共查询到20条相似文献,搜索用时 15 毫秒
1.
Eun Hye Jho Kyungsu Kang Sarangerel Oidovsambuu Eun Ha Lee Sang Hoon Jung Il-Shik Shin Chu Won Nho 《BMB reports》2013,46(10):513-518
We investigated the protective effects of Gymnaster koraiensis against oxidative stress-induced hepatic cell damage. We used two different cytotoxicity models, i.e., the administration of tert-butyl hydroperoxide (t-BHP) and acetaminophen, in HepG2 cells to evaluate the protective effects of G. koraiensis. The ethyl acetate (EA) fraction of G. koraiensis and its major compound, 3,5-di-O-caffeoylquinic acid (DCQA), exerted protective effects in the t-BHP-induced liver cytotoxicity model. The EA fraction and DCQA ameliorated t-BHP-induced reductions in GSH levels and exhibited free radical scavenging activity. The EA fraction and DCQA also significantly reduced t-BHP-induced DNA damage in HepG2 cells. Furthermore, the hexane fraction of G. koraiensis and its major compound, gymnasterkoreayne B (GKB), exerted strong hepatoprotection in the acetaminopheninduced cytotoxicity model. CYP 3A4 enzyme activity was strongly inhibited by the extract, hexane fraction, and GKB. The hexane fraction and GKB ameliorated acetaminophen-induced reductions in GSH levels and protected against cell death. [BMB Reports 2013; 46(10): 513-518] 相似文献
2.
《Biochimica et Biophysica Acta (BBA)/General Subjects》2019,1863(5):870-882
Mucin-type O-glycosylation plays important roles in various biological processes. It is initiated by a family of 20 conserved UDP-GalNAc: polypeptide N-acetylgalactosaminyltransferases (ppGalNAc-Ts). Unlike most ppGalNAc-Ts localized to the Golgi apparatus, ppGalNAc-T18 is predominantly distributed on the endoplasmic reticulum (ER) and exhibits no ppGalNAc-T catalytic activity in vitro. Herein, we found that ppGalNAc-T18 silencing in cells decreased O-glycosylation levels and activated ER stress leading to apoptosis. After treatment with chemical chaperone 4-phenylbutyric acid (PBA) or forced expression of ppGalNAc-T18 in the ppGalNAc-T18 knockdown cell, these defects could be significantly alleviated, suggesting that ppGalNAc-T18 is important for ER homeostasis and protein O-glycosylation. Furthermore, we found that ppGalNAc-T18 exerts its functions in O-glycosylation and ER stress via a non-catalytic mechanism. These results reveal a novel molecular role of ppGalNAc-Ts that the ER-localized ppGalNAc-T18 could regulate the O-glycosylation and ER homeostasis in a non-catalytic manner. 相似文献
3.
Feng Liu Yalu Cui Fang Yang Zhijue Xu Lin-Tai Da Yan Zhang 《Bioorganic & medicinal chemistry》2019,27(15):3372-3382
Ellagitannin-derived ellagic acid (EA) and colonic metabolite urolithins are functional dietary ingredients for cancer prevention, but the underlying mechanism need elucidation. Mucin-type O-glycosylation, initiated by polypeptide N-acetyl-α-galactosaminyltransferases (ppGalNAc-Ts), fine-tunes multiple biological processes and is closely associated with cancer progression. Herein, we aim to explore how specific tannin-based polyphenols affect tumor behavior of colorectal cancer cells (CRC) by modulating O-glycosylation. Utilizing HPLC-based enzyme assay, we find urolithin D (UroD), EA and gallic acid (GA) potently inhibit ppGalNAc-Ts. In particular, UroD inhibits ppGalNAc-T2 through a peptide/protein-competitive manner with nanomolar affinity. Computational simulations combined with site-directed mutagenesis further support the inhibitors’ mode of action. Moreover, lectin analysis and metabolic labelling reveal that UroD can reduce cell O-glycans but not N-glycans. Transwell experiments prove that UroD inhibits migration and invasion of CRC cells. Our work proves that specific tannin-based polyphenols can potently inhibit ppGalNAc-Ts activity to reduce cell O-glycosylation and lead to lowering the migration and invasion of CRC cells, suggesting that disturbance of mucin-type O-glycosylation is an important mechanism for the function of dietary polyphenols. 相似文献
4.
