Studies on the mechanism of photosystem II photoinhibition I. A two-step degradation of D1-protein

The role of D1-protein in photoinhibition was examined. Photoinhibition of spinach thylakoids at 20°C caused considerable degradation of D1-protein and a parallel loss of variable fluorescence, Q_B-independent electron flow and Q_B-dependent electron flow. The breakdown of D1-protein as well as the loss of variable fluorescence and Q_B-independent electron flow were largely prevented when thylakoids were photoinhibited at 0°C. The Q_B-dependent electron flow markedly decreased under the same conditions. This inactivation may represent the primary event in photoinhibition and could be the result of some modification at the Q_B-site of D1-protein. Evidence for this comes from fluorescence relaxation kinetics following photoinhibition at 0°C which indicate a partial inactivation of Q_A ^--reoxidation. These results support the idea of D1-protein breakdown during photoinhibition as a two step process consisting of an initial inactivation at the Q_B-site of the protein followed by its degradation. The latter is accompanied by the loss of PS II-reaction centre function.Abbreviations Asc ascorbate - p-BQ 1, 4-benzoquinone - DAD diaminodurene - DPC diphenylcarbazide - DQH₂ duroquinole - Fecy ferricyanide - MV methylviologen - Q_A primary quinone acceptor of PS II - Q_B secondary quinone acceptor of PS II - SiMo silicomolybdate     

Effect of the addition of microbial surfactants on hydrocarbon degradation in a soil population in a stirred reactor

Summary The hydrocarbon degradation rate could be doubled by the addition of sophorose lipids as biosurfactants in a model system containing 10% soil and a 1.35% hydrocarbon mixture of tetradecane, pentadecane, hexadecene, 1,2,4-trimethylcyclohexane, pristane (2,6,10,14-tetramethylpentadecane) phenyldecane and naphthalene suspended in mineral salts medium. The adaptation phases for two degradation phases were shortened, and the extent of degradation and final biomass were increased. The added biosurfactants were degraded after they had facilitated degradation of all hydrocarbon components.     

Studies on the total synthesis of an A7,B7-dicarbainsulin. III. Assembly of segments and generation of biological activity

As a further contribution to the synthesis of an insulin analogue with a stable A7-B7 interchain bond, the synthesis of A(8-21) by solution methods, and of B(9-25) as well as [7-(2,7-diaminosuberic acid)]B(1-8) by solid phase methods is described. In the latter compound, the amino group of the diaminosuberic acid residue was acylated with A(1-6), and the resulting "U-peptide" sequentially elongated with the C-terminal A- and finally B-chain sequences. The conversion of the product into the disulfide moiety gave a mixture which could not be resolved by currently available methods. However, the low biological activity of the crude product indicates that the A7-B7 disulfide bond is not crucially important for the activity of insulin.     

Carnivora: the primary structure of the beach marten (Martes foina, Mustelidae) hemoglobin

The primary structures of alpha- and beta-chains from the hemoglobin of the Beach Marten (Martes foina, Carnivora) are presented. The globin chains were separated on CM-cellulose in 8M urea buffer. The amino-acid sequences were established by automatic liquid- and gas-phase Edman degradation of the intact chains and the tryptic peptides from oxidized chains. Comparison of the sequences with human hemoglobin shows 21 exchanges in the alpha- and 12 in the beta-chains. The differences concerning heme and interchain contact sites as well as the substitution alpha 77 (EF6)Pro----Ala are discussed. The latter is observed for the first time in a mammalian hemoglobin. The sequences are compared with those of other Carnivora. The beta-chains of Martes foina and Pteronura brasiliensis (Giant Otter) are found to be identical, but their alpha-chains differ in 7 positions. The surprising small numbers of exchanges between the hemoglobin from Beach marten and that from Lesser and Greater Panda are discussed.     

