Noguchi T, Inoue H, Tanaka T
J Biol Chem. 1986 Oct 15;261(29):13807-12.
The complete nucleotide sequences of rat M1- and M2-type pyruvate kinase mRNAs were determined by sequencing the cDNAs and by analyses of S1 nuclease mapping and primer extension. The sequences have an identical molecular size of about 2220 nucleotides excluding a poly(A) tail and include 1593-nucleotide coding region. Their nucleotide sequences are identical except for 160-nucleotide sequences within the coding regions. The amino acid sequences of the M1- and M2-type subunits deduced from the cDNA sequences differ by only 45 residues within domain C, which constitutes the main region responsible for intersubunit contact. The sequence of this region of the M2-type shows higher homology than that of the M1-type with the corresponding sequence of the L-type. Since the M2- and L-types are allosteric enzymes, unlike to the M1-type, the residues common to the M2- and L-types, but not the M1-type may be important for mediating the allosteric properties. Genomic clones encoding both M1- and M2-type isozyme mRNAs were isolated. By partial sequence analysis of a clone lambda MPK37 four exons were identified, of which two adjacent exons coded the M1- and M2-specific sequences, respectively. The two remaining exons present downstream coded amino acids common to the two isozymes. Thus, we conclude that the M1- and M2-type isozymes of pyruvate kinase are produced from the same gene probably by alternative RNA splicing.
通过对cDNA进行测序以及S1核酸酶图谱分析和引物延伸分析，确定了大鼠M1型和M2型丙酮酸激酶mRNA的完整核苷酸序列。这些序列的分子大小相同，约为2220个核苷酸（不包括聚腺苷酸尾），包含1593个核苷酸的编码区。它们的核苷酸序列除了编码区内160个核苷酸序列外完全相同。从cDNA序列推导的M1型和M2型亚基的氨基酸序列在结构域C内仅相差45个残基，结构域C是负责亚基间接触的主要区域。M2型该区域的序列与L型相应序列的同源性高于M1型。由于M2型和L型是别构酶，与M1型不同，M2型和L型共有的残基而非M1型的残基可能对介导别构性质很重要。分离出了编码M1型和M2型同工酶mRNA的基因组克隆。通过对克隆λMPK37的部分序列分析，鉴定出4个外显子，其中两个相邻外显子分别编码M1型和M2型特异性序列。位于下游的另外两个外显子编码两种同工酶共有的氨基酸。因此，我们得出结论，丙酮酸激酶的M1型和M2型同工酶可能是由同一基因通过选择性RNA剪接产生的。
