Hermann Ragg

Light-induced changes of enzyme activities in parsley cell suspension cultures: Effects of inhibitors of RNA and protein synthesis☆

Abstract Light-induced increases in the activities of phenylalanine ammonia-lyase, p -coumarate:CoA ligase, and chalcone-flavanone isomerase in cell suspension cultures of Petroselinum hortense were similarly affected by several inhibitors of transcription and translation. In particular, strong inhibition by low doses of actinomycin D or cycloheximide indicated that both RNA and cytoplasmic protein synthesis were involved in the stimulatory effects of light. The increases in enzyme activities were preceded by a lag period of about 2.5 hr. When actinomycin D or cycloheximide was added at different times after the onset of light, characteristic differences in the inhibitory effects on the increases in phenylalanine ammonia-lyase activity were observed. The results suggested that RNA synthesis commenced immediately upon illumination, while an enhanced rate of synthesis of specific protein(s) did not begin before the end of the lag period. By comparison, the activities of some enzymes not involved in phenylpropanoid metabolism and not affected by light were also not affected by actinomycin D or cycloheximide at the concentrations used. This seems to exclude the possibility of nonspecific effects of these inhibitors on enzyme activities under the conditions of experimentation.

A general method to identify plant structural genes among genomic DNA clones using transposable element induced mutations

SummarySeveral genomic clones from Petroselinum hortense, Zea mays and Antirrhinum majus all homologous to cloned Petroselinum chalcone synthase cDNA were isolated using the λgt WES cloning system.Clones containing the chalcone synthase structural gene were identified by hybridization to cDNA from Petroselinum hortense, genomic wildtype, mutant and revertant DNA.Among the 5 different clones from Petroselinum hortense, PH3 is the most likely candidate to contain at least a portion of the chalcone synthase gene.None of the 4 Zea mays clones appeared to contain part of the chalcone synthase gene.Among the 2 different clones from Antirrhinum majus, AM3 contains the portion of the chalcone synthase structural gene which is altered in the mutant nivea recurrens (nivrec). This mutant is considered to be due to the integration of a transposable element. In revertants of nivrec to niv+ the wildtype locus is restored molecularly.

Expression of human antithrombin III in Saccharomyces cerevisiae and Schizosaccharomyces pombe

Recombinant plasmids were constructed that direct the synthesis of human antithrombin III in bakers yeast, Saccharomyces cerevisiae, and the fission yeast, Schizosaccharomyces pombe. The signal sequence of antithrombin III was recognized by both yeast species, and antithrombin III was secreted into the medium. When the signal sequence was replaced by a sequence of ten arbitrary amino acids, the product expressed from such a construct stayed inside the cell. Antithrombin III was glycosylated by the bakers and fission yeast and was immunologically identical to antithrombin III isolated from human plasma. Antithrombin III isolated from the culture media of recombinant yeasts was biologically active, as could be shown by progressive inhibitor activity and heparin cofactor activity.

Translation of poly(A)-containing and poly(A)-free messenger RNA for phenylalanine ammonia-lyase, a plant-specific protein, in a reticulocyte lysate

Abstract Polyribosomal RNA from light-induced parsley cell suspension cultures was incubated in a rabbit-reticulocyte lysate. The radioactive proteins formed in vitro were treated with a specific antiserum prepared for purified phenylalanine ammonia-lyase, an enzyme which is not found in animals. Analysis of the immunoprecipitate by gel electrophoresis in the presence of dodecyl sulfate showed a single peak of radioactivity at the position of the subunits of the enzyme. No detectable amount of this protein was synthesized with RNA from parsley cells which had been kept in the dark. The polyribosomal RNA was fractionated into poly(A) + and poly(A) − RNA. Both fractions contained messenger RNA for phenylalanine ammonia-lyase, as determined with the reticulocyte lysate. The total template activity of each RNA fraction was assessed in a cell-free extract from wheat germ. About 20–40% of the total mRNA activity was associated with the poly(A) + RNA, and 60–80% with the poly(A) − RNA. Both fractions stimulated the synthesis of radioactive proteins with molecular weights ranging from 10 000 to 80 000; the size distributions of these proteins on polyacrylamide gels were very similar. The results indicate that (i) the mammalian cell-free system accepted plant mRNA for translation, (ii) considerable amounts of translatable, polyribosomal mRNA for phenylalanine ammonia-lyase were present only in irradiated parsley cells, and (iii) large portions of the enzyme-specific and the total mRNAs of the polyribosomes consisted of poly(A) − RNA.

Inhibition of furin by serpin Spn4A from Drosophila melanogaster

The serpin gene Spn4 from Drosophila melanogaster encodes multiple isoforms with alternative reactive site loops (RSL). Here, we show that isoform Spn4A inhibits human furin with an apparent k assoc of 5.5 × 106 M−1 s−1. The serpin forms SDS‐stable complexes with the enzyme and the RSL of Spn4A is cleaved C‐terminally to the sequence –Arg–Arg–Lys–Arg↓ in accord with the recognition/cleavage site of furin. Immunofluorescence studies show that Spn4A is localized in the endoplasmic reticulum (ER), suggesting that the inhibitor is an interesting tool for investigating the cellular mechanisms regulating furin and for the design of agents controlling prohormone convertases.

