LeGros H L, Halim A B, Geller A M, Kotb M
Veterans Affairs Medical Center, Memphis, Tennessee 38104, USA.
J Biol Chem. 2000 Jan 28;275(4):2359-66. doi: 10.1074/jbc.275.4.2359.
MAT II, the extrahepatic form of methionine adenosyltransferase (MAT), consists of catalytic alpha(2)/alpha(2') subunits and a noncatalytic beta subunit, believed to have a regulatory function. The full-length cDNA that encodes the beta subunit of human MAT II was cloned and found to encode for a 334-amino acid protein with a calculated molecular weight of 37,552. Analysis of sequence homology showed similarity with bacterial enzymes that catalyze the reduction of TDP-linked sugars. The beta subunit cDNA was cloned into the pQE-30 expression vector, and the recombinant His tagged protein, which was expressed in Escherichia coli, was recognized by antibodies to the human MAT II, to synthetic peptides copying the sequence of native beta subunit protein, and to the rbeta protein. There is no cross-reactivity between the MAT II alpha(2) or beta subunits. None of the anti-beta subunit antibodies reacted with protein extracts of E. coli host cells, suggesting that these bacteria have no beta subunit protein. Interestingly, the rbeta subunit associated with E. coli as well as human MAT alpha subunits. This association changed the kinetic properties of both enzymes and lowered the K(m) of MAT for L-methionine. Together, the data show that we have cloned and expressed the human MAT II beta subunit and confirmed its long suspected regulatory function. This knowledge affords a molecular means by which MAT activity and consequently the levels of AdoMet may be modulated in mammalian cells.
MAT II是蛋氨酸腺苷转移酶（MAT）的肝外形式，由催化性α(2)/α(2')亚基和一个非催化性β亚基组成，据信该β亚基具有调节功能。编码人MAT IIβ亚基的全长cDNA被克隆出来，发现其编码一种334个氨基酸的蛋白质，计算分子量为37,552。序列同源性分析表明，它与催化TDP连接糖还原的细菌酶具有相似性。将β亚基cDNA克隆到pQE - 30表达载体中，在大肠杆菌中表达的重组His标签蛋白可被抗人MAT II抗体、与天然β亚基蛋白序列相同的合成肽抗体以及抗rβ蛋白抗体识别。MAT II的α(2)亚基或β亚基之间没有交叉反应。抗β亚基抗体均未与大肠杆菌宿主细胞的蛋白提取物发生反应，这表明这些细菌没有β亚基蛋白。有趣的是，rβ亚基与大肠杆菌以及人MATα亚基相关联。这种关联改变了两种酶的动力学特性，并降低了MAT对L - 蛋氨酸的K(m)值。总之，这些数据表明我们已经克隆并表达了人MAT IIβ亚基，并证实了其长期以来被怀疑的调节功能。这一知识为在哺乳动物细胞中调节MAT活性以及进而调节AdoMet水平提供了一种分子手段。
