scholarly journals Cellular Localization and Biochemical Characterization of a Chimeric Fluorescent Protein Fusion ofArabidopsisCellulose Synthase-Like A2 Inserted into Golgi Membrane

2014 ◽  
Vol 2014 ◽  
pp. 1-7 ◽  
Author(s):  
Monica De Caroli ◽  
Marcello S. Lenucci ◽  
Gian-Pietro Di Sansebastiano ◽  
Michela Tunno ◽  
Anna Montefusco ◽  
...  
Keyword(s):  
Cellular Localization ◽  
Biochemical Characterization ◽  
Fluorescent Protein ◽  
Specific Activity ◽  
Proteinase K ◽  
Golgi Membrane ◽  
Protein Fusion ◽  
Enzyme Specific Activity ◽  
Tm Domain

Cellulose synthase-like(Csl) genes are believed to encode enzymes for the synthesis of cell wall matrix polysaccharides. The subfamily ofCslAis putatively involved in the biosynthesis ofβ-mannans. Here we report a study on the cellular localization and the enzyme activity of anArabidopsisCslA family member, AtCslA2. We show that the fluorescent protein fusion AtCslA2-GFP, transiently expressed in tobacco leaf protoplasts, is synthesized in the ER and it accumulates in the Golgi stacks. The chimera is inserted in the Golgi membrane and is functional since membrane preparations obtained by transformed protoplasts carry out thein vitrosynthesis of a14C-mannan starting from GDP-d-[U-14C]mannose as substrate. The enzyme specific activity is increased by approximately 38% in the transformed protoplasts with respect to wild-type. Preliminary tests with proteinase K, biochemical data, and TM domain predictions suggest that the catalytic site of AtCslA2 faces the Golgi lumen.

scholarly journals Rem2, a new member of the Rem/Rad/Gem/Kir family of Ras-related GTPases

2000 ◽  
Vol 347 (1) ◽  
pp. 223-231 ◽  
Author(s):  
Brian S. FINLIN ◽  
Haipeng SHAO ◽  
Keiko KADONO-OKUDA ◽  
Nan GUO ◽  
Douglas A. ANDRES
Keyword(s):  
Cellular Localization ◽  
Biochemical Characterization ◽  
Fluorescent Protein ◽  
Dissociation Constants ◽  
Intrinsic Rate ◽  
Biochemical Properties ◽  
Cell Physiology ◽  
Gtp Binding ◽  
C Terminus ◽  
Green Fluorescent

Here we report the molecular cloning and biochemical characterization of Rem2 (for Rem, ad and G-related 2), a novel GTP-binding protein identified on the basis of its homology with the Rem, Rad, Gem and Kir (RGK) family of Ras-related small GTP-binding proteins. Rem2 mRNA was detected in rat brain and kidney, making it the first member of the RGK family to be expressed at relatively high levels in neuronal tissues. Recombinant Rem2 binds GTP saturably and exhibits a low intrinsic rate of GTP hydrolysis. Surprisingly, the guanine nucleotide dissociation constants for both Rem2 and Rem are significantly different than the majority of the Ras-related GTPases, displaying higher dissociation rates for GTP than GDP. Localization studies with green fluorescent protein (GFP)-tagged recombinant protein fusions indicate that Rem2 has a punctate, plasma membrane localization. Deletion of the C-terminal seven amino acid residues that are conserved in all RGK family members did not affect the cellular distribution of the GFP fusion protein, whereas a larger deletion, including much of the polybasic region of the Rem2 C-terminus, resulted in its redistribution to the cytosol. Thus Rem2 is a GTPase of the RGK family with distinctive biochemical properties and possessing a novel cellular localization signal, consistent with its having a unique role in cell physiology.

scholarly journals OASTL-A1 functions as a cytosolic cysteine synthase and affects arsenic tolerance in rice

2020 ◽  
Vol 71 (12) ◽  
pp. 3678-3689 ◽  
Author(s):  
Chengcheng Wang ◽  
Lihua Zheng ◽  
Zhong Tang ◽  
Shengkai Sun ◽  
Jian Feng Ma ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Tissue Expression ◽  
Protein Fusion ◽  
Outer Cortex ◽  
Cysteine Synthase ◽  
Arsenic Tolerance ◽  
Tissue Expression Analysis ◽  
Green Fluorescent

