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24xi--Methylcholestane-3beta, 5alpha, 6beta, 12beta, 25-pentol 25-monoacetate has been isolated from an Alyconarian and its structure was established in part through extensive high resolution mass spectral and nmr studies and partly through the nonidentity of one of its degradation products with 24xi-methylcholestane-3beta, 5alpha, 6beta, 12alpha-tetrol synthesized from deoxycholic acid.

A proteinase inhibitor for elastases was isolated from extracts of the sea anemone Anemonia sulcata and purified to apparent homogeneity. The procedure comprises ethanolic extraction of the deep-frozen animals followed by gel filtration on Sephadex G-50 and by ion exchange chromatography on DEAE-Sephadex A-25 and SP-Sephadex C-25 and by hydroxylapatite chromatography. The slightly acidic inhibitor (isoelectric point 5.9) is a small protein consisting of 48 amino-acid residues without tryptophan and phenylalanine. The single chain molecule contains two methionines and no free sulfhydryl group but six cysteines presumably forming disulfide bonds. Reaction with cyanogen bromide abolishes the inhibitory properties. The inhibitor exhibits a rather narrow specificity for elastases. It strongly inhibits porcine pancreatic elastase in a permanent fashion with an equilibrium dissociation constant Ki of about 10(-10)M and somewhat weaker the elastase from human leucocytes with a Ki of about 10(-7)M. No obvious inhibition is observed of other serine proteinase such as bovine trypsin, bovine chymotrypsin, subtilisin from Bacillus subtilis and cathepsin G from human leucocytes when tested with synthetic substrates.

Amino acid sequence of neurotoxin I isolated from the anemone Radianthus macrodactylus, and consisted of 48 amino acid residues, including six cysteines, was determined by analysis of products of its trypsin and chymotrypsin digestion.

Intraspecific aggressive interactions have been observed in the scleractinian coral Stylophora pistillata. Extensive field observations suggest that intraspecific interactions result in the formation of necrosis in one of the two competing colonies. The formation of necroses is primarily restricted to those layers that are interspersed with polyps. In a second phase the non-living, central corallum is destroyed by foreign microorganisms. In some cases the necroses are resealed again, either by a living septal layer or by a calcareous plug. Evidence is presented indicating that the enzyme carbonic anhydrase has a role in these activities. First, it is involved in the apical growth of the colony. Using a semiquantitative assay procedure, the highest enzyme level was determined in polyps at the tips of the branches; lower levels were measured in polyps of the foot/trunk region. Secondly, the enzyme was found to be induced in those areas that are in close contact with non-isogeneic tissue. From these data we conclude that carbonic anhydrase is involved in the process of autolytic digestion in response to intraspecific contact of two non-clonemates.

We tested the hypothesis that kinetic differences among allelic variants of glucose-6-phosphate isomerase (GPI; D-glucose-6-phosphate ketol-isomerase, EC 5.3.1.9) from the sea anemone Metridium senile differentially modulate glucose metabolism at the glycolysis-pentose-shunt branch point. Fractional contribution of pentose shunt and absolute flux of glucose in glycolysis were measured in fasted or fed anemones acclimated to 5 degrees C or 15 degrees C. When fed, anemones of genotype Gpiss routed a greater fraction of glucose through the shunt than did Gpiff anemones; the effect was more pronounced at 5 degrees C than at 15 degrees C. This confirms predictions from kinetic and population data and is consistent with thermal selection maintaining the variation. Relative levels of shunt metabolism increased at 5 degrees C, compared with 15 degrees C, in fed anemones regardless of genotype, but the proportion of glucose metabolized by the pentose shunt was unchanged by temperature in fasted anemones. Glucose flux through the shunt was constant at approximately 5 pmol.mg-1.hr-1 in fed anemones at 5 degrees C and 15 degrees C and in fasted anemones at 15 degrees C, indicating apparently near-perfect thermal acclimation of the absolute flux of glucose through the shunt in fed, but not in fasted, anemones. Rates of glucose oxidation and flux through the shunt in freshly collected anemones were similar to those of anemones fed and acclimated at 15 degrees C in the laboratory. If these differences affect organismal-level processes, Gpi variation could contribute to Darwinian fitness in thermally varying environments.

