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Papers by Sebastian Trejo

Protein Journal, 2010

Penduliflorain I, a new plant endopeptidase, was isolated and characterized from Hohenbergia pend... more Penduliflorain I, a new plant endopeptidase, was isolated and characterized from Hohenbergia penduliflora. Crude extract was obtained from stems. A partially purified enzyme preparation was obtained by ethanol precipitation. This preparation showed maximum activity between pH 7.5 and 8.5, was stable at ionic strength (20% decrease in proteolytic activity could be detected after 2 h in 0.4 M sodium chloride solution), and exhibited high thermal stability (inactivation required heating for 20 min at 75 °C). Inhibition and activation assays indicated the cysteine nature of the enzymatic preparation. Penduliflorain I was purified by anion exchange chromatography (Q-Sepharose HP) by FPLC system. Homogeneity was confirmed by mass spectroscopy. Molecular mass of the enzyme was 23 412.847 Da (MALDI-TOF–MS). Kinetic parameters were determined for PFLNA (K m = 0.3227 mM and k cat = 4.27 s−1). The N-terminal sequence (AVPQSIDWRDYGAVTTDKNQ) of isolated protease showed considerable similarity to other cysteine proteases obtained from stems or fruits of different Bromeliaceae species.

Acta Biochimica Et Biophysica Sinica, 2009

Most of the species belonging to Asclepiadaceae family usually secrete an endogenous milk-like fl... more Most of the species belonging to Asclepiadaceae family usually secrete an endogenous milk-like fluid in a network of laticifer cells in which sub-cellular organelles intensively synthesize proteins and secondary metabolites. A new papain-like endopeptidase (asclepain c-II) has been isolated and characterized from the latex extracted from petioles of Asclepias curassavica L. (Asclepiadaceae). Asclepain c-II was the minor proteolytic component in the latex, but showed higher specific activity than asclepain c-I, the main active fraction previously studied. Both enzymes displayed quite distinct biochemical characteristics, confirming that they are different enzymes. Crude extract was purified by cation exchange chromatography (FPLC). Two active fractions, homogeneous by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and mass spectrometry, were isolated. Asclepain c-II displayed a molecular mass of 23,590 Da, a pI higher than 9.3, maximum proteolytic activity at pH 9.4-10.2, and showed poor thermostability. The activity of asclepain c-II is inhibited by cysteine proteases inhibitors like E-64, but not by any other protease inhibitors such as 1,10-phenantroline, phenylmethanesulfonyl fluoride, and pepstatine. The Nterminal sequence (LPSFVDWRQKGVVFPIRNQGQ CGSCWTFSA) showed a high similarity with those of other plant cysteine proteinases. When assayed on N-a-CBZ-amino acid-p-nitrophenyl esters, the enzyme exhibited higher preference for the glutamine derivative. Determinations of kinetic parameters were performed with N-a-CBZ-L-Gln-p-nitrophenyl ester as substrate: K m 5 0.1634 mM, k cat 5 121.48 s 21 , and k cat /K m 5 7.4 3 10 5 s 21 /mM.

Acta Biochimica Et Biophysica Sinica, 2009

Most of the species belonging to Asclepiadaceae family usually secrete an endogenous milk-like fl... more Most of the species belonging to Asclepiadaceae family usually secrete an endogenous milk-like fluid in a network of laticifer cells in which sub-cellular organelles intensively synthesize proteins and secondary metabolites. A new papain-like endopeptidase (asclepain c-II) has been isolated and characterized from the latex extracted from petioles of Asclepias curassavica L. (Asclepiadaceae). Asclepain c-II was the minor proteolytic component in the latex, but showed higher specific activity than asclepain c-I, the main active fraction previously studied. Both enzymes displayed quite distinct biochemical characteristics, confirming that they are different enzymes. Crude extract was purified by cation exchange chromatography (FPLC). Two active fractions, homogeneous by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and mass spectrometry, were isolated. Asclepain c-II displayed a molecular mass of 23,590 Da, a pI higher than 9.3, maximum proteolytic activity at pH 9.4-10.2, and showed poor thermostability. The activity of asclepain c-II is inhibited by cysteine proteases inhibitors like E-64, but not by any other protease inhibitors such as 1,10-phenantroline, phenylmethanesulfonyl fluoride, and pepstatine. The Nterminal sequence (LPSFVDWRQKGVVFPIRNQGQ CGSCWTFSA) showed a high similarity with those of other plant cysteine proteinases. When assayed on N-a-CBZ-amino acid-p-nitrophenyl esters, the enzyme exhibited higher preference for the glutamine derivative. Determinations of kinetic parameters were performed with N-a-CBZ-L-Gln-p-nitrophenyl ester as substrate: K m 5 0.1634 mM, k cat 5 121.48 s 21 , and k cat /K m 5 7.4 3 10 5 s 21 /mM.