Musca domestica (Diptera: Muscidae), the housefly, exhibits unique immune defences and can produce antimicrobial peptides upon stimulation with bacteria. Based on the cDNA library constructed using the suppression subtractive hybridization (SSH) method, a 198-bp antimicrobial peptide gene, which we named MDAP-2, was amplified by rapid amplification of cDNA ends (RACE) from M. domestica larvae stimulated with Salmonella pullorum (Enterobacteriaceae: Salmonella). In the present study, the full-length MDAP-2 gene was cloned and inserted into a His-tagged Escherichia coli prokaryotic expression system to enable production of the recombinant peptide. The recombinant MDAP-2 peptide was purified using Ni-NTA HisTrap FF crude column chromatography. The bacteriostatic activity of the recombinant purified MDAP-2 protein was assessed. The results indicated that MDAP-2 had in vitro antibacterial activity against all of the tested Gram − bacteria from clinical isolates, including E. coli (Enterobacteriaceae: Escherichia), one strain of S. pullorum (Enterobacteriaceae: Salmonella), and one strain of Pasteurella multocida. DNA sequencing and BLAST analysis showed that the MDAP-2 antimicrobial peptide gene was not homologous to any other antimicrobial peptide genes in GenBank. The antibacterial mechanisms of the newly discovered MDAP-2 peptide warrant further study. 相似文献
5.
Functional role of N-glycosylation from ADAM10 in processing,localization and activity of the enzyme
Cristina Escrevente Vanessa A. Morais Sascha Keller Cláudio M. Soares Peter Altevogt Júlia Costa 《Biochimica et Biophysica Acta (BBA)/General Subjects》2008
A disintegrin and metalloprotease 10 (ADAM10) is a type I transmembrane glycoprotein with four potential N-glycosylation sites (N267, N278, N439 and N551), that cleaves several plasma membrane proteins. In this work, ADAM10 was found to contain high-mannose and complex-type glycans. Individual N-glycosylation site mutants S269A, T280A, S441A, T553A were constructed, and results indicated that all sites were occupied. T280A was found to accumulate in the endoplasmic reticulum as the non-processed precursor of the enzyme. Furthermore, it exhibited only residual levels of metalloprotease activity in vivo towards the L1 cell adhesion molecule, as well as in vitro, using a ProTNF-alpha peptide as substrate. S441A showed increased ADAM10 susceptibility to proteolysis. Mutation of N267, N439 and N551 did not completely abolish enzyme activity, however, reduced levels were found. ADAM10 is sorted into secretory vesicles, the exosomes. Here, a fraction of ADAM10 from exosomes was found to contain more processed N-linked glycans than the cellular enzyme. In conclusion, N-glycosylation is crucial for ADAM10 processing and resistance to proteolysis, and results suggest that it is required for full-enzyme activity. 相似文献
6.
7.
Many insects pass the winter in an arrested developmental stage called diapause, either as eggs, as pupae, or even as adults. Exposure to the prolonged cold of winter is required to permit awakening from diapause in the spring. In the diapause eggs of the silkworm Bombyx mori, a metalloglycoprotein, esterase A4 (EA4), has been suggested to serve as a cold-duration clock because its characteristic ATPase activity is transiently elevated at the end of the necessary cold period. This timer property of EA4 is known to start with the dissociation of an inhibitory peptide (called “peptidyl inhibitory needle”) under cold conditions, but its time-measuring mechanism is completely unknown. Here we present the crystal structures and functional properties of EA4 with and without glycosylation. We show that EA4 is a homodimeric ATPase, with each subunit consisting of a copper-zinc superoxide dismutase fold. There is an additional short N-terminal region that is capable of binding one more copper ion, suggesting a timer mechanism in which this ion is involved. The sugar chain appears to reinforce the binding of peptidyl inhibitory needle, which may in turn stabilize the initial conformation of the N-terminal domain, explaining the requirement for glycosylation and for the peptide to set the clock. 相似文献
8.