Abstract Arsenic (As) contamination in paddy soil can cause phytotoxicity and elevated As accumulation in rice grains. Arsenic detoxification is closely linked to sulfur assimilation, but the genes involved have not been described in rice. In this study, we characterize the function of OASTL-A1, an O-acetylserine(thiol) lyase, in cysteine biosynthesis and detoxification of As in rice. Tissue expression analysis revealed that OsOASTL-A1 is mainly expressed in roots at the vegetative growth stage and in nodes at the reproductive stage. Furthermore, the expression of OsOASTL-A1 in roots was strongly induced by As exposure. Transgenic rice plants expressing pOsOASTL-A1::GUS (β-glucuronidase) indicated that OsOASTL-A1 was strongly expressed in the outer cortex and the vascular cylinder in the root mature zone. Subcellular localization using OsOASTL-A1:eGFP (enhanced green fluorescent protein) fusion protein showed that OsOASTL-A1 was localized to the cytosol. In vivo and in vitro enzyme activity assays showed that OsOASTL-A1 possessed the O-acetylserine(thiol) lyase activity. Knockout of OsOASTL-A1 led to significantly lower levels of cysteine, glutathione, and phytochelatins in roots and increased sensitivity to arsenate stress. Furthermore, the osoastl-a1 knockout mutants reduced As accumulation in the roots, but increased As accumulation in shoots. We conclude that OsOASTL-A1 is the cytosolic O-acetylserine(thiol) lyase that plays an important role in non-protein thiol biosynthesis in roots for As detoxification.

scholarly journals Characterization of the Streptococcal C5a Peptidase Using a C5a-Green Fluorescent Protein Fusion Protein Substrate

2000 ◽  
Vol 182 (11) ◽  
pp. 3254-3258 ◽  
Author(s):  
D. K. Stafslien ◽  
P. P. Cleary
Keyword(s):  
Green Fluorescent Protein ◽  
Fusion Protein ◽  
Fluorescent Protein ◽  
High Sensitivity ◽  
Dependent Manner ◽  
Protein Fusion ◽  
Group B Streptococci ◽  
Mutant Proteins ◽  
Green Fluorescent

ABSTRACT A glutathione-S-transferase (GST)–C5a–green fluorescent protein (GFP) fusion protein was designed for use as a substrate for the streptococcal C5a peptidase (SCPA). The substrate was immobilized on a glutathione-Sepharose affinity matrix and used to measure wild-type SCPA activity in the range of 0.8 to 800 nM. The results of the assay demonstrated that SCPA is highly heat stable and has optimal activity on the synthetic substrate at or above pH 8.0. SCPA activity was unaffected by 0.1 to 10 mM Ca2+, Mg2+, and Mn2+ but was inhibited by the same concentrations of Zn2+. The assay shows high sensitivity to ionic strength; NaCl inhibits SCPA cleavage of GST-C5a-GFP in a dose-dependent manner. Based on previously published computer homology modeling, four substitutions were introduced into the putative active site of SCPA: Asp130-Ala, His193-Ala, Asn295-Ala, and Ser512-Ala. All four mutant proteins had over 1,000-fold less proteolytic activity on C5a in vitro, as determined both by the GFP assay described here and by a polymorphonuclear cell adherence assay. In addition, recombinant SCPA1 and SCPA49, from two distinct lineages of Streptococcus pyogenes (group A streptococci), and recombinant SCPB, fromStreptococcus agalactiae (group B streptococci), were compared in the GFP assay. The three enzymes had similar activities, all cleaving approximately 6 mol of C5a mmol of SCP−1liter−1 min−1.

scholarly journals The MDM2 Ubiquitination Signal in the DNA-Binding Domain of p53 Forms a Docking Site for Calcium Calmodulin Kinase Superfamily Members

2007 ◽  
Vol 27 (9) ◽  
pp. 3542-3555 ◽  
Author(s):  
Ashley L. Craig ◽  
Jennifer A. Chrystal ◽  
Jennifer A. Fraser ◽  
Nathalie Sphyris ◽  
Yao Lin ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Specific Activity ◽  
Phosphorylation Site ◽  
Fusion Peptide ◽  
Transactivation Domain ◽  
Docking Site ◽  
Calmodulin Kinase ◽  
Green Fluorescent

