Isolation of a protein subunit from microtubules.

Sea-urchin sperm tails (Strongylocentrotus purpuratus) were obtained by amputation in synthetic sea water and were purified by differential centrifugation. Most of the arms of the outer nine doublets and soluble matrix proteins were removed by this treatment. The central pairs of microtubules were dissolved by dialysis against EDTA at pH 7.5. The extract contained essentially a single component, with a sedimentation constant of 6S, in amounts sufficient to account for the protein content of the central pairs. Incubation of the extract with colchicine-(3)H gave binding levels approaching 0.5-1.0 mole of colchicine per 10(5) g protein. Sucrose-gradient analysis showed that the bound-radioactivity profile coincided with the optical-density profile of the 6S protein. It is concluded that the 6S colchicine-binding protein is a subunit of microtubules.

Properties of the protein subunit of central-pair and outer-doublet microtubules of sea urchin flagella.

The subunit protein has been isolated from the central-pair and outer-doublet microtubules of sea urchin sperm tails. Both proteins have a sedimentation constant of 6S and a molecular weight of 120,000. Both are converted to a 60,000 molecular weight species by denaturation in 6 M guanidine hydrochloride and reduction with mercaptoethanol. The reduced-alkylated proteins have the same R(f) on disc electrophoresis, and the same amino acid composition, which is very similar to that of muscle actin. The central-pair protein has one binding site for colchicine per 120,000 g. Both proteins appear to have a guanine nucleotide binding site, but the ability to bind GTP in solution has been demonstrated only for the central-pair protein. Although 1 mole of guanine nucleotide is bound per 60,000 g to outer-doublet tubules, the protein obtained by dissolving the doublets at pH 10.5 has lost the guanine nucleotide-binding site and also shows little or no colchicine-binding activity. Comparison of the properties of the isolated protein with electron microscopic evidence on structure of microtubules suggests that the chemical subunit (M = 120,000) consists of two of the 40 A morphological subunits.

Amino acid sequence of a major globin from the sea cucumber Paracaudina chilensis.

The sea cucumber Paracaudina chilensis (Echinodermata) contains three major globins I, II and III in coelomic cells. The complete amino acid sequence of globin I has been determined. It is composed of 157 amino acid residues, is acetylated at the N-terminus, and has a characteristic N-terminal extension of 9-10 residues when compared with vertebrate globins. The sequence of Paracaudina globin I showed slightly higher homology with human alpha globin (25%) rather than with the invertebrate Anadara alpha globin (22%). Paracaudina globin I also showed strong homology (59%) with globin D from another sea cucumber, Molpadia arenicola (Mauri, F.C. (1985) Ph.D. dissertation, University of Texas). The globin sequences from the phylum Echinodermata have an important position in the molecular evolution of the globins, because they are the invertebrate group most closely related to the vertebrates.

Oxygen equilibrium and subunit aggregation of a holothurian hemoglobin.

The hemoglobin of the sea cucumber Cucumaria miniata Brandt has a mol. wt of about 36000 in the oxy- form with a s20,w equal to 2.9 and a subunit molecular weight of 18000 by sodium dodecylsulfate gel electrophoresis. This pigment aggregates when deoxygenated to an oligomer with a s20,w equal to 4.7, an aggregation which is reversible upon subsequent oxygenation. The hemoglobin shows a sigmoid binding equilibrium with "n" equal to 1.8 and a decrease in oxygen affinity with an increase in pigment concentration. This hemoglobin is compared with other hemoglobins showing oxygenation-linked subunit aggregation.

Mössbauer spectroscopy of iron-containing dermal granules from Molpadia intermedia.

Dermal granules containing hydrous ferric oxide cores from Molpadia intermedia were studied by Mössbauer spectroscopy from 1.5 to 300 K and in magnetic fields up to 80 kOersted at 4.2 K. A magnetic phase transition to an antiferromagnetically ordered state is observed at 10 K. The results are compared with the magnetic behavior of micellar cores of ferritin from eukaryotes and iron-storage materials from prokaryotes.

The hemolysin-producer coelomocytes in Holothuria polii.

Using sodium metrizoate discontinuous gradients, two hemolysin-producer amebocyte populations have been separated from total circulating Holothuria polii coelomocytes. The amebocytes of population 1 are responsible for the production of the calcium-dependent and temperature-labile hemolysin, whereas those of population 2 produce the calcium-independent and temperature-stable one. The intracytoplasmic hemolysins were evidenced also by immunofluorescence. Petaloid and filipodial amebocytes were the only positive cell types.

Chlorinated insecticides and PCBs in Asteroidea and Holothuroidea species from Rijeka Bay, Yugoslavia.

