The erythrocruorin of Eisenia fetida. I. Properties and subunit structure.

The erythrocruorin of Eisenia fetida possesses a relative molecular mass, determined by sedimentation equilibrium, of (3.82 +/- 0.05) . 10(6). According to the iron and heme contents, 0.218 +/- 0.008% and 2.34 +/- 0.02% by mass, respectively, it contains 144 hemes per molecule. The dimensions of the molecule observed by electron microscopy are 25.0 X 16.5 nm (diameter X height). SDS-polyacrylamide gel electrophoresis indicates that the erythrocruorin consists of six subunits (Mr 14,900, 15,300, 17,200, 19,700, 31,600 and 40,000). Oxygen binding studies showed that E. fetida erythrocruorin has a high oxygen affinity (P50 = 2.8 Torr at pH 7.5), exhibits a slight bohr effect and possesses a high cooperativity with the Hill coefficient h = 3.7-4.8. Treatment of the erythrocruorin either by freezing and thawing or by aging or exposure to alkaline pH converts it irreversibly into a state of lower cooperativity with h = 2.0-2.6. A model of the subunit structure of the erythrocruorin is proposed which takes into account the physiochemical and oxygen-binding properties of the erythrocruorin and the subunits obtained upon its dissociation.

X-ray fluorescence spectrometric determination of the metal content of the extracellular hemoglobin of Tubifex tubifex.

X-ray fluorescence spectrometric analysis of Tubifex hemoglobin, using the energy-dispersive and wavelength-dispersive spectrometers, demonstrated that in addition to Fe, Ca and small amounts of Zn, Cu and Pb were also present. Assuming that Tubifex hemoglobin contains 160 atoms of Fe per molecular weight of 3.9 10(6), the molar ratios of Fe:Ca:Zn:Cu:Pb were calculated to be 160:70:2:8: 2. The iron content of the hemoglobin was also determined spectrophotometrically using the formation of Fe(II)-1,10-phenanthroline and Fe(II)-4-(2-pyridylazo)-resorcinol complexes: it was found to be 0.240+/-0.010% by weight and was unaffected by prior dialysis against 10 mM EDTA solution at neutral pH. Thus, it is unlikely that Tubifex hemoglobin contains any non-heme iron.

Insulin in insects and annelids.

The fruitfly, Drosophila melanogaster, and the earthworm, Annelida oligocheta, were extracted with acid-ethanol by a classic method for recovering insulin from the pancreas. When each extract was filtered on a Sephadex G-50 column, a distinct peak of insulin immunoreactivity. The material in this peak had reactivity insulin (equivalent to 0.1 to 2 ng of insulin/g wet weight) was recovered in the region typical of insulin bioassay, measuring stimulation of glucose oxidation or lipogenesis by isolated rat adipocytes. The bioactivity was partially or largely neutralized by anti-insulin antibodies. In concordance with previous work showing the presence of material very similar to insulin in the blowfly and molluscs, we have confirmed the presence of insulin in insects and extended the observation to the earthworms. These findings suggest that insulin is more widespread in invertebrates than was previously thought. In a companion study (Proc. Natl. Acad. Sci. USA 77:6184-88, 1980), we have demonstrated material similar to insulin in unicellular organisms.

Assembly of erythrocruorin from the earthworm Octolasium complanatum.

The spontaneous assembly of the earthworm erythrocruorin molecule (60 S) from its 1/12 subunits (10 S) obtained by alkaline dissociation is a long debated problem, since the 60 S to 10 S dissociation step has been regarded as essentially irreversible or as only partially reversible when freshly dissociated solutions are used. Erythrocruorin from the earthworm Octolasium complanatum has been reassembled from its 10 S subunits. "Age" of the subunits, pH, and divalent cation concentration are the factors that influence the assembly reaction. Of primary importance is the age of the subunits, i.e. their exposure time to the alkaline dissociating pH. Parallel sedimentation velocity and sodium dodecyl sulfate/polyacrylamide gel electrophoresis experiments on the dissociated and reassembled solutions indicate that two processes take place at alkaline pH values: disulfide exchange and limited proteolysis. These processes, whose relative importance differs in the various preparations, might be responsible for the loss of reassociating capacity of the 10 S subunits. With freshly dissociated subunits, reassembly up to 80% may be achieved at pH 6.2 to 6.5 in the absence of divalent cations; the presence of 25 to 50 mM-Ca2+ renders the reaction essentially pH-independent in the range 6.2 to 8. The effect of Ca2+ is discussed in the light of the presence of structure-stabilizing binding sites for divalent cations at the 10 S intersubunit's contact regions.

