Poster: Enzymology

Abs # 227: Investigation of hydrolytic enzymes present in the pitcher fluid of nepenthes burkei

Presenter:	Stephenson, Paul T., PStephenson@Rollins.edu
Authors	Stephenson, Paul T. (A)   Hogan, Jamie  (A)   Tucker, Kevin  (A)   Andretta, Anthony  (A)
Affiliations:	(A): Rollins College Department of Biology

Nepenthes burkei, is a member of a group of carnivorous plants typically described as "passive trappers" which produce modified leaves termed a pitchers. The pitcher contains an aqueous solution of hydrolases enabling the plant to digest trapped prey and obtain supplemental nitrogen. We used non-denaturing SDS-PAGE activity gels to characterize proteinase activity in pitcher fluid samples. Proteinase activity was examined in pitchers during a 22 day time course experiment in which the plants had been provided either with 0.1 mg/mL BSA as a digestible substrate or sterile water as a control. Additionally, samples were analyzed from newly opened pitchers to determine whether the pitcher fluid possessed proteolytic activity at opening. The results of this study suggest N. burkei pitchers produce at least five proteinases of differing molecular weights (126, 64.6, 50.3, 35.5, and 31.5 kD). It was observed that whereas the pattern of proteolytic activity was consistent for samples from individual pitchers, samples from different pitchers often displayed variant proteolytic activities. This suggests that different pitchers may be secreting proteinases of different molecular weights. Best proteinase activity was observed at pH 3 and inhibition of activity was observed when pitcher fluid samples were treated with pepstatin. This is consistent with previous studies which showed that Nepenthes pitchers secrete aspartic proteinases. Proteinase inhibition was also observed when samples were treated with E-64 suggesting that cysteine proteinases may also be present in Nepenthes pitcher fluid. The presence of RNases in pitcher fluid was confirmed using "drop spot" analysis and non-denaturing SDS-PAGE revealed a single 21.4 kD band of RNase activity.