Proteolysis in the axis of the germinating pea seed

Autoproteolytic, caseolytic and haemoglobin degrading activities, carboxypeptidase and aminopeptidase activities have all been measured in extracts prepared from the radicle of germinating pea seeds (Pisum sativum L.). With increasing time from the beginning of imbibition, the spectrum of protein degrading enzyme activities changed in a complex manner. As a proportion of total autoproteolytic activity, acid proteinases declined, while sulphydryl-and serine-active site endopeptidases accounted for increased proportions of the total activity. The distribution of protein degrading enzyme activities in the root tip compared with the balance of the root was determined after 4 days, at the onset of cell division in the root apex. On a fresh weight basis the tip was enriched ca. 2-fold in protein concentration and all of the exopeptidases. Autoproteolytic activity was concentrated in the tip to a lesser degree, and haemoglobin degrading activity not at all. In contrast, the root tip was depleted in caseolytic activity.Abbreviations AP aminopeptidase - BSA bovine serum albumin - CP carboxypeptidase - DAN diazoacetyl-D, L-norleucine - ME 2-mercaptoethanol - NEM N-ethyl maleimide - PMSF phenyl methyl sulphonyl fluoride - TCA trichloroacetic acid     

Carboxypeptidases of germinating triticale grains

Presence of five carboxypeptidases was found in endosperm of germinating triticale grains, while two of them in scutellum. Changes of their activities during four days of germination suggest that carboxypeptidase II plays an important role at initial stage of germination, while carboxypeptidases I and III - at subsequent stages of the process. High activity of carboxypeptidase II both in scutellum and endosperm of dry grains accompanied by its decrease during germination, and on the other hand, the appearance of carboxypeptidases I and III activities at the 2^nd and 3^rd day of the process seems to confirm such functions of these enzymes. Experiments with GA₃ indicated that carboxypeptidase I was synthesized in scutellum, and carboxypeptidase III — in aleurone layer. Carboxypeptidases I and II cleave N-CBZ-Phe-Ala, and carboxypeptidase III — N-CBZ-Ala-Met and N-CBZ-Ala-Phe as substrates with the highest rate.     

Localization of carboxypeptidase I in germinating barley grain

Activity measurements and Northern blot hybridizations were used to study the temporal and spatial expression of carboxypeptidase I in germinating grains of barley (Hordeum vulgare L. cv Himalaya). In the resting grain no carboxypeptidase I activity was found in the aleurone layer, scutellum, or starchy endosperm. During germination high levels of enzyme activity appeared in the scutellum and in the starchy endosperm but only low activity was found in the aleurone layer. No mRNA for carboxypeptidase I was observed in the resting grain. By day 1 of germination the mRNA appeared in the scutellum where its level remained high for several days. In contrast, little mRNA was observed in the aleurone layer. These results indicate that the scutellum plays an important role in the production of carboxypeptidase I in germinating barley grain.     

Enzymic mechanisms of starch breakdown in germinating rice seeds: 7. Amylase formation in the epithelium

The time sequence analysis of the starch digestion pattern of the thin sectioned germinating rice (Oryza sativa L.) seed specimens using the starch film method showed that at the initial stage amylase activity was almost exclusively localized in the epithelium septum between the scutellum and endosperm. Starch breakdown in the endosperm tissues began afterward; amylase activity in the aleurone layers was detectable only after 2 days. Polyacrylamide gel electrofocusing (pH 4 to 6) revealed nearly the same zymogram patterns between endosperm and scutellum extracts, although additional amylase bands appeared in the endosperm extracts at later germination stages (4 to 6 days). These are presumably attributable to the newly synthesized enzyme molecules in the aleurone cells.     

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 resting seeds did not inhibit the pepsin-like acid pine proteinase that accounts for a minor part of the proteolytic activity of endosperm extracts at pH 3.7. Neither did they have any effect on the acid pine carboxypeptidase or trypsin and chymotrypsin. Fresh extracts of endosperms of germinating seeds contained relatively high proteinase activity (assayed directly) and moderate inhibitor activity (assayed after treatment at 60°C). When fresh extracts were dialysed at 50°C for 48 h their proteinase activities increased considerably while the corresponding inhibitor activities disappeared. It is concluded that the decrease of inhibitors during dialysis is due to enzymatic inactivation and that the corresponding increase of proteinase activities is at least partly due to the destruction of the inhibitors.     

