bluuebunny/crossref_metadata_2025_split huggingface dataset:
Identification and characterization of the principal proteins of the fat‐globule membrane from guinea‐pig milk
Authors: JOHNSON Virginia G. , GREENWALT Dale E. , HEID Hans W. , MATHER Ian H. , MADARA Patricia J. 
Published: March 2005

The milk‐fat‐globule membrane (MFGM) was isolated from guinea‐pig milk and the membrane‐associated proteins and glycoproteins characterized by electrophoretic techniques. Major components of the membrane included PAS‐I, a sialoglycoprotein of Mr≥ 200000, the redox enzyme xanthine oxidase and the glycoprotein, butyrophilin. Membrane preparations also contained two other glycoproteins, GP‐80 and GP‐55, of Mr 80000 and 55000, respectively.Comparison of guinea‐pig xanthine oxidase and butyrophilin with proteins from bovine MFGM by peptide mapping procedures, showed that the two proteins in both species were similar, but not identical. GP‐55 may also be related to glycoproteins of Mr 45000 and 48000 in the bovine membrane.The integral and peripheral components of guinea‐pig MFGM were identified by treating membrane preparations with sodium carbonate solutions at high pH and by partitioning the membrane proteins in solutions of Triton X‐114. By these criteria xanthine oxidase and GP‐55 appeared to be peripheral components and GP‐80 an integral protein of the membrane. PAS‐I and butyrophilin displayed hydrophilic properties in Triton X‐114 solutions, but could not be removed from membrane preparations with sodium carbonate. Possible reasons for these ambiguous data are discussed.The observed similarity between several of the proteins of guinea‐pig and bovine MFGM implies that these proteins may have specific functions related to milk secretion in mammary tissue, e.g. in the budding of milk‐fat globules or the exocytosis of milk protein and lactose at the apical surface.
The redox interconversion mechanism of Saccahromyces cerevisiae glutathione reductase
Authors: PINTO M. Carmen , MATA Ana M. , LÓPEZ‐BAREA Juan 
Published: March 2005

The changes undergone by pure yeast glutathione reductase during redox interconversion have been studied. Both the active and inactive forms of the enzyme had similar molecular masses, suggesting that the inactivation is probably due to intramolecular modification(s).
The glutathione reductase and transhydrogenase activities were similarly inactivated by NADPH and reactivated by GSH, while the diaphorase activity remained unaltered during redox interconversion of glutathione reductase. These results suggest that the inactivation site could be located far from the NADPH‐binding site, although interfering with transhydrogenase activity, perhaps by conformational changes.
The inactivation of glutathione reductase by 0.2 mM NADPH at pH 8 was paralleled by a gradual decrease in the absorbance at 530 nm and a simultaneous increase in the absorbance at 445 nm, while the reactivation promoted by GSH was initially associated with reversal of these spectral changes. The inactive enzyme spectrum retained some absorbance between 500 nm and 700 nm, showing a shoulder at 580–600 nm. Upon treatment of the enzyme with NADPH at pH 6.5 the spectrum remained unchanged, while no redox inactivation was observed under these conditions. It is suggested that the redox inactivation could be associated with the disappearance of the charge‐transfer complex between the proximal thiolate and oxidized FAD in the two‐electron‐reduced enzyme.
The inactive enzyme was reactivated by low GSSG concentrations, moderate dithiol concentrations, and high monothiol concentrations. These results and the spectral changes described above support the hypothesis attributing the redox interconversion to formation/disappearance of an erroneous disulfide between one of the half‐cystines located at the GSSG‐binding site and another cysteine nearby.
Chemical modification of the bifunctional human serum pseudocholinesterase
Authors: BOOPATHY Rathanam , BALASUBRAMANIAN Aiylam S. 
Published: March 2005

