Maculotoxin: a neurotoxin from the venom glands of the octopus Hapalochlaena maculosa identified as tetrodotoxin.

Maculotoxin, a potent neurotoxin isolated from the posterior salivary glands of the blue-ringed octopus. Hapalochlaena maculosa, has now been identified as tetrodotoxin. This is the first reported case in which tetrodotoxin has been found to occur in a venom.

Optical activity of octopus metarhodopsins.

The optical activity of octopus rhodopsin, acid metarhodopsin and alkaline metarhodopsin was studied by a sensitive and rapid CD apparatus. For sometime it has been thought that cephalopod metarhodopsins do not have any optical activity associated with their main absorption band. However, the present work shows that acid metarhodopsin in digitonin has a positive CD band at 498 nm and a negative CD band at 436 nm and alkaline metarhodopsin has a negative CD band at 381 nm. Detergent affected the wavelength of the CD peak of the visual pigments though the pattern of the spectrum was similar. From these results it is concluded that the conformation of all-trans retinal in octopus metarhodopsin is influenced by the asymmetric conformation of the protein near the retinal and therefore inducing optical activity.

Octopus rhodopsin. Amino acid sequence deduced from cDNA.

The primary structure of rhodopsin from the octopus Paroctopus defleini has been determined by parallel analysis of the protein and corresponding cDNA. The amino acid sequence is most similar to the recently cloned Drosophila opsins. Similarities to bovine and human opsins are also evident. The transmembrane topology of octopus rhodopsin is discussed.

High concentrations of dimethylamine and methylamine in squid and octopus and their implications in tumour aetiology.

The levels of the common secondary amines in various squid, in octopus and in 17 other seafoods were determined by HPLC. Ammonia and dimethylamine were found in all of the seafoods tested and some of them also contained methylamine and/or ethylamine. Particularly high levels of dimethylamine (946-2043 ppm) and methylamine (38-255 ppm) were detected in various species of squid and in the octopus. Reaction of nitrite in acidic medium with aqueous extract of squid yielded appreciable amounts of N-nitrosodimethylamine. The optimum pH for this reaction was around 2.4. Dimethylamine in dried squid tissues was readily extracted with water or 1% sodium carbonate solution. Heat treatment of dried squid at 200 degrees C was found to increase its amine content dramatically. It appeared that pyrolytic decarboxylation of some amino acids might cause this increase. Squid is a popular seafood in Japan and other oriental countries. The high incidence of stomach cancer in Japan and China is thought by epidemiologists to be associated with traditional Japanese and Chinese diets. Our present finding that squid and other seafoods contain unusually high levels of dimethylamine and other amines adds to the evidence that dietary factors may have an important role in the aetiology of stomach cancer and other gastro-intestinal tumours.

Histochemical detection of anionic components in the cephalopod brain.

Brains obtained from three species of mediterranean cephalopods (Loligo, Sepia, Octopus) were fixed in Bouin's solution. Paraffin sections were cut sequentially at the frontal plane and used for Alcian blue staining (critical electrolyte concentration method), colloidal iron hydroxide staining procedure, the periodic acid Schiff's reagent method, and the lead tetra-acetate-Schiff method. The stained sections were evaluated at 2 regions of different histological composition: the palliovisceral ganglion and the optic lobe. A high concentration of anionic components was found in synaptic regions of the neuropil whereas neuronal cell bodies showed a relatively weak staining of these constituents. There was a significant reaction of the perineuronal glia nets in the cellular rind of the palliovisceral ganglion. From the comparison of staining patterns obtained with the 4 methods in this study and literature data it can be concluded that the detected anionic sites are mainly carboxyl groups of acidic proteins and/or glycoproteins. Sulphate groups may be present in lower concentrations. Their distribution reveals that the role of anionic components other than sialic acids in the invertebrate brain might be discussed in the context of synaptic transmission similar to that in vertebrates. The possible involvement of the glia cell population has to be taken into consideration.

Heavy metals and organochlorine residues in marine animals and coastal sediments of the Tirrenean sea around Naples.

A survey of selected heavy metals (Cd, Pb, Hg) and organo-chlorine hydrocarbons (DDT and PCB compounds) concentrations in marine animals and of mercury, DDT and PCB in coastal sediments, began in 1973. The sampling was confined to the Bay of Naples, and to the coast along Santa Maria di Castellabate, a small village about 100 km south of Naples which has recently declared a marine park. The results of this investigation indicate that contamination by the above pollutants is low; the extent of contamination in the bay of Naples, compared to that of the marine park, shows that the level of heavy metals and organochlorine hydrocarbons in the benthic organisms is nearly the same within the two areas, whereas that of the sediments of the bay is significantly higher than in the park.

Hemocyanin of Octopus vulgaris. The molecular weight of the minimal functional subunit in 3 M urea.

