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 [14C]gelatin as substrate. At pH 8.0, the activity profile of the enzyme shows a steady rise throughout the period of germination, whereas the activity at the acidic pH is very low up to day 5 and then increases sharply reaching a peak on day 11, followed by an equally sharp decline. The winged-bean acidic protease (WbAP) has been purified to apparent homogeneity, as attested by a single protein band on both PAGE and SDS/PAGE. WbAP is a monomeric enzyme with a molecular mass of 35 kDa and a pH optimum of 6.0. It is a thiol protease that does not belong to the papain family and it has tightly bound Ca2+ as shown by 45Ca(2+)-exchange studies. Besides gelatin and casein, it hydrolyses a 29 kDa winged-bean protein, indicating a prospective physiological role for it in storage-protein mobilization. Immunoblot analysis shows that it occurs only in the seeds and sprouting tubers of this plant and also that it is synthesized in developing seeds just before desiccation. It appears that the newly synthesized enzyme is inactive, and activation takes place around day 6 of germination. However, neither the mechanism of activation nor the signal that triggers it is clearly understood.
Usha
R
R
Indian Institute of Chemical Biology, Calcutta, India.
Singh
M
M
D002118:Calcium / Q000378:metabolism; D002413:Cations, Divalent; D002848:Chromatography, DEAE-Cellulose; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000302:isolation & purification* / Q000378:metabolism; D007887:Fabaceae / Q000201:enzymology*; D018525:Germination; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D010946:Plants, Medicinal; D011480:Protease Inhibitors / Q000494:pharmacology; D013379:Substrate Specificity
D002413:Cations, Divalent; D011480:Protease Inhibitors; D010450:Endopeptidases; D002118:Calcium