Extracts prepared from resting seeds of Scots pine, Pinus sylvestris L., rapidly hydrolysed two peptides, Leu–Tyr and Ala–Gly, at pH 8.6 and 7.8, respectively. In gel chromatography on Sephadex G‐100 the two activities were eluted as separate peaks, which indicates that they are due to two different peptidases. The seeds were allowed to germinate at 20°C, the activities of the two enzymes were assayed separately on extracts from the endosperm and seedling tissues at different stages of germination, and compared with corresponding changes in dry weight and total nitrogen. Both enzyme activities were relatively high in the endosperm of resting seeds, and they increased about 2‐ and 3‐fold during germination (expressed as enzyme units per seed), the increases coinciding with the time of rapid reserve protein mobilization. Both enzymes were also abundant in the embryos of resting seeds, and during germination their activities increased even more rapidly than those in the endosperm. The possible role of these two “alkaline peptidases” in reserve protein hydrolysis is discussed.
Salmia, M. A. 1981. Proleinase activities in resting and germinating seeds of Scots pine, Pinus sylvestris. Plant, Resting seeds uf Scots pine contained a moderate amount of acid proteinase activity, about 90% of wliich was inhibited by pepstatin A and about 10% by p-hydroxymercuribenzoate. In gel chromatography on Sephacr>'i S-200 the proteinase activity showed a eomplex elution pattern with poorly separated peaks at positions corresponding to mol. wts. 100,000 and 30,000 and several shoulders. The results suggested that pine proteinases I and II, which arc the main proteinases in the endosperms of germinating seeds (Sa)mia 1981: Physiol, Plant. 51: 253-258), were not present in the resting seeds. -Seedling extracts showed a low level of acid proteinase activity, which separated into several peaks in chromatography on Sephacryl S-200, As none of the peaks had the catalytic properties of proteinase 1 or II, it seems that these endosperma! enzymes are also lacking in the seedling tissues.in the endosperms of germinating seeds the aetivity of the pepstatin-sensitive acid proteinasc(s) remained at a constant level throughout the period of reserve protein mobilization (lasting up to the stage when the length of dark-grown seedlings was 60 mm). Proteinases I and II were absent from resting seeds, showed a small increase up to the 20-mm stage, and then increased rapidly up to the 60-mm stage. -Resting embrj'os contained relatively higher acid proteinase activity than resting endosperms, and again about 90% of it was inhibited by pepstatin A and about 10% by p-hydroxymereuribenzoate. During germination the former activity decreased, the latter activity remained at approximately the same level, and the activity of the other acid proteinases inereased eontinuously with the growth of the seedling. -It is concluded that the pepstatin-sensitive proteinase(s), which is not affected by endogenous proteinase inhibitors, plays a central role in the initiation of reserve protein mobilization in both the embryo and the endosperm. Proteinases I and 11, on the other hand, seem to account for the greater part of reserve protein breakdown in the main protein storage tissue, the endosperm.
Methods were developed to determine proteinase activity in germinating seeds of Scots pine. The assays were based on the liberation of TCA‐soluble peptides from haemoglobin at pH 3.7 and from casein at pH 5.4 and pH 7.0; the reaction products were determined by the Lowry method. — Endosperms separated from seeds at the time of rapid storage protein mobilization (seedling length between 20 and 50 mm) showed high proteinase activities in all three assays. Experiments with different inhibitors suggested that at least four enzymes were involved. One of the enzymes resembled mammalian and microbial pepsin‐like acid proteinases: the pH optimum was 3.7 and the enzyme was inhibited by pepstatin.—The proteinase activities in the endosperms were high enough to account for the mobilization of the reserve proteins during germination. Moreover the activities at pH 3.7.5.4. and 7.0 in the endosperms were 10‐, 25‐, and 50‐fold the corresponding activities in the growing seedlings (a “reference” tissue). Consequently, it seems that both the acid and neutral proteinases take part in the mobilization of storage proteins in the germinating seed.
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