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Concanavalin A (Con A) is a tetrameric lectin which is synthesized in the cotyledons of developing jack-bean (Canavalia ensiformis (L.) D.C.) seeds and accumulates in the protein bodies of storage-parenchyma cells. The polypeptides of Con A have a molecular weight of 27000 and a relative molecular mass (Mr) of 30000 when analyzed by gel electrophoresis on denaturing polyacrylamide gels. In-vitro translation of RNA isolated from immature jack-bean cotyledons shows that Con A is synthesized as a polypeptide with Mr 34000. In-vivo pulse labeling of cotyledons with radioactive amino acids or glucosamine also resulted in the formation of a 34000-Mr polypeptide. In-vivo labeling with radioactive amino acids in the presence of tunicamycin yielded an additional polypeptide of 32000 Mr. Together these results indicate that Con A is cotranslationally processed by the removal of a signal sequence and the addition of an oligosaccharide side chain of corresponding size. Analysis of the structure of the oligogosaccharide side chain was accomplished through glycosidase digestion of glycopeptides isolated from [(3)H]glucosamine-labeled Con A. Incubation of the labeled glycopeptides with endoglycosidase H, α-mannosidase or β-N-acetylglucosaminidase, followed by gel filtration, allowed us to deduce that the oligosaccharide side chain of pro-Con A is a high-mannose oligosaccharide. Pulse-chase experiments with labeled amino acids are consistent with the interpretation that the glycosylated precursor of Con A is processed to mature Con A (Mr=30000). The 4000 decrease in Mr is interpreted to result from the removal of a small glycopeptide. The implications of the conversion of a glycoprotein pro-Con A to mature Con A are discussed in the context of the unique circular permutation of the primary structure of Con A.

We have investigated the accumulation and intracellular localization of soybean (Glycine max IL.] Merr. cv Forrest) a-galactosidase-hemagglutinin during seed development. Cotyledon tissue was embedded in Lowicryl K4M and immunocytochemical localization was accomplished through treating thin sections with a-galactosidase antisera followed by indirect labeling with protein A coupled to colloidal gold. Gold particles were localized on the Golgi apparatus and protein bodies. We interpret this to indicate that a-galactosidase-hemagutinin is transferred to and transported through the Golgi apparatus and finally deposited within the protein body by a Golgi apparatus-mediated process.a-Galactosidase (EC 3.2.1.22) is widely distributed among plant species and is generally considered to participate in the degradation of reserve raffinose-type oligosaccharides and cell wall galactomannans (for review, see 25). The enzyme, depending on its source, displays a preference for one of these substrates (for review, see 25). The a-galactosidase from legume seed cotyledons exhibits a substrate preference for the raffinose-type oligosaccharide and shows only minimal activity toward galactomannans (5, 10). Thus, the presumed function of the cotyledon a-galactosidase is to degrade the raffinose-type oligosaccharides during germination and early seedling growth (18). a-Galactosidase from mature soybean seeds is a tetrameric protein (10) with a mol wt of 160,000 D at pH 4.0 (4). At pH 7.0, the protein dissociates into subunits with mol wt of 38,000 and 40,000 D (4). Although both the monomeric and tetrameric forms are enzymically active, they display different kinetic properties (4). At pH 4.0, the enzyme displays a transitory type of hemagglutinin activity and is referred to as a-galactosidasehemagglutinin (4). Whether the hemagglutinin activity is physiologically relevent is unknown. The a-galactosidase from soybean is virtually identical in biochemical and physical properties to a-galactosidases from the cotyledons of other legume species which may or may not display hemagglutinin activity (8,9). Soybean a-galactosidase-hemagglutinin is discrete from the wellcharacterized seed lectin, SBA2 (4). Because of its lectin-like properties and because it is immunologically related to legume lectins which bind galactose (8, 9), the a-galactosidases of mung bean and soybean seeds have been of particular interest to lectin researchers.'Supported by National Science Foundation grant PCM 8205148. The present study employs high-resolution electron microscope immunocytochemical observations to examine the developmental accumulation and intracellular localization of a-galactosidase in soybean cotyledons. Our findings indicate that soybean a-galactosidase is localized in protein bodies within storage parenchyma cells. We also demonstrate that a-galactosidase is localized in the Golgi apparatus of developing seed storage parenchyma cells supporting the proposal that the Golgi apparatus mediates the deposition of the legume protein body matrix ...