Yasuyuki Matsumoto Qing Zhang Kaoru Akita Hiroshi Nakada Kazunori Hamamura Akiko Tsuchida Tetsuya Okajima Keiko Furukawa Takeshi Urano Koichi Furukawa 《The Journal of biological chemistry》2013,288(33):24264-24276
We demonstrated previously that ppGalNAc-T13 (T13), identified as an up-regulated gene with increased metastasis in a DNA microarray, generated trimeric Tn (tTn) antigen (GalNAcα1-Ser/Thr)3 on Syndecan 1 in highly metastatic sublines of Lewis lung cancer. However, it is not known how tTn antigen regulates cancer metastasis. Here, we analyzed the roles of tTn antigen in cancer properties. tTn antigen on Syndecan 1 increased cell adhesion to fibronectin in an integrin-dependent manner. Furthermore, cell adhesion to fibronectin induced phosphorylation of focal adhesion kinase and paxillin in T13-transfectant cells. In the search of Syndecan 1-interacting molecules, it was demonstrated that tTn antigen-carrying Syndecan 1 interacted with integrin α5β1 and matrix metalloproteinase 9 and that these molecules shifted to a glycolipid-enriched microdomain/rafts along with increased metastatic potential in T13-transfectant cells. We also identified a tTn substitution site on Syndecan 1, demonstrating that tTn on Syndecan 1 is essential for the interaction with integrin α5β1 as well as for the reaction with mAb MLS128. These data suggest that high expression of the ppGalNAc-T13 gene generates tTn antigen on Syndecan 1 under reduced expression of GM1, leading to enhanced invasion and metastasis via the formation of a molecular complex consisting of integrin α5β1, Syndecan 1, and MMP-9 in the glycolipid-enriched microdomain/rafts. 相似文献
9.
Hua-Shan Yi Cai-Xia Pan Chun Pan Juan Song Yan-Fen Hu La Wang Min-Hui Pan Cheng Lu 《Biochemical and biophysical research communications》2014
In this study we identified a potential pro-apoptotic caspase gene, Bombyx mori(B. mori)ICE-2 (BmICE-2) which encoded a polypeptide of 284 amino acid residues, including a 169QACRG173 sequence which surrounded the catalytic site and contained a p20 and a p10 domain. BmICE-2 expressed in Escherichia coli (E. coli) exhibited high proteolytic activity for the synthetic human initiator caspase-9 substrates Ac-LEHD-pNA, but little activity towards the effector caspase-3 substrates Ac-DEVD-pNA. When BmICE-2 was transiently expressed in BmN-SWU1 silkworm B. mori cells, we found that the high proteolytic activity for Ac-LEHD-pNA triggered caspase-3-like protease activity resulting in spontaneous cleavage and apoptosis in these cells. This effect was not replicated in Spodoptera frugiperda 9 cells. In addition, spontaneous cleavage of endogenous BmICE-2 in BmN-SWU1 cells could be induced by actinomycin D. These results suggest that BmICE-2 may be a novel pro-apoptotic gene with caspase-9 activity which is involved apoptotic processes in BmN-SWU1 silkworm B. mori cells. 相似文献
10.
Abdel-Azeem S. Abdel-Baki Esam Almalki Lamjed Mansour Saleh Al-Quarishy 《The Korean journal of parasitology》2016,54(1):61-66
It has been known that Arak, Salvadora persica, has a number of medicinal properties. We tried to investigate in vitro scolicidal effect of root extracts of this plant against protoscolices from hydatid cysts of Echinococcus granulosus. Protoscolices were aseptically collected from sheep livers containing hydatid cysts. S. persica root extract was used in 10, 30, and 50 mg/ml concentration for 10, 20, and 30 min. The viability of protoscolices was ascertained by 0.1% eosin staining. Scolicidal activity of S. persica extract at a concentration of 10 mg/ml was 36.3%, 50.3%, and 70.8% after 10, 20, and 30 min of exposure, respectively. The scolicidal effect of this extract at a concentration of 30 mg/ml was 52.9%, 86.7%, and 100% after 10, 20, and 30 min of exposure, respectively. S. persica extract at a concentration of 50 mg/ml, meanwhile, killed 81.4%, 100%, and 100% of protoscolices after 10, 20, and 30 min, respectively. Also, the cytotoxic potential of S. persica was assessed on human liver cells (HepG2) using trypan blue exclusion test. No cytotoxic effect was observed on HepG2 cell line. The present study confirmed for the first time that the ethanolic extract of S. persica has high scolicidal power in vitro. However, in vivo effect of this material remains to be studied for treatment of echinococcosis in humans and herbivorous animals. 相似文献
11.