Oxygen binding to Octolasium complanatum erythrocruorin. Modulation of homo- and heterotropic interactions by cations.

The functional properties of erythrocruorin from Octolasium complanatum (a common earthworm of Central Italy) have been characterized in great detail. Special attention has been given to the reciprocal effects of the various ligands, namely oxygen, cations and protons. The data obtained under a variety of experimental conditions bring out the dominant role played by cations in the modulation of both homotropic and heterotropic interactions. In this respect, the most interesting observation concerns the unusual interplay between protons and cations that occurs in this erythrocruorin, the first respiratory pigment in which the Bohr effect is due totally to the O2-linked binding of an allosteric effector. The oxygen binding data collected under the various experimental conditions have been analyzed in terms of a modified two-state model, which takes into account the fact that allosteric effectors may also influence the ligand binding properties of the state that they stabilize. The analysis shows that the number of interacting sites necessary for the observed co-operativity in O2 binding is much smaller than the number of heme groups carried by the whole molecule, in accordance with previous findings on hemocyanins, the other class of giant respiratory pigments. Moreover, the analysis indicates that the dimensions of these "functional constellations" are under the control of allosteric effectors.

Protein analysis of earthworm coelomic fluid. III. Isolation and characterization of several bacteriostatic molecules from Eisenia fetida andrei.

A bacteriostatic activity in Eisenia fetida andrei cell free coelomic fluid is described. This activity is detected by growth inhibition of a bacteria Bacillus megaterium. Gel filtration analysis revealed eleven coelomic fluid protein fractions designated A, B,..J. Antibacterial activity was mainly found within fractions B and C. Chromatofocusing resolved fractions B-C into five different peaks named alpha BC, beta BC,... epsilon BC. Antibacterial activity appeared mediated by three different proteins characterized by their molecular weights (20,000, 40,000 and 45,000) and their isoelectric points (4.9, 5.75 and 6.0). These bacteriostatic proteins possess either hemolysis or hemagglutination activities. The polymorphic aspect of this humoral antibacterial defense is discussed.

Neuronal localization of pancreatic polypeptide (PP) and vasoactive intestinal peptide (VIP) immunoreactivity in the earthworm (Lumbricus terrestris).

Nerve fibres and cell bodies displaying vasoactive intestinal polypeptide (VIP) or pancreatic polypeptide (PP) immunoreactivity were demonstrated in ganglia of the earthworm (Lumbricus terrestris). VIP cell bodies were found in the most anterior ganglion of the ventral nerve cord, the subpharyngeal ganglion. Immunoreactive nerves were seen running in the center of the cord until about the 10th segment. PP cell bodies were found in the cerebral ganglion where VIP was lacking, in the subpharyngeal ganglion and in more posteriorly located ganglia of the ventral nerve cord. PP nerve fibres could be followed below the 10th segment of the cord.

Inhibition by lombricine from earthworm (Lumbricus terrestris) of the growth of spontaneous mammary tumours in SHN mice.

The effects of lombricine extracted and purified from earthworm (Lumbricus terrestris) skin on the growth of palpable sizes (approximately 5 mm) of spontaneous mammary tumours were studied in SHN mice. In Experiment 1, daily subcutaneous injections of lombricine (0.3 mg/0.05 ml olive oil) inhibited markedly the growth of tumours associated with the retardation of the growth of preneoplastic mammary hyperplastic alveolar nodules. In 1H-NMR spectra, the experimental mice had lower serum levels of lactic acid and glucose than the control. On the other hand, urine of the former group contained higher levels of allantoin, creatine and creatinine than that of the latter. In Experiment 2, lombricine given as diet at the concentration of 120 mg/kg also inhibited the growth of tumours, though to a lesser degree than the injection. The treatment had little effect on 1H-NMR spectra of either serum or urine and normal and preneoplastic mammary gland growth. All results indicate that the inhibition by lombricine of the growth of mammary tumours is at least partly due to the maintenance of homeostasis of the body including the regulation of the excess uptake of glucose as a source of energy and nutrition.

