bluuebunny/crossref_metadata_2025_split huggingface dataset:
Evolutionary relationships among pyridoxal‐5′‐phosphate‐dependent enzymes
Authors: ALEXANDER Frederick W. , SANDMEIER Erika , MEHTA Perdeep K. , CHRISTEN Philipp 
Published: March 2005

Pyridoxal‐5′‐phosphate‐dependent enzymes catalyze manifold reactions in the metabolism of amino acids. A comprehensive comparison of amino acid sequences has shown that most of these enzymes can be assigned to one of three different families of homologous proteins. The sequences of the enzymes of each family were aligned and their homology confirmed by profile analysis. Scrutiny of the reactions catalyzed by the enzymes showed that their affiliation with one of the three structurally defined families correlates in most cases with their regio‐specificity.In the largest family, the covalency changes of the substrate occur at the same carbon atom that carries the amino group forming the imine linkage with the coenzyme. This family was thus named α family. It comprises glycine hydroxymethyltransferase, glycine C‐acetyltransferase, 5‐aminolevulinate synthase, 8‐amino‐7‐oxononanoate synthase, all aminotransferases (with the possible exception of subgroup III), a number of other enzymes relatively closely related with the aminotransferases and very likely a certain group of amino acid decarboxylases as well as tryptophanase and tyrosine phenol‐lyase which, however, catalyze β‐elimination reactions. The β family includes L‐ and D‐serine dehydratase, threonine dehydratase, the β subunit of tryptophan synthase, threonine synthase and cysteine synthase. These enzymes catalyze β‐replacement or β‐elimination reactions. The γ family incorporates O‐succinylhomoserine (thiol)‐lyase, O‐acetylhomoserine (thiol)‐lyase, and cystathionine γ‐lyase, which catalyze γ‐replacement or γ‐elimination reactions, as well as cystathionine β‐lyase.The α and γ family might be distantly related with one another, but are clearly not homologous with the β family. Apparently, the primordial pyridoxal‐5′‐phosphate‐dependent enzymes were regio‐specific catalysts, which first specialized for reaction specificity and then for substrate specificity. The following pyridoxal‐5′‐phosphate‐dependent enzymes seem to be unrelated with the α, β or γ family by the criterion of profile analysis: alanine racemase, selenocysteine synthase, and many amino acid decarboxylases. These enzymes may represent yet other families of B6 enzymes.
Aluminium fluoride and magnesium, activators of heterotrimeric GTP‐binding proteins, affect high‐affinity binding of the fungal toxin fusicoccin to the fusicoccin‐binding protein in oat root plasma membranes
Authors: DE BOER Albertus H. , VAN DER MOLEN Gineke W. , PRINS Hidde B. A. , KORTHOUT Henrie A. A. J. , VAN DER HOEVEN Paulus C. J. 
Published: March 2005

The fusicoccin‐binding protein was solubilised from purified oat root plasma membranes. The solubilised protein retained full binding activity, provided that protease inhibitors were included. Sodium fluoride reduced the high‐affinity [3H]fusicoccin binding to almost zero in a concentration‐dependent way, with an optimum at approximately 20 mM sodium fluoride. The presence of magnesium (> 100 μM) was required for the inhibitory action of fluoride, whereas addition of low amounts of aluminium (25 μM) shifted the fluoride optimum to lower concentrations. Fluoride changes the biochemical properties of the binding protein in a reversible manner, because the inhibition was both prevented and reversed by 1 M ammonium sulphate.The combined effects of aluminium, fluoride and magnesium are reminiscent of the action of activated GTP‐binding proteins. Since no functional assay for GTP‐binding‐protein activity in plants is available yet, GTP‐binding‐protein activation by fluoride and magnesium was deduced from competition with binding of [γ‐35S]GTP[S] to purified plasma membranes. Indeed, fluoride (20 mM) completely blocked the specific binding of [γ‐35S]GTP[S].It is concluded that the inhibitory effect of fluoride upon the binding of fusicoccin is indirect and mediated through activated GTP‐binding proteins. A hypothesis on the mechanism of fusicoccin action is presented wherein the fusicoccin‐binding protein is one component of a signal‐transduction chain, two or more steps downstream of a heterotrimeric GTP‐binding protein.
The metabolism of 2‐fluoro‐2‐deoxy‐d‐glucose in human chondrocytes and its incorporation into keratan sulfate proteoglycans
Authors: FEDDERS Gönna , KOCK Rüdiger , VAN DE LEUR Eddy , GREILING Helmut 
Published: March 2005

