PURIFICATION, IMMUNOLOGICAL AND STRUCTURAL CHARACTERIZATION, AND PARTIAL AMINO ACID SEQUENCES

We report the first complete purifications of the cytosolic and plastid isozymes of triose phosphate isomerase (TPI; EC 53.1.1) from higher plants including spinach (Spinacia oleracea), lettuce (Lactuca sativa), and celery (Apium graveolens). Both isozymes are composed of two isosubunits with approximate molecular weight of 27,000; in spinach and lettuce the plastid isozyme is 200 to 400 larger than the cytosolic isozyme. The two isozymes, purified from lettuce, had closely similar amino acid compositions with the exception of methionine which was four times more prevalent in the cytosolic isozyme. Partial amino acid sequences from the N-terminus were also obtained for both lettuce TPIs. Nine of the 13 positions sequenced in the two proteins had identical amino acid residues. The partial sequences of the plant proteins showed high similarity to previously sequenced animal TPIs. Immunological studies, using antisera prepared independently against the purified plastid and cytosolic isozymes from spinach, revealed that the cytosolic isozymes from a variety of species formed an immunologically distinct group as did the plastid isozymes. However, both plastid and cytosolic TPIs shared some antigenic determinants. The overall similarity of the two isozymes and the high similarity of their partial amino acid sequences to those of several animals indicate that TPI is a very highly conserved protein. Higher plants possess two isozymes of each glycolytic enzyme, one located in the plastids and the other in the cytosol (12, 17, 23). The kinetic and regulatory properties of many of the isozymes have been characterized (3, 12, 23), but few of them have been purified to homogeneity or examined structurally. Insofar as their genetics has been studied, each isozyme is coded by an independent nuclear gene (17, 30). The gene pairs could have evolved from duplications of ancestral nuclear genes in the lineage that led to the plants, or the genes specifying the plastid isozymes may have been transferred to the nucleus from the genome of the prokaryotic symbiont thought to have given rise to the plastids (29). The duplication model predicts greater structural similarity between the plastid and cytosolic isozymes of each glycolytic enzyme than between either of them and the single glycolytic enzyme of cyanobacteria or of Prochloron (22), the contemporary organisms probably most like the putative endosymbiont. The endosymbiont-gene transfer model predicts greater similarity between the plastid isozyme and the prokaryotic enzyme, and has been supported by immunological studies (33). We undertook structural studies of the plastid and cytosolic ' Supported by National Science Foundation Grant BSR82-15363. isozymes of TPI2 (EC 5.3.1.1) to seek further evidence on this issue. The two TPI isozymes in flowering plants are coded by independent nuclear genes (15, 25). TPI catalyzes the reversible isomerization of G3P and DHAP. The cytosolic TPI isozyme acts primarily in glycolysis and gluconeogenesis whereas the plastid TPI functions in these pathways and also has an essential role in the generative phase of photosynthesis (12, 27). Since neither TPI isozyme has previously been fully purified from plant tissue, we developed a protocol for such purification. In addition to describing it, we report results of structural and immunological studies of both purified isozymes from spinach, lettuce, and celery. We also present the sequence of the first 1 amino acids from the N-terminus of the cytosolic TPI isozyme and that of the first 13 N-terminal amino acids of the plastid TPI, both from lettuce. MATERIALS AND METHODS Spinach (Spinacia oleracea), lettuce (Lactuca sativa), celery (Apium graveolens), and broccoli (Brassica oleracea) were purchased locally. Certain species of Onagraceae were grown in the greenhouse as described (16). Subcellular Localization. Previous reports have established that TPI in plants occurs in the cytosol and in the plastid fraction (3, 31). We employed two methods to identify the subceilular location of each of the two TPI isozymes observed in the plant species we examined. (a) Cell fractionation: Intact chloroplasts from green leaves were prepared as previously described (26). The preparations were examined by starch gel electrophoresis followed by activity staining. The chloroplast fraction contained only one isozyme and, therefore, this isozyme was identified as the plastid isozyme. The crude extract contained both this isozyme and the cytosolic isozyme (Fig. 1). Our results for spinach confirmed a previous determination of the plastid and cytosolic TPI isozymes (31). (b) Immunological identification: The crude extract from each species was mixed (3:1) with antispinach plastid TPI antiserum (see below). After incubation for 30 min at 4°C, an aliquot was withdrawn and subjected to electrophoresis on starch gel at pH 8.3 (see below), after which the gels were developed for TPI activity. Since most antibody molecules are positively charged at this pH, TPI molecules which are bound to them will not migrate to their usual anodal position on the gel. By this criterion, the antispinach plastid TPI antiserum bound only one of the two TPI isozymes in the crude extracts and it was the same one present in the plastid fractions (Fig. 1). The 2 Abbreviations: TPI, triose phosphate isomerase; G3P, glyceraldehyde-3-P; DHAP, dihydroxyacetone phosphate; G3PDH, glyceraldehyde-3-P dehydrogenase; ELISA, enzyme-linked immunosorbent assay; PAGE, polyacrylamide gel electrophoresis; PGI, phosphoglucose isom-