bluuebunny/crossref_metadata_2025_split huggingface dataset:
Synergism between coenzyme and alcohol binding to liver alcohol dehydrogenase
Authors: ANDERSSON Pia , KVASSMAN Jan , OLDÉN Bertil , PETTERSSON Gösta 
Published: March 2005

Heterotropic cooperativity effects in the binding of alcohols and NAD+ or NADH to liver alcohol dehydrogenase have been examined by equilibrium measurements and stopped‐flow kinetic studies. Equilibrium data are reported for benzyl alcohol, 2‐chloroethanol, 2,2‐dichloroethanol, and trifluoroethanol binding to free enzyme over the pH range 6–10.
Binary‐complex formation between enzyme and alcohols leads to inner‐sphere coordination of the alcohol to catalytic zinc and shows a pH dependence reflecting the ionization states of zinc‐bound water and the zinc‐bound alcohol. The affinity of the binding protonation state of the enzyme for unionized alcohols increases approximately, by a factor of 10 on complex formation between enzyme and NAD+ or NADH.
The rate and kinetic cooperativity with coenzyme binding of the alcohol association step indicates that, enzyme‐bound alcohols participate in hydrogen bonding interactions which affect the rates of alcohol and coenzyme equilibration with the enzyme without providing any pronounced contribution to the net energetics of alcohol binding.
The pKa values determined for alcohol deprotonation at the binary‐complex level are linearly dependent on those of the free alcohols, and can be readily reconciled with the pKa values attributed to ionization of zinc‐bound water. Alcohol coordination to catalytic zinc provides a major contribution to the pKa shift which ensures that the substrate is bound predominantly as an alcoholate ion in the catalytically productive ternary complex at physiological pH. The additional pKa shift contributed by NAD+ binding is less pronounced, but may be of particular mechanistic interest since it increases the acidity of zinc‐bound alcohols relatively to that of zinc‐bound water.
Chemical modification of sulfhydryl groups in p‐hydroxybenzoate hydroxylase from Pseudomonas fluorescens
Authors: VAN BERKEL Willem J. H. , WEIJER Wicher J. , MÜLLER Franz , JEKEL Peter A. , BEINTEMA Jaap J. 
Published: March 2005

The cysteine residues in p‐hydroxybenzoate hydroxylase from Pseudomonas fluorescens were modified with several cysteine reagents. One of the five sulfhydryl groups reacts rapidly and specifically with N‐ethylmaleimide without inactivation of the enzyme. Cysteine‐116 was found to be the reactive cysteine by isolation of a labeled tryptic peptide.The enzyme is easily inactivated by mercurial compounds. The original activity can be fully restored by treatment of the modified enzyme with sulfhydryl‐containing compounds. The rate of incorporation of mercurial compounds is pH‐independent and is pseudo‐first‐order up to 90–95% loss of activity. The reaction shows saturation kinetics. The substrate p‐hydroxybenzoate protects the enzyme from fast inactivation. The mercurial compounds themselves inhibit the inactivation reaction at concentrations higher than 80 μM. A spin‐labeled derivative of p‐chloromercuribenzoate reacts fairly specifically with only Cys‐152 on use of enzyme prelabeled with N‐ethylmaleimide, in contrast to p‐chloromercuribenzoate which reacts with additional cysteine residues, i.e. Cys‐211 and Cys‐158. From these results it is concluded that modification of Cys‐152 decreases drastically the affinity of the enzyme for the substrate. The results strongly indicate that the substrate binding site and Cys‐152 are interdependent. This observation is not obvious when the three‐dimensional data only are considered. The modified enzyme exhibits a somewhat higher affinity for NADPH than the native enzyme.Modification of N‐ethylmaleimide‐prelabeled enzyme by p‐chloromercuribenzoate leads to absorbance difference spectra showing maxima at 250 nm, 290 nm and 360 nm. The intensities of the absorbance difference maxima at 290 nm and 360 nm are strongly dependent on the pH value of the solution. The intensities are very low at low pH values and increase with increasing pH values, reaching a maximum at about pH = 9. The ionizing group shows a pK value of about 7.6. The maximal molar difference absorption coefficient at 290 nm is 3200 M‐1 cm‐1 at pH 9, suggesting that tyrosine residues ionize under the conditions of modification of the enzyme.The results are discussed in the light of the known three‐dimensional structure.
Nature and location of amide‐bound (R)‐3‐acyloxyacyl groups in lipid A of lipopolysaccharides from various gram‐negative bacteria
Authors: WOLLENWEBER Horst‐Werner , SEYDEL Ulrich , LINDNER Buko , LÜDERITZ Otto , RIETSCHEL Ernst Theodor 
Published: March 2005

