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endopeptidase Isoenzyme Characteristics in Cucumis sativus Leaves During Dark-induced Senescence

Journal of Integrative Plant Biology 2007年第4期49卷 507-514页

作者： Peng Zhang Fei Wang Lie-Feng Zhang Qi Rui Lang-Lai Xu College of Life Sciences Nanjing Agricultural University Nanjing 210095 China

The changes and characteristics of endopeptidase （EP） isoenzymes in cucumber （Cucumis sativus L.） leaves during dark-induced senescence were investigated by activity staining after gradient-polyacrylamide gel electrophoresis （G-PAGE） containing co-polymerized gelatin as substrate. The results showed that both the chlorophyll and the protein contents of leaves were decreased, and the protein degradation was correlated with the increase of proteolytic activity during the course of leaf senescence. Meanwhile, nine cucumber endopeptidases isoenzymes （CEP） with 140, 120, 106, 94, 76, 55, 46, 39 and 35 kDa molecular weights were detected. Four of these, CEP2, 3, 4 and CEP9 appeared all the time, but the changes of the activity were different during incubation. Another four CEPs （CEP5, 6, 7 and CEP8） whose activities increased with dark-induced time were only detected in senescent leaves. Furthermore, the biochemical properties of these nine CEP were also characterized. All the CEPs had high activities from 35 ℃ to 45 ℃, and the optimum temperature was found to be 40 ℃. However, the activities of CEPs were not detected below 25 ℃ or over 60 ℃. The activity bands appeared at a wide range of pH from 5.0 to 9.0, but the optimum pH was found at 7.0. No CEPs were detected at pH 4 or pH 10. By inhibition analysis we concluded that CEP2, 3, 4 and CEP9 were serine endopeptidases and CEP6 was a kind of cysteine protease. It is suggested that serine endopeptidases might play a major role in cucumber leaf senescence, and for the first time, six senescencerelated endopeptidases （CEP1, 5, 6, 7, 8 and 9） were found in cucumber leaves.

关键词： Cucumis sativus cucumber endopeptidase leaf senescence.

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Relationship Between endopeptidase and H_2O_2 During the Aging of Wheat Leaf

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Acta Botanica Sinica 2001年第2期43卷 127-131页

作者：张志刚芮琪徐朗莱南京农业大学理学院南京210095

The relationship between hydrogen peroxide (H 2O 2) and endopeptidase(EP) in wheat ( Triticum aestivum L. cv. Yanmai 158) leaves was studied during natural and artificial aging. Rapid accumulation of endogenous H 2O 2 and marked increase of EP activity were observed during the later phase of aging. A new EP isozyme with higher activity was detected by electrophoresis on polyacrylamide gels containing denatured heamoglobin. With the increase of exogenous H 2O 2, the activity of EP increased at first and then decreased.

关键词： endopeptidase H 2O 2 wheat leaf aging

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Agricultural Science & Technology 2010年第1期11卷 31-32,106页

作者：刘清岱王金菊李红涛赵昱张治州天津科技大学食品工程与生物技术学院天津300457 南开大学生命科学学院天津300071 哈尔滨工业大学威海分校山东威海264209

[Objective]To study endopeptidases in Spirodela polyrrhiza half-fronds during senescence and their characters. [Method]Changes in endopeptidase isoenzymes of the Spirodela polyrrhiza half-fronds during senescence were detected by gelatin-SDS-PAGE electrophoresis,and their types were analyzed with protease inhibitors. [Result]Six endopeptidases were detected in the Spirodela polyrrhiza half-fronds during senescence. Among them,HEP1,HEP2,HEP4 and HEP6 （high molecular-weight endoprotease） were senescence-related endopeptidases. [Conclusion]The metalloendopeptidase plays significant roles at the early stage of senescence,and the cysteine endopeptidase are the most abundant at the late stage of senescence.

