Study Of Polyamine Metabolic Pathway In Euplotes

Polyamines are low molecular weight aliphatic compounds with two or more amino groups,and they are short-chain aliphatic amines.Polyamines are widely distributed in prokaryotic and eukaryotic cells,and are important metabolic regulators,which can directly or indirectly affect cell activity.Almost all organisms contain polyamine biosynthesis pathway,but some parasitic protozoa have lost all or part of polyamine biosynthesis pathway.Previous studies showed that the polyamine biosynthesis pathways in ciliates are diverse.Euplotes is a single-celled eukaryote and free-living ciliate,which has a special evolutionary position.In this study,Euplotes was used as experimental material,and the genes related to polyamine biosynthesis pathway in Euplotes were analyzed by bioinformatics.The kinds and contents of polyamines in Euplotes were detected by high performance liquid chromatography(HPLC).The source of polyamines in Euplotes was also discussed.The results obtained are as follows:1.Identification of genes related to polyamine biosynthesis pathway in EuplotesThe genes related to polyamine biosynthesis in five species of Euplotes and other six ciliates whose macronucleus genomes were identified by homologous sequence alignment.The results showed that all the genes of polyamine biosynthesis pathway in Euplotes were lost,but all of them contained two enzymes needed for the hypusine modification of eIF5A.2.Establishment of high performance liquid chromatography method and determination of polyamines content in EuplotesIn order to verify the existence of polyamines in Euplotes,a method using high performance liquid chromatography was developed:benzoyl chloride pre-column derivation—liquid-liquid extraction of polyamines derivatives—reversed phase high performance liquid chromatography detection of intracellular polyamines in ciliates.This method was used to detect intracellular polyamines in Euplotes octocarinatus and Euplotes amieti.The optimum derivative conditions and chromatographic conditions were determined as follows:the dosage of benzoyl chloride was 30 μL,the reaction time was 20 min at 37℃,the mobile phase ratio is methanol:water=60:40,the elution time was 20 min,the flow rate was 1 mL/min,and the detection wavelength was 254 nm.In the chromatograms of polyamine standards obtained by this method,the separation degrees of two adjacent polyamine derivatives were 1.5,4.4,2.2 and 1.8,respectively.All four polyamine standards and the internal derivatives could be effectively separated.Under the optimum experimental condition,the standard curves of putrescine,cadaverine,norspermidine and spermidine were drawn,and the regression equations are Y=0.0184x-0.0362(R2=0.9954),Y=0.0094x+0.018(R2=0.9973),Y=0.0143x+0.0182(R2=0.9991),and Y=0.0078x+0.0511(R2=0.9971),respectively.Subsequently,this method was used to detect polyamines in Tetrahymena thermophilus,and the results showed that there were only putrescine and spermidine,which were consistent with the previous reported by Koei Hamana et al using HPLC fluorescence method.These resluts indicated that this method was feasible and could be used in subsequent experiments.The intracellular polyamines of E.octocarinatus and E.amieti were detected by using this method.The results showed that E.octocarinatus and E.amieti both contained putrescine,norspermidine and spermidine.The contents of putrescine,norspermidine and spermidine in E.octocarinatus were 4.122±0.125 μmol/g,0.169±0.061 μmol/g and 0.043± 0.066 μmol/g,respectively.However,the contents of putrescine,norspermidine and spermidine in E.amieti were 4.344± 0.093 μmol/g,0.725± 0.059 μmol/g and 0.104± 0.031 μmol/g,respectively.3.The source of polyamines in EuplotesThe sources of polyamines in human include cellular biosynthesis,food and intestinal microbiota.Euplotes are heterotrophic organisms,which feed on unicellular algae and bacteria in the natural environment.Moreover,Euplotes harbour endosymbionts.Since polyamines cannot be synthesized by themselves,it is speculated that polyamines may be provided by food or endosymbionts.In our laboratory,Chlorogoium elongatum was used to feed Euplotes.At first,polyamines in Chlorogoium elongatum were detected,including putrescine,demethylspermidine and spermidine.Then,polyamines in Escherichia coli were detected,and three polyamines were found,namely putrescine,cadaverine and spermidine.Their unique polyamines were norspermidine and cadaverine,respectively.When using Chlorogoium elongatum as sole food source,putrescine,spermidine and spermidine were detected in Euplotes.When a mixture of C.elongatum and E.coli were used as as food source,four polyamines,namely putrescine,cadaverine,norspermidine and spermidine,can be detected in Euplotes.The types of intracellular polyamines of Euplotes changed with the changes of food.The genomes of endosymbionts Polynucleobacter necessarius and Fluviibacter phosphoraccumulans both contain polyamine biosynthesis related lysine/arginine/ornithine decarboxylases(LAOdc)genes.Gene cloning,protein expression and enzymatic characterization of LAOdc gene of these two endosymbionts showed that LAOdc protein of endosymbionts were bifunctional ornithine/lysine decarboxylase,but their substrate affinity for L-lysine were much higher than that of L-ornithine.The enzymatic properties of these two decarboxylases were characterized.The optimum pH values of Pn-LAOdc and Fp-LAOdc were 5.5 and 6.5 and the optimum temperature were 55℃ and 60℃,respectively.The Km values of L-lysine were 1.442 mM and 1.088 mM,and the Vmax values were 3.472 μmol/min and 2.636μmol/min,respectively.These results show that food can provide polyamines for Euplotes.The endosymbionts of Euplotes can synthesize cadaverine and putrescine,but it still needs further study to verify that whether symbionts can provide polyamines for Euplotes.