Hartmut Follmann

IDENTIFICATION OF TWO THYLAKOID-ASSOCIATED PHOSPHATASES WITH PROTEIN PHOSPHATASE ACTIVITY IN CHLOROPLASTS OF THE SOYBEAN (GLYCINE MAX)

Abstract Two thylakoid-associated phosphatases from soybean (Glycine max) have been purified and characterized. Both enzymes were prepared by ultrasonication of thylakoids, (NH4)2SO4 fractionation, gel filtration, cation, and anion exchange chromatography. Phosphatase I and II copurified during the first steps of purification and were separated by ion exchange chromatography on CM cellulose. The phosphatases have a molecular mass of 70 kDa. They exhibit a pH optimum at 5.5 and 6, respectively. Mg2+ ions are not required for activity. Several phosphorylated substrates are hydrolyzed, including phenyl and 4-nitrophenyl phosphate, 3-phosphoglycerate, and fructose 1,6-bisphosphate. The thylakoid-associated phosphatases also possess phosphoprotein phosphatase activity towards 32P-labelled casein and chloroplast LHC II as substrates. Fluoride inhibited the hydrolysis of nitrophenylphosphate and [32P]casein but not the protein phosphatase activity towards 32P-LHC II. The protein serine/threonine phosphatase inhibitor okadaic acid did not affect these phosphatases at all.

Age-dependent DNA labeling and deoxyribonucleotide synthesis in wheat seeds.

Utilisation of ribonucleosides as precursors of DNA biosynthesis was studied in germinating wheat embryos because the reductive pathway leading to deoxyribonucleotides is very difficult to demonstrate in extracts of higher plants in vitro. [5-3H]Cytidine and [6-3H]uridine are incorporated into wheat DNA (RNA-free) via ribonucleotide reduction without intermediate scission of the glycosidic bond. This reaction is observed at 20-30 h after the onset of germination only in aged (2-4-year-old) seeds while the embryos isolated from fresh grains show very little cytidine incorporation; in contrast, thymidine incorporation into DNA between 10 and 18 h of germination is not age dependent. Fresh wheat contains a soluble, heat-stable inhibitor fraction, most probably a modified oligonucleotide, which efficiently prevents cytidine incorporation when added to old embryos together with the labeled nucleoside. This material also inhibits purified Escherichia coli ribonucleotide reductase and is thought to be part of the control system for ribonucleotide reduction in wheat; it may gradually decay during storage of the seeds. Dry wheat embryos do not contain deoxyribonucleoside triphosphates. Pool sizes of dATP and dTTP in germinating embryos were found to reach 1 pmol/microgram DNA at 10-15 h of germination (i.e. before ribonucleotide reduction) and were independent of the age of seeds. These data suggest that wheat contains other preformed dexoyribonucleoside derivatives which are phosphorylated at an early time and can initially sustain DNA synthesis. Induction of measurable ribonucleotide reductase activity in fresh winter wheat was for the first time accomplished by 15 days of vernalization of the seeds at +2 degrees C.

Stimulation of protochlorophyllide oxidoreductase by thioredoxin

Summary Protochlorophyllide oxidoreductase [E.C.1.3.1.33] catalyses the reduction of protochlorophyllide to chlorophyllide. Pigment mutant C-2A’ of the alga Scenedesmus obliquus possesses this enzyme when grown in darkness or light. The mutant accumulates protochlorophyllide and a trace of chlorophyll in darkness. In the light it synthesizes chlorophyll in amounts comparable with that of the wild type. Reduced plastidal thioredoxin f of Scenedesmus obliquus, which is formed in this mutant during greening prior to chlorophyll synthesis, stimulates protochlorophyllide oxidoreductase activity in vitro in light and darkness. The cytoplasmic thioredoxins and E. coli thioredoxin are less active. This system is considered to be a new case of thiorexodin-mediated control of chloroplastic enzymes, and a new factor in the regulation of chlorophyll biosynthesis.

Novel thioredoxin targets in Dictyostelium discoideum identified by two-hybrid analysis: interactions of thioredoxin with elongation factor 1α and yeast alcohol dehydrogenase

Abstract Thioredoxins (Trx) are ubiquitous dicysteine proteins capable of modulating enzymes and other cellular targets through specific disulfide-dithiol redox changes. They are unique in that a large number of very diverse metabolic systems are addressed and redox-regulated in bacteria, animal, and plant cells, but the finite number of thioredoxin interaction partners is still unknown. Two-hybrid methodology should provide a rational way to establish thioredoxin functions in a given organism. We report a search for physiological target proteins of thioredoxin1 in the social amoeba Dictyostelium discoideum, which possesses three developmentally regulated thioredoxin genes, all of which lack functional characterisation. A two-hybrid approach identified at least seven bona fide thioredoxin partners, including oxidoreductases, proteins of the ribosomal translation apparatus, and the cytoskeletal protein filopodin. With the exception of ribonucleotide reductase, none of these systems had previously been linked to specific redox modulation. Molecular interactions in two of the new thioredoxin/target protein couples were verified by biochemical studies: (1) thioredoxin1 and the abundant elongation factor 1α from D. discoideum form the mixed heterodisulfide characteristic of the thioredoxin mechanism of action; and (2) reduced thioredoxin, but not glutathione, strongly inhibits yeast alcohol dehydrogenase catalysis of ethanol oxidation.

DNA Methylation and DNA Methyltransferase in Wheat

Plant DNA is distinguished from the deoxyribonucleic acid of all other eukaryotic organisms by its substantial amount of the modified base, 5-methylcytosine (mCyt). mCyt is not detectable in most fungi and in invertebrates, it constitutes only up to 1% of the bases in vertebrate DNA, but it contributes 5–8 mol% of the total base composition in average plant DNA. Thus, one fifth to one third (20–35%) of the cytosine residues are modified in all plants, and nearly all cytosines are replaced by the methylated analog in some special plant DNA fractions.