Membrane excitability expressed in human neuroblastoma cell hybrids

The voltage-sensitive Na+ channel is responsible for the action potential of membrane electrical excitability in neuronal tissue. Three methods were used to demonstrate the presence of neurotoxin-responsive Na+ channels in two hybrid cell lines resulting from the fusion of excitable human neuroblastoma cells with mouse fibroblasts. Only one of the two electrically active hybrid cell lines maintained the sensitivity of the neuroblastoma parent to tetrodotoxin (TTX). The other hybrid, although electrically active, was not responsive to TTX or scorpion venom. Comparisons of the patterns of expression of membrane excitability and of chromosome complements in these human neuroblastoma cell hybrids suggest that the phenotype of membrane excitability is composed of genetically distinct elements.     

Oxidation-reduction thermodynamics of the acceptor quinone complex in whole-membrane fragments from Chloroflexus aurantiacus

Oxidation-reduction thermodynamic equilibria involving the quinone-acceptor complex have been examined in whole-membrane fragments from Chloroflexus aurantiacus. The primary quinone acceptor was titrated by monitoring the amount of cytochrome c554 photooxidized by a flash of light as a function of the redox potential. In contrast to previous data obtained in purified plasma membranes, in which the primary quinone acceptor exhibited a midpoint potential equal to -50 mV at pH 8.2, in whole-membrane fragments it titrated at -210 mV (pH 8.0), with a pH dependence of -60 mV/pH up to a pK value of 9.3. o-Phenanthroline, an inhibitor of electron transfer from the primary to the secondary quinone acceptor, shifted the Em/pH curve of the primary acceptor to higher redox potentials. The midpoint potential of the secondary quinone acceptor and its dependence on pH has been determined by comparing the kinetics of the charge recombination processes within the reaction center complex in the presence and in the absence of o-phenanthroline. It is concluded that both the primary and the secondary quinone acceptors interact with a proton, with pK values of 9.3 and of approximately 10.2 respectively. At physiological pH the electron appears to be stabilized on the secondary with respect to the primary quinone acceptor by approximately 60 meV.     

FAST (Flexible Analysis by Software Tool) and CHAMP (CHemico-physical AMinoacidic Parameter data bank): a new tool to investigate protein structure

A flexible package designed to study protein structure is described.The package is devoted to the analysis of protein sequencesby drawing structural profiles of specific structure-relatedamino acid parameters. An Aminoacidic Parameters Data Bank (CHAMP)containing 32 different series of physico-chemical parametersof amino acids is available. Sequences can be loaded from anyASCII format data bank or from keyboard. The program possessesa routine which enables easy updating of the protein data bankand CHAMP Data Bank. FAST reads statistical correlations betweentwo plots in order to identify structural similarities. Plotscan be printed, saved or used for correlation, comparison orgraph overlap by using common spreadsheets (e.g. Lotus 123).Plots can be smoothed by a running mean or a running median.The program also has a special feature—a global flexibilityanalysis of proteins. The package runs on IBM or compatiblesand requires DOS 3.0 or later. Received on June 20, 1989; accepted on August 2, 1989     

Conservation of the alternative oxidase and enhancement of cyanide-resistant respiration in suspension-cultured pear fruit cells by inhibitors of macromolecular synthesis

The contribution of the alternative pathway to the respiration of suspension-cultured pear ( Pyrus communis cv. Passa Crasanne) cells was enhanced, often severalfold, within 2 to 4 days following the addition of cycloheximide, actinomycin D, or 2-(4-methyl-2,6-dinitroanalino)- N -methyl propionamide (D-MDMP). Concomitant inhibition of cellular protein synthesis by cycloheximide and actinomycin D was transient and incomplete. However, inhibition by D-MDMP was virtually complete (>97%) and persisted over several days. [³⁵S]-labelling and polyacrylamide gel separation indicated that cycloheximide precluded the appearance of discernable new proteins in mitochondria. Probes with monoclonal antibodies revealed a conservation of alternative oxidase protein levels in the mitochondria of inhibitor-treated cells. The data, appraised within the complexities of cell-culture dynamics, lead to the conclusion that the observed increases in capacity for cyanide-resistant respiration are the consequence, likely indirect, of inhibited protein synthesis with resultant retention and activation of constitutive alternative oxidase.     

Kinetics and stereochemistry of the microsomal epoxide hydrolase-catalyzed hydrolysis of cis-stilbene oxides

The microsomal epoxide hydrolase (mEH)-catalyzed hydrolysis of cis-4,4′–dimethylstilbene oxide ( 1a ), cis-4,4′-diethylstilbene oxide ( 1b ), cis-4,4′-diisopropylstilbene oxide ( 1c ), and cis-4,4′-dichlorostilbene oxide ( 1d ) have been investigated using rabbit liver microsomal preparations. The kinetic parameters, K_m and V_max, and the absolute stereochemistry of the reactions have been determined and compared with those of cis-stilbene oxide ( 1e ). All epoxides 1a – d are hydrolyzed by mEH with high product enantioselectivity to give (R,R)-(+)-diols with ee ≥ 90%. The presence of the substituents on the phenyl rings markedly reduces the rates of mEH catalyzed hydrolysis with respect to cis-stilbene oxide, by increasing K_m and reducing V_max in the cases of 1a , 1b , and 1d , or reducing only the V_max in the case of 1c . The very low V_max, together with a persistent ability to fit into the mEH active site, make all these epoxides, and particularly 1c , inhibitors of cis-stilbene oxide hydrolysis. The kinetic and stereochemical results are interpreted on the basis of the proposed topology of the mEH active site. © 1994 Wiley-Liss, Inc.