The small, monomeric Ca2+-binding photoprotein, aequorin, emits blue light by an intramolecular reaction when mixed with Ca2+. The photoprotein is made up of coelenterazine and molecular oxygen, bound noncovalently to apoaequorin (apoprotein). The chemical steps leading to light emission, involving the oxidative degradation of coelenterazine, have been studied extensively, but little is known about the active site and how the molecule catalyzes the oxidation of coelenterazine. The three-dimensional structure of the protein has not been determined and therefore answers to these questions have remained unavailable. The present paper describes a procedure for preparing fairly large amounts of apoaequorin and aequorin for X-ray crystallographic studies. It consists of fusing the apoaequorin cDNA to the signal peptide coding sequence of the outer membrane protein A of Escherichia coli, which is under the control of the lipoprotein promoter. When the cDNA was expressed in E. coli, a large excess of the recombinant protein was produced and released into the culture medium. Purification of the protein was accomplished by acid precipitation and DEAE-cellulose chromatography. The procedure yielded 7.4 mg of recombinant apoaequorin with a purity greater than 95% from 200 ml of culture medium. On regeneration with coelenterazine, the recombinant aequorin was fully active with Ca2+.

The amino-acid sequence of the proteinase inhibitor specific for elastases from the sea anemone Anemonia sulcata was determined from performic-acid oxidized inhibitor and from three cyanogen bromide fragments of reduced and carboxymethylated inhibitor. The molecule consists of a single polypeptide chain formed from 48 amino-acid residues and is stabilized by three intramolecular disulfide bridges. After cyanogen bromide cleavage of the native protein at methionines 10 and 28 followed by chymotryptic cleavage two fragments each containing a single disulfide bridge were isolated. These indicated the location of three intramolecular disulfide linkages between Cys4 and Cys34 (part of A-loop), Cys8 and Cys27 (B-loop) and Cys16 and Cys48 (C-loop). The sequential homology and the disulfide pattern identified the elastase inhibitor as a Kazal-type inhibitor in which, however, not only the CysI-CysII segment is rather short but interestingly the Cys4-Cys34 disulfide anchoring point (i.e. CysI-CysV) in the C-loop is shifted by one turn in the alpha-helical segment towards the C-terminus. Thus, the elastase inhibitor is a non-classical Kazal-type inhibitor with respect to the positioning of the half-cystines. The inhibitor molecule was modelled based on the known three-dimensional structure of the silver pheasant ovomucoid third domain. The shortened amino-terminal segment was arranged in such a manner to allow disulfide bridge formation between the first cysteine Cys4 and the replaced Cys34 under maintenance of a suitable binding loop conformation. The characteristic ovomucoid scaffold consisting of a central alpha-helix, an adjacent three-stranded beta-sheet and the proteinase-binding loop cross-connected through disulfide bridges CysI-CysV and CysIII-CysVI was conserved.

