Mapping of the epitope/paratope interactions of a monoclonal antibody directed against adenosine 3′,5′-monophosphate

Naß, Norbert; Colling, Christiane; Cramer, Matthias; Genieser, H G; Butt, Elke; Winkler, Elisabeth; Jaenicke, Lothar; Jastorff, Bernd

doi:10.1042/bj2850129

Cited by 8 publications

(4 citation statements)

References 25 publications

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“…Three cGMPS analogs were tested that contained a modification either in the 2Ј-position of the ribose ring (38 and 40) or in the 8-position of the purine ring (41). The binding data confirm the above discussed stereoselectivity of the mono-substituted sulfur analogs (37 and 38).…”

Section: Table II Substitutions In the C-2/n-3 Region Of The Purine Ringsupporting

confidence: 51%

Structural Features of the Noncatalytic cGMP Binding Sites of Frog Photoreceptor Phosphodiesterase Using cGMP Analogs

Hebert¹,

Schwede²,

Jastorff³

et al. 1998

Journal of Biological Chemistry

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The cGMP-specific phosphodiesterase (PDE) of retinal photoreceptors is a central regulatory enzyme in the visual transduction pathway of vertebrate vision. Although the mechanism of activation of PDE by transducin is well understood, the role of the noncatalytic cGMP binding sites located on the catalytic subunits of PDE remains obscure. We report here for the first time the molecular basis of the noncovalent interactions between cGMP and the high affinity, noncatalytic cGMP binding sites of frog photoreceptor PDE.None of the tested cGMP analogs were able to bind with greater affinity than cGMP itself, and the noncatalytic sites were unable to bind cAMP. The major determinant for discrimination of cGMP over cAMP is in the N-1/C-6 region of the purine ring of cGMP where hydrogen bonding probably stabilizes the selective binding of cGMP. Substitutions at the C-2 position demonstrate that this region of the molecule plays a secondary but significant role in stabilizing cGMP binding to PDE through hydrogen bond interactions. The unaltered hydrogen at the C-8 position is also important for high affinity binding. A significant interaction between the binding pocket and the ribose ring of cGMP occurs at the 2-hydroxyl position. Steric constraints were greatest in the C-8 and possibly the C-6/N-1 regions, whereas the C-2/N-3 and C-2 regions tolerated bulky substituents better. Several lines of evidence indicate that the noncatalytic site binds cGMP in the anti-conformation. The numerous noncovalent interactions between cGMP and the noncatalytic binding pocket of the photoreceptor PDE described in this study account for both the high affinity for cGMP and the high level of discrimination of cGMP from other cyclic nucleotides at the noncatalytic site.Visual excitation of vertebrate retinal photoreceptors begins with the absorption of light by the visual pigment (opsin) which is followed by activation of a G-protein, transducin. This results in activation of a photoreceptor-specific cGMP phosphodiesterase (PDE).1 Increased cGMP hydrolysis lowers the cytoplasmic cGMP concentration which is believed to cause closure of the cGMP-gated ion channel in the plasma membrane and the generation of an electrical response (for reviews, see Refs. 1-5).The rod photoreceptor PDE holoenzyme is a heterotetramer consisting of two non-identical catalytic subunits (␣ and ␤) and two small subunits (␥) that serve to inhibit catalytic activity. Cone photoreceptors have a highly homologous PDE that consists of two identical catalytic subunits (␣Ј) and two conespecific ␥Ј subunits. In addition to containing the active site for cGMP hydrolysis, the catalytic subunits of rod and cone PDEs also possess noncatalytic cGMP binding sites (6). Two other classes of PDEs, namely the cGMP-stimulated PDE (PDE2) and the cGMP-specific PDE (PDE5), are also known to contain noncatalytic binding sites for cGMP. In the case of PDE2, binding of cGMP at the noncatalytic site allosterically activates cyclic nucleotide hydrolysis at the active site (7,8). For PDE5, the...

