European journal of biochemistry最新文献_第2页

The catalytic mechanism of glutamyl-tRNA synthetase of Escherichia coli. Evidence for a two-step aminoacylation pathway, and study of the reactivity of the intermediate complex. 大肠杆菌谷氨酰胺- trna合成酶的催化机制。两步氨基酰化途径的证据，以及中间复合物反应性的研究。

European journal of biochemistry Pub Date : 2005-03-03 DOI: 10.1111/J.1432-1033.1980.TB06004.X

D. Kern, J. Lapointe

{"title":"The catalytic mechanism of glutamyl-tRNA synthetase of Escherichia coli. Evidence for a two-step aminoacylation pathway, and study of the reactivity of the intermediate complex.","authors":"D. Kern, J. Lapointe","doi":"10.1111/J.1432-1033.1980.TB06004.X","DOIUrl":"https://doi.org/10.1111/J.1432-1033.1980.TB06004.X","url":null,"abstract":"The sequence of substrate binding and of end-product dissociation at the steady state of the catalytic process of tRNAGlu aminoacylation by glutamyl-tRNA synthetase from Escherichia coli has been investigated using bisubstrate kinetics, dead-end and end-product inhibition studies. The nature of the kinetic patterns indicates that ATP and tRNAGlu bind randomly to the free enzyme, whereas glutamate binds only to the ternary enzyme · tRNAGlu· ATP complex. Binding of ATP to the enzyme hinders that of tRNAGlu and vice versa. After interconversion of the quaternary enzyme · substrates complex the end-products dissociate in the following order: PPi first, AMP second and Glu-tRNA last. \u0000 \u0000 \u0000 \u0000In addition to its role as substrate and as effector with ATP for the binding of glutamate, tRNAGlu promotes the catalytically active enzyme state. Whereas at saturating tRNAGlu concentration the catalysis is rate-determining, this conformational change can be rate-determining at low tRNAGlu concentrations. \u0000 \u0000 \u0000 \u0000The results are discussed in the light of the two-step aminoacylation pathway catalyzed by this synthetase.","PeriodicalId":11817,"journal":{"name":"European journal of biochemistry","volume":"1 1","pages":"137-50"},"PeriodicalIF":0.0,"publicationDate":"2005-03-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"89962276","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 23

ADP-ribosylated histone H1. Isolation from Ehrlich-ascites-tumor-cell nuclei and partial characterization. adp核糖化组蛋白H1。埃利希-腹水-肿瘤细胞核分离及部分鉴定。

European journal of biochemistry Pub Date : 2005-03-03 DOI: 10.1111/J.1432-1033.1981.TB06173.X

H. C. Braeuer, P. Adamietz, U. Nellessen, H. Hilz

{"title":"ADP-ribosylated histone H1. Isolation from Ehrlich-ascites-tumor-cell nuclei and partial characterization.","authors":"H. C. Braeuer, P. Adamietz, U. Nellessen, H. Hilz","doi":"10.1111/J.1432-1033.1981.TB06173.X","DOIUrl":"https://doi.org/10.1111/J.1432-1033.1981.TB06173.X","url":null,"abstract":"Conjugates between histone H1 and (ADP-ribose)n, formed in isolated nuclei of Ehrlich ascites tumor cells, were purified free of unmodified H1 using perchloric acid extraction, ion-exchange and boronate-cellulose chromatography. The isolated conjugates comprised 0.6% of the total protein-bound ADP-ribose residues, and about 1% of the histone H1 population. \u0000 \u0000 \u0000 \u0000Electrophoretic analysis in acid/urea gels revealed the presence of multiple components migrating slower than unmodified histone H1. They could be made visible by staining for protein or by fluorography of the [3H]ADP-ribose residues. The neighboring bands appeared to differ from cach other by a single ADP-ribose residue. In most preparations a mean chain length of 2–3 was found. Removal of the ADP-ribosyl groups by treatment with alkali or phosphodiesterase shifted the bands to the position of unmodified histone H1. On higher-resolving gels the bands split into doublets representing different degrees of phosphorylation. The same microheterogeneity was also observed with the non-ADP-ribosylated control. This indicated that phosphorylation of histone H1 did not significantly influence the acceptor properties for ADP-ribosyl transfer. \u0000 \u0000 \u0000 \u0000Studies on the lability of the (ADP-ribose)n protein linkage showed that about 20% of the ADP-ribose was linked by an NH2OH/NaOH-sensitive bond, 70% by an NH2OH-resistant/NaOH-sensitive bond, and the residual 10% apparently by an additional, NaOH-resistant bond. \u0000 \u0000 \u0000 \u0000Cleavage of the (ADP-ribose)n- histone H1 conjugates by N-bromosuccinimide and gel eleetrophoretic analysis of the two fragments revealed that by far the most ADP-ribose residues were linked (presumably at multiple sites) to the C-terminal fragment. Furthermore, a large fraction of the conjugates carried ADP-ribosyl groups exclusively either at the C-terminal fragment or at the N-terminal fragment.","PeriodicalId":11817,"journal":{"name":"European journal of biochemistry","volume":"38 1","pages":"63-8"},"PeriodicalIF":0.0,"publicationDate":"2005-03-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"86960370","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 16

