PLANT PHYSIOLOGY , Vol 101, Issue 3 881-889, Copyright © 1993 by American Society of Plant Biologists
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METABOLISM AND ENZYMOLOGY |
Development of Limit Dextrinase in Germinated Barley (Hordeum vulgare L.) (Evidence of Proteolytic Activation)
M. A. Longstaff and J. H. Bryce
Department of Biological Sciences, Heriot-Watt University, Riccarton, Edinburgh, Edinburgh EH14 4AS, Scotland
Barley (Hordeum vulgare L.) that had been malted for 5 d developed only a
small amount of bound (inactive) limit dextrinase, and very little free
(active) enzyme was detected. Continuation of malting for up to 10 d only
slightly increased the amount of both bound and free forms. Grain grown
under conditions of ample moisture (wet grown) for 5 d produced a much
higher amount of bound enzyme but a similarly low amount of free enzyme
compared to malting conditions. After 10 d of growth there was a decrease
in the amount of bound enzyme and a large increase in the amount of free
enzyme, such that almost all of the enzyme was present in the free form. A
more detailed study of limit dextrinase development in wet-grown grains
revealed that a bound form was rapidly produced soon after germination.
Five to 6 d after germination the amount of bound enzyme decreased rapidly
and a very low amount was found in grains 9 d after germination. Meanwhile,
a free form appeared slightly later and its initial rate of development was
slow. At about 5 d after germination, precisely when the bound enzyme began
to decrease, the free form increased rapidly, so that by 9 d after
germination nearly all the enzyme was in the free form. The release of
bound limit dextrinase in vitro occurred by proteolytic modification
through the action of cysteine proteinases that were kept active or
activated by the presence of reduced thiols in the extraction medium. The
presence of cysteine proteinases was confirmed by inhibition studies using
the inhibitors iodoacetamide, N-ethylmaleimide, antipain, and leupeptin. In
addition, most of the bound form of limit dextrinase was soluble in 0.2 M
sodium acetate buffer (pH 5.0) following extraction at 30[deg]C for 16 h
and centrifugation at 3000g.
