Poster: Enzymology

Abs # 259: Purification of proaleurain maturation enzyme.

Presenter:	Halls, Coralie E, cadolph@mail.wsu.edu
Authors	Halls, Coralie E (A)   Ole, Ostergaard  (A)   Rogers, John C (A)
Affiliations:	(A): Washington State University

Proaleurain is a cysteine aminopeptidase proenzyme expressed in most plant tissues. It traffics through the secretory pathway from the Golgi apparatus to a prevacuolar compartment (PVC) for the lytic vacuole. It is processed to active form by another protease, a unique maturation mechanism for cysteine proteases. Our data indicate that this "proaleurain clipping enzyme" is specifically localized to the PVC, and its identification could provide a biochemical marker for that compartment. Therefore we have substantially purified the clipping enzyme from cauliflower florets. The central problem has been progressive loss of activity with increasing purity through ammonium sulfate precipitation, MonoS cation exchange and bacitracin affinity chromatography. Suprisingly, enzyme activity after bacitracin step could be increased 50-100 fold by treatment with 2% SDS followed by 10x dilution into 0.2% NP-40, but required pH£4.5. Renaturation of the clipping enzyme from SDS-PAGE gel slices demonstrated a size of 25-32 kDa. Proteins comprising the five bands in this size range have been identified by Q-TOF mass spectrometry. Two proteases are present, a serine carboxypeptidase and a cysteine protease containing a granulin domain. Additionally present is a previously uncharacterized ~30 kDa Künitz-type protease inhibitor. We postulate that SDS-activation could result either from dissociation of the Künitz inhibitor from the clipping protease, or from unfolding of an inhibitory domain away from the clipping protease active site. Either mechanism could limit the clipping protease activity to the acidic environment of the PVC. Further studies are underway to define which protease is responsible for the clipping activity, and what mechanism of inhibition pertains.