Recovery of volatile fatty acids from biological materials for direct analysis by gas chromatography.

A simple and efficient methodology for preparing aqueous extracts of volatile fatty acids from biological materials, for direct analysis by gas chromatography is described. Peak areas and responses relative to n-butyric acid were used to calculate concentrations of the individual acids. An example is given for analysis of the volatile fatty acids found in the blood of the lugworm Aerinocola marina.

Immunohistochemical localization of substance P and ACTH-like activity in the central nervous system of the earthworm Lumbricus terrestris L.

The peroxidase-antiperoxidase (PAP) method was used for the immunohistological demonstration of substances P and ACTH in the cerebral and subesophageal ganglia of the earthworm, Lumbricus terrestris L. With rabbit antibody to substance P a positive immunoreaction was found in nerve cells smaller than the type A neurons of the cerebral ganglion. Their perikarya and nerve processes as well as their terminal enlargements in the synaptic zone were immunoreactive. ACTH-like activity was visible in a larger perikaryon type of the A neurons. Their nerve processes did not show any reaction. However, a part of the nerve terminals of the synaptic zone and a few neurons of type B of the cerebral ganglion, further some cells of the subesophageal ganglion reacted positively.

X-ray microprobe analysis of chloragosomes of untreated and of EDTA-treated Lumbricus terrestris by using fresh air-dried smears.

The chloragosomes of Lumbricus terrestris were analysed by an electron microscope X-ray microphobe, using both air-dried smear preparations and conventional electron microscopic sections. In untreated worms the air-dried chloragosomes showed prominent peaks for Ca and P, distinct peaks for S and Zn, while Na, K, and Cl peaks were barely detectable. In air-dried smears the S peak was constantly more prominent than in EM sections, indicating that chloragosomes contain easily soluble S-compounds. By long-term CaNa2EDTA treatment of the worms, the Ca and P content of the chloragosomes could be significantly decreased, empirically proving, that the storage and mobilisation of chloragosomal Ca and P are closely related.

Accumulation of heavy metals in a lake ecosystem.

The concentrations of Cu, Zn, Fe, Mn, Ni, Cd, Pb and Co have been determined in water, bottom sediments, plankton, zoobenthos and ichthyofauna of mesotropic Lake Piaseczno located in eastern Poland. In water, sediments, plankton and benthos the most abundant heavy metals were Fe, Zn and Mn, whereas in fish Zn, Cu, and Mn were most abundant. The amount of heavy metals in the biotic components was dependent upon their concentration in water and partly upon the concentration in bottom sediments. A considerably less important role in the translocation of heavy metals is probably played by trophic interactions.

Pesticide pollution of soil, water and air in Delhi area, India.

In India organochlorine insecticides such as DDT and HCH constitute more than 70% of the pesticides used at present. Its continued use has given interest to monitor for the last few years the extent of organochlorine insecticide residues in soil, water, air and rain water in Delhi area. Out of the 50 samples each of soil and earthworms collected from different parts 48 samples showed that soil and earthworms contained 0-2.61 and 0-37.74 mg Kg-1 of total DDT residues respectively. The area near the vicinity of the DDT factory showed high levels of DDT residues. A two-year survey of the Yamuna river in Delhi showed that water contained an average of 0.24 ug L-1 and the bottom sediment had 0.24 mg Kg-1 of total DDT residues. The fishes collected from the Yamuna river showed very high bioaccumulation of DDT residues. The air and rainwater samples monitored from 3 different areas for two years indicated that DDT was more near the DDT factory area while HCH was more near a commercial complex. These results indicate that the overall organochlorine-residue levels in Delhi is not alarming. It probably indicates that DDT and HCH are more rapidly dissipated and degraded in a tropical country like India.