CuI‐semiquinone radical species in plant copper‐amine oxidasesMedda, Rosaria; Padiglia, Alessandra; Bellelli, Andrea; Pedersen, Jens Z.; Finazzi Agrò, Alessandro; Floris, Giovanni
doi: 10.1016/S0014-5793(99)00675-4pmid: 10403363
The intermediate CuI‐semiquinone radical species in the catalytic mechanism of copper‐amine oxidase from Lens esculenta and Pisum sativum seedlings has been studied by optical, Raman resonance and ESR spectroscopies and by stopped‐flow and temperature‐jump measurements. Treatment of highly purified enzyme preparations with good, poor or suicide substrates, under anaerobic and aerobic conditions, at different pH values and temperatures, makes it possible to generate, detect and characterize this free radical intermediate.
New substrates of DNA polymerasesVictorova, Lyubov; Sosunov, Vasily; Skoblov, Alexander; Shipytsin, Alexander; Krayevsky, Alexander
doi: 10.1016/S0014-5793(99)00615-8pmid: 10403364
Bis‐(2′‐deoxynucleoside) 5′,5′‐tetraphosphates and bis‐(2′‐deoxynucleoside) 5′,5′‐triphosphates were shown to be a new type of substrate for several DNA polymerases of human, bacterial and viral origin. Their substrate properties depend both on their structure and on the nature of the enzyme. They are incorporated by both termini in correspondence with the template nucleotide program in the active center. The results obtained support the mechanism of their direct incorporation rather than prior hydrolysis to dNTP. The highest activity of these compounds was observed for HIV reverse transcriptase. The probable biological significance of the reaction is discussed.
Formation of adenosine 5′‐tetraphosphate from the acyl phosphate intermediate: a difference between the MurC and MurD synthetases of Escherichia coliBouhss, Ahmed; Dementin, Sébastien; van Heijenoort, Jean; Parquet, Claudine; Blanot, Didier
doi: 10.1016/S0014-5793(99)00684-5pmid: 10403366
The mechanism of the Mur synthetases of peptidoglycan biosynthesis is thought to involve in each case the successive formation of an acyl phosphate and a tetrahedral intermediate. The existence of the acyl phosphates for the MurC and MurD enzymes from Escherichia coli was firmly established by their in situ reduction by sodium borohydride followed by acid hydrolysis, yielding the corresponding amino alcohols. Furthermore, it was found that MurD, but not MurC, catalyses the synthesis of adenosine 5′‐tetraphosphate from the acyl phosphate, thereby substantiating its existence and pointing out a difference between the two enzymes.
The small GTPases Rab5a, Rab5b and Rab5c are differentially phosphorylated in vitroChiariello, Mario; Bruni, Carmelo B; Bucci, Cecilia
doi: 10.1016/S0014-5793(99)00686-9pmid: 10403367
Rab GTPases play a fundamental role in the regulation of membrane traffic. Three different Rab5 isoforms have been reported but no differences in their function in endocytosis have been discovered. As the Rab5 isoforms show a conserved consensus site for Ser/Thr phosphorylation, we investigated whether this site was phosphorylated. Here, we report that the three Rab5 proteins are differentially recognized by different kinases. Rab5a is efficiently phosphorylated by extracellular‐regulated kinase 1 but not by extracellular‐regulated kinase 2, while cdc2 kinase preferentially phosphorylates Ser‐123 of Rab5b. These findings strongly suggest that phosphorylation could be important to differentially regulate the function of the Rab5 isoforms.
The lipoate synthase from Escherichia coli is an iron‐sulfur proteinOllagnier-de Choudens, Sandrine; Fontecave, Marc
doi: 10.1016/S0014-5793(99)00694-8pmid: 10403368
Lipoate synthase catalyzes the last step of the biosynthesis of lipoic acid in microorganisms and plants. The protein isolated from an overexpressing Escherichia coli strain was purified from inclusion bodies. Spectroscopic (UV‐visible and electron paramagnetic resonance) properties of the reconstituted protein demonstrate the presence of a (2Fe‐2S) center per protein. As observed in biotin synthase, these clusters are converted to (4Fe‐4S) centers during reduction under anaerobic conditions. The possible involvement of the cluster in the insertion of sulfur atoms into the octanoic acid backbone is discussed.
