Single-channel analysis of the anion channel-forming protein from the plant pathogenic bacterium Clavibacter michiganense ssp. nebraskense

The anion channel protein from Clavibacter michiganense ssp. nebraskense (Schürholz, Th. et al. 1991, J. Membrane Biol. 123: 1-8) was analyzed at different concentrations of KCl and KF. At 0.8 M KCl the conductance G(V_m) increases exponentially from 21 pS at 50 mV up to 53 pS at V_m = 200 mV, 20°C. The concentration dependence of G(V_m) corresponds to a Michaelis-Menten type saturation function at all membrane voltage values applied (0-200 mV). The anion concentration K_0.5, where G(V_m) has its half-maximum value, increases from 0.12 M at 50 mV to 0.24 M at 175 mV for channels in a soybean phospholipid bilayer. The voltage dependence of the single channel conductance, which is different for charged and neutral lipid bilayers, can be described either by a two-state flicker (2SF) model and the Nernst-Planck continuum theory, or by a two barrier, one-site (2B1S) model with asymmetric barriers. The increase in the number of open channels after a voltage jump from 50 mV to 150 mV has a time constant of 0.8 s. The changes of the single-channel conductance are much faster (<1 ms). The electric part of the gating process is characterized by the (reversible) molar electrical work ΔG^θ_el = ρZ_gFV_m ≈ -1.3 RT, which corresponds to the movement of one charge of the gating charge number |Z_g| = 1 across the fraction ρ = ΔV_m/V_m = 0.15 of the membrane voltage V_m = 200 mV. Unlike with chloride, the single channel conductance of fluoride has a maximum at about 150 mV in the presence of the buffer PIPES (≥5 mM, pH 6.8) with K_0.5 ≈ 1 M. It is shown that the decrease in conductance is due to a blocking of the channel by the PIPES anion. In summary, the results indicate that the anion transport by the Clavibacter anion channel (CAC) does not require a voltage dependent conformation change of the CAC.     

Characterization of the non-specific lipid transfer protein EP2 from carrot (Daucus carota L.)

The extracellular protein EP2 was previously identified as non-specific lipid transfer protein based on its cDNA-derived amino acid sequence. Here, the purification of the EP2 protein from the medium of somatic embryo cultures is described. After two cycles of ion-exchange and gel permeation chromatography, a single silver-stained protein band with an apparent molecular mass of 10 kDa was observed on SDS-PAGE. This protein band was recognized by the antiserum raised against a EP2--galactosidase fusion-protein. Employing a fluorescent phospholipid analog, it was shown that the purified EP2 protein is capable of binding phospholipids and is able to enhance their transfer between artificial membranes. Employing a gel permeation assay, it could be demonstrated that the EP2 protein is also capable of binding palmitic and oleic acid as well as oleyl-CoA. Because in plants these fatty acids are used as precursor molecules for cutin, these results are in support of the proposed role of the EP2 protein to transport cutin monomers from their site of synthesis through the cell wall of epidermal cells to sites of cutin polymerization.     

Anaerobic degradation of dimethylsulfoniopropionate to 3-S-methylmercaptopropionate by a marine Desulfobacterium strain

Dimethylsulfoniopropionate, an osmolyte of marine algae, is thought to be the major precursor of dimethyl sulfide, which plays a dominant role in biogenic sulfur emission. The marine sulfate-reducing bacterium Desulfobacterium strain PM4 was found to degrade dimethylsulfoniopropionate to 3-S-methylmercaptopropionate. The oxidation of one of the methyl groups of dimethylsulfoniopropionate was coupled to the reduction of sulfate; this process is similar to the degradation betaine to dimethylglycine which was described earlier for the same strain. Desulfobacterium PM4 is the first example of an anaerobic marine bacterium that is able to demethylate dimethylsulfoniopropionate.Abbreviations DMSP dimethylsulfoniopropionate - DMS dimethyl sulfide - MMPA 3-S-methylmercaptopropionate     

Polarity of meiotic gene conversion is 5′ to 3′ within the niaD gene of Aspergillus nidulans

We have examined polarity of meiotic gene conversion in the niiA-niaD gene cluster of Aspergillus nidulans in two-point crosses. The type and position of the mutations represented by the niaD alleles and the correlation between the relative frequency of gene conversion and the physical position of these mutations were determined. We show that polarity of meiotic gene conversion is 5 to 3 (transcribed strand) within the niaD gene. Additional crosses involving a niiA allele and a niaD allele show little polarity of gene conversion, which suggests that the recombination events leading to restoration of the niaD gene are initiated upstream of the coding region of the niaD gene but within the niiA-niaD gene cluster, possibly within the intergenic promoter region.     

Purification and properties of a phenol oxidase derived from suspension cultures of Mucuna pruriens

From cells of Mucuna pruriens, grown in suspension, a monophenol monooxygenase (EC 1.14.18.1) was purified to homogeneity, as deduced from sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme appeared to have a native molecular weight of 90000±5000 dalton, and consisted of two subunits, each of 42000±1000 dalton. High-performance liquid chromatography with electrochemical detection for specific measurement of catecholes, was used to determine separately the tyrosinehydroxylating and catecholase activities of the enzyme. For the enzymatic activities, pH optima of, respectively, 7.5 and 5.5–6.5 were found; the effects of some inhibitors on both activities appeared to be different. Michaelis-Menten characteristics for some mono-and o-dihydroxysubstrates were determined.Abbreviations DEAE diethylaminoethyl - HPLC high-performance liquid chromatography - L-DOPA L-3,4-dihydroxyphenylalanine     

Cloning of the natural gene for the sweet-tasting plant protein thaumatin

Five different clones, homologous to the structural gene for the sweet-tasting plant protein thaumatin, have been isolated from leaf DNA of Thaumatococcus daniellii Benth. Restriction maps, hybridization studies, S1-nuclease mapping and R-loop formation revealed that the thaumatin genes isolated belong to one multigene family, and have two very small introns situated at different positions in the various structural genes. A similar situation prevails in a number of seed storage genes. This suggests a similarity between the sweet-tasting protein thaumatin and seed storage proteins.     

Teuthiden aus dem Barrême der Insel Maio (Kapverdische Inseln)

The teuthids of the collection R. Stahlecker, 1929, from the Isle of Maio (Cape Verde Islands) are described.Neololigosepia stahleckeri n. gen. n. sp. andMaioteuthis morroensis n. gen. n. sp. are one of the first teuthids from the Lower Cretaceous (Barremian).