Studies on the protolytic reactions coupled with water cleavage in photosystem II membrane fragments from spinach

The protolytic reactions of PSII membrane fragments were analyzed by measurements of absorption changes of the water soluble indicator dye bromocresol purple induced by a train of 10 s flashes in dark-adapted samples. It was found that: a) in the first flash a rapid H⁺-release takes place followed by a slower H⁺-uptake. The deprotonation is insensitive to DCMU but is completely eliminated by linolenic acid treatment of the samples; b) the extent of the H⁺-uptake in the first flash depends on the redox potential of the suspension. In this time domain no H⁺-uptake is observed in the subsequent flashes; c) the extent of the H⁺-release as a function of the flash number in the sequence exhibits a characteristic oscillation pattern. Multiphasic release kinetics are observed. The oscillation pattern can be satisfactorily described by a 1, 0, 1, 2 stoichiometry for the redox transitions S_i S_i+1 (i=0, 1, 2, 3) in the water oxidizing enzyme system Y. The H⁺-uptake after the first flash is assumed to be a consequence of the very fast reduction of oxidized Q₄₀₀(Fe³⁺) formed due to dark incubation with K₃[Fe(CN)₆]. The possible participation of component Z in the deprotonation reactions at the PSII donor side is discussed.Abbreviations A protonizable group at the PSII acceptor side - BCP Bromocresol Purple - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - FWHM Full Width at Half Maximum - Q_A, Q_B primary and secondary plastoquinone at PSII acceptor side - Q₄₀₀ redox group at PSII-acceptor side (high spin Fe²⁺) - P680 Photoactive chlorophyll of PSII reaction center - S_i redox states of the catalytic site of water oxidation - Z redox component connecting the catalytic site of water oxidation with the reaction center     

Biochemical characterization and crystallization of porin from Rhodopseudomonas blastica

Oligomeric porin of the phototrophic bacterium Rhodopseudomonas blastica DSM 2131 was obtained from cell envelopes by differential temperature extraction in the presence of detergent and salt. The isolated porin exhibited strong porin activity after reconstitution into lipid bilayer membranes. The effective channel diameter for the trimer was estimated as 1.5 nm from single channel conductance measurements in the presence of 1 M KCl. Moderate cation-selectivity was observed. Oligomeric porin migrated as a single band (apparent molecular weight 81 kDa) on sodium dodecyl sulfate polyacrylamide gelelectrophoresis when solubilized below 70 °C. The oligomers were converted into monomers on heating to 70 °C or above forming two bands with apparent molecular weight of 36 kDa and 35 kDa. The oligomer was not sensitive to EDTA. Its molecular weight was determined to be 119.3 kDa by analytical ultracentrifugation. The isoelectric point was 5.7. Circular dichroism data indicated a high content of -sheet structure. Gasphase sequencing of the N-terminal residues revealed the sequence: NH₂-Glu-Ile-Ser-Leu-Asn-Gly-Tyr-Gly-Arg-Phe. Crystals with a maximal side length of 300 m and diffracting to 0.32 nm resolution were obtained with the porin oligomer in the presence of C8E4 and 1,2,3-heptanetriol by using the vapor phase equilibration technique.Abbreviations C8E4 n-octyl tetraoxyethylene - M_r apparent molecular weight - Octyl-POE n-octyl polyoxyethylene - LDAO N,N-dimethyl dodecyl aminoxide - LPS lipopolysaccharide - PAGE polyacrylamide gel-electrophoresis - PEG polyethylene glycol     

Monoclonal antibodies Ki-S3 and Ki-S5 yield new data on the 'Ki-67'proteins

Abstract. The monoclonal antibody (mab) Ki-67 has been used for about 10 years, mainly in tissue sections, to monitor proliferating cells, but so far only very little is known about the proteins it recognizes. The new mabs Ki-S3 and Ki-S5 detect proliferating cells in frozen and paraffin-embedded tissues. They recognize proteins with the same molecular mass as Ki-67 in Western blot and for the first time also in immunoprecipitation experiments. With these mabs we were able to enrich and purify the Ki-67 proteins. Protein sequencing of four peptides of the digested proteins corresponded to the cDNA-deduced amino acid sequence already published for the Ki-67 proteins.
Since we were able to immunoprecipitate the Ki-67 proteins, we performed various immunoprecipitation experiments to obtain more information about the nature of these proteins. After radiolabelling L428 cells with [³⁵S]-methionine we were able to immunoprecipitate the Ki-67 proteins after only 5 min of labelling time. In turnover experiments the Ki-67 proteins could not be detected 3 h after the end of labelling. These data indicate a halt-life of the Ki-67 proteins of about 90 min.
Labelling experiments with [³²P]-orthophosphate revealed that the Ki-67 proteins are phosphorylated. After dephosphorylation was blocked with okadaic acid or cell growth was arrested by means of Colcemid, the phosphorylation of the Ki-67 proteins was greatly increased, indicating that the Ki-67 proteins are phosphorylated via serine and threonine, and that the phosphorylation of the Ki-67 proteins increases in cycling cells. Labelling experiments with [³H]-mannose and [³H]-glucose revealed that the protein is weakly N -glycosylated.     

