Light-stimulated proton transport into the vacuoles of leaf mesophyll cells does not require energization by the tonoplast pyrophosphatase

Photosynthetic characteristics of transgenic tobacco (Nicotiana tabacum L.) plants with a soluble pyrophosphatase in the cytosol of their leaf cells were compared to those of wild-type plants. Although the development of the transgenic plants was somewhat retarded compared to the wild type, as shown by stunted growth and delayed flowering, photosynthetic responses were comparable in transgenic and wild-type leaves of similar physiological age. In particular, light-dependent proton transport into the vacuoles of leaf mesophyll cells was not decreased in leaves of the transgenic plants, which did not contain pyrophosphate in the cytosol owing to the presence of a soluble pyrophosphase. This shows that light-stimulated proton pumping did not require the pumping activity of the tonoplast pyrophosphatase. Apparently, light-stimulated proton pumping can be based solely on the activity of the tonoplast ATPase.Abbreviation CDCF 5-(and 6-)arboxy-2,7-dichlorofluorescein This work was supported within the Sonderforschungsbereiche 176 and 251 of the University of Würzburg.     

A site accessible to extracellular TEA+ and K+ influences intracellular Mg2+ block of cloned potassium channels

The members of the RCK family of cloned voltage-dependent K⁺ channels are quite homologous in primary structure, but they are highly diverse in functional properties. RCK4 channels differ from RCK1 and RCK2 channels in inactivation and permeation properties, the sensitivity to external TEA, and to current modulation by external K⁺ ions. Here we show several other interesting differences: While RCK1 and RCK2 are blocked in a voltage and concentration dependent manner by internal Mg²⁺ ions, RCK4 is only weakly blocked at very high potentials. The single-channel current-voltage relations of RCK4 are rather linear while RCK2 exhibits an inwardly rectifying single-channel current in symmetrical K⁺ solutions. The deactivation of the channels, measured by tail current protocols, is faster in RCK4 by a factor of two compared with RCK2. In a search for the structural motif responsible for these differences, point mutants creating homology between RCK2 and RCK4 in the pore region were tested. The single-point mutant K533Y in the background of RCK4 conferred the properties of Mg²⁺ block, tail current kinetics, and inward ion permeation of RCK2 to RCK4. This mutant was previously shown to be responsible for the alterations in external TEA sensitivity and channel regulation by external K⁺ ions. Thus, this residue is expected to be located at the external side of the pore entrance. The data are consistent with the idea that the mutation alters the channel occupancy by K⁺ and thereby indirectly affects internal Mg²⁺ block and channel closing.Abbreviations TEA tetraethylammonium - EGTA Ethylene glycol-bis (-aminoethyl ether) N,N,N,N-tetraacetic acid - 2S3B model 2-site 3-barrier model Correspondence to: S. H. Heinemann     

Reversible cross-linking of crystalline bacterial surface layer glycoproteins through their glycan chains

After periodate oxidation and incubation with dithiodipropionic acid dihydrazide cross-linking of the crystalline surface layer (S-layer) glycoproteins of Clostridium thermohydrosulfuricum L111-69 and Bacillus alvei CCM 2051 was achieved specifically through the glycan chains. The cross-linked S-layers were used for the immobilization of chemically synthesized, spacer-linked, tumour-associated T-disaccharide [Gal(13)GalNAc]. Electron microscopical evaluation of the resulting conjugates showed densely packed, multilayered S-layer structures loaded with the immobilized ligand. After reductive cleavage of the disulphide bond of dithiodipropionic acid by dithiothreitol, monomeric haptenated S-layer conjugates could be obtained. Both the cross-linked and the monomeric type of conjugate might be useful for assessment of specific immune responses, which, in general, can be elicited by those artificial antigens. Correspondence to: P. Messner     

Screening for microorganism with cell wall lytic activity to produce protoplast-forming enzymes

Several different bacteria and fungi capable of degrading yeast cell walls were isolated in the course of a screening programme. One Streptomyces and one Acremonium strain were found to degrade yeast cell walls extremely well. Both isolates produced enzymes in liquid culture that could be used for protoplasting of Sporobolomyces salmonicolor (DSM 70851) and Rhodotorula rubra (DSM 70403). This fact is quite remarkable as, so far, S. salmonicolor could not be protoplasted by commercially available enzymes. Correspondence to: W. Kaul     

Different genomic structure of mouse and human Lmp7 genes: characterization of MHC-encoded proteasome genes

The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession numbers L11045 (human LMP7) and L11145 (mouse Lmp-7).     

