Cloning of the amphibolic Calvin cycle/OPPP enzyme d-ribulose-5-phosphate 3-epimerase (EC 5.1.3.1) from spinach chloroplasts: functional and evolutionary aspects

Exploiting the differential expression of genes for Calvin cycle enzymes in bundle-sheath and mesophyll cells of the C4 plant Sorghum bicolor L., we isolated via subtractive hybridization a molecular probe for the Calvin cycle enzyme d-ribulose-5-phosphate 3-epimerase (R5P3E) (EC 5.1.3.1), with the help of which several full-size cDNAs were isolated from spinach. Functional identity of the encoded mature subunit was shown by R5P3E activity found in affinity-purified glutatione S-transferase fusions expressed in Escherichia coli and by three-fold increase of R5P3E activity upon induction of E. coli overexpressing the spinach subunit under the control of the bacteriophage T₇ promoter, demonstrating that we have cloned the first functional ribulose-5-phosphate 3-epimerase from any eukaryotic source. The chloroplast enzyme from spinach shares about 50% amino acid identity with its homologues from the Calvin cycle operons of the autotrophic purple bacteria Alcaligenes eutrophus and Rhodospirillum rubrum. A R5P3E-related eubacterial gene family was identified which arose through ancient duplications in prokaryotic chromosomes, three R5P3E-related genes of yet unknown function have persisted to the present within the E. coli genome. A gene phylogeny reveals that spinach R5P3E is more similar to eubacterial homologues than to the yeast sequence, suggesting a eubacterial origin for this plant nuclear gene.Abbreviations R5P3E d-ribulose-5-phosphate 3-epimerase - RPI ribose-5-phosphate isomerase - TKL transketolase - PRK phosphoribulokinase - GAPDH glyceraldehyde-3-phosphate dehydrogenase - FBP fructose-1,6-bisphophatase - FBP fructose 1,6-bisphosphate - G6PDH glucose-6-phosphate dehydrogenase - 6PGDH 6-phosphogluconate dehydrogenase - OPPP oxidative pentose phosphate pathway - Rubisco ribulose-1,5-bisphosphate carboxylase/oxygenase - FBA fructose-1,6-bisphophate aldolase - IPTG isopropyl -d-thiogalactoside - GST glutathione S-tranferase - PBS phosphate-buffered saline - TPI triosephosphate isomerase     

Selective dimerization of cysteines in glycopeptides and phosphopeptides

Summary To study the influence of phosphorylation and oxidation on the repeat domains of human Tau protein, we faced the challenge to selectively dimerize two cysteine-containing peptides in the presence of a nearby phosphate group. To this end, we were able to demonstrate the utility of a selective dimerization approach by forming disulfide bonds in unprotected phosphopeptides and extended the methodology to unprotected glycopeptides. Activation of one cysteine of a peptide chain with 2,2-dithiodipyridine and coupling this thiopyridyl-peptide to another peptide chain, containing an unprotected cysteine residue, yielded the mixed dimers in high purities and reasonable yields. Phosphate or sugar side chains on either peptide component remained unaffected during the activation and dimerization processes. While for mixed dimers the activated peptides were isolated by chromatography, homodimers were obtained by a simple one-pot reaction after 1 h. We demonstrate that cysteines can be dimerized in unprotected phosphopeptides and glycopeptides, without any side reactions affecting these posttranslational modifications.Abbreviations DCM dichloromethane - DMF N,N-dimethylformamide - DTP 2,2-dithiodipyridine - Fmoc 9-fluorenylmethyloxycarbonyl - HPLC high-performance liquid chromatography - MALDI matrix-assisted laser desorption/ionization - MS mass spectrometry - NFT neurofibrillary tangles - PHF paired helical filaments - PKC protein kinase C - RP reversed phase - human Tau protein - TFA trifluoroacetic acid Parts of this paper were presented at the 24th European Peptide Symposium in Edinburgh, Scotland, U.K., September 8–13, 1996.     

The function of mitochondria-rich cells (chloride cells) in teleost gills

Summary The CCs are the site of Cl^– transport in teleosts in sea water. The gills of freshwater teleosts contain at least two types of mitochondria-rich cell, the type and the type (Pisam and Rambourg, 1991). During seawater acclimation, the cells vanish and the cells are transformed and proliferate, and accessory cells appear in addition. This gives rise to the question of the function of cells in fish living in fresh water.According to the studies reviewed here, although they deal only with extrabranchial epithelia, the majority of evidence indicates that CCs (or MRCs) function as sites of active Ca²⁺ transport in freshwater teleosts. Moreover, some experimental results suggest that CCs are the Cl^– uptake site in freshwater teleosts. The main problem in characterizing the CC function is that they have not yet been adequately described from the biochemical standpoint. This applies particularly to their metabolic pattern and the composition of their apical and basolateral membranes, including their integrated proteins and cell-cell junctions.Experiments with organ tissue cultures such as gill organ cultures from Oncorhynchus mykiss (McCormick and Bern, 1989) and opercular membrane cultures from Oreochromis mossambicus (McCormick, 1990) will almost certainly yield important results. Primary cell cultures of CCs would be even better for characterizing CCs. Such a cell culture of rainbow trout respiratory cells has already been established (Pärt et al., 1993).     