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Four legume species (four genera) were examined and found to contain hemagglutinins with properties similar to those which we have previously described for the enzymnic hemagglutinin in Vigna radiata. Examination of extracts by gel filtration and ion exchange chromatography showed that an a-galactosidase activity exactly co-purified with a hemagglutinin activity in each of the four species. The a-galactosidase activities in the four species were virtually identical to each other with respect to substrate and inhibitor specificity as well as kinetic behavior. Additionally the hemagglutnin activities in all four species displayed very similar carbohydrate specificities. The inhibitor specificities displayed by the enzymes and the hemagglutinins were qualitatively and quantitatively very nearly identical to each other. The remarkable similarities of these proteins, both to each other and to the previously described Vigna enzymic hemagglutinin, suggest that each of these plants may contain a homologue from a specific class of enzymic hemagglutinin.In a previous report we described an a-galactosidase from Vigna radiata which possessed hemagglutinin properties (4). The Vigna hemagglutinin displayed a unique property which we refer to as "clot-dissolving activity." Apparently, under the appropriate conditions, this protein is capable of enzymically altering those erythrocyte receptors with which it interacts, resulting in a dissolution ofcell aggregates. The disaggregated erythrocytes are permanently altered and are no longer agglutinable by Vigna hemagglutinin (although they remain agglutinable by many other legume lectins). Clot-dissolving activity is readily observed, even with crude extracts, as a disappearance (dissolution) of erythrocyte aggregates at a rate which is directly proportional to the concentration of extract used. That is, with very concentrated extracts, agglutination is rapid and complete but very quick (minutes) to disappear; with dilute extracts, full agglutination develops more slowly and then disappears only after prolonged (hours) incubation.Due to our general interest in hemagglutinins, we wondered if legume species other than Vigna contained similar clot-dissolving agglutinins. We, therefore, surveyed a variety of legume seed extracts for hemagglutinin and a-galactosidase activities and, in those cases where agglutination was seen, we carefully looked to see if any clot dissolution occurred. We readily detected clotdissolving activity in several species, including two which contained previously well characterized non-clot-dissolving hemagglutinins.In this report we describe some general properties of extracts from four different legume species (four genera) each of which contains a clot-dissolving hemagglutinin. Our results suggest that all four species contain a-galactosidase-hemagglutinins very similar to that isolated from V. radiata.

The leguminous tree Sophora japonica contains a family of closely related, but distinct, lectins. Different members of this family are independently expressed in seeds, leaves, and bark (CN Hankins, J Kindinger, LM Shannon 1987 Plant Physiol 83: 825-829; 1988 Plant Physiol 86: 67-10). The inter-, and intracellular distribution of the bark and leaf lectins was studied by indirect postembedding immunogold electron microscopy.AMdehyde fixed bark and leaves postifixed with OS04 and embedded in LR White resin permitted sensitive and specific immunogold labeling while maintaining cellular ultrastructure. The The leaf, bark, root, and stem tissue of several different legumes have been described as containing lectins or other immunologically CRM2 (8, 9, 14, 18, 20, 22; see Ref. 3 for review).Partial amino acid sequences of these nonseed lectins or CRM indicate that each of these proteins is closely related to, but not identical to the seed lectin of the same plant (8,9,14). The lectins of legume seeds have been documented to be localized in storage vacuoles or protein bodies (see Ref. 3 for review). In contrast, immunologically related nonseed legume lectins have been localized by light microscopic immunocytochemistry in the vacuoles of soybean root tips (22), the cell wall in Dolichos stems and leaves (4), and the cytoplasm of Dolichos tissue culture and leaf cells (4, 13). The apparent variability of the localization of nonseed legume lectins contrasts with the electron microscopic localization of lectin in the vacuoles of adult wheat roots (17) and the light microscopic localization of lectin in protein-storage vacuoles of the Elder tree (Sambucus) bark (7).Recent amino-terminal protein sequence and immunological cross-reaction data have shown that the seed, leaf, and bark lectins of the legume tree Sophora japonica are distinct but closely related gene products (8, 9). We have used high resolution electron microscopic immunocytochemistry to localize leaf and bark lectins. These observations demonstrate that the leaf and bark lectins are sequestered in protein-storage vacuoles. ' Research in laboratory of L. M. S. is supported by the National Science Foundation.2 Abbreviations: CRM, cross-reacting materials; TBST, tris-buffered saline containing Tween-20. MATERIALS AND METHODSSophora japonica leaf samples were obtained from a tree growing on the U.C. Riverside campus (8) (19). After labeling, the grids were washed with TBST, then with double distilled water, and were stained with 5% aqueous uranyl acetate for 20 min followed by alkaline lead citrate (33 mg ml-1) for 5 min. The grids were examined and photographed with a Hitachi H500 electron microscope.

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