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101.
Sato H Higashimoto Y Sakamoto H Sugishima M Takahashi K Palmer G Noguchi M 《Journal of inorganic biochemistry》2007,101(10):1394-1399
The heme oxygenase (HO) reaction consists of three successive oxygenation reactions, i.e. heme to alpha-hydroxyheme, alpha-hydroxyheme to verdoheme, and verdoheme to biliverdin-iron chelate. Of these, the least understood step is the conversion of verdoheme to biliverdin-iron chelate. For the cleavage of the oxaporphyrin ring of ferrous verdoheme, involvement of a verdoheme pi-neutral radical has been proposed. To probe this hypothetical mechanism in the HO reaction, we performed electrochemical reduction of ferrous verdoheme complexed with rat HO-1 under anaerobic conditions. On the basis of the electrochemical spectral changes, the midpoint potential for the one-electron reduction of the oxaporphyrin ring of ferrous verdoheme was found to be -0.47+/-0.01 V vs the normal hydrogen electrode (NHE). Because this potential is far lower than those of both flavins of NADPH-cytochrome P450 reductase, and of NADPH, it is concluded that the one-electron reduction of the oxaporphyrin ring of ferrous verdoheme is unlikely to occur and that the formation of the pi-neutral radical cannot be the initial step in the degradation of verdoheme by HO. Rather, it appears more reasonable to consider an alternative mechanism in which binding of O(2) to the ferrous iron of verdoheme is the first step in the degradation of verdoheme. 相似文献
102.
ErbB3-binding protein (Ebp1) promotes cell survival by preventing apoptotic DNA fragmentation through a complex with active nuclear Akt. Ebp1 phosphorylation by protein kinase C (PKC)-delta mediates its binding to nuclear Akt. In this study, we show that Ebp1 itself acts as a substrate of active caspase 3 during the programmed cell death. PKC-delta phosphorylation on Ebp1 protects it from apoptotic degradation initiated in cell-free apoptotic solution. Moreover, Ebp1 is evidently cleaved in PKC-delta-deficient cells but not in wild-type cells. Ebp1 translated from first ATG is resistant to apoptotic cleavage; by contrast, Ebp1 from second and third ATG demonstrates robust degradation, and PKC phosphorylation on S360 suppresses its cleavage by active caspase 3. Ebp1 can be digested at both D53 and D196 sites, but cleavage at D196 appears to be a prerequisite for its further degradation at D53 site. Compared with wild-type Ebp1, D196A mutant markedly protects cells from apoptosis. Thus, PKC-delta antagonizes apoptosis through phosphorylating Ebp1 and protects it from apoptotic degradation. 相似文献
103.
Song HW Cauffman K Chan AP Zhou Y King ML Etkin LD Kloc M 《Differentiation; research in biological diversity》2007,75(6):519-528
The early development of metazoans is mainly regulated by differential translation and localization of maternal mRNAs in the embryo. In general, these processes are orchestrated by RNA-binding proteins interacting with specific sequence motifs in the 3'-untranslated region (UTR) of their target RNAs. Hermes is an RNA-binding protein, which contains a single RNA recognition motif (RRM) and is found in various vertebrate species from fish to human. In Xenopus laevis, Hermes mRNA and protein are localized in the vegetal region of oocytes. A subpopulation of Hermes protein is concentrated in a specific structure in the vegetal cortex, called the germ plasm (believed to contain determinants of the germ cell fate) where Hermes protein co-localizes with Xcat2 and RINGO/Spy mRNAs. The level of total Hermes protein decreases during maturation. The precocious depletion of Hermes protein by injection of Hermes antisense morpholino oligonucleotide (HE-MO) accelerates the process of maturation and results in cleavage defects in vegetal blastomeres of the embryo. It is known that several maternal mRNAs including RINGO/Spy and Mos are regulated at the translational level during meiotic maturation and early cleavage in Xenopus. The ectopic expression of RINGO/Spy or Mos causes resumption of meiotic maturation and cleavage arrests, which resemble the loss of Hermes phenotypes. We found that the injection of HE-MO enhances the acceleration of maturation caused by the injection of RINGO/Spy mRNA, and that Hermes protein is present as mRNP complex containing RINGO/Spy, Mos, and Xcat2 mRNAs in vivo. We propose that as an RNA-binding protein, Hermes may be involved in maturation, cleavage events at the vegetal pole and germ cell development by negatively regulating the expression of RINGO/Spy, Mos, and Xcat2 mRNAs. 相似文献
104.
