Decker R S, Mohrenweiser H W
Mol Cell Biochem. 1986 Jun;71(1):31-44. doi: 10.1007/BF00219326.
Proliferating cells derived from hominoid species contain electrophoretically separable forms of triosephosphate isomerase (TPI), including a constitutive isozyme and major and minor cell proliferation specific isozymes. Genetic studies have shown that the constitutive and inducible isozymes are products of the same structural gene. A procedure has been developed for the rapid isolation of the constitutive and major proliferation specific TPI isozymes from human lymphoblastoid B cells. [35S]methionine labeled isozymes were purified through several steps of polyacrylamide gel electrophoresis in sufficient quantities for turnover studies and preliminary structural analysis. The intact isozymes were subjected to 23 steps of automated Edman degradation; both preparations yield a [35S] PTH-methionine only at cycle 14, as expected if the protein is TPI. Neither isozyme contains a blocked NH2-terminus and length heterogeneity at the amino terminal does not exist. A comparison of the two purified isozymes on 2-D PAGE confirms that the constitutive isozyme consists of only type 1 subunits while the major proliferation specific isozyme is composed of a type 1 subunit and a unique type 2 subunit. The type 1 and type 2 subunits differ by at least four charge units under native, nondenaturing conditions of electrophoresis but do not differ in molecular mass. The difference between the type 1 and type 2 subunits is covalent, as the difference in isoelectric point between the two subunits is stable to both 2% SDS and 8 M urea. The expression of TPI-2 does not correlate with the existence of the labile asparagine residues. Turnover studies indicate that the level of each subunit is regulated by differences in rates of synthesis rather than degradation but a precursor-product relationship between the subunits was not observed. Thus the mechanism for synthesis of TPI-2 must operate either during mRNA processing or nascent peptide synthesis and then only in cells from hominoid species.
源自类人猿物种的增殖细胞含有电泳可分离的磷酸丙糖异构酶（TPI）形式，包括一种组成型同工酶以及主要和次要的细胞增殖特异性同工酶。遗传学研究表明，组成型和诱导型同工酶是同一结构基因的产物。现已开发出一种从人淋巴母细胞B细胞中快速分离组成型和主要增殖特异性TPI同工酶的方法。通过聚丙烯酰胺凝胶电泳的几个步骤纯化了[35S]甲硫氨酸标记的同工酶，其数量足以进行周转研究和初步结构分析。完整的同工酶经过23步自动Edman降解；两种制剂仅在第14个循环产生[35S]PTH-甲硫氨酸，正如预期该蛋白为TPI时的情况。两种同工酶均不含有封闭的NH2末端，并且在氨基末端不存在长度异质性。在二维聚丙烯酰胺凝胶电泳上对两种纯化的同工酶进行比较证实，组成型同工酶仅由1型亚基组成，而主要增殖特异性同工酶由1型亚基和独特的2型亚基组成。在天然、非变性电泳条件下，1型和2型亚基的电荷至少相差四个单位，但分子量没有差异。1型和2型亚基之间的差异是共价的，因为两个亚基之间的等电点差异对2%十二烷基硫酸钠和8M尿素均稳定。TPI-2的表达与不稳定天冬酰胺残基的存在无关。周转研究表明，每个亚基的水平是由合成速率而非降解速率的差异调节的，但未观察到亚基之间的前体-产物关系。因此，TPI-2的合成机制必须在mRNA加工或新生肽合成过程中起作用，并且仅在类人猿物种的细胞中起作用。
