Isolation and characterization of hieronymain II, another peptidase isolated from fruits of Bromelia hieronymi Mez (Bromeliaceae).
From unripe fruits of Bromelia hieronymi Mez (Bromeliaceae), a partially purified protease preparation was obtained by acetone fractionation of the crude extract. Purification was achieved by anionic exchange chromatography (FPLC) on Q-Sepharose HP followed by cationic exchange chromatography (SP-Sepharose HP). Homogeneity of the new enzyme, named hieronymain II, was confirmed by SDS-PAGE and mass spectroscopy (MALDI-TOF-TOF). The molecular mass of was 23,411 Da, and maximum proteolytic activity (more than 90% of maximum activity) was achieved at pH 7.5-9.0 on casein and at pH 7.30-8.3 on Z-Phe-Arg-p-nitroanilide. The enzyme was completely inhibited by E-64 and iodoacetic acid and activated by the addition of cysteine. The N-terminal sequence of hieronymain II (AVPQSIDWRVYGAV) was compared with those of 12 plant cysteine proteases which showed more than 70% of identity. Kinetic enzymatic assays were made on Z-Phe-Arg-p-nitroanilide (Km = 0.72mM, kcat = 1.82 seg(-1) , kcat/ Km = 2.54seg(-1) mM(-l)). No detectable activity could be found on PFLNA or Z-Arg-Arg-p-nitroanilide.
Bruno
Mariela A
MA
Laboratorio de Investigación de Proteínas Vegetales (LIPROVE), Departamento de Ciencias Biológicas, Facultad de Ciencias Exactas, Universidad Nacional de La Plata, C.C. 711, La Plata B1900AVW, Argentina. brunomaricla@biol.unlp.edu.ar
Trejo
Sebastián A
SA
Avilés
Xavier F
XF
Caffini
Néstor O
NO
López
Laura M I
LM
D000595:Amino Acid Sequence; D031231:Bromelia / Q000201:enzymology*; D002852:Chromatography, Ion Exchange; D003545:Cysteine / Q000737:chemistry / Q000494:pharmacology; D003546:Cysteine Endopeptidases / Q000737:chemistry* / Q000187:drug effects / Q000302:isolation & purification; D004789:Enzyme Activation; D005638:Fruit / Q000201:enzymology; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010940:Plant Proteins / Q000819:agonists / Q000737:chemistry* / Q000302:isolation & purification; D017386:Sequence Homology, Amino Acid; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D010940:Plant Proteins; D003546:Cysteine Endopeptidases; D003545:Cysteine