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Presents an introduction to articles, published in the December 2004 issue of the journal.

We have shown recently that in human T lymphocytes, leptin stimulates activity and expression of the endocannabinoid-degrading enzyme fatty acid amide hydrolase (FAAH), through STAT3 (signal transducer and activator of transcription 3) and its CRE (cAMP response element)-like transcriptional target in the FAAH promoter[Maccarrone, M., Di Rienzo, M., Finazzi-Agrò, A.,&Rossi, A. (2003)J. Biol. Chem. 278, 13318–13324]. We have also shown that progesterone, alone or additively with leptin, up-regulates theFAAHgene in human T-cells, through the Ikaros transcription factor[Maccarrone, M., Bari, M., Di Rienzo, M., Finazzi-Agrò, A.,&Rossi, A. (2003)J. Biol. Chem. 278, 32726–32732]. Here, we extend these observations to immortalized human lymphoma U937 cells, where stimulation of FAAH by leptin (up to≈ 300% of the controls) involves binding to a leptin receptor (Kd = 2.0 ± 0.1 nm,Bmax = 382 ± 5 fmol·mg protein−1, apparent molecular mass of≈ 110 kDa), and stimulation by progesterone involves an intracellular receptor of≈ 120 kDa. Unlike FAAH, the other proteins of the endocannabinoid system are not modulated by the two hormones. Interestingly, human neuroblastoma CHP100 cells also have a leptin receptor (≈ 110 kDa,Kd = 2.2 ± 0.2 nm,Bmax = 339 ± 8 fmol·mg protein−1), a progesterone receptor (≈ 120 kDa), STAT3 and Ikaros, yet their FAAH is not activated by leptin or progesterone. These data, corroborated by transient expression and electrophoretic mobility-shift assays, demonstrate an unprecedented cell-specific regulation of theFAAHgene, which has important implications for the control of tone and activity of AEA along the neuroimmune axis. [ABSTRACT FROM AUTHOR]

Metformin (1,1-dimethylbiguanide) is an antihyperglycaemic drug used to normalize glucose concentrations in type 2 diabetes. Furthermore, antioxidant benefits have been reported in diabetic patients treated with metformin. This work was aimed at studying the scavenging capacity of this drug against reactive oxygen species (ROS) like·OH and-free radicals. ROS were produced by gamma radiolysis of water. The irradiated solutions of metformin were analyzed by UV/visible absorption spectrophotometry. It has been shown that hydroxyl free radicals react with metformin in a concentration-dependent way. The maximum scavenging activity was obtained for concentrations of metformin ≥ 200 µmol·L−1, under our experimental conditions. An estimated value of 107 L·mol−1·s−1 has been determined for the second order rate constantk(·OH + metformin). Superoxide free radicals and hydrogen peroxide do not initiate any oxidation on metformin in ourin vitroexperiments. [ABSTRACT FROM AUTHOR]

Saccharomyces cerevisiaeis a widely used host in the production of therapeutic peptides and proteins. Here we report the identification of a novel endoprotease inS. cerevisiae.It is encoded by theCYM1gene and is specific for the C-terminus of basic residues of heterologously expressed peptides. Gene disruption ofCYM1not only reduced the intracellular proteolysis, but also enhanced the secretion of heterologously expressed peptides such as growth hormone, pro-B-type natriuretic peptide and pro-cholecystokinin. Cym1p resembles metalloendoproteases of the pitrilysin family with the HXXEH(X)E(71–77)catalytic domain as seen in insulysin, nardilysin and human metalloprotease 1. It is a nuclear encoded protease that localizes to mitochondria without a hydrophobic N-terminal signal sequence or a C-terminal tail-anchor. The protease does not require post-translational processing prior to activation and it contains cytosolic activity that processes peptides designated for the secretory pathway prior to translocation into the endoplasmic reticulum. [ABSTRACT FROM AUTHOR]

Bifidobacterium bifidumis a useful probiotic agent exhibiting health-promoting properties and containsd-aspartate as an essential component of the cross-linker moiety in the peptidoglycan. To help understandd-aspartate biosynthesis inB. bifidumNBRC 14252, aspartate racemase, which catalyzes the racemization ofd- andl-aspartate, was purified to homogeneity and characterized. The enzyme was a monomer with a molecular mass of 27 kDa. This is the first report showing the presence of a monomeric aspartate racemase. Its enzymologic properties, such as its lack of cofactor requirement and susceptibility to thiol-modifying reagents in catalysis, were similar to those of the dimeric aspartate racemase fromStreptococcus thermophilus. The monomeric enzyme, however, showed a novel characteristic, namely, that its thermal stability significantly increased in the presence of aspartate, especially thed-enantiomer. The gene encoding the monomeric aspartate racemase was cloned and overexpressed inEscherichia colicells. The nucleotide sequence of theaspartate racemasegene encoded a peptide containing 241 amino acids with a calculated molecular mass of 26 784 Da. The recombinant enzyme was purified to homogeneity and its properties were almost the same as those of theB. bifidumenzyme. [ABSTRACT FROM AUTHOR]

High-pressure NMR experiments performed on the histidine-containing phosphocarrier protein (HPr) fromStaphylococcus carnosushave shown that residue Ile14, which is located in the active-centre loop, exhibits a peculiarly small pressure response. In contrast, the rest of the loop shows strong pressure effects as is expected for typical protein interaction sites. To elucidate the structural role of this residue, the mutant protein HPr(I14A), in which Ile14 is replaced by Ala, was produced and studied by solution NMR spectroscopy. On the basis of 1406 structural restraints including 20 directly detected hydrogen bonds, 491HN-15N, and 251HN-1Hα residual dipolar couplings, a well resolved three-dimensional structure could be determined. The overall fold of the protein is not influenced by the mutation but characteristic conformational changes are introduced into the active-centre loop. They lead to a displacement of the ring system of His15 and a distortion of the N-terminus of the first helix, which supports the histidine ring. In addition, the C-terminal helix is bent because the side chain of Leu86 located at the end of this helix partly fills the hydrophobic cavity created by the mutation. Xenon, which is known to occupy hydrophobic cavities, causes a partial reversal of the mutation-induced structural effects. The observed structural changes explain the reduced phosphocarrier activity of the mutant and agree well with the earlier suggestion that Ile14 represents an anchoring point stabilizing the active-centre loop in its correct conformation. [ABSTRACT FROM AUTHOR]

The role of vertebrate histone proteins or histone derived peptides as innate immune effectors has only recently been appreciated. In this study, high levels of core histone proteins H2A, H2B, H3 and H4 were found in hemocytes from the Pacific white shrimp,Litopenaeus vannamei. The proteins were identified by in-gel digestion, mass spectrometry analysis, and homology searching. TheL. vannameihistone proteins were found to be highly homologous to histones of other species. Based on this homology, histone H2A was cloned and its N-terminus was found to resemble the known antimicrobial histone peptides buforin I, parasin, and hipposin. Consequently, a 38 amino acid synthetic peptide identical to the N-terminus of shrimp H2A was synthesized and assayed, along with endogenous histones H2A, H2B, and H4, for growth inhibition againstMicrococcus luteus. Histone H2A, purified to homogeneity, completely inhibited growth of the Gram-positive bacterium at 4.5 µmwhile a mixture of histones H2B and H4 was active at 3 µm. In addition, a fraction containing a fragment of histone H1 was also found to be active. The synthetic peptide similar to buforin was active at submicromolar concentrations. These data indicate, for the first time, that shrimp hemocyte histone proteins possess antimicrobial activity and represent a defense mechanism previously unreported in an invertebrate. Histones may be a component of innate immunity more widely conserved, and of earlier origin, than previously thought. [ABSTRACT FROM AUTHOR]