Acta Biochimica Et Biophysica Sinica, 2009

Most of the species belonging to Asclepiadaceae family usually secrete an endogenous milk-like fl... more Most of the species belonging to Asclepiadaceae family usually secrete an endogenous milk-like fluid in a network of laticifer cells in which sub-cellular organelles intensively synthesize proteins and secondary metabolites. A new papain-like endopeptidase (asclepain c-II) has been isolated and characterized from the latex extracted from petioles of Asclepias curassavica L. (Asclepiadaceae). Asclepain c-II was the minor proteolytic component in the latex, but showed higher specific activity than asclepain c-I, the main active fraction previously studied. Both enzymes displayed quite distinct biochemical characteristics, confirming that they are different enzymes. Crude extract was purified by cation exchange chromatography (FPLC). Two active fractions, homogeneous by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and mass spectrometry, were isolated. Asclepain c-II displayed a molecular mass of 23,590 Da, a pI higher than 9.3, maximum proteolytic activity at pH 9.4-10.2, and showed poor thermostability. The activity of asclepain c-II is inhibited by cysteine proteases inhibitors like E-64, but not by any other protease inhibitors such as 1,10-phenantroline, phenylmethanesulfonyl fluoride, and pepstatine. The Nterminal sequence (LPSFVDWRQKGVVFPIRNQGQ CGSCWTFSA) showed a high similarity with those of other plant cysteine proteinases. When assayed on N-a-CBZ-amino acid-p-nitrophenyl esters, the enzyme exhibited higher preference for the glutamine derivative. Determinations of kinetic parameters were performed with N-a-CBZ-L-Gln-p-nitrophenyl ester as substrate: K m 5 0.1634 mM, k cat 5 121.48 s 21 , and k cat /K m 5 7.4 3 10 5 s 21 /mM.

Acta Biochimica Et Biophysica Sinica, 2009

Most of the species belonging to Asclepiadaceae family usually secrete an endogenous milk-like fl... more Most of the species belonging to Asclepiadaceae family usually secrete an endogenous milk-like fluid in a network of laticifer cells in which sub-cellular organelles intensively synthesize proteins and secondary metabolites. A new papain-like endopeptidase (asclepain c-II) has been isolated and characterized from the latex extracted from petioles of Asclepias curassavica L. (Asclepiadaceae). Asclepain c-II was the minor proteolytic component in the latex, but showed higher specific activity than asclepain c-I, the main active fraction previously studied. Both enzymes displayed quite distinct biochemical characteristics, confirming that they are different enzymes. Crude extract was purified by cation exchange chromatography (FPLC). Two active fractions, homogeneous by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and mass spectrometry, were isolated. Asclepain c-II displayed a molecular mass of 23,590 Da, a pI higher than 9.3, maximum proteolytic activity at pH 9.4-10.2, and showed poor thermostability. The activity of asclepain c-II is inhibited by cysteine proteases inhibitors like E-64, but not by any other protease inhibitors such as 1,10-phenantroline, phenylmethanesulfonyl fluoride, and pepstatine. The Nterminal sequence (LPSFVDWRQKGVVFPIRNQGQ CGSCWTFSA) showed a high similarity with those of other plant cysteine proteinases. When assayed on N-a-CBZ-amino acid-p-nitrophenyl esters, the enzyme exhibited higher preference for the glutamine derivative. Determinations of kinetic parameters were performed with N-a-CBZ-L-Gln-p-nitrophenyl ester as substrate: K m 5 0.1634 mM, k cat 5 121.48 s 21 , and k cat /K m 5 7.4 3 10 5 s 21 /mM.