Microbial metabolism of quinoline and related compounds. IV. Degradation of isoquinoline by Alcaligenes faecalis Pa and Pseudomonas diminuta 7

From sewage and soil isoquinoline-degrading organisms were enriched. Two strains could be isolated which were able to utilize isoquinoline as sole carbon source. The bacteria were tentatively identified as Alcaligenes faecalis and Pseudomonas diminuta with respect to their morphological and physiological characters. When growing on isoquinoline both strains excrete a metabolite into the medium which was identified as 1-oxo-1,2-dihydroisoquinoline. Alcaligenes faecalis was cultivated in continuous culture on 1-oxo-1,2-dihydroisoquinoline to improve growth on isoquinoline and degradative activity.     

High gradient magnetic cell separation with MACS.

A flexible, fast and simple magnetic cell sorting system for separation of large numbers of cells according to specific cell surface markers was developed and tested. Cells stained sequentially with biotinylated antibodies, fluorochrome-conjugated avidin, and superparamagnetic biotinylated-microparticles (about 100 nm diameter) are separated on high gradient magnetic (HGM) columns. Unlabelled cells pass through the column, while labelled cells are retained. The retained cells can be easily eluted. More than 10(9) cells can be processed in about 15 min. Enrichment rates of more than 100-fold and depletion rates of several 1,000-fold can be achieved. The simultaneous tagging of cells with fluorochromes and very small, invisible magnetic beads makes this system an ideal complement to flow cytometry. Light scatter and fluorescent parameters of the cells are not changed by the bound particles. Magnetically separated cells can be analysed by fluorescence microscopy or flow cytometry or sorted by fluorescence-activated cell sorting without further treatment. Magnetic tagging and separation does not affect cell viability and proliferation.     

Coupled reductive and oxidative degradation of 4-chloro-2-nitrophenol by a co-immobilized mixed culture system

Summary The restriction of oxygen transfer in Ca-alginate beads used for the immobilization of microbial cells was applied to a coupled reductive and oxidative microbial degradation of the xenobiotic 4-chloro-2-nitrophenol (CNP). The conversion of CNP by Enterobacter cloacae under anaerobic conditions led to the formation of 4-chloro-2-aminophenol (CAP, 81%) and 4-chloro-2-acetaminophenol (CAAP, 16%) after 50 h incubation. CAP, the main reduction product, was further degraded under aerobic conditions by Alcaligenes sp. TK-2, a hybrid strain isolated by conjugative in-vivo gene transfer. Whereas both degradation steps excluded one another in homogeneous systems with free cells, a coupled reductive and oxidative degradation of CNP was observed in one aerated reactor system after co-immobilization of both strains in Ca alginate. The diameter of the alginate beads used for immobilization was recognized as one main factor determining the properties of this mixed culture system. Offprint requests to: H.-J. Rehm     

The photocycle and the structure of iron containing bacteriorhodopsin —a kinetic and Mössbauer spectroscopy investigation

Bacteriorhodopsin (bR), converted by deionization to the blue form was reconstituted to the active purple membrane by the addition of Fe²⁺ or Fe³⁺ ions. ⁵⁷Fe Mossbauer spectra of these samples were measured at different pH values (pH 3.9, pH 5.0 and pH 7.0) and at temperatures ranging from 4 K to 300 K. The hyperfine parameters reveal two iron environments with oxygen atoms in the neighbourhood of iron. Iron type 1 is in the 3⁺ high spin state. It is bound to acid side chains of the protein and/or the phosphate groups of the lipids. Iron type 2 is in the 2⁺ high spin state and is linked to carboxy groups of the protein in a rather unspecific way. Dynamics as measured by Mossbauer spectroscopy show that the purple membrane becomes flexible only above 220 K. At the interface between membrane and bulk water the mobility is comparable to that of proteins with hydrophilic surfaces. The photocycle of Fe ³⁺-bR is slowed down compared to native bR. 3–5 Fe³⁺/bR are sufficient to inhibit the photocycle turnover by one order of magnitude. This specific effect is also found with Cr³⁺, though it is less pronounced. Mössbauer spectra of Fe³⁺-bR at 4 K reveal that iron nuclei are spin-coupled, indicating their close spatial proximity. It is proposed that iron trinuclear clusters interact with the proton uptake site of bR. Offprint requests to: M. Engelhard