We have cloned, over-expressed and purified enolase fromPlasmodium falciparumstrain NF54 inEscherichia coliin active form, as an N-terminal His6-tagged protein. The sequence of the cloned enolase from the NF54 strain is identical to that of strain 3D7 used in full genome sequencing. The recombinant enolase (r-Pfen) could be obtained in large quantities (≈ 50 mg per litre of culture) in a highly purified form (> 95%). The purified protein gave a single band at≈ 50 kDa on SDS/PAGE. MALDI-TOF analysis gave a mean ± SD mass of 51396 ± 16 Da, which is in good agreement with the mass calculated from the sequence. The molecular mass of r-Pfen determined in gel-filtration experiments was≈ 100 kDa, indicating thatP. falciparumenolase is a homodimer. Kinetic measurements using 2-phosphoglycerate as substrate gave a specific activity of≈ 30 U·mg−1 andKm2PGA = 0.041 ± 0.004 mm. The Michaelis constant for the reverse reaction (KmPEP) is 0.25 ± 0.03 mm. pH-dependent activity measurements gave a maximum at pH 7.4–7.6 irrespective of the direction of catalysis. The activity of this enzyme is inhibited by Na+, whereas K+ has a slight activating effect. The cofactor Mg2+ has an apparent activation constant of 0.18 ±0.02 mm. However, at higher concentrations, it has an inhibitory effect. Polyclonal antibody raised against pure recombinantP. falciparumenolase in rabbit showed high specificity towards recombinant protein and is also able to recognize enolase from the murine malarial parasite,Plasmodium yoelii, which shares 90% identity with theP. falciparumprotein. [ABSTRACT FROM AUTHOR]

Thec-junbelongs to the family of proto-oncogenes and encodes for the protein Jun, a component of transcription factor AP-1 involved in regulation of the expression of genes indispensable for cell proliferation and differentiation. While the role ofc-junin the regulation of such genes has been well examined, the regulation ofc-junin proliferating cells is not fully understood. We have earlier reported that the−148 to−124 region ofc-junis involved in the positive regulation ofc-juntranscription, and interacts with a positive regulatory factor (rat liverjunregulatory protein; RLjunRP)present in rat liver. In this investigation, we report that this region is differentially recognized in proliferating liver as evidenced by the formation of a complex, different from that observed with normal liver extract. The new complex appears as early as 2 h after partial hepatectomy and its appearance coincides with the rise inc-junmRNA levels after partial hepatectomy. In regenerating rat liver nuclear extract, an additional protein of≈ 40 kDa (rRLjunRP) interacts with a pre-existing dimer of RLjunRP complexed with the−148 to−124 region ofc-junto form a slow-migrating complex. rRLjunRP appears to pre-exist in the cytosol and translocate to the nucleus as indicated by the continued presence of the retarded complex in nuclear extract prepared from partially hepatectomized rats treated with cycloheximide. UV crosslinking studies, South-Western blot analysis, SDS/PAGE of affinity-purified factor(s), and 2D-PAGE analysis clearly demonstrate that the additional factor induced in response to growth stimulus is an≈ 40 kDa, that binds with the dimer of RLjunRP and enhances thec-juntranscription. [ABSTRACT FROM AUTHOR]

Retinoid receptors are ligand-dependent transcription factors belonging to the nuclear receptor superfamily. Retinoic acid (RARα,β,γ) and retinoid X (RXRα,β,γ) receptors mediate the retinoid/rexinoid signal to the transcriptional machineries by interacting at the first level with coactivators or corepressors, which leads to the recruitment of enzymatically active noncovalent complexes at target gene promoters. It has been shown that the interaction of corepressors with nuclear receptors involves conserved LXXI/HIXXXI/L consensus sequences termed corepressor nuclear receptor (CoRNR) boxes. Here we describe the use of nondenaturing electrospray ionization mass spectrometry (ESI-MS) to determine the characteristics of CoRNR box peptide binding to the ligand binding domains of the RARα–RXRα heterodimer. The stability of the RARα–RXRα–CoRNRternary complexes was monitored in the presence of different types of agonists or antagonists for the two receptors, including inverse agonists. These results show unambiguously the differential impact of distinct retinoids on corepressor binding. We show that ESI-MS is a powerful technique that complements classical methods and allows one to: (a) obtain direct evidence for the formation of noncovalent NR complexes; (b) determine ligand binding stoichiometries and (c) monitor ligand effects on these complexes. [ABSTRACT FROM AUTHOR]

Presents a correction to the author affiliations, published in the previous issue.

Presents a correction to an article, published in the previous issue of the "European Journal of Biochemistry."

The classical metabolic control theory[Kacser, H.&Burns, J.A. (1973)Symp. Soc. Exp. Biol.27, 65–104; Heinrich, R.&Rapoport, T. (1974)Eur. J. Biochem.42, 89–95.] does not take into account experimental evidence for correlations between enzyme concentrations in the cell. We investigated the implications of two causes of linear correlations: competition between enzymes, which is a mere physical adaptation of the cell to the limitation of resources and space, and regulatory correlations, which result from the existence of regulatory networks. These correlations generate redistribution of enzyme concentrations when the concentration of an enzyme varies; this may dramatically alter the flux and metabolite concentration curves. In particular, negative correlations cause the flux to have a maximum value for a defined distribution of enzyme concentrations. Redistribution coefficients of enzyme concentrations allowed us to calculate the‘combined response coefficient’ that quantifies the response of flux or metabolite concentration to a perturbation of enzyme concentration. [ABSTRACT FROM AUTHOR]

Transcription of yeast phospholipid biosynthesis structural genes, which contain an inositol-sensitive upstream activating sequence in their promoters, responds to the availability of the soluble precursors inositol and choline and to changes in phospholipid metabolism. TheINO1gene is deregulated (derepressed when inositol is present) under the conditions of increased phosphatidylcholine (PtdCho) turnover, as occurs in thesec14Δ cki1Δ strain (SEC14encodes the major yeast phosphatidylinositol transfer protein;CKI1encodes choline kinase of the cytidine diphosphate choline pathway of PtdCho biosynthesis). Five proteins (Sfhp) share sequence homology with phosphatidylinositol transfer protein Sec14p. Two (Sfh2p and Sfh4p), when overexpressed largely complement the otherwise essential Sec14p requirement concerning growth and secretion. In this study, we analysed the ability of Sec14 homologues to correct the defect in regulation of phospholipid biosynthesis resulting from defective or missing Sec14p. We also analysed how PtdCho turnover relates to the transcriptional regulation of phospholipid biosynthesis. The results show that (a) none of the Sec14 homologues was able to substitute for Sec14p in its regulatory aspects of phospholipid biosynthesis, (b) removal of phospholipase D activity corrected the aberrantINO1gene regulation in yeast strains with otherwise high PtdCho turnover, and (c) increased steady-state phosphatidic acid levels correlated with derepressed levels of theINO1gene. Overall, the results support the model in which high phosphatidic acid levels lead to derepression of the genes of phospholipid biosynthesis[Henry, S.A.&Patton-Vogt, J.L. (1998)Prog. Nucleic Acid Res. Mol. Biol.61, 133–179]. [ABSTRACT FROM AUTHOR]

The crystal structure of the complex between human immunodeficiency virus type 1 (HIV-1) protease and a peptidomimetic inhibitor of ethyleneamine type has been refined toRfactor of 0.178 with diffraction limit 2.5 Å. The peptidomimetic inhibitor Boc-Phe-Ψ[CH2CH2NH]-Phe-Glu-Phe-NH2 (denoted here as OE) contains the ethyleneamine replacement of the scissile peptide bond. The inhibitor lacks the hydroxyl group which is believed to mimic tetrahedral transition state of proteolytic reaction and thus is suspected to be necessary for good properties of peptidomimetic HIV-1 protease inhibitors. Despite the missing hydroxyl group the inhibition constant of OE is 1.53 nmand it remains in the nanomolar range also towards several available mutants of HIV-1 protease. The inhibitor was found in the active site of protease in an extended conformation with a unique hydrogen bond pattern different from hydroxyethylene and hydroxyethylamine inhibitors. The isostere nitrogen forms a hydrogen bond to one catalytic aspartate only. The other aspartate forms two weak hydrogen bridges to the ethylene group of the isostere. A comparison with other inhibitors of this series containing isostere hydroxyl group inRorSconfiguration shows different ways of accommodation of inhibitor in the active site. Special attention is devoted to intermolecular contacts between neighbouring dimers responsible for mutual protein adhesion and for a special conformation of Met46 and Phe53 side chains not expected for free protein in water solution. [ABSTRACT FROM AUTHOR]