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Section: Table II Substitutions In the C-2/n-3 Region Of The Purine Ringsupporting

confidence: 51%

Structural Features of the Noncatalytic cGMP Binding Sites of Frog Photoreceptor Phosphodiesterase Using cGMP Analogs

Hebert¹,

Schwede²,

Jastorff³

et al. 1998

Journal of Biological Chemistry

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“…Since they are hydrolysed only relatively well by c-N-I, we suggest that the c-N-I isoenzyme prefers 5h-AMP in anti-conformation when recognized and bound by the enzyme. Our experience with 8-modified cAMP-derivatives [24] supports this conclusion because 8-bromo and 8-amino(methyl) modifications resulted in a reduction of affinity and thus enzymic activity in anti-type cAMP receptors (catabolite gene activator-protein from Escherichia coli and cellsurface receptors from Dictyostelium discoideum) but an increase in affinity for syn-type receptors (cAMP-dependent protein kinase type). At this moment we cannot comment on c-N-II preferences for syn-or anti-conformations as no 8-derivative of 5h-IMP was used.…”

Section: Nucleobase Moiety Modificationsmentioning

confidence: 54%

“…Isoenzymes, receptors and other binding proteins can be compared and characterized using a series of systematically modified analogues of the natural ligands. We have developed this approach for cAMP-and cGMP-binding proteins and were able to identify several characteristic types of molecular interactions ( [24] ; for review see also [25,26]).…”

Section: Introductionmentioning

confidence: 99%

Structure-activity relationship of cytoplasmic 5′-nucleotidase substrate sites

Składanowski

Hoffmann

Krass

et al. 1996

Biochemical Journal

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Various 5'-nucleotidases (EC 3.1.3.5) exist in vertebrate tissues. The sequence and cDNA cloning of the membrane-bound ecto-5'-nucleotidase (e-N) and one of the cytosolic isoenzymes, IMP-preferring (c-N-II), but not the cytosolic AMP-preferring form (c-N-I), have been reported. While c-N-II has a broad tissue distribution, c-N-I is found only in vertebrate heart. The published data on substrate specificity involve mainly the naturally occurring nucleoside monophosphates, without a systematic structure-activity relationship study. In the present study we have used a series of AMP and IMP analogues to examine the structure-activity relationship for c-N-I and c-N-II in detail. The rank order of activity of the test compounds differed substantially between c-N-I and c-N-II. c-N-I and c-N-II varied with respect to the following interactions with substrate: (1) hydrogen-bond formation with the substituent in the 6-position of the purine ring (a donor-type with c-N-I and an acceptor-type with c-N-II); and (2) hydrophobic attraction of the 6-position unsubstituted purine ring (more pronounced with c-N-I than with c-N-II). No better substrate than 5'-AMP was found for c-N-I. We propose that c-N-I functions as an AMP-binding protein in the myocardial cell with an important role during ischaemic ATP breakdown when AMP accumulates rapidly.

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“…The correlation for cyclic nucleotides was further tested using some additional log k ‘ w data from other references. − Good relationships are obtained as well; however, some of the different data sets were parallely shifted (data not shown) and fitted only within those groups of compounds which had been determined together in the respective isocratic runs. Since the isocratic study of Braumann and Jastorff 12 appeared to be the most precise and comprehensive source available for log k ‘ w data of cyclic nucleotides, it was used to calibrate the corresponding data set.…”

Section: Resultsmentioning

confidence: 99%

Determination of Lipophilicity by Gradient Elution High-Performance Liquid Chromatography

1997

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A novel method for the determination of lipophilicity using a simple HPLC protocol based on gradient elution chromatography is presented and compared to the common isocratic log k'(w) procedure. Linear relationships with high correlation coefficients between both methods for biologically active nucleosides and cyclic nucleotides as well as for environmentally relevant aromatic hydrocarbons were found. A mathematical fit to support the empirically determined linear relationship is presented. It is shown that the observed relationship between log k'(w) and the apparent capacity factor (k'(g)) determined by gradient elution is derivable by theoretical considerations as well. Since the gradient method is much less time-consuming compared to other procedures, it represents a convenient alternative for determining lipophilicity data in the future.

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Mapping of the epitope/paratope interactions of a monoclonal antibody directed against adenosine 3′,5′-monophosphate

Cited by 8 publications

References 25 publications

Structural Features of the Noncatalytic cGMP Binding Sites of Frog Photoreceptor Phosphodiesterase Using cGMP Analogs

Structural Features of the Noncatalytic cGMP Binding Sites of Frog Photoreceptor Phosphodiesterase Using cGMP Analogs

Structure-activity relationship of cytoplasmic 5′-nucleotidase substrate sites

Determination of Lipophilicity by Gradient Elution High-Performance Liquid Chromatography

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