In the presence of 3 M urea Octopus vulgaris (Mollusca) hemocyanin dissociates completely, giving a single functional component which reassociates in the original aggregate after removal of urea. The molecular weight of this subunit has been determined by gel filtration, by viscosity measurements, and by ultracentrifugation. The values obtrained with the different methods range from 247,000 to 262,000. Electron microscopy shows that the reassociation of the functional subunit after removal of urea is complete and gives the typical cylindrical structure of the native protein. This component is the minimal functional subunit which can be obtained from Octopus hemocyanin without splitting covalent bonds. It is suggested that this component is made by five protomers (50,000 daltons) containing one oxygen binding site each bound covalently through, perhaps, the carbohydrate moieties of the protein.

Removal of copper from Octopus vulgaris haemocyanin. Preparation of the half-apo and apo derivatives.

The two copper ions bound in the active site of Octopus vulgaris haemocyanin can be removed by cyanide. The two metal ions react with the ligand sequentially. In this paper the preparation of Octopus half-apo-haemocyanin, containing at the active site a single copper ion, is described. Moreover, the conditions to obtain Octopus apo-haemocyanin, containing less than 3% of copper still bound, are given.

Physicochemical characterization of the ribosomal RNA species of the Mollusca. Molecular weight, integrity and secondary-structure features of the RNA of the large and small ribosomal subunits.

1. The rRNA species of the Cephalopoda Octopus vulgaris and Loligo vulgaris were found to have unexpectedly high sedimentation coefficients and molecular weights. In 0.1 M-NaCl the L-rRNA (RNA from large ribosomal subunit) has the same s20 value as the L-rRNA of the mammals (30.7S), whereas the S-rRNA (RNA from small ribosomal subunit) sediments at a faster rate (20.1S) than the S-rRNA of both the mammals and the fungi (Neurospora crassa) (17.5S). The molecular weights of the L-rRNA were determined by gel electrophoresis in formamide and found to be 1.66 X 10(6) (Octupus) and 1.89 X 10(6) (Loligo); the mol.wt. of the S-rRNA of both species is 0.96 X 10(6), i.e. much larger than that of the mammals (0.65 X 10(6)) and almost coincident with that of the '23S' RNA of the prokaryotes. 2. By contrast, the less evolved Gastropoda and Lamellibranchiata (Murex trunculus and Macrocallista chione) have S-rRNA and L-rRNA species with mol.wts. of 0.65 X 10(6) and approx. 1.40 X 10(6).3. All the mature L-rRNA molecules of the cephalopoda are composed of two unequal fragments held together by regions of hydrogen-bonding having a similar, low, thermal stability in the two species; the molecular weights of the two fragments composing the L-rRNA are estimated to be 0.96 X 10(6) and 0.88 X 10(6) (Loligo) and 0.96 X 10(6) and 0.65 X 10(6) (Octupus). THe S-rRNA of both species is a continuous chain with exactly the same molecular weight (0.96 X 10(6)) as the heavier of the two fragments of the L-rRNA. 4. The secondary-structure features of the L-rRNA and S-rRNA species of the Caphalopoda were investigated by thermal 'melting' analysis in 4.0 M-guanidinium chloride; 60-70% of the residues are estimated to form short, independently 'melting' bihelical segments not more than 10 base-pairs in length. 5. Bases are unevenly distributed between non-helical and bihelical portions of the rRNA molecules, G and C residues being preferentially concentrated in bihelical comains. 6. The secondary-structure regions of the L-rRNA species of Octopus and Loligo are heterogenous, including two discrete fractions of independently 'melting' species that give rise to biphasic 'melting' profiles: a fraction consisting of shorter (G + C)-poorer segments (60-68% G + C, not more than 5 base-pairs in length) and a fraction consisting of longer (G + C)-richer segments (80-88% G + C, 5-10 base-pairs in length). No evidence for heterogeneity has been detected in the S-rRNa.

The identification of dansyl sarcosine and its occurrence in molluscs.

An unknown dansyl derivative was identified as dansyl sarcosine. In molluscs, sarcosine was found to be largely localized to the nervous system. Examination of individual snail neurones, regions of the octopus brain and the squid giant synapse showed dramatic variations in sarcosine levels.

Phosphoglycopeptide, a major constituent of the spermatophoric plasma of the octopus (Octopus dofleini martini).

A phosphoglycopeptide, accounting for approximately 90% of the characteristically high content of acid-soluble organically-bound phosphorus in the octopus spermatophoric plasma (4 mg P/ml), was identified. Electrophoretic and chromatographic purification, followed by chemical and enzymic hydrolysis, yielded D-galactose phosphate as a degradation product. The galactose and peptide moieties of the compound were linked via a phosphoryl rather than a glycosidic linkage but the peptide was devoid of aromatic amino acids.