Inhibition of yeast tRNATrp aminoacylation by 4-methyltryptophan

The effect of the tryptophan analogue 4-methyltryptophan in Saccharomyces cerevisiae has been investigated. 4-Methyltryptophan inhibits the aminoacylation of tryptophan specific transfer ribonucleic acid (tRNA^Trp). The mode of inhibition is competitive and the analogue is not charged onto tRNA^Trp. Thus 4-methyltryptophan application depletes the cells from charged tRNA^Trp. As a consequence cell growth and protein synthesis are strongly reduced. 4-Methyltryptophan is degraded efficiently in culture media inoculated with the wild type strain; the effects of 4-methyltryptophan were therefore found to be transient.     

Rate-controlling steps of oxidative phosphorylation in rat liver mitochondria. A synoptic approach of model and experiment

The contribution of different steps to the control of oxidative phosphorylation in isolated rat liver mitochondria was investigated by a combination of experiments and computer simulations. The parameters of the mathematical model of phosphorylating mitochondria were derived from experimental data. The model correctly describes the competition between ATP utilization inside and outside mitochondria for the ATP generated in mitochondria. On the basis of the good agreement between experiments and simulations, the contribution of different steps to the control of respiration was estimated by computing their control strengths, i.e., the influence of their activities on the rate of respiration. The rate-controlling influences vary depending on the load of oxidative phosphorylation. The predominant steps are: in the fully active state (State 3) — the hydrogen supply to the respiratory chain; in the resting state (State 4) — the proton leak of the mitochondrial inner membrane; in states of non-maximum ATP export — the adenine nucleotide translocator. Titrations of respiration with phenylsuccinate, antimycin, oligomycin and carboxyatractyloside completely support these conclusions.     

A topological model for the haemolysin translocator protein HlyD

Summary A topological model for HlyD is proposed that is based on results obtained with gene fusions of lacZ and phoA to hlyD. Active H1yD-LacZ fusion proteins were only generated when lacZ was fused to hlyD. within the first 180 by (60 amino acids). H1yD-PhoA proteins exhibiting alkaline phosphatase (AP) activity were obtained when phoA was inserted into hlyD. between nucleotides 262 (behind amino acid position 87) and 1405 (behind amino acid position 468, only 10 amino acids away from the C-terminus of HlyD Active insertions of phoA into the middle region of hlyD. were not observed on in vivo transposition but such fusions exhibiting AP activity could be constructed by in vitro techniques. A fusion protein that carried the PhoA part close to the C-terminal end of HlyD proved to be the most stable HlyD-PhoA fusion protein. In contrast to the other, rather unstable, HlyD-PhoA⁺ fusions, no proteolytic degradation product of this HlyD-PhoA protein was observed and nearly all the alkaline phosphatase activity was membrane bound. Protease accessibility and cell fractionation experiments indicated that the alkaline phosphatase moiety of this fusion protein was located in the periplasm as for all other HlyD-PhoA⁺ proteins. These data and computer-assisted predictions suggest a topological model for HlyD with the N-terminal 60 amino acids located in the cytoplasm, a single transmembrane segment from amino acids 60 to 80 and a large periplasmic region extending from amino acid 80 to the C-terminus. Neither the HlyD fusion proteins obtained nor a mutant HlyD protein that had lost the last 10 amino acids from the C-terminus of HlyD exhibited translocator activity for HlyA or other reporter proteins carrying the HlyA signal sequence. The C-terminal 10 amino acids of HlyD showed significant similarity with the corresponding sequences of other HlyD-related proteins involved in protein secretion.     

Asynchronous mitotic domains during blastoderm formation inMusca domestica L. (Diptera)

Summary We describe the mitotic cleavage patterns during blastoderm stage of the house flyMusca domestica L. Nuclear divisions up to mitotic stage 11 are apparently synchronous. Beginning with stage 12, nuclear divisions in the posterior third of the embryo lag behind, resulting first in a parasynchronous and finally in an asynchronous cleavage pattern. Thus a stage exists where all nuclei in the anterior region have completed 14 nuclear division cycles, while those in the posterior region have completed only 13 cycles. The border region between these nuclei is well defined and lies at 35% EL (egg length), the expression border of a gap gene. This border region is about 4–5 nuclei wide and shows a specialized mitotic behaviour.