Group 1B human pancreatic secretory phospholipase A2 (hp-sPLA2), a digestive enzyme synthesized by pancreatic acinar cells and present in pancreatic juice, do not have antibacterial activity towards Escherichia coli. Our earlier results suggest that the N-terminal first ten amino acid residues of hp-sPLA2 constitute major portion of the membrane binding domain of full-length enzyme and is responsible for the precise orientation of enzyme on the membrane surface by inserting into the lipid bilayers (Pande et al. (2006) Biochemistry, 45,12436–12447). In this study we report the antibacterial properties of a peptide (AVWQFRKMIK-CONH2; N10 peptide), which corresponds to the N-terminal first ten amino acid residues of hp-sPLA2, against E. coli. Full-length hp-sPLA2, which contains this peptide sequence as N-terminal α-helix, did not showed detectable antibacterial activity. Presence of physiological concentration of salt or preincubation of N10 peptide with soluble anionic polymer inhibits the antibacterial activity indicating the importance of electrostatic interaction in binding of peptide to bacterial membrane. Addition of peptide resulted in destabilization of outer as well as inner cytoplasmic membrane of E. coli suggesting bacterial membranes to be the main target of action. N10 peptide exhibits strong synergism with lysozyme and potentiates the antibacterial activity of lysozyme. The peptide was inactive against human erythrocyte. Our result shows for the first time that a peptide fragment of hp-sPLA2 possesses antibacterial activity towards E. coli and at subinhibitory concentration and can potentiate the antibacterial activity of membrane active enzyme. These observations suggest that N10 peptide may play an important role in the antimicrobial activity of pancreatic juice. 相似文献
12.
Lipopeptide MSI-843 consisting of the nonstandard amino acid ornithine (Oct-OOLLOOLOOL-NH2) was designed with an objective towards generating non-lytic short antimicrobial peptides, which can have significant pharmaceutical applications. Octanoic acid was coupled to the N-terminus of the peptide to increase the overall hydrophobicity of the peptide. MSI-843 shows activity against bacteria and fungi at micromolar concentrations. It permeabilizes the outer membrane of Gram-negative bacterium and a model membrane mimicking bacterial inner membrane. Circular dichroism investigations demonstrate that the peptide adopts α-helical conformation upon binding to lipid membranes. Isothermal titration calorimetry studies suggest that the peptide binding to membranes results in exothermic heat of reaction, which arises from helix formation and membrane insertion of the peptide. 2H NMR of deuterated-POPC multilamellar vesicles shows the peptide-induced disorder in the hydrophobic core of bilayers. 31P NMR data indicate changes in the lipid head group orientation of POPC, POPG and Escherichia colitotal lipid bilayers upon peptide binding. Results from 31P NMR and dye leakage experiments suggest that the peptide selectively interacts with anionic bilayers at low concentrations (up to 5 mol%). Differential scanning calorimetry experiments on DiPOPE bilayers and 31P NMR data from E.coli total lipid multilamellar vesicles indicate that MSI-843 increases the fluid lamellar to inverted hexagonal phase transition temperature of bilayers by inducing positive curvature strain. Combination of all these data suggests the formation of a lipid-peptide complex resulting in a transient pore as a plausible mechanism for the membrane permeabilization and antimicrobial activity of the lipopeptide MSI-843. 相似文献
13.
