核苷二磷酸激酶A的异构及其分子机制

对核苷二磷酸激酶A(NDPKA)的异构及其分子机制进行研究.还原和非还原SDSPAGE观察重组人核苷二磷酸激酶A(rhNDPKA)的异构;RPHPLC分析rhNDPKA异构体的反相色谱行为,并测定rhNDPKA异构体的酶活性;多角度激光散射法测定rhNDPKA异构体在溶液中的表观分子量;飞行质谱分析异构体的质量肽谱.结果发现,rhNDPKA在非还原SDSPAGE上表现为4条带,对应于NDPKA的氧化型、还原型、氧化型二聚体和还原型二聚体,其分子量分别为18.1kD、21.3kD、35.2kD和38.3kD.RPHPLC发现,还原型rhNDPKA和氧化型rhNDPKA疏水性有差异.新鲜制备的rhNDPKA在纯水溶液中,经空气氧化后,逐渐由还原型向氧化型过渡,而还原剂或生理盐水可使rhNDPKA稳定于还原型或氧化型.酶活测定结果表明,还原型rhNDPKA比活性为1965±166Umg,氧化型rhNDPKA比活性为974±53Umg.多角度激光散射检测发现,还原型rhNDPKA在溶液中仍可形成六聚体.质量肽谱结果证明,在氧化型rhNDPKA中,C4和C145位巯基形成二硫键,而C109位巯基游离存在.根据本文所确定的NDPKA单体中的二硫键位置,推导出rhNDPKA单体异构体和二聚体异构体的变构原理,这为进一步研究NDPKA的多能性调节机制打下了良好基础.

Isomerization and Its Mechanism of Nucleoside Diphosphate Kinase A

Nucleoside diphosphate kinase A (NDPK-A) has been implicated as a multifunctional protein and eukaryotic enzyme. To study the isomerization and its mechanism of NDPK-A, the isomerization of recombinant human nucleoside diphosphate kinase A (rhNDPK-A) was observed in reduced and unreduced SDS-PAGE. RP-HPLC was applied to analyze the chemical character and enzymatic activity of rhNDPK-A isomers. Multiangle laser light-scattering method (MALS) was applied to measure the apparent molecular weight of rhNDPK-A isomers in solution. The peptide maps of rhNDPK-A isomers were analyzed by matrix assisted laser desorption ionization time-of-flight MS (MALDI-TOF MS). It was found that rhNDPK-A displayed to be four bands in unreduced SDS-PAGE with estimated molecular weight of 18.1 kD, 21.3 kD, 35.2 kD and 38.3 kD, respectively. The four bands of rhNDPK-A correspond to its four kind of isomers, i.e. oxidative rhNDPK-A, reductive rhNDPK-A, dimer of oxidative rhNDPK-A, dimer of reductive rhNDPK-A. It was observed that there was difference in wash-time between reductive and oxidative rhNDPK-A, and the reductive rhNDPK-A isomerized into oxidative form in water under the oxidation of oxygen in air. Enzymatic activity assay revealed that the specific enzymatic activity of reductive rhNDPK-A was 1965±166 U/mg, while oxidative rhNDPK-A was 974±53 U/mg. Measurement of the apparent molecular weights of rhNDPK-A isomers in solution demostrated that the isomerization of oxidative rhNDPK-A into reductive form does not change its character of aggregating into hexamer. Peptide mapping proved that the Cys-4 and Cys-145 formed into disulfide bond and Cys-109 was free in reductive rhNDPK-A. These results revealed the position of disulfide bond in NDPK-A and the isomeric formtion mechanism among rhNDPK-A monomers and dimers, which put a solid foundation for the mechanism of NDPK's multifunction.