scholarly journals A Pipeline towards the Biochemical Characterization of the Arabidopsis GT14 Family

2021 ◽  
Vol 22 (3) ◽  
pp. 1360
Author(s):  
Lingling Xuan ◽  
Jie Zhang ◽  
Weitai Lu ◽  
Pawel Gluza ◽  
Berit Ebert ◽  
...  
Keyword(s):  
Biochemical Characterization ◽  
Reversed Phase ◽  
Arabinogalactan Protein ◽  
Analysis Pipeline ◽  
Enzymatic Assays ◽  
New Family ◽  
A Cell ◽  
Arabidopsis Thaliana Genome ◽  
Animal Genomes

Glycosyltransferases (GTs) catalyze the synthesis of glycosidic linkages and are essential in the biosynthesis of glycans, glycoconjugates (glycolipids and glycoproteins), and glycosides. Plant genomes generally encode many more GTs than animal genomes due to the synthesis of a cell wall and a wide variety of glycosylated secondary metabolites. The Arabidopsis thaliana genome is predicted to encode over 573 GTs that are currently classified into 42 diverse families. The biochemical functions of most of these GTs are still unknown. In this study, we updated the JBEI Arabidopsis GT clone collection by cloning an additional 105 GT cDNAs, 508 in total (89%), into Gateway-compatible vectors for downstream characterization. We further established a functional analysis pipeline using transient expression in tobacco (Nicotiana benthamiana) followed by enzymatic assays, fractionation of enzymatic products by reversed-phase HPLC (RP-HPLC) and characterization by mass spectrometry (MS). Using the GT14 family as an exemplar, we outline a strategy for identifying effective substrates of GT enzymes. By addition of UDP-GlcA as donor and the synthetic acceptors galactose-nitrobenzodiazole (Gal-NBD), β-1,6-galactotetraose (β-1,6-Gal4) and β-1,3-galactopentose (β-1,3-Gal5) to microsomes expressing individual GT14 enzymes, we verified the β-glucuronosyltransferase (GlcAT) activity of three members of this family (AtGlcAT14A, B, and E). In addition, a new family member (AT4G27480, 248) was shown to possess significantly higher activity than other GT14 enzymes. Our data indicate a likely role in arabinogalactan-protein (AGP) biosynthesis for these GT14 members. Together, the updated Arabidopsis GT clone collection and the biochemical analysis pipeline present an efficient means to identify and characterize novel GT catalytic activities.

Biochemical Characterization of the Histidine Triad Protein PhtD as a Cell Surface Zinc-Binding Protein of Pneumococcus

Biochemistry ◽  
2011 ◽  
Vol 50 (17) ◽  
pp. 3551-3558 ◽  
Author(s):  
Elodie Loisel ◽  
Suneeta Chimalapati ◽  
Catherine Bougault ◽  
Anne Imberty ◽  
Benoit Gallet ◽  
...  
Keyword(s):  
Cell Surface ◽  
Biochemical Characterization ◽  
Binding Protein ◽  
Zinc Binding ◽  
A Cell

scholarly journals Biochemical Characterization of a Novel Redox-Regulated Metacaspase in a Marine Diatom

2021 ◽  
Vol 12 ◽  
Author(s):  
Shiri Graff van Creveld ◽  
Shifra Ben-Dor ◽  
Avia Mizrachi ◽  
Uria Alcolombri ◽  
Amanda Hopes ◽  
...  
Keyword(s):  
Cell Death ◽  
Biochemical Characterization ◽  
Site Directed Mutagenesis ◽  
Marine Diatom ◽  
Type Iii ◽  
Calcium Dependent ◽  
Evolutionary Origins ◽  
Cell Extracts ◽  
A Cell

