Diversity of joro spider toxins

In a study to solve a mystery of venom toxicity of the joro spider, Nephila clavata, we purified and identified novel spider toxins such as clavamine, spidamine and joramine. Chemical analyses, bioassays and physical analyses were specifically elaborated in these procedures. The structure-activity relationship of the spider toxins was discussed biologically and chemically in comparison with the other spider toxins. We considered that the diversity of the joro spider toxins by reserving 2,4-dihydroxyphenylacetyl-L-asparaginylcadaverine as a common moiety gave rise to an important insight into not only the toxic reaction, but also the ontology.     

Characterization of three endogenous peptide inhibitors for multiple metalloproteinases with fibrinogenolytic activity from the venom of Taiwan habu (Trimeresurus mucrosquamatus)

Three small peptide components were isolated and purified from the venom of Taiwan habu (Trimeresurus mucrosquamatus), which show specific activity to inhibit the strong proteolytic activity of multiple metalloproteinases present in the crude venom. Using multiple chromatographies coupled with successive ultrafiltrations, three inhibitors, i.e. pyroglutamate-lysine-tryptophan (pyroGlu-Lys-Trp), pyroglutamate-asparagine-tryptophan (pyroGlu-Asn-Trp) and pyroglutamate-glutamine-tryptophan (pyroGlu-Gln-Trp) were obtained in good yields and high homogeneity. The yields of these peptide fractions were estimated to be about 0.65 mg, 0.55 mg and 0.42 mg from 250 mg total lyophilized crude venom, which corresponded to the approximate concentrations of 8.4 mM, 7.3 mM and 5.4 mM respectively in venom secretion. Detailed and unambiguous structural determination was established by amino acid analyses, mass spectrometry and microsequencing of purified peptides. Further functional characterization of these three tripeptides showed that they could weakly inhibit three metalloproteinases previously isolated from the same venom. The inhibitory activities were similar among these tripeptides and their IC50 (concentration for 50% inhibition) were estimated in a range of 0.20-0.95 mM, which is much more effective than citrate, another venom protease inhibitor of low molecular-weight component. Since these tripeptides are the endogenous peptide inhibitors present in the lumen of venom glands, it is conceivable that they may act as a self-defensive mechanism against the auto-digestive deleterious effect of the strong metalloproteinases in vivo, particularly several zinc-dependent metalloproteinases present in crotalid and viperid venoms.     

A new alpha-conotoxin which targets alpha3beta2 nicotinic acetylcholine receptors

We have isolated a 16-amino acid peptide from the venom of the marine snail Conus magus which potently blocks nicotinic acetylcholine receptors (nAChRs) composed of alpha3beta2 subunits. This peptide, named alpha-conotoxin MII, was identified by electrophysiologically screening venom fractions against cloned nicotinic receptors expressed in Xenopus oocytes. The peptide's structure, which has been confirmed by mass spectrometry and total chemical synthesis, differs significantly from those of all previously isolated alpha-conotoxins. Disulfide bridging, however, is conserved. The toxin blocks the response to acetylcholine in oocytes expressing alpha3beta2 nAChRs with an IC50 of 0.5 nM and is 2-4 orders of magnitude less potent on other nAChR subunit combinations. We have recently reported the isolation and characterization of alpha-conotoxin ImI, which selectively targets homomeric alpha7 neuronal nAChRs. Yet other alpha-conotoxins selectively block the muscle subtype of nAChR. Thus, it is increasingly apparent that alpha-conotoxins represent a significant resource for ligands with which to probe structure-function relationships of various nAChR subtypes.     

HPLC detection of serotonin within the rat cochlea

This study was performed to analyse the cochlear concentrations of serotonin (5-HT) and 5-hydroxyindole-3-acetic acid (5-HIAA), their sources and modifications induced by noise exposure. Superior cervical ganglionectomy did not modify these concentrations. However, removal of the blood by aortic perfusion reduced significantly (about 76%) the cochlear concentration of 5-HT without affecting the 5-HIAA concentration. These results indicate that blood constitutes an important source of 5-HT to the cochlea, opposite to the superior cervical ganglion. Exposure to noise at 90 dB SPL did not modify the total cochlear concentrations of 5-HT and 5-HIAA, or the concentrations remaining after removal of the blood, suggesting that 5-HT could have a modulatory role in the cochlea distinct from that of olivocochlear neurotransmitters.     

