Yves Le Gal

Phenoloxidase (E.C. 1.14.18.1) from the byssus gland of Mytilus edulis: Purification, partial characterization and application for screening products with potential antifouling activities

Although a total ban on the use of TBT coatings is not expected in the short term, there is a growing need for environmentally safe antifouling systems. To assist in the rapid screening of a large number of potential antifouling substances, a method that is simple, efficient and inexpensive is required. The production of byssus threads by the blue mussel, Mytilus edulis, has often been studied for testing the antifouling efficacy of various compounds. The present study reports a new antifouling assay based on the inhibition of purified M. edulis phenoloxidase activity. The method has the advantage of being specific, reliable, sensitive and rapid.

Characterization of the vasoactive intestinal polypeptide (VIP) in the gut of fishes

Abstract 1. 1. The presence of VIP was investigated in the dogfish, Scyliorhinus canicula, the ballanwrasse. Lubrus berggylta and the bib. Trisopterus luscus, using a specific radioreceptorassay. 2. 2. Pure porcine VIP and gut extracts of fishes yielded similar dilution curves. 3. 3. In the dogfish, the highest concentration of VIP was found in the hindgut. In contrast, in the two teleostei studied, the highest levels of VIP were in the first part of the gut. 4. 4. The biologically active VIP measured by radioreceptorassay correlated well with the molecule determined using a specific radioimmunoassay. 5. 5. Our results support the hypothesis of the appearance of VIP early in evolution.

Purification and characterization of an aminopeptidase from tuna (Thunnus albacares) pyloric caeca

An aminopeptidase was purified from a water soluble fraction of tuna pyloric caeca by heat treatment, Zn2+ fractionation, ion exchange on a DEAE cellulose column, gel filtration on Fractogel TSK-55, and immobilized metal ion affinity chromatography (IMAC) on IDA(Cu2+/Zn2+)-Sepharose 6B. The molecular mass of the enzyme was estimated to be 150,000 on Sephacryl S-300 HR, and was found to be near 72,000 by SDS-PAGE. The aminopeptidase, which is a glycoprotein rich in acidic amino acids, is optimally active at pH 8.8 and 65 degrees C. The enzyme activity was not affected by Mg2+, Zn2+, Ca2+, Mn2+, Co2+, PMSF, iPr2FP, 4-hydroxymercuribenzoic acid, iodoacetamide, puromycin, and cysteine but it was strongly inhibited by metal chelators (EDTA and o-phenanthroline), amastatin, Hg2+, Cd2+, and Cu2+. The enzyme was also inhibited by some L-amino acids. Kinetic parameters of the enzyme were determined with some aminoacyl-p-nitroanilides and aminoacyl-beta-naphthylamides. L-Alanine-p-nitroanilide and L-alanine-beta-naphthylamide were hydrolysed most rapidly while the highest hydrolytic coefficient (kcat/Km) value was obtained with L-methionine-p-nitroanilide. The apoaminopeptidase was prepared and reconstitution of an active enzyme was carried out using metal chelating interaction chromatography on an IDA-Sepharose 6B column charged with a metal ion. Full activity was restored with Zn2+, Co2+, Cu2+ and Al3+. Zn(2+)-Enzyme was the most thermostable form of the aminopeptidase. Reversal inhibition by Cu2+ and Cd2+ was also examined. When the aminopeptidase was partially deglycosylated by a treatment with N-glycosidase F some of its physical properties differed from that of the native enzyme: its electrophoretic mobility was reduced and its stability to denaturation by SDS and by ionic strength were lower than those of the untreated enzyme. All together, our results indicate that the tuna pyloric caeca aminopeptidase is distinct from the peptide hydrolases characterized in the literature.

Purification of a functional competitive antagonist for calcitonin gene related peptide action from sardine hydrolysates

Calcitonin gene related peptide (CGRP) related molecules were purified from sardine hydrolysates prepared using 0.1% alcalase and two hours of hydrolysis. Gel exclusion chromatography and HPLC performed purification of these molecules. The purified molecules were characterised using specific CGRP radioimmunoassays and radioreceptoraasays. From 22 mg of crude extract, we obtained 14 µg of CGRP related molecules, the molecular weight determined by mass spectrophotometry was 6000 daltons. The biological activity of these molecules was analysed using the ability of CGRP to stimulate the adenylate cyclase activity in rat liver membranes. The purified molecules induced an inhibition of the CGRP stimulated adenylate cyclase activity, this effect was specific as no such effect was observed on the glucagon stimulated adenylate cyclase activity measured in the same rat liver membrane preparation. These results suggest that the purified molecules may act as antagonists for peptides that bind to CGRP receptors in rat liver membranes. These new antagonists may be of particular importance in various aspects of CGRP action in vertebrates.

