HUMAN-KIDNEY THIOPURINE METHYLTRANSFERASE - PURIFICATION AND BIOCHEMICAL-PROPERTIES
HUMAN-KIDNEY THIOPURINE METHYLTRANSFERASE - PURIFICATION AND BIOCHEMICAL-PROPERTIES
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DOI：
10.1016/0006-2952(83)90582-8
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发表时间：
1983-01-01
影响因子：
5.8
通讯作者：
WEINSHILBOUM, RM
中科院分区：
文献类型：
--
作者：
WOODSON, LC;WEINSHILBOUM, RM
硫嘌呤甲基转移酶（TPMT）催化硫嘌呤和硫嘧啶的S-甲基化。人肾TPMT纯化超过300倍，并测定其生化性质。TPMT是可溶性的，并且具有约1的MW。36，000道尔顿，通过凝胶过滤色谱法估计。纯化的TPMT的最适pH为6.7。反应的2种共底物6-巯基嘌呤（6-MP）和S-腺苷-L-甲硫氨酸（SAM）的真实Km值分别为0.30 mM和2.7 μ M。6-硫鸟嘌呤和2-硫氧嘧啶，2个其他甲基受体底物的表观Km值分别为0.55和2.0 mM。脂肪族硫醇化合物是TPMT的不良底物或未甲基化。当底物为SAM时，S-腺苷-L-同型半胱氨酸对TPMT为竞争性抑制剂，而6-甲巯基嘌呤对6-MP为非竞争性抑制剂。纯化的TPMT既不激活，也不抑制1 mM的Ca 2+或Mg 2+，但暴露于试剂，如N-乙基马来酰亚胺和ethacrynic酸，与SH基团相互作用的失活酶。托酚酮抑制TPMT，Ki为apprx。0.85最后，基于亚细胞分布、底物特异性、动力学特征和对抑制剂的不同敏感性，可以将人肾TPMT活性与人肾巯基甲基转移酶（EC2.1.1.9）活性区分开。
Thiopurine methyltransferase (TPMT) catalyzes the S-methylation of thiopurines and thiopyrimidines. Human kidney TPMT was purified over 300-fold and its biochemical properties were determined. TPMT was soluble and had a MW of .apprx. 36,000 daltons as estimated by gel filtration chromatography. The pH optimum of the purified TPMT was 6.7. True Km values for 6-mercaptopurine (6-MP) and S-adenosyl-L-methionine (SAM), the 2 cosubstrates for the reaction, were 0.30 mM and 2.7 .mu.M, respectively. Apparent Km values for 6-thioguanine and 2-thiouracil, 2 other methyl acceptor substrates, were 0.55 and 2.0 mM, respectively. Aliphatic thiol compounds were either poor substrates for TPMT or were not methylated. S-Adenosyl-L-homocysteine was a competitive inhibitor of TPMT when the varied substrate was SAM and 6-methylmercaptopurine was a noncompetitive inhibitor with respect to 6-MP. Purified TPMT was neither activated nor inhibited by 1 mM Ca2+ or Mg2+, but exposure to reagents such as N-ethylmaleimide and ethacrynic acid that interact with SH groups inactivated the enzyme. Tropolone inhibited TPMT with a Ki of .apprx. 0.85 mM. Finally, human kidney TPMT activity could be distinguished from human kidney thiol methyltransferase (EC 2.1.1.9) activity on the basis of subcellular distribution, substrate specificity, kinetic characteristics and differential sensitivity to inhibitors.