A Thio-based Phospholipase Assay Employed for the Study of Lipolytic Acyl Hydrolase in the Ephemeral Corolla of Ipomoea tricolor

Abstract

Summary A thioester-analogue of phosphatidylcholine was employed for the kinetic measurement of phospholipase activity in enzyme preparations from the corolla of Ipomoea tricolor. The corresponding lipolytic acyl hydrolase activity is constitutively high; its potential is sufficient for the almost instantaneous deacylation of all phospholipds in the tissue and, indeed, upon the homogenization of corolla tissue phospholipids are degraded rapidly. In the living cells, the enzyme seems to be functionally latent, probably because it is located in the vacuoles. Partial purifications comprising ammonium sulphate precipitation and chromatograhies (anion exchange, gel permeation and chromatofocussing) yielded preparations suitable for the characterization of lipolytic acyl hydrolase with regard to pH-optima (pH 5.5–6 and pH 8), size (70 and 120 kDalton) and isolelectric point (pH 4.3). The activity is inhibited by Ca-ions. The lipolytic acyl hydrolase of I. tricolor is considered to be part of the latent vacuolar lyric potential, which upon the collapse of subcellular compartmentation is responsible for the rapid degradation of residual cell constituents.