It has previously been demonstrated [Eur. J. Biochem. 124, 191–198 (1982) and 137, 15–22 (1983)] that the lipid A component of Salmonella and Proteus lipopolysaccharides contains amide‐linked (R)‐3‐acyloxyacyl residues. In the present study lipid A of other gram‐negative bacteria was analysed for the presence of amide‐bound 3‐acyloxyacyl residues. It was found that such residues are constituents of all lipid A tested (Agrobacterium tumefaciens, Chromobacterium violaceum, Pseudomonas aeruginosa, Xanthomonas sinensis, Bacteriodes fragilis, Vibrio cholerae, Fusobacterium nucleatum, Rhodospirillum tenue, Acinetobacter calcoaceticus, and Escherichia coli). Amide‐linked (R)‐3‐acyloxyacyl groups, therefore, represent common and ubiquitous structural elements of bacterial lipid A.The composition of 3‐acyloxyacyl groups differed considerably among different bacteria. As amide‐bound (R)‐3‐hydroxy fatty acids straight chain and isobranched acyl groups with 10–17 carbon atoms were identified. The most frequently encountered fatty acids, substituting the 3‐hydroxyl group of 3‐hydroxy fatty acids, were nonhydroxylated straight chain and isobranched acyl residues with 10–17 carbon atoms as well as (S)‐2‐hydroxy fatty acids with 12 carbon atoms.In some cases, using laser desorption mass spectrometry, the distribution of 3‐acyloxyacyl residues over the two available glucosamine amino groups of the lipid A backbone was investigated.
Nucleotide sequence of cloned cDNAs coding for preprosecapin, a major product of queen‐bee venom glands
Authors: VLASAK Reinhard , KREIL Günther 
Published: March 2005

Total mRNA from queen‐bee venom glands was transcribed into cDNA and cloned into the PstI and ClaI site of pBR322. The nucleotide sequence of two clones (pUM9/24 and pBMC1) with inserts derived from mRNAs for preprosecapin is presented. The two inserts, which are identical in their overlapping regions, have a coding region encompassing 77 triplets. The cloned cDNAs differ in the length of the 3′‐untranslated region, which is about twice as long in clone pUM9/24. The postulated amino acid sequence of preprosecapin starts with a signal peptide containing an estimated 32 residues, followed by a pro part which terminates with a single arginine. The end‐product secapin is located at the COOH end of the precursor. Activation of prosecapin must, therefore, differ from the processing of promelittin synthesized in the same gland.Cell‐free translation of total venom gland mRNA in the presence of radioactive histidine yields only two major products, the smaller of which is presumably preprosecapin. Edman degradation of total translation products yields peaks of radioactive tyrosine and histidine at the 4th and 10th cycles respectively. This confirms the sequence of preprosecapin deduced from the cDNA clones. The content of secapin in queen‐bee venom must be higher than it is in worker bee venom.
β‐d‐Galactoside transport in Escherichia coli
Authors: KÖNIG Bernhard , SANDERMANN Heinrich 
Published: March 2005

