Hominoid triosephosphate isomerase: characterization of the major cell proliferation specific isozyme

Abstract

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.