Biochemical analysis of the interaction between elongation factor 1α and α/β‐tubulins from a ciliate, Tetrahymena pyriformisNakazawa, Masumi; Moreira, David; Laurent, Jacqueline; Le Guyader, Hervé; Fukami, Yasuo; Ito, Keisuke
doi: 10.1016/S0014-5793(99)00692-4pmid: 10403369
The interaction between elongation factor 1α (EF‐1α) and α/β‐tubulins has been analyzed in vivo and in vitro. An association of both α‐ and β‐tubulins with EF‐1α in the lysate of Tetrahymena pyriformis was detected by co‐immunoprecipitation analysis. In contrast, in vitro biomolecular interaction analysis with glutathione S‐transferase (GST) fusion proteins revealed that GST‐β‐tubulin, but not GST‐α‐tubulin, can bind to GST‐EF‐1α. Two β‐tubulin binding sites have been identified to reside in the domains I and III of EF‐1α. In addition, β‐tubulin itself seems to have two distinct interaction sites for each of the domains. Since domain II of EF‐1α did not interact with β‐tubulin, we have re‐evaluated the phylogenetic status of ciliates using EF‐1α sequences devoid of domain II. The phylogenetic tree thus obtained was significantly different from that inferred from the whole sequence of EF‐1α, suggesting the presence of functional constraints on the molecular evolution of EF‐1α.
Coupling of a targeting peptide to plasmid DNA by covalent triple helix formationNeves, Carole; Byk, Gerardo; Scherman, Daniel; Wils, Pierre
doi: 10.1016/S0014-5793(99)00674-2pmid: 10403371
The nuclear localization signal (NLS) of the SV40 large T antigen efficiently induces nuclear entry of proteins. We have developed a strategy for covalent coupling of one or a controlled number of NLS peptides to plasmid DNA at a specific site by triple helix formation. A psoralen‐oligonucleotide‐NLS peptide conjugate was synthesized and characterized by proteolysis with trypsin. This conjugate was used to covalently associate one NLS peptide to plasmid DNA by triple helix formation and photoactivation. The oligonucleotide‐NLS peptide conjugate interacted with the NLS‐receptor importin α. The reporter gene was expressed after transfection of the modified plasmid in NIH 3T3 cells, indicating no loss of the gene expression functionality of the plasmid. On the other hand, no increase in expression was observed as a result of the NLS peptide. This site‐specific coupling technology can be used to couple to a plasmid other ligands targeting to a specific receptor.
Two novel delta‐endotoxin gene families cry26 and cry28 from Bacillus thuringiensis ssp. finitimusWojciechowska, J.A; Lewitin, E; Revina, L.P; Zalunin, I.A; Chestukhina, G.G
doi: 10.1016/S0014-5793(99)00650-Xpmid: 10403372
Genes cry26Aa1 and cry28Aa1 were cloned from Bacillus thuringiensis ssp. finitimus strain B‐1166 VKPM. This strain forms insecticidal crystal bodies either outside or inside the exosporium. The deduced amino acid sequence of the cry26Aa1 gene product included seven residues determined to be an N‐terminal part of a chymotrypsin‐treated delta‐endotoxin isolated from the same strain. Earlier this protein was detected in both free and spore‐associated types of crystals [Revina et al., Biokhimia (1999) in press]. Neither BtI nor BtII promoter sequences were found upstream of the open reading frames in both genes. Southern hybridization has shown that the surroundings of both genes at least 3 kb upstream and downstream of the open reading frames are unique. We suggest that the protein Cry26Aa1 in both types of crystal bodies is synthesized under the control of one and the same genomic locus.