Activation of the action potential Na+ ionophore of cultured neuroblastoma cells by veratridine and batrachotoxin.

The activation of the action potential Na+ ionophore by veratridine and batrachotoxin is time- and concentration-dependent and completely reversible. Batrachotoxin acts more slowly than veratridine. The concentration dependence of activation at equilibrium suggests reversible interaction of each toxin with a single class of independent sites having dissociation constants at physiologic ion concentrations of 80 plus or minus 13 muM for veratridine and 0.4 plus or minus muM for batrachotoxin. The maximum velocity of Na+ uptake at 50 mM Na+ is 128 plus or minus 12 nmol/min/mg in the presence of batrachotoxin compared to 48 plus or minus 4 nmol/min/mg in the presence of veratridine. Treatment of cells with excess veratridine in addition to batrachotoxin inhibits batrachotoxin-dependent 22-Na+ uptake. The concentration dependence of this inhibition suggests that it reflects competitive displacement of batrachotoxin from its binding site by veratridine. The activation by veratridine and batrachotoxin is inhibited in a competitive manner by divalent cations. The inhibition by divalent cations exhibits significant ion specificity with Mn-2+ greater than Co-2+ greater than Ni-2+ greater than Ca-2+ greater than Mg-2+ greater than Sr-2+. The inhibition constants (KI) for Ca-2+ are 0.84 mM for veratridine-dependent 22-Na+ uptake and 1.2 mM for batrachotoxin-dependent 22-Na+ uptake. The activation by veratridine and batrachotoxin is inhibited in a noncompetitive manner by tetrodotoxin. The apparent KD for tetrodotoxin as 11 plus or minus 1 nM in the presence of 150 mM Na+ and approximately 8.5 nM in 50 mM Na+. Divalent cations do not affect the apparent KD for tetrodotoxin. A hypothesis is presented which suggests that batrachotoxin, veratridine, and divalent cations interact with an activation site associated with the action potential Na+ ionophore, whereas tetrodotoxin interacts with a physically and functionally independent site involved in the transport of monovalent cations by the ionophore.     

THE ANATOMY AND RELATIONSHIPS OF ELACHISINA DALL (GASTROPODA: RISSOACEA)

Species of Elachisina are marine and extend from the Caribbeanand western North and Central America to the Philippines, Hawaiiand New Zealand. The anatomy and other morphological featuresof Elachisina ( = Microdocluis) floridana (Rehder) from theFlorida Keys are described and compared with rissoacean familiesjudged to be most closely allied. This species has a uniquecombination of characters which do not correspond to those character-setsused to define any known rissoacean family. Important featuresinclude a metapodial as well as pallial tentacles, a wide ctenidiumwith low, triangular filaments, an osphradium which is shortrelative to the ctenidium, an S-shaped loop of the intestinewhich extends into the pallial roof, long dorsal folds in themidoesophagus, a rather loosely organised nervous system, anopen prostate gland from which the pallial vas deferens exitsat its anterior extremity, glandular lobes on the penis, a non-glandularfold on the right side of the ventral channel of the capsulegland and a fold on the left side in the posterior end of thechannel, a pair of seminal receptacles lying between the albumenand capsule glands, a bursa which lies alongside the capsulegland and opens posteriorly to the ventral channel and a U-shaped,expanded section of the oviduct proximal to the albumen glandand seminal receptacles. This combination of characters is consideredto be sufficiently distinct to arrant the erection of a newfamily-level taxon to accommodate the small group of speciesjudged to be congeneric with E. floridana. The Elachisinidaeappears to be most closely related to the Rissoidae, Iravadiidaeand Hydrobiidae. (Received 19 October 1983;     

Inactivation of Escherichia coli pyruvate formate-lyase by hypophosphite: evidence for a rate-limiting phosphorus-hydrogen bond cleavage

Recently, Knappe and co-workers [Knappe, J., Neugebauer, F. A., Blaschkowski, H. P., & Ganzler, M. (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 1332] have shown that the catalytically active form of pyruvate formate-lyase from Escherichia coli is associated with a protein-bound organic free radical which is quenched upon enzyme inactivation by oxygen or hypophosphite. Our interest in the chemical mechanism of this unusual enzymatic reaction has led us to investigate several key aspects of the inactivation of the lyase by hypophosphite and its relationship to the normal enzymatic reaction. We report here that the inactivation of both the free and acetylated forms of the lyase is subject to a primary kinetic isotope effect using [2H2]hypophosphite. This suggests that phosphorus-hydrogen bond cleavage is at least partially rate limiting during inactivation. In addition, the inactivated enzyme can be fully reactivated. We have also determined a Vmax/Km isotope effect of 3.6 +/- 0.7 for pyruvate formation from [2H]formate and acetyl coenzyme A. Thus, carbon-hydrogen bond cleavage is partially rate limiting in the normal reverse reaction. On the basis of our findings, the previous work of Knappe and co-workers, the likelihood that hypophosphite is a formate analogue, the known susceptibility of both hypophosphite and formate to homolysis, and a chemical precedent for homolytic cleavage of pyruvate, we offer a preliminary mechanistic proposal for the lyase reaction.