ABSTRACT Genetic and biochemical studies have shown that Ser20 phosphorylation in the transactivation domain of p53 mediates p300-catalyzed DNA-dependent p53 acetylation and B-cell tumor suppression. However, the protein kinases that mediate this modification are not well defined. A cell-free Ser20 phosphorylation site assay was used to identify a broad range of calcium calmodulin kinase superfamily members, including CHK2, CHK1, DAPK-1, DAPK-3, DRAK-1, and AMPK, as Ser20 kinases. Phosphorylation of a p53 transactivation domain fragment at Ser20 by these enzymes in vitro can be mediated in trans by a docking site peptide derived from the BOX-V domain of p53, which also harbors the ubiquitin signal for MDM2. Evaluation of these calcium calmodulin kinase superfamily members as candidate Ser20 kinases in vivo has shown that only CHK1 or DAPK-1 can stimulate p53 transactivation and induce Ser20 phosphorylation of p53. Using CHK1 as a prototypical in vivo Ser20 kinase, we demonstrate that (i) CHK1 protein depletion using small interfering RNA can attenuate p53 phosphorylation at Ser20, (ii) an enhanced green fluorescent protein (EGFP)-BOX-V fusion peptide can attenuate Ser20 phosphorylation of p53 in vivo, (iii) the EGFP-BOX-V fusion peptide can selectively bind to CHK1 in vivo, and (iv) the Δp53 spliced variant lacking the BOX-V motif is refractory to Ser20 phosphorylation by CHK1. These data indicate that the BOX-V motif of p53 has evolved the capacity to bind to enzymes that mediate either p53 phosphorylation or ubiquitination, thus controlling the specific activity of p53 as a transcription factor.

scholarly journals Biochemical characterization of androgen receptor-interacting protein 4

2006 ◽  
Vol 393 (3) ◽  
pp. 789-795 ◽  
Author(s):  
Andrii Domanskyi ◽  
Katja T. Virtanen ◽  
Jorma J. Palvimo ◽  
Olli A. Jänne
Keyword(s):  
Androgen Receptor ◽  
Biochemical Characterization ◽  
Specific Activity ◽  
Protein Complexes ◽  
Sequence Similarity ◽  
Protein Family ◽  
Interacting Protein ◽  
Attachment Sites

ARIP4 [AR (androgen receptor)-interacting protein 4] is a member of the SNF2-like family of proteins. Its sequence similarity to known proteins is restricted to the centrally located SNF2 ATPase domain. ARIP4 is an active ATPase, and dsDNA (double-stranded DNA) and ssDNA (single-stranded DNA) enhance its catalytic activity. We show in the present study that ARIP4 interacts with AR and binds to DNA and mononucleosomes. The N-terminal region of ARIP4 mediates interaction with AR. Kinetic parameters of the ARIP4 ATPase are similar to those of BRG-1 and SNF2h, two members of the SNF2-like protein family, but the specific activity of ARIP4 protein purified to >90% homogeneity is approximately ten times lower, being 120 molecules of ATP hydrolysed by an ARIP4 molecule per min in contrast with approx. 1000 ATP molecules hydrolysed per min by ATP-dependent chromatin remodellers. Unlike other members of the SNF2 family, ARIP4 does not appear to form large protein complexes in vivo or remodel mononucleosomes in vitro. ARIP4 is covalently modified by sumoylation, and mutation of six potential SUMO (small ubiquitin-related modifier) attachment sites abolished the ability of ARIP4 to bind DNA, hydrolyse ATP and activate AR function. We conclude that, similar to its closest homologues in the SNF2-like protein family, ATRX (α-thalassemia, mental retardation, X-linked) and Rad54, ARIP4 does not seem to be a classical chromatin remodelling protein.

scholarly journals Dynamic localization of two tobamovirus ORF6 proteins involves distinct organellar compartments

2013 ◽  
Vol 94 (1) ◽  
pp. 230-240 ◽  
Author(s):  
Vladimir A. Gushchin ◽  
Nina I. Lukhovitskaya ◽  
Dmitri E. Andreev ◽  
Kathryn M. Wright ◽  
Michael E. Taliansky ◽  
...  
Keyword(s):  
Mosaic Virus ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Tobacco Rattle Virus ◽  
Tomato Mosaic Virus ◽  
Protein Fusion ◽  
Orf6 Gene ◽  
Orf6 Protein

