bluuebunny/crossref_metadata_2025_split huggingface dataset:
The uncoupling protein dimer can form a disulfide cross‐link between the mobile C‐terminal SH groups
Authors: KLINGENBERG Martin , APPEL Maria 
Published: March 2005

Isolated uncoupling protein (UCP) can be cross‐linked, by various disulfide‐forming reagents, to dimers. The best cross‐linking is achieved with Cu2+‐phenanthroline oxidation. Because cross‐linking is independent of UCP concentration and prevented by SDS addition, a disulfide bridge must be formed between the two subunits of the native dimer. Cross‐linking is prevented by SH reagent and reversed by SH‐reducing reagents. In mitochondria, cross‐linking of UCP with disulfide‐forming agents is even more efficient than in isolated state. It proves that UCP is a dimer in mitochondria, before isolation. Disulfide‐bridge formation does not inhibit GTP‐binding to UCP. Cross‐linked UCP re‐incorporated in proteoliposomes either before or after cross‐linking fully retains the H+‐transport function.Rapid cross‐linking by membrane impermeant reagents indicates a surface localization of the C‐terminus in soluble UCP and projection to the outer surface in mitochondria. Intermolecular disulfide‐bridge formation in a dimer requires juxtaposition of identical cysteines at the twofold symmetry axis. A rigid juxtaposition of cysteines is unlikely, unless intended for a native disulfide bridge. The absence of such a bridge in UCP suggests that juxtaposition of cysteines is generated by high mobility. In order to localize the cysteine involved, cross‐linked UCP was cleaved by BrCN. The CB‐7 C‐terminal peptide, which contains cysteines at positions 287 and 304, disappears. Limited trypsinolytic cleavage, previously shown to occur at Lys‐292, removed cross‐linking in UCP both in the solubilized and mitochondrially bound state. The cleaved C‐terminal peptide of 11 residues contains only cystein‐304 which, thus, should be the only one (out of 7 cysteines in UCP) involved in the S—S bridge formation. Obviously, the C‐terminal location of the cysteine, because of its high mobility, permits juxtapositioning for cross‐linking. This agrees with predictions from hydrophobicity analysis that the last 14 residues in UCP protrude from the membrane.
Inhibition and active‐site modelling of prolidase
Authors: KING Glenn F. , CROSSLEY Maxwell J. , KUCHEL Philip W. 
Published: March 2005

Consideration of the active‐site model of prolidase led us to examine azetidine, pyrrolidine and piperidine substrate analogs as potential in vivo inhibitors of the enzyme. One of these, N‐benzyloxycarbonyl‐L‐proline, was shown to be a potent competitive inhibitor of porcine kidney prolidase (Ki= 90μM); its rapid protein‐mediated permeation of human and sheep erythrocytes suggests that it may be effective in vivo. The higher homolog, N‐benzyloxycarbonyl‐L‐pipecolic acid, was also a potent inhibitor of the enzyme while the antihypertensive drugs, captoprilR and enalaprilatR, were shown to have mild and no inhibitor effects, respectivelyAnalysis of inhibitor action and consideration of X‐ray crystallographic data of relevant Mn2+ complexes allowed the active‐site model of prolidase to be further refined; a new model is presented in which the substrate acts as a bidentate ligand towards the active‐site manganous ion. Various aspects of the new model help to explain why Mn2+ has been ‘chosen’ by the enzyme in preference to other biologically avialable metal ions.
Biochemical characterization of φX174‐protein‐E‐mediated lysis of Escherichia coli
Authors: WITTE Angela , LUBITZ Werner 
Published: March 2005

Energetic and permeability properties of Escherichia coli cells were determined prior to and during lysis caused by expression of the cloned gene E of bacteriophage φX174. Before onset of cell lysis the transmembrane gradients for K+, Na+ or Mg2+/ions, the level of ATP and the membrane potential, were unaffected. All these parameters changed simultaneously at the time of lysis onset, as monitored by measurements of culture turbidity as well as by determining the various specifications over a period of 1 min. During cell lysis chromosomal DNA was fragmented whereas plasmid DNA was liberated in its intact supercoiled form. Cytoplasmic constituents were released almost entirely, as indicated by the activity of β‐galactosidase in the supernatant fraction of protein‐Elysed cells.Periplasmic enzymes were only found in limited amounts in the cell supernatant and most remained associated with the cell ghosts. Such ghosts exhibited no gross cell damage or morphological alterations when compared with intact E. coli by light microscopy. All parameters investigated indicated that protein‐E‐mediated lysis of E. coli is caused by the formation of a transmembrane tunnel structure through the envelope complex of the bacterium.
Comparison of computer simulations of the F‐type and L‐type non‐oxidative hexose monophosphate shunts with 31P‐NMR experimental data from human erythrocytes
Authors: MCINTYRE Lisa M. , THORBURN David R. , BUBB William A. , KUCHEL Philip W. 
Published: March 2005

