Regulatory Role of the GTP-Binding Protein, Go, in the Mechanism of Exocytosis in Adrenal Chromaffin Cells

To elucidate the possible involvement of GTP-binding proteins (G proteins) in the mechanism of exocytosis, we studied effects of pertussis toxin (PTX), guanosine 5'-O-(3-thiotriphosphate) (GTP-gamma-S), and antibodies against the G proteins (Gi and G(o)) on the secretory function of bovine adrenal chromaffin cells. Pretreatment of chromaffin cells with PTX resulted in an increase in acetylcholine-evoked catecholamine release. High K(+)-, histamine-, or gamma-aminobutyric acid-evoked catecholamine release was also potentiated by PTX pretreatment. The concentration of extracellular Ca2+ required for maximal release by 10(-4) M acetylcholine was decreased significantly in PTX-treated cells. In digitonin-permeabilized cells, PTX pretreatment resulted in a decrease of the half-maximal concentration (Km) of Ca2+ required for exocytosis with no significant change in the maximal stimulation (Vmax). Exposure of permeabilized cells to GTP-gamma-S (a nonhydrolyzable GTP analogue) inhibited Ca(2+)-dependent exocytosis by reducing the affinity for Ca2+. The effects of PTX pretreatment were mimicked by treatment of permeabilized cells with polyclonal antibodies selective for the alpha subunit of the PTX-sensitive G protein, G(o). Treatment with similar antibodies against the alpha subunit of Gi had no effect. These findings suggest that G(o) directly controls the Ca(2+)-triggered process in the machinery of exocytosis by lowering the affinity of the unknown target for Ca2+.     

Glutamate Inhibits Adenylate Cyclase Activity in Dispersed Rat Hippocampal Cells Directly via an N-Methyl-d-Aspartate-Like Metabotropic Receptor

Three major subtypes of glutamate receptors that are coupled to cation channels--N-methyl-D-aspartate (NMDA), kainate, and alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptors--are known as ionotropic receptors in the mammalian CNS. Recently, an additional subtype that is coupled to GTP binding proteins and stimulates (or inhibits) metabolism of phosphoinositides has been proposed as a metabotropic receptor. Incubation of dispersed hippocampal cells from adult rats with glutamate or NMDA decreased forskolin-stimulated cyclic AMP (cAMP) accumulation; half-maximal effects were obtained with 5.6 +/- 2.2 and 6.4 +/- 2.3 microM, respectively. Kainate and quisqualate were less potent. The effect of glutamate was antagonized by 2,3-diaminopropionate and 2-amino-5-phosphonovalerate, NMDA/glutamate receptor antagonists, but not by 0.5 microM Joro spider toxin, a specific blocker of the AMPA receptor. The inhibitory effect of glutamate on cAMP formation was not blocked by 2 microM tetrodotoxin or by the absence of Ca2+. In hippocampal membranes, glutamate, similar to carbachol, inhibited adenylate cyclase activity in a GTP-dependent manner. These findings suggest that the glutamate inhibition of adenylate cyclase is direct and is not due to a result of the release of other neurotransmitters. The effect of glutamate on cAMP accumulation was observed in an assay medium containing 0.7 mM MgCl2, which is known to inhibit both ionotropic NMDA receptor/channels in the hippocampus and metabotropic NMDA receptors in the cerebellum. The inhibitory effect of glutamate was abolished by pertussis toxin treatment.(ABSTRACT TRUNCATED AT 250 WORDS)     

Differential sensitivity of pneumolysin-induced channels to gating by divalent cations