The β‐d‐galactoside transport protein (y‐gene product) of Escherichia coli, strain T 206, was solubilized in 85–95% yield using the organic solvents hexamethylphosphoric triamide at pH 7.5 or butan‐1‐ol at pH 4.2. The transport protein obtained with the former solvent could be incorporated into a defined lipid/protein aggregate of density 1.12 g/ml, but no β‐d‐galactoside binding was restored. Diacylglycerol kinase regained activity in the same lipid/protein aggregates. In control experiments, liposomes formed from hexamethylphosphoric triamide were found to be active in the valinomycin‐mediated uptake of Rb+ ions.β‐d‐Galactoside binding (3.6–5.7 nmol/mg protein) as well as diacylglycerol kinase activity [7 nmol min–1 (mg protein)–1] was reconstituted into proteoliposomes from butan‐1‐ol solution by adaptation of a published procedure [Wright, J. K. et al. (1982) Eur. J. Biochem. 124, 545–552]. A microparticulate nature of the butan‐1‐ol‐solubilized transport protein could be excluded by gel permeation chromatography on a newly synthesized matrix, hydroxypropyl‐Sephacryl S‐300.
Phosphorylation in vivo and in vitro of the arginine‐ornithine periplasmic transport protein of Escherichia coli
Authors: CELIS Roberto T. F. 
Published: March 2005

The arginine‐ornithine periplasmic binding protein, an essential component of the arginine‐ornithine transport system of Escherichia coli, was isolated in a phosphorylated form and in a non‐phosphorylated form from the periplasmic fluid, after incubation of intact cells with (32P)orthophosphate under conditions similar to those used for arginine transport studies. The binding protein could also be labeled with 32Pi by incubation in vitro of the periplasmic fluid with [γ‐32P]ATP, or by incubation in vitro of the purified binding protein with radioactive ATP, Mg2+ and a phosphokinase enzyme released by osmotic‐shock treatment. The two forms of the protein were separated by DEAE‐Sephacel chromatography.By several different criteria, which included binding studies, analyses of the amino acid composition of the two forms of the protein, analysis by sodium dodecyl sulfate/polyacrylamide gel electrophoresis and testing for other components of the periplasmic space with affinities for inorganic phosphate, it was concluded that the 32P‐labeled protein corresponds to a phosphorylated form of the arginine‐ornithine‐binding protein. The phosphorylation reaction required Mg2+ and a phosphokinase from the periplasmic fluid. The dissociation constant of the phosphorylated protein for arginine was 5.0 μM (dissociation constant of the unmodified protein equals 0.1 μM), suggesting that the chemically modified protein is the active form of the molecule which releases the ligand for its translocation through the cytoplasmic membrane. The pH‐stability profile of the phosphoprotein has a ‘U’‐shape characteristic of acyl phosphates. Reaction of the phosphorylated binding protein with hydroxylamine at pH 5.4, also released Pi from the phosphoprotein. These properties suggest that the phosphoryl group of the phosphoprotein is linked covalently to a carboxyl function of the protein. This information indicates that ATP is a direct energy donor for the active transport of arginine and ornithine in E. coli, and a step of phosphorylation of the arginine‐ornithine‐binding protein appears to be involved in the utilization of the phosphate bond energy by the arginine‐ornithine transport system.
Mechanisms of activation and secretion of a cell‐associated precursor of an exocellular protease of Pseudomonas aeruginosa 34362A
Authors: FECYCZ Irene T. , CAMPBELL James N. 
Published: March 2005