A cysteine-rich collagenous protein from bovine placenta. Isolation of its constituent polypeptide chains and some properties of the non-denatured protein. 从牛胎盘中提取的富含半胱氨酸的胶原蛋白。其组成多肽链的分离及非变性蛋白的一些性质。

European journal of biochemistry Pub Date : 2005-03-03 DOI: 10.1111/J.1432-1033.1981.TB06165.X

R. Jander, J. Rauterberg, B. Voss, D. von Bassewitz

{"title":"A cysteine-rich collagenous protein from bovine placenta. Isolation of its constituent polypeptide chains and some properties of the non-denatured protein.","authors":"R. Jander, J. Rauterberg, B. Voss, D. von Bassewitz","doi":"10.1111/J.1432-1033.1981.TB06165.X","DOIUrl":"https://doi.org/10.1111/J.1432-1033.1981.TB06165.X","url":null,"abstract":"A high-molecular-weight collagenous protein was isolated, after limited pepsin digestion, from the endometrial villi of bovine placenta; it resembled the protein isolated from human aortas by Chung et al. [Chung, E., Rhodes, R. K. and Miller, E. J. (1976) Biochem. Biophys. Res. Commun. 71, 1167–1174] and from human placenta by Furuto and Miller [Furuto, D. K. and Miller, E. J. (1980) J. Biol. Chem. 255, 290–295]. After denaturation and reductive cleavage of disulfide bridges three chains with Mr of 40000–55000 were liberated. Two of the chains could be separated by ion-exchange chromatography; the third chain aggregated under non-denaturing conditions and could be isolated by sequential molecular-sieve chromatography in denaturing and non-denaturing buffers. Their amino acid composition resembled that of basement membrane collagens with respect to the high content of completely glycosylated hydroxylysine but differed with respect to their relatively low hydroxyproline content and high values of cysteine, tyrosine and glucosamine. A relatively low glycine content indicated regions of non-collagenous structure. Reduction under non-denaturing conditions and another pepsin treatment removed non-collagenous regions containing mainly hydrophobic amino acid residues. The denaturation temperature of the component was considerably reduced by this treatment, indicating stabilization of triple-helical structures by disulfide bridges in the native molecule. \u0000 \u0000 \u0000 \u0000Fibrillar aggregates were precipitated with ATP which showed in the electron microscope a typical crossstriation pattern different to those of segment-long-spacing crystallites obtained from other collagen types. Immunofluorescence tests demonstrated the protein in blood vessels and interstitial areas of the renal medulla but not in basement membranes of glomeruli and tubuli.","PeriodicalId":11817,"journal":{"name":"European journal of biochemistry","volume":"9 1","pages":"17-25"},"PeriodicalIF":0.0,"publicationDate":"2005-03-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"79110345","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 62

Studies on energy supply for genetic processes. Requirement for membrane potential in Escherichia coli infection by phage T4. 遗传过程的能量供应研究。T4噬菌体感染大肠杆菌对膜电位的要求。

European journal of biochemistry Pub Date : 2005-03-03 DOI: 10.1111/J.1432-1033.1983.TB07126.X

