bluuebunny/crossref_metadata_2025_split huggingface dataset:
Lipid vesicles which can bind to protein kinase C and activate the enzyme in the presence of EGTA
Authors: EPAND Richard M. , STAFFORD Alan R. , LESTER David S. 
Published: March 2005

Maximal protein kinase C activity with vesicles of phosphatidic acid and 1,2‐dioleoyl‐sn‐glycerol is observed in the absence of added Ca2+. Addition of phosphatidylcholine to these vesicles restores some calcium dependence of enzyme activity. 1,2‐Dioleoyl‐sn‐glycerol eliminates the Ca2+‐dependence of protein kinase C activity found with phosphatidic acid alone. Phorbol esters do not mimic the action of 1,2‐dioleoyl‐sn‐glycerol in this respect. This suggests that the 1,2‐dioleoyl‐sn‐glycerol effect is a result of changes it causes in the physical properties of the membrane rather than to specific binding to the enzyme. The effect of 1,2‐dioleoyl‐sn‐glycerol on the phosphatidic‐acid‐stimulated protein kinase C activity is dependent on the molar fraction of 1,2‐dioleoyl‐sn‐glycerol used and results in a gradual shift from Ca2+ stimulation at low 1,2‐dioleoyl‐sn‐glycerol concentrations to calcium inhibition at higher concentrations of 1,2‐dioleoyl‐sn‐glycerol. Phosphatidylserine‐stimulated activity is also shown to be largely independent of the calcium concentration at higher molar fractions of 1,2‐dioleoyl‐sn‐glycerol. Thus, with certain lipid compositions, protein kinase C activity becomes independent of the calcium concentration or requires only very low, stoichiometric binding of Ca2+ to high affinity sites on the enzyme.Protein kinase C can bind to phosphatidic acid vesicles more readily than it can bind to phosphatidylserine vesicles in the absence of calcium. Addition of 1,2‐dioleoyl‐sn‐glycerol to phosphatidylserine vesicles promotes the partitioning of protein kinase C into the membrane in the absence of added Ca2+. There is no isozyme specificity in this binding. These results suggest that a less‐tightly packed headgroup region of the bilayer causes increased insertion of protein kinase C into the membrane. This is a necessary but not sufficient condition for activation of the enzyme in the presence of EGTA.
Site‐specific mutagenesis of Escherichia coli asparaginase II
Authors: WEHNER Andreas , HARMS Etti , JENNINGS Michael P. , BEACHAM Ifor R. , DERST Christian , BAST Peter , RÖHM Klaus H. 
Published: March 2005

Site‐specific mutagenesis was used to replace the three histidine residues of Escherichia coli asparaginase II (EcA2) with other amino acids. The following enzyme variants were studied: [H87A]EcA2, [H87L]EcA2, [H87K]EcA2, [H183L]EcA2 and [H197L]EcA2. None of the mutations substantially affected the Km for l‐aspartic acid β‐hydroxamate or impaired aspartate binding. The relative activities towards l‐Asn, l‐Gln, and 1‐aspartic acid β‐hydroxamate were reduced to the same extent, with residual activities exceeding 10% of the wild‐type values. These data do not support a number of previous reports suggesting that histidine residues are essential for catalysis.Spectroscopic characterization of the modified enzymes allowed the unequivocal assignment of the histidine resonances in 1H‐NMR spectra of asparaginase II. A histidine signal previously shown to disappear upon aspartate binding is due to His183, not to the highly conserved His87. The fact that [H183L]EcA2 has normal activity but greatly reduced stability in the presence of urea suggests that His183 is important for the stabilization of the native asparaginase tetramer. 1H‐NMR and fluorescence spectroscopy indicate that His87 is located in the interior of the protein, possibly adjacent to the active site.
H2‐forming methylenetetrahydromethanopterin dehydrogenase, a novel type of hydrogenase without iron‐sulfur clusters in methanogenic archaea
Authors: ZIRNGIBL Carmen , VAN DONGEN Walter , SCHWÖRER Beatrix , VON BÜNAU Rudolf , RICHTER Monika , KLEIN Albrecht , THAUER Rudolf K. 
Published: March 2005

