Search

By quantitative sodium dodecyl sulfate-polyacrylamide gel electrophoresis, paramyosin:myosin heavy chain molecular ratios were calculated for three molluscan muscles:Aequipecten striated adductor, Mercenaria opaque adductor, and Mytilus anterior byssus retractor; and four arthropodan muscles:Limulus telson, Homarus slow claw. Balanus scutal depressor, and Lethocerus air tube retractor. These ratios correlate positively with both thick filament dimensions and maximum active tension development in these tissues. The role of paramyosin in these muscles is discussed with respect to the following characteristics: force development, "catch," and extreme reversible changes in length.

By sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunodiffusion, we identified paramyosin in two smooth invertebrate "catch" muscles (Mytilus anterior byssus retractor and Mercenaria opaque adductor) and five invertebrate striated muscles (Limulus telson levator, Homarus claw muscle, Balanus scutal depressor, Lethocerus air tube retractor, and Aequipecten striated adductor). We show that (a) the paramyosins in all of these muscles have the same chain weights and (b) they are immunologically similar. We stained all of these muscles with specific antibody to Limulus paramyosin using the indirect fluorescent antibody technique. Paramyosin was localized to the A bands of the glycerinated striated muscles, and diffus fluorescence was seen throughout the glycerinated fibers of the smooth catch muscles. The presence of paramyosin in Homarus claw muscle, Balanus scutal depressor, and Lethocerus air tube retractor is shown here for the first time. Of the muscles in this study, Limulus telson levator is the only one for which the antiparamyosin staining pattern has been previously reported.

The high molecular weight hemocyanin found in the hemolymph of the horseshoe crab, Limulus polyphemus, is composed of at least eight different kinds of subunits. Ion exchange chromatography at high pH in the presence of EDTA yields five major zones, hemocyanins I to V, three of which are electrophoretically heterogeneous. The subunits have similar molecular weights, 65,000 to 70,000, and their amino acid compositions are remarkably similar to each other and to other arthropod and molluscan hemocyanins. Digestion of the native subunits of Limulus hemocyanin by formic acid or trypsin shows considerable structural diversity which is supported by cyanogen bromide cleavage patterns and by peptide mapping of the tryptic peptides prepared from denatured hemocyanin subunits. The structural differentiation of the subunits is accompanied by functional differentiation, as shown in previous investigations of their O2 and CO affinities (Sullivan, B., Bonaventura, J., and Bonaventura, C. (1974) Proc. Natl. Acad. Sci. U.S.A. 71, 2558-2562; Bonaventura, C., Bonaventura, J., Sullivan, B., and Bourne, S. (1975) Biochemistry 13, 4784-4789). The subunit diversity of Limulus hemocyanin suggests that other electrophoretically heterogeneous hemocyanins may be composed of structurally distinct subunits.

Two ecdysteroids were isolated from juveniles and adults as well as from eggs of the pycnogonid Pycnogonum litorale Ström and characterized by bioassay, thin layer chromatography, gas liquid chromatography, and high performance liquid chromatography. One of the hormones could be identified as 20-hydroxyecdysone whereas the other behaves in HPLC in a way that is similar to but not identical with ecdysone. This unidentified ecdysone-like hormone exceeded 20-hydroxyecdysone in all developmental stages (1.67:1 to 3:1). Determination of moulting hormone titers by a radioimmunoassay showed highest amounts of hormones in juvenile females (150 ng/mg tissue dry wt). The results are compared with those obtained from other arthropods and are discussed in view of the evolution of hormonal systems.

The investigations were carried out with prothoracic glands of Galleria mellonella. The influence of various fixation media on the preservation of the ultrastructure and on the immunocytochemical staining of the steroid ecdysone was studied. An immunocytochemical staining technique for ecdysone is described allowing the ultrastructural localization of ecdysone in different cellular compartments.

The sulfated mucopolysaccharide composition of 22 species of invertebrates belonging to the phyla Arthropoda, Mollusca, Annelida, Tunicata, Echinodermata, Coelenterata, and Porifera was analyzed. It is shown that all the species contain variable amounts of one or more types of sulfated mocopolysaccharides, most of which similar to the ones found in vertebrates. It is shown also that each species has a characteristic composition, differing from each other regarding the relative amount and type of chondroitin sulfates A, B, and C, heparitin sulfate, and heparin. The possible biological role of the sulfated mucopolysaccharides in cell recognition or aggregation or both is discussed in view of the present findings.

A novel cyanogenetic compound benzoyl cyanide, was isolated from the defensive secretion of 3 polydesmoid millipeds (Pseudopolydesmus seratus, Apheloria corrugata and A. trimaculata). The secretion of the 3 species also contains mandelonitrile and benzaldehyde, and that of P. serratus contains mandelonitrile benzoate, benzoic acid, isovaleric acid, myristic acid and stearic acid.