Identification and oligosaccharide structure analysis of rhodopsin glycoforms containing galactose and sialic acid

The N-linked oligosaccharides of frog (Rana pipiens) rhodopsin were analysed by sequential exoglycosidase digestion and gel filtration chromatography, following reductive tritiation. In addition, selected tryptic glycopeptides obtained from frog retinal rod outer segment membranes were examined by electrospray mass spectrometry (ES-MS), fast atom bombardment mass spectrometry (FAB-MS), amino acid sequence and composition analysis, and carbohydrate composition analysis. The amino acid sequence data demonstrated that the glycopeptides were derived from rhodopsin and confirmed the presence of two N-glycosylation sites, at residues Asn2 and Asn15. The predominant glycan (approximately 60% of total) had the structure GlcNAc beta 1-2Man alpha 1-3(Man alpha 1-6) Man beta 1-4GlcNAc beta 1-4GlcNAc-(Asn), with the remaining structures containing 1-3 additional hexose residues, as reported previously for bovine rhodopsin. Unlike bovine rhodopsin, however, a sizable fraction of the total glycans of frog rhodopsin also contained sialic acid (NeuAc), with the sialylated oligosaccharides being present exclusively at the Asn2 site. FAB-MS analysis of oligosaccharides released from the Asn2 site gave, among other signals, an abundant quasimolecular ion corresponding to a glycan of composition NeuAc1Hex6HexNAc3 (where Hex is hexose and HexNAc is N-acetylhexosamine), consistent with a hybrid structure. The potential biological implications of these results are discussed in the context of rod outer segment membrane renewal.     

Quantifying the venom dose of the spider Cupiennius salei using monoclonal antibodies

The variation in venom dose with prey size of the neotropical wandering spider Cupieinnius salei was examined experimentally. Monoclonal antibodies were raised against the venom toxins of C. salei. Mab 9H3, recognizing the main toxin CSTX-1, was used to quantify the venom by enzyme-linked immunosorbent assay (ELISA). Crickets (Achta domesticus) in four size classes were randomly offered to sixteen mature female spiders at 14d intervals. The prey items were removed from spiders five minutes after the initial bite and subsequently homogenized for ELISA measurements. The quantity of venom expended was significantly related to the size of prey, ranging from 0.15 microl for the smallest (100 110 mg) to 1.53 microl for the largest (600-660 mg) crickets. Adaptations to prey size were also reflected in capturing behavior. None of the smallest, but almost 50% of the largest crickets were wrapped in silk following the spiders bite. Some other behavioral features may reduce the energetic costs of venom production. In 22% of the smallest crickets no venom was detectable, with the majority showing mechanical damage as a result of fang contact. This indicates. that C. salei does not rely exclusively on its venom when feeding on small prey. Some other aspects such as the site of the bite and the speed of paralyzation and their consequences associated with the amount of venom expended are discussed.     

Octoxynol presence in bear-bile

The presence of octoxynol from dried bear-bile was examined. Octoxynol was coextracted when glycolipids by Folch-Suzuki partition method. Octoxynol formed mixed-micelles with glycosphingolipids. The glycolipids were purified by DEAE-Sephadex A-25 column chromatography. The fractions containing mixed micelles were obtained from linear gradient solvent of 0.05M-0.5M ammonium acetate in methanol. HPLC ( Bondapak-NH(2) - linked to a Bondapak-C(18) column) chromatogram showed five peaks. Two possible structures for the fourth peak fraction were proposed as (CH(3))(3)C-CH(2)-C(CH(3))(2)-C(6)H(4)-OR and (CH(3))(3)C-C(CH(3))(2)-CH(2)-C(6)H(4)-OR by NMR spectroscopy. The structure was further confirmed by electrospray tandem mass spectrometry (ESI MS/MS). The spectrum showed a protonated molecule at m/z 559 and three different series of ions with mass difference of 44 were detected in the MS/MS spectrum. Therefore, the structure of the fourth peak fraction from HPLC was confirmed as octoxynol, (CH(3))(3)C-CH(2)-C(CH(3))(2)-C(6)H(4)-(OCH(2)-CH(2))n-OH, based on mass spectrometry and NMR spectroscopy.     

