Catharanthus roseus (periwinkle) produces a wide range of terpenoid indole alkaloids, including several pharmaceutically important compounds, from the intermediate strictosidine. The complete mRNA sequence for the enzyme strictosidine synthase (SSS) was determined. Comparison of the primary structure of the encoded protein with the amino-terminal sequence of purified SSS indicated the presence of a signal peptide of 31 amino acids in the putative primary translation product. SSS is encoded by a single-copy gene indicating that isoenzymes reported by others are formed post-translationally from a single precursor. The sss gene and the tryptophan decarboxylase gene (tdc), encoding another enzyme essential for indole alkaloid biosynthesis, are coordinately regulated. In plants steady-state mRNA levels are highest in roots. In cell suspension cultures the genes are rapidly down-regulated by auxin. In contrast, both genes are strongly induced by fungal elicitors such as Pythium aphanidermatum culture filtrate or yeast extract. Induction is a rapid, transcriptional event occurring independent of de novo protein synthesis. These results show that a first important regulatory step in the complex process leading to indole alkaloid accumulation in C. roseus suspension cells is transcription of the biosynthetic genes.

Magainins, positively charged peptides present in the skin of Xenopus laevis, are known to permeabilize free-energy transducing membranes. Structural studies in otherwise protein-free model systems show alpha-helical magainins parallel to the membrane water interface. However, functional studies in biological membranes suggest that magainins operate as oligomeric complexes. Here we investigate whether magainins function as oligomers in protein-free liposomes also. We report that they do exhibit strong positive heterocooperativity. The magainins, magainin 2 and PGLa, act synergistically. Both activity and cooperativity are enhanced by net negative charge of the liposomal membranes. A transmembrane electric potential, negative inside, enhanced the activity of the peptides. We propose a model in which (i) binding to the surface of the membrane, mainly guided by electrostatic interactions, occurs and (ii) the bound form is in equilibrium with an n-meric complex of magainins spanning the membrane.

General rightsIt is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s), other than for strictly personal, individual use, unless the work is under an open content license (like Creative Commons). Disclaimer/Complaints regulationsIf you believe that digital publication of certain material infringes any of your rights or (privacy) interests, please let the Library know, stating your reasons. In case of a legitimate complaint, the Library will make the material inaccessible and/or remove it from the website. Please Ask the Library: http://uba.uva.nl/en/contact, or a letter to: Library of the University of Amsterdam, Secretariat, Singel 425, 1012 WP Amsterdam, The Netherlands. You will be contacted as soon as possible. Download date: 10 May 2018Eur. J. Biochem. 228, 257-264 (1995) 0 FEBS 1995 Xenopus laevis skin secretion contains a mixture of magainins, which are small positively charged oligopeptides with antimicrobial activity. In this study, we show that two of these peptides, i.e. magainin-2 and PGLa, are much more active in biological functions when added together than when added alone. This synergy applies for the antimicrobial activity of these peptides, and for the toxic effects on tumor cells. We show that this peptide combination is also synergistic when permeabilizing protein-free liposomes for glucose, when dissipating the membrane potential in cytochrome oxidase liposomes and Escherichia coli, and, reversibly, when stimulating respiration in the liposomes. The occurrence of synergy in these diverse systems (complex and simple) suggests that the biological synergy results from synergy in the primary activity of the magainin peptides, namely the permeabilization of free-energy transducing membranes, possibly by forming a multimeric transmembrane pore of mixed peptide composition. The antimicrobial activity of X. laevis skin secretions may be greatly enhanced by the application of this binary weapon.

The membrane-bound flavoprotein NADPH:cytochrome P-450 (cytochrome c) reductase, that functions in electron transfer to cytochrome P-450 monooxygenases, was purified from a cell suspension culture of the higher plant Catharanthus roseus. Anti-serum raised against the purified protein was found to inhibit NADPH:cytochrome c reductase activity as well as the activities of the cytochrome P-450 enzymes geraniol 10-hydroxylase and trans-cinnamate 4-hydroxylase, which are involved in alkaloid biosynthesis and phenylpropanoid biosynthesis, respectively. Immunoscreening of a C. roseus cDNA expression library resulted in the isolation of a partial NADPH: cytochrome P-450 reductase cDNA clone, which was identified on the basis of sequence homology with NADPH:cytochrome P-450 reductases from yeast and animal species. The identify of the cDNA was confirmed by expression in Escherichia coli as a functional protein capable of NADPH-dependent reduction of cytochrome c and neotetrazolium, two in vitro substrates for the reductase. The N-terminal sequence of the reductase, which was not present in the cDNA clone, was determined from a genomic NADPH: cytochrome P-450 reductase clone. It was demonstrated that the reductase probably is encoded by a single copy gene. A sequence comparison of this plant NADPH:cytochrome P-450 reductase with the corresponding enzymes from yeast and animals species showed that functional domains involved in binding of the cofactors FMN, FAD and NADPH are highly conserved between all kingdoms. In C. roseus cell cultures a rapid increase of the reductase steady state mRNA level was observed after the addition of fungal elicitor preparations that are known to induce cytochrome P-450-dependent biosynthetic pathways.