A periodic structure in the 'pen' chitin of the squid Loligo vulgaris

The organization of the chitin-proteoglycan in Loligo vulgaris pen was examined ultrastructurally and related to the molecular order indicated by X-ray diffraction. There is a centrosymmetric striated repeat of 22 nm in the system which is based upon dark and light bands of unequal width. The banding is orientated perpendicular to the direction of the major molecular axis of the chitin fibres. The chitin molecules are laid down in sheets with a mutual, though irregular, twist to produce a laminated 'plywood' material.     

High-yield expression, purification, and characterization of the recombinant diisopropylfluorophosphatase from Loligo vulgaris

Organophosphate degrading enzymes are of great interest in light of their ability to detoxify chemical warfare agents. The diisopropylfluorophosphatase (DFPase) from Loligo vulgaris is characterized by its high stability and broad substrate specifity. Here we report the production of large amounts of active, recombinant DFPase using an Escherichia coli expression system. The enzyme was purified to homogeneity using a combination of immobilized metal affinity and ion exchange chromatography. CD-spectroscopy indicates a well folded protein with a high amount of beta-sheet structure. Limited proteolysis was used to gain a deeper insight into the structural organization of the protein. DFPase possesses an internal protease-sensitive region located between amino acids 146 and 149. The two proteolytic fragments remain as a tight complex retaining a DFPase activity comparable to the intact enzyme. Overexpression clones for each fragment were constructed with the expression resulting in the formation of inclusion bodies. Upon isolation and refolding active protein is only formed when both fragments are present. Thus, the two proteolytic fragments are probably part of a stable single-domain protein with multiple contacts between them and only one protease accessible surface loop.     

The isolation and characterization of the major glutathione S-transferase from the squid Loligo vulgaris

1. The major glutathione S-transferase (GST) from the common squid Loligo vulgaris has been purified and shown to be a homodimer of subunit molecular mass 24,000 and pI 6.8. 2. It has high activity towards 1-chloro-2,4-dinitrobenzene, p-nitrobenzyl chloride, 4-hydroxynon-2-enal and linoleic acid hydroperoxide, low activity with 1,2-dichloro-4-nitrobenzene and no activity with ethacrynic acid, trans-4-phenyl-3-buten-2-one and 1,2-epoxy-3-(p-nitrophenoxy)propane. 3. The L. vulgaris GST did not cross-react with any of the available polyclonal antibodies raised against mammalian GSTs. 4. Forty amino acids of its N-terminal sequence have been determined. 5. Its activities and primary structure are compared with related proteins from other species.     

The ultrastructure and innervation of muscles controlling chromatophore expansion in the squid,Loligo vulgaris

Squid chromatophores are organs of colour change, consisting of a pigment sac opened by contraction of 10–24 radial muscle fibres. The ultrastructure and innervation of these muscle fibres were examined by electron microscopy and diagramatic reconstructions made on the basis of serial ultra-thin sections. At the proximal end of the fibre, nearest the pigment sac a cortical myofilament zone surrounds 2 cores containing mitochrondria; further along the fibre these merge to form one central core. The myofilament zone forms a groove containing a nerve bundle consisting of 2 to 4 axons per muscle fibre. The axons are surrounded by glial cell processes, and either originate from a neighbouring fibre, or join the fibre at some point along its length. Axons twist around each other, forming a series of synapses with the muscle fibre. As many as 6–37 synapses exist along the length of each muscle fibre; the mean synapse interval is 9.05 m, but the largest may be 123 m. At the distal end of the muscles, the nerve is located towards the middle of the fibre, which it penetrates as the muscle splits up. Electron-lucent vesicles are present in all synaptic regions, but electron-dense vesicles are only found towards the distal end of the fibre. There is thus a possibility that more than one neurotransmitter is present in the nerves innervating chromatophores. Electron-lucent and dense-cored vesicles are not colocalised.This work was carried out during the tenure of a BBSRC CASE studentship     

Mutational and structural studies of the diisopropylfluorophosphatase from Loligo vulgaris shed new light on the catalytic mechanism of the enzyme