Purification,characterization and gene cloning of isoeugenol-degrading enzyme from <Emphasis Type="Italic">Pseudomonas putida</Emphasis> IE27 总被引:1,自引:0,他引:1
An isoeugenol-degrading enzyme was purified to homogeneity from Pseudomonas putida IE27, an isoeugenol-assimilating bacterium. The purified enzyme was a 55 kDa monomer and catalyzed the initial step of isoeugenol
degradation, the oxidative cleavage of the side chain double-bond of isoeugenol, to form vanillin. Another reaction product
of isoeugenol degradation besides vanillin was identified to be acetaldehyde. The values of Km and k
cat for isoeugenol were 175 μM and 5.18 s–1, respectively. The purified enzyme catalyzed the incorporation of an oxygen atom from either molecular oxygen or water into
vanillin, suggesting that the isoeugenol-degrading enzyme is a kind of monooxygenase. The gene encoding the isoeugenol-degrading
enzyme and its flanking regions were isolated from P. putida IE27. The amino acid sequence of the enzyme was similar to those of lignostilbene-α,β-dioxygenases, carotenoid monooxygenases
and 9-cis-epoxycarotenoid dioxygenases. 相似文献
105.
Andberg M Jäntti J Heilimo S Pihkala P Paananen A Koskinen AM Söderlund H Linder MB 《Protein science : a publication of the Protein Society》2007,16(8):1751-1761
Improved ways to cleave peptide chains at engineered sites easily and specifically would form useful tools for biochemical research. Uses of such methods include the activation or inactivation of enzymes or the removal of tags for enhancement of recombinant protein expression or tags used for purification of recombinant proteins. In this work we show by gel electrophoresis and mass spectroscopy that salts of Co(II) and Cu(II) can be used to cleave fusion proteins specifically at sites where sequences of His residues have been introduced by protein engineering. The His residues could be either consecutive or spaced with other amino acids in between. The cleavage reaction required the presence of low concentrations of ascorbate and in the case of Cu(II) also hydrogen peroxide. The amount of metal ions required for cleavage was very low; in the case of Cu(II) only one to two molar equivalents of Cu(II) to protein was required. In the case of Co(II), 10 molar equivalents gave optimal cleavage. The reaction occurred within minutes, at a wide pH range, and efficiently at temperatures ranging from 0 degrees C to 70 degrees C. The work described here can also have implications for understanding protein stability in vitro and in vivo. 相似文献
106.
Sun Z Lu W Tang Y Zhang J Chen J Deng H Li X Liu JN 《Protein expression and purification》2007,55(2):312-318
Urodilatin is a 32-amino acid peptide hormone synthesized in kidney to regulate natriuresis and diuresis. It has been shown clinically useful for the treatment of acute decompensated heart failure. A synthetic deoxyoligonucleotide encoding urodilatin was cloned into a pET32a vector immediately after the thioredoxin encoding sequence with a hexa-hisditine tag and an enterokinase recognition site incorporated in between. The fusion protein was overexpressed in Escherichia coli, which constituted 28% of the total cell proteins. More than 85% of Trx-urodilatin was soluble and purified nearly homogenous by Ni-Sepharose affinity chromatography. Urodilatin was then released from the fusion protein by the enterokinase treatment and separated from the fusion partner by the subtractive chromatography using Ni-Sepharose once again. The urodilatin sample was further purified with reverse phase HPLC. Via a biological activity assayed in vitro, it was found that urodilatin had a potent vasodilatory effect on rabbit aortic strips with an EC50 of (2.02+/-0.36)x10(-6)mg/ml, which was similar to that of the synthetic urodilatin standard. The method described here promises to produce about 4.5mg fully active recombinant urodilatin with homogeneity over 97% from one liter shaking flask culture of E. coli. 相似文献
107.
M. A. Molchanova V. A. Andryushina T. S. Savinova T. S. Stytsenko N. V. Rodina N. E. Voishvillo 《Russian Journal of Bioorganic Chemistry》2007,33(3):354-358
A product of microbiological cleavage of the sterols side chain, androsta-1,4-diene-3,17-dione, is toxic for bacteria, in particular, actinobacteria of the genera Mycobacterium and Arthrobacter. Sterols were transformed into androsta-1,4-diene-3,17-dione by culturing the M. neoaurum VKPM Ac-1656 strain in a high yield, provided that a sorbent was used for elimination of contact between the bacterial cells and the product. Unlike the cholesterol side chain, the more branched chains of phytosterols were cleaved in the presence of M. neoaurum at a high rate only under turbulent stirring of the culture medium, which intensified the formation of hydrocarbonate ion from NaNI3 in situ. 相似文献
108.