The effect of chemical modification on the pseudocholinesterase and aryl acylamidase activities of purified human serum pseudocholinesterase was examined in the absence and presence of butyrylcholine iodide, the substrate of pseudocholinesterase. Modification by 2‐hydroxy‐5‐nitrobenzyl bromide, N‐bromosuccinimide, diethylpyrocarbonate and trinitrobenzenesulfonic acid caused a parallel inactivation of both pseudocholinesterase and aryl acylamidase activities that could be prevented by butyrylcholine iodide. With phenyglyoxal and 2,4‐pentanedione as modifiers there was a selective activation of pseudocholinesterase alone with no effect on aryl acylamidase. This activation could be prevented by butyrylcholine iodide. N‐Ethylmaleimide and p‐hydroxy‐mercuribenzoate when used for modification did not have any effect on the enzyme activities. The results suggested essential tryptophan, lysine and histidine residues at a common catalytic site for pseudocholinesterase and aryl acylamidase and an arginine residue (or residues) exclusively for pseudocholinesterase.The use of N‐acetylimidazole, tetranitromethane and acetic anhydride as modifiers indicated a biphasic change in both pseudocholinesterase and aryl acylamidase activities. At low concentrations of the modifiers a stimulation in activities and at high concentrations an inactivation was observed. Butyrylcholine iodide or propionylcholine chloride selectively protected the inactivation phase without affecting the activation phase. Protection by the substrates at the inactivation phase resulted in not only a reversal of the enzyme inactivation but also an activation. Spectral studies and hydroxylamine treatment showed that tyrosine residues were modified during the activation phase. The results suggested that the modified tyrosine residues responsible for the activation were not involved in the active site of pseudocholinesterase or aryl acylamidase and that they were more amenable for modification in comparison to the residues responsible for inactivation.Two reversible inhibitors of pseudocholinesterase, namely ethopropazine and imimpramine, were used as protectors during modification. Unlike the substrate butyrylcholine iodide, these inhibitors could not protect against the inactivation resulting from modification by 2‐hydroxy‐5‐nitrobenzyl bromide, N‐bromosuccinimide and trinitrobenzenesulfonic acid. But they could protect against the activation of pseudocholinesterase and aryl acylamidase by low concentrations of N‐acetylimidazole and acetic anhydride thereby suggesting that the binding site of these inhibitors involves the non‐active‐site tyrosine residues.
Mammalian fatty acid synthetase is a structurally and functionally symmetrical dimer
Authors: SMITH Stuart , STERN Alan , RANDHAWA Zafar I. , KNUDSEN Jens 
Published: March 2005

We have explored a comprehensive experimental approach to determine whether the two condensing‐enzyme active centers of the mammalian fatty acid synthetase are simultaneously functional. Our strategy involved utilization of trypsinized fatty acid synthetase, which is a nicked homodimer composed of two pairs of 125 + 95‐kDa polypeptides. These core polypeptides lack the chain‐terminating thioesterase domains but retain all other functional domains of the native enzyme and can assemble long‐chain acyl moieties at a rate equal to that of the native enzyme. The 4′‐phosphopantetheine content of these enzyme preparations, estimated from the amount of β‐alanine present, from the amount of taurine formed by performic acid oxidation and from the amount of carboxymethylcysteamine formed by alkylation with iodo[2‐14C]acetate, was typically 0.86 mol/mol 95‐kDa polypeptide. The stoichiometry of long‐chain acyl‐enzyme synthesis, measured with radiolabeled precursors, indicated that 0.84 mol acyl‐chains were assembled/mol 95‐kDa polypeptide. When the small amount of apoenzyme present is taken into account, this stoichiometry translates to 1.94 acyl chains per holoenzyme dimer. The 125‐kDa polypeptide of one subunit could be cross‐linked to the 95‐kDa polypeptide of the other subunit by 1,3‐dibromo‐2‐propanone yielding a single molecular species of 220 kDa. Cross‐linking was accompanied by a loss of condensing‐enzyme activity. This result is consistent with a structurally symmetrical model for the animal fatty acid synthetase [J. K. Stoops and S. J. Wakil (1981) J. Biol. Chem. 256, 5128–5133] in which the juxtaposed 4′‐phosphopantetheine and cysteine thiols of opposing subunits that form the two potential catalytic centers for condensing activity are readily susceptible to cross‐linking. Both half‐maximal cross‐linking and 50% inhibition of activity were observed with 1 mol 1,3‐dibromo‐2‐propanone bound/mol enzyme. After assembly of long‐chain acyl moieties on the 4′‐phosphopantetheine residues, no vacant condensing‐enzyme active sites were demonstrable either by cross‐linking with 1,3‐dibromo‐2‐propanone or by formation of carboxymethylcysteamine on treatment with iodoacetate.These results are consistent with a structurally and functionally symmetrical model for the mammalian fatty acid synthetase in which the two condensation sites are simultaneously active.
Nuclear magnetic resonance studies of the old yellow enzyme
Authors: BEINERT Wolf‐Dieter , RÜTERJANS Heinz , MÜLLER Franz , BACHER Adelbert 
Published: March 2005