The active site, the substrate binding site, and the metal binding sites of the diisopropylfluorophosphatase (DFPase) from Loligo vulgaris have been modified by means of site-directed mutagenesis to improve our understanding of the reaction mechanism. Enzymatic characterization of mutants located in the major groove of the substrate binding pocket indicates that large hydrophobic side chains at these positions are favorable for substrate turnover. Moreover, the active site residue His287 proved to be beneficial, but not essential, for DFP hydrolysis. In most cases, hydrophobic side chains at position 287 led to significant catalytic activities although reduced relative to the wild-type enzyme. With respect to the Ca-1 binding site, where catalysis occurs, various mutants indicated that the net charge at this calcium-binding site as well as the relative positions of the charged calcium ligands is crucial for catalytic activity. The importance of the electrostatic potential at the active site was furthermore revealed by various mutations of residues lining the interior of the central water-filled tunnel, which traverses the entire protein structure. In this respect, the structural features of residue His181, which is located at the opposite end of the DFPase tunnel relative to the active site, were characterized extensively. It was concluded that a tunnel-spanning hydrogen bond network, which includes a large number of apparently slow exchanging water molecules, relays any modifications in the electrostatics of the system to the active site, thus affecting the catalytic reactivity of the enzyme.     

The importance of dynamic effects on the enzyme activity: X-ray structure and molecular dynamics of onconase mutants

Onconase (ONC), a member of the RNase A superfamily extracted from oocytes of Rana pipiens, is an effective cancer killer. It is currently used in treatment of various forms of cancer. ONC antitumor properties depend on its ribonucleolytic activity that is low in comparison with other members of the superfamily. The most damaging side effect from Onconase treatment is renal toxicity, which seems to be caused by the unusual stability of the enzyme. Therefore, mutants with reduced thermal stability and/or increased catalytic activity may have significant implications for human cancer chemotherapy. In this context, we have determined the crystal structures of two Onconase mutants (M23L-ONC and C87S,des103-104-ONC) and performed molecular dynamic simulations of ONC and C87S,des103-104-ONC with the aim of explaining on structural grounds the modifications of the activity and thermal stability of the mutants. The results also provide the molecular bases to explain the lower catalytic activity of Onconase compared with RNase A and the unusually high thermal stability of the amphibian enzyme.     

The relationship between quaternary structure and enzyme activity

Methods have been developed to study the influence of quaternary structure on enzyme activity. Some enzymes which normally exist as stable oligomers remain catalytically active when the subunits are dissociated by artificial means.     

Enzymatic catalysis in microemulsions: enzyme reuse and product recovery

A technique for enzyme reuse and product recovery from enzymatic catalysis in microemulsions is demonstrated. The enzymatic reaction is performed in a homogeneous isotropic microemulsion; AOT (sodium bis-(2-ethyl- hexyl)sulfosuccinate)/isooctane/buffer or C(12)E(5)(penta ethylene glycol dodecyl ether)/heptane/buffer. By small temperature changes the systems are shifted to two phase regions, where an oil-rich phase, containing the product, coexists with a water-rich phase containing surfactant and enzyme. The oil-rich phase may be replaced by an oil solution containing new substrate. Thus, the reaction may be continued and the enzyme reused. This procedure was repeated nine times in the present study. Data on phase behavior in presence and in absence of protein, partitioning of the components and a radioactive-labelled protein between the phases, and the repeated use of horse liver alcohol dehydrogenase (HLADH) in the microemulsions are presented.     

Fine structure and absorption of ferritin in the digestive organs of Loligo vulgaris and L. Forbesi (Cephalopoda,Teuthoidea)

The digestive organs possibly involved in food absorption in Loligo vulgaris and L. forbesi are the caecum, the intestine, the digestive gland, and the digestive duct appendages. The histology and the fine structure showed that the ciliated organ, the caecal sac, and the intestine are lined with a ciliated epithelium. The ciliary rootlets are particularly well developed in the ciliated organ, apparently in relation to its function of particle collection. Mucous cells are present in the ciliated organ and the intestine. Histologically, the digestive gland appears rather different from that of other cephalopods. However, the fine structure of individual types of squid digestive cell is actually similar to that of comparable organs in other species, and the squid cells undergo the same stages of activity. Digestive cells have a brush border of microvilli, and numerous vacuoles, which sometimes contain “brown bodies.” However, no “boules” (conspicuous protein inclusions of digestive cells in other species) could be identified in their cytoplasm; instead only secretory granules are present. In the digestive duct appendages, numerous membrane infoldings associated with mitochondria are characteristic features of the epithelial cells in all cephalopods. Two unusual features were observed in Loligo: first, the large size of the lipid inclusions in the digestive gland, in the caecal sac, and in the digestive duct appendages; and second, the large number of conspicuous mitochondria with well-developed tubular cristae. When injected into the caecal sac, ferritin molecules can reach the digestive gland and the digestive duct appendages via the digestive ducts, and they are taken up by endocytosis in the digestive cells. Thus, it appears that the digestive gland of Loligo can act as an absorptive organ as it does in other cephalopods.     

