Biochemical Properties Of Phenoloxidase From Penaeus Chinensis And Effects Of Several Immunostimulants On Penaeus Phenoloxidase And Hemocytes

Using I-DOPA as a specific substrate, phenoloxidase (PO) from Penaeus chinensis hemolymph was purified by gel-filtration and ion-exchange chromatography, and characterized in terms of its molecular weight and enzymatic properties in this study. It was found that prophenoloxidase (proPO), isolated as a monomeric protein, had a molecular weight of 87.5 kDa, and a 77 kDa phenoloxidase molecule was often contained in preparations. The 87.5 kDa proPO had a very low enzymatic activity on 0.02 mol/L L-DOPA, whereas the 77 kDa PO had a very high enzymatic activity on it. Enzymatic activity of PO against L-DOPA was optimal at pH 6.0 and temperature of 40 , with an apparent Km value of 1.99 mmol/L. The PO activity was extremely sensitive to ascorbic acid, cysteine, and dithiothreitol, very sensitive to thio urea, but not sensitive to sodium sulfite, citric acid, and benzoic acid. All these indicate that the PO purified in this study do be a kind of phenoloxidase. Combined with its specific enzymatic activity on substrate of monophenol and L-DOPA, it implies that the PO belongs to a tyrosinase-type phenoloxidase. Besides, the PO was very sensitive to EDTA and some metal ions, and its activity was strongly enhanced by Mg2+ and strongly inhibited by EDTA and Cu2+. The PO is very sensitive to some trypsin-specific inhibitors such as SBTI, p-APMSF and bestatin, but almost not affected by trypsin-specific inhibitor TLCK at all, indicates that it is like a tyrosine-type protease. The results on EDTA and some metal ions, combined with the excellent recovery effect of Cu2+ on DETC-inhibited PO activity, indicate that this PO is a kind of Cu2+ metalloenzyme.The effect of polysaccharides and vibrios on the hemocytes and PO fromshrimp was investigated. Under the stimulation of p-glucan, the number of all hemocytes, the number of semigranular cells, the amount of PO synthesized and PO activity increased about 83.4 %, 100.4 %, 104.7 % and 79.4%, respectively. Under the stimulation of LPS, the number of all hemocytes, the number of semigranular cells, the amount of PO synthesized and PO activity increased about 52.0 %, 67.3 %, 81.3 % and 113.1 %, respectively. Under the stimulation of inactivated Vibrio harveyi, the number of all hemocytes, the number of semigranular cells, the amount of PO synthesized and PO activity increased about 73.4 %, 57.2 %, 30.1 % and 37.8 %, respectively. Under the stimulation of inactivated Vibrio anguillarum, the number of all hemocytes, the number of semigranular cells, the amount of PO synthesized and PO activity increased about 111.3 %, 102.9 %, 40.4 % and 46.3 %, respectively. The highest percentege of semigranular cells in all kinds of hemocytes after stimulation implies that it plays a key role in the innate immune system of Penaeus chinensis. Morever, under the stimulation of polysaccharides and vibrios, the amount of PO increases greatly, but the unit activity of PO remains almost unchanged. It can be concluded that the elevated ability of Penaeus immune system is realized via increasing PO production instead of increasing of unit enzyme activity of PO. Since the biggest number of hemocytes, semigranular cells and PO production have been obtained after -1,3-glucan and LPS stimulation, it implies that the -1,3-glucan and LPS would be the most efficient immunostimulant on shrimp, Penaeus chinensis, among the immunostimulants used.