◽  
Gel Filtration ◽  
Cell Suspension Cultures ◽  
Suspension Cultures ◽  
Exchange Chromatography ◽  
High Group ◽  
Ph Optimum ◽  
Glucose Molecule ◽  
Transfer Potential

Abstract A soluble enzyme catalyzing the transfer of the glucose moiety from UDP-glucose to the nitrogen atom of nicotinic acid was detected in protein preparations from heterotrophic cell suspension cultures of parsley (Petroselinum hortense Hoffm.). Enzyme activity was enriched 22-fold by ammonium sulfate precipitation, gel filtration and ion exchange chromatography. The UDP-glucose : nicotinic acid-N-glucosyltransferase showed a pH-optimum at pH 7.8-8.2 and a temperature optimum at 30 °C. The apparent KM values were determined to be 170 μM for nicotinic acid and 1.2 mM for the cosubstrate. The native enzyme had a molecular mass of about 46 kDa. The glucosyltransferase reaction was shown to be reversible. The transfer of the glucose molecule from nicotinic acid-N-glucoside to uridinediphosphate yielding uridinediphosphoglucose and nicotinic acid could be demonstrated indicating that nicotinic acid-N-glucoside has a high group-transfer potential.

scholarly journals Purification and properties of porphobilinogen deaminase from Arabidopsis thaliana.

1994 ◽  
Vol 299 (3) ◽  
pp. 895-902 ◽  
Author(s):  
R M Jones ◽  
P M Jordan
Keyword(s):  
Arabidopsis Thaliana ◽  
Gel Filtration ◽  
Chain Termination ◽  
Porphobilinogen Deaminase ◽  
Ph Optimum ◽  
Heat Stable ◽  
Purification And Properties ◽  
Sds Page ◽  
Plant Arabidopsis Thaliana

Porphobilinogen deaminase (EC 4.3.1.8) has been purified to homogeneity (16,000-fold) from the plant Arabidopsis thaliana in yields of 8%. The deaminase is a monomer of M(r) 35,000, as shown by SDS/PAGE, and 31,000, using gel-filtration chromatography. The pure enzyme has a Vmax. of 4.5 mumol/h per mg and a Km of 17 +/- 4 microM. Determination of the pI and pH optimum revealed values of 5.2 and 8.0 respectively. The sequence of the N-terminus was found to be NH2-XVAVEQKTRTAI. The deaminase is heat-stable up to 70 degrees C and is inhibited by NH3 and hydroxylamine. The enzyme is inactivated by arginine-, histidine- and lysine-specific reagents. Incubation with the substrate analogue and suicide inhibitor, 2-bromoporphobilinogen, results in chain termination and in inactivation.

scholarly journals Identification and characterization of plant glycerophosphodiester phosphodiesterase

2004 ◽  
Vol 379 (3) ◽  
pp. 601-607 ◽  
Author(s):  
Benoît van der REST ◽  
Norbert ROLLAND ◽  
Anne-Marie BOISSON ◽  
Myriam FERRO ◽  
Richard BLIGNY ◽  
...  
Keyword(s):  
Arabidopsis Thaliana ◽  
Cell Wall ◽  
Cell Walls ◽  
Expression System ◽  
Gel Filtration ◽  
Cell Suspension Cultures ◽  
Cell Wall Proteins ◽  
Ammonium Sulphate Precipitation ◽  
Carrot Cell ◽  
Glycerophosphodiester Phosphodiesterase

GPX-PDE (glycerophosphodiester phosphodiesterase; EC 3.1.4.46) is a relatively poorly characterized enzyme that catalyses the hydrolysis of various glycerophosphodiesters (glycerophosphocholine, glycerophosphoethanolamine, glycerophosphoglycerol, glycerophosphoserine and bis-glycerophosphoglycerol), releasing sn-glycerol 3-phosphate and the corresponding alcohol. In a previous study, we demonstrated the existence of a novel GPX-PDE in the cell walls and vacuoles of plant cells. Since no GPX-PDE had been identified in any plant organism, the purification of GPX-PDE from carrot cell walls was attempted. After extraction of cell wall proteins from carrot cell suspension cultures with CaCl2, GPX-PDE was purified up to 2700-fold using, successively, ammonium sulphate precipitation, gel filtration and concanavalin A–Sepharose. Internal sequence analysis of a 55 kDa protein identified in the extract following 2700-fold purification revealed strong similarity to the primary sequence of GLPQ, a bacterial GPX-PDE. To confirm the identity of plant GPX-PDE, an Arabidopsis thaliana cDNA similar to that encoding the bacterial GPX-PDE was cloned and overexpressed in a bacterial expression system, and was used to raise antibodies against the putative Arabidopsis thaliana GPX-PDE. Immunochemical assays performed on carrot cell wall proteins extracted by CaCl2 treatment showed a strong correlation between GPX-PDE activity and detection of the 55 kDa protein, validating the identity of the plant GPX-PDE. Finally, various properties of the purified enzyme were investigated. GPX-PDE is a multimeric enzyme, specific for glycerophosphodiesters, exhibiting a Km of 36 µM for glycerophosphocholine and active within a wide pH range (from 4 to 10). Since these properties are similar to those of GLPQ, the bacterial GPX-PDE, the similarities between plant and bacterial enzymes are also discussed.

