Biochemical characterization and cellular localization of the cholinergic system in the chicken retina

Ubiquitin fold modifier 1 (UFM1) is a member of the ubiquitin-like protein family. UFM1 undergoes a cascade of enzymatic reactions including activation by UBA5 (E1), transfer to UFC1 (E2) and selective conjugation to a number of target proteins via UFL1 (E3) enzymes. Despite the importance of ufmylation in a variety of cellular processes and its role in the pathogenicity of many human diseases, the molecular mechanisms of the ufmylation cascade remains unclear. In this study we focused on the biophysical and biochemical characterization of the interaction between UBA5 and UFC1. We explored the hypothesis that the unstructured C-terminal region of UBA5 serves as a regulatory region, controlling cellular localization of the elements of the ufmylation cascade and effective interaction between them. We found that the last 20 residues in UBA5 are pivotal for binding to UFC1 and can accelerate the transfer of UFM1 to UFC1. We solved the structure of a complex of UFC1 and a peptide spanning the last 20 residues of UBA5 by NMR spectroscopy. This structure in combination with additional NMR titration and isothermal titration calorimetry experiments revealed the mechanism of interaction and confirmed the importance of the C-terminal unstructured region in UBA5 for the ufmylation cascade.

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Biochemical characterization of Plasmodium falciparum CTP:phosphoethanolamine cytidylyltransferase shows that only one of the two cytidylyltransferase domains is active

Biochemical Journal ◽

10.1042/bj20121480 ◽

2013 ◽

Vol 450 (1) ◽

pp. 159-167 ◽

Cited By ~ 9

Author(s):

Sweta Maheshwari ◽

Marina Lavigne ◽

Alicia Contet ◽

Blandine Alberge ◽

Emilie Pihan ◽

...

Keyword(s):

Plasmodium Falciparum ◽

Cellular Localization ◽

Biochemical Characterization ◽

De Novo ◽

Membrane Lipids ◽

Polyclonal Antibodies ◽

Homology Modelling ◽

Site Directed Mutagenesis ◽

Recombinant Enzymes ◽

Parasite Life Cycle

The intra-erythrocytic proliferation of the human malaria parasite Plasmodium falciparum requires massive synthesis of PE (phosphatidylethanolamine) that together with phosphatidylcholine constitute the bulk of the malaria membrane lipids. PE is mainly synthesized de novo by the CDP:ethanolamine-dependent Kennedy pathway. We previously showed that inhibition of PE biosynthesis led to parasite death. In the present study we characterized PfECT [P. falciparum CTP:phosphoethanolamine CT (cytidylyltransferase)], which we identified as the rate-limiting step of the PE metabolic pathway in the parasite. The cellular localization and expression of PfECT along the parasite life cycle were studied using polyclonal antibodies. Biochemical analyses showed that the enzyme activity follows Michaelis–Menten kinetics. PfECT is composed of two CT domains separated by a linker region. Activity assays on recombinant enzymes upon site-directed mutagenesis revealed that the N-terminal CT domain was the only catalytically active domain of PfECT. Concordantly, three-dimensional homology modelling of PfECT showed critical amino acid differences between the substrate-binding sites of the two CT domains. PfECT was predicted to fold as an intramolecular dimer suggesting that the inactive C-terminal domain is important for dimer stabilization. Given the absence of PE synthesis in red blood cells, PfECT represents a potential antimalarial target opening the way for a rational conception of bioactive compounds.

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

Biochemical Journal ◽

10.1042/bj3470223 ◽

2000 ◽

Vol 347 (1) ◽

pp. 223-231 ◽

Cited By ~ 37

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.

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Cellular localization and biochemical characterization of a novel calcium-dependent protein kinase from tobacco

Cell Research ◽

10.1038/sj.cr.7290330 ◽

2005 ◽

Vol 15 (8) ◽

pp. 604-612 ◽

Cited By ~ 13

Author(s):

Yun WANG ◽

Mei ZHANG ◽

Ke KE ◽

Ying Tang LU

Keyword(s):

Protein Kinase ◽

Cellular Localization ◽

Biochemical Characterization ◽

Dependent Protein Kinase ◽

Calcium Dependent ◽

Dependent Protein ◽

Calcium Dependent Protein Kinase

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Cellular localization of the microsomal antigen and the thyroid peroxidase antigen

Acta Endocrinologica ◽

10.1530/acta.0.114s057 ◽

1987 ◽

Vol 116 (1_Suppl) ◽

pp. S57-S62 ◽

Cited By ~ 10

Author(s):

A. Pinchera ◽

S. Mariotti ◽

L. Chiovato ◽

P. Vitti ◽

G. Lopez ◽

...