Developmental expression of a catalase inhibitor in maize

The expression of an endogenous catalase inhibitor has been studied during development of Zea mays. In the 3-day seedling, the inhibitor is expressed primarily in the scutellum and in the aleurone layer of the endosperm. These tissues also show the highest catalase activity at this stage. Inhibitor expression has also been studied temporally in the scutellum, roots, and shoot over the first 12 days of germination. Inhibitor expression shows an inverse relationship with catalase activity in the scutellum and in the shoot. The relationship is less rigid in the root, due probably to the low levels of inhibitor found in that tissue. The role of the inhibitor in catalase regulation is discussed.     

Endopeptidases of Triticale Seeds

The changes in endopeptidase activity in different parts of germinating triticale cv. Malno were investigated. Haemoglobin, gliadin, azocasein and azoalbumin were used as substrates. During the first day of germination the activity of haemoglobin hydrolyzing endopeptidases predominated while after the second day, mainly in the endosperm, a rapid increase in endopeptidases activity preferring gliadin hydrolysis was observed. In all the investigated tissues azocaseinolytic activities increased with the successive days of germination. Similar changes were observed using azoalbumin with one exception: in the embryo axis this activity decreased with the progression of germination. Separation of endopeptidases on the DEAE Sepharose CL-6B reveals three activity peaks in extract from dry seeds and four peaks in extract from 3 d germinated seeds. The obtained peaks differed in substrate specificity and in sensitivities to class-specific inhibitors.     

In Vitro Stability and Inactivation of Peptide Hydrolases Extracted from Phaseolus vulgaris L.

Endopeptidase activity against azocasein had a higher temperatureoptimum (50°C) in leaf extracts than in cotyledon extracts(37°C). The temperature optima for aminopeptidase (46°C)and for carboxypeptidase (53°C) were similar in leaf andcotyledon extracts. The endopeptidase activity showed an excellentstability in crude extracts from leaves even at 37°C, whilethe endopeptidase in cotyledon extracts was less stable. Carboxypeptidasewas very stable in both leaf and cotyledon extracts. Aminopeptidasewas the least stable of the enzymes investigated and its inactivationrate depended on the source of the extract. A moderate stabilitywas observed in extracts of leaves or of ungerminated seeds,but this enzyme was rapidly inactivated in cotyledon extractsat pH 5.4. At pH 7.5 aminopeptidase remained active longer thanat pH 5.4. From experiments with mixed extracts it could beconcluded that in cotyledons an aminopeptidase inactivatingfactor was formed during germination. This factor was heat sensitive,excluded by Sephadex G-25, precipitated by 75% ammonium sulfateand inhibited by tosyl-L-lysine chloromethyl ketone. These datasuggest that the factor is a protein and considering the similarproperties it appears possible that it is the endopeptidaseformed during germination. (Received May 15, 1981; Accepted July 18, 1981)     

Peptidohydrolases of Soybean Root Nodules : IDENTIFICATION, SEPARATION, AND PARTIAL CHARACTERIZATION OF ENZYMES FROM BACTEROID-FREE EXTRACTS

Nodule extracts prepared from Glycine max var Woodworth possessed endopeptidase, aminopeptidase, and carboxypeptidase activities. Three distinct endopeptidase activities could be resolved by disc-gel electrophoresis at pH 8.8. According to their order of increasing electrophoretic mobility, the first of these enzymes hydrolyzed azocasein and n-benzoyl-l-Leu-beta-naphthylamide, while the second hydrolyzed n-benzoyl-l-Arg-beta-naphthylamine (Bz-l-Arg-betaNA), n-benzoyl-l-Arg-p-nitroanilide (Bz-l-Arg-pNA), and azocasein. The third endopeptidase hydrolyzed Bz-l-Arg-betaNA, Bz-l-Arg-pNA, and hemoglobin. Fractions of these enzymes extracted from electrophoresis gels were shown to have pH optima from 7.5 to 9.8. All of the endopeptidases were completely inhibited by diisopropylphosphorofluoridate, demonstrating that they were serine proteases.Aminopeptidase activity was measured using amino acyl-beta-naphthylamides. Electrophoresis of nodule extracts at pH 6.8 resolved the aminopeptidase activity of nodule extracts into at least four fractions based on mobility and on activities toward amino acyl-beta-naphthylamides. The major activity of two of the aminopeptidases was directed toward l-Leu- and l-Met-beta-naphthylamide, while the other two aminopeptidases exhibited broader specificity and were capable of hydrolyzing a large number of amino acyl-beta-naphthylamides. Two of the aminopeptidases extracted from electrophoresis gels were classified as thiol type enzymes, and all four aminopeptidases had neutral to basic pH optima.     