A stress-responsive glyoxalase I from the parasitic nematode Onchocerca volvulus

2001 ◽  
Vol 353 (3) ◽  
pp. 445-452 ◽  
Author(s):  
Alexandra SOMMER ◽  
Peter FISCHER ◽  
Kristina KRAUSE ◽  
Kay BOETTCHER ◽  
Peter M. BROPHY ◽  
...  
Keyword(s):  
Affinity Purification ◽  
Functional Characterization ◽  
Gel Filtration ◽  
Transcript Level ◽  
Binding Pocket ◽  
Onchocerca Volvulus ◽  
Glyoxalase I ◽  
Gene Transcript ◽  
Three Dimensional Model ◽  
Calculated Molecular Mass

Glyoxal, methylglyoxal and other physiological α-oxoaldehydes are formed by the lipid peroxidation, glycation and degradation of glycolytic intermediates. They are detoxified enzymically by the glyoxalase system. To investigate the physiological function of glyoxalase I in parasitic organisms, the cDNA for glyoxalase I from the filarial nematode Onchocerca volvulus (designated Ov-GloI) has been cloned and characterized. The isolated cDNA contains an open reading frame of 579bp encoding a protein with a calculated molecular mass of 21930Da. Owing to the high degree of sequence identity (60%) with human glyoxalase I, for which the X-ray structure is available, it has been possible to build a three-dimensional model of Ov-GloI. The modelled core of Ov-GloI is conserved compared with the human glyoxalase I; however, there are critical differences in the residues lining the hydrophobic substrate-binding pocket of Ov-GloI. A 22kDa protein was obtained by heterologous expression in Escherichia coli. A homogeneous enzyme preparation was obtained by affinity purification and functional characterization of the recombinant enzyme included the determination of kinetic constants for methylglyoxal and phenylglyoxal as well as inhibition studies. Gel filtration demonstrated a dimeric structure. To assess the role of Ov-GloI as a potential vaccine candidate or serodiagnostic tool, the serological reactivity of the recombinant Ov-GloI was analysed with sera from microfilaria carriers and specific IgG1 antibodies were detected. The effects of oxidative insult, namely plumbagin and xanthine/xanthine oxidase, on the gene transcript level of Ov-GloI were investigated. By using a semi-quantitative PCR ELISA it was shown that Ov-GloI is expressed at elevated levels under conditions of oxidative stress.

Isolation and Characterization of Plasminogen Activator from Pig Leucocytes

1974 ◽  
Vol 31 (01) ◽  
pp. 072-085 ◽  
Author(s):  
M Kopitar ◽  
M Stegnar ◽  
B Accetto ◽  
D Lebez
Keyword(s):  
Molecular Weight ◽  
Plasminogen Activator ◽  
Gel Electrophoresis ◽  
Gel Filtration ◽  
Ph Optimum ◽  
Isolation And Characterization ◽  
Ph Range ◽  
Inhibition Studies ◽  
Final Purification

SummaryPlasminogen activator was isolated from disrupted pig leucocytes by the aid of DEAE chromatography, gel filtration on Sephadex G-100 and final purification on CM cellulose, or by preparative gel electrophoresis.Isolated plasminogen activator corresponds No. 3 band of the starting sample of leucocyte cells (that is composed from 10 gel electrophoretic bands).pH optimum was found to be in pH range 8.0–8.5 and the highest pH stability is between pH range 5.0–8.0.Inhibition studies of isolated plasminogen activator were performed with EACA, AMCHA, PAMBA and Trasylol, using Anson and Astrup method. By Astrup method 100% inhibition was found with EACA and Trasylol and 30% with AMCHA. PAMBA gave 60% inhibition already at concentration 10–3 M/ml. Molecular weight of plasminogen activator was determined by gel filtration on Sephadex G-100. The value obtained from 4 different samples was found to be 28000–30500.

scholarly journals Functional Characterization of DEAD-Box RNA Helicases in Arabidopsis thaliana under Abiotic Stress Conditions

2008 ◽  
Vol 49 (10) ◽  
pp. 1563-1571 ◽  
Author(s):  
Jin Sun Kim ◽  
Kyung Ae Kim ◽  
Tae Rin Oh ◽  
Chul Min Park ◽  
Hunseung Kang
Keyword(s):  
Arabidopsis Thaliana ◽  
Abiotic Stress ◽  
Functional Characterization ◽  
Stress Conditions ◽  
Rna Helicases ◽  
Dead Box

scholarly journals Cloning and functional characterization of mammalian homologues of the COPII component Sec23.

