CHITIN SYNTHASE IN ENCYSTING ENTAMOEBA-INVADENS
CHITIN SYNTHASE IN ENCYSTING ENTAMOEBA-INVADENS
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DOI：
10.1042/bj2800641
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发表时间：
1991-12-15
影响因子：
4.1
通讯作者：
GILLIN, FD
中科院分区：
文献类型：
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作者：
DAS, S;GILLIN, FD
尽管入侵内阿米巴的囊壁含有甲壳素，但这种结构聚合物在包囊过程中的合成以前从未被描述过。在这里，我们报告了刺激寄生虫包囊的条件导致几丁质合成酶(CHS)活性增加，通过掺入来自UDP-GlcNAc的[H-3]GlcNAc([H-3]N-乙酰氨基葡萄糖)来衡量。放射性标记产物可被三氯乙酸或乙醇沉淀，被纯化的几丁质酶消化为放射性的壳二糖和GlcNAc，经鉴定为甲壳素。细胞分选表明，大约。60%的酶在高速上清液中。PH活性图谱显示，可溶性CHS在pH为6.0时有一最适pH，而颗粒态CHS在pH为7.0-7.5有一峰值。两种酶的活性均依赖于二价金属离子，尤其是Mn2+和Mn2++Co2+。与其他生物的CHS相比，侵染E.inadens的颗粒CHS和可溶性CHS均未被胰酶处理激活。洋地黄素和磷脂酰丝氨酸也能刺激可溶性和颗粒CHS，而磷脂酰乙醇胺只刺激可溶性CHS。UDP、UDP-葡萄糖和UDP-GalNAc对酶活性有抑制作用，而类似物多辛-D和尼可霉素则不抑制该酶的活性。这是首次报道了一种在原始真核细胞内阿米巴属囊化过程中受发育调节的酶。
Although the cyst wall of Entamoeba invadens contains chitin, synthesis of this structural polymer during encystation has not been described before. Here we report that conditions which stimulate encystation of the parasite lead to increased chitin synthase (ChS) activity, measured by incorporation of [H-3]GlcNAc ([H-3]N-acetylglucosamine) from UDP-GlcNAc. The radiolabelled product was precipitable by trichloroacetic acid or ethanol and identified as chitin because it was digested by purified chitinase to radioactive chitobiose and GlcNAc. Cell fractionation indicated that approx. 60% of the enzyme is in the high-speed supernatant. pH-activity profiles showed that soluble ChS has an optimum at 6.0, whereas particulate ChS has a peak at pH 7.0-7.5. Both the activities were dependent on bivalent metal ions, especially Mn2+ and Mn2+ plus Co2+. In contrast with the ChS of other organisms, neither the particulate nor the soluble ChS of E. invadens was activated by trypsin treatment. Soluble and particulate ChS were also stimulated by digitonin and phosphatidylserine, whereas phosphatidylethanolamine stimulated only the soluble ChS. The enzyme activities were inhibited by UDP, UDP-glucose and UDP-GalNAc, but not by the analogues Polyoxin-D or Nikkomycin. This is the first report of an enzyme which is developmentally regulated during encystation of the primitive eukaryotic genus Entamoeba.