E. Kalasauskaĭte, D. Kadisaite, R. Daugelavičius, L. Grinius, A. Jasaitis

{"title":"Studies on energy supply for genetic processes. Requirement for membrane potential in Escherichia coli infection by phage T4.","authors":"E. Kalasauskaĭte, D. Kadisaite, R. Daugelavičius, L. Grinius, A. Jasaitis","doi":"10.1111/J.1432-1033.1983.TB07126.X","DOIUrl":"https://doi.org/10.1111/J.1432-1033.1983.TB07126.X","url":null,"abstract":"In this study the hypothesis considering the requirement for an electrochemical proton gradient in the injection of phage T4 DNA into Escherichia coli cell has been verified experimentally. The phage caused a reversible depolarization of cell membrane, while phage 'ghosts' induced an irreversible depolarization. The phage infection was strictly dependent on E. coli membrane potential value when phage/cell ratio was 5 and higher. When the ratio was close to 1, the decrease in the membrane potential up to -100 mV caused practically no effect on the phage infection. The infection inhibition was observed when the membrane potential was lowered below this 'threshold' value. On the other hand, the decrease in the membrane potential caused no effect on the phage infection under conditions promoting a concomitant increase in the value of the transmembranous pH gradient. The phage DNA transfer through the membrane of ATPase-deficient cells was reversibly inhibited by switching off the respiratory chain - the sole generator of a protonmotive force in these mutant cells. The membrane should be kept in the energized state during the phage DNA entrance into the cell. Adsorption of the phage on E. coli was followed by the reversible release of the respiratory control. Thus the results presented here indicate the requirement of the electrochemical proton gradient across the plasma membrane for injection of phage T4 DNA into E. coli. They support the concept postulating an expenditure of host cell metabolic energy for phage T4 DNA transfer through the membrane.","PeriodicalId":11817,"journal":{"name":"European journal of biochemistry","volume":"9 1","pages":"123-30"},"PeriodicalIF":0.0,"publicationDate":"2005-03-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"77246115","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 40

Chicken-gizzard actin. Interaction with skeletal-muscle myosin. 鸡肫肌动蛋白。与骨骼肌肌球蛋白的相互作用。

European journal of biochemistry Pub Date : 2005-03-03 DOI: 10.1111/J.1432-1033.1980.TB06024.X

E. Próchniewicz, H. Strzelecka-Gołaszewska

{"title":"Chicken-gizzard actin. Interaction with skeletal-muscle myosin.","authors":"E. Próchniewicz, H. Strzelecka-Gołaszewska","doi":"10.1111/J.1432-1033.1980.TB06024.X","DOIUrl":"https://doi.org/10.1111/J.1432-1033.1980.TB06024.X","url":null,"abstract":"Interaction of actin from chicken gizzard and from rabbit skeletal muscle with rabbit skeletal muscle myosin was compared by measuring the rate of superprecipitation, the activation of the Mg-ATPase and inhibition of K-ATPase activity of myosin and heavy meromyosin, and determination of binding of heavy meromyosin in the absence of ATP. Both the rate of superprecipitation of the hybrid actomyosin and the activation of myosin ATPase by gizzard actin are lower than those obtained with skeletal muscle actin. The activation of myosin Mg-ATPase by the two actin species also shows different dependence on substrate concentration: with gizzard actin the substrate inhibition starts at lower ATP concentration. The double-reciprocal plots of the Mg-ATPase activity of heavy meromyosin versus actin concentration yield the same value of the extrapolated ATPase activity at infinite actin concentration (V) for the two actins and nearly double the actin concentration needed to produce half-maximal activation (Kapp) in the case of gizzard actin. A corresponding difference in the abilities of the two actin species to inhibit the K-ATPase activity of heavy meromyosin in the absence of divalent cations was also observed. The results are discussed in terms of the effect of substitutions in the amino acid sequence of gizzard and skeletal muscle actins on their interaction with myosin.","PeriodicalId":11817,"journal":{"name":"European journal of biochemistry","volume":"17 1","pages":"305-12"},"PeriodicalIF":0.0,"publicationDate":"2005-03-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"79506891","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 20

Expression in yeast of a novel phospholipase A1 cDNA from Arabidopsis thaliana. 拟南芥新型磷脂酶A1 cDNA在酵母中的表达。

European journal of biochemistry Pub Date : 2004-09-01 DOI: 10.1111/j.1432-1033.2004.04317.x

Alexandre Noiriel, Pierre Benveniste, Antoni Banas, Sten Stymne, Pierrette Bouvier-Navé

