"Comparison of two cysteine endopeptidases from Pseudananas macrodontes (Morr.) Harms. (Bromeliaceae)"

L.M.I. LÓPEZ, C. SEQUEIROS, A. BRULLO, S. TREJO, B. MARAS, D. BARRA, C.L. NATALUCCI Y N.O. CAFFINI

Portoroz, Eslovenia

Conferencia; International Conference on Cysteine Proteinases and their Inhibitors; 2000

Two papain-like cysteine peptidases isolated from fruits of Pseudananas macrodontes (Morr.) Harms, a species closely related to pineapple (Ananas comosus L.) have been purified and characterized. The enzymes, named macrodontain I and macrodontain II, were purified by acetone fractionation followed by anion exchange chromatography (FPLC, Q Sepharose Fast Flow). Macrodontain I is the main proteolytic fraction (62.2% of total activity), but macrodontain II exhibits higher specific activity (13.2 vs. 9.3 caseinolytic units/mg) with casein as substrate. Optimum pH range (more than 90% of maximum activity with casein) was achieved at pH 6.1–8.5 for macrodontain I and pH 7.5–8.5 for macrodontain II. Both enzymes were inhibited by E-64 and other cysteine peptidases inhibitors but not affected by inhibitors of the other catalytic types of peptidases. Homogeneity was confirmed by bidimensional electrophoresis and mass spectroscopy (MS). Molecular mass were 23,459 and 23,703 (MS) and isoelectric points (IEF) were 6.1 and 5.9 for macrodontain I and II, respectively. Activity on synthetic substrates (N–a–CBZ–L–amino acid p–nitrophenyl esters and N-benzoyl-Phe-Val-Arg-p-nitroanilide) was tested for both enzymes. The N-terminal sequence of macrodontain I (AVPQSIDWRDYGAVNEVKNQGPCGG CW) and macrodontain II (AVPQSIDWRDYGAVNEVKNQNPCGSCWAFAAI) showed a high degree of identity (92.6%). Both proteases showed a great deal of sequence similarity to other pineapple stem-derived cysteine endopeptidases. Macrodontain I possesses 85.2% identity with stem bromelain, and 77.8% identity with ananain, while macrodontain II possesses 88.9% identity with stem bromelain, and 77.8% identity with ananain. It seems clear that the Bromeliaceae endopeptidases are more closely related to each other than to other members of the papain family, suggesting relatively recent divergence.