Acid-base titration of hemocyanin from Octopus vulgaris Lam.
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Amides↗
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A resonance Raman study of the C=N configurations of octopus rhodopsin, bathorhodopsin, and isorhodopsin.
The resonance Raman spectra of octopus rhodopsin, bathorhodopsin, and isorhodopsin at 120 K have been obtained as well as those of pigments regenerated with isotopically labeled retinals near the C14-C15 bond. Deuteration of the Schiff base nitrogen induces relatively large changes in the C-C stretch region between 1100 and 1300 cm-1, including a large frequency shift of the C14-C15 stretch mode located at 1206-1227 cm-1 in the three octopus species, as revealed by the Raman spectra of their 14,15-(13)C2 derivatives. Such results are different compared to those of the bovine pigments, in which no significant frequency shift of the C14-C15 stretch mode was observed upon Schiff base N deuteration. In an earlier Raman study of a Schiff base model compound which contained only one single bond adjacent to two double bonds, we have found that the stretch mode of this C-C single bond at 1232 cm-1 shifts up by 15 cm-1 and its intensity is also greatly reduced upon Schiff base N deuteration when the C=N configuration is anti [Deng et al., (1994) J. Phys. Chem. 98, 4776-4779]. The same study has also shown that when the C=N configuration is syn, the C-C stretch mode should be at about 1150 cm-1. Since the C14-C15 stretch mode frequency is relatively high in the spectra of octopus rhodopsin and bathorhodopsin (> 1200 cm-1) and since the normal mode pattern near the Schiff base is similar to the model, we suggest that the C=N configuration in these two species is anti. The different responses of the C14-C15 stretch mode to the Schiff base nitrogen deuteration in bovine and octopus pigments are due to the fact that the coupled C14-C15 stretch and the C12-C13 stretch motions in the model compound or in bovine rhodopsin are altered in octopus rhodopsin so that the stretch motion of the C14-15 bond is more localized, similar to the C-C stretch motion in the small Schiff base model compound. In clear contrast with the bovine rhodopsin Raman spectrum, which is very similar to that for the 11-cis-retinal Schiff base, the drastically different octopus rhodopsin spectrum indicates large protein perturbations on the C11=C12-C13 moiety, either by steric or by electrostatic interactions. Further studies are required to determine if such spectral differences indicate a difference of the energy conversion mechanism in the primary photochemical event of these two pigments.
Animals↗
Simple purification and functional reconstitution of octopus photoreceptor Gq, which couples rhodopsin to phospholipase C.
In invertebrate photoreceptors, illuminated rhodopsin activates multiple G proteins, which are assumed to initiate multiple phototransduction cascades. In this paper, we focused on one of the phototransduction cascades, which utilizes rhodopsin, a Gq-like G protein, and phospholipase C (PLC). A Gq-like G protein from octopus photoreceptors was successfully purified to apparent homogeneity as an active form by simple two-step chromatography. The purified G protein had an alpha beta gamma-trimeric structure consisting of 44-kDa alpha, 37-kDa beta, and 9-kDa gamma subunits. The 44-kDa alpha subunit was assigned to the Gq class by western blot with antiserum against mammalian Gq alpha and by partial amino acid sequencing of its proteolytic fragments. Light-dependent binding of GTP gamma S was observed when the purified octopus Gq was reconstituted with octopus rhodopsin that had been integrated into phospholipid vesicles. Octopus Gq activated PLC beta 1 purified from bovine brain dose-dependently in the presence of A1F4-. Finally, light- and GTP-dependent activation of PLC beta 1 was observed in a reconstitution system consisting of octopus rhodopsin, Gq, and bovine PLC beta 1.
Aluminum Compounds↗
Structural dynamics of water and the peptide backbone around the Schiff base associated with the light-activated process of octopus rhodopsin.
