Structure and biochemistry of phloem-proteins isolated from Cucurbita maxima

Phloem proteins (P-proteins) were isolated from exudates of both the fruit skin and the stem of the pumpkin, Cucurbita maxima, and the cucumber, Cucumis sativus, and were purified by ammonium sulphate precipitation and chromatography over Sephadex, DEAE-cellulose, and CM-cellulose. It was found that the fruit skin phloem is an excellent source for large-scale preparations of P-proteins which are biochemically and structurally identical with those from stem exudates. Besides fractionation on columns the P-protein preparation was characterized by electrophoresis in polyacrylamide gels (in SDS or acidic urea solutions) and on cellulose acetate, by precipitation studies using salts, acids and thiol reagents, by centrifugation techniques, by determination of amino acid composition, by cyanogen bromide cleavage, by immunodiffusion and immunoelectrophoresis using anti-P-protein-sera obtained from mice, and by electron microscopy of negatively stained and ultrathin sectioned preparations of native and reaggregated P-proteins. P-proteins were identified as a class of at least eight different but related basic (IEPs from pH 9.6 to 10.4) proteins and polypeptides of molecular weights 14000, 17000, 44000, 59000, 116000 and 158000 D which are rich in lysine, leucine, glycine, glutamic acid (plus glutamine) and aspartic acid (plus asparagine). Structurally they were pleomorphic and formed, at various proportions, floccules, fibrils, doughnuts and 100 Å lamellae with a membrane-like ultrastructure. The P-proteins showed in several properties (amino acids, IEPS, retention on CM-cellulose, antigenic sites, molecular weights of the smaller components) a strong similarity to proteins extracted from a ribosomal fraction prepared from cucurbit seedlings, in particular to a weakly basic subtraction of ribosomal proteins. It is hypothesized that P-proteins are formed during sieve element maturation by aggregation and oxidative disulphide cross-linking of preexisting proteins which, at least in the Cucurbitaceae, are basic and may include ribosomal or ribosome-associated proteins. Possible functions of these aggregates are discussed.