Programmed cell death (PCD) in marine microalgae was suggested to be one of the mechanisms that facilitates bloom demise, yet its molecular components in phytoplankton are unknown. Phytoplankton are completely lacking any of the canonical components of PCD, such as caspases, but possess metacaspases. Metacaspases were shown to regulate PCD in plants and some protists, but their roles in algae and other organisms are still elusive. Here, we identified and biochemically characterized a type III metacaspase from the model diatom Phaeodactylum tricornutum, termed PtMCA-IIIc. Through expression of recombinant PtMCA-IIIc in E. coli, we revealed that PtMCA-IIIc exhibits a calcium-dependent protease activity, including auto-processing and cleavage after arginine. Similar metacaspase activity was detected in P. tricornutum cell extracts. PtMCA-IIIc overexpressing cells exhibited higher metacaspase activity, while CRISPR/Cas9-mediated knockout cells had decreased metacaspase activity compared to WT cells. Site-directed mutagenesis of cysteines that were predicted to form a disulfide bond decreased recombinant PtMCA-IIIc activity, suggesting its enhancement under oxidizing conditions. One of those cysteines was oxidized, detected in redox proteomics, specifically in response to lethal concentrations of hydrogen peroxide and a diatom derived aldehyde. Phylogenetic analysis revealed that this cysteine-pair is unique and widespread among diatom type III metacaspases. The characterization of a cell death associated protein in diatoms provides insights into the evolutionary origins of PCD and its ecological significance in algal bloom dynamics.

scholarly journals MORPHOLOGICAL AND BIOCHEMICAL CHARACTERIZATION OF A CELL DEATH PROCESS INDUCED BY E3, A NEW SYNTHETIC DIARYLSULFONILUREA ANALOGUE

2001 ◽  
Vol 1 (S3) ◽  
pp. 50-50
Author(s):  
M. Alonso ◽  
M. Miglaccio ◽  
I. Encio ◽  
A. Asumendi ◽  
V. Martinez-Merino ◽  
...  
Keyword(s):  
Cell Death ◽  
Biochemical Characterization ◽  
Death Process ◽  
Cell Death Process ◽  
A Cell

scholarly journals Antibacterial peptides of the moth Galleria mellonella.

2001 ◽  
Vol 48 (4) ◽  
pp. 1191-1195 ◽  
Author(s):  
P Mak ◽  
D Chmiel ◽  
G J Gacek
Keyword(s):  
Antimicrobial Peptides ◽  
Biochemical Characterization ◽  
Galleria Mellonella ◽  
Reversed Phase ◽  
Unique Sequence ◽  
Antibacterial Peptides ◽  
Reversed Phase Chromatography ◽  
Viable Bacteria

The work describes purification and biochemical characterization of two inducible antimicrobial peptides from the hemolymph of Galleria mellonella. The peptides were isolated by a sequence of reversed-phase chromatography steps from the hemolymph of larvae immunized with viable bacteria. The first peptide is a member of the cecropin family while the second one is rich in proline residues and has a unique sequence.

scholarly journals Structural and Biochemical Characterization of CIB1 Delineates a New Family of EF-hand-containing Proteins

2004 ◽  
Vol 280 (9) ◽  
pp. 8407-8415 ◽  
Author(s):  
Holly R. Gentry ◽  
Alex U. Singer ◽  
Laurie Betts ◽  
Cheng Yang ◽  
Joseph D. Ferrara ◽  
...  
Keyword(s):  
Biochemical Characterization ◽  
New Family ◽  
Ef Hand

Purification and Biochemical Characterization of Trypsin Inhibitor from Oyster

2012 ◽  
Vol 577 ◽  
pp. 119-124
Author(s):  
Yuan Hui Zhao ◽  
Ming Yong Zeng ◽  
Xia Li
Keyword(s):  
Liquid Chromatography ◽  
Trypsin Inhibitor ◽  
High Performance ◽  
Biochemical Characterization ◽  
Gel Filtration ◽  
Reversed Phase ◽  
Phase Liquid ◽  
Lung Adenocarcinoma A549 ◽  
Head Inhibitor