A Ca2+-triggered luciferin-binding protein (BP-LH2) from the bioluminescent marine coelenterate, Renilla reniformis, has been purified by conventional methods. One kilogram of processed animals yields approximately 2.7 mg of pure protein with an overall yield of 55%. Physicochemical studies show that BP-LH2 is a globular protein containing one single polypeptide chain with one disulfide bond. Ultracentrifugation studies, amino acid analysis, and sodium dodecyl sulfate-gel electrophoresis show that BP-LH2 has an average molecular weight of 18,500. BP-LH2 has a Stokes radius of 23 A, a sedimentation coefficient, S020,w, of 2.3 S, and an isoelectric point of 4.3. The acidic nature of the protein was confirmed by amino acid analysis, which showed that 27% of the residues are acidic. The protein contains no carbohydrate, phosphate, or tryptophan. There is one noncovalently bound molecule of coelenterate type luciferin resulting in distinct protein spectral properties with absorption maxima at 276 nm (epsilon 0.1% 276 = 1.31) and 446 nm (episoln 0.1% 446 = 0.47) and a fluorescence emission at 520 nm (uncorrected). In the presence of Ca2+, BP-LH2 will react with Renilla luciferase to give the characteristic in vitro blue bioluminescence. Ca2+ binding produces a distinct change in the spectral properties of BP-LH2 including a 4-fold enhancement of tyrosine fluorescence at 332 nm and a 5-fold fluorescence enhancement at 520 nm. In addition, the visible absorption maximum shifts from 446 nm to 420 nm. The fluorescence enhancement at 320 nm occurs over the range from 1 to 10 micrometer Ca2+. BP-LH2 has two Ca2+-binding sites with an estimated Kd of 0.02 micrometer, in 10 muM Tris at pH 7.2. BP-LH2 was compared to several well studied Ca2+-binding proteins and was found to possess similar Ca2+-binding and physicochemical properties. This study clearly demonstrates that BP-LH2 is capable of triggering a bioluminescent flash in response to an intracellular Ca2+ transient.

High-field pulsed Fourier-transform nuclear magnetic resonance spectroscopy (NMR) was used to quantify the adenylate levels of sea anemones (Aiptasia pulchella) with and without symbiotic dinoflagellates (Symbiodinium sp.). Animals were fed to repletion, then starved in darkness for up to six days before collection of in vivo NMR spectra. The host adenylate ratio of ATP: (ATP + ADP) declined significantly with increasing periods of starvation in both symbiotic and aposymbiotic hosts (P less than 0.05). However, the decline in the animal adenylate ratio was significantly more rapid in animals bearing symbiotic algae (P less than 0.05). This suggests that symbiotic algae in darkness cause more rapid depletion of host energy reserves, possibly by drawing on host pools of organic substrates. In vivo NMR spectroscopy was also used to evaluate the effect on A. pulchella of photosynthesis by zooxanthellae. Symbiotic anemones were fed to repletion, then starved under high irradiance (300 to 320 mu Ein m-2 s-1) or low irradiance (70 to 80 mu Ein m-2 s-1) conditions for up to five days. The host adenylate ratio declined significantly (P less than 0.01) with starvation under both treatments, but no significant difference was detected between treatments (P greater than 0.35). Blotted wet weight of anemones under high and low irradiance declined by 50% over eight days of starvation, but there was no significant difference in the rate of weight loss by anemones in the two treatments. There results suggest that translocation of photosynthate from symbiotic zooxanthellae does not significantly affect host adenylate ratio or have a sparing effect on host biomass during starvation in this symbiotic sea anemone.

This paper reports the results of a pharmacological survey of drug action on muscle preparations of the anthozoan Bunodosoma caissarum. A critical review of previous results of drug action on Coelenterate neuromuscular junction is also given. The preparations responded only to body wall homogenates and acetylcholine. Sympathomimeticamines (adrenaline and nor-adrenaline), indolalkylamines (tryptamine, serotonin) and other drugs (tyramine, histamine, 1-glutamate and GABA) showed no action. The view that the Coelenterates do not employ acetylcholine as a neurotransmitter is discussed.

The southern Japan's soft coral, Sarcophyton glaucum, was found to contain several polyhydroxylates steroids. One of the minor components was isolated and its structure was established as 24 xi-methylcholestane-1 beta, 3 beta, 5 alpha, 6 beta, 25-pentol 25-monoacetate from spectral evidence and from comparison with a reference compound, 5 alpha-spirostan-1 beta, 3 beta, 5 alpha, 6 beta-tetrol, which was synthesized from ruscogenin. A mixture of 1 beta, 3 beta, 5 alpha, 6 beta-tetra hydroxy C27- and C28-sterols was also isolated.