ORF6 is a small gene that overlaps the movement and coat protein genes of subgroup 1a tobamoviruses. The ORF6 protein of tomato mosaic virus (ToMV) strain L (L-ORF6), interacts in vitro with eukaryotic elongation factor 1α, and mutation of the ORF6 gene of tobacco mosaic virus (TMV) strain U1 (U1-ORF6) reduces the pathogenicity in vivo of TMV, whereas expression of this gene from two other viruses, tobacco rattle virus (TRV) and potato virus X (PVX), increases their pathogenicity. In this work, the in vivo properties of the L-ORF6 and U1-ORF6 proteins were compared to identify sequences that direct the proteins to different subcellular locations and also influence virus pathogenicity. Site-specific mutations in the ORF6 protein were made, hybrid ORF6 proteins were created in which the N-terminal and C-terminal parts were derived from the two proteins, and different subregions of the protein were examined, using expression either from a recombinant TRV vector or as a yellow fluorescent protein fusion from a binary plasmid in Agrobacterium tumefaciens. L-ORF6 caused mild necrotic symptoms in Nicotiana benthamiana when expressed from TRV, whereas U1-ORF6 caused severe symptoms including death of the plant apex. The difference in symptoms was associated with the C-terminal region of L-ORF6, which directed the protein to the endoplasmic reticulum (ER), whereas U1-ORF6 was directed initially to the nucleolus and later to the mitochondria. Positively charged residues at the N terminus allowed nucleolar entry of both U1-ORF6 and L-ORF6, but hydrophobic residues at the C terminus of L-ORF6 directed this protein to the ER.

Biochemical characterization a digestive trypsin in the midgut of large cabbage white butterfly, Pieris brassicae L. (Lepidoptera: Pieridae)

2017 ◽  
Vol 108 (4) ◽  
pp. 501-509
Author(s):  
A. Sharifloo ◽  
A. Zibaee ◽  
J. Jalali Sendi ◽  
K. Talebi Jahroumi
Keyword(s):  
Biochemical Characterization ◽  
Specific Activity ◽  
Pieris Brassicae ◽  
Larval Midgut ◽  
Posterior Midgut ◽  
Diethylaminoethyl Cellulose ◽  
Cabbage White Butterfly ◽  
Fast Flow

AbstractA comprehensive study on digestive trypsin was undertaken in the larval midgut of Pieris brassicae L. Results of enzymatic compartmentalization showed a significantly higher activity of crude trypsin in the anterior larval midgut rather than posterior-midgut. Using Diethylaminoethyl cellulose fast flow column chromatography a purified trypsin was obtained by specific activity of 21 U mg−1 protein, recovery of 22%, purification fold of 28-fold and molecular weight of 25 kDa. This purified enzyme showed the highest activity at pH 8 and the corresponding temperature of 40°C. However, the specific inhibitors used including 4-(2-Aminoethyl) benzenesulfonyl fluroride hydrochloride, N-p-Tosyl-L-lysine methyl ester hydrochloride and Soybean Trypsin Inhibitor significantly lowered the activity of the purified enzyme in vitro. Moreover, the activity of trypsin and likewise the nutritional indices were significantly altered in the larval midgut feeding upon the leaves treated by 1 mM concentration of each inhibitor in comparison with control. Determination of enzymatic characteristics of insect trypsins is crucial in paving the path for controlling pests by potential natural compounds via transgenic plants.

scholarly journals Production and analysis of a mammalian septin hetero-octamer complex

2020 ◽  
Author(s):  
Barry T. DeRose ◽  
Robert S. Kelley ◽  
Roshni Ravi ◽  
Bashkim Kokona ◽  
Elias T. Spiliotis ◽  
...  
Keyword(s):  
Cell Biology ◽  
Biochemical Characterization ◽  
Fluorescent Protein ◽  
Mechanistic Study ◽  
Central Position ◽  
Protein Fusion ◽  
Recombinant Production ◽  
And Function ◽  
Order Structures

AbstractThe septins are filament-forming proteins found in diverse eukaryotes from fungi to vertebrates, with roles in cytokinesis, shaping of membranes and modifying cytoskeletal organization. These GTPases assemble into rod-shaped soluble hetero-hexamers and hetero-octamers in mammals, which polymerize into filaments and higher order structures. While the cell biology and pathobiology of septins are advancing rapidly, mechanistic study of the mammalian septins is limited by a lack of recombinant hetero-octamer materials. We describe here the production and characterization of a recombinant mammalian septin hetero-octamer of defined stoichiometry, the SEPT2/SEPT6/SEPT7/SEPT3 complex. Using a fluorescent protein fusion to the complex, we observed filaments assembled from this complex. In addition, we used this novel tool to resolve recent questions regarding the organization of the soluble septin complex. Biochemical characterization of a SEPT3 truncation that disrupts SEPT3-SEPT3 interactions is consistent with SEPT3 occupying a central position in the complex while the SEPT2 subunits are at the ends of the rod-shaped octameric complexes. Consistent with SEPT2 being on the complex ends, we find that our purified SEPT2/SEPT6/SEPT7/SEPT3 hetero-octamer copolymerizes into mixed filaments with separately purified SEPT2/SEPT6/SEPT7 hetero-hexamer. We expect this new recombinant production approach to lay essential groundwork for future studies into mammalian septin mechanism and function.