Effect of anoxia on striatal monoamine metabolism in immature rat brain compared with that of hypoxia: an in vivo microdialysis study

We examined in 5-day-old rats the effects of either anoxia or 8% hypoxia on extracellular monoamines such as dopamine (DA), 3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), norepinephrine (NE), 5-hydroxytryptamine (5-HT), and 5-hydroxyindole-3-acetic acid (5-HIAA) using in vivo microdialysis and subsequent HPLC. After stabilization 64 animals were exposed to 100% nitrogen for 16 min and 40 animals to 8% oxygen for 128 min. Both anoxia and hypoxia produced acute increase in the striatal extracellular DA (anoxia: P < 0.001, hypoxia: P < 0.01). Especially in anoxia, DA levels increased transiently to 2000-times the basal levels and 6-times higher than those in hypoxia. NE also increased in both anoxia and hypoxia. DOPAC and HVA decreased during hypoxia (P < 0.01 and P < 0.001, respectively), while those in anoxia were unchanged. In anoxia, decrease tendency of their levels were in short duration and that of 5-HIAA was followed by gradual increase (P < 0.001). These data demonstrated that brief exposure to anoxia or hypoxia had significant influence on striatal monoamine metabolism in immature brain and the pattern of change of monoamine in anoxia was different from that in hypoxia.     

The action of Taiwan cobra venom on methionine enkephalin: a useful assay for oligopeptidase content

The pentapeptide methionine enkephalin is readily hydrolysed by the oligopeptidase activity contained in Taiwan cobra (Naja naja atra) venom. It is a significantly better substrate than the peptides previously used to identify the presence of this enzyme, but it retains many of the sequence characteristics shared by these other peptides. Analysis of the manner of hydrolysis by means of high-performance liquid chromatography and electrospray mass spectrometry revealed the simultaneous actions of at least two types of oligopeptidase on the neuropeptide, producing two routes of initial breakdown. By one route, an endopeptidase cleaved the Gly-Phe bond of enkephalin first to release Tyr-Gly-Gly and Phe-Met. By the other route, an aminopeptidase was able to release Tyr and Gly-Gly-Phe-Met by cleaving the Tyr-Gly bond first. From amongst the various peptide fragments produced, Tyr-Gly-Gly was subject to immediate aminopeptidase action to release Tyr and Gly-Gly. The free Tyr produced in these reactions was in turn quickly transformed by the L-amino acid oxidase in the venom. The kinetic qualities of the enkephalin hydrolysis, and the conversions of the fragments Tyr-Gly-Gly and Tyr, were measured. Methionine enkephalin has potential as a routine assay for venom oligopeptidases, either in testing the venoms from other species or in attempts to purify these enzymes. Moreover, the ease of hydrolysis of this bioactive peptide, coupled with the revelation of the other enzymic steps involving the fragments generated, may provide important clues as to the possible role of the oligopeptidases (and L-amino acid oxidase) in the venom.     

Structural studies of the O-antigenic polysaccharide of Fusobacterium necrophorum

The O-specific polysaccharide component of the lipopolysaccharide produced by Fusobacterium necrophorum is of the teichoic acid type, with repeating units connected by phosphoric diester linkages. Dephosphorylation of the polysaccharide by treatment with aqueous hydrogen fluoride yielded a carbohydrate composed of a trisaccharide linked to an acidic component. This product, and the polysaccharide, were investigated by chemical methods and 1H-, 13C-, 31P- and 15N-NMR spectroscopy and the former also by fast-atom-bombardment mass spectrometry. It is proposed that the polysaccharide is composed of repeating units having the following structure, in which Fuc represents fucose (6-deoxy-galactose), Am represents an acetamidino group and Sug 2,4-diamino-2,4,6-trideoxy-D-glucose ('bacillosamine') acetylated at the 2-position and acylated with a (S)-3-hydroxybutanoic acid at the 4-position. The acid was identified as a 2-amino-2-deoxy-2-C-methyl-pentonic acid (2-amino-2-methyl-3,4,5-trihydroxypentanoic acid). The configuration of this acid remains to be determined. [formula: see text]     

Effect of N-methyl-D-aspartate and potassium on striatal monoamine metabolism in immature rat: an in vivo microdialysis study