The apoenzyme of NADPH oxidoreductase, ‘old yellow enzyme’, was reconstituted with selectively 13C‐enriched flavin mononucleotides and investigated by 13C NMR spectroscopy. The 13C NMR results confirm the results obtained by 15N NMR spectroscopy and yield additional information about the coenzyme‐apoenzyme interaction. A strong deshielding of the C(2) and C(4) atoms of enzyme‐bound FMN both in the oxidized and reduced state is observed, which is supposed to be induced by hydrogen‐bond formation between the protein and the two carbonyl groups at C(2) and C(4) of the isoalloxazine ring system. The chemical shifts of all 13C resonances of the flavin in the two‐electron‐reduced state indicate that the N(5) atom is sp3‐hybridized.From 31P NMR measurements it is concluded that the FMN phosphate group is not accessible to bulk solvent. The unusual 31P chemical shift of FMN in old yellow enzyme seems to indicate a different binding mode of the FMN phosphate group in this enzyme as compared to the flavodoxins.The 13C and 15N NMR data on the old‐yellow‐enzyme–phenolate complexes show that the atoms of the phenolate are more deshielded whereas the atoms of the enzyme‐bound isoalloxazine ring are more shielded upon complexation. A non‐linear correlation exists between the chemical shifts of the N(5) and the N(10) atoms and the pKa value of the phenolate derivative bound to the protein. Since the chemical shifts of N(5), N(10) and C(4a) are influenced most on complexation it is suggested that the phenolate is bound near the pyrazine ring of the isoalloxazine system. 15N NMR studies on the complex between FMN and 2‐aminobenzoic acid indicate that the structure of this complex differs from that of the old‐yellow‐enzyme–phenolate complexes.
Role of plasma membrane transport in hepatic glutamine metabolism
Authors: HÄUSSINGER Dieter , SOBOLL Sibylle , MEIJER Alfred J. , GEROK Wolfgang , TAGER Joseph M. , SIES Helmut 
Published: March 2005

In livers of fed rats and in perfused livers supplied with a physiological portal glutamine concentration of 0.6 mM, the mitochondrial and cytosolic glutamine concentrations are 20 mM and 7 mM, respectively, thus, the mitochondrial/cytosolic glutamine concentration gradient is 2–3.
Uptake and release of glutamine by periportal and perivenous hepatocytes occurs predominantly by an Na+‐dependent transport system (so‐called system ‘N’).
Histidine in near‐physiological concentrations inhibits both glutamine uptake by periportal hepatocytes and its release by perivenous hepatocytes. This is not due to an inhibition of glutamine‐metabolizing enzymes by histidine or its metabolites. With physiological portal glutamine concentrations (0.6 mM), stimulation of glutaminase flux or of glutamine transaminase flux is followed by a decreased of hepatic glutamine levels to about 80% or 30%, respectively, glutamine levels are further decreased to 50% or 20% in the presence of histidine.
When glutamine is synthesized endogeneously (no glutamine added), the histidine‐induced inhibition of glutamine release is paralleled by a 210% increase of the hepatic tissue level of glutamine.
In experiments with and without methionine sulfoximine and in the absence of added glutamine, the glutamine content in the small perivenous hepatocyte population containing glutamine synthetase is estimated to be about 3.5 μmol/g wet weight and that in the periportal hepatocytes as low as 0.1 μmol/g wet weight.
In contrast to the prevailing view, it is concluded that glutamine transport across the plasma membrane of hepatocytes is a potential regulatory site in glutamine degradation and synthesis, especially under the influence of effectors like histidine.
Phase equilibria in four lysophosphatidylcholine/water systems
Authors: ARVIDSON Gösta , BRENTEL Ingvar , KHAN Ali , LINDBLOM Göran , FONTELL Krister 
Published: March 2005

The phase equilibria in four lysophosphatidylcholine/water systems were investigated at different temperatures. Each of the 1‐palmitoyl‐, 1‐stearoyl‐, 1‐oleoyl‐ and 1‐linoleoyl‐sn‐glycero‐3‐phosphocholines was dispersed in heavy water at different concentrations. The phase structures were determined by 2H‐, 14N‐ and 31P‐NMR, polarization microscopy and low‐angle X‐ray diffraction.The phase diagrams of the oleoyl and linoleoyl systems were quite similar. At room temperature and with decreasing water content the isotropic micellar solution was followed by a hexagonal phase and then a cubic phase. Finally the lamellar phase appeared before the region of hydrated crystals. The same sequence of phases was observed in the stearoyl system at elevated temperatures. The palmitoyl system differed from the others: here a cubic phase followed after the micellar solution, then came a hexagonal phase and after this a lamellar phase. In general the lysophosphatidylcholines seem to behave similarly to the many soaps and detergents as they show the same sequence of isotropic micellar solution, hexagonal phase, lamellar phase with interspersed cubic phases.The presently established phase diagrams demonstrate that the major lysophosphatidylcholines which may be generated by phospholipase A2 in mammalian cell membranes, viz. 1‐palmitoyl‐ and 1‐stearoyl‐glycerophosphocholines differ greatly in their packing properties. The extraordinary ability of 1‐palmitoyl‐glycerophosphocholine to form a cubic phase in equilibrium with a micellar solution is of particular interest with regard to the possible occurrence of cubic structures in biomembranes during the process of fusion.
Mitochondrial aldehyde dehydrogenase from human liver
Authors: HEMPEL John , KAISER Rudolf , JÖRNVALL Hans 
Published: March 2005

