An α-Galactosidase with Hemagglutinin Properties from Soybean Seeds

Abstract

Soybean (Glycine max L.) seeds contain a galactose-binding protein which displays two activities: (a) an a-galactosidase activity and (b) a hmgghtinin activity. The a-glactosidase-hemagglutinin was puifid to homogeneity by conventional protein purification procedures and also by afiiy chromatography. This protein can be easily separated from soybean agglutinin, the N-acetyl-D-galactosamine-specific lectin in soybean Further, these two agglutinins show no nologial relatedness. The agalactosidase-hemagglutinin can be reversibly converted by pH changes from a tetramerc form which displays both enzymk and hemglutinin activities to a monomeric form which displays enzynic activity only. Although both the monomerc and tetrameric forms are euzymically active, they display diferent pH optima and carbohydrate specificities.Previously, we reported that partially purified a-galactosidase preparations from several legumes, including soybeans, appeared to display associated hemagglutinin properties (4, 5). Only in mung beans, however, has it been conclusively demonstrated that both enzyme and hemagglutinin activities are associated with a single protein species (2, 5). We reasoned that purification and further characterization ofthe soybean a-galactosidase-hemagglutinin would be useful for several reasons. (1) We have seen no reports (other than our own) indicating that soybean contains a hemagglutinin activity other than that which is inhibited by Nacetyl-galactosamine. (2) A soybean a-galactosidase has been purified and characterized (6) Enzyme Assay. a-Galactosidase activity was assayed by following the initial rate of hydrolysis of p-nitrophenyl a-galactoside. The reaction mixture contained 3.3 ,imol of substrate, enzyme and Mcllvaine's phosphate:citrate buffer (1:4), pH 4.5 (8). The 1-ml reaction mixture was incubated at 30°C for the specified time and terminated by adding 3.0 ml of 0.5 M Na2CO3, pH 10.5. One unit of a-galactosidase activity was defined as the amount of enzyme which yielded 1 nmol of product (p-nitrophenol) per min at 30°C. The molar extinction coefficient ofp-nitrophenol is 18,300 at 405 nm (1). The kinetic parameters, Km and V,,, were calculated from Lineweaver-Burk plots.Hemagglutinin Assays. Routine assays were performed with trypsinized rabbit red blood cells as described previously (5), except that assays were incubated at 0 to SoC and pH 6.0 for 30 min, unless otherwise specified. N-acetylgalacto8amine (20 mM) was included in all assay buffers to inhibit soybean agglutinin.Protein Purification. Flour was obtained by grinding dry seeds in a Wiley mill fitted with a 40-mesh screen. Flour (200 g) was suspended in 2 L of 0.1 M sodium phosphate buffer, pH 6.0, containing phenyl-methyl-sulfonyl-fluoride. Following 1 h at room temperature, the suspension was filtered through four layers of cheese-cloth and the filtrate was centrifuged 30 min at 16,400g.The supernatant solution was incubated at 0 to SoC for 18 h and additional protein precipitates were removed by centrifugation as before. The cry...

Multiple forms of cotyledonary b-galactosidases from Vigna unguiculata quiescent seeds

“…The retained peak was eluted with a linear NaCl gradient (0.2 to 1.0 M). Absorbance at 280 nm and β-galactosidase activity were determined in each fraction , and the ones that showed the highest activities (peaks DS-I and DS-II) were dialyzed against distilled water for 24 h, concentrated and applied on a Lactosyl-Sepharose affinity column (16 x 190 mm) equilibrated with McIlvaine buffer, pH 4.0, diluted 1:4, containing 0.1 mM EDTA and 1.0 mM 2-ME at 4°C (Campillo & Shannon 1982). The flow rate was adjusted to 36 mL.h -1 ; fractions of 4.8 mL were eluted with the equilibrium buffer and the retained fractions were eluted with the same buffer containing 100 mM lactose and 0.5 M NaCl.…”

Defense enzyme activities and biochemical variations of Pelargonium zonale in response to nanosilver application and dark storage

“…CAT activity was measured by the decrease in absorbance at 240 nm for 1 min. 2.9.5. β-GLU activity The β-GLU (EC 3.2.1.21) activity was determined by a method described by Del Campillo and Shannon (1982) with some modifications. Fresh leaf (0.5 g) was homogenized with 1 mL of 100 mM citrate buffer adjusted to pH 5.0 with KOH.…”

Accumulation and Subcellular Localization of α-Galactosidase-Hemagglutinin in Developing Soybean Cotyledons

“…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).…”

“…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.…”