Effects of N-methyl-D-aspartate (NMDA) and potassium on 5-day-old rat's brain were examined. We measured extracellular striatal monoamines such as dopamine (DA), 3,4 dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), and 5-hydroxyindole-3-acetic acid (5-HIAA) using intracerebral microdialysis. After 3 h stabilization, pups received varying concentrations of NMDA (1-3 mM) and potassium (200-800 mM) by intrastriatal perfusion for 32 minutes. Increasing the concentration of NMDA and potassium induced a dose related DA increase (p < 0.001), whereas DOPAC, HVA, and 5-HIAA decreased significantly. Five days later the same animals were sacrificed and the weight reduction of their cerebral hemispheres was measured. The weight of the drug perfused side was significantly reduced compared with that of the contralateral one. We examined next the relationship between the level of maximum DA and the relative hemisphere weight reduction. The DA peak was highly correlated with the hemisphere weight reduction (r = 0.70, n = 52, p < 0.001 in the NMDA group, r = 0.83, n = 30, p < 0.001 in the potassium group, respectively). These data show that each treatment alter striatal monoamine metabolism in immature rat brain and that the extracellular DA peak is a potential early indicator to estimate brain injury.     

Endotoxemic-like shock induced by Loxosceles spider venoms: pathological changes and putative cytokine mediators

The systemic symptoms, tissue lesions and release of cytokines were analysed in four isogenic mouse strains with distinct haplotypes injected with various doses of Loxosceles intermedia spider venom. The estimated LD50 were 24.5 microg for C57Bl/6, 17.6 microg for BALB/c, 6.3 microg for C3H/HeJ and 4.6 microg for A/Sn mice. Prostration, acute cachexia, hypothermia, neurological disorders and hemoglobinuria were the signals preceding death. Accumulation of eosinophilic material inside the proximal and distal renal tubules and acute tubular necrosis were the most common histopathological findings. Death was prevented by previous treatment of venom with specific antivenom serum. The protein F35 purified from the whole venom retained the ability to induce the symptoms of the whole venom. The cytokines tumor necrosis factor (TNF), interleukins IL-6 and IL-10 and the radical nitric oxide were detected in serum at different levels after venom injection. These findings indicate that the state of shock produced in mice by whole endotoxin-free L. intermedia venom or by its purified fraction, protein F35, mimics the endotoxemic shock, that susceptibility to the systemic effects of the venom varies among mice of different haplotypes and that the pattern of in vivo cytokine release resembles that of endotoxemic shock.     

Using matrix-assisted laser desorption ionization mass spectrometry to map the quinol binding site of cytochrome bo3 from Escherichia coli

The cytochrome bo3 ubiquinol oxidase contains at least one and possibly two binding sites for ubiquinol/ubiquinone. Previous studies used the photoreactive affinity label 3-[3H]azido-2-methyl-5-methoxy-6-geranyl-1,4-benzoquinone (azido-Q), a substrate analogue, to demonstrate that subunit II contributes to at least one of the quinol binding sites. In the current work, mass spectroscopy is used to identify a peptide within subunit II that is photolabeled by the azido-Q. Purified cytochrome bo3 was photolabeled as previously described using azido-Q that was not tritiated (i.e., not radiolabeled). Subunit II was then isolated from an SDS-PAGE gel and proteolyzed in situ with trypsin. The resulting peptides were eluted from the gel and then identified using matrix-assisted laser desorption ionization mass spectrometry. The resulting mass spectrum was compared to that obtained by analysis of subunit II that had not been exposed to the photolabel. Using the amino acid sequence, each peak in the mass spectrum of the unlabeled subunit II could be assigned to an expected trypsin fragment. Two additional peaks were observed in the mass spectrum of the photolabeled subunit with m/z 1931.9 and 2287.7. Subtraction of the mass of azido-Q from the peak at m/z 1931.9 results in a mass equivalent to that of a peptide consisting of amino acids 165-178. The assignment of the peak at m/z 2287.7 cannot be made unequivocally and may correspond either to the covalent attachment of azido-Q to peptide 254-270 or to a peptide resulting from incomplete proteolysis. The labeled peptide, 165-178, is within the water-soluble domain of subunit II, whose X-ray structure is known. This peptide is located near the site where CuA is located in the homologous cytochrome c oxidases and can be placed near the interface between subunits I and II.     

