Ara12 subtilisin-like protease from Arabidopsis thaliana: purification, substrate specificity and tissue localization.
A C-terminal portion of Ara12 subtilisin-like protease (residues 542-757) was expressed in Escherichia coli cells as a fusion protein bound to maltose binding protein. Polyclonal antisera raised against the expressed protein were used to examine the tissue specificity and subcellular localization of Ara12. The protease was found predominantly in the silique and stem of plants, but was hardly detectable in leaf and not seen in root tissue. The distribution observed using immunological techniques is different from that seen by an RNA analysis study, which demonstrated similar mRNA abundance in the stem and leaves. Using immunogold labelling, Ara12 was shown to have an extracellular localization and was found in the intercellular spaces in stem tissue. Ara12 protease was purified to homogeneity from Arabidopsis thaliana cell suspension cultures by anion exchange and hydrophobic interaction chromatography. Proteolytic activity of Ara12 was inhibited by a number of serine protease inhibitors, but was almost unaffected by inhibitors of other catalytic classes of proteases. Optimal proteolytic activity was displayed under acidic conditions (pH 5.0). Ara12 activity was relatively thermostable and was stimulated in the presence of Ca2+ ions. Substrate specificity studies were conducted using a series of internally quenched fluorogenic peptide substrates. At the P1 position of substrates, hydrophobic residues, such as Phe and Ala, were preferred to Arg, whilst at the P1' position, Asp, Leu and Ala were most favoured. Possible functions of Ara12 are discussed in the light of the involvement of a number of plant subtilisin-like proteases in morphogenesis.
Hamilton
John M U
JM
School of Biological and Biomedical Sciences, University of Durham, South Road, Durham DH1 3LE, UK.
Simpson
David J
DJ
Hyman
Stefan C
SC
Ndimba
Bongani K
BK
Slabas
Antoni R
AR
D017360:Arabidopsis / Q000201:enzymology*; D029681:Arabidopsis Proteins / Q000302:isolation & purification / Q000378:metabolism*; D001483:Base Sequence; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism; D002850:Chromatography, Gel; D017931:DNA Primers; D004229:Dithiothreitol / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D007150:Immunohistochemistry; D011480:Protease Inhibitors / Q000494:pharmacology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013379:Substrate Specificity; D013381:Subtilisins / Q000302:isolation & purification / Q000378:metabolism*; D013696:Temperature
D029681:Arabidopsis Proteins; D017931:DNA Primers; D011480:Protease Inhibitors; D013381:Subtilisins; C095313:ara12 protein, Arabidopsis; D002118:Calcium; D004229:Dithiothreitol