Biosynthesis of acid phosphatase of baker's yeast. Factors influencing its production by protoplasts and characterization of the secreted enzyme

Abstract

1. 1. The secretion of acid phosphatase (orthophosphoric monoester phosphohydrolase, EC 3.1.3.2) by protoplasts prepared from baker's yeast has been studied. Secretion into the incubation medium begins after a lag period, is linear for 2 to 3 h and stops after about 5 h. During the linear phase the secretion rate is about 50 molecules of enzyme/s per protoplast. Renewed vigorous secretion can be induced by refreshing the medium or replenishing it with glucose. 2. 2. Secretion of acid phosphatase only occurs when (1) the protoplasts are prepared from log-phase yeast cells containing less than 3 μmoles Pi per 108 cells; (2) the protoplasts are incubated in a medium containing less than 10 μM Pi, i.e. less than 10−2 μmole P1 per 108 protoplasts; (3) the medium contains at least 56 μmoles glucose per 108 protoplasts. 3. 3. The secreted acid phosphatase appears to be identical with the enzyme located in the cell wall of the intact yeast (a mannan-protein complex). It has the same pH optimum, the same Km towards the substrates p-nitrophenyl phosphate and β-glycerophosphate, the same atypical transphosphorylation behaviour, is inhibited in the same way by molybdate ions, and its synthesis requires a low-phosphate medium and an unimpeded production of mannan. 4. 4.Throughout the secretion of acid phosphatase, and also after its termination, a small amount of enzyme remains firmly bound to the protoplasts, even after lysis. After a transient decrease in the beginning this membrane-bound fraction remains constant.