The incorporation of 2‐fluoro‐2‐deoxy‐d‐[14C]glucose in proteoglycans was investigated in a cell culture system, where human articular chondrocytes were cultured in high‐cell‐density thin‐layer soft agarose. The proteoglycans were solubilized from the culture medium and the cell layer fraction by extracting with a guanidine hydrochloride buffer and purified by an ion‐exchange‐chromatography (DEAE‐Sepharose CL‐6B). With enzymic decomposition experiments concerning the glycosaminoglycan side‐chains it could be shown that 65–69% were digestible by keratanase, whereas 21–29% of the 14C‐labeled proteoglycans were digested with chondroitinase AC/ABC. The main constituent of the 2‐fluoro‐2‐deoxy‐d‐[14C]glucose‐metabolites present in the glycosaminoglycan side chains of the proteoglycans was 2‐fluoro‐2‐deoxy‐d‐[14C]galactose. Therefore, 2‐fluoro‐2‐deoxy‐d‐glucose was preferentially incorporated into keratan sulfate.We investigated the effect of non‐radioactive 2‐fluoro‐2‐deoxy‐d‐glucose on UDP‐sugar and proteoglycan biosynthesis after incubation periods of 1–30 h. A high 2‐fluoro‐2‐deoxy‐d‐glucose concentration in the culture medium did not influence the pool size of UDP‐N‐acetylhexosamines, but UDP‐d‐glucose, UDP‐d‐galactose, UDP‐d‐glucuronic acid, UDP‐2‐fluoro‐2‐deoxy‐d‐glucose, UDP‐2‐fluoro‐2‐deoxy‐d‐galactose and UDP‐2‐fluoro‐2‐deoxy‐d‐glucuronic acid accumulated in the chondrocytes time dependently. In a pulse/chase experiment the retarded synthesis of fluorinated UDP‐sugars was proved. The half‐lives (t1/2) for UDP‐2‐fluoro‐2‐deoxy‐d‐glucose and UDP‐2‐fluoro‐2‐deoxy‐d‐galactose were about 7.7 h and 13.3 h, respectively. UDP‐2‐fluoro‐2‐deoxy‐d‐glucuronic acid could be found with delay. The incubation with 2‐fluoro‐2‐deoxy‐d‐glucose and [14C]glucosamine resulted in a decreased radioactive labelling of chondroitin sulfate and keratan sulfate.
The requirement of matrix ATP for the import of precursor proteins into the mitochondrial matrix and intermembrane space
Authors: STUART Rosemary A. , GRUHLER Albrecht , van der KLEI Ida , GUIARD Bernard , KOLL Hans , NEUPERT Walter 
Published: March 2005

