0000000000324097
AUTHOR
Juhani Mikola
Mobilization of proline in the starchy endosperm of germinating barley grain.
In germinating grains of barley, Hordeum vulgare L. cv. Himalaya, free proline accumulated in the starchy endosperm during the period of rapid mobilization of reserve proteins. When starchy endosperms were separated from germinating grains and homogenized in a dilute buffer of pH 5 (the pH of the starchy endosperm), the liberation of proline continued in these suspensions. The process was completely inhibited by diisopropylfluorophosphate, indicating that it was totally dependent on serine carboxy-peptidases. The carboxypeptidases present in the starchy endosperms of germinating grains were fractionated by chromatography on DEAE-cellulose. Four peaks were obtained, all with different activi…
Occurrence and Heterogeneity of Chymotrypsin Inhibitors in Vegetative Tissues of Barley
Inhibitors of chymotrypsin and the alkaline proteinase of Aspergillus oryzae were present in the shoots of barley seedlings and weak activities were also detected in the shoot tops of 6-week-old plants. Treatments which induce inhibitor formation in tomato and potato leaves had no effect when tested on mature leaves, seedlings, or young tillers of barley. Fractionation experiments with isoelectric focusing showed that the barley leaves contained several proteinase inhibitors acting on both chymotrypsin and the Aspergillus proteinase, and one inhibitor which acted only on the Aspergillus enzyme. All of these inhibitors were different from the five Aspergillus proteinase inhibitors which are …
Localization and Activity of a Carboxypeptidase in Germinating Seeds of Scots Pine, Pinus sylvestris
Extracts prepared from the endosperm of germinating seeds of Scots pine, Pinus sylvestris L., hydrolysed two typical carboxypeptidase substrates, Z-Phe-Ala and Z-Phe-Phe, with pH optima at 4.2 and 5.0. The activities were completely destroyed by diisopropylfluorophosphate. Identical heat inactivation curves and elution patterns in gel chromatography on Sephadex G-200 suggest that the two activities are due to a single enzyme. In resting seeds very low carboxypeptidase activity was present in both the endosperm and the embryo. During germination on agar gel at 20°C in the dark the activities, expressed as enzyme units per seed, increased in the seedling and particularly in the endosperm up t…
Transport-related amino acid metabolism in germinating barley grains
When eight [14C]-labelled amino acids were separately injected into the endosperm of germinating (4 days at 20°C) barley (Hordeum vulgare L. cv. Himalaya) grains, the label was rapidly taken up by the scutellum and further transported to the shoot and roots. Some of the amino acids (leucine, lysine and asparagine) were transported in an intact form through the scutellum to the seedling, whilst glutamic acid and aspartic acid were largely converted to glutamine in the scutellum. Proline was mainly transported unchanged, but a small part of the label appeared in glutamine. Arginine was mostly broken down in the scutellum, possibly providing ammonia for the synthesis of glutamine. During furth…
Activities of Two Peptidases in Resting and Germinating Seeds of Scots Pine, Pinus sylvestris
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 dur…
Characterization of the Proteinases Present in Germinating Seeds of Scots Pine, Pinus sylvestris
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.—Th…
Apparent transinhibition of peptide uptake in the scutellum of barley grain
The uptake of glycylsarcosine (Gly‐Sar) into scutella separated from germinating grains of barley (Hordeum vulgare L. cv. Himalaya) is inhibited by other peptides; in most cases the inhibition is not purely competitive but of a mixed type (simultaneous increase in the apparent Km and decrease in Vmax) (Sopanen, T. 1979. FEBS Lett. 108: 447–450). The aim of the present experiments was to elucidate the mechanism of the mixed inhibition by studying how peptides already taken up into the cells affect the uptake of Gly‐Sar. When scutella were preincubated in the presence of various peptides, 11 of the 13 peptides tested inhibited the subsequent uptake of Gly‐Sar by 10 to 45%. The inhibition, stu…
Localization and Activity of Naphthylamidases in Germinating Seeds of Scots Pine, Pinus sylvestris
Extracts prepared from endosperms of germinating seeds of Scots pine, Pinus sylvestris L., rapidly hydrolysed the β-naphthylamides of L-phenylalanine and L-leucine optimally at pH 6.5 and that of L-arginine at pH 7.7. Disc electrophoresis followed by activity staining showed that the activities were due to two naphthylamidases (aminopeptidases) with different substrate specificities. Seeds were allowed to germinate at 20°C on agar gel in the dark and the activities on the three substrates were assayed from separated endosperms and seedlings at various stages of germination. The activities in the endosperm of resting seeds were relatively high and they remained unchanged throughout the perio…
Inhibitors of endogenous proteinases in the seeds of Scots pine, Pinus sylvestris
Extracts of resting pine seeds inhibited the proteinase activities present in extracts of endosperms of germinating seeds (hydrolysis of haemoglobin at pH 3.7 and hydrolysis of casein at pH 5.4 and 7.0). Heating the extracts of resting seeds at 60°C destroyed their own proteinase activity but their proteinase inhibitor activity decreased by only 25 to 30%. Some properties of the inhibitor(s) were studied using extracts treated at 60°C. The inhibitor activities were non-dialysable. the inhibition increased linearly with increasing inhibitor concentration up to 80% of total proteinase activity, and the maximal inhibition was 80% at pH 3.7. 90% at pH 5.4. and 97% at pH 7.0. The extracts of res…