Paramyosin in invertebrate muscles. II. Content in relation to structure and function.

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.

Paramyosin in invertebrate muscles. I. Identification and localization.

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.

Specific localization of scallop gill epithelial calmodulin in cilia.

Calmodulin has been isolated and characterized from the gill of the bay scallop aequipecten irradians. Quantitative electrophoretic analysis of epithelial cell fractions show most of the calmodulin to be localized in the cilia, specifically in the detergent- solubilized membrane-matrix fraction. Calmodulin represents 2.2 +/- 0.3 percent of the membrane-matrix protein or 0.41 +/- 0.5 percent of the total ciliary protein. Its concentration is at least 10(-4) M if distributed uniformly within the matrix. Extraction in the presence of calcium suggests that the calmodulin is not bound to the axoneme proper. The ciliary protein is identified as a calmodulin on the basis of its calcium- dependent binding to a fluphenazine-sepharose affinity column and its comigration with bovine brain calmodulin on alkaline-urea and SDS polyacrylamide gels in both the presence and absence of calcium. Scallop ciliary calmodulin activates bovine brain phosphodiesterase to the same extent as bovine brain and chicken gizzard calmodulins. Containing trimethyllysine and lacking cysteine and tryptophan, the amino acid composition of gill calmodulin is typical of known calmodulins, except that it is relatively high in serine and low in methionine. Its composition is less acidic than other calmodulins, in agreement with an observed isoelectric point approximately 0.2 units higher than that of bovine brain. Comparative tryptic peptide mapping of scallop gill ciliary and bovine brain calmodulins indicates coincidence of over 75 percent of the major peptides, but at least two major peptides in each show no near-equivalency. Preliminary results using ATP-reactivated gill cell models show no effect of calcium at micromolar levels on ciliary beat or directionality of the lateral cilia, the cilia which constitute the vast majority of those isolated. However, ciliary arrest will occur at calcium levels more than 150 muM. Because calmodulin usually functions in the micromolar range, its role in this system is unclear. Scallop gill ciliary calmodulin may be involved in the direct regulation of dyneintubule sliding, or it may serve some coupled calcium transport function. At the concentration in which it is found, it must also at least act as a calcium buffer.

Isoguanosine: isolation from an animal.

Isoguanosine (oxyadenosine or crotonoside), previously known to occur in nature only in the croton bean, was isolated from an animal, the marine nudibranch mollusk Diaulula sandiegensis.

Molluscan gastrin: concentration and molecular forms.

Blood and gastrointestinal tissues of the sea hare Aplysia californica and the land snail Otala lactea contain immunoreactive gastrin in heterogeneous forms similar to those of mammals. The observation that blood concentrations in terms of porcine gastrin standard are comparable to those of pig, man, and dog suggests significant homology between the structures of molluscan and mammalian gastrins.

Doridosine: a new hypotensive N-methylpurine riboside from the nudibranch Anisodoris nobilis.

A new N-methylpurine riboside (doridosine), probably N1-Methylisoguanosine, was isolated from the digestive glands of a nudibranch. Doridosine produces prolonged hypotension and bradycardia in anesthetized rats, decreases the rate and the amplitude of contraction of guinea pig atria in vitro, and causes the heart rate in anesthetized mice to be reduced by 50 percent for many hours after which the animals recover completely.

Octopamine: presence in single neurons of Aplysia suggests neurotransmitter function.

Octopamine has been identified and measured in individual neurons from Aplysia californica. Neither dopamine nor norepinephrine was detected in these cells. Thus, in Aplysia there may be separate populations of catecholaminergic and monophenolaminergic cells. Octopamine may have functions of its own in the central nervous system of mollusks.

An antibody to the Drosophila period protein recognizes circadian pacemaker neurons in Aplysia and Bulla.