The proliferation of smooth muscle cells (SMC) is a key event in the development of atherosclerosis. In addition to growth factors or cytokines, we have shown previously that n-3 polyunsaturated fatty acids (PUFAs) act in opposition to n-6 PUFAs by modulating various steps of the inflammatory process. We have investigated the molecular mechanisms by which the incorporation of the n-6 PUFA, arachidonic acid, increases the proliferation of rat SMC treated with interleukin-1β, while the n-3 PUFAs eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), elicit no mitogenic response. Incorporation of EPA or DHA into SMC, which are then activated by interleukin-1β to mimic inflammation, decreases promoter activity of the cyclin D1 gene and phosphorylation of the retinoblastoma protein. Together, our data demonstrate that n-3 effects are dependent on the Ras/Raf-1/extracellular signal regulated kinase (ERK)/mitogen-activated protein kinase pathway, and that down-regulation of the cyclin D1 promoter activity is mediated by the specific binding of the early growth response factor-1. Finally, we have shown that the incorporation of EPA and DHA also increased the concentration of caveolin-1 and caveolin-3 in caveolae, which correlated with n-3 PUFA inhibition of SMC proliferation through the mitogen-activated protein kinase pathway. We provide evidence indicating that, in contrast to n-6 PUFAs, n-3 PUFAs exert antiproliferative effects on SMC through the mitogen-activated protein kinase/ERKpathway. [ABSTRACT FROM AUTHOR]

The X-ray structure of native cellobiohydrolase IB (CBH IB) from the filamentous fungusTalaromyces emersonii, PDB 1Q9H, was solved to 2.4 Å by molecular replacement. 1Q9H is a glycoprotein that consists of a large, single domain with dimensions of≈ 60 Å × 40 Å × 50 Å and an overallβ-sandwich structure, the characteristic fold of Family 7 glycosyl hydrolases (GH7). It is the first structure of a native glycoprotein and cellulase from this thermophilic eukaryote. The long cellulose-binding tunnel seen in GH7 Cel7A fromTrichoderma reeseiis conserved in 1Q9H, as are the catalytic residues. As a result of deletions and other changes in loop regions, the binding and catalytic properties ofT. emersonii1Q9H are different. The gene (cel7) encoding CBH IB was isolated fromT. emersoniiand expressed heterologously with an N-terminal polyHis-tag, inEscherichia coli. The deduced amino acid sequence ofcel7is homologous to fungal cellobiohydrolases in GH7. The recombinant cellobiohydrolase was virtually inactive against methylumberiferyl-cellobioside and chloronitrophenyl-lactoside, but partial activity could be restored after refolding of the urea-denatured enzyme. Profiles ofcel7expression inT. emersonii, investigated by Northern blot analysis, revealed that expression is regulated at the transcriptional level. Putative regulatory element consensus sequences for cellulase transcription factors have been identified in the upstream region of thecel7genomic sequence. [ABSTRACT FROM AUTHOR]

A small anaerobically induced tomato transcription factor was isolated from a subtractive library. This factor, designated ABZ1 (anaerobic basic leucine zipper), is anaerobically induced in fruits, leaves and roots and encodes a nuclear localized protein. ABZ1 shares close structural and sequence homology with the S-family of small basic leucine zipper (bZIP) transcription factors that are implicated in stress response. Nuclear localization of ABZ1 is mediated by the basic region and occurs under normoxic conditions. ABZ1 binds to G-box-like target sites as a dimer. Binding can be abolished by heterodimerization with a truncated protein retaining the leucine zipper but lacking the DNA binding domain. The protein binds in a sequence specific manner to the CaMV 35S promoter which is down regulated when ABZ1 is coexpressed. This correlates with the anaerobic down regulation of the 35S promoter in tomato and tobacco. These results may suggest that small bZIP proteins are involved in the negative regulation of gene expression under anaerobic conditions. [ABSTRACT FROM AUTHOR]

Titin is known to interact with actin thin filaments within the I-band region of striated muscle sarcomeres. In this study, we have used a titin fragment of 800 kDa (T800) purified from striated skeletal muscle to measure the effect of this interaction on the functional properties of the actin–myosin complex. MALDI-TOF MS revealed that T800 contains the entire titin PEVK (Pro, Glu, Val, Lys-rich) domain. In the presence of tropomyosin–troponin, T800 increased the sliding velocity (both average and maximum values) of actin filaments on heavy-meromyosin (HMM)-coated surfaces and dramatically decreased the number of stationary filaments. These results were correlated with a 30% reduction in actin-activated HMM ATPase activity and with an inhibition of HMM binding to actin N-terminal residues as shown by chemical cross-linking. At the same time, T800 did not affect the efficiency of the Ca2+-controlled on/off switch, nor did it alter the overall binding energetics of HMM to actin, as revealed by cosedimentation experiments. These data are consistent with a competitive effect of PEVK domain-containing T800 on the electrostatic contacts at the actin–HMM interface. They also suggest that titin may participate in the regulation of the active tension generated by the actin–myosin complex. [ABSTRACT FROM AUTHOR]

The article presents a list of authors who contributed their research papers to the November 2004 issue of the "European Journal of Biochemistry." Some of the authors included in this list are: H. Akeboshi; C. Astier; S. Bousserouel; D.L. Catalano Dupuy; T.V. Demidkina; J. Konvalinka; B. Hove-Jensen; M. Greenwell; K. Fukuyama; M. Mahmoud; A. Nishikawa; M. Raymondjean; B. Samuelsson; G. Zuccarello; I.A. Yamskov; E. Vinogradov; R. Thompson; A. Shimonishi; L.L. Brown; P. Chaussepied; H.K. Das; F. Gabriel; K. Honma; K. Kishi; D. Pyring.

The mammalian small heat shock proteinαB-crystallin can be phosphorylated at three different sites, Ser19, Ser45 and Ser59. We compared the intracellular distribution of wild-type, nonphosphorylatable and all possible pseudophosphorylation mutants ofαB-crystallin by immunoblot and immunocytochemical analyses of stable and transiently transfected cells. We observed that pseudophosphorylation at two (especially S19D/S45D) or all three (S19D/S45D/S59D) sites induced the partial translocation ofαB-crystallin from the detergent-soluble to the detergent-insoluble fraction. Double immunofluorescence studies showed that the pseudophosphorylation mutants localized in nuclear speckles containing the splicing factor SC35. TheαB-crystallin mutants in these speckles were resistant to mild detergent treatment, and also to DNase I or RNase A digestion, indicating a stable interaction with one or more speckle proteins, not dependent on intact DNA or RNA. We further found that FBX4, an adaptor protein of the ubiquitin-protein isopeptide ligase SKP1/CUL1/F-box known to interact with pseudophosphorylatedαB-crystallin, was also recruited to SC35 speckles when cotransfected with the pseudophosphorylation mutants. Because SC35 speckles also react with an antibody againstαB-crystallin endogenously phosphorylated at Ser45, our findings suggest thatαB-crystallin has a phosphorylation-dependent role in the ubiquitination of a component of SC35 speckles. [ABSTRACT FROM AUTHOR]