The 500‐residue amino acid sequence of the subunit of mitochondrial human liver aldehyde dehydrogenase is reported. It is the first structure determined for this enzyme type from any species, and is based on peptides from treatments with trypsin, CNBr, staphylococcal Glu‐specific protease, and hydroxylamine. The chain is not blocked (in contrast to that of the acetylated cytosolic enzmye form), but shows N‐terminal processing heterogeneity over the first seven positions. Otherwise, no evidence for subunit microheterogeneities was obtained. The structure displays 68% positional identity with that of the corresponding cytosolic enzyme, and comparisons allow functional interpretations for several segments.A region with segments suggested to participate in coenzyme binding is the most highly conserved long segment of the entire structure (positions 194–274). Cys‐302, identified in the cytosolic enzyme in relation to the disulfiram reaction, is also present in the mitochondrial enzyme. A new model of the active site appears possible and involves a hydrophobic cleft. Near‐total lack of conservation of the N‐terminal segments may reflect a role of the N‐terminal region in signaling the transport of the mitochondrial protein chains. Non‐conservation of interior regions may reflect the differences between the two enzyme forms in subunit interactions, explaining the lack ofheterotetrameric molecules. The presence of some internal repeat structures is also noted as well as apparently general features of differences between cytosolic and mitochondrial enzymes.
Identification of 2‐keto‐3‐deoxy‐1,7‐dicarboxyheptonic acid as a constituent of the lipopolysaccharide of Acinetobacter calcoaceticus NCTC 10305
Authors: BRADE Helmut , RIETSCHEL Ernst Th. 
Published: March 2005

A 3‐deoxyaldulosonic acid, not previously described, was identified as a component of the lipopolysaccharide of Acinetobacter calcoaceticus NCTC 10305: 2‐keto‐3‐deoxy‐1,7‐dicarboxyheptonic acid. This compound, whose steric configuration has not been determined, was released from lipopolysaccharide after acid hydrolysis as monosaccharide and in the form of a glucose‐substituted disaccharide. It was identified by combined gasliquid chromatography/mass spectrometry using various derivatization procedures. The presence of a 2‐keto‐3‐deoxyaldulosonic acid was established from the typical fragmentation pattern of the C1–C6 moiety which was identical to that of similarly similarly derivatized authentic 2‐keto‐3‐deoxy‐D‐Mannooctonic acid (dOclA). Location of the keto and deoxy function at C2 and C3, respectively, was deduced from the fragmentation pattern of the deuterolabelled derivatives after reduction of the keto and carboxy groups. The presence of two carboxy groups was ascertained after carboxy reduction with sodium boro‐(2H)hydride of the carbonyl‐reduced and permethylated derivative whereby the molecular masses were found to differ by 4 Da. Further proof for the presence of two carboxy groups was obtained by alkaline transesterification of the carbonyl‐reduced and permethylated derivative with ethanolic sodium ethylate: it was shown that two methyl ester groups had been transesterified since the molecular mass was shifted higher by 28 Da. Substitution by a D‐glucopyranosyl residue in position 4 was established by methylation analysis performed on the carbonyl‐ and carboxyl‐reduced and permethylated dissaccharide. The D‐configuration of the glucosyl residue was determined by its reactivity with D‐glucose oxidase.Thus, the structure of the disaccharide is 4‐O‐glucopyranosyl‐2‐keto‐3‐deoxy‐1,7‐dicarboxyheptonic acid.
Activation of glycolysis by insulin with a sequential increase of the 6‐phosphofructo‐2‐kinase activity, fructose‐2,6‐bisphosphate level and pyruvate kinase activity in cultured rat hepatocytes
Authors: PROBST Irmelin , UNTHAN‐FECHNER Kirsten 
Published: March 2005

