title: Biochemical and immunological properties of alkaline invertase isolated from sprouting soybean hypocotyls.
abstract: Alkaline invertase from sprouting soybean (Glycine max) hypocotyls was purified to apparent electrophoretic homogeneity by consecutive use of DEAE-cellulose, green 19 dye, and Cibacron blue 3GA dye affinity chromatography. This protocol produced about a 100-fold purification with about a 11% yield. The purified protein had a specific activity of 48 mumol of glucose produced mg-1 protein min-1 (pH 7.0) and showed a single protein band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (PAGE) (58 kDa) and in native PAGE, as indicated by both protein and activity staining. The native enzyme molecular mass was about 240 kDa, suggesting a homotetrameric structure. The purified enzyme exhibited hyperbolic saturation kinetics with a Km (sucrose) near 10 mM and the enzyme did not utilize raffinose, maltose, lactose, or cellibose as a substrate. Impure alkaline invertase preparations, which contained acid invertase activity, on contrast, showed biphasic curves versus sucrose concentration. Combining equal activities of purified alkaline invertase with acid invertase resulted in a biphasic response, but there was a transition to hyperbolic saturation kinetics when the activity ratio, alkaline: acid invertase, was increased above unity. Alkaline invertase activity was inhibited by HgCl2, pridoxal phosphate, and Tris with respective Ki values near 2 microM, 5 microM, and 4 mM. Glycoprotein staining (periodic acid-Schiff method) was negative and alkaline invertase did not bind to two immobilized lectins, concanavalin A and wheat germ agglutinin; hence, the enzyme apparently is not a glycoprotein. The purified alkaline invertase, and a purified soybean acid invertase, was used to raise rabbit polyclonal antibodies. The alkaline invertase antibody preparation was specific for alkaline invertase and cross-reacted with alkaline invertases from other plants. Neither purified soybean alkaline invertases nor the crude enzyme from several plants cross-reacted with the soybean acid invertase antibody.
authors.affiliation: Department of Biochemistry, University of Georgia, Athens 30602.
authors.forename: J Q
authors.initials: JQ
authors.lastname: Chen
authors.forename: C C
authors.initials: CC
authors.lastname: Black
mesh_terms: D000917:Antibody Formation; D003429:Cross Reactions; D006023:Glycoproteins; D006026:Glycoside Hydrolases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification*; D007527:Isoenzymes / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification*; D007700:Kinetics; D008970:Molecular Weight; D012639:Seeds / Q000254:growth & development; D013025:Glycine max / Q000201:enzymology* / Q000254:growth & development; D013045:Species Specificity; D013395:Sucrose / Q000378:metabolism*; D014018:Tissue Distribution; D043324:beta-Fructofuranosidase
chemical_list: D006023:Glycoproteins; D007527:Isoenzymes; D013395:Sucrose; D006026:Glycoside Hydrolases; D043324:beta-Fructofuranosidase