The molecular mechanisms of the pacemakers underlying circadian rhythms are not well understood. One molecule that presumably functions in the circadian clock of Drosophila is the product of the period (per) gene, which dramatically affects biological rhythms when mutated. An antibody specific for the per protein labels putative circadian pacemaker neurons and fibers in eyes of two marine gastropods, Aplysia and Bulla. As was found for the Drosophila per protein, there is a daily rhythm in the levels of the per-like antigen in Aplysia eyes. Thus, certain molecular features of the per protein, as well as aspects of the temporal regulation of its expression, may be conserved in circadian pacemakers of widely divergent species.

Aromatic amino-acids and subunit assembly in the hemoglobins from Scapharca inaequivalvis: a fluorescence and CD study of the apoproteins.

The dimeric and tetrameric hemoglobins from the mollusc Scapharca inaequivalvis have a unique assembly that places the heme-carrying E and F helices in the inside of the molecule. These helices form the intersubunit contact in the dimer, which represents the structural unit since the tetramer is a dimer of dimers. The E and F helices are highly conserved and contain about 70% of the phenylalanine and tyrosine residues, while the tryptophan residues are near the tetramer contact. The spectroscopic properties (circular dichroism and intrinsic fluorescence) of the aromatic amino-acid residues in the two globins indicate that heme removal brings about a larger conformational change in the tetrameric than in the dimeric protein and that the tryptophan residues acquire a more rigid conformation in the tetramer.

The quaternary structure of a molluscan (Helisoma trivolvis) extracellular hemoglobin.

The hemoglobin (erythrocruorin) of the planorbid mollusc Helisoma trivolvis has a molecular weight of 1.7-10(6) and a sedimentation coefficient (s0 20, w) of 33.8 S at pH 7.0. At pH 2.0, the pigment consists of 32 S and 13 S material. The hemoglobin exists as a 350 000 molecular weight submultiple in 6 M guanidine and can be further dissociated into a 175-200 000 dalton polypeptide in 6M guanidine, 0.1 M 2-mercaptoethanol or by sodium dodecyl sulfate gel electrophoresis of globin, performic acid oxidized globin or carboxymethylated globin. Electron microscope observations show a ten-membered ring structure measuring 200 A in diameter. It is proposed that Helisoma hemoglobin consists of a 1.7-10(6) dalton circular assembly of ten 175-200 000 dalton polypeptide chains. The amino acid composition of the pigment is reported. The hemoglobin contains one heme per 18-19 000 g protein. Limited proteolysis of the intact pigment shows 60 000, 40 000 and 17 000-18 500 dalton components when analyzed by sodium dodecylsulfate gel electrophoresis. It is likely that the 175-200 000 dalton polypeptide consists of a linear arrangement of 8-12 heme-containing domains, each domain having a molecular weight of 18-19 000.

Amino acid sequence of dimeric myoglobin from Cerithidea rhizophorarum.

The complete amino acid sequence of the dimeric myoglobin from Cerithidea rhizophorarum, a common gastropodic mollusc on the Japanese coast having an elongated many-whorled shell, has been determined. The monomer is composed of 151 amino acid residues, is acetylated at the amino terminus, and 75 residues out of 151 are homologous with the monomer of myoglobin from the whelk Busycon canaliculatum. Unlike Aplysia myoglobin, which lacks the distal histidine, Cerithidea myoglobin contains three histidines in its monomer, His-66 being assigned to the distal position, and in its oxymyoglobin form its stability properties show a very strong pH dependence.

Oxidation reaction of Scapharca inaequivalvis hemoglobins with nitrite.

The oxidation reaction with nitrite of the dimeric and tetrameric hemoglobins from the mollusc Scapharca inaequivalvis has been studied kinetically and at equilibrium. In line with previous findings obtained with ferricyanide as oxidant, in both proteins the stable oxidation product is a hemichrome, although the nitrite-methemoglobin complex is formed in significant amount when excess nitrite is employed. The reaction kinetics are characterized by a lag period followed by an autocatalytic phase, as in the case of human hemoglobin. However, with respect to human hemoglobin, in the two molluscan proteins the lag phase is prolonged significantly due to the instability of their met-form, an obligatory intermediate for the onset of autocatalysis. All the data obtained in spectrophotometric, EPR and sedimentation velocity experiments under a variety of experimental conditions conform to the reaction mechanism proposed for human hemoglobin (Spagnuolo et al., Biochim. Biophys. Acta 911 (1987) 59-63) provided hemichrome formation and nitrite binding are taken into account.

