Peter Gröbner

Enzymes involved in the dynamic equilibrium of core histone acetylation of Physarum polycephalum

DEAE‐Sepharose chromatography of extracts from plasmodia of the myxomycete Physarum polycephalum revealed the presence of multiple histone acetyltransferases and histone deacetylases. A cytoplasmic histone acetyltransferase B, specific for histone H4, and two nuclear acetyltransferases A1 and A2 were identified; A1 acetylates all core histones with a preference for H3 and H2A, whereas A2 is specific for H3 and also slightly for H2B. Two histone deacetylases, HD1 and HD2, could be discriminated. They differ with respect to substrate specificity and pH dependence. For the first time the substrate specificity of histone deacetylases was determined using HPLC‐purified individual core histone species. The order of acetylated substrate preference is H2A>H3>‐H4>H2B for HD1 and H3>H2A>H4 for HD2, respectively; HD2 is inactive with H2B as substrate. Moreover histone deacetylases are very sensitive to butyrate, since 2 mM butyrate leads to more than 50% inhibition of enzyme activity.

Thymidine kinase enzyme variants in Physarum polycephalum; change of pattern during the synchronous mitotic cycle.

Thymidine kinase activity (EC 2.7.1.75) exhibits periodic changes in macroplasmodia of Physantm polycephalum correlated with the synchronous nuclear division cycle [ 1,2]. Studies with inhibitors of RNA and protein synthesis suggest that production of this enzyme is mainly restricted to a small segment (1 h) of the 10 h nuclear cycle starting shortly prior to the onset of the synchronous mitosis [3]. Preliminary attempts to isolate the enzyme from plasmodial extracts have shown that thymidine kinase activity appears in several fractions behaving differently during purification procedures. This communication reports on the separation of at least three enzyme variants by isoelectric focusing. The absolute and relative quantities of these variants change characteristically during the synchronous mitotic cycle. The results suggest that during the induction period a single enzyme species is produced which undergoes post-transcriptional modifications giving rise to multiple enzyme variants.

MvaL1 autoregulates the synthesis of the three ribosomal proteins encoded on the MvaL1 operon of the archaeon Methanococcus vannielii by inhibiting its own translation before or at the formation of the first peptide bond

The control of ribosomal protein synthesis has been investigated extensively in Eukarya and Bacteria. In Archaea, only the regulation of the MvaL1 operon (encoding ribosomal proteins MvaL1, MvaL10 and MvaL12) of Methanococcus vannielii has been studied in some detail. As in Escherichia coli, regulation takes place at the level of translation. MvaL1, the homologue of the regulatory protein L1 encoded by the L11 operon of E. coli, was shown to be an autoregulator of the MvaL1 operon. The regulatory MvaL1 binding site on the mRNA is located about 30 nucleotides downstream of the ATG start codon, a sequence that is not in direct contact with the initiating ribosome. Here, we demonstrate that autoregulation of MvaL1 occurs at or before the formation of the first peptide bond of MvaL1. Specific interaction of purified MvaL1 with both 23S RNA and its own mRNA is confirmed by filter binding studies. In vivo expression experiments reveal that translation of the distal MvaL10 and MvaL12 cistrons is coupled to that of the MvaL1 cistron. A mRNA secondary structure resembling a canonical L10 binding site and preliminary in vitro regulation experiments had suggested a co‐regulatory function of MvaL10, the homologue of the regulatory protein L10 of the β‐operon of E. coli. However, we show that MvaL10 does not have a regulatory function.

Thymidine kinase enzyme variants in the life cycle of Physarum polycephalum

Abstract Thymidine kinase activity and the enzyme variant patterns, obtained by isoelectric focusing in polyacrylamide gels, at several stages in the life cycle of Physarum polycephalum were studied. It was shown that there is no difference in the thymidine kinase enzyme variant pattern between plasmodia of different strains, in particular between apogamic (haploid) and heterothallic (diploid) strains. Whenever mitosis occurs in the life cycle the total thymidine kinase activity sharply increases and most of the enzyme activity at this point is represented by enzyme variants with low isoelectric points. In the amoebae of different strains there is a single, unique thymidine kinase enzyme variant which is not observed in the other stages of the life cycle.

A heme-nonapeptide tracer for electron microscopy

SummaryA heme-nonapeptide (H-9-P)1, applicable to electron microscopic cytochemistry via peroxidase-like activity, was prepared by passing horse heart cytochrome c through a column with Sepharose and covalently attached trypsin. After purification by column chromatography (Sephadex G50 Superfine, Biogel P-2) a maximal yield of ∼50% and purity of >99% was achieved. A concise schedule allows for inexpensive preparation of H-9-P with standard laboratory equipment. H-9-P has the following properties: Its structure is (14) Cys-Ala-Gln-Cys-His-Thr-Val-Glu-Lys (22) with heme attached to Cys (14) and (17). MW=1630, pI=4.95, λE(max)pH 7= 397.5 nm, ε22 °C, pH 7397.5 nm = 1.11 × 105 [Liter/Mole x cm]. With the use of a diaminobenzidine-H2O2-medium — as applied for cytochemistry — we determined spectrophotometrically a pHopt=12.5 and an apparent K5 = 3.14 × 10− 3 [M]. Glutardialdehyde leads to considerable de-activation and, according to SDS-polyacrylamide-gel-electrophoresis, to diffuse crosslinking accompanied by a shift of the active pH-region towards neutral pH values. An attempt was made to optimize the cytochemical assay. The peroxidase-like activity of H-9-P is well comparable to that of other heme-tracers; only horseradish peroxidase has a higher turnover number. When injected to mice or added to cell suspensions, even high concentrations of H-9-P did not entail any signs of toxicity.