A novel hydrogenase has recently been found in methanogenic archaea. It catalyzes the reversible dehydrogenation of methylenetetrahydromethanopterin (CH2─H4MPT) to methenyltetrahydromethanopterin (CH═H4MPT+) and H2 and was therefore named H2‐forming methylenetetrahydromethanopterin dehydrogenase. The hydrogenase, which is composed of only one polypeptide with an apparent molecular mass of 43 kDa, does not mediate the reduction of viologen dyes with either H2 or CH2─H4MPT. We report here that the purified enzyme from Methanobacterium thermoautotrophicum exhibits the following other unique properties: (a) the colorless protein with a specific activity of 2000 U/mg (Vmax) did not contain iron‐sulfur clusters, nickel, or flavins; (b) the activity was not inhibited by carbon monoxide, acetylene, nitrite, cyanide, or azide; (c) the enzyme did not catalyze an isotopic exchange between 3H2 and 1H+; (d) the enzyme catalyzed the reduction of CH═H4MPT+ with 3H2 generating [methylene‐3H]CH2─H4MPT; and (e) the primary structure contained at most four conserved cysteines as revealed by a comparison of the DNA‐deduced amino acid sequence of the proteins from M. thermoautotrophicum and Methanopyrus kandleri. None of the four cysteines were closely spaced as would be indicative for a (NiFe) hydrogenase or a ferredoxintype iron‐sulfur protein.Properties of the H2‐forming methylenetetrahydromethanopterin dehydrogenase from Methanobacterium wolfei are also described indicating that the enzyme from this methanogenic archaeon is very similar to the enzyme from M. thermoautotrophicum with respect both to molecular and catalytic properties.
Differentiation‐inducing‐factor dechlorinase, a novel cytosolic dechlorinating enzyme from Dictyostelium discoideum
Authors: NAYLER Oliver , INSALL Robert , KAY Robert R. 
Published: March 2005

Differentiation‐inducing‐factor 1 (DIF‐1) is a dichlorinated alkyl phenone {1‐[(3,5‐dichloro‐2,6‐dihydroxy‐4‐methoxy)phenyl]hexan‐1‐one} from Dictyostelium discoideum, that induces amoebae to differentiate into stalk cells. It was shown previously that DIF‐1 is rapidly metabolized into a series of more polar compounds by living cells [Traynor, D. & Kay, R. R. (1991) J. Biol. Chem. 266, 5291–5297]. The first step in DIF‐1 metabolism is the formation of DIF metabolite 1 (now known to be DIF‐3) by a monodechlorination. We report here the discovery of the enzyme activity catalyzing this dechlorination. A very sensitive enzyme assay was developed, using [3H]DIF‐1 and a TLC system to separate DIF‐1 from the product, DIF‐3. DIF‐1 3(5)‐dechlorinase is present in the high‐speed supernatant of cell lysates, and uses glutathione, at physiological concentrations, as cofactor. Kinetic measurements indicate a Km for DIF‐1 of about 70 nM. The enzyme activity is inhibited by DIF‐2 (the pentan‐1‐one analogue of DIF‐1), with a median inhibitor concentration (IC50) of 1 μM, and DIF‐3 (IC50= 5 μM), which presumably act as substrates, but other compounds structurally related to DIF‐1 were much less effective. Aurothioglucose, an inhibitor of selenocysteine enzymes, inhibited DIF‐1 3(5)‐dechlorinase with IC50= 100 nM.DIF‐1 3(5)‐dechlorinase activity is developmentally regulated. It is essentially absent from growing cells and increases at the end of aggregation to reach a first peak of activity at the first finger stage, with a further rise at culmination.
Splicing of the platelet‐derived‐growth‐factor A‐chain mRNA in human malignant mesothelioma cell lines and regulation of its expression
Authors: LANGERAK Anthonie W. , DIRKS Ron P. H. , VERSNEL Marjan A. 
Published: March 2005

Platelet‐derived‐growth‐factor (PDGF) A‐chain transcripts differing in the presence or absence of an alternative exon‐derived sequence have been described. In some publications, the presence of PDGF A‐chain transcripts with this exon‐6‐derived sequence was suggested to be tumour specific. However, in this paper it was shown by reverse‐transcription polymerase‐chain‐reaction (PCR) analysis that both normal mesothelial cells and malignant mesothelioma cell lines predominantly express the PDGF A‐chain transcript without the exon‐6‐derived sequence. This sequence encodes a cell‐retention signal, which means that the PDGF A‐chain protein is most likely to be secreted by both cell types. In cultured normal mesothelial cells, the secreted PDGF A‐chain protein might be involved in autocrine growth stimulation via PDGF α receptors. However, human malignant mesothelioma cell lines only possess PDGF β receptors. If this also holds true in vivo, the PDGF A‐chain protein produced and secreted by malignant mesothelial cells might have a paracrine function.In a previous paper, we described elevated expression of the PDGF A‐chain transcript in human malignant mesothelioma cell lines, compared to normal mesothelial cells. In this paper, the possible reason for this elevation was studied. First, alterations at the genomic level were considered, but cytogenetic and Southern‐blot analysis revealed neither consistent chromosomal aberrations, amplification nor structural rearrangement of the PDGF A‐chain gene in the malignant cells. Possible differences in transcription rate of the PDGF A‐chain gene, and stability of the transcript between normal and malignant cells, were therefore studied. The presence of a protein‐synthesis inhibitor, cycloheximide, in the culture medium did not significantly influence the PDGF A‐chain mRNA level in normal mesothelial and malignant mesothelioma cell lines. Furthermore, nuclear run‐off analysis showed that nuclear PDGF A‐chain mRNA levels varied in both cell types to the same extent as the levels observed in Northern blots. Taken together, this suggests that increased transcription is the most probable mechanism for the elevated mRNA level of the PDGF A‐chain gene in human malignant mesothelioma cell lines.
The interaction of acetate and formate with cobalt carbonic anhydrase
Authors: BERTINI Ivano , LUCHINAT Claudio , PIERATTELLI Roberta , VILA Alejandro J. 
Published: March 2005