The three-dimensional structure of porin from Rhodobacter capsulatus at 3 A resolution

The crystal structure of porin from Rhodobacter capsulatus strain 37b4 has been solved at 3.0 A (1 A = 0.1 nm) resolution by multiple isomorphous replacement and solvent-flattening. The three pores of the trimer are well defined in the electron density map. Each pore consists of a 16-stranded beta-barrel which traverses the membrane as a tube. Near its center the tube is narrowed by chain segments protruding from the inner wall of the barrel that form an eye-let with an irregular cross-section of about 6 A by 10 A. The eye-let has an axial length of about 10 A; it defines the exclusion limit for diffusing particles.     

Studies on the electrogenic action of acetylcholine with Torpedo marmorata electric organ: V. Qualitative correlation between pharmacological effects and equilibration processes of the cholinergic receptor protein as revealed by the structural probe quinacrine

When the differential fluorescence emission at 515 nm of receptor-rich membrane fragments from Torpedo marmorata labelled with quinacrine is followed by energy transfer, addition of a high concentration of an agonist such as 0.4 mm-carbamylcholine or 0.4 mm-phenyltrimethylammonium causes a fast (unresolved) increase of fluorescence intensity followed by a slow (minute range) decrease, which leads to a final stable level. On the other hand, a stepwise addition of agonist at low concentrations gives only a slow fluorescence increase. Antagonists, such as flaxedil and d-tubocurarine, at all the concentrations tested, bring about exclusively slow fluorescence increases. Decamethonium at 0.4 mm gives no slow reaction but only a fast (unresolved) increase without transient overshoot.Addition of a local anaesthetic reduces the amplitude of the fluorescence response to carbamylcholine. The α-toxin from Naja nigricollis does not cause any change of fluorescence intensity but blocks the effect of the cholinergic agonists and antagonists.Dissolution of the membrane fragments by a non-ionic detergent abolishes the fast transient reaction triggered by the agonists but preserves the slow ones observed in the presence of agonists or antagonists. The data are interpreted in terms of a three-state model, a revised version of the model of Katz & Thesleff (1957): the fast transient reaction brought about by the agonists being assigned to the “activation” of the receptor-ionophore complex and the slow one to its “desensitization”.     

The development and characterization of an E. coli O25B bioconjugate vaccine

Extraintestinal pathogenic Escherichia coli (ExPEC) cause a wide range of clinical diseases such as bacteremia and urinary tract infections. The increase of multidrug resistant ExPEC strains is becoming a major concern for the treatment of these infections and E. coli has been identified as a critical priority pathogen by the WHO. Therefore, the development of vaccines has become increasingly important, with the surface lipopolysaccharide constituting a promising vaccine target. This study presents genetic and structural analysis of clinical urine isolates from Switzerland belonging to the serotype O25. Approximately 75% of these isolates were shown to correspond to the substructure O25B only recently described in an emerging clone of E. coli sequence type 131. To address the high occurrence of O25B in clinical isolates, an O25B glycoconjugate vaccine was prepared using an E. coli glycosylation system. The O antigen cluster was integrated into the genome of E. coli W3110, thereby generating an E. coli strain able to synthesize the O25B polysaccharide on a carrier lipid. The polysaccharide was enzymatically conjugated to specific asparagine side chains of the carrier protein exotoxin A (EPA) of Pseudomonas aeruginosa by the PglB oligosaccharyltransferase from Campylobacter jejuni. Detailed characterization of the O25B-EPA conjugate by use of physicochemical methods including NMR and GC-MS confirmed the O25B polysaccharide structure in the conjugate, opening up the possibility to develop a multivalent E. coli conjugate vaccine containing O25B-EPA.