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.
ABSTRACFFive N-acetyl-galactosamine-specific lectins were isolated from the bark of the legume tree Sophora japonica. These
A number of well characterized legume lectins including the enzymic lectin from Vigna radiata were examined for immunological relatedness. The immunological cross-reactions observed indicate that most of the legume lectins, including Vigna lectin, are evolutionarily closely related proteins. The possibility that these proteins are homologs with enzymic functions is discussed.Recently we discovered a protein in mung bean seeds which possessed both lectin and enzymic properties (5). This finding led us to ask if this enzymic lectin was related to any of the other legume lectins which have been described. The possible significance of a relationship, with respect to understanding the functional role(s) of plant lectins, is obvious.Lectins appear to be widely distributed among leguminous plants (13) and most of what we now know about these proteins is the result of studies with lectins isolated from species of this plant family. Collectively, the legume lectins display many similar properties. For example, most of those thus far studied are metal ion-binding, tetrameric glycoproteins possessing identical or nearly identical subunits (8, 10). Based upon their common source and upon their possessing similar physical, chemical, and biological properties, many of the legume lectins might be homologs (4).Immunochemistry provides a powerful method for determining relationships among proteins. Different proteins can be functionally similar and yet antigenetically totally unrelated. However, when proteins are found to be antigenetically similar there is a strong probability of both an evolutionary and a functional relationship. We have begun experiments designed to assess the immunological relatedness both among legume lectins and between these proteins and the enzymic lectin from mung beans.Initial studies in this laboratory (6) indicated that many of the plant lectins which have been previously studied are immunologically related and, moreover, that there may be a unique portion of these proteins that is evolutionarily highly conserved.In this report we provide evidence that the enzymic lectin from mung beans is immunologically closely related to several other legume lectins and that these lectins are members of a group of related proteins that encompass the majority of the legume lectins which have been studied. MATERIALS AND METHODSLectins. The lectins used in this study were derived from the 'This research was supported in part by National Science Foundation Grant PCM 77-17612. plant species listed in Table I. The sources of the lectins and their hapten sugar specificities are indicated. All of these species with the exception of Ricinus communis (castor bean) are from the family Leguminosae. This list represents the majority of the legume lectins which have been well characterized.Antisera. High titer rabbit antisera were raised against the lectins from Vigna radiata, Sophora japonica, Bandeiraea simplicifolia, Bauhiniapurpurea alba, Glycine max, and Phaseolus lunatus by methods described previously (7). The le...
ABSTIRACITwo lectins, Leaf Lectin I and Leaf Lectin II (LLI and LLII) were purified from the leaves of Sophora japonica. Like This property (pH-sensitivity) has been observed in several other cases, however, even in a legume lectin of very different structure (12).In previous studies, we noted that crude extracts of Sophora leaves contained hemagglutinin activity and also displayed strong immunological cross-reactions with antisera raised against the Sophora seed lectin. In this report, we describe the purification and some of the properties of two lectins from Sophora leaves. MATERIALS AND METHODSThe tissues used in these studies were harvested from a single specimen of Sophora japonica, growing on the campus of the University of California, Riverside. Seeds were harvested when fully desiccated and stored at room temperature after removal from their pods. Fully expanded, mature leaves were harvested, washed with distilled H20, and if not extracted immediately, were frozen with liquid N2 and stored at -20C for later use.Extraction. All procedures were carried out at 0 to 5C unless otherwise specified. Leaves (200 g) were blended 2 min in a 4 L Waring Blendor with 1 L of distilled H20 containing 10 mM 3-mercaptoethanol and then squeezed through four layers of cheesecloth. The extract was then centrifuged (l0,OOOg) for 20 min to yield a clarified crude extract. The pH ofthe crude extract was lowered to 4.0 by the dropwise addition ofglacial acetic acid while stirring and after 30 min the resulting precipitate removed by centiifugation and discarded as before, yielded the pH supernatant. An equal volume of ice-cold acetone was added dropwise to the pH supernatant over a period of 30 min while stirring on ice, then allowed to stand on ice for 30 min. The material precipitated by acetone was collected by centrifugation (50OOg x 10 min) and suspended in 100 ml of distilled H20. A substantial amount of insoluble material was removed by centrifugation (5000g, 10 min) and discarded.
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