Imamura T Yamamoto N Tamura A Murabayashi S Hashimoto S Shimada H Taguchi S 《Biochemical and biophysical research communications》2008,369(2):609-615
Activity improvement of an antimicrobial peptide, thanatin, has been achieved up to 4-fold higher than natural original one by site-specific chemical modifications with tert-butyl group at two cysteine residues which form an intramoleular disulfide bridge. The chemically modified thanatin (C11tBu/C18tBu) exhibited improved antimicrobial activity toward Gram-positive bacteria, Micrococcus luteus, whereas lowered activity toward Gram-negative bacteria, Escherichia coli. This finding suggests that disulfide-bridge formation is not only indispensable for exhibition of antimicrobial activity of thanatin but also closely related to the activity specificity towards bacteria. NMR analysis indicates that thanatin acts against E.coli stereospecifically by taking advantage of its C-terminal β-hairpin structure, while the activity against M. luteus does not relate to structures and correlates very well to side-chain hydrophobicity. 相似文献
14.
Huimin Meng Zhangxun Wang Xiangyun Meng Ling Xie Bo Huang 《Genetics and molecular biology》2015,38(3):381-389
Entomopathogenic fungi can produce a series of chitinases, some of which function synergistically with proteases and other hydrolytic enzymes to degrade the insect cuticle. In the present study, the chitinase gene Ifu-chit2 from Isaria fumosorosea was investigated. The Ifu-chit2 gene is 1,435-bp long, interrupted by three short introns, and encodes a predicted protein of 423 amino acids with a 22 residue signal peptide. The predicted Ifu-Chit2 protein is highly homologous to Beauveria bassiana chitinase Bbchit2 and belongs to the glycohydrolase family 18. Ifu-Chit2 was expressed in Escherichia coli to verify chitinase activity, and the recombinant enzyme exhibited activity with a colloidal chitin substrate. Furthermore, the expression profiles of Ifu-chit2 were analyzed at different induction times under in vivo conditions. Quantitative real-time PCR analysis revealed that Ifu-chit2 expression peaked at two days post-induction. The expression of chitinase Ifu-chit2 in vivo suggests that the chitinase may play a role in the early stage of pathogenesis. 相似文献
15.
Background and Aims
The classification and phylogeny of Eurasian (EA) Aster (Asterinae, Astereae, Asteraceae) remain poorly resolved. Some taxonomists adopt a broad definition of EA Aster, whereas others favour a narrow generic concept. The present study aims to delimit EA Aster sensu stricto (s.s.), elucidate the phylogenetic relationships of EA Aster s.s. and segregate genera.Methods
The internal and external transcribed spacers of nuclear ribosomal DNA and the plastid DNA trnL-F region were used to reconstruct the phylogeny of EA Aster through maximum parsimony and Bayesian analyses.Key Results
The analyses strongly support an Aster clade including the genera Sheareria, Rhynchospermum, Kalimeris (excluding Kalimeris longipetiolata), Heteropappus, Miyamayomena, Turczaninowia, Rhinactinidia, eastern Asian Doellingeria, Asterothamnus and Arctogeron. Many well-recognized species of Chinese Aster s.s. lie outside of the Aster clade.Conclusions
The results reveal that EA Aster s.s. is both paraphyletic and polyphyletic. Sheareria, Rhynchospermum, Kalimeris (excluding K. longipetiolata), Heteropappus, Miyamayomena, Turczaninowia, Rhinactinidia, eastern Asian Doellingeria, Asterothamnus and Arctogeron should be included in Aster, whereas many species of Chinese Aster s.s. should be excluded. The recircumscribed Aster should be divided into two subgenera and nine sections. Kalimeris longipetiolata, Aster batangensis, A. ser. Albescentes, A. series Hersileoides, a two-species group composed of A. senecioides and A. fuscescens, and a six-species group including A. asteroides, should be elevated to generic level. With the Aster clade, they belong to the Australasian lineages. The generic status of Callistephus should be maintained. Whether Galatella (including Crinitina) and Tripolium should remain as genera or be merged into a single genus remains to be determined. In addition, the taxonomic status of A. auriculatus and the A. pycnophyllus–A. panduratus clade remains unresolved, and the systematic position of some segregates of EA Aster requires further study. 相似文献16.