An inactive precursor to the active exocellular protease 1 of Pseudomonas aeruginosa is cell‐associated and located primarily in the periplasmic space. We have studied factors that bring about activation of the precursor in vitro in order to shed some light on the process of its activation and secretion in vivo. A variety of diverse procedures were shown to effect irreversible activation. Several mild non‐enzymatic procedures were effective, such as dialysis of an ammonium sulfate precipitate against neutral buffers, gel filtration (Sephadex G‐100), and ion‐exchange chromatography (DEAE‐cellulose). Activation also resulted following treatment with anionic detergents (sodium dodecyl sulfate, N‐lauroyl sarcosine) and deoxycholate. Limited exposure to any of several proteases with different specificities also resulted in activation. The kinetics of detergent‐catalyzed activation reveals a long lag followed by rapid activation, suggesting at least a two‐stage process. The precursor and the mature protease 1 have indistinguishable molecular masses (33 kDa), as measured by sodium dodecyl sulfate/polyacrylamide gel electrophoresis of these proteins purified by immunoabsorbance chromatography under denaturing conditions. Further, both precursor and protease have identical N‐terminal alanine. Our results suggest that it is improbable that activation is the result of proteolytic processing of the precursor itself, but rather that it may involve the removal of a non‐covalently associated inhibitor molecule. Hydrophobic interaction chromatography on octyl‐Sepharose revealed that activation was accompanied by a significant change in the hydrophobicity, pointing to a significant change in the conformation of the precursor and the mature protease.A mutant has been studied which accumulates activatable precursor in the periplasm but releases no active enzyme into the culture medium, supporting the hypothesis that secretion through the inner and outer membranes proceed by different mechanisms. Comparison of outer membranes of protease‐secreting strains (34 362A and PAKS 1) and a protease‐negative mutant (PAKS 18) which accumulates precursor has shown that there is a change in the outer membrane protein profile in the latter.
Glycosphingolipids in insects
Authors: DENNIS Roger D. , GEYER Rudolf , EGGE Heinz , MENGES Hans , STIRM Stefan , WIEGANDT Herbert 
Published: March 2005

The presence of glycosphingolipids in the pupae of the blowfly, Calliphora vicina, was established. The thin layer chromatographic pattern of the total neutral glycolipids revealed the presence of more than 13 components, the major one being ceramide monohexoside. By the use of high performance liquid chromatography, the three simplest components were isolated and their chemical structures determined: Glc(β1–1)Cer, Man(β1–4)‐Glc(β1–1)Cer [with minor component Gal(β1–4)Glc(β1–1)Cer] and GlcNAc(β1–3)Man(β1–4)Glc(β1–1)‐Cer.The ceramide composition of the parent insect glycosphingolipids is dominated by the 20:0 fatty acid, arachidic acid, and the sphingoid tetradecasphing‐4‐enine.
Direct evidence by carbon‐13 NMR spectroscopy for the erythro configuration of the sphingoid moiety in Gaucher cerebroside and other natural sphingolipids
Authors: SARMIENTOS Francesco , SCHWARZMANN Günter , SANDHOFF Konrad 
Published: March 2005

By comparison of carbon‐13 NMR data from Gaucher cerebroside and from other natural sphingolipids with those from synthetic L‐threo‐ceramide, D‐glucosyl‐L‐threo‐ceramide, D‐erythro‐ceramide and D‐glucosyl‐D‐erythro‐ceramide, we have been able to obtain directly, for the first time, unequivocal evidence for the erythro configuration of the sphingosine moiety in natural sphingolipids. Gaucher cerebroside gave the same data as D‐glucosyl‐D‐erythro‐ceramide. In contrast, significant shift differences have been observed for sphingosine C‐3 (1.9 ppm) and C‐4(1.2 ppm) when comparing the spectral data of Gaucher cerebroside with those of D‐glucosyl‐L‐threo‐ceramide.Similar differences have also been found between the carbon‐13 NMR data of synthetic D‐erythro‐ceramide and those of L‐threo‐ceramide. In addition, we have observed that threo‐sphingosine C‐5 and C‐4 of ceramide and D‐glucosyl‐ceramide resonate near 129.5 ppm and 133.5 ppm, respectively, with a signal separation of about 4 ppm. On the other hand, these signals from the spectra of synthetic D‐erythro‐ceramide and D‐glucosyl‐D‐erythro‐ceramide as well as from those of gangliosides, lactosyl‐ceramide, galactosyl‐ceramide and sphingomyelin are found near 130 ppm and 135 ppm, respectively, thus being apart by about 5 ppm, irrespective of the degree of glycosylation of the ceramide portion. Thus, by carbon‐13 NMR spectroscopy the erythro configuration of the sphingoid base in natural sphingolipids is directly demonstrated.
Synthesis and secretion of hemopexin in primary cultures of rat hepatocyts
Authors: KATZ Norbert R. , GOLDFARB Valentina , LIEM Heng , MULLER‐EBERHARD Ursula 
Published: March 2005