Comparative histochemistry as a springboard for the discovery of biogenic amines and active peptides in molluscan tissues and amphibian skin.

Histochemistry has been the springboard for the identification in molluscan tissues (hypobranchial body and salivary glands) and amphibian skin of a number of biogenic amines, choline esters and active peptides. Among the biogenic amines 5-HT and related indolealkylamines, octopamine, tyramine and several histamines are to be listed; among the choline esters murexine, dihydromurexine, senecioylcholine and acryloylcholine; among the peptides an array of compounds belonging to at least ten different peptide families. 5-HT, octopamine, tyramine and histamine are present, together with representatives of some peptide families in the posterior salivary glands of octopods, the hypobranchial body of prosobranchiate molluscs and the amphibian skin. This points to the possible occurrence in the above structures of cells belonging to the Pearse's APUD series, intended in its broadest sense. Immunocytochemical studies and even ultrastructural studies on amine and peptide storing cells in molluscan tissues and amphibian skin are virtually lacking. This is certainly a gap that deserves to be filled, considering that these studies may help in elucidating problems of general relevance in invertebrates and vertebrates.

Histochemical evidence for catecholamines as neurotransmitters in the statocyst of Octopus vulgaris.

Formaldehyde-induced fluorescence (Falck-Hillarp technique) provided histochemical evidence for the presence of catecholamines in the sensory epithelia (macula and crista) of the Octopus statocyst. A specific bright green fluorescence occurred in the neuronal plexus beneath the receptor cell layers of the epithelia and in the appropriate nerves. The histochemical findings are discussed with reference to the well-known neuronal and synaptic organization of the epithelia and to relevant results in cephalopods as well as in other molluscs. All data support the hypothesis that in the receptor systems of the Octopus statocyst catecholamines (probably dopamine and/or noradrenaline) act as neurotransmitters in the efferent fibre system.

Luminescence of deoxyhemocyanin and deoxytyrosinase.

The deoxy form of hemocyanins and tyrosinases from certain species displays a weak low-energy luminescence when solutions of the protein are irradiated with light at approximately 290 nm. The emission most likely results from a copper-to-imidazole charge transfer state as shown by studies with a synthetic copper(I) complex having three imidazole ligands.

Structure and function of the carboxyl-terminal oxygen-binding domain from the subunit of Octopus dofleini hemocyanin.

The C-terminal domain, Od-1, of the 7-domain subunit of Octopus dofleini hemocyanin has been prepared by partial trypsinolysis followed by ion-exchange chromatography. It binds oxygen reversibly and is homogeneous in molecular weight. Its physical properties have been compared with those of the subunit. The domain molecular weight is found by sedimentation equilibrium to be 4.7 X 10(4), in excellent agreement with the result recently obtained in our laboratory from cDNA sequencing of this domain [Lang, W. H. (1988) Biochemistry (preceding paper in this issue)]. It has a sedimentation coefficient of 3.8 S. Both the molecular weight and sedimentation coefficient are consistent with the domain constituting approximately one-seventh of the Mr 3.5 X 10(5) subunit. Its amino acid composition and carbohydrate content differ significantly from that of the whole subunit, confirming the heterogeneity in domains previously established on an immunological basis. Circular dichroism predicts similar secondary structure for the domain and subunit. The domain does not self-associate in the presence of Mg2+ but does bind to the whole molecule in a ratio of approximately 1 domain/subunit. The oxygen affinity of this domain is quite low. It shows intrinsic magnesium and Bohr effects similar to those of the whole molecule but of greatly reduced magnitude.

The structure of arthropod and mollusc hemocyanins.

The hemocyanins from molluscs and from arthropods differ in the size of their polypeptide chains. A variety of physical techniques including sodium dodecyl sulfate polyacrylamide gel electrophoresis and column chromatography in sodium dodecyl sulfate and guanidine HCl indicate that the polypeptide chain of mollusc hemocyanin has a molecular weight of 290,000. These results were corroborated by quantitative end group analyses. Several experiments designed to count the number of tryptophan and methionine-containing peptides in the hemocyanin from the whelk Busycon canaliculatum indicate that sequence homology within the polypeptide chain of the mollusc hemocyanins accounts for their large size. Digestion of the native protein with subtilisin produces a 50,000-dalton fragment in high yield which corresponds to one binding site for oxygen. On the other hand, the polypeptide chain molecular weight of lobster hemocyanin is 76,000 to 78,000 and this seems to be a general property of all arthropod hemocyanins. The pigment from lobster consists of two very similar polypeptide chains which are not present in equal amount. Analysis of the cysteine-containing and of the tryptophan-containing tryptic peptides confirms the value of the molecular weight. However, separation of fragments which contain methionine indicates that there is sequence homology withing the polypeptide chain of this protein. It is concluded that the mollusc and arthropod hemocyanins have little structural similarity.