Characterization of serological properties of polyclonal antibodies produced against enzymes involved in the L-selective cleavage of hydantoin derivatives

Summary Polyclonal antibodies were produced against the highly purified enzymes L-hydantoinase, hydantoin-racemase and L-N-carbamoylamino acid amidohydrolase of Arthrobacter aurescens DSM 3747. In order to exploit these antibodies for basic research (molecular biology) or bioengineering (process development), the serological properties had to be characterized. Both, the hydantoinase- and carbamoylase-antibodies were observed to be monofunctional, whereas the hydantoin-racemase-antibody was found to be additionally specific against the L-hydantoinase. Monospecificity was realized after affinity chromatography. Investigations on serological crossreactions with several linear- and cyclic amidases (e.g. hydantoinases) as well as hydantoin-racemases are demonstrated in this paper.Deticated to Prof. Dr. Klaus Mosbach on the occation of his 60th birthday.     

Cloning,sequence analysis and expression of a cDNA encoding active phosphoenolpyruvate carboxylase of the C3 plant Solanum tuberosum

A cDNA coding for phosphoenolpyruvate carboxylase (PEPC) was isolated from a cDNA library from Solanum tuberosum and the sequence of the cDNA was determined. It was inserted into a bacterial expression vector and a PEPC^- Escherichia coli mutant could be complemented by the cDNA construct. A functional fusion protein could be synthesized in E. coli. The properties of this PEPC protein clearly resembled those of typical C3 plant enzymes.     

Axonal Glycoproteins with Immunoglobulin- and Fibronectin Type III-Related Domains in Vertebrates: Structural Features, Binding Activities, and Signal Transduction

Abstract: The L1- and F11-like axonal glycoproteins, implicated in neurite outgrowth and fasciculation, are members of the Ig superfamily comprising multiple fibronectin type III-like domains. Their Ig-like and fibronectin type III-related domains are likely to be composed of seven β-strands arranged in two opposing β-sheets of highly similar topology. Whereas the F11-like molecules lack a transmembrane sequence and are anchored in the plasma membrane by a glycosylphosphatidylinositol, the L1 -like molecules comprise cytoplasmic domains with highly conserved sequence motifs. Most of the latter proteins occur in different isoforms generated by alternative pre-mRNA splicing, which has not been documented for molecules of the F11 subgroup. L1 -like proteins undergo heterophils as well as homophilic interactions, whereas only the former mode of binding was observed for F11 -like proteins. Evidence is accumulating that these Ig superfamily molecules with fibronectin type III-like domains are interacting in a complex manner with each other and molecules of the extracellular matrix. Investigations assigning structure to function reveal that their individual extracellular domains serve distinct binding activities. Recent studies also suggest that L1 and NCAM are implicated in the transduction of transmembrane signals.     

A SIGNAL GLYCOPROTEIN WITH α-d-MANNOSYL RESIDUES IS INVOLVED IN THE WOUND-HEALING RESPONSE OF ANTITHAMNION SPARSUM (CERAMIALES,RHODOPHYTA)1

A variety of fluorescein isothiocyanate-labeled lectins specific for different sugar moieties were examined as probes for the wound-healing response in the filamentous red alga Antithamnion sparsum Tokida. Among them, only concanavalin A (ConA) and Lens culrinaris agglutinin (LCA), which have specificity to α-D-mannosyl residues, bound specifically to repair cells during the wound-healing process. When ConA or LCA was added at various time intervals after wounding, it first bound (3 h post-wounding) as a thin layer at the tips of the adjacent cells. Later (4–5 h post-wounding) labeling also appeared at the tips of the repair cells. Intense labeling at these sites continued throughout the healing process until repair cell fusion, at which time the lectin labeling was reduced to a narrow ring around the area of fusion. When added to plants prior to wounding and continually monitored, these same lectins acted as inhibitors to the wound-healing response. Other control lectins showed no inhibitory effects. A crude extract solution obtained from decapitated filaments stimulated the wound-healing response, and a lectin-binding component bound strongly to a protein-binding transfer membrane. These results suggest that the labeled compound is a glycoprotein that has α-D-mannosyl residues and is similar to the repair hormone rhodomorphin found in Griffithsia pacifica Kylin.