Specific labelling of the active site of the phosphate translocator in spinach chloroplasts by 2,4,6-trinitrobenzene sulfonate

Phosphate transport across the chloroplast envelope is rapidly inactivated by the amino-group reagent 2,4,6-trinitrobenzene sulfonate. Subsequent exposure to [³H]NaBH₄ leads to an incorporation of the trinitrophenyl moiety into envelope membrane preparations. From the membrane proteins only a polypeptide with 29000 dalton molecular weight is labelled. The inactivation of phosphate transport and the incorporation of radioactivity are both specifically reduced by the presence of substrates.The results lead to the conclusion that a polypeptide with a molecular weight of 29000 dalton and containing a lysyl residue at the substrate binding site is involved in the phosphate translocator function.     

Free Amino Acid Composition of Phloem Sap and Growing Fruit of Lycopersicon esculentum

Pure phloem sap of tomato leaves was collected by stylectomy.Glutamine and glutamate were the predominant free amino acidstranslocated by the phloem stream. In developing fruits glutaminecontent increased significantly, reaching 35% of the total freeamino acids. Comparison in the amino acid composition betweenthe two tissues are discussed. (Received October 6, 1997; Accepted January 27, 1998)     

Asymmetric amyloid fibril elongation: a new perspective on a symmetric world

Amyloids are insoluble, fibrous proteins formed through the aggregation of misfolded proteins. They accumulate in the tissue of individuals with degenerative diseases, such as Parkinson's and Alzheimer's. The purpose of this study was to determine whether fibril growth from an initial model fibril seed is unidirectional or bidirectional. The prevailing theory on amyloid formation is that a symmetric fibril elongates equally from both ends. This study provides evidence to the contrary; the process occurs predominately unidirectionally, demonstrating that amyloid fibrils may be asymmetric and propagate mostly in one direction. Alexa Fluor 568 labeled insulin fibrils were seeded into a native insulin solution and allowed to elongate at 65°C while the kinetics of fibril growth was monitored. The resulting elongated fibrils were labeled with thioflavin-T, and the fluorescent images of the fibrils show that a majority of the elongated fibrils propagated along only one end of the seed, with the remaining labeled fibrils having bidirectional elongation or no elongation. Using a crystallographic model, we offer a structural explanation for asymmetric growth of the insulin fibrils. Thus, instead of the current view that fibrils grow symmetrically from both ends of the fibril, this is the first evidence that insulin amyloid fibrils formed in solution are asymmetric and appear to grow from only one end.     

Influence of spectral heterogeneity of prodan and laurdan solutions on the transfer of electronic energy to octadecyl rhodamine B

Fluorescence quenching and resonance energy transfer have been studied by steady-state fluorescence spectroscopy. The experimental and theoretical values for the rate constants of the electronic energy transfer (kET) and critical radius (R0) were determined for prodan and laurdan as donors and octadecyl rhodamine B as acceptor. The spectroscopic data show, that prodan and laurdan in solution create an inhomogeneous spectroscopic medium in which multi-channel luminescence phenomena take place. This finding indicated that the modified form of the Stern-Volmer relation should be used for analyzing fluorescence quenching data. Results of performed studies point out, that dipole-dipole interaction is responsible for the resonance energy transfer from prodan and laurdan to octadecyl rhodamine B. The relative quenching efficiencies of both dyes depend on polarity of the medium and are higher for more polar solvent (AcN).     

Biochemical and structural insights into bacterial organelle form and biogenesis

Many heterotrophic bacteria have the ability to make polyhedral structures containing metabolic enzymes that are bounded by a unilamellar protein shell (metabolosomes or enterosomes). These bacterial organelles contain enzymes associated with a specific metabolic process (e.g. 1,2-propanediol or ethanolamine utilization). We show that the 21 gene regulon specifying the pdu organelle and propanediol utilization enzymes from Citrobacter freundii is fully functional when cloned in Escherichia coli, both producing metabolosomes and allowing propanediol utilization. Genetic manipulation of the level of specific shell proteins resulted in the formation of aberrantly shaped metabolosomes, providing evidence for their involvement as delimiting entities in the organelle. This is the first demonstration of complete recombinant metabolosome activity transferred in a single step and supports phylogenetic evidence that the pdu genes are readily horizontally transmissible. One of the predicted shell proteins (PduT) was found to have a novel Fe-S center formed between four protein subunits. The recombinant model will facilitate future experiments establishing the structure and assembly of these multiprotein assemblages and their fate when the specific metabolic function is no longer required.     

Photo-catalytic preparation of silver-coated TiO2 particles for antibacterial applications

Colloidal silver has been known to have unique antimicrobial activity that may be useful in the construction of antibacterial materials (self-cleaning materials) to aid in the fight against bacteria-related infections. In this study, silver-coated TiO₂ (Ag/TiO₂) particles prepared through the photo-reduction of Ag⁺ were investigated as an antibacterial agent against Escherichia coli and Staphylococcus aureus. The deposition of Ag onto the surface was confirmed with SEM and EDS analysis of the post-reaction particles. It was also determined that the initial concentration of Ag⁺ in solution played a significant role in the effective size of the post-irradiation particles. The antibacterial effectiveness of the Ag/TiO₂ was evaluated through the determination of the minimum inhibitory concentration (MIC) of AgTiO₂ for each species of bacteria. The MIC values for the Ag/TiO₂, on both E. coli and S. aureus, were much lower than the MIC values for Ag metal, and quite comparable to the MIC values for AgNO₃. A disc diffusion/antibiotic sensitivity test was also performed using the Ag/TiO₂ particles and the results compared with the results obtained for Ag metal, AgNO₃ and common antibacterial agents; tetracycline, chloramphenicol, erythromycin, and neomycin. The zone of inhibition diameters for the Ag/TiO₂ particles were found to be comparable with those of the other antimicrobial agents.     

Radiolabeling of the hydrophobic components of lung surfactant with 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine

Studies of the metabolism and distribution of lung surfactant are aided by use of radiolabeled surfactant or surfactant components. These studies have often made use of [3H]- or [14C]phosphatidylcholine. Analysis of the lung content of surfactant containing these beta-emitting labels usually requires tissue digestion, use of scintillation fluids, and significant correction for quenching of photon production. Because use of a gamma-emitting isotope would obviate these requirements, we have investigated the use of 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine ([125I]TID), a lipophilic photoactivatable compound, to radiolabel pulmonary surfactant. Our results indicate that, during photoactivation, products of [125I]TID are produced that result in radiolabeling of both the lipid and protein components of extracted porcine surfactant. Separation of radiolabeled surfactant from hydrophobic nonlabelling photolysis products was accomplished by gel chromatography. Exposure of surfactant (34 mumol/ml) to [125I]TID under labeling conditions resulted in incorporation of 45.3 +/- 5.1% of the radiolabel. Incorporation of radiolabel in the various phospholipids of lung surfactant was approximately equivalent. Lipophilic surfactant apoproteins were also radiolabeled. Finally, both in vitro and in vivo testing of radiolabeled surfactant (0.1 microCi/mg) revealed full retention of surface tension lowering ability.