ANTIGENIC RELATIONSHIPS BETWEEN CHLOROPLAST AND CYTOSOLIC FRUCTOSE-1,6-BISPHOSPHATASES

Cytosolic fructose-1,6-biphosphatases (FBPase, EC 3.1.3.11) from pea ( Pisum sativum L cv Lincoln) and spinach (Spinacia oleracea L. cv Winte r Giant) did not cross-react by double immunodiffusion and western blo tting with either of the antisera raised against the chloroplast enzym e of both species; similarly, pea and spinach chloroplast FBPases did not react with the spinach cytosolic FBPase antiserum. On the other ha nd, spinach and pea chloroplast FBPases showed strong cross-reactions against the antisera to chloroplast FBPases, in the same way that the pea and spinach cytosolic enzymes displayed good cross-reactions again st the antiserum to spinach cytosolic FBPase. Crude extracts from spin ach and pea leaves, as well as the corresponding purified chloroplast enzymes, showed by western blotting only one band (44 and 43 kD, respe ctively) in reaction with either of the antisera against the chloropla st enzymes. A unique fraction of molecular mass 38 kD appeared when ei ther of the crude extracts or the purified spinach cytosolic FBPase we re analyzed against the spinach cytosolic FBPase antiserum. These mole cular sizes are in accordance with those reported for the subunits of the photosynthetic and gluconeogenic FBPases. Chloroplast and cytosoli c FBPases underwent increasing inactivation when increasing concentrat ions of chloroplast or cytosolic anti-FBPase immunoglobulin G (IgG), r espectively, were added to the reaction mixture. However, inactivation s were not observed when the photosynthetic enzyme was incubated with the IgG to cytosolic FBPase, or vice versa. Quantitative results obtai ned by enzyme-linked immunosorbent assays (ELISA) showed 77% common an tigenic determinants between the two chloroplast enzymes when tested a gainst the spinach photosynthetic FBPase antiserum, which shifted to 6 4% when assayed against the pea antiserum. In contrast, common antigen ic determinants between the spinach cytosolic FBPase and the two chlor oplast enzymes were less than 10% when the ELISA test was carried out with either of the photosynthetic FBPase antisera, and only 5% when th e assay was performed with the antiserum to the spinach cytosolic FBPa se. These results were supported by sequencing data: the deduced amino acid sequence of a chloroplast FBPase clone isolated from a pea cDNA library indicated a 39,253 molecular weight protein, with a homology o f 85% with the spinach chloroplast FBPase but only 48.5% with the cyto solic enzyme from spinach.