Summary The induction of channels across planar lipid bilayers by purified, recombinant pneumolysin (a hemolytic protein from Streptococcus pneumoniae) has been studied by measuring increases in electrical conductivity. Pneumolysin-induced channels exhibit a wide range of single channel conductances (<50 pS to >1 nS at 0.1 m KCl). Channels can be categorized on the basis of their K⁺:C^– selectivity: the smallest channels are strongly cation selective, with t₊ (the cation transference number) approaching 1.0; the largest channels are unselective (t₊ 0.5). Channels tend to remain open at all voltages (–150 to 150 mV); only the smallest channels exhibit any rectification.In the presence of divalent cations (1–5 mm Zn²⁺; 10–20 mm Ca²⁺), small (<50 pS) and medium-sized (50 pS to 1 nS) channels are closed in a voltage-dependent manner (more closure at higher voltages); at 0 voltage channels reopen. Overall selectivity is reduced by divalent cations, compatible with small, selective channels being closed preferentially to large, nonselective ones.It is concluded that a single molecular species (pneumolysin) induces multiple-sized channels that can be categorized by cation: anion selectivity and by their sensitivity to closure by divalent cations.We are grateful to Dr. G. J. Boulnois and T. J. Mitchell forfruitful discussion and supplies of pneumolysin, and to G. M. Alder for technical assistance. YEK is grateful to Dr. A. A. Lev for leave of absence and to the USSR Academy of Sciences and the Global Network for Molecular and Cell Biology (UNESCO) for support of travel and accommodation, respectively. The work was supported by the Cell Surface Research Fund.     

SAXS study of the snake toxin α-crotamine

-crotamine is a small toxic protein (42 amino acid residues with three disulphide bridges) present in the venom of Crotallus durissus terrificus. Molecular parameters (R _g=13.7 Å, S=3,000 Ų, V=9,200 ų and D _max=40 Å) were derived from SAXS curves obtained from a solution of this protein at pH=4.5. An excellent agreement between the experimental distance distribution curve and that calculated from a model consisting of two lobes linked by the Cys(18)-Cys(30) disulphide bridge.     

Solution conformation of leiurotoxin I (scyllatoxin) by H nuclear magnetic resonance Resonance assignment and secondary structure

A proton NMR study at 500 MHz of leiurotoxin I in water is presented. Nearly complete sequence-specific assignments of the individual backbone and side-chain proton resonances were achieved using through-bond and through-space connectivities obtained from standard two-dimensional NMR techniques. The secondary structure of this toxin is inferred from a combination of short-range nuclear Overhauser enhancements, scalar couplings and proton/deuteron exchange rates. Three disulflde bridges locate the N-terminal part (that is -helical from residue 6 to 16) on one side of a C-terminal two stranded antiparallel β sheet (from Leu¹⁸ to Val²⁹). The latter features a tight turn at Gly²³-Asp²⁴.     

镰刀菌毒素的研究 Ⅱ.T-2毒素的提取、纯化及结构鉴定

本文报道了三线镰刀菌M-20(Fusarium tricinctum)接种于已灭菌的玉米粒培养基中,于13℃培养30天后,提取培养物所得粗毒素。经二次硅胶柱层析纯化后,经丙酮-正己烷结晶,可获得白色针状结晶。根据它的熔点(151—2℃)R_f0.56,LD_(50)2.5,mg/kg。分子量466,FD质谱测定,~1H核磁共振谱及EI质谱,确证结晶为T-2毒素。     

Mutated form (G52E) of inactive diphtheria toxin CRM197: molecular simulations clearly display effect of the mutation to NAD binding

Mutated form (G52E) of diphtheria toxin (DT) CRM197 is an inactive and nontoxic enzyme. Here, we provided a molecular insight using comparative molecular dynamics (MD) simulations to clarify the influence of a single point mutation on overall protein and active-site loop. Post-processing MD analysis (i.e. stability, principal component analysis, hydrogen-bond occupancy, etc.) is carried out on both wild and mutated targets to investigate and to better understand the mechanistic differences of structural and dynamical properties on an atomic scale especially at nicotinamide adenine dinucleotide (NAD) binding site when a single mutation (G52E) happens at the DT. In addition, a docking simulation is performed for wild and mutated forms. The docking scoring analysis and docking poses results revealed that mutant form is not able to properly accommodate the NAD molecule.