Isolation, properties and N-terminal amino acid sequence of a factor V activator from Vipera lebetina (Levantine viper) snake venom

A factor V activator (VLFVA) was separated from Vipera lebetina venom by gel filtration on Sephadex G-100 superfine, followed by chromatography on CM-cellulose and on heparin-agarose. This enzyme (VLFVA) with a molecular mass of 28.4 kDa, as determined by matrix assisted laser desorption ionization time-of-flight mass spectrometry, is a single-chain glycoprotein containing seven residues of neutral sugars, seven residues of hexosamines and three residues of neuraminic acid per molecule. The treatment with N-glycosidase F lowered the molecular mass approximately 6%. The N-terminal sequencing of VLFVA up to the 30th residue evidenced a high homology with Vipera russelli factor V activator RVV-Vgamma (90% identity). Aside from factor V, no other protein substrate for VLFVA has yet been identified. VLFVA hydrolyzes several synthetic arginine ester substrates, such as benzoylarginine ethyl ester (BAEE), tosylarginine methyl ester (TAME) and amide substrates such as Pro-Phe-Arg-MCA. The arginine ester hydrolase activity of the enzyme is markedly lower than that of the crude venom. The ability of VLFVA to activate factor V and its activity to BAEE and TAME were inhibited by the serine proteinase inhibitor, diisopropylfluorophosphate. VLFVA is thermostable protein, heating for 20 min at 70 degrees C does not alter the arginine esterase activity of the enzyme.     

Makatoxin I, a novel toxin isolated from the venom of the scorpion Buthus martensi Karsch, exhibits nitrergic actions

Buthus martensi Karsch venom exhibits nitrergic action in rat anococcygeus muscle (ACM). We have purified a novel toxin, makatoxin I (MkTx I), which exhibits nitrergic action, to homogeneity from this venom by a combination of gel-filtration, cation-exchange chromatography, and reverse-phase chromatography. Its purity was assessed by capillary electrophoresis and mass spectrometry. Its molecular weight was found to be 7031.71 +/- 2.88 as calculated from electrospray mass spectrographic data. The complete amino acid sequence was elucidated by sequencing of reduced and S-pyridylethylated toxin and a carboxyl-terminal peptide, P55-64, generated by the cleavage of toxin with endoproteinase Lys-C. The complete sequence of MkTx I is GRDAYIADSENCTYTCALNPYCNDLCTKNGAKSGYCQWAGRYGNACWCIDLPDKVPIRISGSCR. This toxin is composed of 64 amino acid residues and contains 8 half-cystine residues. Structurally, MkTx I has high similarity to Bot I and Bot II when compared with toxins from other scorpion species. The effects of MkTx I on nitrergic responses were investigated using the rat isolated ACM mounted in Krebs solution (37 degrees C, 5% CO2 in O2). MkTx I (2 microg/ml) markedly relaxed the carbachol-precontracted ACM; the relaxation was inhibited by the stereoselective inhibitor of nitric oxide synthase, Nomega-nitro-L-arginine methyl ester (50 microM). Thus, MkTx I is the first alpha-toxin that can mediate nitrergic responses in the rat isolated ACM.     

High performance liquid chromatographic profiling of tryptophan and related indoles in body fluids and tissues of carcinoid patients

A high performance liquid chromatographic method with quaternary gradient elution and fluorometric detection was developed for profiling of tryptophan (TRP), 5-hydroxytryptophan, serotonin (5-HT) and 5-hydroxyindole-3-acetic acid (5-HIAA) in urine, platelet-rich plasma and (tumour) tissue of patients with carcinoid tumours. Prior to injection, urine samples were diluted and filtered. Platelet-rich plasma and tissue homogenates were prepurified by C18 solid phase extraction. Detection limits were approx. 2 pmol. Results of urinary 5-HT and 5-HIAA compared favourably with those of single component analyses. No consistent diurnal variations were found for TRP, 5-HT and 5-HIAA in 12-h urine samples from 15 healthy adults. Abstinence of 5-HT-rich foods reduced urinary levels of 5-HT and 5-HIAA. C18 extraction of indoles from protein-containing matrices was studied in platelet-rich plasma. Although time-consuming and complicated for daily routine use, the present approach offers particular advantages over single component analyses in the study of TRP metabolism in patients with carcinoid tumours.     

Structural conservation in prokaryotic and eukaryotic potassium channels

Toxins from scorpion venom interact with potassium channels. Resin-attached, mutant K+ channels from Streptomyces lividans were used to screen venom from Leiurus quinquestriatus hebraeus, and the toxins that interacted with the channel were rapidly identified by mass spectrometry. One of the toxins, agitoxin2, was further studied by mutagenesis and radioligand binding. The results show that a prokaryotic K+ channel has the same pore structure as eukaryotic K+ channels. This structural conservation, through application of techniques presented here, offers a new approach for K+ channel pharmacology.     