The role of ATP in the matrix for the import of precursor proteins into the various mitochondrial subcompartments was investigated by studying protein translocation at experimentally defined ATP levels. Proteins targeted to the matrix were neither imported or processed when matrix ATP was depleted. Import and processing of precytochrome b2, (pb2), a precursor carrying a bipartite presequence, into the intermembrane space was also strongly dependent on matrix ATP. Preproteins, consisting of 220 or more residues of pb2 fused to dihydrofolate reductase, showed the same requirement for matrix ATP, whereas the import of shorter fusion proteins (up to 167 residues of pb2) was largely independent of matrix ATP. For those intermembrane‐space‐targeted proteins that did need matrix ATP, the dependence could be relieved either by unfolding these proteins prior to import or by introducing a deletion into the mature portion of the protein thereby impairing the tight folding of the cytochrome b5 domain.These results suggest the following: (a) The import of matrix‐targeted preproteins, in addition to a membrane potential ΔΨ, requires matrix ATP [most likely to facilitate reversible binding of mitochondrial heat‐shock protein 70 (mt‐Hsp70) to incoming precursors], for two steps, securing the presequence on the matrix side of the inner membrane and for the completion of translocation; (b) in the case of intermembrane‐space‐targeted precursors with bipartite signals, the function of ATP/mt‐Hsp70 is not obligatory, as components of the intermembrane‐space‐sorting pathway may substitute for ATP/mt‐Hsp70 function (however, if a tightly folded domain is present in the precursor, ATP/mt‐Hsp70 is indispensable); (c) unfolding on the mitochondrial surface of tightly folded segments of preproteins is facilitated by matrix‐ATP/mt‐Hsp70.
Molecular organization at the glycoprotein‐complex‐binding site of dystrophin
Authors: SUZUKI Atsushi , YOSHIDA Mikiharu , HAYASHI Kensuke , MIZUNO Yuji , HAGIWARA Yasuko , OZAWA Eijiro 
Published: March 2005

Direct interaction between the C‐terminal portion of dystrophin and dystrophin‐associated proteins was investigated. The binding of dystrophin to each protein was reconstituted by overlaying bacterially expressed dystrophin fusion proteins onto the blot membranes to which dystrophin‐associated proteins were transferred after separation by SDS/PAGE with the following results. (a) Among the components of the glycoprotein complex which links dystrophin to the sarcolemma, a 43‐kDa dystrophin‐associated glycoprotein binds directly to dystrophin. Although at least one of the binding sites of this protein resides within the cysteine‐rich domain of dystrophin, a contribution of additional amino acid residues within the first half of the C‐terminal domain was also suggested for more secure binding. (b) Two other proteins also directly bind to dystrophin. Their binding sites are suggested to reside within the last half of the C‐terminal domain which is alternatively spliced depending on the tissue type.Previously, based on the enzyme digestion experiments, we showed that the binding site for the glycoprotein complex on dystrophin is present within the cysteine‐rich domain and the first half of the C‐terminal domain [Suzuki, A., Yoshida, M., Yamamoto, H. & Ozawa, E. (1992) FEBS Lett. 308, 154–160]. Here, we have extended this work and found that the region which is involved in interaction with the complex is widely extended to the entire length of this part of the molecule. On the basis of the present results, we propose a model of molecular architecture at the binding site for the complex on dystrophin.
Formylmethanofuran dehydrogenases from methanogenic Archaea Substrate specificity, EPR properties and reversible inactivation by cyanide of the molybdenum or tungsten iron‐sulfur proteins
Authors: BERTRAM Peter A. , KARRASCH Marion , SCHMITZ Ruth A. , BÖCHER Reinhard , ALBRACHT Simon P. J. , THAUER Rudolf K. 
Published: March 2005