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 to the stage when the reserves of the endosperm were completely depleted. The time of rapid increase of activity in the endosperm did not coincide with the onset of storage protein mobilization. On the contrary, the major part of the increase occurred after the bulk of endosperm nitrogen had already been transferred to the seedling. The results suggest that the carboxypeptidase does not play a major role in the mobilization of storage proteins in germinating pine seeds. On the other hand, it probably functions in the proteolytic reactions associated with the senescence of the reserve-depleted endosperm.     

Proteolytic and Trypsin Inhibitor Activity in Germinating Jojoba Seeds (Simmondsia chinensis)

Changes in proteolytic activity (aminopeptidase, carboxypeptidase, endopeptidase) were followed during germination (imbibition through seedling development) in extracts from cotyledons of jojoba seeds (Simmondsia chinensis). After imbibition, the cotyledons contained high levels of sulfhydryl aminopeptidase activity (APA) but low levels of serine carboxypeptidase activity (CPA). CPA increased with germination through the apparent loss of a CPA inhibitor substance in the seed. Curves showing changes in endopeptidase activity (EPA) assayed at pH 4, 5, 6, 7, and 8 during germination were distinctly different. EPA at pH 4, 5, 6, and 7 showed characteristics of sulfhydryl enzymes while activity at pH 8 was probably due to a serine type enzyme. EPA at pH 6 was inhibited early in germination by one or more substances in the seed. Activities at pH 5 and later at pH 6 were the highest of all EPA throughout germination and increases in these activities were associated with a rapid loss of protein from the cotyledons of the developing seedling.     

Appearance of Endoproteolytic Enzymes during the Germination of Barley

Barley endoproteolytic enzymes are important to germination because they hydrolyze endosperm storage proteins to provide precursors for new protein synthesis. We recently developed an electrophoretic method utilizing gel-incorporated protein substrates to study the endoproteinases of 4-d-germinated barley (Hordeum vulgare L. cv Morex) grain. This work extends those findings to determine the temporal pattern of the appearance of the endoproteinases during germination, the sensitivities of the proteinases to class-specific proteinase inhibitors, and where, in germinating caryopses, the proteinases reside. Six endoproteinase activity bands (representing a minimum of seven enzymes) were present in 5-d-germinated barley grain extracts subjected to electrophoresis in nondenaturing gels at pH 8.8. The activities of two of the enzyme bands (“neutral” proteinases) increased as the pH was increased from 3.8 to 6.5. The activities of the remaining four (“acidic”) bands diminished abruptly as the pH increased above 4.7. Two proteinase bands hydrolyzed gelatin but not edestin, four of the proteinases hydrolyzed both gelatin and edestin at nearly the same rates, and one enzyme degraded only edestin. One neutral endoproteinase was sensitive to diisopropyl fluorophosphate inhibition, and the other was not inhibited by any of inhibitors tested. Four of acidic enzymes were cysteine proteinases [inhibited by trans-epoxysuccinyl-l-leucylamido(4-guanidino)butane and N-ethylmaleimide]; the other was an aspartic acid endoproteinase (sensitive to pepstatin). Only the aspartic proteinase was detected in either ungerminated or steeped barley grain. During the germination (malting) process, the aspartic endoproteinase activity decreased until the second day of germination and then increased until germination day 5. The first endoproteinase(s) induced during germination was a neutral enzyme that showed activity on the 1st day of the germination phase after steeping. Most of the endoproteinases became active on the 2nd or 3rd germination day, but one cysteine proteinase was not detected until the 5th day. Acid cysteine proteinases were present in the aleurone, scutellum, and endosperm tissues but not in shoots and roots. The aleurone layer and endosperm contained almost exclusively band B1 neutral proteinases, whereas the scutellum, shoots, and roots contained both B1 and B2 bands. This work shows that germinating barley contains a complex set of proteinases whose expression is temporally and spatially controlled. But, at the same time, it also shows that this electrophoretic method for separating and studying individual enzymes of this complex will allow us to more readily characterize and purify them.     

