Domain swapping reveals that the C- and N-terminal domains of DnaG and DnaB, respectively, are functional homologues

A panel of 11 monoclonal antibodies specific to alpha- or beta-tubulin subunits was used to study the location of tubulin molecules in cytoplasmic microtubules. Specificity of antibodies was confirmed by immunoblotting and immunofluorescence experiments on fixed cells. The limited proteolysis of tubulin with trypsin and chymotrypsin followed by immunoblotting demonstrated that the antibodies discriminated between structural domains of both subunits. Epitope mapping of isolated alpha-tubulin revealed that a set of antibodies against the N-terminal domain of the alpha-subunit (TU-01, TU-02, TU-03, TU-09, 6–11B-1) recognized at least four different antigenic determinants. Immunofluorescence staining of unfixed detergent-extracted cells showed that antibodies to determinants on C-terminal domains labelled microtubules, but these were not decorated with antibodies to N-terminal domains. The same results were obtained after microinjection of antibodies into living cells. The unchanged distribution of microtubules in injected cells was confirmed by double-label immunofluorescence with polyclonal antibodies. The data indicate that while parts of C-terminal domains of both subunits are exposed on the exterior of the microtubules, considerable regions of the N-terminal domains are either not exposed on the surface of cytoplasmic microtubules, or are masked by interacting proteins.

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Human plasma and cerebrospinal fibronectins differ in the accessibility of the epitopes on the N-terminal domains.

Acta Biochimica Polonica ◽

10.18388/abp.2010_2413 ◽

2010 ◽

Vol 57 (3) ◽

Cited By ~ 1

Author(s):

Małgorzata Pupek ◽

Anna Lemańska-Perek ◽

Jolanta Jasonek ◽

Iwona Kątnik-Prastowska

Keyword(s):

Cerebrospinal Fluid ◽

Monoclonal Antibodies ◽

Human Plasma ◽

Antigenic Epitope ◽

Cell Binding ◽

Cerebrospinal Fluid Analysis ◽

Fluid Analysis ◽

C And N ◽

The Mean ◽

Terminal Domains

Three monoclonal antibodies specific to the central cell-binding and the C- and N-terminal domains of fibronectin (FN) were used to test antigenic epitope accessibility on human plasma and cerebrospinal fibronectins. In the plasma group, the mean N-terminal FN domain immunoreactivity was about one fourth that of the cell-binding and C-terminal domains, whereas in cerebrospinal fluid they were nearly equal. In the presence of 0.5-6 M urea N-terminal domain immunoreactivity in the plasma increased 3-6-fold, but it decreased 0.7-3-fold in the cerebrospinal fluid. Analysis of fibronectin domain immunoreactivities of the cell-binding and N-terminal domains by a panel of specific monoclonal antibodies may reveal N-terminal fibronectin domain accessibility for reaction with biological partner ligand(s) and/or processes in which FN could be implicated. Such determinations may have important clinical implications.

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Characterization of Interaction of C- and N-Terminal Domains in LIM15/DMC1 and RAD51 from a Basidiomycetes, Coprinus cinereus

Biochemical and Biophysical Research Communications ◽

10.1006/bbrc.2000.3250 ◽

2000 ◽

Vol 275 (1) ◽

pp. 97-102 ◽

Cited By ~ 15

Author(s):

Takayuki Nara ◽

Taichi Yamamoto ◽

Kengo Sakaguchi

Keyword(s):

Coprinus Cinereus ◽

C And N ◽

Terminal Domains

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Crystal Structures of the Human Doublecortin C- and N-terminal Domains in Complex with Specific Antibodies

Journal of Biological Chemistry ◽

10.1074/jbc.m116.726547 ◽

2016 ◽

Vol 291 (31) ◽

pp. 16292-16306 ◽

Cited By ~ 9

Author(s):

Dominique Burger ◽

Martine Stihle ◽

Ashwani Sharma ◽

Paola Di Lello ◽

Jörg Benz ◽

...

Keyword(s):

Crystal Structures ◽

Specific Antibodies ◽

C And N ◽

Terminal Domains

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Regulatory Properties and Interaction of the C- and N-Terminal Domains of BetP, an Osmoregulated Betaine Transporter fromCorynebacterium glutamicum†

Biochemistry ◽

10.1021/bi801325r ◽

2008 ◽

Vol 47 (46) ◽

pp. 12208-12218 ◽

Cited By ~ 41

Author(s):

Vera Ott ◽

Joachim Koch ◽

Kira Späte ◽

Susanne Morbach ◽

Reinhard Krämer

Keyword(s):

C And N ◽

Regulatory Properties ◽

Terminal Domains

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Substrate-specifying determinants of the nucleotide pyrophosphatases/phosphodiesterases NPP1 and NPP2

Biochemical Journal ◽

10.1042/bj20040465 ◽

2004 ◽

Vol 381 (1) ◽

pp. 71-77 ◽

Cited By ~ 47

Author(s):

Anisoara CIMPEAN ◽

Cristiana STEFAN ◽

Rik GIJSBERS ◽

Willy STALMANS ◽

Mathieu BOLLEN

Keyword(s):

Substrate Specificity ◽

Metal Binding ◽

Catalytic Domain ◽

Polypeptide Chain ◽

Catalytic Site ◽

Domain Swapping ◽

Lysophospholipase D ◽

Terminal Domains

The nucleotide pyrophosphatases/phosphodiesterases NPP1 and NPP2/autotaxin are structurally related eukaryotic ecto-enzymes, but display a very different substrate specificity. NPP1 releases nucleoside 5′-monophosphates from various nucleotides, whereas NPP2 mainly functions as a lysophospholipase D. We have used a domain-swapping approach to map substrate-specifying determinants of NPP1 and NPP2. The catalytic domain of NPP1 fused to the N- and C-terminal domains of NPP2 was hyperactive as a nucleotide phosphodiesterase, but did not show any lysophospholipase D activity. In contrast, chimaeras of the catalytic domain of NPP2 and the N- and/or C-terminal domains of NPP1 were completely inactive. These data indicate that the catalytic domain as well as both extremities of NPP2 contain lysophospholipid-specifying sequences. Within the catalytic domain of NPP1 and NPP2, we have mapped residues close to the catalytic site that determine the activities towards nucleotides and lysophospholipids. We also show that the conserved Gly/Phe-Xaa-Gly-Xaa-Xaa-Gly (G/FXGXXG) motif near the catalytic site is required for metal binding, but is not involved in substrate-specification. Our data suggest that the distinct activities of NPP1 and NPP2 stem from multiple differences throughout the polypeptide chain.

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