Formylmethanofuran dehydrogenases, which are found in methanogenic Archaea, are molybdenum or tungsten iron‐sulfur proteins containing a pterin cofactor. We report here on differences in substrate specificity, EPR properties and susceptibility towards cyanide inactivation of the enzymes from Methanosarcina barkeri, Methanobacterium thermoautotrophicum and Methanobacterium wolfei.The molybdenum enzyme from M. barkeri (relative activity with N‐formylmethanofuran = 100%) was found to catalyze, albeit at considerably reduced apparent Vmax, the dehydrogenation of N‐furfurylformamide (11%), N‐methylformamide (0.2%), formamide (0.1%) and formate (1%). The molybdenum enzyme from M. Wolfei could only use N‐furfurylformamide (1%) and formate (3%) as pseudosubstrates. The molybdenum enzyme from M. thermoautotrophicum and the tungsten enzymes from M. thermoautotrophicum and M. wolfei were specific for N‐formylmethanofuran.The molybdenum formylmethanofuran dehydrogenases exhibited at 77 K two rhombic EPR signals, designated FMDred and FMDox, both derived from Mo as shown by isotopic substitution with 97Mo. The FMDred signal was only displayed by the active enzyme in the reduced form and was lost upon enzyme oxidation; the FMDox signal was displayed by an inactive form and was not quenched by O2. The tungsten isoenzymes were EPR silent.The molybdenum formylmethanofuran dehydrogenases were found to be inactivated by cyanide whereas the tungsten isoenzymes, under the same conditions, were not inactivated. Inactivation was associated with a characteristic change in the molybdenum‐derived EPR signal. Reactivation was possible in the presence of sulfide.
Characterization of the ATP‐dependent leukotriene C4 export carrier in mastocytoma cells
Authors: LEIER Inka , JEDLITSCHKY Gabriele , BUCHHOLZ Ulrike , KEPPLER Dietrich 
Published: March 2005

The biosynthesis of leukotriene C4 (LTC4) must be followed by an export of this mediator into the extracellular space where it interacts with receptors. Using mastocytoma cells we have demonstrated the existence of a primary‐active, ATP‐dependent transport mediating this export of LTC4 [Schaub, T., Ishikawa, T. & Keppler, D. (1991) FEBS Lett. 279, 83–86]. The following inhibitors served to characterize further this transport system in plasma membrane vesicle from mastocytoma cells: Probenecid, an inhibitor of organic anion transport, induced half‐maximal inhibition of the ATP‐dependent LTC4 transport at 71 μ. Cyclosporin A and its non‐immunosuppressive analog PSC 833 inhibited the ATP‐dependent transport with Ki values of 4.5 μM and 30μM, respectively. The LTD4 receptor antagonist 3‐([{3‐(2‐[7‐chloro‐2‐quinolinyl]ethenyl)phenyl}‐{(3‐dimethylamino‐3‐oxopropyl)‐thio}methyl]thio)propanoic acid (MK 571) was the most potent competitive inhibitor of the export carrier with a Ki value of 0.8 μM.The transport inhibitor MK 571 served as competitor in the photoaffinity labeling of LTC4‐binding membrane proteins using [3]LTC4 as the photolabile ligand. Proteins with molecular masses of about 190 kDa and 35 kDa were predominantly labeled. In addition, a minor [3H]LTC4 labeling was observed in the molecular mass range of 100kDa.The [3H]LTC4 labeling of the 190‐kDa protein was competed for by MK 571. The labeled proteins resisted extraction from the membrane with 2% sodium taurocholate suggesting that they are integral membrane proteins. Treatment of the membrane proteins with peptide N‐glycosidase F resulted in the appearance of an additional labeled polypeptide of about 140 kDa suggesting that the 190‐kDa protein is a glycoprotein. Photoaffinity labeling with 8‐azio[α‐32P]ATP predominantly labeled the LTC4‐binding 35‐kDa protein. The [3H]LTC4‐labeled 190‐kDa protein showed a mean isoelectric point at pH 6.3 with a range of pH 5.8–6.7, while the 35‐kDa protein had an isoelectric point at pH 6.8.Specific labeling of a 190‐kDa membrane glycoprotein by the glutathione conjugate LTC4, which is competed for by a potent inhibitor of the ATP‐dependent LTC4 export carrier, pinpoints its involvement in the ATP‐dependent transport of LTC4 and related conjugates.
Heterogeneity of the rat NADH‐cytochrome‐b5‐reductase transcripts resulting from multiple alternative first exons
Authors: MOTA VIEIRA Luisa , KAPLAN Jean‐Claude , KAHN Axel , LEROUX Alena 
Published: March 2005

