Contamination of oat mesophyll protoplasts by apoplastic polyamine oxidase

Abstract

Mesophyll protoplasts isolated from peeled oat (Avena sativa L. cv Victory) leaves with 1% (w/v) Cellulysin in 20 mM KPO4, pH 5.5 and 0.6 M sorbitol retain about 6% of the polyamine oxidase (PAO, EC 1.4.3.4) activity of the whole peeled leaf. However, more than 99% of the oat leaf PAO activity is apoplastic and can be extracted by vacuum infiltration with 200 mM NaCl and this procedure extracts no activity for the cytoplasmic marker enzyme glucose‐6‐phosphate dehydrogenase (G6PD, EC 1.1.1.49). By these criteria we consider PAO in oat leaves to be totally apoplastic and PAO found in the isolated protoplast to be contamination. The degree of protoplast contamination by PAO depends on the pH and ionic strength of the isolating and washing medium. It can be eliminated by washing protoplasts in 0.6 M sorbitol with 100 mM KPO4, pH 6.5. Pellets of lysed protoplasts incubated with dialyzed apoplastic enzymes in 5 mM KPO4, pH 5.5 adsorb about 87% of the added PAO activity but only about 25% of the added peroxidase (EC 1.11.1.7) activity. The adsorbed activity can be solubilized from the pellet by extraction with 1 M NaCl. The results demonstrate that weakly ionically bound cell wall enzymes may contaminate protoplasts isolated and purified by conventional techniques.