Secretion of hemopexin (20% carbohydrate) and its dependence on glycosylation was studied in primary rat hepatocyte cultures in comparison to the secretion of transferrin (5% carbohydrate).

In pulse‐chase experiments with [35S]methionine half of the labeled hemopexin was secreted in 30 min. By contrast, it took approximately 50 min for secretion of half of the transferrin.
Tunicamycin treatment of cultures significantly delayed the secretion of hemopexin but not that of transferrin.
During the pulse period a proiod a prominent intracellular precursor of hemopexin, smaller than the mature protein, was evident.

It is concluded that the extent of glycosylation of a secretory protein is not necessarily a determinant of the transit time required for intracellular processing and secretion. In the case of hemopexin the glycosylation apparently facilitates the secretion although it is not an absolute prerequisite for the exocytosis of this protein.
Decrease of fatty acid oxidation, ketogenesis and gluconeogenesis in isolated perfused rat liver by phenylalkyl oxirane carboxylate (B 807‐27) due to inhibition of CPT I (EC 2.3.1.21)
Authors: WOLF Horst P. O. , ENGEL Dieter W. 
Published: March 2005

Sodium 2‐[5‐(4‐chlorophenyl)‐pentyl]‐oxirane‐2‐carboxylate (B 807‐27 or POCA) inhibits ketogenesis from endogenous and exogenous long‐chain fatty acids and 14CO2 production from [U‐14C]palmitate, but not from [U‐14C]palmitoylcarnitine or octanoate, and inhibits gluconeogenesis from lactate and pyruvate in perfused livers of starved rats.
Inhibition of ketogenesis, 14CO2 production and gluconeogenesis was complete at concentrations of 10 μmol/1 POCA, but onset was more rapid for inhibition of ketogenesis and CO2 production than for gluconeogenesis. Infusion of octanoate abolished inhibition of all three processes.
Experiments with isolated rat liver mitochondria showed that carnitine palmitoyltransferase I (EC 2.3.1.21) is inhibited by POCA‐CoA.The inhibitory process is dependent on time and concentration, and more pronounced in mitochondria from fed than from fasted rats. Concentrations required for 50% inhibition after 20 min preincubation with POCA‐CoA are 0.02, 0.06 and 0.1 μmol/l in liver mitochondria from fed, 24‐h‐fasted and 48‐h‐fasted rats, respectively. The inhibitor appears to be tightly bound to the enzyme.
The extent of inhibition of carnitine palmitoyltransferase I correlates well with the hypoglycaemic and hypoketonaemic effects of the compounds in fasted rats. We conclude that specific inhibition of the enzyme leads, due to inhibition of long‐chain fatty acid utilization, to depressed ketogenesis and gluconeogenesis and, in consequence, to hypoglycaemic and hypoketonaemia in vivo under gluconeogenic and ketogenic conditions.
Precursors of ricin and Ricinus communis agglutinin
Authors: LORD J. Michael 
Published: March 2005