The interaction of formate and acetate ions with cobalt‐substituted carbonic anhydrase (CA) has been investigated through 13C‐NMR and one‐dimensional and two‐dimensional 1H‐NMR spectroscopy. 13C data on formate are consistent with a regularly coordinated ligand, as previously proposed for the acetate anion [Bertini, I., Luchinat, C. & Scozzafava, A. (1977) J. Chem. Soc. Dalton Trans., 1962–1965]. 1H‐NOE experiments on both anions give evidence of through‐space interactions between ligand protons and protein protons. The latter are assigned to specific residues in the active cavity through nuclear Overhauser effect spectroscopy (NOESY) experiments. The 13C‐derived and 1H‐derived constrains allow reliable docking of these ligands in the active‐site cavity. The resulting geometries are similar to one another and consistent with five‐coordinated structures around the metal ion, as previously proposed from electronic spectroscopy [Bertini, I., Canti, G., Luchinat, C. & Scozzafava, A. (1978) J. Am. Chem. Soc. 100, 4873–4877].The results are discussed in light of the current debate on anion binding to metal ions in carbonic anhydrase [Lindahl, M., Svensson, A. & Liljas, A. (1992) Proteins, in the press]; Bertini, I., Luchinat, C., Pierattelli, R. & Vila, A. J. (1992) Inorg. Chem., in the press; Banci, L. & Merz, K. (1992) unpublished results] and, in particular, of the proposed long Zn‐O distance found in the recent X‐ray results on the formate adduct [Hakanson, K., Carlsson, M., Svensson, A. & Liljas, A. (1992) J. Mol. Biol., in the press].
Plasma‐membrane‐independent pool of the α subunit of the stimulatory guanine‐nucleotide‐binding regulatory protein in a low‐density‐membrane fraction of S49 lymphoma cells
Authors: SVOBODA Petr , KVAPIL Petr , INSEL Paul A. , RANSNÄS Lennart A. 
Published: March 2005

We report that compartmentalisation of the stimulatory guanine‐nucleotide‐binding regulatory protein (Gs) exists in S49 lymphoma cells. In addition to the previously reported cytosolic form of the α subunit of Gs (Gsα) [Ransnäs, L. A., Svoboda P., Jasper, J. R. & Insel, P. A. (1989) Proc. Natl Acad. Sci. USA 86, 7900–7903], three membrane‐bound forms of Gsα were identified through ratezonal centrifugation in sucrose density gradients, Gsα‐specific anti‐peptide serum and an adenylate cyclase complementation assay. The sedimentation profile of the first pool of Gsα in the high‐density portion of the gradient (1.13–1.16 g/cm3) is identical with that of β‐adrenergic‐receptor binding, Na/K‐ATPase and adenylate cyclase activity, and may therefore be identified as plasma‐membrane fragments. The second pool, which was recovered in the middle portion of the gradient (1.09–1.11 g/cm3), contains a much lower total amount of Gsα and correlates with the endoplasmic reticulum (microsomal) enzyme markers, NADPH–cytochrome‐c reductase and glucose‐6‐phosphatase.The identity of the third pool of Gsα located at the top of the gradient (1.06–1.08 g/cm3), is unknown. The Golgi apparatus marker, UDPgalactose:N‐acetylglucosamine glycosyltransferase, was partially recovered in this area; however, this enzyme was also present in the high‐density portion of the gradient. Complete absence of specific adenylate cyclase and Na/K‐ATPase activity indicates that this low‐density (light) membrane form of Gsα is distinct from any plasma‐membrane fragments. Furthermore, sedimentation at 100000 ×g proves its particulate (membrane) character. The light membrane form of Gsα subunit is functionally active in an adenylate cyclase complementation assay using cyc− membranes devoid of Gsα.Overall, our data indicates that a substantial portion of Gsα is localized in membrane pools other than plasma membrane.
The binding of mutant actins to profilin, ATP and DNase I
Authors: DRUMMOND Douglas R. , HENNESSEY Emma S. , SPARROW John C. 
Published: March 2005