关键词： Spirodela polyrrhiza Half-fronds Senescence endopeptidase

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Digesting gluten with oral endopeptidases to improve the management of celiac disease

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World Journal of Gastroenterology 2024年第26期30卷 3201-3205页

作者： Katelin Durham Mirac Nedim Ince Department of Internal Medicine Division of Gastroenterology and HepatologyUniversity of IowaCarver College of Medicineand the Iowa City Veterans Affairs Medical CenterIowa CityIA 52242United States

In our editorial,we want to comment on the article by Stefanolo et al titled“Effect of Aspergillus niger prolyl endopeptidase in patients with celiac disease on a long-term gluten-free diet”.Celiac disease is an immune-mediated disorder triggered by dietary gluten in genetically predisposed *** avoiding gluten can permit patients to live symptom-free,ongoing voluntary or involuntary exposure to gluten is common and associated with persistent villous atrophy in small bowel *** villous atrophy predisposes patients to life threatening complications,such as osteoporotic fractures or malignancies,therapeutic adjuncts to gluten-free diet become important to improve patients’quality of life and,if these adjuncts can be shown to improve villous atrophy,avoid *** administration of enzyme preparations,such as endopeptidases that digest gluten and mitigate its antigenicity to trigger inflam-mation,is one clinical strategy under *** article is about the utility of one endopeptidase isolated from Aspergillus *** critique findings of this clinical trial and also summarize endopeptidase-based as well as other strategies and how they can complement gluten-free diet in the management of celiac disease.

关键词： Celiac disease Gluten-free diet endopeptidase Villous atrophy Aspergillus niger Adjunct therapy

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Characterization of endopeptidases in Wheat Leaves During Dark-induced Senescence

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Acta Botanica Sinica 2003年第9期45卷 1049-1054页

作者：芮琪徐朗莱南京农业大学生命科学学院南京210095

The characterization of senescence-associated endopeptidase (EP) isoenzymes in wheat (Triticum aestivum L. cv. Yangmai 158) leaves during dark-induced senescence was performed. It was found that there was much higher endoproteolytic activity in dark-induced wheat leaves than in control. Six endopeptidase isoenzymes (EP1-EP6) were identified by natural gradient-polyacrylamide gel electrophoresis (PAGE) co-polymerized gelatin in the gel, five of which (EP1, EP2, EP4, EP5 and EP6) were only detected in senescing leaves. Treatment with 6-benzyl aminopurine (6-BA) delayed the expression of these EP isoenzymes and abscisic acid (ABA) accelerated it. The activity of EP3 could be detected at a wider range of pH and temperature levels while EP4, EP5 and EP 6 could be only detected at pH 4-5 and 30 -45 degreesC, EP1 and EP2 at pH 3-5 and 30-45 degreesC. All of the EP isoenzymes showed high thermal stability, especially EP3, EP5 and EP6 which still had activitiy even by incubation at 55 degreesC for 1 h. By using different class-specific inhibitors, EP1 and EP2 were characterized as metal-dependent cysteine-proteases, EP4 as a serine-protease.

关键词： endopeptidase leaf senescence wheat

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Activity Gel Analysis of endopeptidases in Rose Petals

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Horticultural Plant Journal 2016年第2期2卷 114-120页

作者： ZHAO Xiting ZHANG Changqing ZHU Yuting LI Tianzhong GAO Junping College of Life Sciences Henan Normal University Department of Ornamental Horticulture and Landscape China Agricultural University Department of Pomology China Agricultural University

