The SpvB protein from Salmonella enterica was recently discovered as an actin-ADP-ribosylating toxin. SpvB is most likely delivered via a type-III secretion system into eukaryotic cells and does not have a binding/translocation component. This is in contrast to the family of binary actin-ADP-ribosylating toxins from various Bacillus and Clostridium species. However, there are homologies in amino acid sequences between the C-terminal domain of SpvB and the catalytic domains of the actin-ADP-ribosylating toxins such as C2 toxin from Clostridium botulinum and iota toxin from Clostridium perfringens. We compared the biochemical properties of the catalytic C-terminal domain of SpvB (C/SpvB) with the enzyme components of C2 toxin and iota toxin. The specificity of C/SpvB concerning the modification of G- or F-actin was comparable to the C2 and iota toxins, although there were distinct differences regarding the recognition of actin isoforms. C/SpvB and iota toxin modify both muscle alpha-actin and nonmuscle beta/gamma-actin, whereas C2 toxin only modifies beta/gamma-actin. In contrast to the iota and C2 toxins, C/SpvB possessed no detectable glycohydrolase activity in the absence of a protein substrate. The maximal reaction rates were comparable for all toxins, whereas variable K(m) values for NAD were evident. We identified arginine-177 as the modification site for C/SpvB with the actin homologue protein Act88F from Drosophila.

P2X receptors are cation channels gated by extracellular ATP and related nucleotides. Because of the widespread distribution of P2X receptors and the high subtype diversity, potent and selective antagonists are needed to dissect their roles in intact tissues. Based on suramin as a lead compound, several derivates have been described that block recombinant P2X receptors with orders of magnitude higher potency than suramin. Here we characterized the suramin analogue 4,4′,4″,4‴-(carbonylbis(imino-5,1,3-benzenetriylbis(carbonylimino)))tetrakis-benzene-1,3-disulfonic acid (NF449) with respect to its potency to antagonize ATP or αβ-methyleneadenosine 5′-trisphosphate-induced inward currents of homomeric rat P2X1–P2X4 receptors or heteromeric P2X1+5 and P2X2+3 receptors, respectively. NF449 most potently blocked P2X1 and P2X1+5 receptors with IC50 values of 0.3nM and 0.7nM, respectively. Three to four orders of magnitude higher NF449 concentrations were required to block homomeric P2X3 or heteromeric P2X2+3 receptors (IC50 1.8 and 0.3μM, respectively). NF449 was least potent at homomeric P2X2 receptors (IC50 47μM) and homomeric P2X4 receptors (IC50>300μM). Altogether, these results characterize NF449 as the so far most potent and selective antagonist of receptors incorporating the P2X1 subunit such as the P2X1 homomer and the P2X1+5 heteromer.