Keyword(s):

Cellular Localization ◽

Biochemical Characterization ◽

Human Thyroid ◽

Thyroid Peroxidase ◽

Immunofluorescence Analysis ◽

Thyroid Cells ◽

Hla Dr ◽

Microsomal Antigen ◽

Dependent Mechanism

Abstract. Evidence has been accumulated that human thyroid microsomal/microvillar autoantigen (M) is expressed both in the cytoplasm and on the surface of thyroid follicular cells. The availability of this autoantigen to the immune system, possibly associated with abnormally expressed HLA-DR antigens may be relevant both to the triggering and to maintenance of thyroid autoimmune reactions. Preliminary biochemical characterization of M suggested that it was a glycoprotein with a mol. wt. of about 100–110 kD. recent studies carried out in our laboratories taking advantage of monoclonal antibodies provided evidence that the structure presently referred as M-Ag is represented by thyroid peroxidase (TPO). The identity between TPO and M is further supported by four-layer immunofluorescence analysis showing a complete overlap of the two antigens both in the surface and in the cytoplasm of thyroid cells and by the observation that the expression of M and TPO is similarly modulated by TSH, possibly through a cAMP-dependent mechanism.

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Myo3A, One of Two Class III Myosin Genes Expressed in Vertebrate Retina, Is Localized to the Calycal Processes of Rod and Cone Photoreceptors and Is Expressed in the Sacculus

Molecular Biology of the Cell ◽

10.1091/mbc.e02-06-0317 ◽

2003 ◽

Vol 14 (3) ◽

pp. 1058-1073 ◽

Cited By ~ 40

Author(s):

Andréa C. Dosé ◽

David W. Hillman ◽

Cynthia Wong ◽

Lorraine Sohlberg ◽

Jennifer Lin-Jones ◽

...

Keyword(s):

Cellular Localization ◽

Biochemical Characterization ◽

Expression Patterns ◽

Tail Domain ◽

Class Iii ◽

Feature Characteristic ◽

Filament Bundles ◽

Myosin Motors ◽

Rod And Cone Photoreceptors

The striped bass has two retina-expressed class III myosin genes, each composed of a kinase, motor, and tail domain. We report the cloning, sequence analysis, and expression patterns of the long (Myo3A) and short (Myo3B) class III myosins, as well as cellular localization and biochemical characterization of the long isoform, Myo3A. Myo3A (209 kDa) is expressed in the retina, brain, testis, and sacculus, and Myo3B (155 kDa) is expressed in the retina, intestine, and testis. The tails of these two isoforms contain two highly conserved domains, 3THDI and 3THDII. Whereas Myo3B has three IQ motifs, Myo3A has nine IQ motifs, four in its neck and five in its tail domain. Myo3A localizes to actin filament bundles of photoreceptors and is concentrated in the calycal processes. An anti-Myo3A antibody decorates the actin cytoskeleton of rod inner/outer segments, and this labeling is reduced by the presence of ATP. The ATP-sensitive actin association is a feature characteristic of myosin motors. The numerous IQ motifs may play a structural or signaling role in the Myo3A, and its localization to calycal processes indicates that this myosin mediates a local function at this site in vertebrate photoreceptors.

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Biochemical characterization and cellular localization of serine protease in myopathic hamster

Journal of Molecular and Cellular Cardiology ◽

10.1016/0022-2828(81)90294-7 ◽

1981 ◽

Vol 13 (12) ◽

pp. 1035-1049 ◽

Cited By ~ 17

Author(s):

T Kuo

Keyword(s):

Serine Protease ◽

Cellular Localization ◽

Biochemical Characterization

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Identification of a family of casein kinases in Paramecium: biochemical characterization and cellular localization

Biochemical Journal ◽

10.1042/bj2960729 ◽

1993 ◽

Vol 296 (3) ◽

pp. 729-735 ◽

Cited By ~ 16

Author(s):

C E Walczak ◽

R A Anderson ◽

D L Nelson

Keyword(s):

Cellular Localization ◽

Biochemical Characterization ◽

Gel Filtration ◽

Casein Kinase ◽

Significant Activity ◽

Paramecium Tetraurelia ◽

Casein Kinase I ◽

Whole Cells ◽

Casein Kinases ◽

Molecular Masses

Protein phosphorylation is believed to play a role in the regulation of ciliary motility in the protozoan Paramecium tetraurelia. Five protein kinases from Paramecium, activated by cyclic nucleotides or Ca2+, have been characterized previously. We report here the identification of a family of second-messenger-independent casein kinases in Paramecium. Casein kinase activity was enriched in the soluble fraction of cilia, but there was also significant activity tightly associated with axonemes. Three ciliary casein kinase activities (soluble CKS1 and CKS2, and axonemal CKA) were separated by chromatography and characterized. The native forms of all three were monomeric, with molecular masses of 28-45 kDa as judged by in-gel kinase assays and sizing by gel filtration. CKS2 was inhibited by heparin, but CKA was unaffected and CKS1 was stimulated. All three activities preferred acidic substrates such as casein and phosvitin, but they could be distinguished by their preference for other substrates. Antibodies against mammalian casein kinase I recognized CKS1 and CKS2 in immunoblots (43 kDa), but did not stain CKA. The antibodies to casein kinase I were used to probe other cellular fractions. A 65 kDa antigen (particulate casein kinase, CKP) was enriched in particulate fractions of whole cells. This 65 kDa protein was found in isolated cell cortices, but was not present in the infraciliary lattice. This report represents the first biochemical identification of a casein kinase I family in protozoa.

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