Anemones

Anelliening, /. anellation (see anellieren). Anemoninsaure,/. auemoninic acid. Anemon-kampher, m. anemonin, -sSure, /. anemonic acid. 

Citral Lemon grass oil, mandarin oil Chelidonic acid Asparagus, Celandine, anemone... 

Protoanemonin, which has been isolated from Anemone pulsatilla and Ranunculus spp., was reported to inhibit root growth by slowing down metabolism and blocking mitosis 35). Erickson and Rosen 35) observed cytological effects in corn root tips at concentrations of 10M and lower. Cells undergoing division appeared to accumulate in the interphase or prophase stages. Metaphase, anaphase, and telophase stages were not observed. Cytoplasmic and vacuolar structures were disturbed and the presence of mitochondria could not be demonstrated in treated tissue. Thimann and Bonner 141) reported that protoanemonin was 10 to 30 times more inhibitory than coumarin in coleoptile and split pea stem tests, and that BAL prevented the inhibitory action. 

Sea Anemone Pofypeptide Toxins Affecting Sodium Channels... 

Over 40 different types of polypeptide toxins have been found in marine animals (i). Many of these toxins are exquisitely selective in their actions, affecting a single process or receptor at minute concentrations. So far the sea anemone and gastropod Conus) toxins have attracted the most attention as molecular probes of ion channels. In this chapter, we discuss several approaches which are being used to investigate, at the molecular level, the interactions of the sea anemone neurotoxic polypeptides with sodium channels. 

Amino acid sequences of eleven homologous sea anemone polypeptides have been elucidated. All possess three disulfide bonds. The six half-cysteine residues always occur in the same positions (7,8). Initial studies concerning the toxin secondary and tertiary structures relied upon circular dichroism, laser Raman, and, to a lesser extent, fluorescence spectral measurements (15—18). The circular dichroism spectra of the four toxins so far examined are essentially superimpos-able and thus indicate a common secondary structure. The only peak observed, a negative ellipticity at 203 nm, largely results from a non-regular ("random")... 

Figure 1. Most cx)mmon (consensus) sequences of the two types of sea anemone toxins. Bold letters represent residues which both toxin types have in common. Letters above each sequence are nonconservative substitutions, while letters below each sequence are conservative substitutions. A nonconservative substitution was defined as one in which (a) electronic charge changed, (b) a hydrogen-bonding group was introduced or removed, (c) the molecular size of the sidechain was changed by at least 50%, or (d) the secondary structure propensity was changed drastically from b to h or vice versa (Ref.

A multivariate analytical approach has recently been applied to smaller molecules, including peptides (26,27). Recently, we have applied this approach to the sea anemone type 1 toxins. About 90% of the differences observed in... 

Figure 2. Relative toxicity (LD50 and LD qq) estimates for actiniid sea anemone toxins upon crabs (Carcimis maenas) and mice. Values for Anemonia sulcata (As) and Anthopleura xanthogrammica (Ax) toxins are from ref. 24 data for Condylactis gigantea and Phyl-lactis flosculifera toxins are unpublished (Kem). The arrows indicate that the real mouse LD q values for Cg II and Pf II must exceed the values indicated in the Ogure. Although insufficient data are presently available to quantitatively define a relationship between mammalian and crustacean toxicity, it seems that there is usually an inverse relationship, which may be approximately defined by the stipple zone.

Comparisons between these toxins allow delineation of the variability of each position in the sequence. For instance, the residues which are extremely invariant (conservative) for both types of sea anemone toxin are the half-cystines, certain glycyl residues which are expected to be involved in )9-turns, and only a few other residues - Asp 5 or 6, Arg 13 or 14, and Tryp 30 or 31 (the numbering depends upon the toxin type) — expected to be important for folding or receptor binding. Rather surprising is the variation in the residues which NMR studies (22,23) have shown are involved in formation of the four stranded )9-pleated sheet. 

Some chemical modification studies on the sea anemone toxins have unfortunately been less than rigorous in analyzing the reaction products. Consequently, results from many of these studies can only provide suggestions, rather than firm conclusions, regarding the importance of particular sidechains. Many such studies also have failed to determine if the secondary and tertiary structures of the toxin products were affected by chemical modification. 

Numerous organisms, both marine and terrestrial, produce protein toxins. These are typically relatively small, and rich in disulfide crosslinks. Since they are often difficult to crystallize, relatively few structures from this class of proteins are known. In the past five years two dimensional NMR methods have developed to the point where they can be used to determine the solution structures of small proteins and nucleic acids. We have analyzed the structures of toxins II and III of RadiarUhus paumotensis using this approach. We find that the dominant structure is )9-sheet, with the strands connected by loops of irregular structure. Most of the residues which have been determined to be important for toxicity are contained in one of the loops. The general methods used for structure analysis will be described, and the structures of the toxins RpII and RpIII will be discussed and compared with homologous toxins from other anemone species. 

Despite the vast amount of data on the pharmacological properties, very little about the conformation of the proteins has b n known until recent NMR studies. 2D-NMR results have provided detailed information about the secondary structure of several related anemone toxins. ATX I from Anemonia sulcata (3,4) and AP-A from Anthopleura xanthogrammica (5,6) have been studied by Gooley and Norton, and more recently Widmer et al. have further purified the A. sulcata toxins and obtained complete sequence specific assignments for ATX la (7). Our laboratory, on the other hand, has studied the structures of RpII and RpIII from RadiarUhiis paumotensis (8). 

Schweitz et al. purified four related toxins (Rpl, RpII, RpIII, and RpIV) from sea anemone Radianthus paumotensis (Rp) and studied their pharmacological properties (9). During the course of initial NMR studies, the reported sequence of RpII was found to have errors, and was redetermined (8). Subsequently Metrione et al. determined the sequence of RpIII as well (10). T e other two Rp toxin sequences are yet to be determined. Sequences of the Rp, and several other sea anemone toxins, are shown in Table I. We have used a two letter code to denote the species consistently and this notation differs from the earlier designations of Norton and Wiithrich groups. In our notation. As la and Ax I correspond to ATX la and AP-A, respectively. From alignment of the cystines in these sequences, it is clear that Rp toxins have three disulfide bonds, as do the other toxins. 

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