Transition-state analysis of a Vmax mutant of AMP nucleosidase by the application of heavy-atom kinetic isotope effects

The transition state of the Vmax mutant of AMP nucleosidase from Azotobacter vinelandii [Leung, H. B., & Schramm, V. L. (1981) J. Biol. Chem. 256, 12823-12829] has been characterized by heavy-atom kinetic isotope effects in the presence and absence of MgATP, the allosteric activator. The enzyme catalyzes hydrolysis of the N-glycosidic bond of AMP at approximately 2% of the rate of the normal enzyme with only minor changes in the Km for substrate, the activation constant for MgATP, and the Ki for formycin 5'-phosphate, a tight-binding competitive inhibitor. Isotope effects were measured as a function of the allosteric activator concentration that increases the turnover number of the enzyme from 0.006 s-1 to 1.2 s-1. The kinetic isotope effects were measured with the substrates [1'-3H]AMP, [2'-2H]AMP, [2'-2H]AMP, [9-15N]AMP, and [1',9-14C, 15N]AMP. All substrates gave significant kinetic isotope effects in a pattern that establishes that the reaction expresses intrinsic kinetic isotope effects in the presence or absence of MgATP. The kinetic isotope effect with [9-15N]AMP decreased from 1.034 +/- 0.002 to 1.021 +/- 0.002 in response to MgATP. The [1'-3H]AMP isotope effect increased from 1.086 +/- 0.003 to 1.094 +/- 0.002, while the kinetic isotope effect for [1',9-14C, 15N]AMP decreased from 1.085 +/- 0.003 to 1.070 +/- 0.004 in response to allosteric activation with MgATP. Kinetic isotope effects with [1'-14C]AMP and [2'-2H]AMP were 1.041 +/- 0.006 and 1.089 +/- 0.002 and were not changed by addition of MgATP.(ABSTRACT TRUNCATED AT 250 WORDS)     

Sensitivity to voltage-independent inhibition determined by pore-lining region of the acetylcholine receptor.

Some noncompetitive inhibitors (e.g., ganglionic blockers) exhibit selectivity for the inhibition of neuronal nicotinic acetylcholine receptors (nAChRs). This study characterizes the mechanism of selective long-term inhibition of neuronal and muscle-neuronal chimeric nAChRs by bis(2,2,6,6-tetramethyl-4-piperidinyl) sebacate (bis-TMP-10 or BTMPS), a bifunctional form of the potent ganglionic blocker tetramethylpiperidine. Long-term inhibition of neuronal nAChRs by bis-TMP-10 has been previously demonstrated to arise, at least in part, from the binding of the bis compound to neuronal beta-subunits. In this study, long-term inhibition is demonstrated to be dependent upon the presence of sequence element(s) within the pore-lining second transmembrane domain (tm2) of neuronal beta-subunits; however, the inhibitor binding site itself does not appear to be contained within the segment of the channel pore influenced by the membrane electric field. Specifically, our results imply that bis-TMP-10 interacts with an activation-sensitive element, the availability of which may be regulated by a sequence in the tm2 domain. Furthermore, we demonstrate a compound length requirement for long-term inhibition that would be consistent with binding to multiple sites located on the extracellular portion of the receptor.     

Identification of a 220-kDa membrane tumor-associated antigen by human anti-UK114 monoclonal antibodies selected from the immunoglobulin repertoire of a cancer patient

Human monoclonal antibodies (HuMAb) specific for a 14-kDa perchloric acid-soluble protein (defined as UK114) were produced by somatic fusion of the human-mouse myeloma K6H6/B5 with Epstein-Barr virus-transformed peripheral B lymphocytes from a cancer patient previously treated with UK101 preparations, containing the UK114 protein. Three IgM-secreting clones were selected on the criteria of specificity for the purified UK114 protein immobilized onto plastic and adapted to grow in a serum-free medium. The reactivity of these antibodies showed a broad distribution pattern restricted to fresh tumor tissues and tumor cell lines, mainly of the adenocarcinoma type. None of the normal cells, nonmalignant cell lines, and normal tissues surrounding the neoplastic lesions were reactive. The immunochemical analysis of the target antigens showed that the HuMAb recognize a molecule of 220 kDa selectively expressed by the surface of tumor cells, as well as a cytoplasmic 14-kDa protein. The 220-kDa antigen was different from other tumor-associated antigens with similar molecular mass and, so far, unique. In the presence of human complement, two of three HuMAb are cytotoxic for tumor cells expressing the 220-kDa surface antigen. The tumor specificity and the lytic ability attributed to these HuMAb are promising features for the exploration of future clinical applications.     