Antibiotics as Inhibitor of Glutathione S-transferase: Biological Evaluation and Molecular Structure Studies

2021 ◽  
Vol 22 ◽  
Author(s):  
Adnan Ayna ◽  
Luqman Khosnaw ◽  
Yusuf Temel ◽  
Mehmet Ciftci
Keyword(s):  
Biochemical Characterization ◽  
Specific Activity ◽  
Biological Evaluation ◽  
Glutathione S Transferase ◽  
Scopolamine Butylbromide ◽  
Glutathione S Transferases ◽  
Concentration Graphs ◽  
Compound Concentration

Background: The glutathione S-transferases (GSTs) are family of enzymes that are notable for their role in phase II detoxification reactions. Antibiotics have been reported to have several adverse effects on the activity of the enzymes in mammals. Aim: The aim of this study was structural and biochemical characterization of rat erythrocyte GST and understanding the effects of gentamicin, clindamycin, cefazolin, ampicillin and scopolamine butylbromide on the activity of human erythrocyte GST using rat as a model. Methods: The enzyme was purified by GSH-agarose affinity chromatography. In vitro GST enzyme activity was measured at 25°C using CDNB as a model substrate. IC50 of drugs were measured by activity %–vs compound concentration graphs. Lineweaver–Burk graphs were drawn to determine the inhibition type and Ki constants for the drugs. The structure of the enzyme was predicted via Protein Homology/analogY Recognition Engine. Results: In this study, GST was purified from rat erythrocyte with a specific activity of 6.3 EU/mg protein, 44 % yield and 115 fold. Gentamicin and clindamycin inhibited the enzymatic activity with IC50 of 1.69 and 6.9 mM and Ki of 1.70 and 2.36 mM, respectively. Ampicillin and scopolamine butylbromide were activator of the enzyme while the activity of the enzyme was insensitive to cefazolin. The enzyme was further characterized by homology modeling and sequence alignment revealing similarities with human GST. Conclusion: Collectively, it could be concluded that gentamicin and clindamycin are the inhibitors of erythrocyte GST.

scholarly journals Cx26 keratitis ichthyosis deafness syndrome mutations trigger alternative splicing of Cx26 to prevent expression and cause toxicity in vitro

2019 ◽  
Vol 6 (8) ◽  
pp. 191128 ◽  
Author(s):  
Jonathan Cook ◽  
Elizabeth de Wolf ◽  
Nicholas Dale
Keyword(s):  
Alternative Splicing ◽  
Branch Point ◽  
Splice Site ◽  
Cellular Localization ◽  
Fluorescent Protein ◽  
Point Mutations ◽  
Future Research ◽  
Cryptic Splice Site ◽  
Toxicity In Vitro

The Cx26 mRNA has not been reported to undergo alternative splicing. In expressing a series of human keratitis ichthyosis deafness (KID) syndrome mutations of Cx26 (A88V, N14K and A40V), we found the production of a truncated mRNA product. These mutations, although not creating a cryptic splice site, appeared to activate a pre-existing cryptic splice site. The alternative splicing of the mutant Cx26 mRNA could be prevented by mutating the predicted 3′, 5′ splice sites and the branch point. The presence of a C-terminal fluorescent protein tag (mCherry or Clover) was necessary for this alternative splicing to occur. Strangely, Cx26 A88V could cause the alternative splicing of co-expressed WT Cx26—suggesting a trans effect. The alternative splicing of Cx26 A88V caused cell death, and this could be prevented by the 3′, 5′ and branch point mutations. Expression of the KID syndrome mutants could be rescued by combining them with removal of the 5′ splice site. We used this strategy to enable expression of Cx26 A40V-5′ and demonstrate that this KID syndrome mutation removed CO 2 sensitivity from the Cx26 hemichannel. This is the fourth KID syndrome mutation found to abolish the CO 2 -sensitivity of the Cx26 hemichannel, and suggests that the altered CO ­2 -sensitivity could contribute to the pathology of this mutation. Future research on KID syndrome mutations should take care to avoid using a C-terminal tag to track cellular localization and expression or if this is unavoidable, combine this mutation with removal of the 5′ splice site.

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