Purification and characterization of an aminopeptidase from the chloroplast stroma of barley leaves by chromatographic and electrophoretic methods.
Aminopeptidases catalyze the cleavage of amino acids from the amino terminus of protein or peptide substrates. Although some aminopeptidase activities have been found in plant chloroplasts, the identity of these proteins remains unclear. In this work, we report the purification to apparent homogeneity of a soluble aminopeptidase from isolated barley chloroplasts which preferentially degraded alanyl-p-nitroanilide (Ala-pNA). After organelle isolation in a density gradient and precipitation of soluble proteins with ammonium sulfate, the proteins were purified in three consecutive steps including hydrophobic interaction, gel permeation and ion-exchange chromatographies. The purified enzyme appeared as a single band with a Mr of approximately 84000 in sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. The Mr of the native enzyme was estimated to be approximately 93000 by gel permeation chromatography, suggesting that the protein is a monomer. Mass spectrometry analysis of tryptic digests indicates that the primary structure of the protein has not been reported previously. The enzyme was characterized as a metalloprotease as it could be totally inhibited by 1,10-phenanthroline. Strong inhibition could also be observed using the specific aminopeptidase inhibitors amastatin and bestatin. Besides Ala-pNA, the purified protein could also cleave with decreasing activity glycyl-pNA, leucyl-pNA, lysyl-pNA, methionyl-pNA and arginyl-pNA. The possible physiological role of this enzyme in the chloroplast stroma is discussed.
Desimone
M
M
lnstitut für Biologie II, Albert-Ludwigs-Universität, Freiburg, Germany. desimone@sun2.ruf.uni-freiburg.de
Krüger
M
M
Wessel
T
T
Wehofsky
M
M
Hoffmann
R
R
Wagner
E
E
D000626:Aminopeptidases / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism; D002736:Chloroplasts / Q000201:enzymology*; D002853:Chromatography, Liquid / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D001467:Hordeum / Q000201:enzymology*; D018515:Plant Leaves / Q000201:enzymology; D013379:Substrate Specificity
D004791:Enzyme Inhibitors; D000626:Aminopeptidases