Using immunocytochemistry, gastrin/CCK-like immunoreactivity is found in sensory nerve cells in the ectoderm of the mouth region of hydra and in nerve cells in the endoderm of all body regions of the sea anemone tealia. These results are corroborated by radioimmunoassay: One hydra contains at least 5 fmole and one tealia at least 2 nmole gastrin/CCK-like immunoreactivity. Reactivities towards gastrin and CCK antisera with different specificities suggest that the coelenterate gastrin/CCK-like peptide contains the C-terminal amino-acid sequence common to mammalian gastrin and CCK. In addition the radioimmunochemical data indicate that the coelenterate peptide also contains an amino-acid sequence that resembles the sequence 20-30 of porcine CCK-33, but that no other sequences of gastrin are present. Thus, it is probably more CCK-like than gastrin-like.

Analysis of substances involved in light-emitting reactions among bioluminescent coelenterates has revealed a pronounced uniformity in the structural features of initial reactants, i.e., "luciferins" and photo-protein chromophores, as well as the light-emitter product. This product is structurally identical among the different classes of coelenterates: Hydrozoa (the jellyfish, Aequorea), Anthozoa (the sea cactus, Cavernularia; sea pansy, Renilla; and sea pen, Leioptilus), and very likely also the Scyphozoa (the jellyfish, Pelagia). In each of these instances the reaction product, namely, 2-(p-hydroxy-pnenylacetyl)amino-3-benzyl-5-(p-hydroxyphenyl) pyrazine, is the actual light-emitter, whether it occurs in a Ca2+-triggered photoprotein type of luminescence, or in a "luciferin-luciferase" type. The evidence indicates that in certain coelenterates, e.g., Cavernularia, these two types are equally significant, whereas in others (Renilla and Leioptilus) the "luciferin-luciferase" type predominates over the Ca-triggerable photoprotein type, and finally that only the photoprotein type functions in the luciferaseless jellyfish, Aequorea. In all instances investigated, the structure of the light-emitter prior to the luminescence reaction appears to be essentially the same as that of the chromophore of unreacted aequorin. The product of the luminescence reaction is absent in extracts of non luminous species. However, a product very similar to that of luminescent coelenterates occurs also in representatives of other phyla, including the cephalopod molluscs, e.g., the "firefly squid" Watasenia and probably various ctenophores as well.

The regional differences in the sensitivity of protein synthesis and free radical processes to temperature, trypsin, urea and LiCl were studied in Obelia flexuosa by means of autoradiography. The regional differences were also determined with respect to the rate of incorporation and excretion of a labelled aminoacid under the normal conditions. The metabolic reactions of larvae can be divided in primary (the first 30 min following the effect) and subsequent adaptive ones. The primary reactions are characterized by the greater sensitivity of the anterior larval regions to all factors under study. The subsequent reactions are characterized by synchronous and unidirectional metabolic starts in both the anterior and posterior larval regions, the starts being bigger in the anterior regions. The restoration of the normal ratios of metabolic activities of the opposite larval regions does not always correlate with the restoration of the normal absolute level of metabolism. The adaptive reactions are better expressed for protein synthesis, rather than for free radical processes. The anterior larval region has the greatest metabolic non-stability by a series of indices.

A mathematical model has been suggested to describe the kinetics of the prostanoid biosynthesis in the Plexaura homomalla coral. It allows to predict the changes in prostaglandin A2 concentration at various pH and concentration of sodium ions citrate when the latter is not too high. The degradation of prostaglandin biosynthesis intermediates is shown to proceed as two consecutive first-order reactions. For the second step the reaction rate grows into the raise of prostaglandin A2 concentration, apparently due to autocatalytic character of the process.