The role of serpins in the surveillance of the secretory pathway

Abstract.Serpins (serine protease inhibitors) constitute a class of proteins with an unusually wide spectrum of different functions at extracellular sites and within the nucleocytoplasmic compartment that extends from protease inhibition to hormone transport and regulation of chromatin organization. Recent investigations reveal a growing number of serpins acting in secretory pathway organelles, indicating that they are not simply cargo destined for export, but fulfill distinct roles within the classical organellecoupled trafficking system. These findings imply that some serpins are part of a quality control system that monitors the export and possibly import routes of eukaryotic cells. The molecular targets of these serpins are often unknown, opening new avenues for future research.

The Spn4 gene from Drosophila melanogaster is a multipurpose defence tool directed against proteases from three different peptidase families

By alternative use of four RSL (reactive site loop) coding exon cassettes, the serpin (serine protease inhibitor) gene Spn4 from Drosophila melanogaster was proposed to enable the synthesis of multiple protease inhibitor isoforms, one of which has been shown to be a potent inhibitor of human furin. Here, we have investigated the inhibitory spectrum of all Spn4 RSL variants. The analyses indicate that the Spn4 gene encodes inhibitors that may inhibit serine proteases of the subtilase family (S8), the chymotrypsin family (S1), and the papain-like cysteine protease family (C1), most of them at high rates. Thus a cohort of different protease inhibitors is generated simply by grafting enzyme-adapted RSL sequences on to a single serpin scaffold, even though the target proteases contain different types and/or a varying order of catalytic residues and are descendents of different phylogenetic lineages. Since all of the Spn4 RSL isoforms are produced as intracellular residents and additionally as variants destined for export or associated with the secretory pathway, the Spn4 gene represents a versatile defence tool kit that may provide multiple antiproteolytic functions.

Multiple gains of spliceosomal introns in a superfamily of vertebrate protease inhibitor genes

BackgroundIntron gains reportedly are very rare during evolution of vertebrates, and the mechanisms underlying their creation are largely unknown. Previous investigations have shown that, during metazoan radiation, the exon-intron patterns of serpin superfamily genes were subject to massive changes, in contrast to many other genes.ResultsHere we investigated intron dynamics in the serpin superfamily in lineages pre- and postdating the split of vertebrates. Multiple intron gains were detected in a group of ray-finned fishes, once the canonical groups of vertebrate serpins had been established. In two genes, co-occurrence of non-standard introns was observed, implying that intron gains in vertebrates may even happen concomitantly or in a rapidly consecutive manner. DNA breakage/repair processes associated with genome compaction are introduced as a novel factor potentially favoring intron gain, since all non-canonical introns were found in a lineage of ray-finned fishes that experienced genomic downsizing.ConclusionMultiple intron acquisitions were identified in serpin genes of a lineage of ray-finned fishes, but not in any other vertebrates, suggesting that insertion rates for introns may be episodically increased. The co-occurrence of non-standard introns within the same gene discloses the possibility that introns may be gained simultaneously. The sequences flanking the intron insertion points correspond to the proto-splice site consensus sequence MAG↑N, previously proposed to serve as intron insertion site. The association of intron gains in the serpin superfamily with a group of fishes that underwent genome compaction may indicate that DNA breakage/repair processes might foster intron birth.

Ancestry and evolution of a secretory pathway serpin

BackgroundThe serpin (serine protease inhibitor) superfamily constitutes a class of functionally highly diverse proteins usually encompassing several dozens of paralogs in mammals. Though phylogenetic classification of vertebrate serpins into six groups based on gene organisation is well established, the evolutionary roots beyond the fish/tetrapod split are unresolved. The aim of this study was to elucidate the phylogenetic relationships of serpins involved in surveying the secretory pathway routes against uncontrolled proteolytic activity.ResultsHere, rare genomic characters are used to show that orthologs of neuroserpin, a prominent representative of vertebrate group 3 serpin genes, exist in early diverging deuterostomes and probably also in cnidarians, indicating that the origin of a mammalian serpin can be traced back far in the history of eumetazoans. A C-terminal address code assigning association with secretory pathway organelles is present in all neuroserpin orthologs, suggesting that supervision of cellular export/import routes by antiproteolytic serpins is an ancient trait, though subtle functional and compartmental specialisations have developed during their evolution. The results also suggest that massive changes in the exon-intron organisation of serpin genes have occurred along the lineage leading to vertebrate neuroserpin, in contrast with the immediately adjacent PDCD10 gene that is linked to its neighbour at least since divergence of echinoderms. The intron distribution pattern of closely adjacent and co-regulated genes thus may experience quite different fates during evolution of metazoans.ConclusionThis study demonstrates that the analysis of microsynteny and other rare characters can provide insight into the intricate family history of metazoan serpins. Serpins with the capacity to defend the main cellular export/import routes against uncontrolled endogenous and/or foreign proteolytic activity represent an ancient trait in eukaryotes that has been maintained continuously in metazoans though subtle changes affecting function and subcellular location have evolved. It is shown that the intron distribution pattern of neuroserpin gene orthologs has undergone substantial rearrangements during metazoan evolution.

Zinc ions promote the interaction between heparin and heparin cofactor II

The effects of bivalent cations on heparin binding, structure, and thrombin inhibition rates of heparin cofactor II were examined. Zn2+ – and to a lesser extent Cu2+ and Ni2+ – enhanced the interaction between heparin cofactor II and heparin as demonstrated by heparin affinity chromatography and surface plasmon resonance experiments. Metal chelate chromatography and increased intrinsic protein fluorescence in the presence of Zn2+ indicated that heparin cofactor II has metal ion‐binding properties. The results are compatible with the hypothesis that Zn2+ induces a conformational change in heparin cofactor II that favors its interaction with heparin.