Annual Progress Report on Malting Barley Research March, 2002

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two serine proteinases that we had characterized earlier. Unlike the proteinases S and T,
this enzyme was not inhibited by a barley extract when tested using the experimental
conditions that readily detected the S and T enzyme inhibitions. This was interesting,
because it indicates that if this enzyme is produced during FHB-infection, the barley may
not be able to stop it from functioning. However, we have not yet determined whether
this new enzyme is actually produced by the fungus in infected barley grain. The
properties of the enzyme will be studied in the near future, if time allows.
Fusarium proteinases are produced in FHB-infested barley. Polyclonal antibodies against
the purified S and T serine proteinases were produced in rabbits at the University of
Wisconsin Medical School Animal Care Unit. These antibodies have been used to show
that the proteinases do occur inside Fusarium infected grain (as opposed to only being
present in the Fusarium culture medium, from which they were purified). Both antibodies
specifically recognized their respective S and T antigens in extracts that were prepared
from artificially inoculated, field grown, barley grain. The tested grain samples were
obtained from the VTT Microbiology group in Finland. The enzymes were detected only
in material that was heavily Fusarium infested, implying that the fungus produced them
only after it was well established within the kernel. On the other hand, this may simply
indicate that the sensitivity of the method that we used was not adequate for recognizing
very small amounts of the proteinases. To determine exactly where the proteinases are
produced within the infected grains, the antibodies are now being used to carry out
immunomicroscopic studies. This study is being carried out in collaboration with Dr
Salla Marttila, a researcher at the Swedish University of Agricultural Sciences in Alnarp,
Sweden. Preliminary immunolocalization experiments have shown that the proteinase T
was produced during all of the infection stages when Fusarium was applied to barley
plants that were grown in a glass house. Similar studies using the proteinase S antibodies
have not yet been carried out. A manuscript reporting the results of studies that we have
carried out with infected grain from the field experiment is now being prepared.
Inhibitors of the Fusarium proteinases
Previously, we reported how we had purified five barley proteins that inhibited the
activity of proteinase S. These were purified by size-exclusion and ion exchange
chromatographies and reverse phase-HPLC. By improving these purification methods we
have been able to isolate several other proteins that also inhibited this enzyme. We have
also purified a single protein that inhibited the proteinase T. The N-terminal amino acid
sequences of the various purified inhibitor samples were analyzed at the UTMB. The
proteinase S inhibitors were the barley α-amylase/subtilisin inhibitor (BASI) and the
chymotrypsin/subtilisin inhibitors (CI) 1A, 1B and 2A. Some of the inhibitors were CIs
that contained all of their amino acids and some were the same molecules from which
various numbers of amino acid residues had been removed. The proteinase T inhibitor
was a protein called the Bowman-Birk inhibitor (BBI). All of these inhibitors have been
described previously in the literature, but it was not known that they affected any of the
Fusarium proteinases. Previous studies of the kinetic properties of the CI inhibitors by
other reseachers had shown that they were slow-binding inhibitors that form a tight
complex with bovine chymotrypsin or bacterial subtilisin. We have now shown that they
use a similar mechanism to inhibit the Fusarium proteinase S. The interaction of the BBI