Metallothionein (MT) and the related metal-binding proteins in tissues of lower vertebrates and invertebrates were isolated by gel filtration chromatography (conventional liquid and high performance liquid chromatography). The metal-binding proteins including MT were detected by determining the metals bound to the proteins by atomic absorption spectrophotometry. This analytical procedure was recommendable as the first step to characterize unknown metal-binding proteins in diverse living bodies. As MT in the lower vertebrates, avian (Japanese quail), reptilian (tortoise), amphibian (8 species of frogs and 2 species of tailed amphibians) and fish MTs were isolated and characterized. The number of isoMTs was different among animals belonging not only to different classes but also to different species in the same family. One of the isoMTs in the avian, reptilian and amphibian MTs was shown to contain one histidinyl residue. Cadmium(Cd)-binding proteins were induced in three species of insect (Insecta, Arthropoda) larvae (midge, fleshfly and silkworm) by loading of Cd. Distribution of Cd in insect tissues was different among the three species though the alimentary canal was commonly the most important tissue for the storage of Cd in all species. Further, Cd in the soluble fractions was bound to inducible proteins of different molecular sizes in the three species. Cd-binding protein was also detected in the waterflea (Crustacea, Arthropoda) after loading of Cd. Cd-binding proteins induced in the earthworm (Oligochaeta, Annelida) by loading of Cd were a mixture of proteins with three different molecular sizes. One of the Cd-binding proteins with an apparent molecular size of 7,000 was shown to have characteristics of MT.

The alpha 2-macroglobulins are large molecular weight proteinase-binding proteins that inhibit the ability of proteinases to hydrolyze protein substrates without suppressing activity against amide or ester substrates. They are also able to protect the active site of bound proteinases from active site inhibitors of suitably high molecular weight. The ability to protect the amidolytic activity of trypsin from the macromolecular active site inhibitor, soybean trypsin inhibitor, was used to demonstrate an alpha 2-macroglobulinlike activity in the blood of the horseshoe crab, Limulus polyphemus and the crustaceans Libinia emarginata (the spider crab) and Cancer borealis (the Jonah crab). The alpha 2-macroglobulinlike activities of L. polyphemus and L. emarginata are sensitive to methylamine, but that of C. borealis is relatively insensitive. The molecular weights (mw) of the trypsin-protecting proteins in L. emarginata and C. borealis, estimated from gelfiltration studies, are, respectively, 480 X 10(3) and 460 X 10(3), and are significantly smaller than that of L. polyphemus (Mr = 570 X 10(3)).

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.

1. A phylogenetic study of oxytocin (OXT)-like immunoreactive cells was performed by the PAP method in the central nervous system of invertebrates. 2. The immunoreactivity was detected in the nerve cells of Hydra magnipapillata of the Coelenterata; Neanthes japonica and Pheretima communissima of the Annelida; Oncidium verrucosum, Limax marginatus and Meretrix lamarckii of the Mollusca; and Baratha brassica of the Arthropoda. 3. No immunoreactive cells were found in Bipalium sp. of the Platyhelminthes; Pomacea canaliculata, Aplysia kurodai, Bradybaena similaris and Achatina fulica of the Mollusca; and Gnorimosphaeroma rayi, Procambarus clarkii, Hemigrapsus sanguineus, Helice tridens and Gryllus bimaculatus of the Arthropoda; Asterina pectinifera of the Echinodermata; and Halocynthia roretzi of the Protochordata. 4. These results demonstrate that an OXT-immunoreactive substance is widely present not only in vertebrates but also in invertebrates. 5. OXT seems to have been introduced into these invertebrates at an early stage of their phylogenetic history.

1. A phylogenetic study of arg-vasotocin (AVT)/arg-vasopressin (AVP)-like immunoreactive cells was performed by the PAP method in the central nervous system of invertebrates. 2. The immunoreactivity was detected in the nerve cells of Hydra magnipapillata of the Coelenterata; Neanthes japonica and Pheretima communissima of the Annelida; Pomacea canaliculata, Aplysia kurodai, Oncidium verrucosum, Bradybaena similaris, Achatina fulica, Limax marginatus and Meretrix lamarckii of the Mollusca; Gnorimosphaeroma rayi, Hemigrapsus sanguineus, Gryllus bimaculatus and Baratha brassicae of the Arthropoda; Asterina pectinifera of the Echinodermata; and Halocynthia roretzi of the Protochordata. 3. No immunoreactivity was detected in Bipalium sp. of the Platyhelminthes, or in Procambarus clarkii and Helice tridens of the Arthropoda. 4. From these results, it appears that AVT/AVP is a phylogenetically ancient peptide which is present in a wide variety of invertebrates. 5. The actions of AVT/AVP and its presence in invertebrates are discussed.

Examination and measurement of electron micrographs of negatively stained hemocyanin molecules from Cherax destructor show that the predominant aggregated forms, the 16S and 24S components, are typical structures for arthropod hexamers and dodecamers, respectively. In Cherax hemocyanin the hexamers are formed from the monomeric (Mr congruent to 75,000) subunits, M1 and M2, while the dodecamers contain in addition a dimeric (Mr congruent to 150,000) subunit, M3'. Studies of the composition of solutions of the subunits M1 and m2 to which calcium ions have been added at pH 7.8 show that, under these conditions, reassembly occurs to particles indistinguishable from native hexamers. It is noteworthy that dodecamers are not seen since this confirms the previous suggestion that incorporation of the dimeric subunit in the assembly process is necessary for their formation. The results obtained from Cherax hemocyanin are related to those of previous structural studies of arthropod hemocyanins. In particular, the possible controlling role of certain specific subunits in arthropod hemocyanin oligomers containing more than one kind of subunit is illustrated with a model for the Cherax dodecamer, in which the dimeric subunit is shared between the two halves of the molecule.