In this paper, the purification and biochemical characterization of the endogenous oyster (Crassostrea gigas) trypsin inhibitor were researched. A oyster trypsin inhibitor（OTI）has been purified by successive ammonium sulfate precipitation, gel filtration, affinity chromatography and high performance reversed-phase liquid chromatography. OTI has a molecular weight of approximately 5036 Da estimated by high performance size exclusive liquid chromatography. OTI was heat-, acid- and basic-stable competitive trypsin inhibitor. And OTI was double-head inhibitor with the inhibition constant (Ki) value of 1.644×10-2 mmol L-1. OTI was composed of nine kinds of amino acid, and rich in cysteine, alanine and glutamic acid. Furthermore, OTI can inhibit the proliferations of human lung adenocarcinoma A549 cell and human cervical cancer Hela cell

scholarly journals A Cell Cycle-Regulated Toxoplasma Deubiquitinase, TgOTUD3A, Targets Polyubiquitins with Specific Lysine Linkages

mSphere ◽  
2016 ◽  
Vol 1 (3) ◽  
Author(s):  
Animesh Dhara ◽  
Anthony P. Sinai
Keyword(s):  
Cell Cycle ◽  
Toxoplasma Gondii ◽  
Biochemical Characterization ◽  
Recent Analysis ◽  
Dependent Manner ◽  
Polyubiquitin Chains ◽  
Content Type ◽  
A Cell ◽  
Cycle Dependent

ABSTRACT The role of ubiquitin-mediated processes in the regulation of the apicomplexan cell cycle is beginning to be elucidated. The recent analysis of the Toxoplasma “ubiquitome” highlights the importance of ubiquitination in the parasite cell cycle. The machinery regulating the ubiquitin dynamics in T. gondii has remained understudied. Here, we provide a biochemical characterization of an OTU (ovarian tumor) family deubiquitinase, TgOTUD3A, defining its localization and dynamic expression pattern at various stages of the cell cycle. We further establish that TgOTUD3A has activity preference for polyubiquitin chains with certain lysine linkages—such unique activity has not been previously reported in any apicomplexan. This is particularly important given the finding in this study that Toxoplasma gondii proteins are modified by diverse lysine-linked polyubiquitin chains and that these modifications are very dynamic across the cell cycle, pointing toward the sophistication of the “ubiquitin code” as a potential mechanism to regulate parasite biology. The contribution of ubiquitin-mediated mechanisms in the regulation of the Toxoplasma gondii cell cycle has remained largely unexplored. Here, we describe the functional characterization of a T. gondii deubiquitinase (TGGT1_258780) of the ovarian-tumor domain-containing (OTU) family, which, based on its structural homology to the human OTUD3 clade, has been designated TgOTUD3A. The TgOTUD3A protein is expressed in a cell cycle-dependent manner mimicking its mRNA expression, indicating that it is regulated primarily at the transcriptional level. TgOTUD3A, which was found in the cytoplasm at low levels in G1 parasites, increased in abundance with the progression of the cell cycle and exhibited partial localization to the developing daughter scaffolds during cytokinesis. Recombinant TgOTUD3A but not a catalytic-site mutant TgOTUD3A (C229A) exhibited activity against poly- but not monoubiquitinated targets. This activity was selective for polyubiquitin chains with preference for specific lysine linkages (K48 > K11 > K63). All three of these polyubiquitin linkage modifications were found to be present in Toxoplasma, where they exhibited differential levels and localization patterns in a cell cycle-dependent manner. TgOTUD3A removed ubiquitin from the K48- but not the K63-linked ubiquitinated T. gondii proteins independently of the modified target protein, thereby exhibiting the characteristics of an exodeubiquitinase. In addition to cell cycle association, the demonstration of multiple ubiquitin linkages together with the selective deubiquitinase activity of TgOTUD3A reveals an unappreciated level of complexity in the T. gondii “ubiquitin code.” IMPORTANCE The role of ubiquitin-mediated processes in the regulation of the apicomplexan cell cycle is beginning to be elucidated. The recent analysis of the Toxoplasma “ubiquitome” highlights the importance of ubiquitination in the parasite cell cycle. The machinery regulating the ubiquitin dynamics in T. gondii has remained understudied. Here, we provide a biochemical characterization of an OTU (ovarian tumor) family deubiquitinase, TgOTUD3A, defining its localization and dynamic expression pattern at various stages of the cell cycle. We further establish that TgOTUD3A has activity preference for polyubiquitin chains with certain lysine linkages—such unique activity has not been previously reported in any apicomplexan. This is particularly important given the finding in this study that Toxoplasma gondii proteins are modified by diverse lysine-linked polyubiquitin chains and that these modifications are very dynamic across the cell cycle, pointing toward the sophistication of the “ubiquitin code” as a potential mechanism to regulate parasite biology.