The 3D-solution structure ofNeurospora crassaCu6-metallothionein (NcMT) polypeptide backbone was determined using homonuclear, multidimensional1H-NMR spectroscopy. It represents a new metallothionein (MT) fold with a protein chain where the N-terminal half is left-handed and the C-terminal half right-handedly folded around a copper(I)-sulfur cluster. As seen with other MTs, the protein lacks definable secondary structural elements; however, the polypeptide fold is unique. The metal coordinationand the cysteine spacing defines this unique fold. NcMT is only the second MT in the copper-bound form to be structurally characterized and the first containing the -CxCxxxxxCxC- motif. This motif is found in a variety of mammalian MTs and metalloregulatory proteins. Thein vitroformation of the Cu6NcMT identical to the native Cu6NcMT was dependent upon the prior formation of the Zn3NcMT and its titration with Cu(I). The enhanced sensitivity and resolution of the 800 MHz1H-NMR spectral data permitted the 3D structure determination of the polypeptide backbone without the substitution and utilization of the NMR active spin 1/2 metals such as113Cd and109Ag. These restraints have been necessary to establish specific metal to cysteine restraints in 3D structural studies on this family of proteins when using lower field, less sensitive1H-NMR spectral data. The accuracy of the structure calculated without these constraints is, however, supported by the similarities of the 800 MHz structures of theα-domain of mouse MT1 compared to the one recalculated without metal–cysteine connectivities. [ABSTRACT FROM AUTHOR]

Trehalose synthase (TreS) catalyzes the reversible interconversion of trehalose (glucosyl-α,α-1,1-glucose) and maltose (glucosyl-α1-4-glucose). TreS was purified from the cytosol ofMycobacterium smegmatisto give a single protein band on SDS gels with a molecular mass of≈ 68 kDa. However, active enzyme exhibited a molecular mass of≈ 390 kDa by gel filtration suggesting that TreS is a hexamer of six identical subunits. Based on amino acid compositions of several peptides, thetreSgene was identified in theM. smegmatisgenome sequence, and was cloned and expressed in active form inEscherichia coli.The recombinant protein was synthesized with a (His)6 tag at the amino terminus. The interconversion of trehalose and maltose by the purified TreS was studied at various concentrations of maltose or trehalose. At a maltose concentration of 0.5 mm, an equilibrium mixture containing equal amounts of trehalose and maltose (42–45% of each) was reached during an incubation of about 6 h, whereas at 2 mmmaltose, it took about 22 h to reach the same equilibrium. However, when trehalose was the substrate at either 0.5 or 2 mm, only about 30% of the trehalose was converted to maltose in≥ 12 h, indicating that maltose is the preferred substrate. These incubations also produced up to 8–10% free glucose. TheKm for maltose was≈ 10 mm, whereas for trehalose it was≈ 90 mm. Whileβ,β-trehalose, isomaltose (α1,6-glucose disaccharide), kojibiose (α1,2) or cellobiose (β1,4) were not substrates for TreS, nigerose (α1,3-glucose disaccharide) andα,β-trehalose were utilized at 20 and 15%, respectively, as compared to maltose. The enzyme has a pH optimum of about 7 and is inhibited in a competitive manner by Tris buffer.[3H]Trehalose is converted to[3H]maltose even in the presence of a 100-fold or more excess of unlabeled maltose, and[14C]maltose produces[14C]trehalose in excess unlabeled trehalose, suggesting the possibility of separate binding sites for maltose and trehalose. The catalytic mechanism may involve scission of the incoming disaccharide and transfer of a glucose to an enzyme-bound glucose, as[3H]glucose incubated with TreS and either unlabeled maltose or trehalose results in formation of[3H]disaccharide. TreS also catalyzes production of a glucosamine disaccharide from maltose and glucosamine, suggesting that this enzyme may be valuable in carbohydrate synthetic chemistry. [ABSTRACT FROM AUTHOR]

This paper reports the results obtained using the osmotic stress method applied to the purified cathodic and anodic hemoglobins (Hbs) from the catfishHoplosternum littorale, a species that displays facultative accessorial air oxygenation. We demonstrate that water potential affects the oxygen affinity ofH. littoraleHbs in the presence of an inert solute (sucrose). Oxygen affinity increases when water activity increases, indicating that water molecules stabilize the high-affinity state of the Hb. This effect is the same as that observed in tetrameric vertebrate Hbs. We show that both anodic and cathodic Hbs show conformational substrates similar to other vertebrate Hbs. For both Hbs, addition of anionic effectors, especially chloride, strongly increases the number of water molecules bound, although anodic Hb did not exhibit sensitivity to saturating levels of ATP. Accordingly, for both Hbs, we propose that the deoxy conformations coexist in at least two anion-dependent allosteric states, To and Tx, as occurs for human Hb. We found a single phosphate binding site for the cathodic Hb. [ABSTRACT FROM AUTHOR]

Presents a correction to the November 2004 issue of the "European Journal of Biochemistry."

The kinetic data on sugarcane (Saccharumspp. hybrids) sucrose synthase (SuSy, UDP-glucose:d-fructose 2-α-d-glucosyltransferase, EC 2.4.1.13) are limited. We characterized kinetically a SuSy activity partially purified from sugarcane variety N19 leaf roll tissue. Primary plot analysis and product inhibition studies showed that a compulsory order ternary complex mechanism is followed, with UDP binding first and UDP-glucose dissociating last from the enzyme. Product inhibition studies showed that UDP-glucose is a competitive inhibitor with respect to UDP and a mixed inhibitor with respect to sucrose. Fructose is a mixed inhibitor with regard to both sucrose and UDP. Kinetic constants are as follows:Km values (mm, ± SE) were, for sucrose, 35.9 ± 2.3; for UDP, 0.00191 ± 0.00019; for UDP-glucose, 0.234 ± 0.025 and for fructose, 6.49 ± 0.61.values were, for sucrose, 227 mm; for UDP, 0.086 mm; for UDP-glucose, 0.104; and for fructose, 2.23 mm. Replacing estimated kinetic parameters of SuSy in a kinetic model of sucrose accumulation with experimentally determined parameters of the partially purified isoform had significant effects on model outputs, with a 41% increase in sucrose concentration and 7.5-fold reduction in fructose the most notable. Of the metabolites included in the model, fructose concentration was most affected by changes in SuSy activity: doubling and halving of SuSy activity reduced and increased the steady-state fructose concentration by about 42 and 140%, respectively. It is concluded that different isoforms of SuSy could have significant differential effects on metabolite concentrationsin vivo, therefore impacting on metabolic regulation. [ABSTRACT FROM AUTHOR]

The conduction of protons through human Nox2 has previously been shown to be dependent upon His115. Alignment of sequences for both animal and plant Nox proteins indicated that histidines 115 and 119 are both highly conserved, while His111 was conserved among animal homologues of Nox1–4. To investigate the possible role that these histidine residues might play in the conduction of protons through Nox2, we have introduced both paired and single mutations into these histidine residues. Each construct was used to generate a CHO cell line in which the expression of the mutated Nox2 was assessed. Nox2 was expressed in each of the CHO cell lines generated, however, the level of expression of H111/115L in CHO cells was lower and that of H111L very much reduced, compared to that of wild-type Nox2. The arachidonic acid activated proton flux was absent in the CHO cell lines expressing the mutations of H111/115L, H111/119L or H115/119L, compared to that observed for wild-type Nox2. Similarly only a small efflux of protons was observed from CHO cells expressing either H119L or H111L. In all cases the expected proton flux was elicited through the addition of the protonophore, carbonyl cyanide m-chlorophenylhydrazone. Conclusions regarding the role of His111 in the conduction of protons cannot be drawn due to the reduced expression. We can, however, conclude that His119, in addition to His115, is required for the conduction of protons through Nox2. His119 has been identified as a highly conserved residue for which no function has previously been proposed. [ABSTRACT FROM AUTHOR]

Extracellular ATP triggers catecholamine secretion from PC12 cells by activating ionotropic purine receptors. Repeated stimulation by ATP leads to habituation of the secretory response. In this paper, we use amperometric detection to monitor the habituation of PC12 cells to multiple stimulations of ATP or its agonist. Cells habituate to 30 µmATP slower than they do to 300 or 600 µmATP. Modifying external Mg2+ affects the response of cells to 30 µmATP, but does not affect habituation, suggesting that habituation does not necessarily correspond to either stimulus intensity or cellular response. Mg2+ affects the initial response of PC12 cells to 2MeSATP in a manner similar to ATP. Increasing external[Mg2+] to 3.0 mm, however, eliminates habituation to 2MeSATP. This habituation can be partially restored by costimulation with 100 µmUTP. Background application of UTP increases habituation to both ATP and 2MeSATP. This suggests that ATP-sensitive metabotropic (P2Y) receptors play a role in the habituation process. Finally, although Ca2+ influx through voltage-operated calcium channels does not appear to contribute to secretion during ATP stimulation, blocking these channels with nicardipine increases habituation. This suggests a role for voltage-operated calcium channels in the habituation process. [ABSTRACT FROM AUTHOR]

