The novel heteromeric bivalent ligand SB9 potently antagonizes P2Y(1) receptor-mediated responses.

Effects of 6-[(4,6,8-trisulfo-1-naphthyl)iminocarbonyl-1, 3-(4-methylphenylene)iminocarbonyl-1, 3-phenylene-azo]-pyridoxal-5'-phosphate (SB9), a heterodimeric bivalent ligand consisting of pyridoxal-5'-phosphate and the suramin monomer, were studied on contractions of the rat vas deferens elicited by alpha beta-methylene ATP (alpha beta meATP; mediated by P2X(1)-like receptors), contractions of the guinea-pig ileal longitudinal smooth muscle elicited by adenosine 5'-O-(2-thiodiphosphate) (ADP beta S mediated by P2Y(1)-like receptors), and the degradation of ATP by ecto-nucleotidases in folliculated Xenopus laevis oocytes. SB9 (0.1-10 microM) antagonized contractile responses produced by alpha beta meATP or ADP beta S in a concentration-dependent manner. Schild analysis yielded linear regression lines of unit slope, indicating competitive antagonism. From the rightward shifts of the agonist concentration-response curves pA(2) values of 6.05+/-0.13 (vas deferens) and 6.98+/-0.07 (ileum) were derived. In both preparations, SB9 behaved as a slow onset, slow offset antagonist. Incubation of three oocytes in the presence of ATP produced an increase in inorganic phosphate (P(i)) over a 30-min period, which amounted to 35.1+/-1.9 microM P(i) from 100 microM ATP. SB9 (10-1000 microM) reduced this degradation (pIC(50)=4.33+/-0.10). The results illustrate that SB9 is a high-affinity P2Y(1) receptor antagonist with a remarkable selectivity for P2Y(1) vs. P2X(1) receptors (about 10-fold) and ecto-nucleotidases (447-fold). These properties make it unique among the pyridoxal-5'-phosphate and suramin derivatives reported to date.

The novel pyridoxal-5'-phosphate derivative PPNDS potently antagonizes activation of P2X(1) receptors.

Pyridoxal-5'-phosphate-6-(2'-naphthylazo-6'-nitro-4',8'-disulfonat e) (PPNDS) potently antagonized P2X(1) receptor-mediated responses in rat vas deferens (pK(B)=7.43) and Xenopus laevis oocytes (pIC(50)=7. 84). It showed lower (up to 20,000-fold) inhibitory potency on ecto-nucleotidase in Xenopus oocytes and on P2Y(1) receptors in guinea-pig ileum (pA(2)=6.13). PPNDS did not interact with alpha(1A)-adrenoceptors, adenosine A(1) and A(2B), histamine H(1) and muscarinic M(3) receptors. Thus, PPNDS is a novel, specific P2 receptor antagonist and represents the pyridoxal-5'-phosphate derivative with the highest potency at P2X(1) receptors.

Associations between erythrocyte band 3 protein and aldolase in detergent solution. Determining their stoichiometry by analytical ultracentrifugation.

The cytoplasmic domain of band 3, the predominant polypeptide of the erythrocyte membrane, represents a binding site for certain glycolytic enzymes. We have studied the association between human band 3 protein and aldolase, in order to clarify the role of the different band 3 oligomers as ligand binding sites. The experiments were performed on mixtures of solubilized band 3 and aldolase in solutions of a nonionic detergent, nonaethyleneglycol lauryl ether. The main technique applied was sedimentation equilibrium analysis in an analytical ultracentrifuge. In addition, nonequilibrium centrifugation techniques were used. To facilitate the evaluations, the aldolase was labelled with a dye. The following results were obtained. (1) With unmodified band 3, aldolase is bound exclusively or at least predominantly to the band 3 tetramer (but not to monomers or dimers). (2) The band 3 tetramer can bind up to four aldolase tetramers. (3) The band 3 tetramer/aldolase complex is unstable on the time scale of the techniques used. (4) Stable band 3 dimers (stabilized either covalently or noncovalently) can also associate with aldolase and can bind up to two aldolase tetramers. The results described, together with those reported previously, point at a prominent role of the band 3 tetramer in ligand binding.

PPADS, a novel functionally selective antagonist of P2 purinoceptor-mediated responses.

We have characterized PPADS (pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid) as a novel antagonist which selectively blocks P2 purinoceptor-mediated responses in rabbit vas deferens at pre- and postjunctional sites. PPADS did not interact with alpha 1-adrenoceptors, muscarinic M2 and M3 receptors, histamine H1 and adenosine A1 receptors. Thus, PPADS is a novel and useful pharmacological tool to study co-transmission in tissues where ATP and co-existing neurotransmitters act in concert.

The influence of Mg2+ on anion binding to sarcoplasmic reticulum membranes as detected by 35Cl-NMR.

35Cl-NMR spectroscopy has been used to study the competition between anions, including nucleotides, on skeletal muscle sarcoplasmic reticulum membranes. Different chloride binding sites can be distinguished according to their Mg2+ sensitivity. Phosphate binding is enhanced by Mg2+ whereas the anion transport inhibitor pyridoxalphosphate-6-azophenyl-2'-sulfonic acid (PPAPS) binding is not. The affinity of the enzyme for the Mg-adenylyl imidodiphosphate (MgAMP-PNP) complex is decreased whereas that for MgATP is increased. Three sets of binding sites can be discriminated from which chloride is displaced by different anions with varying efficiency. High affinity binding of AMP-PNP and PPAPS occurs at the same site, that can also be occupied by phosphate. Low-affinity binding of PPAPS and AMP-PNP also coincides, but in a site where phosphate binding is negligible. ATP and ADP bind to both sites. In the presence of Mg2+ a third anion binding site can be occupied by phosphate but neither by AMP-PNP nor PPAPS.