For an evaluation of the extent of pollution in Rijeka Bay, Yugoslavia by chlorinated hydrocarbons an investigation of levels in sediments and the most abundant benthic organisms (Asteroidea and Holothuroidea species) has been carried out. Samples were collected at several stations in October 1981. The concentrations of chlorinated insecticides and polychlorinated biphenyls (PCBs) in Asteroidea species were from 0.5 to 46.0 micrograms kg-1 for DDTtotal, and from 2.5 to 435 micrograms kg-1 (wet weight) for PCBs. For the PCBs in Holothuroidea species the values ranged from 4.4 to 37.2 micrograms kg-1 and for DDTtotal from 0.9 to 48.5 micrograms kg-1. The contamination of Asteroidea and Holothuroidea samples from Rijeka Bay with persistent chlorinated hydrocarbons was compared with other data for the same species from other parts of the world. Accumulation of chlorinated hydrocarbons by Asteroidea and Holothuroidea species from sediments of Rijeka Bay is discussed with respect to concentration factors (CF). The highest CF (70.0) (dry biota/dry sediment) was obtained for the intestines of Asteroidea species for PCBs and a value of only 2.6 was obtained for DDTtotal for the skin and muscle of Holothuroidea species. The relatively low bioaccumulation of PCBs and DDTs from sediments in the Rijeka Bay by the Asteroidea and Holothuroidea species is reassuring, because of the problem of dredging and ocean dumping of sediments polluted with persistent chlorinated hydrocarbons.

[Single copy and repetitive nucleotide sequences in the genome of Echinodermata. II. Nucleotide sequence divergence of DNA of echinoderms]. / Unikal'nye i povtoriaiushchiesia nukleotidnye posledovatel'nosti v genome iglokozhikh. II. Divergentsiia nukleotidnykh posledovate l'nostei DNK u iglokozhikh.

The degree of divergence of short and long repetitive DNA sequences and single copy DNA of five Echinodermata species (sea urchins, starfish, sea-cucumber) was studied by the method of molecular hybridization. Different fractions of 3H-DNA of the sea urchin Strongylocentrotus intermedius were hybridized with the DNA of other species. Thermal stability of the hybridized DNA molecules was determined. The results obtained suggest that short repetitive sequences were most conservative during the evolution of Echinodermata. Single copy DNA fractions of closely related sea urchin species (S. intermedius and S. nudus) have more homologous sequences than long repetitive DNA fractions of the same species. The DNA of evolutionary distant species (sea urchin and starfish) have more homologous long repetitive sequences than the single copy ones. All DNA fractions of S. intermedius have sequences hybridized with the DNA of all other species studied: short repetitive sequences--55%, long repetitive sequences--20%, single copy sequences--12%.

[Single copy and repetitive DNA sequences of Echinodermata. I. Arrangement of DNA sequences of different multiplicity]. / Unikal'nye i povtoriaiushchiesia nukleotidnye posledovatel'nosti v genome iglokozhikh. I. Organizatsiia nukleotidnykh posledovatel'nostei razlichnoi stepeni mnozhestvennosti v DNK iglokozhikh.

Arrangement of repetitive and single copy DNA sequences in the DNA of 8 Echinodermata species (sea urchins, starfishes and sea-cucumber) has been studied. Comparison of the reassociation kinetics of short and long DNA fragments assayed by hydroxyapatite binding indicates that the pattern of DNA sequence organization of all these species is similar to the so called Xenopus pattern found in genomes of most animals and plants. Interspecies differences consist mainly in the quantities of sequences of various repetition degrees and their interspersion with each other and with single copy sequences. Measurements of the size of S1 nuclease resistant reassociated repetitive sequences show variability in the relative quantities of long and short repetitive sequences of different species. Difference in the arrangement of single copy and repetitive sequences between Echinodermata species are not related to their evolutionary proximity.

[A comparative study of the biogenic amine receptors in the muscle tissue of mollusks, echinoderms and tunicates]. / Sravnitel'noe izuchenie retseptorov biogennykh aminov v myshechnoi tkani molliuskov, iglokozhikh i obolochnikov.

Smooth muscles of Mollusca, Echinodermata and Tunicata contain one class of adrenoreceptors with the dissociation constant and maximal specific binding 2.5 and 82 in Anodonta, 2.3 and 320 in Holothuria, 4.9 pM and 232.5 fmol/mg of protein in ascidia. Catecholamines and their antagonists can be ranged in the row as follows: isoproterenol greater than adrenalin greater than propranolol-noradrenaline greater than phentolamine. Negative regulation of the beta-adrenoreceptor affinity to isoproterenol by means of guanine nucleotides (GN) was shown. The muscular tissues of Mollusca, Echinodermata and Tunicata have only one class of the serotonin receptors with the dissociation constant and maximal specific binding 120 and 13.2 in Anodonta, 88 and 192 in Holothuria, 2.6 pM and 54 fmol/mg of protein in ascidia. The GTP negative regulation of serotonin receptors affinity to the hormone was found. The GN regulation of the above receptors affinity to agonist suggests that muscle tissue of the above species has specific GTP [correction of GTR]-binding proteins capable of coupling with these receptors.

[Holotoxin content in the tissues of the holothurian Stichopus japonicus at different seasons of the year and their effect on oocyte maturation]. / Soderzhanie golotoksinov v tkaniakh goloturii Stichopus japonicus v raznye sezony goda i ikh vliianie na sozrevanie ootsitov.

Seasonal variations and distribution of holotoxins in tissues of the sea cucumber Stichopus japonicus have been studied. It was shown that holotoxin content in the ovaries is higher than that in other organs during the breeding season. In the course of a year, the amount of glycosides in other holothurian organs changed a little. It has been found out that high concentrations of glycosides inhibit oocyte maturation in the holothurian Stichopus japonicus.