In order to understand the mechanisms responsible for the generation of different isoforms (membrane‐bound and soluble) of NADH‐cytochrome b5 reductase, and the different clinical forms of recessive congenital methemoglobinemia due to the deficiency of this enzyme in humans (type I, without mental retardation; type II, with mental retardation), we have looked for mRNA heterogeneity in various rat tissues.We have found four types of mRNAs, each with a different first exon (1L, 1R, IX and 1Y), all of which were precisely spliced to join the common second exon. Our results are consistent with a 5′→3′‘scanning’ mechanism for splice‐site selection. The previously characterized 1L and 1R transcripts arise from the alternative use of either a ubiquitous promoter (Pr‐L) or an erythroid‐specific promoter (Pr‐R). In addition, the X and Y RNA species are novel transcripts which are expressed ubiquitously and at a relatively low level. The first alternative exons 1X and 1Y are non‐coding, such that the AUG codon present in the common second exon is functional, as it is in the R mRNA. Thus, the X and Y mRNAs are expected to be translated in vivo into a ubiquitous soluble enzyme. Consequently, the rat NADH‐cytochrome‐b5‐reductase gene is expressed through the use of at least four different promoters, which are probably subjected to different forms of regulation. This model of gene expression in rat could be important in understanding the basis for the different types of the NADH‐cytochrome‐b5‐reductase enzyme and their deficiency in man.
Human β1,4 galactosyltransferase and α2,6 sialyltransferase expressed in Saccharomyces cerevisiae are retained as active enzymes in the endoplasmic reticulum
Authors: KREZDORN Christian H. , KLEENE Ralf B. , WATZELE Manfred , IVANOV Svetoslav X. , HOKKE Cornelis H. , KAMERLING Johannis P. , BERGER Eric G. 
Published: March 2005

Biosynthesis and intracellular transport of recombinant human full‐length β1,4 galactosyltransferase (GT) and full‐length α2,6 sialyltransferase (ST) were investigated in Saccharomyces cerevisiae. Recently, enzymic activity of recombinant GT (rGT) in crude homogenates of S. cerevisiae could successfully be demonstrated [Krezdorn, C., Watzele, G., Kleene, R. B., Ivanov, S. X. & Berger, E. G. (1993) Eur. J. Biochem. 212, 113–120]. In the present work, we show that, in yeast strains transformed with plasmid pDPSIA containing the cDNA coding for human ST, rST enzymic activity using asialo‐fetuin or N‐acetyllactosamine as acceptor substrates could readily be detected. Analysis by 1H‐NMR spectroscopy of the disaccharide product of rGT, as recently reported, and the trisaccharide product of rST demonstrated that only the expected glycosidic linkages were formed. Following mechanical disruption of yeast cells, both enzymes sedimented with a fraction enriched in membranes of the endoplasmic reticulum (ER) and were activated by Triton X‐100 3–5‐fold. rGT and rST could be immunoprecipitated from their [35S]Met‐labelled transformed yeast extracts using polyclonal antibodies raised against fusion proteins consisting of β‐galactosidase‐GT or β‐galactosidase‐ST, respectively, expressed in Escherichia coli. For rGT a single glycosylated form of apparent molecular mass 48 kDa was reported, but for rST two main bands corresponding to apparent molecular masses of 48 kDa and 44 kDa, respectively, were detected. Immunoprecipitation from either tunicamycin‐treated [35S]Met‐labelled transformed yeast cells or labelling with radioactive sugars both indicated that the 44‐kDa form of rST was non‐glycosylated and that the 48‐kDa form of rST was core N‐glycosylated. In addition, core glycosylation of both recombinant enzymes demonstrated that they were competent for translocation across the ER membranes. However, the 44‐kDa form of rST was converted to the 48‐kDa glycosylated form only slowly, suggesting a mechanism of posttranslational translocation. Absence of hyperglycosylation of rST and rGT in wild type and lack of the Golgi‐specific man‐α1,6‐man epitope suggest that the recombinant enzymes did not enter the yeast Golgi apparatus.These results indicated that both rGT and rST are retained as enzymically active enzymes in the ER of yeast and suggest a ribonucleoprotein‐independent import of rST into the ER.
Characterisation and mode of in vitro replication of pea chloroplast OriA sequences
Authors: REDDY Malireddy K. , CHOUDHURY Nirupam Roy , KUMAR Dhirendra , MUKHERJEE Sunil K. , TEWARI K. K. 
Published: March 2005