Poor resolution for the identification of endopeptidase(EP) activity in activity gel assays is a critical issue in the analysis of the postharvest physiology of rose petals. In this paper, major factors influencing EP activity gel assays were evaluated. The results showed that a phosphate(NaH_2PO_4/Na_2HPO_4) buffer favors the detection of clear EP bands, as compared to Tris–HCl buffer. Removal of salts and pigments with Sephadex G-25 columns was vital to the measurement of EP activity in rose petal extracts. For optimal resolution of bands, we show that before electrophoresis,samples should be treated for 10 min at 40 °C. Additionally, electrophoresis should be done in 12% SDS–PAGE co-polymerized with 0.15%(w/v) gelatin. After electrophoresis, the optimal incubation temperature and pH are 42 °C and 7.0, respectively. Using our optimized assay, Rh-EP1,Rh-EP2, Rh-EP3, three proteases with molecular masses of 200, 123.5, and 97.4 kD, respectively, were detected in rose petals. Experiments using EP class-specific inhibitors revealed that Rh-EP2 and Rh-EP3 were both serine proteases, while Rh-EP1 was a metalloprotease. In this study, we also measured changes in EP activity during flower opening, senescence, and water deficit stress(WDS) using our optimized activity gel assay,and found that Rh-EP3 may be more relevant to senescence in roses compared with Rh-EP1 or Rh-EP2. Changes occurring to EPs after WDS were similar to those during the period from flower opening to senescence, and Rh-EP3 activities were greatly increased by WDS treatment. Collectively, our results suggest that significant increases in Rh-EPs activities, especially increases in Rh-EP3 activity, may contribute to the flower senescence induced under WDS treatment.

关键词： rose petal endopeptidase activity gel

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Peptide asparaginyl ligases——renegade peptide bond makers

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Science China Chemistry 2020年第3期63卷 296-307页

作者： James P.Tam Ning-Yu Chan Heng Tai Liew Shaun J.Tan Yu Chen School of Biological Sciences Nanyang Technological UniversitySingapore 637551Singapore Synzymes and Natural Products Center Nanyang Technological UniversitySingapore 637551Singapore NTU Institute of Structural Biology Nanyang Technological UniversitySingapore 636921Singapore

Making peptide bonds is tightly controlled by genetic code and machinery which includes cofactors,ATP,and *** this regard,the stand-alone and genetic-code-independent peptide ligases constitute a new family of renegade peptide-bond makers.A prime example is butelase-1,an Asn/Asp(Asx)-specific ligase that structurally belongs to the asparaginyl endopeptidase ***-1 specifically recognizes a C-terminal Asx-containing tripeptide motif,Asn/Asp-Xaa-Yaa(Xaa and Yaa are any amino acids),to form a site-specific Asn-Xaa peptide bond either intramolecularly as cyclic proteins or intermolecularly as modified *** work in the past five years has validated that butelase-1 is a potent and versatile *** we review the advances in ligases,with a focus on butelase-1,and their applications in engineering bioactive peptides and precision protein modifications,antibody-drug conjugates,and live-cell labeling.

关键词： asparaginyl endopeptidase Asn-specific ligation bioorthogonal ligation butelase chemoenzymatic ligation live-cell labeling protein engineering protein modification site-specific labeling tandem ligation

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Childhood chronic gastritis and duodenitis: Role of altered sensory neuromediators

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World Journal of Gastroenterology 2016年第37期22卷 8349-8360页

作者： Ali Islek Aygen Yilmaz Gulsum Ozlem Elpek Nuray Erin Department of Pediatric Gastro-enterology Akdeniz University School of Medicine Department of Pathology Akdeniz University School of Medicine Department of Medical Pharmacology Akdeniz University School of Medicine

AIM To investigate the roles of the neuropeptides vasoactive intestinal peptide(VIP), substance P(SP), and calcitonin gene-related peptide(CGRP) in chronic gastritis and duodenitis in *** Biopsy samples from the gastric and duodenal mucosa of 52 patients and 30 control subjects were obtained. Samples were taken for pathological examination, immunohistochemical staining, enzyme activity measurements and quantitative measurements of tissue peptide *** We observed differential effects of the disease on peptide levels, which were somewhat different from previously reported changes in chronic gastritis in adults. Specifically, SP was increased and CGRP and VIP were decreased in patients with gastritis. The changes were more prominent at sites where gastritis was severe, but significant changes were also observedin neighboring areas where gastritis was less severe. Furthermore, the degree of changes was correlated with the pathological grade of the disease. The expression of CD10, the enzyme primarily involved in SP hydrolysis, was also decreased in patients with *** Based on these findings, we propose that decreased levels of VIP and CGRP and increased levels of SP contribute to pathological changes in gastric mucosa. Hence, new treatments targeting these molecules may have therapeutic and preventive effects.

