Phospholipid-dependence of plant UDP-glucose-sterol-β-D-glucosyltransferase. II. Acetone-mediated delipidation and kinetic studies

Abstract

Etiolated maize coleoptiles microsomes were delipidated by acetone. The resulting acetone powder, when resuspended in buffer and tested with radioactive UDP-glucose has lost the UDP-glucose:sterol β-D-glucosyltransferase (UDPG-SGTase) activity present in the microsomes. Addition of Triton X-100 at solubilizing concentration reveals only a residual activity (1% of the original solubilized microsomal activity). Simultaneous addition of Triton and sterols restores a low activity (20% of the solubilized microsomal activity). The subsequent addition of the coarse acetonic extract, or phospholipids purified from it, produces the restoration of 100% of the original activity. These results, together with those of the preceding paper, P. Bouvier-Navé et al., Plant Sci. Lett., 36 (1984) 19, point toward the phospholipid dependence of plant UDPG-SGTase. Unlike the microsomal or plasma membrane preparations of UDPG-SGTase activity, the acetone powder is a sterol-depleted preparation and thus, allowed a bisubstrate kinetic analysis of UDPG-SGTase mechanism. Reciprocal plots of initial rates of activity indicate clearly that the kinetics follow a sequential mechanism. The sterolic substrate specificity of the enzyme was also studied and is discussed.