Acta Biochimica Et Biophysica Sinica, 2009

Most of the species belonging to Asclepiadaceae family usually secrete an endogenous milk-like fl... more Most of the species belonging to Asclepiadaceae family usually secrete an endogenous milk-like fluid in a network of laticifer cells in which sub-cellular organelles intensively synthesize proteins and secondary metabolites. A new papain-like endopeptidase (asclepain c-II) has been isolated and characterized from the latex extracted from petioles of Asclepias curassavica L. (Asclepiadaceae). Asclepain c-II was the minor proteolytic component in the latex, but showed higher specific activity than asclepain c-I, the main active fraction previously studied. Both enzymes displayed quite distinct biochemical characteristics, confirming that they are different enzymes. Crude extract was purified by cation exchange chromatography (FPLC). Two active fractions, homogeneous by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and mass spectrometry, were isolated. Asclepain c-II displayed a molecular mass of 23,590 Da, a pI higher than 9.3, maximum proteolytic activity at pH 9.4-10.2, and showed poor thermostability. The activity of asclepain c-II is inhibited by cysteine proteases inhibitors like E-64, but not by any other protease inhibitors such as 1,10-phenantroline, phenylmethanesulfonyl fluoride, and pepstatine. The Nterminal sequence (LPSFVDWRQKGVVFPIRNQGQ CGSCWTFSA) showed a high similarity with those of other plant cysteine proteinases. When assayed on N-a-CBZ-amino acid-p-nitrophenyl esters, the enzyme exhibited higher preference for the glutamine derivative. Determinations of kinetic parameters were performed with N-a-CBZ-L-Gln-p-nitrophenyl ester as substrate: K m 5 0.1634 mM, k cat 5 121.48 s 21 , and k cat /K m 5 7.4 3 10 5 s 21 /mM.

Protein Journal, 2010

Penduliflorain I, a new plant endopeptidase, was isolated and characterized from Hohenbergia pend... more Penduliflorain I, a new plant endopeptidase, was isolated and characterized from Hohenbergia penduliflora. Crude extract was obtained from stems. A partially purified enzyme preparation was obtained by ethanol precipitation. This preparation showed maximum activity between pH 7.5 and 8.5, was stable at ionic strength (20% decrease in proteolytic activity could be detected after 2 h in 0.4 M sodium chloride solution), and exhibited high thermal stability (inactivation required heating for 20 min at 75 °C). Inhibition and activation assays indicated the cysteine nature of the enzymatic preparation. Penduliflorain I was purified by anion exchange chromatography (Q-Sepharose HP) by FPLC system. Homogeneity was confirmed by mass spectroscopy. Molecular mass of the enzyme was 23 412.847 Da (MALDI-TOF–MS). Kinetic parameters were determined for PFLNA (K m = 0.3227 mM and k cat = 4.27 s−1). The N-terminal sequence (AVPQSIDWRDYGAVTTDKNQ) of isolated protease showed considerable similarity to other cysteine proteases obtained from stems or fruits of different Bromeliaceae species.