Identification of beta-D-mannosylceramide in hepatopancreas of the fresh-water bivalve, Hyriopsis schlegelii.

A mannosylceramide was isolated by preparative thin-layer chromatography on a 3% borate-impregnated silica gel plate from a monohexosylceramide fraction of the hepatopancreas of the fresh-water bivalve, Hyriopsis schlegelii. It contained only mannose as the sugar component and the ceramide moiety contained mainly sphingosine and palmitic acid. Anomeric configuration of the sugar moiety was determined by enzymatic hydrolysis with beta-D-mannosidase. The concentration of this glycolipid was 5% of the total monohexosylceramide fraction of the hepatopancreas.

Determination of the presence of ceramide aminoethylphosphonate and ceramide N-methylaminoethylphosphonate in marine animals by fast atom bombardment mass spectrometry.

Phosphonosphingolipids from 15 kinds of shellfish were analyzed by fast atom bombardment mass spectrometry to determine the contents of ceramide aminoethylphosphonate (CAEPn) and ceramide N-methylaminoethylphosphonate (CMAEPn). Two pairs of ions, at m/z 126 and 140 in the positive ion mode and at m/z 124 and 138 in the negative ion mode, were used to distinguish between aminoethylphosphonic acid and N-methylaminoethylphosphonic acid in CAEPn and CMAEPn. Interestingly, mollusca in the early stage of evolution have both CAEPn and CMAEPn, while most in the middle stage have only CMAEPn and those in the highest stage have only CAEPn.

Mössbauer spectroscopic studies of the cores of human, limpet and bacterial ferritins.

Ferritin cores from human spleen, limpet (Patella vulgata) haemolymph and bacterial (Pseudomonas aeruginosa) cells have been investigated using 57Fe Mössbauer spectroscopy. The Mössbauer spectra were recorded over a range of temperatures from 1.3 to 78 K, all the spectra are quadrupole-split doublets with similar quadrupole splittings and isomer shifts, characteristic of iron(III), while at sufficiently low temperatures the spectra of all the samples show well-resolved magnetic splitting. At intermediate temperatures, the spectra from the human ferritin exhibit typical superparamagnetic behaviour, while those from the bacterial ferritin show behaviour corresponding to a transition from a magnetically ordered to a paramagnetic state. The spectra from the limpet ferritin show a complex combination of the two effects. The results are discussed in terms of the magnetic behaviour of small particles. The data are consistent with magnetic ordering temperatures of about 3 and 30 K for the bacterial and limpet ferritin cores, respectively, while the data indicate that the magnetic ordering temperature for the human ferritin cores must be above 50 K. These differences are interpreted as being related to different densities of iron in the cores and to variations in the composition of the cores. The human ferritin cores are observed to have a mean superparamagnetic blocking temperature of about 40 K, while that of the limpet ferritin cores is about 25 K. This difference is interpreted as being due not only to different mean numbers of iron atoms in the two types of core but also to the higher degree of crystallinity in the cores of the human ferritin.

The stabilizing influence of divalent ions and Na+ on the di-decameric structure of Yoldia limatula hemocyanin.