Three novel CXC chemokines were identified in common carp (Cyprinus carpioL.) through homology cloning. Phylogenetic analyses show that one of the three CXC chemokines is an unambiguous orthologue ofCXCL14, whereas both others are orthologues ofCXCL12, and were namedCXCL12aandCXCL12b. Percentages of amino acid identity between each of these carp chemokines and their human and mouse orthologues are markedly higher than those reported previously for other carp CXC chemokines, suggestive of involvement in vital processes, which have allowed for relatively few structural changes. Furthermore, all three novel carp CXC chemokines are expressed during early development, in contrast to established immune CXC chemokines. In noninfected adult carp,CXCL12bandCXCL14are predominantly expressed in the brain.CXCL12ais highly expressed in kidney and anterior kidney, but its expression is still more abundant in brain than any other carp CXC chemokine. Clearly, these chemokines must play key roles in the patterning and maintenance of the (developing) vertebrate central nervous system. [ABSTRACT FROM AUTHOR]

The SUMO proteins are a class of small ubiquitin-like modifiers. SUMO is attached to a specific lysine side chain on the target protein via an isopeptide bond with its C-terminal glycine. There are at least four SUMO proteins in humans, which are involved in protein trafficking and targeting. A truncated human SUMO-2 protein that contains residues 9–93 was expressed inEscherichia coliand crystallized in two different unit cells, with dimensions of a = b = 75.25 Å, c = 29.17 Å and a = b = 74.96 Å, c = 33.23 Å, both belonging to the rhombohedral space groupR3. They diffracted X-rays to 1.6 Å and 1.2 Å resolution, respectively. The structures were determined by molecular replacement using the yeast SMT3 protein as a search model. Subsequent refinements yieldedR/ Rfree values of 0.169/0.190 and 0.119/0.185, at 1.6 Å and 1.2 Å, respectively. The peptide folding of SUMO-2 consists of a half-openβ-barrel and two flankingα-helices with secondary structural elements arranged asββαββαβ in the sequence, identical to those of ubiquitin, SMT3 and SUMO-1. Comparison of SUMO-2 with SUMO-1 showed a surface region near the C terminus with significantly different charge distributions. This may explain their distinct intracellular locations. In addition, crystal-packing analysis suggests a possible trimeric assembly of the SUMO-2 protein, of which the biological significance remains to be determined. [ABSTRACT FROM AUTHOR]

Poly(β-l-malate) (PMLA) has been reported as an unconventional, physiologically important biopolymer in plasmodia of myxomycetes, and has been proposed to function in the storage and transport of nuclear proteins by mimicking the phospho(deoxy)ribose backbone of nucleic acids. It is distributed in the cytoplasm and especially in the nuclei of these giant, multinucleate cells. We report here for the first time an increase in growth rate and a shortening of the cell cycle after the injection of purified PMLA. By comparing two strains ofPhysarum polycephalumthat differed in their production levels of PMLA, it was found that growth activation and cell cycle shortening correlated with the relative increases of PMLA levels in the cytoplasm or the nuclei. Growth rates of a low PMLA producer strain (LU897 × LU898) were increased by 40–50% while those of a high producer strain (M3CVIII) were increased by only 0–17% in comparison with controls. In both strains, shortening of the cell cycle occurred to a similar extent (7.2–9.5%), and this was associated with similar increases in nuclear PMLA levels. The effects showed saturation dependences with regard to the amount of injected PMLA. A steep rise of intracellular PMLA shortly after injection was followed by the appearance of histone H1 in the cytoplasm. The increase in growth rate, the shortening of the cell cycle duration and the appearance of H1 in the cytoplasm suggest that PMLA competes with nucleic acids in binding to proteins that control translation and/or transcription. Thus, PMLA could play an important role in the coordination of molecular pathways that are responsible for the synchronous functioning of the multinucleate plasmodium. [ABSTRACT FROM AUTHOR]

HIC1(hypermethylated in cancer 1) is a transcriptional repressor containing five Krüppel-like C2H2 zinc fingers and an N-terminal dimerization and autonomous repression domain called BTB/POZ. Here, we demonstrate that full-length HIC1 proteins are modified bothin vivoandin vitrowith O-linkedN-acetylglucosamine (O-GlcNAc). This is a highly dynamic glycosylation found within the cytosolic and the nuclear compartments of eukaryotes. Analysis of[3H]Gal-labeled tryptic peptides indicates that HIC1 has three major sites forO-GlcNAc glycosylation. Using C-terminal deletion mutants, we have shown thatO-GlcNAc modification of HIC1 proteins occurred preferentially in the DNA-binding domain. Nonglycosylated and glycosylated forms of full-length HIC1 proteins separated by wheat germ agglutinin affinity purification, displayed the same specific DNA-binding activity in electrophoretic mobility shift assays proving that theO-GlcNAc modification is not directly implicated in the specific DNA recognition of HIC1. Intriguingly, N-terminal truncated forms corresponding to BTB-POZ-deleted proteins exhibited a strikingly differential activity, as the glycosylated truncated forms are unable to bind DNA whereas the unglycosylated ones do. Electrophoretic mobility shift assays performed with separated pools of glycosylated and unglycosylated forms of a construct exhibiting only the DNA-binding domain and the C-terminal tail of HIC1 (residues 399–714) and supershift experiments with wheat germ agglutinin or RL-2, an antibody raised againstO-GlcNAc residues, fully corroborated these results. Interestingly, these truncated proteins areO-GlcNAc modified in their C-terminal tail (residues 670–711) and not in the DNA-binding domain, as for the full-length proteins. Thus, theO-GlcNAc modification of HIC1 does not affect its specific DNA-binding activity and is highly sensitive to conformational effects, notably its dimerization through the BTB/POZ domain. [ABSTRACT FROM AUTHOR]

The tumor suppressor protein, p53, selectively binds to supercoiled (sc) DNA lacking the specific p53 consensus binding sequence (p53CON). Using p53 deletion mutants, we have previously shown that the p53 C-terminal DNA-binding site (CTDBS) is critical for this binding. Here we studied supercoil-selective binding of bacterially expressed full-length p53 using modulation of activity of the p53 DNA-binding domains by oxidation of cysteine residues (to preclude binding within the p53 core domain) and/or by antibodies mapping to epitopes at the protein C-terminus (to block binding within the CTDBS). In the absence of antibody, reduced p53 preferentially bound scDNA lacking p53CON in the presence of 3 kb linear plasmid DNAs or 20 mer oligonucleotides, both containing and lacking the p53CON. Blocking the CTDBS with antibody caused reduced p53 to bind equally to sc and linear or relaxed circular DNA lacking p53CON, but with a high preference for the p53CON. The same immune complex of oxidized p53 failed to bind DNA, while oxidized p53 in the absence of antibody restored selective scDNA binding. Antibodies mapping outside the CTDBS did not prevent p53 supercoil-selective (SCS) binding. These data indicate that the CTDBS is primarily responsible for p53 SCS binding. In the absence of the SCS binding, p53 binds sc or linear (relaxed) DNA via the p53 core domain and exhibits strong sequence-specific binding. Our results support a hypothesis that alterations to DNA topology may be a component of the complex cellular regulatory mechanisms that control the switch between latent and active p53 following cellular stress. [ABSTRACT FROM AUTHOR]

