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 μmol of glucose produced mg −1 protein min − (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 K m (sucrose) near 10 m m 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 HgCl 2, pridoxal phosphate, and Tris with respective K i values near 2 μ m, 5 μ m, and 4 m m. 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 crossreacted 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.