Acta Biochimica Et Biophysica Sinica, 2009

Most of the species belonging to Asclepiadaceae family usually secrete an endogenous milk-like fl... more Most of the species belonging to Asclepiadaceae family usually secrete an endogenous milk-like fluid in a network of laticifer cells in which sub-cellular organelles intensively synthesize proteins and secondary metabolites. A new papain-like endopeptidase (asclepain c-II) has been isolated and characterized from the latex extracted from petioles of Asclepias curassavica L. (Asclepiadaceae). Asclepain c-II was the minor proteolytic component in the latex, but showed higher specific activity than asclepain c-I, the main active fraction previously studied. Both enzymes displayed quite distinct biochemical characteristics, confirming that they are different enzymes. Crude extract was purified by cation exchange chromatography (FPLC). Two active fractions, homogeneous by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and mass spectrometry, were isolated. Asclepain c-II displayed a molecular mass of 23,590 Da, a pI higher than 9.3, maximum proteolytic activity at pH 9.4-10.2, and showed poor thermostability. The activity of asclepain c-II is inhibited by cysteine proteases inhibitors like E-64, but not by any other protease inhibitors such as 1,10-phenantroline, phenylmethanesulfonyl fluoride, and pepstatine. The Nterminal sequence (LPSFVDWRQKGVVFPIRNQGQ CGSCWTFSA) showed a high similarity with those of other plant cysteine proteinases. When assayed on N-a-CBZ-amino acid-p-nitrophenyl esters, the enzyme exhibited higher preference for the glutamine derivative. Determinations of kinetic parameters were performed with N-a-CBZ-L-Gln-p-nitrophenyl ester as substrate: K m 5 0.1634 mM, k cat 5 121.48 s 21 , and k cat /K m 5 7.4 3 10 5 s 21 /mM.

Acta Biochimica Et Biophysica Sinica, 2009

Most of the species belonging to Asclepiadaceae family usually secrete an endogenous milk-like fl... more Most of the species belonging to Asclepiadaceae family usually secrete an endogenous milk-like fluid in a network of laticifer cells in which sub-cellular organelles intensively synthesize proteins and secondary metabolites. A new papain-like endopeptidase (asclepain c-II) has been isolated and characterized from the latex extracted from petioles of Asclepias curassavica L. (Asclepiadaceae). Asclepain c-II was the minor proteolytic component in the latex, but showed higher specific activity than asclepain c-I, the main active fraction previously studied. Both enzymes displayed quite distinct biochemical characteristics, confirming that they are different enzymes. Crude extract was purified by cation exchange chromatography (FPLC). Two active fractions, homogeneous by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and mass spectrometry, were isolated. Asclepain c-II displayed a molecular mass of 23,590 Da, a pI higher than 9.3, maximum proteolytic activity at pH 9.4-10.2, and showed poor thermostability. The activity of asclepain c-II is inhibited by cysteine proteases inhibitors like E-64, but not by any other protease inhibitors such as 1,10-phenantroline, phenylmethanesulfonyl fluoride, and pepstatine. The Nterminal sequence (LPSFVDWRQKGVVFPIRNQGQ CGSCWTFSA) showed a high similarity with those of other plant cysteine proteinases. When assayed on N-a-CBZ-amino acid-p-nitrophenyl esters, the enzyme exhibited higher preference for the glutamine derivative. Determinations of kinetic parameters were performed with N-a-CBZ-L-Gln-p-nitrophenyl ester as substrate: K m 5 0.1634 mM, k cat 5 121.48 s 21 , and k cat /K m 5 7.4 3 10 5 s 21 /mM.

Acta Biochimica Et Biophysica Sinica, 2009

Most of the species belonging to Asclepiadaceae family usually secrete an endogenous milk-like fl... more Most of the species belonging to Asclepiadaceae family usually secrete an endogenous milk-like fluid in a network of laticifer cells in which sub-cellular organelles intensively synthesize proteins and secondary metabolites. A new papain-like endopeptidase (asclepain c-II) has been isolated and characterized from the latex extracted from petioles of Asclepias curassavica L. (Asclepiadaceae). Asclepain c-II was the minor proteolytic component in the latex, but showed higher specific activity than asclepain c-I, the main active fraction previously studied. Both enzymes displayed quite distinct biochemical characteristics, confirming that they are different enzymes. Crude extract was purified by cation exchange chromatography (FPLC). Two active fractions, homogeneous by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and mass spectrometry, were isolated. Asclepain c-II displayed a molecular mass of 23,590 Da, a pI higher than 9.3, maximum proteolytic activity at pH 9.4-10.2, and showed poor thermostability. The activity of asclepain c-II is inhibited by cysteine proteases inhibitors like E-64, but not by any other protease inhibitors such as 1,10-phenantroline, phenylmethanesulfonyl fluoride, and pepstatine. The Nterminal sequence (LPSFVDWRQKGVVFPIRNQGQ CGSCWTFSA) showed a high similarity with those of other plant cysteine proteinases. When assayed on N-a-CBZ-amino acid-p-nitrophenyl esters, the enzyme exhibited higher preference for the glutamine derivative. Determinations of kinetic parameters were performed with N-a-CBZ-L-Gln-p-nitrophenyl ester as substrate: K m 5 0.1634 mM, k cat 5 121.48 s 21 , and k cat /K m 5 7.4 3 10 5 s 21 /mM.