Mathematical modelling was used to predict the behaviour of the two most favoured schemes for the operation of the non‐oxidative hexose monophosphate shunt (HMS), the F‐type and the L‐type pathways. The models simulate the time courses of sugar‐phosphate concentrations when various substrates are metabolized via each pathway. A 31P‐NMR technique, with which to observe time courses of concentrations of sugar phosphates in a human red cell lysate, was developed. The accuracy of each hypothesised scheme was then evaluated by comparing predicted with observed data.The results were more consistent with time courses of sugar‐phosphate levels predicted by the F‐type (classical) pathway than those predicted by the L‐type model. However, the accumulation of sedoheptulose 1,7‐bisphosphate when a haemolysate was incubated with ribose 5‐phosphated showed that the F‐type pathway is not a complete description of the system of reactions. Transaldolase was demonstrated to be essential for the normal metabolism of sugar phosphates by haemolysates. The effects of the heat‐inactivation of transldolase on the metabolism of sugar phosphates were accurately predicted by the F‐type model.The relevance of attempting to describe the reaction of the non‐oxidative HMS as a distinct ‘pathway’ or ‘cycle’ is discussed.
Studies of the functional topography of Escherichia coli RNA polymerase
Authors: GRACHEV Mikhail A. , LUKHTANOV Evgeny A. , MUSTAEV Arkady A. , ZAYCHIKOV Evgeny F. , ABDUKAYUMOV Melis N. , RABINOV Igor V. , RICHTER Vladimir I. , SKOBLOV Yury S. , CHISTYAKOV Pavel G. 
Published: March 2005

A method is proposed for localization of the sites of affinity labelling of the β subunit of Escherichia coli RNA polymerase. The principle of this method is similar to that of the methods of rapid sequencing of nucleic acids.The polypeptide bearing a radioactive affinity label at one of the amino acid residues is subjected to short‐term treatment with cyanogen bromide. The conditions of this reaction are selected in such a way that less than one cleavage occurs on average per polypeptide chain. Two series of radioactive peptides are formed, one involving all the possible N‐terminal peptides and the other the C‐terminal peptides. The distribution of the lengths of these peptides is studied by means of gel electrophoresis and compared with the theoretical ones based on the known amino acid sequence of the β subunit. Obviously, the affinity label resides between the C‐terminus of the shortest N‐terminal radioactive peptide and the N‐terminus of the shortest C‐terminal radioactive peptide. In order to increase reliability and resolution of the method, partial trypsinolysis may be employed.The evidence obtained suggests that lysine residues over the regions 1036–1066, 1234–1242, and histidine‐1237 are situated in the nearest neighbourhood to, or directly involved in the formation of the active center of initiating substrate binding of the β subunit of E. coli RNA polymerase.
Metabolism of aspartate in Mycobacterium smegmatis
Authors: SRITHARAN Venkataraman , WHEELER Paul R. , RATLEDGE Colin 
Published: March 2005

Mycobacterium smegmatis grows best on l‐asparagine as a sole nitrogen source; this was confirmed. [14C]Aspartate was taken up rapidly (46 nmol · mg dry cells−1· h−1 from 1 mM l‐asparagine) and metabolised to CO2 as well as to amino acids synthesised through the aspartate pathway. Proportionately more radioactivity appeared in the amino acids in bacteria grown in medium containing low nitrogen.Activities of aspartokinase and homoserine dehydrogenase, the initial enzymes of the aspartate pathway, were carried by separate proteins. Aspartokinase was purified as three isoenzymes and represented up to 8% of the soluble protein of M. smegmatis. All three isoenzymes contained molecular mass subunits of 50 kDa and 11 kDa which showed no activity individually; full enzyme activity was recovered on pooling the subunits. Km values for aspartate were: aspartokinases I and III, 2.4 mM; aspartokinase II, 6.4 mM. Aspartokinase I was inhibited by threonine and homoserine and aspartokinase III by lysine, but aspartokinase II was not inhibited by any amino acids. Aspartokinase activity was repressed by methionine and lysine with a small residue of activity attributable to unrepressed aspartokinase I. Homoserine dehydrogenase activity was 96% inhibited by 2 mM threonine; isoleucine, cysteine and valine had lesser effects and in combination gave additive inhibition. Homoserine dehydrogenase was repressed by threonine and leucine. Only amino acids synthesised through the aspartate pathway were tested for inhibition and repression. Of these, only one, meso‐diaminopimilate, had no discernable effect on either enzyme activity.
Comparison of the two classes of binding sites (A and B) of type I and type II cyclic‐AMP‐dependent protein kinases by using cyclic nucleotide analogs
Authors: ØGRIED Dagfinn , EKANGER Roald , SUVA Robert H. , MILLER Jon P. , DØSKELAND Stein Ove 
Published: March 2005