Peptide tyrosine phenylalanine: a novel neuropeptide F-related nonapeptide from the brain of the squid, Loligo vulgaris.

A novel nonapeptide, sequence YAIVARPRFamide, was isolated from brain extracts of the squid, L. vulgaris. Designated peptide tyrosine phenylalanine (PYF), the peptide shows marked homology with the C-terminal nonapeptides of pancreatic polypeptide and neuropeptide F (NPF) from a number of sources. If PYF is the C-terminal nonapeptide of squid NPF, then it may be derived by a novel processing mechanism involving specific cleavage between two TYR residues. PYF may be a highly truncated, receptor-active variant of NPF.     

Polymorphisms in human soluble epoxide hydrolase: effects on enzyme activity, enzyme stability, and quaternary structure

Human soluble epoxide hydrolase (hsEH) has been shown to play a role in regulating blood pressure and inflammation. HsEH consists of an N-terminal phosphatase and a C-terminal epoxide hydrolase domain. In the present study, we examined the effects of polymorphisms in the hsEH gene on phosphatase activity, enzyme stability, and protein quaternary structure. The results showed that mutants Lys55Arg, Arg103Cys, Cys154Tyr, Arg287Gln, and the Arg103Cys/Arg287Gln (double mutant) have significantly lower phosphatase activity compared to the most frequent allele (MFA) of hsEH. In addition, the Lys55Arg, Arg103Cys, Cys154Tyr, Arg287Gln, and the double mutant have significantly lower kcat/Km values. The stabilities at 37 degrees C of purified Arg287Gln and Arg103Cys/Arg287Gln mutants were also significantly reduced compared to the MFA. HPLC size-exclusion studies showed that the MFA exists predominantly as a dimer. However, the Arg287Gln and Arg103Cys/Arg287Gln mutants show increased concentration of the monomer. We conclude that the Arg287Gln polymorphism disrupts putative intra- and inter-monomeric salt-bridges responsible for dimerization.     

Purification and characterization of two proteins with inorganic pyrophosphatase activity from Desulfovibrio vulgaris: rubrerythrin and a new, highly active, enzyme

The inorganic pyrophosphatase activity of a soluble extract from the strict anaerobe, sulfate-reducing, Desulfovibrio vulgaris, is readily resolved into two peaks. After purification, two active proteins with very dissimilar properties are obtained. One is the non-heme iron-containing rubrerythrin, with a specific activity of 350 pyrophosphate hydrolyzed, min-1, mg protein-1. The other, a protein of Mr = 39,000, with a specific activity of 12,000.     

Livoneca sinuata (Crustacea; Isopoda; Cymothoidae) on Loligo vulgaris from Turkey, and unusual cymothoid associations

In this paper, an unusual association of Livoneca sinuata (Crustacea; Isopoda; Cymothoidae) with the cephalopod Loligo vulgaris is reported for the first time from the Aegean sea coasts of Turkey. Moreover, a review of all the cases of unusual associations involving cymothoids is performed.     

Solution structure and dynamics of biomolecules from Raman optical activity

Raman optical activity (ROA) measures vibrational optical activity by means of a small difference in the intensity of Raman scattering from chiral molecules in right and left circularly polarized incident laser light. The ROA spectra of a wide range of biomolecules in aqueous solution can now be measured routinely. Because of its sensitivity to the chiral elements of biomolecular structure, ROA provides new information about solution structure and dynamics complementary to that supplied by conventional spectroscopic techniques. This article provides a brief introduction to the theory and practice of ROA spectroscopy followed by a review of recent ROA results on polypeptides, proteins, carbohydrates, nucleic acids and viruses which illustrate how new insight into current problems of structure, folding and function may be obtained from ROA studies.     

Foaming and enzyme activity in fungal cultures grown on sugar beet cosette

Summary Enzyme synthesis, foaming behaviour and its effects were studied using two common cellulolytic fungi;Trichoderma reesei andSporotrichum pulverulentum in a medium containing sugar beet cosette as a cellulosic substrate. Cellulase enzyme activities in the culture broth were found to be higher than the enzyme activities in natural and experimentally forced foam layers.