Subcellular Localization of Alcohol Acetyltransferase in Strawberry Fruit

Abstract

The first step of the extraction of alcohol acetyltransferase (AAT) from fruits is usually achieved by preparation of free-cells with enzymatic maceration of tissues. The free-cells from strawberry tissue were suspended in a suitable buffer and disrupted by centrifugation. The supernatant held a strong activity of AAT. The addition of 0.1% Triton X-100 to the extraction buffer indicated that a minor part of AAT was bound-form. Strawberry pulp was grated in isotonic solution (0.5 M sorbitol and 2.5% ficoll), then fractionated through a sucrose-gradient layer (20-65%) by centrifugation (82,200×g, 3 h). The distribution of AAT in the subcellular fraction of the grated pulp was similar to those of glucose-6-phosphate dehydrogenase (cytosolic enzyme) and anthocyanin (vacuolar pigment). Protoplasts were prepared by enzymatic digestion, and fractionated by stepwise gradient sucrose centrifugation, into the collapsed vacuole part and miniprotoplasts. Miniprotoplasts consisting only of cytosol, were formed from protoplasts during the preparation. Since most activities of AAT were detected in the miniprotoplast fraction, we concluded that most AAT was located in the cytosol.