cAMP analogs, all 96 of which were modified in the adenine moiety, were examined quantitatively for their ability to inhibit the binding of [3H]cAMP to each of the two classes (A and B) of cAMP‐binding sites of type I (rabbit skeletal muscle) and type II (bovine heart) cAMP‐dependent protein kinase. The study showed that analogs can be constructed that have a higher affinity than cAMP for a binding site. N6‐phenyl‐cAMP had 18‐fold increased affinity for site A of RI (AI) and 40‐fold increased affinity for site AII. 2‐chloro‐8‐methylamino‐cAMP had a 7‐fold increased affinity for BI, and 8‐(4‐chlorophenylthio)‐cAMP had 17‐fold increased affinity for BII.Analogs could discriminate between the two classes of binding sites by more than two orders of magnitude in binding affinity: 2‐chloro‐8‐methylamino‐cAMP had 170‐fold higher affinity for BI than for AI, and 2‐n‐butyl‐8‐thiobenzyl‐cAMP had 700‐fold higher affinity for BII than for AII. Analogs could also discriminate between the homologous binding sites of the isozymes: 2‐n‐butyl‐8‐bromo‐cAMP had 260‐fold higher affinity for AI than for AII (22‐fold higher for BII than BI), and 8‐piperidino‐cAMP had 50‐fold higher affinity for BII than for BI (and 50‐fold higher for AI than for AII).The data suggest the following conclusions. (a) Stacking interactions are important for the binding of cAMP to all the binding sites. (b) Subtle differences exist between the sites as to the optimal electron distribution in the adenine ring since modifications that withdraw electrons at C2 and donate at C8 favour binding to BI, and disfavour binding to AI and AII. (c) There are no hydrogen bonds between the adenine ring of cAMP and any of the binding sites. (d) All sites bind cAMP in the syn conformation. (e) The subsites adjacent to the N6 and C8 positions may have nonpolar neighbouring regions since hydrophobic substituents at N6 could increase the affinity for AI and AII and similar substituents at C8 could increase the affinity for BII. Finally, (f) the sites differed in their ability to accomodate bulky substituents at C2 and C8.For all compounds tested, their potency as activators of protein kinases I and II was found to correlate, in a predictable fashion, to their mean affinity for the two classes of binding sites, rather than to the affinity for only one of the sites.
Comparison of the NH2‐terminal amino acid sequences of the four non‐identical subunits of the NAD‐linked hydrogenases from Nocardia opaca 1b and Alcaligenes eutrophus H16
Authors: ZABOROSCH Christiane , SCHNEIDER Klaus , SCHLEGEL Hans G. , KRATZIN Hartmut 
Published: March 2005

The cytoplasmic, NAD‐linked hydrogenase of the Gram‐positive hydrogen‐oxidizing bacterium Nocardia opaca 1b was compared with the analogous enzyme isolated from the Gram‐negative bacterium Alcaligenes eutrophus H16. The hydrogenase of N. opaca 1b was purified by a new procedure applying chromatography on phenyl‐Sepharose and DEAE‐Sephacel with two columns in series. A homogeneous enzyme preparation with a specific activity of 74 μmol H2 oxidized · min−1· mg protein−1 and a yield of 32% was isolated. The A. eutrophus enzyme was purified as previously published. Both enzymes are tetrameric proteins composed of four non‐identical subunits (α, β, γ, δ). The four subunits of both of these enzymes were separated and isolated as single polypeptides by preparative polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate.Immunological comparison of the four subunits of the Nocardia hydrogenase with those of the Alcaligenes enzyme showed that the α, β, γ, and δ subunits of one organism were serologically related to the analogous subunits of the other organism. Among themselves, the four subunits do not have any serological relationship.The eight individual polypeptides were also compared with respect to the NH2‐terminal amino acid sequences determined by automated Edman degradation and to the amino acid compositions. Strong sequence similarities exist between the analogous subunits isolated from the two bacteria. Within the established N‐terminal sequences the similarities between both (α, β, γ, and δ) subunits amount to 63%, 79%, 80% and 65%, respectively. No similarities exist between the different, non‐analogous subunits α, β, γ, and δ.
Apolipoprotein‐E‐gene expression in rat liver during development in relation to insulin and glucagon
Authors: MANGENEY Marise , CARDOT Philippe , LYONNET Stanislas , COUPE Christine , BENAROUS Richard , MUNNICH Arnold , GIRARD Jean , CHAMBAZ Jean , BEREZIAT Gilbert 
Published: March 2005

