Lin M, Turpin D H, Plaxton W C
Department of Biology, Queen's University, Kingston, Ontario, Canada.
Arch Biochem Biophys. 1989 Feb 15;269(1):219-27. doi: 10.1016/0003-9861(89)90103-3.
Pyruvate kinase from the green alga Selenastrum minutum consists of two isoforms (PK1 and PK2) separable by Q-Sepharose chromatography. The two isoforms have been highly purified to respective final specific activities of 42 and 23 (mumol pyruvate produced/min)/mg protein. Purification steps included salt fractionation, anion-exchange, hydrophobic interaction, and gel filtration chromatography. The final enzyme preparations differ significantly in physical and immunological properties. PK1 is heat labile and is completely inactivated following reaction with N-ethylmaleimide. In contrast, PK2 is heat-stable and is only partially inactivated following N-ethylmaleimide treatment. PK1 appears to be homotetrameric with a native molecular mass of about 240 kDa, whereas PK2 appears to be homodecameric with a native molecular mass of approximately 590 kDa. The antigenic reaction of both final PK preparations to rabbit antiserum prepared against homogeneous germinating castor bean endosperm cytosolic pyruvate kinase was tested by immunoprecipitation and Western blotting. The two algal pyruvate kinases are immunologically unrelated as only PK2 cross-reacts with the cytosolic pyruvate kinase antibodies. These data indicate that the S. minutum pyruvate kinase isoforms, PK1 and PK2, are not interconvertible forms of the same protein, but probably represent chloroplastic and cytosolic isozymes, respectively.
来自绿藻微小色球藻的丙酮酸激酶由两种可通过Q-琼脂糖凝胶层析分离的同工型（PK1和PK2）组成。这两种同工型已被高度纯化，各自的最终比活性分别为42和23（微摩尔丙酮酸产生量/分钟）/毫克蛋白质。纯化步骤包括盐分级分离、阴离子交换、疏水相互作用和凝胶过滤层析。最终的酶制剂在物理和免疫特性上有显著差异。PK1对热不稳定，与N-乙基马来酰亚胺反应后会完全失活。相比之下，PK2对热稳定，N-乙基马来酰亚胺处理后仅部分失活。PK1似乎是同源四聚体，天然分子量约为240 kDa，而PK2似乎是同源十聚体，天然分子量约为590 kDa。通过免疫沉淀和蛋白质印迹法检测了两种最终PK制剂与针对同质萌发蓖麻籽胚乳胞质丙酮酸激酶制备的兔抗血清的抗原反应。两种藻类丙酮酸激酶在免疫上不相关，因为只有PK2与胞质丙酮酸激酶抗体发生交叉反应。这些数据表明，微小色球藻丙酮酸激酶同工型PK1和PK2不是同一蛋白质的可相互转化形式，而是可能分别代表叶绿体和胞质同工酶。
