BIOCHEMICAL PROPERTIES OF RECOMBINANT CHOLONESTERASE VARIANT SPECIFIC IN JAPANESE

据报道，人血清丁酰胆碱酯酶(EC3.1.1.8；BCHE)的几个基因变异与肌肉松弛药物琥珀胆碱治疗后长时间的呼吸暂停有关。用地布卡因数(Dn)和氟化数(Fn)对这些BCHE变异体进行定性鉴定。非典型变异体通过对地布卡因抑制的抗性(Low Dn)进行表型鉴定，而耐氟变异体通过对NaF(低Fn和Dn)的抑制进行表型鉴定。已有两种类型的抗氟基因被报道。它们分别是Thr243 Met和Gly390Val的氟化物-1和-2变异体。即使在对日本人群中的非典型BCHE(Asp70Gly)和两种抗氟变异体进行多次筛查后，我们也没有发现这些突变(我们的个人数据)。因此，我们认为这些差异可能源于种族差异。我们发现了一个L330I突变纯合子(密码子330由亮氨酸变为异亮氨酸)，表现出酶活性降低，DN和FN。他的DN和FN表现为FS表型，而不是FF。L330I突变在国外从未见过报道。通过重组BCHE蛋白在人胎肾细胞中的表达，BCHE L330I突变具有低酶活性，低DN和FN。因此，我们认为BCHE L330I突变是一个日本型的抗氟基因。我们还表达了重组CHE蛋白(野生型、330V和330S)。所有330位氨基酸替换的重组子不仅导致BCHE活性的降低，而且导致了DN和FN的异常，降低的程度几乎相同。我们认为330密码子突变的重组BCHE可能具有抗氟基因的特性，提示330密码子在与底物和氟化钠的相互作用中起重要作用。

Several genetic variants of human serum butyrylcholinesterase (EC3.1.1.8 ; BCHE) have been reported to be associated with prolonged apnea after medication of the muscle relaxant drug succinylcholine. These BCHE variants were qualitatively identified by dibucaine number (DN) and fluoride number (FN). Atypical variant is phenotyped by resistance to inhibition by dibucaine (low DN) and fluoride resistant variants were phenotyped by resistance to inhibition by NaF (low FN and DN).Two types of fluoride-resistant gene have been reported. They were Fluoride-1 and -2 variants which were Thr243Met and Gly390Val, respectively. Even after screening several times for the atypical BCHE (Asp70Gly), and two kinds of the fluoride-resistant variants in the Japanese population, we could not find these mutations (our personal data). Therefore, we considered these differences might have originated from ethnic difference.We found an individual homozygous for L330I mutation (alteration from leucine to isoleucine at codon 330) who showed reductions of enzymatic activity, DN and FN.His DN and FN showed FS phenotype rather than FF.The L330I mutation has never been reported in foreign countries.By expression of the recombinant BCHE protein in human fetal kidney cells, The BCHE L330I variant showed low enzymatic activity, low DN and FN.Therefore, we conclude the BCHE L330I mutation is a Japanese type fluoride-resistant gene. We also have expressed recombinant ChE proteins (wild type, 330V and 330S). All of the recombinants with amino acid substitutions at codon 330 caused not only a reduction of BCHE activity but also the abnormal DN and FN.The degrees of the reduction were almost the same. We conclude that the mutant recombinant BCHEs at codon 330 possibly have the properties of the fluoride-resistant gene and suggest that codon 330 is important to interact with substrate and sodium fluoride.