Alcian blue dye normally binds to polyanionic, polymeric substances. Such structures are often associated with calcium binding portions of the organic matrix in calcifying tissues. The organic matrix of spicules prepared from the gorgonian Pseudoplexaura flagellosa (Houttuyn) is alcianophilic. The dye is very tightly bound to the lipoid portion of the insoluble spicule matrix. No acidic substances (sulfated or acidic polysaccharides or phospholipids) were demonstrable in this material, suggesting an unusual but unknown interaction between dye and substrate. On a microscopical basis, inclusion of Alcian blue (or Ruthenium red) is an essential co-requisite to glutaraldehyde fixation. Without the dye the morphological integrity of the spicule is lost on decalcification. The fragmented matrix is still alcianophilic suggesting that the dye may substitute for material solubilized by the decalcifying agents. Examination of post-decalcification supernatants demonstrate that approximately 13% of the matrix is solubilized on demineralization, releasing 93% of the carbohydrate but less than 20% of the protein. Liberated protein takes the form of peptides ranging from 1100-1500 daltons. The composition of these peptides is a function of the demineralizing agent. Acidic demineralizers produce peptides proportionately high in acidic amino acids, that do not bind calcium. Peptides produced by chelator decalcification appear to bind calcium but other evidence strongly suggests that the binding is due to adsorbed chelator rather than by soluble matrix.

The incubation of planulae Obelia loveni in the actinomycin D solution (40 mug/ml) for 3 hrs at 17--20 degrees resulted in the marked inhibition of the process of attachment of planulae to the substrate and their transformation in polyps. A suggestion is put forward that at the final phase of larval development a special RNA is synthesized which participates in the genetically determined control of the complicated morphogenetic phenomenon of attachment and transformation of the planula in polyp.

This paper is a brief review of the diterpenoid chemistry of gorgonian corals of the genus Pseudopterogorgia. Previously described compounds and several new diterpenoids are presented in the context of their potential utilization in the development of anti-inflammatory and analgesic drugs.

Analyses of fatty acids with carbon numbers between C12 and C22 are reported for five Great Barrier Reef sponges. These analyses indicate that phototrophic cyanobacterial symbionts (blue-green algae) present in three of the sponges are chemically distinct, whereas the other two sponges do not contain cyanobacterial symbionts. All the sponges contain other, nonphototrophic bacteria. The fatty acid analyses indicate that the non-phototrophic bacterial populations present in the different sponges are distinct in both their chemical compositions and their abundances. Nonphototrophic bacteria are estimated to account for between 60 and 350 micrograms/g (extractable fatty acids:tissue wet weight), whereas cyanobacteria account for between 10 and 910 micrograms/g. One sponge (Pseudaxinyssa sp.) contains a relatively large amount of the isoprenoid acid, 4, 8, 12-trimethyltridecanoic acid; this acid is presumed to be derived from phytol, a degradation product of chlorophyll. This sponge also contains relatively large amounts of the nonmethylene interrupted fatty acid, octadeca-5,9-dienoic acid. Analyses of interior and cyanobacteria-rich surface tissues of this sponge indicate that these two acids are probably not associated with the symbiotic cyanobacteria.

1. After separation by SDS gel-chromatography, analysis of AEP-containing glycoproteins from M. senile, indicated 66% amino acids with 220 AEP res./1000 res. and 30% carbohydrate for high mol. wt (greater than 10(7) forms and 80% amino acids with 25-50 AEP res./1000 res. and 10% carbohydrate for low mol. wt (2-4 x 10(4) forms. 2. Uronic acids, sulfate, lipid, and sialic acids were absent. 3. Mild base digestion released AEP-hexosamine containing oligosaccharides and destroyed ser-thr residues in the high mol. wt components. 4. Phosphonoglycoproteins appear to be acidic connective tissue components with AEP linked to hexosamine containing oligosaccharide side chains.