In situ measurements of adenylate energy charge and assessment of pollution

Abstract Adenylate energy charge (AEC) measurements were performed on two marine species: Crangon crangon and Nassarius reticulatus sampled in two differently polluted stations, Bay of Seine on the channel and Bay of Concarneau on the Atlantic coast. Significant drops in the AEC and modifications in the nucleotide pool composition were recorded in the Bay of Seine populations. It is proposed that AEC and nucleotide concentration measurements could provide a useful tool in long term in situ monitoring of natural populations.

Characterization of a chymosin-like pepsin from the dogfish Scyliorhinus canicula

1. Pepsin II extracted from the gastric mucosa of Scyliorhinus canicula has been characterized and compared to calf chymosin. 2. The kcat and Km of the dogfish enzyme for the synthetic hexapeptide Leu-Ser-Phe(NO2)-Nle-Ala-Leu-OMe have been determined. The kcat/Km ratio is close to that of calf chymosin. Its milk-clotting efficiency is however 21-fold lower than that of calf chymosin. 3. The proteolytic activity against haemoglobin is optimal at pH 2.5. It clots the milk up to pH 6.8. 4. The dogfish pepsin II shows relatively better activity at low temperatures than calf chymosin.

Glutamine synthetase in the marine coccolithophorid Emiliania huxleyi (Prymnesiophyceae): regulation of activity in relation to light and nitrogen availability

Abstract Regulation of glutamine synthetase (GS; EC 6.3.1.2) in relation to light and nitrogen availability was investigated in Emiliania huxleyi . GS activity was low when effective illumination of the cells, and so photosynthetic activity, was high whereas it was high when effective illumination was lower. Measurements of glutamate dehydrogenase (GDH; EC 1.4.1.3) activities in the same type of culture revealed that NADPH-GDH was only present at a low level at the beginning of the exponential phase. Nitrogen starvation stimulated GS activity and also had an effect on the kinetic properties of GS. The apparent K m of GS for NH 4 + was 1.05 mM when cells were grown on NO 3 − and 0.146 mM when cells were nitrogen-starved. Subsequent to the addition of 1 mM NH 4 + to a NO 3 − -grown culture at the beginning of the exponential phase, GS was progressively inactivated, whereas NADPH-GDH activity largely increased. On the other hand, GS remained active and NADPH-GDH not detectable when ammonium ions were added during the mid-exponential phase. Algae were able to grow in the presence of 2 mM MSX, with NO 3 − as the nitrogen source, though GS was largely inactivated under these conditions. Meanwhile, NADPH-GDH activity doubled. Nitrogen and carbon metabolism appear to be intimately linked in the regulation of Emiliania GS activity. The results also suggest a major role for GS in ammonium assimilation in Emiliania huxleyi .

Demonstration of specific receptors for calcitonin in isolated trout gill cells

Abstract 1. 1. Salmon calcitonin binding by isolated gill cells from rainbow trout, Salmo gairdneri has been investigated. 2. 2. The calcitonin receptor interaction is time- and temperature-dependent. 3. 3. 50% of inhibition of the 125 I labeled calcitonin binding is observed in presence of 1.5 ng/ml unlabeled salmon calcitonin. 4. 4. Two types of receptors are described: a high affinity-low capacity site and a low affinity-large capacity site. 5. 5. These studies strongly support the role of calcitonin as a hormone regulating the gill function in physiological conditions.

Isolation of a chromoid from the chloroplast of Chlamydomonas reinhardi

Abstract Chloroplast preparations have been obtained from Chlamydomonas reinhardi mutant CW15 which lacks cell walls. The chloroplast suspensions were largely free of nuclear contamination but the organelles retained their capacity of incorporating thymidine and uracil precursors, in DNA and RNA, respectively, thus assessing the presence of the corresponding pathways. The chromoid was obtained by lysis of the chloroplast and purified by gel filtration. The transcriptional activity of the material was effective without the addition of any exogenous polymerase.

Purification and some properties of a carboxypeptidase B from dogfish Scyliorhinus canicula

Abstract A carboxypeptidase B (CPB) has been purified from dogfish ( Scyliorhinus canicula ) pancreas and partially characterized. The purification procedure included acetone precipitation, ion-exchange chromatography on a CM-cellulose column and gel filtration on Sephadex G-75. The purified enzyme migrates as a single band both on PAGE and SDS-PAGE. Its molecular mass is estimated to be about 32 kDa. The optimum of activity is obtained at pH 7.5–8.2. The enzyme is inhibited by typical metal-chelating agents (EDTA and o -phenanthroline) and by Hg 2+ . It is activated by Co 2+ , l -cysteine and by heat treatment at 40° and 50°C. Kinetic parameters, K m and k cat , of native enzyme, Co 2+ -activated CPB and heat-treated CPB have been determined