{"title":"Expression in yeast of a novel phospholipase A1 cDNA from Arabidopsis thaliana.","authors":"Alexandre Noiriel, Pierre Benveniste, Antoni Banas, Sten Stymne, Pierrette Bouvier-Navé","doi":"10.1111/j.1432-1033.2004.04317.x","DOIUrl":"https://doi.org/10.1111/j.1432-1033.2004.04317.x","url":null,"abstract":"During a search for cDNAs encoding plant sterol acyltransferases, we isolated four full-length cDNAs from Arabidopsis thaliana that encode proteins with substantial identity with animal lecithin : cholesterol acyltransferases (LCATs). The expression of one of these cDNAs, AtLCAT3 (At3g03310), in various yeast strains resulted in the doubling of the triacylglycerol content. Furthermore, a complete lipid analysis of the transformed wild-type yeast showed that its phospholipid content was lower than that of the control (void plasmid-transformed) yeast whereas lysophospholipids and free fatty acids increased. When microsomes from the AtLCAT3-transformed yeast were incubated with di-[1-14C]oleyl phosphatidylcholine, both the lysophospholipid and free fatty acid fractions were highly and similarly labelled, whereas the same incubation with microsomes from the control yeast produced a negligible labelling of these fractions. Moreover when microsomes from AtLCAT3-transformed yeast were incubated with either sn-1- or sn-2-[1-14C]acyl phosphatidylcholine, the distribution of the labelling between the free fatty acid and the lysophosphatidylcholine fractions strongly suggested a phospholipase A1 activity for AtLCAT3. The sn-1 specificity of this phospholipase was confirmed by gas chromatography analysis of the hydrolysis of 1-myristoyl, 2-oleyl phosphatidylcholine. Phosphatidylethanolamine and phosphatidic acid were shown to be also hydrolysed by AtLCAT3, although less efficiently than phosphatidylcholine. Lysophospatidylcholine was a weak substrate whereas tripalmitoylglycerol and cholesteryl oleate were not hydrolysed at all. This novel A. thaliana phospholipase A1 shows optimal activity at pH 6-6.5 and 60-65 degrees C and appears to be unaffected by Ca2+. Its sequence is unrelated to all other known phospholipases. Further studies are in progress to elucidate its physiological role.","PeriodicalId":11817,"journal":{"name":"European journal of biochemistry","volume":"271 18","pages":"3752-64"},"PeriodicalIF":0.0,"publicationDate":"2004-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1111/j.1432-1033.2004.04317.x","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"24672788","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 39

Mechanism for transcriptional synergy between interferon regulatory factor (IRF)-3 and IRF-7 in activation of the interferon-beta gene promoter. 干扰素调节因子(IRF)-3和IRF-7在干扰素- β基因启动子激活中的转录协同作用机制。

European journal of biochemistry Pub Date : 2004-09-01 DOI: 10.1111/j.1432-1033.2004.04310.x

Hongmei Yang, Gang Ma, Charles H Lin, Melissa Orr, Marc G Wathelet

{"title":"Mechanism for transcriptional synergy between interferon regulatory factor (IRF)-3 and IRF-7 in activation of the interferon-beta gene promoter.","authors":"Hongmei Yang, Gang Ma, Charles H Lin, Melissa Orr, Marc G Wathelet","doi":"10.1111/j.1432-1033.2004.04310.x","DOIUrl":"https://doi.org/10.1111/j.1432-1033.2004.04310.x","url":null,"abstract":"The interferon-beta promoter has been studied extensively as a model system for combinatorial transcriptional regulation. In virus-infected cells the transcription factors ATF-2, c-Jun, interferon regulatory factor (IRF)-3, IRF-7 and NF-kappaB, and the coactivators p300/CBP play critical roles in the activation of this and other promoters. It remains unclear, however, why most other combinations of AP-1, IRF and Rel proteins fail to activate the interferon-beta gene. Here we have explored how different IRFs may cooperate with other factors to activate transcription. First we showed in undifferentiated embryonic carcinoma cells that ectopic expression of either IRF-3 or IRF-7, but not IRF-1, was sufficient to allow virus-dependent activation of the interferon-beta promoter. Moreover, the activity of IRF-3 and IRF-7 was strongly affected by promoter context, with IRF-7 preferentially being recruited to the natural interferon-beta promoter. We fully reconstituted activation of this promoter in insect cells. Maximal synergy required IRF-3 and IRF-7 but not IRF-1, and was strongly dependent on the presence of p300/CBP, even when these coactivators only modestly affected the activity of each factor by itself. These results suggest that specificity in activation of the interferon-beta gene depends on a unique promoter context and on the role played by coactivators as architectural factors.","PeriodicalId":11817,"journal":{"name":"European journal of biochemistry","volume":"271 18","pages":"3693-703"},"PeriodicalIF":0.0,"publicationDate":"2004-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1111/j.1432-1033.2004.04310.x","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"24671185","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 54