Photoproteins, which emit light in an oxygen-independent intramolecular reaction initiated by calcium ions, have been isolated from several bioluminescent organisms, including the hydrozoan jellyfish Aequorea and the ctenophore Mnemiopsis. The system of a related anthozoan coelenterate, the sea pansy Renilla reniformis, however, is oxygen dependent, requiring two organic components, luciferin and luciferase. Previously published indirect evidence indicates that photoproteins may contain a Renilla-type luciferin. We have now extracted in high yield a Renilla-type luciferin from three photoproteins, aequorin (45% yield), mnemiopsin (98% yield), and berovin (85% yield). Photoprotein luciferin, released from the holoprotein by mercaptoethanol treatment and separated from apo-photoprotein by gel filtration, no longer responds to calcium but now requires luciferase and O2 for light production. Photoprotein luciferin is identical to Renilla luciferin with respect to reaction kinetics and bioluminescence spectral distribution. In view of these results, the generally accepted hypothesis that the photoprotein chromophore is a protein-stabilized hydroperoxide of luciferin must be modified. We believe, instead, that the chromophore is free luciferin and that oxygen is bound as an oxygenated derivative of an amino-acid side chain of the protein. We propose the general term "coelenterate luciferin" to describe the light-producing chromophore from all bioluminescent coelenterates and ctenophores.

A furanocembranolide, 11 beta, 12 beta-epoxypukalide (1), C21H24O7, was isolated in trace quantities from the gorgonian Leptogorgia setacea. The structure was determined from detailed 1H-nmr and 13C-nmr analyses and comparison of these spectral data with those of the closely related compounds pukalide (2) and lophotoxin (3). The ultimate origin of 1 is discussed briefly.

Metabolic adjustments occurring during air exposure have been studied in a population of Actinia equina submitted to long-lasting emersion periods. Oxygen consumption upon reimmersion seems to account for the discharge of an oxygen debt related to the accumulation of end-products derived from anaerobic pathways. Incorporation of radioactive labelled glucose into both submerged and air exposed animals has allowed identification of these final products. A correspondence between the length of exposure-time and the amount of oxygen debt is encountered, which parallels the evolution of the rate of radioactivity incorporation into amino acids. Increasing levels of amino acid synthesis from glucose in the exposed conditions appear as the main metabolic feature.

Four species of gorgonians: three related pseudoplexaurids Pseudoplexaura porosa, P. flagellosa and P. wagenaari; and the unrelated Pseudopterogorgia americana, are sources of zooxanthellae capable, in purified broken cell preparations, of converting [14C]labeled farnesyl pyrophosphate into squalene. More extensive studies with P. porosa and P. americana zooxanthellae preparations characterize the conversion as enzymatic and demonstrate farnesyl pyrophosphate and reduced pyridine nucleotide as substrates. NADPH and NADH are essentially equivalent. Anaerobic conditions are not required. Despite numerous attempts zooxanthellae formation of sterols, including gorgosterol, from radioactive substrates was unsuccessful. By enzyme studies, we can show only the conversion of mevalonate into squalene as a zooxanthellae capability.

The green-fluorescent protein (GFP) that functions as a bioluminescence energy transfer acceptor in the jellyfish Aequorea has been renatured with up to 90% yield following acid, base, or guanidine denaturation. Renaturation, following pH neutralization or simple dilution of guanidine, proceeds with a half-recovery time of less than 5 min as measured by the return of visible fluorescence. Residual unrenatured protein has been quantitatively removed by chromatography on Sephadex G-75. The chromatographed, renatured GFP has corrected fluorescence excitation and emission spectra identical with those of the native protein at pH 7.0 (excitation lambda max = 398 nm; emission lambda max = 508 nm) and also at pH 12.2 (excitation lambda max = 476 nm; emission lambda max = 505 nm). With its peak position red-shifted 78 nm at pH 12.2, the Aequorea GFP excitation spectrum more closely resembles the excitation spectra of Renilla (sea pansy) and Phialidium (hydromedusan) GFPs at neutral pH. Visible absorption spectra of the native and renatured Aequorea green-fluorescent proteins at pH 7.0 are also identical, suggesting that the chromophore binding site has returned to its native state. Small differences in far-UV absorption and circular dichroism spectra, however, indicate that the renatured protein has not fully regained its native secondary structure.