Acta Biochimica Et Biophysica Sinica, 2009

Most of the species belonging to Asclepiadaceae family usually secrete an endogenous milk-like fl... more Most of the species belonging to Asclepiadaceae family usually secrete an endogenous milk-like fluid in a network of laticifer cells in which sub-cellular organelles intensively synthesize proteins and secondary metabolites. A new papain-like endopeptidase (asclepain c-II) has been isolated and characterized from the latex extracted from petioles of Asclepias curassavica L. (Asclepiadaceae). Asclepain c-II was the minor proteolytic component in the latex, but showed higher specific activity than asclepain c-I, the main active fraction previously studied. Both enzymes displayed quite distinct biochemical characteristics, confirming that they are different enzymes. Crude extract was purified by cation exchange chromatography (FPLC). Two active fractions, homogeneous by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and mass spectrometry, were isolated. Asclepain c-II displayed a molecular mass of 23,590 Da, a pI higher than 9.3, maximum proteolytic activity at pH 9.4-10.2, and showed poor thermostability. The activity of asclepain c-II is inhibited by cysteine proteases inhibitors like E-64, but not by any other protease inhibitors such as 1,10-phenantroline, phenylmethanesulfonyl fluoride, and pepstatine. The Nterminal sequence (LPSFVDWRQKGVVFPIRNQGQ CGSCWTFSA) showed a high similarity with those of other plant cysteine proteinases. When assayed on N-a-CBZ-amino acid-p-nitrophenyl esters, the enzyme exhibited higher preference for the glutamine derivative. Determinations of kinetic parameters were performed with N-a-CBZ-L-Gln-p-nitrophenyl ester as substrate: K m 5 0.1634 mM, k cat 5 121.48 s 21 , and k cat /K m 5 7.4 3 10 5 s 21 /mM.

Acta Biochimica Et Biophysica Sinica, 2009

Most of the species belonging to Asclepiadaceae family usually secrete an endogenous milk-like fl... more Most of the species belonging to Asclepiadaceae family usually secrete an endogenous milk-like fluid in a network of laticifer cells in which sub-cellular organelles intensively synthesize proteins and secondary metabolites. A new papain-like endopeptidase (asclepain c-II) has been isolated and characterized from the latex extracted from petioles of Asclepias curassavica L. (Asclepiadaceae). Asclepain c-II was the minor proteolytic component in the latex, but showed higher specific activity than asclepain c-I, the main active fraction previously studied. Both enzymes displayed quite distinct biochemical characteristics, confirming that they are different enzymes. Crude extract was purified by cation exchange chromatography (FPLC). Two active fractions, homogeneous by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and mass spectrometry, were isolated. Asclepain c-II displayed a molecular mass of 23,590 Da, a pI higher than 9.3, maximum proteolytic activity at pH 9.4-10.2, and showed poor thermostability. The activity of asclepain c-II is inhibited by cysteine proteases inhibitors like E-64, but not by any other protease inhibitors such as 1,10-phenantroline, phenylmethanesulfonyl fluoride, and pepstatine. The Nterminal sequence (LPSFVDWRQKGVVFPIRNQGQ CGSCWTFSA) showed a high similarity with those of other plant cysteine proteinases. When assayed on N-a-CBZ-amino acid-p-nitrophenyl esters, the enzyme exhibited higher preference for the glutamine derivative. Determinations of kinetic parameters were performed with N-a-CBZ-L-Gln-p-nitrophenyl ester as substrate: K m 5 0.1634 mM, k cat 5 121.48 s 21 , and k cat /K m 5 7.4 3 10 5 s 21 /mM.