Kahn A, Marie J, Boivin P
Hum Genet. 1976 Jul 7;33(1):35-46. doi: 10.1007/BF00447284.
By focusing in sucrose, gradient L-type pyruvate kinase from human liver could be separated into 2 major forms (pI 6.28 +/- 0.03 and 5.85 +/- 0.09) and a minor more acid form (pI = 5). These different forms could also be detected by focusing in acrylamide-ampholine slab gel. The major forms were interconvertible, the equilibrium being shifted toward the acid form by fructose 1,6-diphosphate and SH reagents, and toward the alkaline form by proteinic factors extracted by ammonium sulphate fractionation from liver extracts and from hemolysates. These factors seemed to be responsible for the stabilization of the liver crude extract enzyme in its alkaline conformation. By acrylamide slab gel electrofocusing, erythrocyte pyruvate kinase from whole hemolysates exhibited a complex pattern composed of at least 3 introconvertible forms. The in vitro aging of the red blood cells and the storage of the hemolysates resulted in a progressive disappearance of the acid forms and in a strengthening of the alkaline form. Partially purified erythrocyte enzyme focused in 2 major bands, interconvertible under the influence of the same factors as those described for L-type pyruvate kinase. Although closely related, the focusing patterns of L-type and erythrocyte-type were never exactly identical. Double immunodiffusion against antihuman erythrocyte-and L-type pyruvate kinases. Moreover, antihuman M2-type serum was unable to neutralize erythrocyte pyruvate kinase as well as to change its electrophoretic mobility. Consequently, we conclude that both human erythrocyte- and liver L-type pyruvate kinases existed under several conformers interconvertible under the influence of the same ligands or proteinic factors; erythrocyte-type enzyme seems to include L-type subunit and not M1- or M2-type subunits. The erythrocyte- and L-type enzymes, however, are not identical and the nature of the differences between them is discussed.
通过聚焦蔗糖，人肝脏的梯度L型丙酮酸激酶可被分离为2种主要形式（pI 6.28±0.03和5.85±0.09）以及1种酸性更强的次要形式（pI = 5）。这些不同形式也可通过在丙烯酰胺-两性电解质平板凝胶中聚焦来检测。主要形式可相互转化，果糖1,6-二磷酸和SH试剂会使平衡向酸性形式移动，而硫酸铵分级分离从肝脏提取物和溶血产物中提取的蛋白质因子则会使平衡向碱性形式移动。这些因子似乎负责肝脏粗提物酶碱性构象的稳定。通过丙烯酰胺平板凝胶电聚焦，全溶血产物中的红细胞丙酮酸激酶呈现出由至少3种可相互转化形式组成的复杂图谱。红细胞的体外老化和溶血产物的储存导致酸性形式逐渐消失，碱性形式增强。部分纯化的红细胞酶聚焦在2条主要条带，在与L型丙酮酸激酶相同的因子影响下可相互转化。尽管密切相关，但L型和红细胞型的聚焦图谱从未完全相同。针对抗人红细胞和L型丙酮酸激酶进行双向免疫扩散。此外，抗人M2型血清无法中和红细胞丙酮酸激酶，也无法改变其电泳迁移率。因此，我们得出结论，人红细胞和肝脏L型丙酮酸激酶在相同配体或蛋白质因子的影响下以几种可相互转化的构象存在；红细胞型酶似乎包含L型亚基，而非M1或M2型亚基。然而，红细胞型和L型酶并不相同，并对它们之间差异的性质进行了讨论。
