| Previous studies showed that in the scallop Chlamys farreri, bacterialpolysaccharides challenge or viral infection caused a rapid and dramatic increase ofphenoloxidase (PO) activity in hemocytes, indicating that PO is one of the mostsensitive and effective factors in the immune responses of C. farreri (Xing et al.,2008;Lin et al.,2011). In this dissertation, PO of the scallop C. farreri was purified fromhemocytes and characterized biochemically and enzymatically, then the antibacterialactivity of C. farreri PO reaction products generated in vitro was characterized, finally,the difference among C. farreri PO and POs from Argopecten irradians, Ruditapesphilippinarum, Haliotis discus hannai and Scapharca subcrenata on biochemical andenzymatic characteristics were analyzed.(I) Phenoloxidase in the scallop C. farreri: purification, biochemical andenzymatic characterization. PO of the scallop C. farreri was purified fromhemocytes using linear-gradient native PAGE combined with gel permeation columnchromatography, and characterized based on substrate specificity, kinetic parameters,and the effects of temperature, pH, divalent metal ions, and inhibitors on enzymaticactivity. The results show that purified PO had a molecular mass of576kDa in nativePAGE and53kDa in SDS-PAGE, suggesting that C. farreri PO is an aggregatecomposed of one subunit. The analysis of the kinetics indicated that the Kmvalues ofPO for L-DOPA, catechol, dopamine and hydroquinone were0.61mmol/L,0.20mmol/L,0.88mmol/L and0.83mmol/L, respectively, which suggests that C. farreriPO was a laccase-type phenoloxidase, showing the highest affinity for catechol, thelowest affinity for dopamine, and a higher affity for L-DOPA than hydroquinone. ThePO had optimal activities at30oC and a pH of8.0, and its activity was greatlyenhanced by Ca2+and Mg2+, and inhibited by Fe2+. In addition, the PO activity wasinhibited by sodium sulfite, ascorbic acid, sodium diethyldithiocarbamate (DETC),cysteine, citric acid, and ethylenediamine tetraacetic acid disodium (EDTA), which suggests that C. farreri PO was a copper-containing metalloenzyme.(II) Antibacterial activity of C. farreri PO reaction products generated in vitro.Antibacterial activity of C. farreri PO reaction products against Vibrio alginolyticus, V.parahaemolyticus, Aeromonas salmonicida, Edwardsiella tarda, Streptococcusdysgadysgalactiae, Streptococcus iniae and Micrococcus lysodeikticus wascharacterized by observation and determination of bacterial growth after treatingbacteria with purified PO and substrates. The results show that the mixture of PO andsubstrates inhibited bacterial growth, while PO solely had no effect on bacterialgrowth, illustrating that it is PO reaction products not PO have the antibacterialactivity. L-DOPA-derived compounds only showed inhibition in A. salmonicida,while dopamine-derived compounds showed strong inhibitions in the Gram-negativeV. alginolyticus, V. parahaemolyticus and A. salmonicida, indicating thatdopamine-derived compounds are more cytotoxic than L-DOPA-derived compounds.No significant inhibition was found in Gram-positive S. dysgadysgalactiae, S. iniaeand M. lysodeikticus, suggesting that PO reaction compounds are more harmful to thecell components of Vibrio and Aeromonas than those of Streptococcus andMicrococcus. Besides, PO reaction products showed no inhibition in E. tarda, whichwas also Gram-negative, suggesting that the extracellular polysaccharides with strongantioxidant activities, produced by Edwardsiella, might inhibit the PO reaction.Microscopic observation showed that after incubation with PO and dopamine, V.alginolyticus, V. parahaemolyticus and A. salmonicida lacked transparency andmobility, and often appeared as aggregates covered with black pigments.(III) Comparison of C. farreri PO and POs from A. irradians, R. philippinarum,H. discus hannai and S. subcrenata based on biochemical and enzymaticcharacteristics. POs of A. irradians, R. philippinarum, H. discus hannai and S.subcrenata were isolated from hemocytes using linear-gradient native-PAGEcombined with catechol staining, then C. farreri PO was compared and analyzed withthe isolated POs based on substrate specificity, kinetic parameters and the effects ofdivalent metal ions and inhibitors on enzymatic activity. The results of isolation showthat C. farreri was similar to A. irradians, R. philippinarum and H. discus hannai, in which only one PO was detected in hemocytes, and different from S. subcrenata, inwhich four POs were detected. In terms of molecular mass in native-PAGE, C. farreriPO (576kDa) was similar to POs in A.irradians (555kDa) and R. philippinarum(563kDa), and differed greatly from POs in H. discus hannai (228kDa) and S.subcrenata (391,206,174kDa and an unknown one, which were named as S391PO,S206PO, S174PO and SuPO respectively). As far as substrate specificity is concerned,C. farreri PO was similar to POs from A. irradians, R. philippinarum, H. discushannai and S. subcrenata, which could oxidize o-diphenols and p-dipehnols, werelaccase-type phenoloxidase. Kinetic analysis indicated that the Kmvalues of C. farreriPO for L-DOPA, catechol, dopamine and hydroquinone were more similar to that of A.irradians PO, suggesting that the catalytic capability is parallel between the two POs.Metal ions test showed that C. farreri PO and POs from A. irradians, R.philippinarum, H. discus hannai and S. subcrenata were all sensitive to divalent metalions, and all inhibited by Fe2+, both Ca2+-and Mg2+-mediated enhancement of POactivity were only found in C. farreri and A. irradians, and in general the results ofmetal ions test in C. farreri PO are very similar to that in A. irradians PO, whichsuggests that there might be very high similarity on protein structure between the twoPOs. Besides, the strong inhibition of EDTA and DETC indicating that C. farreri POand POs from A. irradians, R. philippinarum, H. discus hannai and S. subcrenatawere all copper-containing metalloenzyme, however, C. farreri PO was more similarto A. irradians PO, S206PO and SuPO, which were not inhibited by thiourea andsodium azide. |
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