Difference Fourier transform infrared spectra were recorded for the formation of the photointermediates and isorhodopsin from octopus rhodopsin at low temperatures. Analysis was done for H bonding of the Schiff base, internal water molecules, and the peptide backbone. The imine hydrogen of the Schiff base was in the same H bonding state throughout the photointermediates and the unphotolyzed state. In contrast, H bonding of the hydrogen of the water molecule whose oxygen might be complexed with the imine hydrogen of the Schiff base was altered upon the formation of bathorhodopsin. The same water molecule was in a different H bonding state in the subsequent intermediates, lumirhodopsin and mesorhodopsin. These intermediates were also characterized by a decrease in the C = N bond order of the Schiff base as a reflection of distorted structure around the Schiff base. The polar N-H bond in these intermediates could be also ascribed to the Schiff base. Some changes in H bonding of water and the perturbation of the polyene chain in lumirhodopsin and mesorhodopsin were also observed in isorhodopsin. Acid metarhodopsin exhibited extensive changes in the H bonding states of the peptide backbone and internal water molecules. A large part of these changes was extinguished in alkaline metarhodopsin with the unprotonated Schiff base, suggesting interaction of the protonated Schiff base with the peptide backbone and intramembrane water molecules in acid metarhodopsin.
Animals↗
Isozyme multiplicity with anomalous dimer patterns in a class III alcohol dehydrogenase. Effects on the activity and quaternary structure of residue exchanges at "nonfunctional" sites in a native protein.
The isozymes of class III alcohol dehydrogenase/glutathione-dependent formaldehyde dehydrogenase from cod were characterized. They exhibited three unexpected properties of general interest. First, these dimeric isozymes, derived from two types of subunit (h and l, for high- and low-activity forms), were recovered from liver preparations in only the homodimeric ll and heterodimeric hl combinations. Dissociation and reassociation of the isolated hl form in vitro also resulted in lower yields of the hh than the ll homodimer, although class III subunits are usually freely associable over wide borders of divergence (human and Drosophila). The h and l primary structures show that both chain types are characteristic of class III enzymes, without large amino acid replacements at positions of known subunit interactions. Hence, the hh dimer partial restriction indicates nontraditional alterations at h-subunit interfaces. The structure provides a possible explanation, in the form of h-chain modifications that may influence the anchoring of a loop at positions of two potentially deamidative beta-aspartyl shifts at distant Asn-Gly structures. Second the ll and hl forms differ in enzymatic properties, having 5-fold different K(m) values for NAD+ at pH 8, different K(m) values for S-(hydroxymethyl)glutathione (10 versus 150 microM), and different specific activities (4.5 versus 41 units/mg), with ll resembling and hl deviating from human and other class III alcohol dehydrogenases. However, functional residues lining substrate and coenzyme pockets in the known conformations of homologous forms are largely identical in the two isozymes [only minor conservative exchanges of Val/Leu116, Val/Leu203, Ile/Val224, and Ile/Val269 (numbering system of the human class I enzyme)], again indicating effects from distantly positioned h-chain replacements. Third, the two isozymes differ a surprising amount in amino acid sequence (18%, the same as the piscine/ human difference), reflecting a remarkably old isozyme duplication or, more probably, discordant accumulation of residue exchanges with greater speed of evolution for one of the subunits (h chain) than is typical for the slowly evolving class III alcohol dehydrogenase.
Aldehyde Oxidoreductases↗
The paradigm that all oxygen-respiring eukaryotes have cytosolic CuZn-superoxide dismutase and that Mn-superoxide dismutase is localized to the mitochondria does not apply to a large group of marine arthropods.