Acute interaction between ethanol and serotonin metabolism in the rat

The effect of acute ethanol on peripheral serotonin (5HT) metabolism was studied in Sprague-Dawley rats. Four hours after a single dose of ethanol (1.0 g/kg) administered into the stomach, a significant increase in the 5HT level in stomach tissue and a decrease in ileum was observed. The level of 5-hydroxyindole-3-acetic acid (5HIAA) was increased in urine, while increased concentrations of 5-hydroxytryptophol (5HTOL) occurred in jejunum, ileum, spleen and urine. After 7-9 h when the blood ethanol concentration had returned to zero, 5HTOL levels were still higher than control values in jejunum, ileum and urine. At 4 h, an elevated ratio of 5HTOL to 5HIAA was observed in urine and ileum (by approximately 2-fold), liver (approximately 3-fold), and spleen (approximately 5-fold), whereas the ratio was reduced in stomach. In urine and spleen, this metabolic shift persisted after 7-9 h. The 5HTOL level in bile was increased by approximately 3.5-fold after 8 h. 5HIAA was not detectable in bile. The present results indicate that the rat has a much higher proportion of 5HTOL formation than man under normal conditions. The rat does not appear to be an ideal model for studying the interaction between ethanol and 5HT metabolism in man.     

Structural studies of the cell envelope lipopolysaccharides from Haemophilus ducreyi strains ITM 2665 and ITM 4747

The structures of the lipopolysaccharides from Haemophilus ducreyi strains ITM 2665 and ITM 4747 have been investigated. Oligosaccharides were obtained from phenol/water-extracted lipopolysaccharide by mild acid hydrolysis and were studied with methylation analysis, fast atom bombardment-mass spectrometry, and NMR spectroscopy. The major oligosaccharide obtained from strain 2665 is a nonasaccharide with the following structure: beta-D-Galp-1-->4-beta-D-GlcNAcp-1-->3-beta-D-Galp-1-->4-D-alpha-D -Hepp- 1-->6-beta-D-Glcp-1-->(L-alpha-D-Hepp-1-->2-L-alpha-D-Hepp-1 -->3)-4-L-alpha- D-Hepp-Kdo, where the reducing terminal 3-deoxy-D-manno-octulosonic acid (or Kdo) exists in reduced anhydro forms. The proposed structure complements the preliminary structure described for Haemophilus ducreyi strain 35000 (Melaugh, W., Phillips, N. J., Campagnari, A. A., Karalus, R., and Gibson, B. W. (1992) J. Biol. Chem. 267, 13434-13439) with the missing anomeric configurations. The saccharide isolated from strain 4747 is a markedly simpler hexasaccharide with the following structure: beta-D-Galp-1-->4-beta-D-Glcp-1-->(L-alpha-D-Hepp-1-->2-L-alpha-D- Hepp- 1-->3)4-L-alpha-D-Hepp-Kdo. Apart from a different phosphorylation of the inner core region the proposed structure is identical to the structure of lipopolysaccharide from an only distantly related bacterium, viz. Haemophilus influenzae nontypable strain 2019 (Phillips, N. J., Apicella, M. A., Griffiss, J. M., and Gibson, B.W. (1992) Biochemistry 31, 4515-4526). The implications of these findings as regards the role of lipopolysaccharide as a virulence factor are discussed.     

Structural studies of the O-antigenic polysaccharide from Escherichia coli O167

The structure of the O-antigenic polysaccharide from Escherichia coli O167:H5 has been investigated. Sugar and methylation analyses, fast-atom-bombardment mass spectrometry and 1H- and 13C-NMR spectroscopy were the main methods used. The structure of the repeating unit of the polysaccharide was found to be: [formula in text]. Oligosaccharide derivatives of the polysaccharide were obtained by HF solvolysis and by a Smith degradation. Furthermore, base treatment of the polysaccharide led to a degraded polymeric material. For the methylated polysaccharide the amide linkage between alanine and the galacturonic acid residue was reductively cleaved with LiBD4 in ethanol, to give, among other things, a 3-O-methyl galactose derivative.