Biosensor analysis of antigen-antibody interactions as a priority step in the generation of monoclonal bispecific antibodies

A biosensor system aimed at real-time measuring molecular interactions among label-free reactants has been used for a comparative analysis of the binding features (i.e., association-dissociation rates and affinity constants) as well as epitope mapping between bivalent monoclonal antibodies and the derived monovalent bispecific monoclonal antibody. The results show that observed different affinities between parental and derived bispecific antibodies concern the association rate constant, whereas the dissociation rate constants are unaltered. The apparent affinity-constant values determined by solid-phase radioimmunoassay yielded figures almost overlapping with those obtained with the biosensor instrument. The results of the present work indicate that the biosensor system has gained a key role not only as a tool for the study of antigen-antibody interactions, but also for setting up the reference parameters for the selection of the best candidates in the generation of bispecific monoclonal antibodies.     

CD157, the Janus of CD38 but with a unique personality

CD157 is a pleiotropic ectoenzyme which belongs to the CD38 family and to the growing number of leukocyte surface molecules known to act independently as both receptors and enzymes. A 45-kDa surface structure with a GPI anchor, the CD157 molecule displays two distinct domains in its extracellular component. The first is implicated in the enzymic activities of the molecule and the second features adhesion/signalling properties. CD157 shares several characteristics with CD38, including a similar amino acid sequence and enzymic functions. Both molecules are involved in the metabolism of NAD(+), and the CD157 gene is synthenic on 4p15 with CD38, with which it also shares a unique genomic organization. Their conservation in phylogeny is striking evidence for their relevance in the life and death cycle of the cell.     

Primary 13C and beta-secondary 2H KIEs for trans-sialidase. A snapshot of nucleophilic participation during catalysis

Trypanosoma cruzi trans-sialidase catalyzes a novel reaction that involves the transfer of sialic acid between host and parasite glycoconjugates. In this paper, we report kinetic isotope effect studies on recombinant trans-sialidase. beta-Dideuterium and primary 13C isotope effects were measured for a good substrate, sialyl-lactose, and a slow substrate, sialyl-galactose, in both acid-catalyzed solvolysis and enzymatic transfer reactions. The beta-dideuterium isotope effect for sialyl-lactose in the acid hydrolysis reaction was 1.113 +/- 0.012. The primary 13C isotope effects for hydrolysis of sialyl-lactose and sialyl-galactose were 1. 016 +/- 0.011 and 1.015 +/- 0.008, respectively. In the enzymatic transfer reactions, the beta-dideuterium and primary 13C effects for sialyl-galactose were 1.060 +/- 0.008 and 1.032 +/- 0.008, respectively. The isotope effects for hydrolysis describe a dissociative SN1-like mechanism, and these data are contrasted by the data for the enzyme-catalyzed reaction. The enzymatic deuterium isotope effects are lower by a factor of 2, but the primary carbon isotope effects are higher by a factor of 2. This pattern describes a mechanism involving nucleophilic participation in the rate-determining transition state.     

The Activity of GAT107, an Allosteric Activator and Positive Modulator of α7 Nicotinic Acetylcholine Receptors (nAChR), Is Regulated by Aromatic Amino Acids That Span the Subunit Interface

GAT107, the (+)-enantiomer of racemic 4-(4-bromophenyl)-3a,4,5,9b-tetrahydro-3H-cyclopenta[c]quinoline-8-sulfonamide, is a strong positive allosteric modulator (PAM) of α7 nicotinic acetylcholine receptor (nAChR) activation by orthosteric agonists with intrinsic allosteric agonist activities. The direct activation produced by GAT107 in electrophysiological studies is observed only as long as GAT107 is freely diffusible in solution, although the potentiating activity primed by GAT107 can persist for over 30 min after drug washout. Direct activation is sensitive to α7 nAChR antagonist methyllycaconitine, although the primed potentiation is not. The data are consistent with GAT107 activity arising from two different sites. We show that the coupling between PAMs and the binding of orthosteric ligands requires tryptophan 55 (Trp-55), which is located at the subunit interface on the complementary surface of the orthosteric binding site. Mutations of Trp-55 increase the direct activation produced by GAT107 and reduce or prevent the synergy between allosteric and orthosteric binding sites, so that these mutants can also be directly activated by other PAMs such as PNU-120596 and TQS, which do not activate wild-type α7 in the absence of orthosteric agonists. We identify Tyr-93 as an essential element for orthosteric activation, because Y93C mutants are insensitive to orthosteric agonists but respond to GAT107. Our data show that both orthosteric and allosteric activation of α7 nAChR require cooperative activity at the interface between the subunits in the extracellular domain. These cooperative effects rely on key aromatic residues, and although mutations of Trp-55 reduce the restraints placed on the requirement for orthosteric agonists, Tyr-93 can conduct both orthosteric activation and desensitization among the subunits.     

Paleontology and Evolution in the News

This is a review of recent media publications and journal articles about evolution and paleontology.