The stabilizing influence of Ca2+, Mg2+, Ba2+ and Na+ on the di-decameric structure of the hemocyanin of the bivalve, Yoldia limatula has been investigated by light-scattering molecular weight measurements and by analytical ultracentrifugation. The molecular weight (Mw) data, examined as a function of decreasing divalent ion and sodium ion concentrations at pH 8.0 and at a constant hemocyanin concentration of 0.10 g.l-1, show biphasic transition profiles, with a sharp initial decline in Mw as the concentration of the stabilizing cations is reduced. The analysis of the molecular weight data is best described in terms of the four-species, di-decamer-decamer-dimer-monomer scheme of association-dissociation equilibria. About 25 to 35 bound divalent ions and about 10 bound Na+ ions per half-molecule or decamer are required in order to account for the initial step of the observed transitions. The subsequent transitions representing the decamer to dimer and the dimer to monomer steps of the reaction account for the additional binding of three to four and two to four cations per dimer and per monomer, respectively. The relatively large number of divalent ions per decamer suggests strong ionic stabilization of the decamer to decamer contacts within the parent di-decameric assembly of Yoldia hemocyanin. This is consistent with earlier observations showing relatively few hydrophobic groups at the decamer to decamer contact areas.

The influence of primary structure changes on the higher order structure of histone H2B.

Structural changes of histones H2B in aqueous media were studied under conditions of varying pH and ionic strength. The techniques involved intrinsic fluorescence of tyrosine residues, extrinsic fluorescence using the hydrophobic probe 8-anilinonaphthalene sulfonate and fluorescence polarization. Secondary structure predictive methods were also used to study potential effects of primary mutations. In spite of many primary structure changes, the predicted effect on a globular structure of H2B is minimal. The results indicate the formation of a major hydrophobic region, the formation of helices and the burial of tyrosine residue 83 (calf), the structural changes of which appear to be identical in all H2B's as ionic strength is increased. The effect of primary structure changes on protein-protein interaction where H2B's are concerned is likely to be negligible, whereas the major effect is likely to be the protein-DNA interaction where H2B could be at least partially responsible for differentiation.

Isolation and characterization of a heparin with high anticoagulant activity from Anomalocardia brasiliana.

The isolation, some structural features, physicochemical properties and pharmacological activities of a heparin from Anomalocardia brasiliana are reported. It is shown that the mollusc heparin is very similar to those present in mammalian tissues with regard to chemical composition, physicochemical properties, pharmacological activities and susceptibility to heparinase and heparitinase II from Flavobacterium heparinum, as well as to the types of products formed by the action of these enzymes. Three significant quantitative differences were observed for the mollusc heparin when compared with the ones from mammalian origin, namely, a higher degree of binding with antithrombin III (45%), higher molecular weight (27-43 kDa) and higher anticoagulant activity (320 I.U./mg). The possible biological role of heparin is discussed in view of the present findings.

Reexamination of the structure of eumelanin.

The generally accepted concept that the black melanin eumelanin is made mostly from 5,6-dihydroxyindole but not from 5,6-dihydroxyindole-2-carboxylic acid (DHIC) was reexamined by comparison of synthetic and natural eumelanins. The analytical methods used were elemental analysis and determination of the carboxyl group by acid treatment to yield CO2 and by permanganate oxidation to yield pyrrole-2,3,5-tricarboxylic acid. It was found that DHIC-derived monomer units comprise only approx. 10% of enzymically prepared dopa-melanins but as much as a half of intact, natural eumelanins. The results also show that dopa-melanins prepared at higher pH retain higher percentages of the carboxyl group of dopa and contain higher percentages of pyrrole units, and that melanins are decomposed to a significant extent on acid treatment, the method commonly used to isolate melanins from natural sources.

Identification of shellfish fatty acids and their effects on lipogenic enzymes.

The fatty acids which are common to and characteristic of shellfish, were identified by mass spectrometry and NMR spectral analyses as being: octadecatetraenoioc acid, eicosapentaenoic acid, docosapentaenoic acid and docosahexaenoic acid. When the fatty acids isolated by high performance liquid chromatography were separately intubated into rats, hepatic glucose-6-phosphate dehydrogenase (EC 1.1.1.49), malic enzyme (EC 1.1.1.40) and acetyl-CoA carboxylase (EC 6.4.1.2) were reduced more effectively as compared with linoleic acid intubation. These enzymes were reduced most markedly by eicosapentaenoic acid-intubation. The fatty acids seem to be effective components in reduction of triacylglycerol and lipogenic enzyme levels in rats fed on shellfish.