The Effects Of Chitosan On The Key Enzymes In Ammonia Assimilation From Non-heading Chinese Cabbage Leaves

The effects of chitosan (CTS) on the key enzymes (glutamine synthetase, GS; glutamine 2-oxoglutarate aminotransferase, GOGAT; glutamate dehydrogenase, GDH) of ammonia assimilation and the protein content were investigated in non-heading Chinese cabbage leaves. The results confirmed that chitosan treatment led to depression of GS activity and increase in GDH activity in the leaves sprayed with chitosan (0.8mg/mL). CTS treatment of the leaves dramatically lowered the Km value of GDH for ammonium ion and elevated that of GS from 71mM to 23mM and 50mM to 67mM, respectively. The contents of GDH and GS isoenzymes showed similar responses to specific GDH and GS activities. Moreover, the further analysis of GDH isoenzymes demonstrated that chitosan treatment also increased the levels of some of the low molecular weight polypeptides (aminating) of GDH. It displayed that GDH pathway might be predominant and GS/GOGAT cycle be secondary in ammonia assimilation by analysis of the activity ratios of GS/GDH and GS/GOGAT. Results indicated that the amination capability was enhanced in the non-heading Chinese cabbage leaves treated with chitosan. The enhanced amination capacity by GDH would in turn lead to the increase of the soluble protein content.GDH and GS activities in non-heading Chinese cabbage leaves were investigated by using different applications (chitosan, CaCl2, EGTA,alone or in combination with chitosan (CTS). The results revealed that CTS, Ca2+, DTT and ATP could increase GDH and GS activities, while EGTA, Verapamil, LaCl3 and Chloropromaize decreased GDH and GS activities. Furthermore, the combinations of CTS with exogenous Ca2+ and the factors elevating [Ca2+]i could raise the activities of GDH and GS, whereas CTS declined the activity levels of GDH and GS after calcium messenger system having been blocked by calcium chelator (EGTA), plasma membrane calcium channel inhibitors (verapamil and LaCl3) and CaM repel Reagent (chloropromaize). It was concluded that Ca2+/CaM might involve in CTS regulating the activities of GDH and GS, correspondingly, which resulted in the improved nutrient qualities and elevated yield of non-heading Chinese cabbage.A novel chitosan (CTS)-binding protein was isolated from the non-heading Chinese cabbage leaves by chitosan affinity chromatography and partially characterized. The overall yield of affinity-purified protein was 8mg/kg fresh leaves for non-heading Chinese cabbage. Results revealed that the protein did not exhibit chitosan activity. PAGE showed only one band coincident with the purified protein. The profile of 53.1kDa purified protein on SDS-PAGE was compared with the native molecular weigh of 106.5kDa, which indicated it was a dimer with identical subunits. After isoelectric focusing, it gave a band at pH 8.25. Agglutination test suggested that the protein was a lectin. A PAGE gel, stained by the petiodic acid-schiff's method to reveal glycoproteins, further displayed that the bindng-protein was a glycoprotein belonging to lectin, which contained 17.4%(w/w) neutral carbohydrate content of the glycoprotein detected by the phenol/H2SO4 method. Peptide mapping was comparable to the reported protein in protein bank. The database homology search (NCBI blast)indicated that the binding-protein shared 70-80% homogeneity to L-aspartate oxidase, Aspartyl/glutamyl-tRNA (Asn/Gln) amidotransferase subunit B, Glutamyl-tRNA reductase, Histidyl-tRNA synthetase.Quantitive RT-PCR approach was applied to examine the effect of CTSon the levels of GDHmRNA and GSmRNA. The results combined with thechanges of GDH and GS activities suggested that the trend of GDHmRNAlevels almost coincided with that of GDH activity, and that GS activity wasnot well correlated with the GSmRNA steady-state level. It indicated thatCTS was likely to regulate two key enzymes, GDH and GS, intranscriptional and posttranslational levels, respectively.