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.

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.

scholarly journals Identification and functional characterization of two bamboo FD gene homologs having contrasting effects on shoot growth and flowering

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Smritikana Dutta ◽  
Anwesha Deb ◽  
Prasun Biswas ◽  
Sukanya Chakraborty ◽  
Suman Guha ◽  
...  
Keyword(s):  
Large Scale ◽  
Forest Ecology ◽  
Phylogenetic Analyses ◽  
Functional Characterization ◽  
Constitutive Expression ◽  
Transcript Level ◽  
In Planta ◽  
Mobility Shift ◽  
Vegetative Development ◽  
Sequence Comparisons

AbstractBamboos, member of the family Poaceae, represent many interesting features with respect to their fast and extended vegetative growth, unusual, yet divergent flowering time across species, and impact of sudden, large scale flowering on forest ecology. However, not many studies have been conducted at the molecular level to characterize important genes that regulate vegetative and flowering habit in bamboo. In this study, two bamboo FD genes, BtFD1 and BtFD2, which are members of the florigen activation complex (FAC) have been identified by sequence and phylogenetic analyses. Sequence comparisons identified one important amino acid, which was located in the DNA-binding basic region and was altered between BtFD1 and BtFD2 (Ala146 of BtFD1 vs. Leu100 of BtFD2). Electrophoretic mobility shift assay revealed that this alteration had resulted into ten times higher binding efficiency of BtFD1 than BtFD2 to its target ACGT motif present at the promoter of the APETALA1 gene. Expression analyses in different tissues and seasons indicated the involvement of BtFD1 in flower and vegetative development, while BtFD2 was very lowly expressed throughout all the tissues and conditions studied. Finally, a tenfold increase of the AtAP1 transcript level by p35S::BtFD1 Arabidopsis plants compared to wild type confirms a positively regulatory role of BtFD1 towards flowering. However, constitutive expression of BtFD1 had led to dwarfisms and apparent reduction in the length of flowering stalk and numbers of flowers/plant, whereas no visible phenotype was observed for BtFD2 overexpression. This signifies that timely expression of BtFD1 may be critical to perform its programmed developmental role in planta.

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.

scholarly journals Identification and Functional Characterization of Protest SWI/SNF and ISWI Complexes

2021 ◽  
Author(s):  
Alejandro Saettone Chipana
Keyword(s):  
Chromatin Remodeling ◽  
Tetrahymena Thermophila ◽  
Affinity Purification ◽  
Functional Characterization ◽  
Peptide Array ◽  
Interacting Proteins ◽  
Silent Chromatin ◽  
Lysine Residues ◽  
Chromatin Remodeling Complexes

The thesis aims to identify and initiate functional characterization of the SWI/SNF and ISWI complexes in Tetrahymena thermophila. Through affinity purification of the conserved subunit Snf5 followed by mass spectrometry (AP-MS), I identified the first SWI/SNF complex in protists. One of the subunits I found is a small bromodomain containing protein named Ibd1. Through AP-MS of Ibd1 I found Ibd1 is versatile and interacts with several additional chromatin remodeling complexes. Bromodomains are known to have affinity for acetylated lysine residues within proteins such as histones. A peptide array experiment suggests that Ibd1 also has affinity for acetylated chromatin. Indirect immunofluorescence (IF) of Ibd1 hints at a role in transcription. My analysis of Tetrahymena Iswi1 shows expression during meiosis, vegetative growth and starvation. IF data shows its localization is consistent with Iswi1 function in mitosis/meiosis or maintenance of silent chromatin. AP-MS of ISW1 discovered several interacting proteins of unknown function.

scholarly journals Structural and Functional Characterization of K339T Substitution Identified in the PB2 Subunit Cap-binding Pocket of Influenza A Virus

2013 ◽  
Vol 288 (16) ◽  
pp. 11013-11023 ◽  
Author(s):  
Yong Liu ◽  
Kun Qin ◽  
Geng Meng ◽  
Jinfang Zhang ◽  
Jianfang Zhou ◽  
...  
Keyword(s):  
Influenza A Virus ◽  
Influenza A ◽  
Functional Characterization ◽  
Binding Pocket

scholarly journals An exome-wide sequencing study of lipid response to high-fat meal and fenofibrate in Caucasians from the GOLDN cohort