In view of the increasing number of reported concentration oscillations in living cells, methods are needed that can identify the causes of these oscillations. These causes always derive from the influences that concentrations have on reaction rates. The influences reach over many molecular reaction steps and are defined by the detailed molecular topology of the network. So-called‘autoinfluence paths’, which quantify the influence of one molecular species upon itself through a particular path through the network, can have positive or negative values. The former bring a tendency towards instability. In this molecular context a new graphical approach is presented that enables the classification of network topologies into oscillophoretic and nonoscillophoretic, i.e. into ones that can and ones that cannot induce concentration oscillations. The network topologies are formulated in terms of a set of uni-molecular and bi-molecular reactions, organized into branched cycles of directed reactions, and presented as graphs. Subgraphs of the network topologies are then classified as negative ones (which can) and positive ones (which cannot) give rise to oscillations. A subgraph is oscillophoretic (negative) when it contains more positive than negative autoinfluence paths. Whether the former generates oscillations depends on the values of the other subgraphs, which again depend on the kinetic parameters. An example shows how this can be established. By following the rules of our new approach, various oscillatory kinetic models can be constructed and analyzed, starting from the classified simplest topologies and then working towards desirable complications. Realistic biochemical examples are analyzed with the new method, illustrating two new main classes of oscillophore topologies. [ABSTRACT FROM AUTHOR]

The structural stability of the large cytoplasmic domain (H4-H5 loop) of mouseα1 subunit of Na+/K+ ATPase (L354–I777), the number and the location of its binding sites for 2′-3′-O-(trinitrophenyl) adenosine 5′-triphosphate (TNP-ATP) andp-nitrophenylphosphate (pNPP) were investigated. C- and N-terminal shortening revealed that neither part of the phosphorylation (P)-domain are necessary for TNP-ATP binding. There is no indication of a second ATP site on the P-domain of the isolated loop, even though others reported previously of its existence by TNP-N3ADP affinity labeling of the full enzyme. Fluorescein isothiocyanate (FITC)-anisotropy measurements reveal a considerable stability of the nucleotide (N)-domain suggesting that it may not undergo a substantial conformational change upon ATP binding. The FITC modified loop showed only slightly diminished phosphatase activity, most likely due to a pNPP site on the N-domain around N398 whose mutation to D reduced the phosphatase activity by 50%. The amino acids forming this pNPP site (M384, L414, W411, S400, S408) are conserved in theα1−4 isoforms of Na+/K+ ATPase, whereas N398 is only conserved in the vertebrates'α1 subunit. The phosphatase activity of the isolated H4-H5 loop was neither inhibited by ATP, nor affected by mutation of D369, which is phosphorylated in native Na+/K+ ATPase. [ABSTRACT FROM AUTHOR]

Duchenne muscular dystrophy represents one of the most common hereditary diseases. Abnormal ion handling is believed to render dystrophin-deficient muscle fibres more susceptible to necrosis. Although a reduced Ca2+ buffering capacity has been shown to exist in the dystrophic sarcoplasmic reticulum, surprisingly no changes in the abundance of the main luminal Ca2+ reservoir protein calsequestrin have been observed in microsomal preparations. To address this unexpected finding and eliminate potential technical artefacts of subcellular fractionation protocols, we employed a comparative subproteomics approach with total mouse skeletal muscle extracts. Immunoblotting, mass spectrometry and labelling of the entire muscle protein complement with the cationic carbocyanine dye‘Stains-All’ was performed in order to evaluate the fate of major Ca2+-binding proteins in dystrophin-deficient skeletal muscle fibres. In contrast to a relatively comparable expression pattern of the main protein population in normal vs. dystrophic fibres, our analysis showed that the expression of key Ca2+-binding proteins of the luminal sarcoplasmic reticulum is drastically reduced. This included the main terminal cisternae constituent, calsequestrin, and the previously implicated Ca2+-shuttle element, sarcalumenin. In contrast, the‘Stains-All’-positive protein spot, representing the cytosolic Ca2+-binding component, calmodulin, was not changed in dystrophin-deficient fibres. The reduced 2D‘Stains-All’ pattern of luminal Ca2+-binding proteins in mdx preparations supports the calcium hypothesis of muscular dystrophy. The previously described impaired Ca2+ buffering capacity of the dystrophic sarcoplasmic reticulum is probably caused by a reduction in luminal Ca2+-binding proteins, including calsequestrin. [ABSTRACT FROM AUTHOR]

Human cytochrome P450 3A4 forms a series of minor testosterone hydroxylation products in addition to 6β-hydroxytestosterone, the major product. One of these, formed at the next highest rate after the 6β- and 2β-hydroxy products, was identified as 1β-hydroxytestosterone. This product was characterized from a mixture of testosterone oxidation products using an HPLC-solid phase extraction-cryoprobe NMR/time-of-flight mass spectrometry system, with an estimated total of≈ 6 µg of this product. Mass spectrometry established the formula as C19H29O3 (MH+ 305.2080). The 1-position of the added hydroxyl group was established by correlated spectroscopy and heteronuclear spin quantum correlation experiments, and theβ-stereochemistry of the added hydroxyl group was assigned with a nuclear Overhauser correlated spectroscopy experiment (1α-H). Of several human P450s examined, only P450 3A4 formed this product. The product was also formed in human liver microsomes. [ABSTRACT FROM AUTHOR]

In this work, evidence for the presence of ferritins in plant mitochondria is supplied. Mitochondria were isolated from etiolated pea stems and Arabidopsis thaliana cell cultures. The proteinswere separated by SDS/PAGE.Aprotein, with an apparent molecular mass of approximately 25-26 kDa (corresponding to that of ferritin), was cross-reacted with an antibody raised against pea seed ferritin. The mitochondrial ferritin from pea stems was also purified by immunoprecipitation. The purified protein was analyzed by MALDITOF mass spectrometry and the results of both mass finger print and peptide fragmentation by post source decay assign the polypeptide sequence to the pea ferritin (P &<0.05). The mitochondrial localization of ferritin was also confirmed by immunocytochemistry experiments on isolated mitochondria and cross-sections of pea stem cells. The possible role of ferritin in oxidative stress of plant mitochondria is discussed. [ABSTRACT FROM AUTHOR]

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 sameincubation 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 phospholipaseA1 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 deg;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. [ABSTRACT FROM AUTHOR]

In this report, we have focused our attention on identifying intracellular mammalian proteins that bind S100A12 in a Ca2+-dependent manner. Using S100A12 affinity chromatography, we have identified cytosolic NADP+-dependent isocitrate dehydrogenase (IDH), fructose-1,6-bisphosphate aldolase A (aldolase), glyceraldehyde-3-phosphate dehydrogenese (GAPDH), annexin V, S100A9, and S100A12 itself as S100A12-binding proteins. Immunoprecipitation experiments indicated the formation of stable complexes between S100A12 and IDH, aldolase, GAPDH, annexin V and S100A9 in vivo. Surface plasmon resonance analysis showed that the binding to S100A12, of S100A12, S100A9 and annexinV,was strictlyCa2+-dependent,whereas that of GAPDH and IDH was only weakly Ca2+-dependent. To localize the site of S100A12 interaction, we examined the binding of a series of C-terminal truncation mutants to the S100A12-immobilized sensor chip. The results indicated that the S100A12-binding site on S100A12 itself is located at the C-terminus (residues 87-92). However, cross-linking experiments with the truncation mutants indicated that residues 87-92 were not essential for S100A12 dimerization. Thus, the interaction between S100A12 and S100A9 or immobilized S100A12 should not be viewed as a typical S100 homo- or heterodimerization model. Ca2+-dependent affinity chromatography revealed that C-terminal residues 75-92 are not necessary for the interaction of S100A12 with IDH, aldolase, GAPDH and annexin V. To analyze the functional properties of S100A12, we studied its action in protein folding reactions in vitro. The thermal aggregation of IDH or GAPDH was facilitated by S100A12 in the absence of Ca2+, whereas in the presence of Ca2+ the protein suppressed the aggregation of aldolase to less than 50%. These results suggest that S100A12 may have a chaperone/antichaperone- like function which is Ca2+-dependent. [ABSTRACT FROM AUTHOR]