During synthesis in vivo the castor bean lectin precursors initially appear in the endoplasmic reticulum as a group of core glycosylated polypeptides of relative molecular mass 64 000–68 000. Pretreatment of intact castor bean endosperm tissue with tunicamycin partially inhibits the cotranslational core glycosylation step and results in the accumulation of a single sized unglycosylated precursor polypeptide of relative molecular mass 59 000. The glycosylated precursors in the endoplasmic reticulum were enzymically converted to the 59 000‐Mr from by incubation with endoglucosaminidase H. Intracellular transport of the glycosylated lectin precursors from the endoplasmic reticulum to a denser vesicle fraction was accompanied by modifications to the oligosaccharide moieties which conferred resistance to the action of endoglucosaminidase H. The post‐translational addition of fucose to the carbohydrate chain was identified as one of the oligosaccharide modification steps. Fucose addition was catalysed by a glycosyltransferase associated with a smooth‐surfaced membrane fraction which was distinct from the endoplasmic reticulum and which was tentatively identified as the Golgi apparatus.Glycosylation was not essential for intracellular transport of the lectin precursors: unglycosylated precursor synthesized in the presence of tunicamycin gave rise to unglycosylated lectin subunits in the protein bodies.
Effects of denaturation and methylation on the degradation of proteins in cultured hepatoma cells and in reticulocyte cell‐free systems
Authors: KATZNELSON Revital , KULKA Richard G. 
Published: March 2005

Radioiodinated, native and denatured bovine serum albumin (albumin) β‐lactoglobulin and cytochrome c were introduced into hepatoma tissue culture cells by erythrocyte‐ghost‐mediated microinjection, and their rates of degradation were compared. Denatured albumin was degraded at 20% of the rate of undenatured albumin, denatured β‐lactoglobulin was degraded three times faster than undenatured β‐lactoglobulin, while denatured and undenatured cytochrome c were degraded at the same rate. Thus, denaturation does not affect the rates of intracellular breakdown of microinjected proteins in a simple predictable way. Exhaustive methylation did not inhibit the degradation of denatured β‐lactoglobulin or albumin, indicating that, like their undenatured counterparts, they are not degraded via the ubiquitin pathway.In reticulocyte lysates, in the presence of ATP, denatured albumin and β‐lactoglobulin were broken down at slightly slower rates than the parent proteins. Exhaustive methylation of both denatured and undenatured proteins completely abolished their ATP‐dependent breakdown. This inhibition is consistent with the hypothesis that free – NH2 groups are required for the attachment of ubiquitin prior to degradation in this system.Removal of an ammonium sulfate fraction from reticulocyte lysates produces a proteolytic system markedly different from the whole lysate [Speiser, S. & Etlinger, J. D. (1983) Proc. Natl Acad. Sci. USA 80, 3577–3580]. In this system both denatured and undenatured albumin and β‐lactoglobulin were degraded essentially independently of ATP. Methylation only slightly decreased the breakdown of denatured proteins, suggesting that they are not degraded via the ubiquitin pathway. A possible explanation of these results is that removal of the ammonium sulfate fraction unmasks an ATP‐independent proteolytic system unrelated to the ubiquitin pathway.
Isolation and characterization of an iron‐containing superoxide dismutase from tomato leaves, Lycopersicon esculentum
Authors: KWIATOWSKI Jan , SAFIANOWSKA Aleksandra , KANIUGA Zbigniew 
Published: March 2005

A cyanide‐insensitive superoxide dismutase was purified from tomato leaves (Lycopersicon esculentum, Mill., var. Venture) to apparent homogeneity. The enzyme had twofold higher specific activity (about 4000 standard units) than ferric superoxide dismutases purified from Brassica campestris [Salin, M. L. and Bridges, S. M. (1980) Arch. Biochem. Biophys. 201, 369–374] and Nuphar luteum [Salin, M. L. and Bridges, S. M. (1982) Plant Physiol. 69, 161–165].The protein had a relative molecular mass of about 42000 and was composed of two equal subunits noncovalently joined. It was negatively charged (pI = 4.6) and contained about 1.45 mol Fe/mol dimer and negligible amounts of Mn, Cu and Zn. Absorption spectrum and sensitivity to NaN3, H2O2 and temperature are also reminiscent of other ferric superoxide dismutases.Comparison of amino acid composition indicated, however, a closer relationship to the Mn‐containing enzymes rather than to other Fe‐containing superoxide dismutases. Two possible ways of Fe‐containing superoxide dismutase acquisition by vascular plants were suggested.