Tomasselli A G, Noda L H
Eur J Biochem. 1980 Feb;103(3):481-91. doi: 10.1111/j.1432-1033.1980.tb05972.x.
AMP phosphotransferase has been purified 186-fold from beef heart mitochondria in an overall yield of 30%. The purified enzyme, Mr 31 500, has a specific activity of 800 U/mg and was found to be a homogeneous protein by constancy of specific activity across the elution peak following passage through an Affi-Gel Blue agarose column and by dodecylsulfate gel electrophoresis. Isoelectrofocusing shows a minor peak of activity that arises again when the major peak is re-focused. The pI of the enzyme is 9.30 and the optimum pH of activity is 5.8 in the forward reaction (formation of ADP) and 8.0 in the reverse reaction. The enzyme requires the presence of a divalent metal ion, namely Mg2+, Co2+, Ca2+ Mn2+, Ni2+ and Zn2+ in decreasing order of activity and is influenced by NaCl. It shows rigorous specificity for AMP (dAMP) was phosphate acceptor but is rather unspecific for the triphosphate donor since, UTP, CTP, ITP and GTP may substitute for ATP (dATP). ATP and MgATP (dATP was not tested) bind to the enzyme with dissociation constants of 0.122 and 0.132 mM respectively, while AMP (dAMP was not tested), UTP, CTP, ITP and GTP binding could not be detected by the dialysis equilibrium technique used. Amino acid analysis gave the following composition as residues: 26 aspartic acid or asparagine, 14 threonine, 17 serine, 27 glutamic acid or glutamine, 24 proline, 19 glycine, 25 alanine, 17 valine, 8 methionine, 14 isoleucine, 31 leucine, 5 tyrosine, 9 phenylalanine, 6 histidine, 25 lysine, 15 arginine, 4 half cystine (no free sulfhydryls) and no tryptophan, giving a total of 286 residues. The purified mitochondrial adenylate kinase is not a contaminating heart cytosolic enzyme. ATP:AMP phosphotransferase was extracted from beef heart cytosol depleted of mitochondria and brought to homogeneity. Determinations of pI, Mr and amino acid composition showed that the heart mitochondrial and cytosolic adenylate kinases are different isozymes.
AMP磷酸转移酶已从牛心线粒体中纯化出来，纯化倍数为186倍，总产率为30%。纯化后的酶，分子量为31500，比活性为800 U/mg，经Affi-Gel Blue琼脂糖柱洗脱峰上比活性恒定以及十二烷基硫酸钠凝胶电泳鉴定，该酶为均一蛋白质。等电聚焦显示有一个次要活性峰，当主要活性峰重新聚焦时该次要峰再次出现。该酶的pI为9.30，正向反应（生成ADP）的最佳活性pH为5.8，反向反应的最佳活性pH为8.0。该酶需要二价金属离子的存在，即Mg2+、Co2+、Ca2+、Mn2+、Ni2+和Zn2+，活性依次降低，且受NaCl影响。它对AMP（dAMP）作为磷酸受体具有严格的特异性，但对三磷酸供体则不太特异，因为UTP、CTP、ITP和GTP可替代ATP（dATP）。ATP和MgATP（未测试dATP）与该酶结合，解离常数分别为0.122和0.132 mM，而通过所用的透析平衡技术未检测到AMP（未测试dAMP）、UTP、CTP、ITP和GTP的结合。氨基酸分析给出的残基组成如下：26个天冬氨酸或天冬酰胺、14个苏氨酸、17个丝氨酸、27个谷氨酸或谷氨酰胺、24个脯氨酸、19个甘氨酸、25个丙氨酸、17个缬氨酸、8个甲硫氨酸、14个异亮氨酸、31个亮氨酸、5个酪氨酸、9个苯丙氨酸、6个组氨酸、25个赖氨酸、15个精氨酸、4个半胱氨酸（无游离巯基）且无色氨酸，总共286个残基。纯化的线粒体腺苷酸激酶不是污染的心脏胞质酶。ATP:AMP磷酸转移酶从不含线粒体的牛心胞质中提取并达到均一性。pI、分子量和氨基酸组成的测定表明，心脏线粒体和胞质腺苷酸激酶是不同的同工酶。