Twenty‐five mutations were created in the Drosophila melanogaster Act88F actin gene by in vitro mutagenesis and the mutant actins expressed in vitro. The affinity of the mutant actins for ATP, profilin and DNase I was determined. They were also tested for conformational changes by non‐denaturing gel electrophoresis. Mutations at positions 364 (highly conserved) and 366 (invariant) caused changes in conformation, reduced ATP binding and increased profilin binding. At position 362 (invariant) only the conservative change from tyrosine to phenylalanine had no effect; other changes at this position affected conformation, ATP and profilin binding. Although only glycine or serine occur naturally at position 368, changes to threonine or glutamine had no effect on the actin. The mutant in which Asp363 was replaced by His and that in which Glu364 was replaced by Lys decreased DNase I binding, yet neither amino acid occurs in the DNase I binding site. Likewise several mutations affect ATP and profilin binding but are distant from the binding sites.We conclude that, although actin has a highly conserved amino acid sequence, individual amino acids can have variable tolerance for substitutions. Also amino acid changes can exert significant effects on the binding of ligands to distant parts of the actin structure.
Purification and paritial characterization of a thermostable trithionate hydrolase from the acidophilic sulphur oxidizer Thiobacillus acidophilus
Authors: MEULENBERG Rogier , PRONK Jack T. , FRANK Johannes , HAZEU Wim , BOS Piet , KUENEN J. Gijs 
Published: March 2005

Cell‐free extracts of Thiobacillus acidophilus catalysed the quantitative conversion of trithionate (S3O62−) to thiosulphate and sulphate. A continuous assay for quantification of experimental results was based on the difference in absorbance between trithionate and thiosulphate at 220 nm.Trithionate hydrolase was purified to near homogeneity from cell‐free extracts of T. acidophilus. The molecular masses of the native enzyme and the subunit were 99 kDa (gel filtration) and 34 kDa (SDS/PAGE). The purified enzyme has a pH optimum of 3.5–4.5 and a temperature optimum of 70°C. Enzyme activity was stimulated by sulphate. The stimulation of the enzyme activity by sulphate was half maximal at a concentration of 0.23 M. The Km for trithionate is 70 μM at 30°C and 270 μM at 70°C. Enzyme activity was lost after 36 days at 0°C, 27 days at 70°C; but after 97 days at 30°C, 40% of the initial activity was still present. The enzyme activity was inhibited by mercury chloride, N‐ethylmaleimide, thiosulphate and tetrathionate. Tetrathionate S4O62− was not hydrolysed by trithionate hydrolase.
Characteristics of energy‐linked proton translocation in liposome reconstituted bovine cytochrome bc1 complex
Authors: COCCO Tiziana , LORUSSO Michele , DI PAOLA Marco , MINUTO Michele , PAPA Sergio 
Published: March 2005

A study is presented on the H+/e− stoichiometry for proton translocation by the isolated cytochrome bc1 complex under level‐flow and steady‐state conditions. An experimental procedure was used which allows the determination of pure vectorial proton translocation in both conditions in a single experiment. The results obtained indicate an H+/e− ratio of 1 at level‐flow and 0.3 at steady‐state. The ratios appear to be independent of the rate of electron transfer through the complex. Making use of pyranine‐entrapped bc1 vesicles, a respiration‐dependent steady‐state ΔpH value of 0.4 was determined in the presence of valinomycin. This value could be either decreased by subsaturating concentrations of the uncoupler carbonyl cyanide m‐chlorophenylhydrazone (CCCP) or increased by introducing bovine serum albumin in the assay mixture. The steady‐state H+/e− ratio appeared to be in linear inverse correlation with the ΔpH. This indicates that ΔpH exerts a control on the proton pump of the bc1 complex at the steady state.The effect of valinomycin‐mediated potassium‐diffusion potential on electron‐transfer and proton‐translocation activities is also shown. The experiments presented show that the H+/e− ratio is unaffected, both at level flow and steady state, by an imposed diffusion potential up to around 100 mV. At higher potential values the level‐flow H+/e− ratio slightly decreased. Measurements as a function of imposed membrane potential of the rate of electron transfer at level flow and of the rate of the pre‐steady‐state reduction of b and c1 cytochromes in the complex indicate activation of electron transfer at potential values of 40–50 mV. This activation appears, however, to involve a rate‐limiting step which remains normally coupled to proton translocation.
Characterization of [3H]acifluorfen binding to purified pea etioplasts, and evidence that protoporphyrinogen oxidase specifically binds acifluorfen
Authors: MATRINGE Michel , MORNET René , SCALLA René 
Published: March 2005

