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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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