RNA polymerase activity and template activity of chromatin after butyrate induced hyperacetylation of histones in Physarum

We have studied the effect of sodium-n-butyrate on endogenous RNA polymerase in Physarum polycephalum. 1 mM butyrate strongly reduces RNA polymerase activity measured in isolated nuclei or chromatin; both RNA polymerase A as well as the alpha-amanitin sensitive RNA polymerase B are equally affected. Despite a concomitant hyperacetylation of histone H4 the template activity of chromatin, as analyzed by in vitro transcription of the chromatin with exogenous RNA polymerase from E. coli or RNA polymerase II from wheat germ, remains unaltered as compared to untreated control chromatin, indicating that there is no positive correlation between histone acetylation and template activity of chromatin for transcription in this organism. The results further indicate, that butyrate acts primarily as a quick but reversible inhibitor of protein synthesis in Physarum; the fast decrease of endogenous RNA polymerase activity after butyrate treatment is due to inhibition of enzyme synthesis rather than inactivation of other factors necessary for transcription.

Acceleration of mitosis induced by mitotic stimulators of Physarum polycephalum

Abstract Extracts of the myxomycete Physarum polycephalum exhibit an accelerating effect on nuclear division which fluctuates during the synchronous nuclear division cycle. Extracts from late G2 phase plasmodia can advance mitosis in recipient test plasmodia by up to 30% of the length of the control cycle. The advancing capacity of extracts is heat- and ammonium sulphate-precipitable, non-dialysable and destroyed by pronase, suggesting that the active substance is a protein. The advance of mitosis is in strong correlation with the applied dose of stimulatory material.

Physarum nitric oxide synthases: genomic structures and enzymology of recombinant proteins.

Physarum polycephalum expresses two closely related, calcium-independent NOSs (nitric oxide synthases). In our previous work, we showed that both NOSs are induced during starvation and apparently play a functional role in sporulation. In the present study, we characterized the genomic structures of both Physarum NOSs, expressed both enzymes recombinantly in bacteria and characterized their biochemical properties. Whereas the overall genomic organization of Physarum NOS genes is comparable with various animal NOSs, none of the exon–intron boundaries are conserved. Recombinant expression of clones with various N-termini identified N-terminal amino acids essential for enzyme activity, but not required for haem binding or dimerization, and suggests the usage of non-AUG start codons for Physarum NOSs. Biochemical characterization of the two Physarum isoenzymes revealed different affinities for L-arginine, FMN and 6R-5,6,7,8-tetrahydro-L-biopterin.

Thymidylate synthetase during synchronous nuclear division cycle and differentiation of Physarum polycephalum

Thymidylate synthetase and thymidine kinase activities in wild type strain M3b and in thymidine kinase-deficient mutant TU63 of Physarum polycephalum are studied. Whenever nuclear division occurs in macroplasmodia of wild type, thymidine kinase and thymidylate synthetase activities sharply increase, although the increase of thymidylate synthetase activity is less pronounced than thymidine kinase activity. This is also true for other investigated nuclear divisions during the life cycle of P. polycephalum. It is shown for the first time that thymidylate synthetase is a periodically fluctuating enzyme during the naturally synchronous nuclear division cycle of P. polycephalum with a peak of specific activity in the S phase. In macroplasmodia, as well as after germination of microsclerotia of M3b, thymidine kinase is the dominant enzyme, whereas at the time of the precleavage mitosis in sporulating macroplasmodia thymidylate synthetase is the predominant enzyme. This study describes and compares both dTMP-synthesizing enzymes during proliferation and differentiation of the same organism.

Response of the dTMP-synthesizing enzymes to differentiation processes in Physarum polycephalum

Synthesis of deoxythymidylate (dTMP) is a rate-limiting step in DNA synthesis; there are two main enzymes which are responsible for dTMP production, thymidylate synthetase (ts) and thymidine kinase (tk). Both enzymes were studied during several differentiation processes of the myxomycete Physarum polycephalum. In all stages of proliferation (microplasmodia, macroplasmodia, germinating microsclerotia and germinating spores) tk is the dominant enzyme in terms of activity, whereas ts is the predominant enzyme in quiescent stages (microsclerotia, sporangia, respectively spores); this is expressed by calculating the tk/ts ratio. This ratio is greater than 1 during proliferation and much less than 1 during quiescence. Our results clearly show that ts is the basic enzyme for dTMP production during all differentiation stages, whereas tk, if required, is shut on and represents an additional potential for dTMP synthesis during rapid proliferation.