The involvement of 6‐phosphofructo‐2‐kinase, fructose 2,6‐bisphosphate [Fru(2,6)P2] and pyruvate kinase in the insulin‐dependent short‐term activation of glycolysis was studied in primary cultures of rat hepatocytes. The short‐term influence of insulin on these parameters was dependent on the insulin concentration used for the longterm culture. Cells were cultured either with 10 nM or 0.1 nM insulin for 48 h, and are referred to as ‘insulin cells’ and ‘control cells’, respectively.1. Insulin cells exhibited a high level of Fru(2,6)P2. Addition of insulin to insulin cells led to an immediate stimulation of glycolysis (two‐fold) and activation of pyruvate kinase. The concentration of Fru(2,6)P2 and activity of 6‐phosphofructo‐2‐kinase remained constant.2. Control cells exhibited a very low level of Fru(2,6)P2 and low activity of 6‐phosphofructo‐2‐kinase directly after the medium change. However, both parameters increased during a 1–2‐h−incubation in the absence of insulin. Although the level of Fru(2,6)P2 thus changed up to tenfold the glycolytic rate remained at a constant value.3. Addition of insulin to control cells led to a 5–8‐fold stimulation of glycolysis but only after a 30–90‐min lag phase. During this lag period insulin strongly increased sequentially the 6‐phosphofructo‐2‐kinase, the level of Fru(2,6)P2 and the pyruvate kinase activity. The activation of the latter enzyme slightly preceded the onset of the insulin‐stimulated glycolysis.4. Addition of insulin to control cells, which were preincubated for 3 h in the absence of insulin and in which the Fru(2,6)P2 level had risen insulin‐independently, led to an immediate increase in glycolysis without a lag phase.It is concluded that in this insulin‐sensitive cell system: (a) the changes of glycolytic flux did not correlate with changes in the level of total Fru(2,6)P2 either in insulin or in control cells; (b) an increase in the Fru(2,6)P2 concentration was not obligatory for the insulin‐dependent stimulation of glycolysis in insulin cells; (c) activation of pyruvate kinase and thus glycolysis by insulin did not proceed unless the Fru(2,6)P2 level had been elevated above a threshold level.The lack of correlation between total Fru(2,6)P2 levels and the glycolytic flux and the apparent existence of a threshold concentration for Fru(2,6)P2 suggest a permissive action for this effector in enzyme interconversion.
Calcium release from intact calmodulin and calmodulin fragment 78–148 measured by stopped‐flow fluorescence with 2‐p‐toluidinylnaphthalene sulfonate
Authors: SUKO Josef , PIDLICH Johann , BERTEL Oswald 
Published: March 2005

Calcium release from high and low‐affinity calcium‐binding sites of intact bovine brain calmodulin (CaM) and from the tryptic fragment 78–148, purified by high‐pressure liquid chromatography, containing only the high‐affinity calcium‐binding sites, was determined by fluorescence stopped‐flow with 2‐p‐toluidinylnaphthalene sulfonate (TNS). The tryptic fragments 1–77 and 78–148 each contain a calcium‐dependent TNS‐binding site, as shown by the calcium‐dependent increase in TNS fluorescence. The rate of the monophasic fluorescence decrease in endogenous tyrosine on calcium dissociation from intact calcium‐saturated calmodulin (Kobs 10.8 s−1 and 3.2 s−1 at 25°C and 10°C respectively) as well as the rate of equivalent slow phase of the biphasic decrease in TNS fluorescence (Ksiowobs 10.6 s−1 and 3.0 s−1 at 25°C and 10°C respectively) and the rate of the solely monophasic decrease in TNS fluorescence, obtained with fragment 78–148 (Kobs 10.7 −1 and 3.5 s−1 at 25°C and 10°C respectively), were identical, indicating that the rate of the conformational change associated with calcium release from the high‐affinity calcium‐binding sites on the C‐terminal half of calmodulin is not influenced by the N‐terminal half of the molecule. The fast phase of the biphasic decrease of TNS fluorescence, observed with intact calmodulin only (Kfastobs 280 s−1 at 10°C) but not with fragment 78–148, is most probably due to the conformational change associated with calcium release from low‐affinity sites on the N‐terminal half.The calmodulin fragments 1–77 and 78–148 neither activated calcium/calmodulin‐dependent protein kinase of cardiac sarcoplasmic reticulum nor inhibited calmodulin‐dependent activation at a concentration approximately 1000‐fold greater (5 μM) than that of the calmodulin required for half‐maximum activation (5.9 nM at 0.8 mM Ca+2 and 5 mM Mg2+) of calmodulin‐dependent phosphoester formation.