A partially purified replicative system of pea chloroplast that replicates recombinant DNAs containing pea chloroplast origin sequences has been characterised. Polymerisation by this system is very fast and insensitive to chain terminators like dideoxynucleotides, arabinosylcytosine 5′‐triphosphate, etc. Both strands of template DNA are synthesized and single‐stranded DNA templates undergo more than one round of replication.When sequences of either of the two chloroplast origins of replication (OriA or OriB) are used as templates, the replicative intermediates are found to have sigma structures. Electron microscopic analysis of the sigma structures restricted with various enzymes reveals that the initiation site of in vitro replication maps near the displacement‐loop regions where replication initiates also in vivo. Although the observed replication initiation in the OriA recombinant template is chloroplast‐DNA‐specific, the mode of replication is different from that observed in vivo with intact ctDNA. However, when the template DNA contains both the OriA and OriB sequences, the in vitro replication proceeds in the theta mode, the mode of replication usually observed in vivo.
Regulation of the erythrocyte Ca2+‐ATPase at high pH
Authors: DE CARVALHO‐ALVES Paulo Cesar , FREIRE Monica M. , BARRABIN Hector , SCOFANO Helena M. 
Published: March 2005

The activation of the Ca2+‐ATPase from erythrocyte membranes at high pH has been investigated. Following alkalinization and in the absence of regulators, the enzyme exhibits a very high affinity for Ca2+ and a decreased maximal velocity. Either addition of calmodulin, addition of acidic phospholipids, or controlled trypsinization decreases the concentration of effector required to elicit half‐maximal activation of the enzyme for calcium to similar values. The increase in affinity for Ca2+, however, is smaller than that observed at neutral pH. The maximal velocity at high pH becomes insensitive to both calmodulin and controlled proteolysis, although calmodulin binds to the protein with similar affinities at pH 7.0 and 8.0, as indicated by similarity in binding to a calmodulin‐Sepharose resin and in dependence on calmodulin concentrations when the pH is increased.In contrast to the attenuated effects of calmodulin and proteolysis, at pH 8.0 the enzyme is susceptible to stimulation by phospholipids, indicating that the pathway for transduction of the signal from phospholipids is distinct from that pathway engaged by calmodulin and/or trypsinization. At pH 8.0, phosphatidylinositol induces the modulatory effect of ATP at the regulatory site but calmodulin does not. We suggest that the intraenzymic connection between the calmodulin‐binding, autoinhibitory peptide and the nucleotide domain of the enzyme is impaired upon alkalinization, which would account for the differing abilities of the activators to modulate the ATP effects.
An inducible arylsulfatase of Volvox carteri with properties suitable for a reporter‐gene system
Authors: HALLMANN Armin , SUMPER Manfred 
Published: March 2005

The multicellular green flagellate Volvox carteri synthesizes a periplasmic arylsulfatase in response to sulfur deprivation. The inducible enzyme has been purified to homogeneity and characterized. The corresponding gene and cDNA have been cloned. Determination of the sequence of genomic clones and comparisons to the cDNA sequence, revealed sixteen introns and seventeen exons that encode a 649‐amino‐acid polypeptide chain.Since the arylsulfatase enzyme is readily assayed using chromogenic substrates, but is not detectable in cells grown in sulfate‐containing medium, the gene encoding arylsulfatase may be useful as a reporter gene in V. carteri. In addition, the highly regulated promoter of the arylsufatase gene suggests its suitability as a tool for producing inducible expression vectors for cloned genes.