Cyclophilins facilitate the peptidyl-prolyl isomerization of a trans-isomer to a cis-isomer in the refolding process of unfolded proteins to recover the natural folding state with cis-proline conformation. To date, only short peptideswith a cis-form proline have been observed in complexes of human and Escherichia coli proteins of cyclophilin A, which is present in cytoplasm. The crystal structures analyzed in this study show two complexes in which peptides having a transform proline, i.e. succinyl-Ala-trans-Pro-Ala-p-nitroanilide and acetyl-Ala-Ala-trans-Pro-Ala-amidomethylcoumarin, are bound on a K163T mutant of Escherichia coli cyclophilin B, the preprotein of which has a signal sequence. Comparison with cis-form peptides bound to cyclophilin A reveals that in any case the proline ring is inserted into the hydrophobic pocket and a hydrogen bond between CO of Pro andNeta2 of Arg is formed to fix the peptide. On the other hand, in the cis-isomer, the formation of two hydrogen bonds of NH and CO of Ala preceding Pro with the protein fixes the peptide, whereas in the trans-isomer formation of a hydrogen bond between CO preceding Ala-Pro and His47 Nepsiv2 via a mediating water molecule allows the large distortion in the orientation of Ala of Ala-Pro. Although loss of double bond character of the amide bond of Ala-Pro is essential to the isomerization pathway occurring by rotating around its bond, these peptides have forms impossible to undergo proton transfer from the guanidyl group of Arg to the prolyl N atom, which induces loss of double bond character. Guanylate cyclase-activating proteins (GCAPs) are neuronal calcium sensors that activate membrane bound guanylate cyclases (EC 4.6.1.2.) of vertebrate photoreceptor cells when cytoplasmic Ca2+ decreases during illumination. GCAPs contain four EF-hand Ca2+-binding motifs, but the first EF-hand is nonfunctional. It was concluded that for GCAP-2, the loss of Ca2+-binding ability of EF-hand 1 resulted in a region that is crucial for targeting guanylate cyclase [Ermilov, A.N.,Olshevskaya, E.V.& Dizhoor,A.M. (2001) J. Biol. Chem. 276, 48143-48148]. In this study we tested the consequences of mutations in EF-hand 1 of GCAP-1 with respect to Ca2+ binding, Ca2+-induced conformational changes and target activation. When the nonfunctional first EF-hand in GCAP-1 is replaced by a functional EF-hand the chimeric mutant CaM-GCAP-1 bound four Ca2+ and showed similar Ca2+-dependent changes in tryptophan fluorescence as the wild-type. CaM- GCAP-1 neither activated nor interacted with guanylate cyclase. Size exclusion chromatography revealed that the mutant tended to form inactive dimers instead of active monomers like the wild-type. Critical amino acids in EFhand 1 ofGCAP-1 are cysteine at position 29 and proline at position 30, as changing these to glycine was sufficient to cause loss of target activation without a loss of Ca2+- induced conformational changes. The latter mutation also promoted dimerization of the protein. Our results show that EF-hand 1 in wild-typeGCAP-1 is critical for providing the correct conformation for target activation. [ABSTRACT FROM AUTHOR]

Malate dehydrogenase (MDH) may be important in carbohydrate and energy metabolism in malarial parasites. The cDNA corresponding to the MDH gene, identified on chromosome 6 of the Plasmodium falciparum genome, was amplified by RT-PCR, cloned and overexpressed in Escherichia coli. The recombinant Pf MDH was purified to homogeneity and biochemically characterized as an NAD+(H)-specific MDH, which catalysed reversible interconversion of malate to oxaloacetate. Pf MDH could not use NADP/NADPH as a cofactor, but used acetylpyridine adenine dinucleoide, an analogue of NAD. The enzyme exhibited strict substrate and cofactor specificity. The highest levels of Pf MDH transcripts were detected in trophozoites while the Pf MDH protein level remained high in trophozoites as well as schizonts. A highly refined model of Pf MDH revealed distinct structural characteristics of substrate and cofactor binding sites and important amino acid residues lining these pockets. The active site amino acid residues involved in substrate binding were conserved in Pf MDH but the N-terminal glycine motif, which is involved in nucleotide binding, was similar to the GXGXXG signature sequence found in Pf LDH and also in α-proteobacterial MDHs. Oxamic acid did not inhibit Pf MDH, while gossypol, which interacts at the nucleotide binding site of oxidoreductases and shows antimalarial activity, inhibited Pf MDH also. Treatment of a synchronized culture of P. falciparum trophozoites with gossypol caused induction in expression of Pf MDH, while expression of Pf LDH was reduced and expression of malate:quinone oxidoreductase remained unchanged. Pf MDH may complement Pf LDH function of NAD/NADH coupling in malaria parasites. Thus, dual inhibitors of Pf MDH and Pf LDH may be required to target this pathway and to develop potential new antimalarial drugs. [ABSTRACT FROM AUTHOR]

The His332 residue of the D1 protein has been identified as the likely ligand of the catalytic Mn ions in the water oxidizing complex (Ferreira, K.N., Iverson, T.M., Maghlaoui, K., Barber, J. & Iwata, S. (2004) Science 303, 1831–1838). However, its function has not been fully clarified. Here we used thermoluminescence and flash-induced chlorophyll fluorescence measurements to characterize the effect of the D1-H333E, D1-H332D and D1-H332S mutations on the electron transport of Photosystem II in intact cells of the cyanobacterium Synechocystis 6803. Although the mutants are not photoautotrophic they all show flash-induced thermoluminescence and chlorophyll fluorescence, which originate from the S2QA– and S2QB– recombinations demonstrating that charge stabilization takes place in the water oxidizing complex. However, the conversion of S2 to higher S states is inhibited and the energetic stability of the S2QA– charge pair is increased by 75, 50 and 7 mV in the D1-H332D, D1-H332E and D1-H332S mutants, respectively. This is most probably caused by a decrease of Em(S2/S1). Concomitantly, the rate of electron donation from Mn to Tyr-Z&bdot; during the S1 to S2 transition is slowed down, relative to the wild type, 350- and 60-fold in the D1-H332E and D1-H332D mutants, respectively, but remains essentially unaffected in D1-H332S. A further effect of the D1-H332E and D1-H332D mutations is the retardation of the QA to QB electron transfer step as an indirect consequence of the donor side modification. Our data show that although the His residue in the D1-332 position can be substituted by other metal binding residues for binding photo-oxidisable Mn it is required for controlling the functional redox energetics of the Mn cluster. [ABSTRACT FROM AUTHOR]

We have used footprinting techniques on a wide range of natural and synthetic footprinting substrates to examine the sequence-selective interaction of the bis-daunorubicin antibiotic WP631 with DNA. The ligand produces clear DNase I footprints that are very different from those seen with other anthracycline antibiotics such as daunorubicin and nogalamycin. Footprints are found in a diverse range of sequences, many of which are rich in GT (AC) or GA (TC) residues. As expected, the ligand binds well to the sequences CGTACG and CGATCG, but clear footprints are also found at hexanucleotide sequences such GCATGC and GCTAGC. The various footprints do not contain any particular unique di-, tri- or tetranucleotide sequences, but are frequently contain the sequence (G/C)(A/T)(A/T)(G/C). All sequences with this composition are protected by the ligand, though it can also bind to some sites that differ from this consensus by one base pair. [ABSTRACT FROM AUTHOR]

This work investigated the kinetics of catabolism and the catabolic fate of the newly synthesized 35S-labelled proteoglycans present in explant cultures of tendon. Tissue from the proximal region of bovine deep flexor tendon was incubated with [35S]sulfate for 6 h and then placed in explant cultures for periods of up to 15 days. The amount of radiolabel associated with proteoglycans and free [35S]sulfate lost to the medium and retained in the matrix was determined for each day in culture. It was shown that the rate of catabolism of radiolabelled small proteoglycans (decorin and biglycan) was significantly slower (T½ > 20 days) compared with the radiolabelled large proteoglycans (aggrecan and versican) that were rapidly lost from the tissue (T½ ≈ 2 days). Both the small and large newly synthesized proteoglycans were lost from the matrix with either intact or proteolytically modified core proteins. When explant cultures of tendon were maintained either at 4 °C or in the presence of the lysosomotrophic agent ammonium chloride, inhibition of the cellular catabolic pathway for small proteoglycans was demonstrated indicating the involvement of cellular activity and lysosomes in the catabolism of small proteoglycans. It was estimated from these studies that approximately 60% of the radiolabelled small proteoglycans that were lost from the tissue were degraded by the intracellular pathway present in tendon cells. This work shows that the pathways of catabolism for large aggregating and small leucine-rich proteoglycans are different in tendon and this may reflect the roles that these two populations of proteoglycans play in the maintenance of the extracellular matrix of tendon. [ABSTRACT FROM AUTHOR]