Activities of some peptidases and proteinases in germinating kidney bean, Phaseolus vulgaris

The activities of aminopeptidase (EC 3.4.11), dipeptidase (EC 3.4.13), carboxypeptidase (EC 3.4.16), naphthylamidase (EC 3.4.11) and proteinases (EC 3.4.21) were assayed in extracts from the cotyledons and the axial tissues of resting and germinating kidney beans ( Phaseolus vulgaris L. cv. Processor).
The activities of the alkaline peptidases (aminopeptidase hydrolyzing Leu-Tyr at pH 9.2 and dipeptidase acting on Ala-Gly at pH 8.5) and naphthylamidases (hydrolyzing Leu-β-naphthylamide at pH 6.4) were high in the cotyledons of resting seeds, but decreased during germination. This decrease was faster than the loss of the total nitrogen. On the contrary, the activities of carboxypeptidase (hydrolyzing carbobenzoxy-Phe-Ala at pH 5.9) and proteinases (acting on haemoglobin at pH 3.7 and on casein at pH 5.4 and 7.0) were low in the resting seeds, but increased during germination reaching their maximal values when the mobilization of nitrogen was highest. It has been suggested that the breakdown of storage proteins is initiated inside the protein bodies by acid proteinases and carboxypeptidases. Although the activities of the alkaline peptidases and naphthylamidases decreased during germination, these were still relatively high and enough for the completion of the proteolytic breakdown. Thus, it is suggested that, as a final step in a chain of events, the main role for the alkaline peptidases in the cotyledons of germinating seeds is to provide amino acids for the growth of the seedling.     

The sterol esters of maize seedlings

1. The composition of the sterol ester fraction of the shoot, root, scutellum and endosperm of 10-day-old maize seedlings was investigated. 2. The scutellum and endosperm together contain 80% of the sterol ester of the seedling. 3. beta-Sitosteryl linoleate is the major sterol ester of the scutellum and endosperm. 4. beta-Sitosteryl and stigmasteryl palmitate, palmitoleate, oleate and linoleate are the major sterol esters of the root. 5. In the shoot phytosterol linoleate is less abundant than phytosterol myristate, palmitate, palmitoleate and oleate. 6. There is a greater proportion of cholesteryl ester in the shoot than in the other tissues of the seedling.     

Localization of major endopeptidase activities in maize endosperms

Using a tissue print method, major endopeptidase activitieswere observed in the aleurone layer and along parts of scutellumsurface 1 d after imbibition. By day 2 the zone of activityhad spread into the subaleurone and starchy parenchyma cellsof the endosperm. Three days later, activity was detected throughoutthe endosperm tissue, but not in the embryo. Endosperm tissues,aleurone layers and scu-tella were dissected from the seedlingsat different stages after imbibition and endopeptidase activitywas analysed by an activity stain after native PAGE. At leastten different endopeptidase activities were detected in theendosperm tissues during the initial 5 d. Activities similarto these ten enzymes were also detected in aleurone layers.These results suggest that the main source of these endopeptidasesin the endosperm is the aleurone layer. The scutellum had adifferent spectrum of endopeptidases. One of these alternativeendopeptidases, which was detected on the first day after theaddition of water, was a metallo-enzyme with electrophoreticproperties similar to an activity found in endosperm tissueshortly after imbibition. Key words: Zea mays, endopeptidase localization, seed germination     

Fractionation of the proteinases present in the endosperm of germinating seed of Scots pine, Pinus sylvestris