Zhang R Zhu G Zhang W Cao S Ou X Li X Bartlam M Xu Y Zhang XC Rao Z 《Protein science : a publication of the Protein Society》2008,17(8):1412-1423
A novel short-chain (S)-1-phenyl-1,2-ethanediol dehydrogenase (SCR) from Candida parapsilosis exhibits coenzyme specificity for NADPH over NADH. It catalyzes an anti-Prelog type reaction to reduce 2-hydroxyacetophenone into (S)-1-phenyl-1,2-ethanediol. The coding gene was overexpressed in Escherichia coli and the purified protein was crystallized. The crystal structure of the apo-form was solved to 2.7 Å resolution. This protein forms a homo-tetramer with a broken 2-2-2 symmetry. The overall fold of each SCR subunit is similar to that of the known structures of other homologous alcohol dehydrogenases, although the latter usually form tetramers with perfect 2-2-2 symmetries. Additionally, in the apo-SCR structure, the entrance of the NADPH pocket is blocked by a surface loop. In order to understand the structure–function relationship of SCR, we carried out a number of mutagenesis–enzymatic analyses based on the new structural information. First, mutations of the putative catalytic Ser-Tyr-Lys triad confirmed their functional role. Second, truncation of an N-terminal 31-residue peptide indicated its role in oligomerization, but not in catalytic activity. Similarly, a V270D point mutation rendered the SCR as a dimer, rather than a tetramer, without affecting the enzymatic activity. Moreover, the S67D/H68D double-point mutation inside the coenzyme-binding pocket resulted in a nearly 10-fold increase and a 20-fold decrease in the kcat/KM value when NADH and NADPH were used as cofactors, respectively, with kcat remaining essentially the same. This latter result provides a new example of a protein engineering approach to modify the coenzyme specificity in SCR and short-chain dehydrogenases/reductases in general. 相似文献
17.
Amol B. Salake Aparna S. Chothe Shrikant S. Nilewar Madhavi Khilare Rutuja S. Meshram Abhishek A. Pandey M. K. Kathiravan 《Journal of chemical biology》2014,7(1):29-35
Fungal infections pose a continuous and serious threat to human health and life. The intrinsic resistance has been observed in many genera of fungi. Many fungal infections are caused by opportunistic pathogens that may be endogenous (Candida infections) or acquired from the environment (Cryptococcus and Aspergillus infections). So, new therapeutic strategies are needed to combat various fungal infections. Fluconazole shows good antifungal activity with relatively low toxicity and is preferred as first line antifungal therapy, but it has suffered from severe drug resistance. So, there is a need to design novel analogues by modification of fluconazole-like structure. A novel series of phenyl(2H-tetrazol-5-yl)methanamine derivatives were synthesized by reaction of α-amino nitrile with sodium azide and ZnCl2 in presence of isopropyl alcohol. They were evaluated for antifungal activity against Candida albicans and Aspergillus niger and subjected to docking study against 1EA1. 相似文献
18.
Material antigenically related to the neuromodulatory peptide FMRFamide was detected and examined in preparations of the soybean cyst nematode, Heterodera glycines, and in the free-living nematodes Caenorhabditis elegans and Panagrellus redivivus. FMRFamide-related peptides were quantified by an enzyme-linked immunosorbent assay. Specific activities were remarkably similar among all of the vermiform members of the three species. FMRFamide-related peptide immunoactivity was present in both sexes and all stages of H. glycines examined. The highest specific activity was present in second-stage juveniles and in males, and the lowest in white and yellow females. Total FMRFamide-related peptide level per individual was highest in brown females, with 90% of the activity associated with the eggs. Peptide levels in these eggs and in second-stage juveniles were comparable and increased in adults, especially in females. Chromatographic analysis of FMRFamide-related peptide preparations from H. glycines juveniles, C. elegans, and P. redivivus revealed distinct qualitative differences between the infective plant parasite and the free-living nematodes. 相似文献
19.