2018 ◽  
Vol 59 (4) ◽  
pp. 722-729 ◽  
Author(s):  
Xin Geng ◽  
Marguerite R. Irvin ◽  
Bertha Hidalgo ◽  
Stella Aslibekyan ◽  
Vinodh Srinivasasainagendra ◽  
...  
Keyword(s):  
Rare Variants ◽  
Transcript Level ◽  
Lipid Lowering ◽  
Gene Transcript ◽  
High Fat ◽  
Heart Study ◽  
Lipid Response ◽  
Coding Variants ◽  
Fat Meal ◽  
Meal Challenge

Our understanding of genetic influences on the response of lipids to specific interventions is limited. In this study, we sought to elucidate effects of rare genetic variants on lipid response to a high-fat meal challenge and fenofibrate (FFB) therapy in the Genetics of Lipid Lowering Drugs and Diet Network (GOLDN) cohort using an exome-wide sequencing-based association study. Our results showed that the rare coding variants in ITGA7, SIPA1L2, and CEP72 are significantly associated with fasting LDL cholesterol response to FFB (P = 1.24E-07), triglyceride postprandial area under the increase (AUI) (P = 2.31E-06), and triglyceride postprandial AUI response to FFB (P = 1.88E-06), respectively. We sought to replicate the association for SIPA1L2 in the Heredity and Phenotype Intervention (HAPI) Heart Study, which included a high-fat meal challenge but not FFB treatment. The associated rare variants in GOLDN were not observed in the HAPI Heart study, and thus the gene-based result was not replicated. For functional validation, we found that gene transcript level of SIPA1L2 is associated with triglyceride postprandial AUI (P < 0.05) in GOLDN. Our study suggests unique genetic mechanisms contributing to the lipid response to the high-fat meal challenge and FFB therapy.

scholarly journals SAUR49 Can Positively Regulate Leaf Senescence by Suppressing SSPP in Arabidopsis

2019 ◽  
Vol 61 (3) ◽  
pp. 644-658 ◽  
Author(s):  
Zewen Wen ◽  
Yuanyuan Mei ◽  
Jie Zhou ◽  
Yanjiao Cui ◽  
Dan Wang ◽  
...  
Keyword(s):  
Leaf Senescence ◽  
Protein Phosphatase ◽  
Functional Characterization ◽  
Transcript Level ◽  
In Planta ◽  
Environmental Signals ◽  
Receptor Like Kinase ◽  
Premature Leaf Senescence ◽  
Natural Leaf ◽  
Two Hybrid

Abstract The involvement of SMALL AUXIN-UP RNA (SAUR) proteins in leaf senescence has been more and more acknowledged, but the detailed mechanisms remain unclear. In the present study, we performed yeast two-hybrid assays and identified SAUR49 as an interactor of SENESCENCE SUPPRESSED PROTEIN PHOSPHATASE (SSPP), which is a PP2C protein phosphatase that negatively regulates Arabidopsis leaf senescence by suppressing the leucine-rich repeat receptor-like protein kinase SENESCENCE-ASSOCIATED RECEPTOR-LIKE KINASE (SARK), as reported previously by our group. The interaction between SAUR49 and SSPP was further confirmed in planta. Functional characterization revealed that SAUR49 is a positive regulator of leaf senescence. The accumulation level of SAUR49 protein increased during natural leaf senescence in Arabidopsis. The transcript level of SAUR49 was upregulated during SARK-induced premature leaf senescence but downregulated during SSPP-mediated delayed leaf senescence. Overexpression of SAUR49 significantly accelerated both natural and dark-induced leaf senescence in Arabidopsis. More importantly, SAUR49 overexpression completely reversed SSPP-induced delayed leaf senescence. In addition, overexpression of SAUR49 reversed the decreased plasma membrane H+-ATPase activity mediated by SSPP. Taken together, the results showed that SAUR49 functions in accelerating the leaf senescence process via the activation of SARK-mediated leaf senescence signaling by suppressing SSPP. We further identified four other SSPP-interacting SAURs, SAUR30, SAUR39, SAUR41 and SAUR72, that may act redundantly with SAUR49 in regulating leaf senescence. All these observations indicated that certain members of the SAUR family may serve as an important hub that integrates various hormonal and environmental signals with senescence signals in Arabidopsis.

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