It is now generally accepted that protoporphyrinogen oxidase is the target‐enzyme for diphenylether‐type herbicides. Recent studies [Camadro, J‐M., Matringe, M., Scalla, R. & Labbe, P. (1991) Biochem. J. 277, 17–21] have revealed that in maize, diphenyl ethers competitively inhibit protoporphyrinogen oxidase with respect to its substrate, protoporphyrinogen IX.In this study, we show that, in purified pea etioplast, [3H]acifluorfen specifically binds to a single class of high‐affinity binding sites with an apparent dissociation constant of 6.2 ± 1.3 nM and a maximum density of 29 ± 5 nmol/g protein. [3H]Acifluorfen binding reaches equilibrium in about 1 min at 30°C. Half dissociation occurs in less than 30 s, indicating that the binding is fully reversible.The specificity of [3H]acifluorfen binding to protoporphyrinogen oxidase is examined. [3H] Acifluorfen binding is inhibited by all the peroxidizing molecules tested. The phthalimide derivative, N(4‐chloro‐2‐fluoro‐5‐isopropoxy)phenyl‐3,4,5,6‐tetra hydrophthalimide, exerts a mixed‐competitive inhibition on this binding. The effects of all these molecules on the binding of [3H]acifluorfen are tightly linked to their capacity to inhibit pea etioplast protoporphrinogen oxidase activity. Furthermore, protoporphyrinogen IX, the substrate of the reaction catalyzed by protoporphyrinogen oxidase, was able to competitively inhibit the binding of [3H]acifluorfen. In contrast, protoporphyrin IX, the product of the reaction, did not inhibit this binding.All these results provide clear evidence that in pea etioplasts, [3H]acifluorfen exclusively binds to protoporphyrinogen oxidase, that the protoporphyrinogen oxidase inhibitors tested so far bind to the same region of the enzyme and that this region overlaps the catalytic site of the enzyme.
Regulation of two rat serine‐protease inhibitor gene promoters by somatotropin and glucocorticoids
Authors: PAQUEREAU Laurent , VILAREM Marie José , ROSSI Valérie , ROUAYRENC Jean François , LE CAM Alphonse 
Published: March 2005

Only two out of the three serine‐protease inhibitor genes (SPI 2.1, 2.2 and 2.3) expressed in rat liver are tightly controlled by somatotropin acting mainly at the transcriptional level, thus making this gene system particularly suitable to study its molecular mechanism of action. In these studies, we analyzed SPI promoter activities in cultured hepatocytes transfected by electroporation or in cell‐free extracts. The proximal SPI 2.1 promoter region contains two somatotropin‐responsive sites which are functional in intact cells. The more distal element that maps at positions – 175 to – 114, and is analogous to the one originally described by Yoon et al. (1990) [Yoon, J. B., Berry, S. A., Seelig, S. & Towle, H. C. (1990) J. Biol. Chem. 265, 19947–19954], behaves as a weak enhancer whose activity is strongly potentiated by proximal 5′ downstream sequences that contains potential CCAAT‐enhancer binding protein (C/EBP) sites. An additional proximal hormone‐sensitive site is located in the close vicinity of the transcription‐start site between positions −41 and +8, and also requires the first C/EBP‐binding element to be active. The distal element appears to contribute more importantly (60%) than the proximal one (40%) to the overall somatotropin stimulation of chimeric gene expression. Nonetheless, both displayed similar dose‐dependence, with half‐maximal and maximal effects occurring at 0.5–1 nM and 5–10 nM, respectively. The somatotropin refractoriness of the SPI 2.3 gene appears to be due to the presence of distal (–2300 to –200) inhibitory element(s) in the promoter. Glucocorticoids exert both positive and negative effects on SPI promoter activity. Their stimulatory action appears to involve sequences located between positions –114 and –82, together with a more distal half glucocorticoid‐responsive element, whereas their inhibitory effect is more likely mediated by sequences located between positions −41 and +8. In vitro transcription assays, performed with promoter‐deletion mutants and competitor oligonucleotides, revealed the presence of a major functional C/EBP site located immediately upstream from the transcription‐start point. Unfortunately, the regulatory features of SPI gene expression observed in intact cells were completely obliterated by breaking down the cell structure, and could not therefore be studied using cell‐free systems.