The enzyme superoxide dismutase (SOD), which catalyzes the dismutation of the superoxide radical, is present in the cytosol and mitochondria of all oxygen-respiring eukaryotes. The cytosolic form contains copper and zinc (CuZnSOD), whereas the mitochondrial form contains manganese (MnSOD). The latter protein is synthesized in the cytosol as a MnSOD precursor, containing an N-terminal mitochondrial-targeting sequence. CuZnSOD is sensitive toward cyanide (CN) and hydrogen peroxide (H2O2), but MnSOD is not. Assays for SOD activity in cytosol from the hepatopancreas of the blue crab, Callinectes sapidus, showed the presence of a CN/H2O2-insensitive form of SOD. No CN/H2O2-sensitive CuZnSOD was found. This unexpected phenomenon was shown to occur in all decapod crustacea (crabs, lobsters, shrimp) examined. The cytosolic and mitochondrial SODs of C. sapidus were purified by means of ion-exchange, size-exclusion, and reverse-phase HPLC. The cytosolic SOD is a homodimeric protein, which exists in a monomer-dimer equilibrium (24 kDa left and right arrow 48 kDa). The protein contains approximately 1 Mn per subunit. No copper or zinc is present. Amino acid sequence analysis identified the novel cytosolic SOD as a MnSOD precursor with an abnormal mitochondrial-targeting sequence. The mitochondrial SOD of C. sapidus is similar to the MnSOD found in other eukaryotes. N-Terminal amino sequences of mitochondrial and cytosolic blue crab MnSOD differ in several positions. The MnSODs are thus encoded for by two different genes. The paradigm that all eukaryotes contain intracellular CuZnSOD and that MnSOD occurs exclusively in the mitochondria appears not to apply to a large group of marine arthropods.
Amino Acid Sequence↗
Ultraviolet resonance Raman examination of the light-induced protein structural changes in rhodopsin activation.
Ultraviolet resonance Raman (UVRR) spectra of rhodopsin and its metarhodopsin I and metarhodopsin II photointermediates have been obtained to examine the molecular mechanism of G-protein-coupled receptor activation. Spectra were acquired using a single-pass capillary flow technique in combination with a Littrow prism UV prefilter detection system. The UVRR difference spectra between rhodopsin and mearhodopsin I exhibit small differences assignalbe to tyrosine residues and no differences due to tryptophan. The UVRR difference spectra between rhodopsin and metarhodopsin II exhibit significant differences for vibrations of both tryptophan and tyrosine residues. Most importantly, there is and intensity decrease of the totally symmetric tryptophan modes at 759, 1008, and 1545 cm-1, an intensity decrease of the tryptophan W7 band at 1357 cm-1, and a frequency shift of the tryptophan W17 ban from 885 to 892 cm-1. These difference features are assigned to one or more tryptophan residues that reside in a hydrophobic, weakly hydrogen-bonding environment in rhodopsin and that are transferred to a less hydrophobic, non-hydrogen-bonding environment during rhodopsin activation. The available evidence suggests that Trp265 makes a dominant contribution to the tryptophan features in this difference spectrum. These results are interpreted with a model for rhodopsin activation in which retinal isomerization alters the interaction of Trp265 with the ionone ring of the retinal chromophore.
Animals↗
The enzymatic properties of Octopus vulgaris hemocyanin: o-diphenol oxidase activity.
Hemocyanin and tyrosinase are dinuclear copper proteins capable of reversibly binding dioxygen. Despite the great similarity of structure and properties of their active site, the two proteins perform different biological functions (oxygen transport/storage versus monooxygenase and oxidase activity). In this paper, we show that Octopus vulgaris hemocyanin exhibits a tyrosinase-like activity; namely, it is capable of utilizing dioxygen for the oxidation of o-diphenol to quinone. The reaction is specific for this isomer of diphenol, the meta and para isomers being unreactive, and is strongly controlled by steric factors. Dioxygen represents a cosubstrate of the reaction, and it is involved in the catalytic turnover by binding to the dinuclear copper site of the protein to form, under steady-state conditions, oxy-Hc, which is the active species. The generation of semiquinone radicals, detected by EPR and by their reaction with N,N,N',N'-tetramethyl-1,4-phenylenediamine, strongly supports a reaction mechanism in which such radicals represent the reaction products of one-electron oxidation of the substrate, quinone being generated by dismutation of semiquinones. Met-Hc is regenerated by the substrate to the deoxy form. To close the catalytic cycle, the proposed reaction mechanism also involves the participation of two transient protein forms with the total oxidation state of the active site (V and IV) intermediate between that of oxy-Hcy, [CuIIO22-CuII]VI, and deoxy-Hc, [CuICuI]II. A mathematical model has been elaborated to describe the reaction kinetics. The differences in reaction mechanisms between hemocyanin and tyrosinase are discussed in terms of accessibility to exogenous molecules of their active sites.
