BIOCHEMICAL AND MOLECULAR CHARACTERIZATION OF NUCLEOSIDE TRIPHOSPHATE HYDROLASE ISOZYMES FROM THE PARASITIC PROTOZOAN TOXOPLASMA-GONDII
BIOCHEMICAL AND MOLECULAR CHARACTERIZATION OF NUCLEOSIDE TRIPHOSPHATE HYDROLASE ISOZYMES FROM THE PARASITIC PROTOZOAN TOXOPLASMA-GONDII
复制标题
DOI：
10.1074/jbc.270.19.11391
复制
发表时间：
1995-05-12
影响因子：
4.8
通讯作者：
TAKEUCHI, T
中科院分区：
文献类型：
--
作者：
ASAI, T;MIURA, S;TAKEUCHI, T
我们以前曾报道过一种新的核苷三磷酸水解酶（NTH）的存在下，迅速繁殖的速殖子形式的强毒株（RH）的弓形虫。经进一步的检测，发现纯化的酶不是单一的酶，而是两种同工酶的混合物，分别命名为NTPase-I和NTPase-II。这两种同工酶经凝胶过滤测得的分子量约为240-270 kDa，经SDS聚丙烯酰胺凝胶电泳测得的分子量约为66-67 kDa，含有四个相同的亚基。两种形式的NTR都能被二硫苏糖醇激活，NTPase-I在ATP水解中的比活性比NTR II高4.5倍。这两种同工酶之间的主要区别在于它们水解三磷酸核苷与二磷酸核苷底物的能力。NTPase-II水解ATP为ADP和ADP为AMP的速率几乎相同，而NTPase-I水解ADP为AMP的速率要慢得多对NTPase-I和NTPase-II的完整cDNA进行测序，发现其编码相同大小的预测开放阅读框，其中628个氨基酸中只有16个在两种同工酶之间不同，这两种形式的NTR都含有一个NH 2-末端疏水信号肽，这与我们以前的发现一致，即这些酶被分泌到寄生虫占据的宿主细胞液泡中。编码NTPase-II的基因在所有弓形虫株中均有表达，而NTPase-I仅限于强毒株。刚地
We have previously reported the presence of a novel nucleoside triphosphate hydrolase (NTPase) in the rapidly multiplying tachyzoite form of a virulent strain (RH) of Toxoplasma gondii. On further examination, it was found that the purified enzyme was not a single enzyme but was a mixture of two isozymes termed NTPase-I and NTPase-II.The two isozymes had similar molecular masses of approximately 240-270 kDa by gel filtration and contained four identical subunits of molecular mass 66-67 kDa by SDS polyacrylamide gel electrophoresis. Both forms of the NTPase were activated by dithiothreitol, and NTPase-I had a specific activity 4.5-fold higher than NTPase II in hydrolysis of ATP, The primary difference between these isozymes lies in their ability to hydrolyze nucleoside triphosphate versus diphosphate substrates. While NTPase-II hydrolyzed ATP to ADP and ADP to AMP at almost the same rate, NTPase-I hydrolyzed ADP to AMP at a much slower rate (0.7% of the rate for ATP).The complete cDNAs for NTPase-I and NTPase-II were sequenced and found to encode the same size predicted open reading frame of which only 16 of 628 amino acids differed between the two isozymes, Both forms of the NTPase contained an NH2-terminal hydrophobic signal peptide, consistent with our previous findings that these enzymes are secreted into the host cell vacuole occupied by the parasite. The gene encoding NTPase-II was found in all strains of Toxoplasma, while the NTPase-I was confined only to virulent strains, Expression of this highly active ATPase (NTPase-I) may contribute to intracellular survival and virulence of T. gondii.