关键词： Gastritis Neuropeptides Childhood Neutral endopeptidase

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Cellular response toβ-amyloid neurotoxicity in Alzheimer's disease and implications in new therapeutics

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Animal Models and Experimental Medicine 2023年第1期6卷 3-9页

作者： Haolin Zhang Xianghua Li Xiaoli Wang Jiayu Xu Felice Elefant Juan Wang Faculty of Environment and Life Beijing University of TechnologyBeijingChina Department of Biology Drexel UniversityPhiladelphiaPennsylvaniaUSA

β-Amyloid(Aβ)is a specific pathological hallmark of Alzheimer's disease(AD).Because of its neurotoxicity,AD patients exhibit multiple brain ***-modifying therapy(DMT)is the central concept in the development of AD thera-peutics today,and most DMT drugs that are currently in clinical trials are anti-Aβdrugs,such as aducanumab and ***,understanding Aβ's neurotoxic mechanism is crucial for Aβ-targeted drug *** its total length of only a few dozen amino acids,Aβis incredibly *** addition to the well-known Aβ_(1-42),N-terminally truncated,glutaminyl cyclase(QC)catalyzed,and pyroglutamate-modified Aβ(pEAβ)is also highly amyloidogenic and far more *** extracel-lular monomeric Aβ_(x-42)(x=1-11)initiates the aggregation to form fibrils and plaques and causes many abnormal cellular responses through cell membrane receptors and receptor-coupled signal *** signal cascades further influence many cel-lular metabolism-related processes,such as gene expression,cell cycle,and cell fate,and ultimately cause severe neural cell ***,endogenous cellular anti-Aβdefense processes always accompany the Aβ-induced microenvironment alterations.Aβ-cleaving endopeptidases,Aβ-degrading ubiquitin-proteasome system(UPS),and Aβ-engulfing glial cell immune responses are all essential self-defense mechanisms that we can leverage to develop new *** review discusses some of the most recent advances in understanding Aβ-centric AD mechanisms and suggests prospects for promising anti-Aβstrategies.

关键词： Alzheimer's disease(AD) astrocytes endopeptidase glutaminyl cyclase(QC) microglia p75 neurotrophin receptor(p75NTR) proteolysis targeting chimeras(PROTACs) β-Amyloid(Aβ)

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Early Morphological and Physiological Events Occurring During Germination of Maize Seeds

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Agricultural Sciences in China 2008年第8期7卷 950-957页

作者： MEI Yu-qin SONG Song-quan Xishuangbanna Tropical Botanical Garden Chinese Academy of Sciences Mengla 666303 P.R.China Institute of Botany Chinese Academy of Sciences Beijing 100093 P.R. China

The early morphological and physiological events occurring during maize （Zea mays cv. Nongda 108） seed imbibition and germination were studied. Water uptake of seeds exhibited a triphasic pattern with a marked increase during the initial phase of imbibition, and then a slow increase, followed by a second substantial increase. Imbibition time for 10 and 50% of seed germination was about 26 and 46 h at 30℃, respectively. The relative conductivity of maize seeds dramatically decreased during the initial phase of imbibition, followed by a substantial increase. Respiratory rate of seeds gradually increased with imbibition. Length of root cap cells decreased during the initial phase and then increased; those of meristematic zone cells increased during the initial phase and then decreased; and those of elongation zone cells and of the whole elongation zone of the radicle gradually increased during germination. The contents of soluble sugars and starch in embryos gradually decreased as the activities of α- and β-amylase strikingly increased with imbibition. In the meantime, protein contents of embryos gradually decreased and free amino acid content increased. The activities of aminopeptidase and endopeptidase increased until 12 h of imbibition and then decreased. It is concluded that germination of maize seeds is mainly completed by extension of cells in the elongation zone of the radicle, and that mobilization of stored reserves in the embryo during the initial phase of imbibition is also an early event during seed germination.

关键词： α-and β-amylase aminopeptidase cellular morphology endopeptidase germination maize seed

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