C Inagaki M Martinez-Maldonado A Schwartz 《Archives of biochemistry and biophysics》1973,158(1):421-434
Binding of [14C]ethaerynic acid [EA]at concentrations of EA from 10?4m to 10?2m to a membrane preparation containing Na+,K+-ATPase activity in vitro occurred in a nonsaturable manner; binding was stimulated by Na+ or K+, but was not affected by Mg2+ and/or ATP. [14C]EA significantly bound to a microsomal preparation with low Na+,K+-ATPase activity as well as to a heat-denatured enzyme; this binding reaction was not stimulated by Na+. These observations suggest that EA binds non-specifically or to nonspecific sites on membrane preparations. Nonselective binding of [14C]EA to subcellular particles after fractionation of slices also suggested the presence of nonspecific EA binding sites in vivo. In vitro [3H]ouabain binding to medullary and cortical Na+,K+-ATPase preparations was partially reduced by pretreatment with EA. On the other hand, [14C]EA binding to Na+,K+-ATPase was not affected by pretreatment of the preparation with ouabain (10?6m to 5 × 10?4m). EA reduced the sensitivity of [3H]ouabain binding to the enzyme preparation to Na4 and K+.EA was infused (0.1, 1.0, and 10 mg/min) into one renal artery of hydropenic dogs. A prompt natriuresis in the infused kidney occurred. Similar changes were observed in the contralateral kidney 20 min after starting the infusion. Both kidneys were removed 30 min after the beginning of the infusion, and Na+,K+-ATPase was isolated from the cortex and the medulla. Enzyme activity from cortex and medulla of either kidney was not significantly different from enzyme activity from cortex and medulla of control, uninfused dogs, regardless of dose of EA or method of enzyme isolation. Furthermore, in vitro binding of [3H]ouabain to Na+,K+-ATPase membrane preparations from cortex and medulla was the same for experimental and control kidneys. In vitro incubation of 2 × 10?3m EA with a membrane preparation caused the same inhibition of ATPase activity when the enzyme was isolated either from control or EA-infused dogs. The inhibition could not be reversed by recentrifugation or rehomogenization of the enzyme. Our results do not support the concept that Na+,K+-ATPase is a pharmacological receptor for ethacrynic acid. 相似文献
20.
Francesca Spyrakis Paolo Felici Alexander S. Bayden Enea Salsi Riccardo Miggiano Glen E. Kellogg Pietro Cozzini Paul F. Cook Andrea Mozzarelli Barbara Campanini 《Biochimica et Biophysica Acta - Proteins and Proteomics》2013,1834(1):169-181
O-acetylserine sulfhydrylase (OASS) catalyzes the synthesis of l-cysteine in the last step of the reductive sulfate assimilation pathway in microorganisms. Its activity is inhibited by the interaction with serine acetyltransferase (SAT), the preceding enzyme in the metabolic pathway. Inhibition is exerted by the insertion of SAT C-terminal peptide into the OASS active site. This action is effective only on the A isozyme, the prevalent form in enteric bacteria under aerobic conditions, but not on the B-isozyme, the form expressed under anaerobic conditions. We have investigated the active site determinants that modulate the interaction specificity by comparing the binding affinity of thirteen pentapeptides, derived from the C-terminal sequences of SAT of the closely related species Haemophilus influenzae and Salmonella typhimurium, towards the corresponding OASS-A, and towards S. typhimurium OASS-B. We have found that subtle changes in protein active sites have profound effects on protein–peptide recognition. Furthermore, affinity is strongly dependent on the pentapeptide sequence, signaling the relevance of P3–P4–P5 for the strength of binding, and P1–P2 mainly for specificity. The presence of an aromatic residue at P3 results in high affinity peptides with Kdiss in the micromolar and submicromolar range, regardless of the species. An acidic residue, like aspartate at P4, further strengthens the interaction and results in the higher affinity ligand of S. typhimurium OASS-A described to date. Since OASS knocked-out bacteria exhibit a significantly decreased fitness, this investigation provides key information for the development of selective OASS inhibitors, potentially useful as novel antibiotic agents. 相似文献