scholarly journals Molecular and Biochemical Characterization of a Distinct Type of Fructose-1,6-Bisphosphatase from Pyrococcus furiosus

2002 ◽  
Vol 184 (12) ◽  
pp. 3401-3405 ◽  
Author(s):  
Corné H. Verhees ◽  
Jasper Akerboom ◽  
Emile Schiltz ◽  
Willem M. de Vos ◽  
John van der Oost
Keyword(s):  
Biochemical Characterization ◽  
Pyrococcus Furiosus ◽  
Distinct Type ◽  
Sequence Motifs ◽  
Conserved Sequence ◽  
New Family ◽  
Fructose 1,6 Bisphosphatase ◽  
Conserved Sequence Motifs ◽  
Fructose 1,6 Bisphosphate

ABSTRACT The Pyrococcus furiosus fbpA gene was cloned and expressed in Escherichia coli, and the fructose-1,6-bisphosphatase produced was subsequently purified and characterized. The dimeric enzyme showed a preference for fructose-1,6-bisphosphate, with a Km of 0.32 mM and a V max of 12.2 U/mg. The P. furiosus fructose-1,6-bisphosphatase was strongly inhibited by Li+ (50% inhibitory concentration, 1 mM). Based on the presence of conserved sequence motifs and the substrate specificity of the P. furiosus fructose-1,6-bisphosphatase, we propose that this enzyme belongs to a new family, class IV fructose-1,6-bisphosphatase.

Purification and characterization of an intracellular aspartyl acid proteinase (pumAi) from Ustilago maydis

2005 ◽  
Vol 51 (2) ◽  
pp. 171-175 ◽  
Author(s):  
Yuridia Mercado-Flores ◽  
Adriana Trejo-Aguilar ◽  
Bernardo Ramírez-Zavala ◽  
Lourdes Villa-Tanaca ◽  
César Hernández-Rodríguez
Keyword(s):  
Biochemical Characterization ◽  
Hydrophobic Interactions ◽  
Gel Filtration ◽  
Specific Inhibitor ◽  
Ustilago Maydis ◽  
Acid Proteinase ◽  
A Cell ◽  
Dimorphic Yeast ◽  
Pepstatin A

The intracellular proteinase pumAi in Ustilago maydis has been associated with yeast-mycelium dimorphic transition. The proteinase was purified from a cell-free extract by ammonium sulfate fractionation and chromatographic steps including hydrophobic interactions on a Phenyl Superose column, ion exchange on a Mono Q column, and gel filtration on Superose 12 columns. The enzyme has a mass of 35.3–36.6 kDa, a pH and temperature optimum of 4.0 and 40 °C, respectively, and a pI of 5.5. The enzyme degraded hemoglobin, gelatin, albumin, and casein, but not collagen, and the enzymatic activity was strongly inhibited by pepstatin A, an aspartyl proteinase-specific inhibitor. The biochemical characteristics of pumAi are similar to other fungal intracellular aspartyl proteinases, however, this is the first biochemical characterization of a basidiomycete proteinase probably associated with dimorphic yeast-mycelium transition.Key words: aspartyl proteinase, yeast-mycelium transition, Ustilago maydis.

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