Molecular phylogeny among catalase–peroxidases, cytochrome c peroxidases, and ascorbate peroxidases was analysed. Sixty representative sequences covering all known subgroups of class I of the superfamily of bacterial, fungal, and plant heme peroxidases were selected. Each sequence analysed contained the typical peroxidase motifs evolved to bind effectively the prosthetic heme group, enabling peroxidatic activity. The N-terminal and C-terminal domains of catalase–peroxidases matching the ancestral tandem gene duplication event were treated separately in the phylogenetic analysis to reveal their specific evolutionary history. The inferred unrooted phylogenetic tree obtained by three different methods revealed the existence of four clearly separated clades (C-terminal and N-terminal domains of catalase–peroxidases, ascorbate peroxidases, and cytochrome c peroxidases) which were segregated early in the evolution of this superfamily. From the results, it is obvious that the duplication event in the gene for catalase–peroxidase occurred in the later phase of evolution, in which the individual specificities of the peroxidase families distinguished were already formed. Evidence is presented that class I of the heme peroxidase superfamily is spread among prokaryotes and eukaryotes, obeying the birth-and-death process of multigene family evolution. [ABSTRACT FROM AUTHOR]

Using 0.4 m imidazole citrate buffer (pH 7.5) containing 0.1 m m l-cysteine, homodimeric starch phosphorylase from Corynebacterium calluane ( CcStP) was dissociated into native-like folded subunits concomitant with release of pyridoxal 5′-phosphate and loss of activity. The inactivation rate of CcStP under resolution conditions at 30 °C was, respectively, four- and threefold reduced in two mutants, Arg234→Ala and Arg242→Ala, previously shown to cause thermostabilization of CcStP [Griessler, R., Schwarz, A., Mucha, J. & Nidetzky, B. (2003) Eur. J. Biochem. 270, 2126–2136]. The proportion of original enzyme activity restored upon the reconstitution of wild-type and mutant apo-phosphorylases with pyridoxal 5′-phosphate was increased up to 4.5-fold by added phosphate. The effect on recovery of activity displayed a saturatable dependence on the phosphate concentration and results from interactions with the oxyanion that are specific to the quarternary state. Arg234→Ala and Arg242→Ala mutants showed, respectively, eight- and > 20-fold decreased apparent affinities for phosphate ( Kapp), compared to the wild-type ( Kapp≈ 6 m m). When reconstituted next to each other in solution, apo-protomers of CcStP and Escherichia coli maltodextrin phosphorylase did not detectably associate to hybrid dimers, indicating that structural complementarity among the different subunits was lacking. Pyridoxal-reconstituted CcStP was inactive but ≈ 60% and 5% of wild-type activity could be rescued at pH 7.5 by phosphate (3 m m) and phosphite (5 m m), respectively. pH effects on catalytic rates were different for the native enzyme and pyridoxal-phosphorylase bound to phosphate and could reflect the differences in p Ka values for the cofactor 5′-phosphate and the exogenous oxyanion. [ABSTRACT FROM AUTHOR]

HAND2/dHAND is a basic helix-loop-helix transcription factor expressed in the heart and neural crest derivatives during embryogenesis. Although dHAND is essential for branchial arch, cardiovascular and limb development, its target genes have not been identified. The regulatory mechanisms of dHAND function also remain relatively unknown. Here we report that Akt/PKB, a serine/threonine protein kinase involved in cell survival, growth and differentiation, phosphorylates dHAND and inhibits dHAND-mediated transcription. AU5-dHAND expressed in 293T cells became phosphorylated, possibly at its Akt phosphorylation motif, in the absence of kinase inhibitors, whereas the phosphatidylinositol 3-kinase inhibitor wortmannin and the Akt inhibitor NL-71-101, but not the p70 S6 kinase inhibitor rapamycin, significantly reduced dHAND phosphorylation. Coexpression of HA-Akt augmented dHAND phosphorylation at multiple serine and threonine residues mainly located in the bHLH domain and, as a result, decreased the transcriptional activity of dHAND. Consistently, alanine mutation mimicking the nonphosphorylation state abolished the inhibitory effect of Akt on dHAND, whereas aspartate mutation mimicking the phosphorylation state resulted in a loss of dHAND transcriptional activity. These changes in dHAND transcriptional activity were in parallel with changes in the DNA binding activity rather than in dimerization activity. These results suggest that Akt-mediated signaling may regulate dHAND transcriptional activity through the modulation of its DNA binding activity during embryogenesis. [ABSTRACT FROM AUTHOR]

The control properties of biochemical pathways can be described by control coefficients and elasticities, as defined in the framework of metabolic control analysis. The determination of these parameters using the traditional metabolic control analysis relationships is, however, limited by experimental difficulties (e.g. realizing and measuring small changes in biological systems) and lack of appropriate mathematical procedures (e.g. when the more practical large changes are made). In this paper, the recently developed lin-log approach is proposed to avoid the above-mentioned problems and is applied to estimate control parameters from measurements obtained in steady state experiments. The lin-log approach employs approximative linear-logarithmic kinetics parameterized by elasticities and provides analytical solutions for fluxes and metabolite concentrations when large changes are made. Published flux and metabolite concentration data are used, obtained from a reconstructed section of glycolysis converting 3-phosphoglycerate to pyruvate [Giersch, C. (1995) Eur. J. Biochem. 227, 194–201]. With the lin-log approach, all data from different experiments can be combined to give realistic elasticity and flux control coefficient estimates by linear regression. Despite the large changes, a good agreement of fluxes and metabolite concentrations is obtained between the measured and calculated values according to the lin-log model. Furthermore, it is shown that the lin-log approach allows a rigorous statistical evaluation to identify the optimal reference state and the optimal model structure assumption. In conclusion, the lin-log approach addresses practical problems encountered in the traditional metabolic control analysis-based methods by introducing suitable nonlinear kinetics, thus providing a novel framework with improved procedures for the estimation of elasticities and control parameters from large perturbation experiments. [ABSTRACT FROM AUTHOR]

In Klebsiella pneumoniae, the nif specific transcriptional activator NifA is inhibited by NifL in response to molecular oxygen and ammonium. Here, we demonstrate complex formation between NifL and NifA (approximately 1 : 1 ratio), when synthesized in the presence of oxygen and/or ammonium. Under simultaneous oxygen- and nitrogen-limitation, significant but fewer NifL–NifA complexes (approximately 1%) were formed in the cytoplasm as a majority of NifL was sequestered to the cytoplasmic membrane. These findings indicate that inhibition of NifA in the presence of oxygen and/or ammonium occurs via direct NifL interaction and formation of those inhibitory NifL–NifA complexes appears to be directly and exclusively dependent on the localization of NifL in the cytoplasm. We further observed evidence that the nitrogen sensory protein GlnK forms a trimeric complex with NifL and NifA under nitrogen limitation. Binding of GlnK to NifL–NifA was specific; however the amount of GlnK within these complexes was small. Finally, two lines of evidence were obtained that under anaerobic conditions but in the presence of ammonium additional NtrC-independent GlnK synthesis inhibited the formation of stable inhibitory NifL–NifA complexes. Thus, we propose that the NifL–NifA–GlnK complex reflects a transitional structure and hypothesize that under nitrogen-limitation, GlnK interacts with the inhibitory NifL–NifA complex, resulting in its dissociation. [ABSTRACT FROM AUTHOR]