Ras oncogene induces beta-galactoside alpha2,6-sialyltransferase (ST6Gal I) via a RalGEF-mediated signal to its housekeeping promoter. Ras癌基因通过ralgef介导的信号向其管家启动子诱导β -半乳糖苷α 2,6-唾液转移酶(ST6Gal I)。

European journal of biochemistry Pub Date : 2004-09-01 DOI: 10.1111/j.1432-1033.2004.04284.x

Martin Dalziel, Fabio Dall'Olio, Arron Mungul, Véronique Piller, Friedrich Piller

{"title":"Ras oncogene induces beta-galactoside alpha2,6-sialyltransferase (ST6Gal I) via a RalGEF-mediated signal to its housekeeping promoter.","authors":"Martin Dalziel, Fabio Dall'Olio, Arron Mungul, Véronique Piller, Friedrich Piller","doi":"10.1111/j.1432-1033.2004.04284.x","DOIUrl":"https://doi.org/10.1111/j.1432-1033.2004.04284.x","url":null,"abstract":"Several oncogenic proteins are known to influence cellular glycosylation. In particular, transfection of codon 12 point mutated H-Ras increases CMP-Neu5Ac: Galbeta1,4GlcNAc alpha2,6-sialyltransferase I (ST6Gal I) activity in rodent fibroblasts. Given that Ras mediates its effects through at least three secondary effector pathways (Raf, RalGEFs and PI3K) and that transcriptional control of mouse ST6Gal I is achieved by the selective use of multiple promoters, we attempted to identify which of these parameters are involved in linking the Ras signal to ST6Gal I gene transcription in mouse fibroblasts. Transformation by human K-Ras or H-Ras (S12 and V12 point mutations, respectively) results in a 10-fold increase in ST6Gal I mRNA, but no alteration in the expression of related sialyltransferases. Using an inducible H-RasV12 expression system, a direct causal link between activated H-Ras expression and elevated ST6Gal I mRNA was demonstrated. The accumulation of the ST6Gal I transcript in response to activated Ras was accompanied by an increase of alpha2,6-sialyltransferase activity and of Neu5Acalpha2,6Gal at the cell surface. Results obtained with H-RasV12 partial loss of function mutants H-RasV12S35 (Raf signal only), H-RasV12C40 (PI3-kinase signal only) and H-RasV12G37 (RalGEFs signal only) suggest that the H-Ras induction of the mouse ST6Gal I gene (Siat1) transcription is primarily routed through RalGEFs. 5'-Rapid amplification of cDNA ends analysis demonstrated that the increase in ST6Gal I mRNA upon H-RasV12 or K-RasS12 transfection is mediated by the Siat1 housekeeping promoter P3-associated 5' untranslated exons.","PeriodicalId":11817,"journal":{"name":"European journal of biochemistry","volume":"271 18","pages":"3623-34"},"PeriodicalIF":0.0,"publicationDate":"2004-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1111/j.1432-1033.2004.04284.x","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"24672355","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 52

Differences in substrate specificities between cysteine protease CPB isoforms of Leishmania mexicana are mediated by a few amino acid changes. 墨西哥利什曼原虫半胱氨酸蛋白酶CPB亚型之间底物特异性的差异是由一些氨基酸变化介导的。

European journal of biochemistry Pub Date : 2004-09-01 DOI: 10.1111/j.1432-1033.2004.04311.x

Maria A Juliano, Darren R Brooks, Paul M Selzer, Hector L Pandolfo, Wagner A S Judice, Luiz Juliano, Morten Meldal, Sanya J Sanderson, Jeremy C Mottram, Graham H Coombs

