PROTEASES OF GERMINATING WINGED-BEAN (PSOPHOCARPUS-TETRAGONOLOBUS) SEEDS - PURIFICATION AND CHARACTERIZATION OF AN ACIDIC PROTEASE

Two major classes of protease are shown to occur in germinating winged -bean (Psophocarpus tetragonolobus) seeds, by assaying extracts at pH 8.0 and pH 5.1 with [C-14]gelatin as substrate. At pH 8.0, the activit y profile of the enzyme shows a steady rise throughout the period of g ermination, whereas the activity at the acidic pH is very low up to da y 5 and then increases sharply reaching a peak on day 11, followed by an equally sharp decline. The winged-bean acidic protease (WbAP) has b een purified to apparent homogeneity, as attested by a single protein band on both PAGE and SDS/PAGE. WbAP is a monomeric enzyme with a mole cular mass of 35 kDa and a pH optimum of 6.0. It is a thiol protease t hat does not belong to the papain family and it has tightly bound Ca2 as shown by Ca-45(2+)-exchange studies. Besides gelatin and casein, i t hydrolyses a 29 kDa winged-bean protein, indicating a prospective ph ysiological role for it in storage-protein mobilization. Immunoblot an alysis shows that it occurs only in the seeds and sprouting tubers of this plant and also that it is synthesized in developing seeds just be fore desiccation. It appears that the newly synthesized enzyme is inac tive, and activation takes place around day 6 of germination. However, neither the mechanism of activation nor the signal that triggers it i s clearly understood.