Differences in crystal properties and ligand affinities of an antifluorescyl fab (4‐4‐20) in two solvent systems

Gibson, Amy L.; Herron, James N.; He, Xiaomin; Patrick, VA; Mason, M. L.; Lin, Jinn-Nan; Kranz, David M.; Voss, Edward W.; Edmundson, Allen B.

doi:10.1002/prot.340030304

Cited by 45 publications

(18 citation statements)

References 18 publications

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“…Antibody concentrations were determined spectrophotometrically using a E 280 nm 1% value of 15 for IgG (23). Fab fragments were prepared from affinity-purified anti-PhK1 antibody according to the procedure of Gibson et al (24) as briefly described here. A sample of affinity-purified antibody (0.5 mg) was dialyzed overnight at pH 7.5 against 50 mM Tris-HCl, 0.15 M NaCl, and 2 mM EDTA.…”

Section: Methodsmentioning

confidence: 99%

Activation and Inhibition of Phosphorylase Kinase by Monospecific Antibodies Raised against Peptides from the Regulatory Domain of the γ-Subunit

Wangsgard

Meixell

Dasgupta

et al. 1996

Journal of Biological Chemistry

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The C terminus of the catalytic gamma-subunit of phosphorylase kinase comprises a regulatory domain that contains regions important for subunit interactions and autoinhibitory functions. Monospecific antibodies raised against four synthetic peptides from this region, PhK1 (362-386), PhK5 (342-366), PhK9 (322-346), and PhK13 (302-326), were found to have significant effects on the catalytic activities of phosphorylase kinase holoenzyme and the gamma delta complex. Antibodies raised against the very C terminus of the gamma-subunit, anti-PhK1 and anti-PhK5, markedly activated both holoenzyme and the gamma delta complex, in the presence and absence of Ca2+. In the presence of Ca2+ at pH 8.2, anti-PhK1 activated the holoenzyme more than 11-fold and activated the gamma delta complex 2.5-fold. Activation of the holoenzyme and the gamma delta complex by anti-PhK5 was 50-70% of that observed with anti-PhK1. Prior phosphorylation of the holoenzyme by the cAMP-dependent protein kinase blocked activation by both anti-PhK1 and anti-PhK5. Antibodies raised against the peptides from the N terminus of the regulatory domain, anti-PhK9 and anti-PhK13, were inhibitory, with their greatest effects on the gamma delta complex. These data demonstrate that the binding of antibodies to specific regions within the regulatory domain of the gamma-subunit can augment or inhibit structural changes and subunit interactions important in regulating phosphorylase kinase activity.

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Section: Methodsmentioning

confidence: 99%

Activation and Inhibition of Phosphorylase Kinase by Monospecific Antibodies Raised against Peptides from the Regulatory Domain of the γ-Subunit

Wangsgard

Meixell

Dasgupta

et al. 1996

Journal of Biological Chemistry

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“…first called for characterization of a clone expressing the high-affinity 4-4-20 antibody. 1,3,6,7 This effort was followed shortly by the development of a panel of idiotypically cross-reactive anti-fluorescein antibodies, 8,9 including 4-4-20, six antibodies of intermediate affinity (10-25, 5-14, 5-27, 9-40, 12-40 and 3-24) and one low affinity IgM antibody (3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13). Except for 5-27, which expressed VL and VH domains from different subgroups, the antibodies of intermediate affinity showed VL and VH gene usage similar to those encoding 4-4-20.…”

Section: Introductionmentioning

confidence: 98%

Three-dimensional Structures of Idiotypically Related Fabs with Intermediate and High Affinity for Fluorescein

Terzyan

Ramsland²,

Voss

et al. 2004

Journal of Molecular Biology

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“…Caging was tested by complexing F-NTS-IBSA with excess mouse monoclonal antibody 4-4-20 (anti-F), which binds F with an affinity constant of 3.4 x 1010 M-1 (18). The antibody-conjugate mixture was added to egg extract containing rat liver nuclei and nuclear uptake of F-NTS-IBSA was assayed (Fig.…”

Section: Resultsmentioning

confidence: 99%

Antibody caging of a nuclear-targeting signal.

Halleck¹,

Rechsteiner²

1990

Proc. Natl. Acad. Sci. U.S.A.

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We have developed a technique for reversibly masking a peptide-targeting signal. A fluoresceinated derivative of the simian virus 40 large tumor antigen nucleartargeting signal was synthesized and cross-linked to bovine serum albumin. The conjugated protein was efficiently transported into rat liver nuclei unless the peptide-targeting signal was sterically hindered by binding of an anti-fluorescein antibody. Addition of free 5-aminofluorescein competed for antibody binding and rapidly restored nuclear accumulation of the derivatized bovine serum albumin. General use of hapten derivatization and anti-hapten antibodies for caging portions of macromolecular surfaces can be extended to a variety of proteins, including antibodies themselves.The ability to initiate biochemical reactions by rapidly revealing hidden functional groups provides a powerful experimental tool with potential applications in many areas of biology. Just over a decade ago, Kaplan et al.(1) synthesized a nitrophenyl ester of the terminal phosphate on ATP. The derivatized ATP molecule is unable to participate in phosphorylation reactions unless the ester is cleaved by photolysis. Such reversible masking or caging has since been applied to a variety of small molecules including Ca2' (2), H+ (3), and other nucleotides (4-6). These compounds have, in turn, been used to study physiological processes ranging from bacterial chemotaxis to signal transduction and muscle contraction.Studies of organelle biogenesis have established the general principle that short stretches of amino acids within a protein largely determine its intracellular location (7,8). A technique for reversibly concealing such peptide signals would be a useful tool, permitting manipulation of the position as well as the function of proteins within cells. To develop such a procedure, we chose the nuclear-targeting signal (NTS) of the simian virus 40 (SV40) tumor antigen.When fused to a cytoplasmic enzyme such as pyruvate kinase, the SV40 NTS is sufficient to misdirect the chimeric protein to the nucleus (9). In addition, synthetic peptides containing the signal can be chemically cross-linked to large nonnuclear proteins, and the resulting conjugates then accumulate in nuclei (10,11). Equally important, the ability to demonstrate nuclear transport in Xenopus egg extracts containing foreign nuclei (12) provides a convenient assay for caging. By reversibly masking the heptapeptide SV40 tumor antigen NTS, we show that the caging approach, once limited to small molecules, can be extended to functional regions on protein surfaces.

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Differences in crystal properties and ligand affinities of an antifluorescyl fab (4‐4‐20) in two solvent systems

Cited by 45 publications

References 18 publications

Activation and Inhibition of Phosphorylase Kinase by Monospecific Antibodies Raised against Peptides from the Regulatory Domain of the γ-Subunit

Activation and Inhibition of Phosphorylase Kinase by Monospecific Antibodies Raised against Peptides from the Regulatory Domain of the γ-Subunit

Three-dimensional Structures of Idiotypically Related Fabs with Intermediate and High Affinity for Fluorescein

Antibody caging of a nuclear-targeting signal.

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