{"title":"Differences in substrate specificities between cysteine protease CPB isoforms of Leishmania mexicana are mediated by a few amino acid changes.","authors":"Maria A Juliano, Darren R Brooks, Paul M Selzer, Hector L Pandolfo, Wagner A S Judice, Luiz Juliano, Morten Meldal, Sanya J Sanderson, Jeremy C Mottram, Graham H Coombs","doi":"10.1111/j.1432-1033.2004.04311.x","DOIUrl":"https://doi.org/10.1111/j.1432-1033.2004.04311.x","url":null,"abstract":"The CPB genes of the protozoan parasite Leishmania mexicana encode stage-regulated cathepsin L-like cysteine proteases that are important virulence factors and are in a tandem array of 19 genes. In this study, we have compared the substrate preferences of two CPB isoforms, CPB2.8 and CPB3, and a H84Y mutant of the latter enzyme, to analyse the roles played by the few amino acid differences between the isoenzymes in determining substrate specificity. CPB3 differs from CPB2.8 at just three residues (N60D, D61N and D64S) in the mature domain. The H84Y mutation mimics an additional change present in another isoenzyme, CPB18. The active recombinant CPB isoenzymes and mutant were produced using Escherichia coli and the S1-S3 and S1'-S3' subsite specificities determined using a series of fluorogenic peptide derivatives in which substitutions were made on positions P3 to P3' by natural amino acids. Carboxydipeptidase activities of CPB3 and H84Y were also observed using the peptide Abz-FRAK(Dnp)-OH and some of its analogues. The kinetic parameters of hydrolysis by CPB3, H84Y and CPB2.8 of the synthetic substrates indicates that the specificity of S3 to S3' subsites is influenced greatly by the modifications at amino acids 60, 61, 64 and 84. Particularly noteworthy was the large preference for Pro in the P2' position for the hydrolytic activity of CPB3, which may be relevant to a role in the activation mechanism of the L. mexicana CPBs.","PeriodicalId":11817,"journal":{"name":"European journal of biochemistry","volume":"271 18","pages":"3704-14"},"PeriodicalIF":0.0,"publicationDate":"2004-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1111/j.1432-1033.2004.04311.x","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"24672784","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 24

Antioxidant defences and homeostasis of reactive oxygen species in different human mitochondrial DNA-depleted cell lines. 不同人类线粒体dna缺失细胞系中活性氧的抗氧化防御和体内平衡。

European journal of biochemistry Pub Date : 2004-09-01 DOI: 10.1111/j.1432-1033.2004.04298.x

Lodovica Vergani, Maura Floreani, Aaron Russell, Mara Ceccon, Eleonora Napoli, Anna Cabrelle, Lucia Valente, Federica Bragantini, Bertrand Leger, Federica Dabbeni-Sala

{"title":"Antioxidant defences and homeostasis of reactive oxygen species in different human mitochondrial DNA-depleted cell lines.","authors":"Lodovica Vergani, Maura Floreani, Aaron Russell, Mara Ceccon, Eleonora Napoli, Anna Cabrelle, Lucia Valente, Federica Bragantini, Bertrand Leger, Federica Dabbeni-Sala","doi":"10.1111/j.1432-1033.2004.04298.x","DOIUrl":"https://doi.org/10.1111/j.1432-1033.2004.04298.x","url":null,"abstract":"Three pairs of parental (rho+) and established mitochondrial DNA depleted (rho0) cells, derived from bone, lung and muscle were used to verify the influence of the nuclear background and the lack of efficient mitochondrial respiratory chain on antioxidant defences and homeostasis of intracellular reactive oxygen species (ROS). Mitochondrial DNA depletion significantly lowered glutathione reductase activity, glutathione (GSH) content, and consistently altered the GSH2 : oxidized glutathione ratio in all of the rho0 cell lines, albeit to differing extents, indicating the most oxidized redox state in bone rho0 cells. Activity, as well as gene expression and protein content, of superoxide dismutase showed a decrease in bone and muscle rho0 cell lines but not in lung rho0 cells. GSH peroxidase activity was four times higher in all three rho0 cell lines in comparison to the parental rho+, suggesting that this may be a necessary adaptation for survival without a functional respiratory chain. Taken together, these data suggest that the lack of respiratory chain prompts the cells to reduce their need for antioxidant defences in a tissue-specific manner, exposing them to a major risk of oxidative injury. In fact bone-derived rho0 cells displayed the highest steady-state level of intracellular ROS (measured directly by 2',7'-dichlorofluorescin, or indirectly by aconitase activity) compared to all the other rho+ and rho0 cells, both in the presence or absence of glucose. Analysis of mitochondrial and cytosolic/iron regulatory protein-1 aconitase indicated that most ROS of bone rho0 cells originate from sources other than mitochondria.","PeriodicalId":11817,"journal":{"name":"European journal of biochemistry","volume":"271 18","pages":"3646-56"},"PeriodicalIF":0.0,"publicationDate":"2004-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1111/j.1432-1033.2004.04298.x","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"24671179","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 56