1996 ◽  
Vol 7 (10) ◽  
pp. 1535-1546 ◽  
Author(s):  
J P Paccaud ◽  
W Reith ◽  
J L Carpentier ◽  
M Ravazzola ◽  
M Amherdt ◽  
...  
Keyword(s):  
Functional Characterization ◽  
Gel Filtration ◽  
Apparent Molecular Weight ◽  
Cdna Clones ◽  
Restrictive Temperature ◽  
Rnase Protection ◽  
Cell Extracts ◽  
Exact Function ◽  
Mammalian Protein

We screened a human cDNA library with a probe derived from a partial SEC23 mouse homologue and isolated two different cDNA clones (hSec23A and hSec23B) encoding proteins of a predicted molecular mass of 85 kDa. hSec23Ap and hSec23Bp were 85% identical and shared 48% identity with the yeast Sec23p. Affinity-purified anti-hSec23A recognized a protein of approximately 85 kDa on immunoblots of human, mouse, and rat cell extracts but did not recognize yeast Sec23p. Cytosolic hSec23Ap migrated with an apparent molecular weight of 350 kDa on a gel filtration column, suggesting that it is part of a protein complex. By immunoelectron microscopy, hSec23Ap was found essentially in the ribosome-free transitional face of the endoplasmic reticulum (ER) and associated vesicles. hSec23Ap is a functional homologue of the yeast Sec23p as the hSec23A isoform complemented the temperature sensitivity of the Saccharomyces cerevisiae sec23-1 mutation at a restrictive temperature of 34 degrees C. RNase protection assays indicated that both hSec23 isoforms are coexpressed in various human tissues, although at a variable ratio. Our data demonstrate that hSec23Ap is the functional human counterpart of the yeast COPII component Sec23p and suggest that it plays a similar role in mammalian protein export from the ER. The exact function of hSec23Bp remains to be determined.

Functional analysis of a stable transcription arrest site in the first intron of the murine adenosine deaminase gene

1993 ◽  
Vol 13 (5) ◽  
pp. 2718-2729
Author(s):  
S F Kash ◽  
J W Innis ◽  
A U Jackson ◽  
R E Kellems
Keyword(s):  
Rna Polymerase Ii ◽  
Adenosine Deaminase ◽  
Functional Characterization ◽  
Gel Filtration ◽  
Point Mutations ◽  
Elongation Factor ◽  
Dependent Manner ◽  
First Intron ◽  
Intron 1

Transcription arrest plays a role in regulating the expression of a number of genes, including the murine adenosine deaminase (ADA) gene. We have previously identified two prominent arrest sites at the 5' end of the ADA gene: one in the first exon and one in the first intron (J. W. Innis and R. E. Kellems, Mol. Cell. Biol. 11:5398-5409, 1991). Here we report the functional characterization of the intron 1 arrest site, located 137 to 145 nucleotides downstream of the cap site. We have determined, using gel filtration, that the intron 1 arrest site is a stable RNA polymerase II pause site and that the transcription elongation factor SII promotes read-through at this site. Additionally, the sequence determinants for the pause are located within a 37-bp fragment encompassing this site (+123 to +158) and can direct transcription arrest in an orientation-dependent manner in the context of the ADA and adenovirus major late promoters. Specific point mutations in this region increase or decrease the relative pausing efficiency. We also show that the sequence determinants for transcription arrest can function when placed an additional 104 bp downstream of their natural position.

Identification, purification, and characterization of phosphotyrosine-specific protein phosphatases from cultured chicken embryo fibroblasts

1984 ◽  
Vol 4 (6) ◽  
pp. 1003-1012
Author(s):  
R L Nelson ◽  
P E Branton
Keyword(s):  
Chicken Embryo ◽  
Protein Phosphatases ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Soluble Form ◽  
Ph Optimum ◽  
Cell Extracts ◽  
Phosphoprotein Phosphatases ◽  
Embryo Fibroblasts ◽  
Chicken Embryo Fibroblasts

Tyrosine phosphorylation catalyzed by a unique class of protein kinases is an important process in both normal cell proliferation and oncogenic transformation. In this study, phosphoprotein phosphatases specific for the dephosphorylation of phosphotyrosine residues were partially purified from secondary chicken embryo fibroblasts, using 32P-labeled immunoglobulin G phosphorylated by pp60src as substrate. Crude cell extracts contained ca. 70% of the activity in the soluble form and ca. 30% associated with a crude membrane fraction. The soluble activity was purified by using DEAE-cellulose and carboxymethyl cellulose column chromatography and gel filtration, and at least three enzyme species of apparent Mr 55,000 (pTPI), 50,000 (pTPII), and 95,000 (pTPIII)--comprising ca. 20, 45, and 35%, respectively, of the total activity--were resolved. All three enzymes possessed somewhat similar properties. They had a pH optimum of about 7.4, they were inhibited by Zn2+, vanadate, ATP, and ADP, and they were unaffected by divalent metal cations, EDTA, and F- under standard assay conditions employing a physiological ionic strength. These properties suggest that they represent a class of enzymes distinct from well-known phosphoseryl-phosphothreonyl-protein phosphatases and that dephosphorylation of phosphotyrosine-containing proteins may be carried out by a unique family of phosphoprotein phosphatases. Transformation by Rous sarcoma virus resulted in a small increase in phosphotyrosyl-protein phosphatase activity.

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