Animals↗
Significant enhancement of monooxygenase activity of oxygen carrier protein hemocyanin by urea.
Oxygenation of a series of p-substituted phenols to the corresponding catechols (phenolase activity) by the (mu-eta2:eta2-peroxo)dicopper(II) species of Octopus hemocyanin has been directly examined for the first time by using a UV-vis spectroscopic method in a 0.5 M borate buffer solution containing 8 M urea under anaerobic conditions. Preliminary kinetic studies have indicated that the reaction involves an electrophilic aromatic substitution mechanism as in the case of phenolase reaction of tyrosinase. The oxygenation of phenols by hemocyanin also proceeded catalytically when the reaction was carried out under aerobic conditions.
Animals↗
Isolation and partial characterization of two types of muscle collagen in some cephalopods.
Collagen from muscle of volador (Illex coindetii), pota (Toradopsis eblanae), and white octopus (Eledone cirrhosa) was characterized in terms of anatomical location, sex, and maturity. Collagen content was higher in arms than in mantle in all three species; there were also significant differences in octopus depending on the age of the individual. Concerning sex, the largest differences in the amount of collagen were found in relation to total protein content. In volador and pota, collagen solubility was higher in the mantle than in the arms, and in the case of pota there were also sex-related differences. In octopus males, solubility was higher in the arms. Two types of collagen, I and V, were identified as the principal constituents in all three species and at both anatomical locations (mantle and arms). The electrophoretic mobility of the alpha2 chain differed in the two types of collagen, but the amino acid compositions of the collagen were similar in the mantle and arms in all three species examined.
Amino Acids↗
Synthesis and study of thiocarbonate derivatives of choline as potential inhibitors of acetylcholinesterase.
Fourteen alkyl and aryl thiocarbonate derivatives of choline were synthesized and studied as potential inhibitors of acetylcholinesterase (AChE). Twelve of the compounds inhibited AChEs derived from calf forebrain, human red blood cells, and octopus brain ranging from low to moderately high inhibition potency. The concentration of each inhibitory compound giving 50% inhibition of enzyme activity (IC50 values, which ranged from 1 x 10(-2) to 8 x 10(-7) M) was determined and is reported; inhibitor constants (Ki values) for the most inhibitory compounds, (1-pentylthiocarbonyl)choline chloride and (1-heptylthiocarbonyl)choline chloride, were calculated from kinetic data and are also reported. The inhibitors are competitive with substrate, and they are not hydrolyzed by the AChE activities. Certain of these new compounds may provide direction for the development of new drugs that have anticholinesterase activity and may be used for the treatment of Alzheimer's disease.
Acetylcholinesterase↗
Inhibition of octopus glutathione transferase by Meisenheimer complex analog, S-(2,4,6-trinitrophenyl) glutathione.
The tight binding of Meisenheimer intermediate with octopus digestive gland glutathione transferase was analyzed with 1,3,5-trinitrobenzene, which forms a trapped Meisenheimer complex with glutathione because there is no leaving group at the ipso carbon. By steady-state enzyme kinetic analysis, an inhibition constant of 1.89 +/- 0.17 microM was found for the transient formed, S-(2,4,6-trinitrophenyl) glutathione. The above inhibition constant is 407-fold smaller than the Km value for the substrate (2,4-dinitrochlorobenzene). Thus, S-(2,4,6-trinitrophenyl) glutathione is considered to be a transition-state analog. The tight binding of this inhibitor to the enzyme provides an explanation for the involvement of the biological binding effect on the rate enhancement in the glutathione transferase-catalyzed SNAr mechanism.
Animals↗
Heat shock protein 70 and heat shock protein 90 expression in light- and dark-adapted adult octopus retinas.