Marilena Manea Gábor Mezo Ferenc Hudecz Michael Przybylski 《Journal of peptide science》2007,13(4):227-236
Trypsin cleaves specifically peptide bonds at the C-terminal side of lysine and arginine residues, except for -Arg-Pro- and -Lys-Pro- bonds which are normally resistant to proteolysis. Here we report evidence for a -Lys-Pro- tryptic cleavage in modified oligotuftsin derivatives, Ac-[TKPKG]4-NH2) (1), using high-resolution mass spectrometry and HPLC as primary methods for analysis of proteolytic reactions. The proteolytic susceptibility of -Lys-Pro- bonds was strongly dependent on flanking residues, and the flexibility of the peptide backbone might be a prerequisite for this unusual cleavage. While -Lys-Gly- bonds in 1 were rapidly cleaved, the modification of these Lys residues by the attachment of a ss-amyloid(4-10) epitope to yield -Lys(X)-Gly derivatives prevented cleavage of this bond, and provided trypsin cleavage of -Lys-Pro- bonds, the pathway of this degradation being independent on the type of Lys-N(epsilon)-side chains (acetyl group, amino acid, peptide). Substitution of the Lys residues by Ala at the P'2 positions decreased the tryptic cleavage, while replacement of the bulky side chain of Thr at the P2 positions strongly increased the cleavage of -Lys-Pro- bonds. Circular dichroism (CD) data of the modified oligotuftsin derivatives are in accord with enhanced flexibility of the peptide backbone, as a prerequisite for increased susceptibility to cleavage of -Lys-Pro- bonds. These results obtained of oligotuftsin derivatives might have implications for the proteolytic degradation of target peptides that require specific conformational preconditions. 相似文献
109.
L1 is the most proliferative autonomous retroelement that comprises about 20% of mammalian genomes. Why L1s have proliferated so extensively in mammalian genomes is an important yet unsolved question. L1 copies are amplified via retrotransposition, in which the DNA cleavage specificity by the L1-encoded endonuclease (EN) primarily dictates sites of insertion. Whereas mammalian L1s show target preference for 5'-TTAAAA-3', other L1-like elements exhibit various degrees of target specificity. To gain insights on diversification of the EN specificity during L1 evolution, ENs of zebrafish L1 elements were analyzed here. We revealed that they form 3 discrete clades, M, F, and Tx1, which is in stark contrast to a single L1 clade in mammalian species. Interestingly, zebrafish clade M elements cluster as a sister group of mammalian L1s and show target-site preference for 5'-TTAAAA-3'. In contrast, elements of the clade F, the immediate outgroup of the clade M, show little specificity. We identified certain clade-specific amino acid residues in EN, many of which are located in the cleft that recognizes the substrate, suggesting that these amino acid alterations have generated 2 types of ENs with different substrate specificities. The distribution pattern of the 3 clades suggests a possibility that the acquisition of target specificity by the L1 ENs improved the L1 fitness under the circumstances in mammalian hosts. 相似文献
110.
Mikalai Lapkouski Watchalee Chuenchor Min-Sung Kim Martin Gellert Wei Yang 《The Journal of biological chemistry》2015,290(23):14618-14625
Mammalian immune receptor diversity is established via a unique restricted set of site-specific DNA rearrangements in lymphoid cells, known as V(D)J recombination. The lymphoid-specific RAG1-RAG2 protein complex (RAG1/2) initiates this process by binding to two types of recombination signal sequences (RSS), 12RSS and 23RSS, and cleaving at the boundaries of RSS and V, D, or J gene segments, which are to be assembled into immunoglobulins and T-cell receptors. Here we dissect the ordered assembly of the RAG1/2 heterotetramer with 12RSS and 23RSS DNAs. We find that RAG1/2 binds only a single 12RSS or 23RSS and reserves the second DNA-binding site specifically for the complementary RSS, to form a paired complex that reflects the known 12/23 rule of V(D)J recombination. The assembled RAG1/2 paired complex is active in the presence of Mg2+, the physiologically relevant metal ion, in nicking and double-strand cleavage of both RSS DNAs to produce a signal-end complex. We report here the purification and initial crystallization of the RAG1/2 signal-end complex for atomic-resolution structure elucidation. Strict pairing of the 12RSS and 23RSS at the binding step, together with information from the crystal structure of RAG1/2, leads to a molecular explanation of the 12/23 rule. 相似文献