When fresh extracts of endosperms separated from germinating seeds of Scots pine were dialysed at 5°C, proteinase activity on haemoglobin at pH 3.7 showed only a small initial increase, proteinase activities on casein at pH 5.4 and at pH 7.0 increased several-fold, and all the corresponding inhibitor activities disappeared (Salmia and Mikola 1980, Physiol. Plant. 48: 126–130). To find out what happens during dialysis, both fresh and dialysed extracts were fractionated by gel chromatography on Sephacryl S-200. – The fresh extracts had a major proteinase peak (mol. wt. 42,000) with high activity at pH 3.7 and moderate activities at pH 5.4 and 7.0 (pine proteinase I) and a smaller peak (mol. wt. 30,000) with high activity at pH 5.4 and 7.0 and smaller activity at pH 3.7 (pine proteinase II). In dialysed extracts the situation was reversed: the peak of proteinase I was very small while the peak of proteinase II was very high. Apparently, proteinase I is largely inactivated during dialysis while the activity of proteinase II increases, at least partly due to destruction of inhibitors. – The two enzymes were -SH proteinases, as they were completely inhibited by p -hydroxymercuribenzoate; both of them were also inhibited by the endogenous proteinase inhibitors of resting pine seeds. Besides these enzymes, the endosperm extracts contained pepsin-like acid proteinase activity, which is not affected by the endogenous inhibitors. This enzyme activity was largely inactivated during dialysis.     

滇重楼种子水浸液对三种植物种子萌发和幼苗生长的影响

利用滇重楼(Paris polyphylla Smith var.yunnanensis(Franch.)Hand.-Mazz.)种子外种皮和胚乳的水浸液对白菜(Brassica pekinensis(Lour.)Rupr.)、绿豆(Vigna radiata(Linn.)Wilczak)、小麦(Triticum aestivum L.)种子进行处理,研究滇重楼种子水浸液对3种植物种子萌发、幼苗生长和保护酶活性的影响,并利用GC-MS方法对滇重楼种子内源抑制物的成分进行分析。结果显示,不同浓度滇重楼外种皮、胚乳水浸液对上述3种受体植物的发芽率、苗高、根长及鲜重均产生影响,其作用强度和水浸液的浓度有关,总体上表现出低促高抑的双重浓度效应。滇重楼种子水浸液对白菜的影响作用最强,对绿豆的影响作用最弱,且胚乳水浸液的影响较外种皮强。不同浓度滇重楼种子外种皮和胚乳水浸液均能影响3种植物幼苗体内保护酶的活性,随着水浸液浓度的升高,叶片中超氧化物歧化酶(SOD)、过氧化物酶(POD)活性总体增加,与对照相比差异显著。白菜、小麦过氧化氢酶(CAT)活性减少,与对照相比差异显著;绿豆过氧化氢酶(CAT)活性增加,但与对照相比无显著差异。利用GC-MS方法从胚乳和外种皮水浸液中分别检出8种和2种物质。研究结果表明滇重楼种子中存在内源抑制物质,可能是导致种子休眠的原因;种子水浸液可能通过影响植物幼苗保护酶的活性进而影响其正常生长;有机酸类物质可能是滇重楼种子内源抑制物之一。     

多裂骆驼蓬中生物碱类物质对小麦种子萌发和幼苗生长的影响

室内培养和盆栽试验结果表明,多裂骆驼蓬生物碱提取液浸种的小麦萌发种子呼吸速率、淀粉酶和蛋白酶活性受抑,种子萌发率下降并随处理浓度提高而降低。幼苗根系活性和叶绿素含量提高,叶片硝酸还原酶（NR）、超氧化物歧化酶（SOD）和过氧化物酶（POD）活性增强,过氧化氢酶（CAT）活性下降;幼苗根体积,株高、根系和地上部干重增加。多裂骆驼蓬生物碱提取液浸种对小麦种子萌发和幼苗生长作用的大小顺序为：总生物碱〉水溶性生物碱〉脂溶性生物碱。     

Acid phosphatase from maize scutellum: Properties as a function of seed germination

Acid phosphatase (optimum pH at 5.2) purified from maize scutellum both in a state of dormancy and during the first 24 hours of seed germination has a M, of ca 65000, contains 6% neutral sugar, maintains its catalytic activity after succinylation of 52 free amino groups per molecule and does not show the apparent movement of optimum pH from 5.4 to 6.7 in the presence of 4 mM fluoride. Kinetic data showed Michaelian behaviour for the enzymatic hydrolysis of PNP-P and an apparent number of Pi bound per molecule equal to one. Our results also suggest that the increased acid phosphatase activity in maize scutellum as a function of seed germination could be the result of modifications in the enzyme molecule.