Light- and dark-adaptation leads to changes in rhabdom morphology and photopigment distribution in the octopus retina. Molecular chaperones, including heat shock proteins (Hsps), may be involved in specific signaling pathways that cause changes in photoreceptor actin- and tubulin-based cytoskeletons and movement of the photopigments, rhodopsin and retinochrome. In this study, we used immunoblotting, in situ RT-PCR, immunofluorescence and confocal microscopy to localize the inducible form of Hsp70 and the larger Hsp90 in light- and dark-adapted and dorsal and ventral halves of adult octopus retinas. The Hsps showed differences in distribution between the light and dark and in dorsal vs. ventral position in the retina. Double labeling confocal microscopy co-localized Hsp70 with actin and tubulin, and Hsp90 with the photopigment, retinochrome. Our results demonstrate the presence of Hsp70 and Hsp90 in otherwise non-stressed light- and dark-adapted octopus retinas. These Hsps may help stabilize the cytoskeleton, important for rhabdom structure, and are perhaps involved in the redistribution of retinochrome in conditions of light and dark.
Adaptation, Ocular↗
The health benefit of seafood.
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Animals↗
Appetitive conditioning in Octopus cyanea.
The performance of Octopus cyanea was studied in 3 appetitive conditioning situations. In Experiment 1, 2 groups were trained in a runway; a large reward produced faster acquisition when reinforcement was consistent and better subsequent performance on a partial schedule than did a small reward. In Experiment 2, activity in the vicinity of a feeder was measured, and in Experiment 3, latency and probability of response were measured in an automated version of a traditional conditioned attack situation (Boycott & Young, 1950). There was evidence of acquisition with continuous reinforcement in both experiments but in neither with partial reinforcement. All of the results can be understood in terms of growth and decline in the strength of stimulus-reinforcer associations with reinforcement and nonreinforcement.
Animals↗
How do octopuses use their arms?
A taxonomy of the movement patterns of the 8 flexible arms of octopuses is constructed. Components consist of movements of the arm itself, the ventral suckers and their stalks, as well as the relative position of arms and the skin web between them. Within 1 arm, combinations of components result in a variety of behaviors. At the level of all arms, 1 group of behaviors is described as postures, on the basis of the spread of all arms and the web to make a 2-dimensional surface whose position differs in the 3rd dimension. Another group of arm behaviors is actions, more or less coordinated and involving several to all arms. Arm control appears to be based on radial symmetry, relative equipotentiality of all arms, relative independence of each arm, and separability of components within the arm. The types and coordination of arm behaviors are discussed with relationship to biomechanical limits, muscle structures, and neuronal programming.
Animals↗
Experimental evidence for spatial learning on octopuses (octopus bimaculoides).
Octopuses forage far from temporary home dens to which they return for shelter. Spatial tasks may assess learning. Octopuses (Octopus bimaculoides) were placed in a novel arena, and their movements were tracked for 72 hr. Movements around the arena decreased across time, consistent with exploratory learning. Next, octopuses were given 23 hr to move around an arena; after a 24-hr delay, their memory of a burrow location was tested. Most remembered the location of the open burrow, demonstrating learning in 1 day. Finally, octopuses were trained to locate a single open escape burrow among 6 possible locations. Retention was tested after a week and was immediately followed by reversal training (location rotated 180 degrees ). Octopuses learned the original location of the burrow, remembering it for a week. Path lengths increased significantly after reversal, gradually improving and showing relearning. Octopuses show exploratory behavior, learning, and retention of spatial information.
Animals↗
When do octopuses play? Effects of repeated testing, object type, age, and food deprivation on object play in Octopus vulgaris.
Studying play behavior in octopuses is an important step toward understanding the phylogenetic origins and function of play as well as the cognitive abilities of invertebrates. Fourteen Octopus vulgaris (7 subadults and 7 adults) were presented 2 Lego objects and 2 different food items on 7 consecutive days under 2 different levels of food deprivation. Nine subjects showed play-like behavior with the Lego objects. There was no significant difference in play-like behavior corresponding to food deprivation, age, and sex of the octopuses. The sequence of behaviors, from exploration to play-like behavior, had a significant influence on the establishment of play-like behavior, as it occurred mostly on Days 3-6 of the 7-day experiment. The pattern of development of play-like activities after a period of exploration and habituation in this study agrees with the hypothesis that object play follows object exploration. A homologous origin of this behavioral trait in vertebrates and invertebrates is highly unlikely, as the last common ancestor might not have had the cognitive capacity to possess this trait.
Animals↗