An apolipoprotein‐E (apo‐E) cDNA probe, cloned by immunoscreening of a λGT11 rat liver cDNA library, was used to further characterize the expression of the apo‐E gene in rat liver during development, in relation to plasma insulin and glucagon levels.The apo‐E mRNA level was low in fetus liver, then abruptly increased at birth and rose further during the suckling period. It returned to the level at birth in 10‐week‐old adults. These variations were paralleled with dramatic changes in plasma glucagon, which rose at birth and remained high during suckling. At the same time, the insulin/glucagon molar ratio fell.Administration of N6, O2‐dibutyryl cAMP to 5‐day‐old rats resulted in a significant induction of liver apo‐E mRNA. Moreover, liver apo‐E mRNA rose in 10‐h‐fasted suckling rats as compared to controls, while plasma glucagon increased and the insulin/glucagon ratio decreased. Conversely, glucose feeding of suckling rats did not induce any increase in liver apo‐E mRNA, the insulin/glucagon ratio was 10‐fold higher than in fasted animals.Our results are consistent with liver apo‐E gene expression being under the control of plasma glucagon and of the glucagon/insulin balance.
Evidence for cytochrome oxidase subunit I and a cytochrome c– subunit II fused protein in the cytochrome ‘c1aa3’ of Thermus thermophilus
Authors: BUSE Gerhard , HENSEL Sieghard , FEE James A. 
Published: March 2005

The terminal cytochrome c1aa3 of the respiratory chain of Thermus thermophilus has been isolated and purified to homogeneity by a novel procedure. The two subunit proteins (55 and 33 kDa) have been characterized chemically. Computer searches with partial amino acid sequences obtained from both subunits show that the larger subunit belongs to the cytochrome oxidase subunit I protein family while the smaller covalently heme‐binding subunit is not a cytochrome c1 but appears to be a fused protein between cytochrome c and cytochrome oxidase subunit II.With respect to the 16‐S rRNA‐derived phylogeny of procaryotes, the results show that the genetic information for an O2‐reacting cytochrome oxidase (EC 1.9.3.1) existed already in early eubacteria.
Purification and characterization of ribonuclease M and mRNA degradation in Escherichia coli
Authors: CANNISTRARO Vincent J. , KENNELL David 
Published: March 2005

A previously unreported endoribonuclease has been identified in Escherichia coli, which has a preference for hydrolysis of pyrimidine‐adenosine (Pyd‐Ado) bonds in RNA. It was purified about 7000‐fold to give a single band after SDS/polyacrylamide gel electrophoresis; the eluted protein gave the same RNase specificity. The sizes of the native and denatured enzymes agreed suggesting that the enzyme exists as a monomer of approximately 26 kDa. It is called RNase M.The only other reported broadly specific endoribonuclease in E. coli is RNase I, a periplasmic enzyme. Based on differences in charge, heat stability and substrate specificity, it was clear that RNase M is not RNase I. The specificity of RNase M was remarkably similar to that of pancreatic RNase A even though the two enzymes differ in charge characteristics and size.Earlier studies had shown that mRNA from the lactose operon of E. coli is hydrolyzed in vivo primarily between Pyd‐Ado bonds [Cannistraro et al. (1986) J. Mol. Biol. 192, 257–274]. We propose that this major RNase activity accounts for these cleavages observed in vivo and that it is the endonuclease for mRNA degradation in E. coli.
Basic proline‐rich proteins of murine parotid glands
Authors: BANNISTER Andrew J. , DIVECHA Nullin , ASHMORE Michael , McDONALD Charles J. 
Published: March 2005

Five major basic polypeptides with characteristics typical of proline‐rich proteins, accumulated in parotid glands after long term isoprenaline treatment of Balb C mice. They were studied by two‐dimensional gel electrophoresis and designated B1°, B2′°, B2°, B3° and B4° on the basis of pI‐dependent mobility. They were not observed in the glands of normal mice and were precipitated when glands were homogenized in 10% trichloroacetic acid unlike the three isoprenaline‐induced proline‐rich proteins of murine parotid glands reported previously.Isoprenaline induced six proline‐rich in vitro translation products which were absent normally. Four of these species had pI‐dependent mobilities almost identical to B1°, B2°, B3° and B4°, indicating not only precursor/product relationships, but also that isoprenaline induced the accumulation of the proteins by regulating the mRNA.Identical salivary counterparts of the basic glandular proline‐rich proteins were not detected whereas a series of smaller and more basic isoprenaline‐induced polypeptides were observed in saliva (major speices B1s–B4s). The glandular proline‐rich proteins were secreted from parotid tissue in vitro and the data indicate that proline‐rich proteins are synthesised as precursors and processed into salivary form in the parotid glands after secretion. The relationships between the B‐type in vitro translation products, parotid gland precursors and salivary proteins were also confirmed immunologically.
Effect of the microenvironment on the tertiary structure of cytochrome P‐450 LM2
Authors: UVAROV Valentin Yu. , TRETIAKOV Vadim E. , LESHCHENKO Albina V. , RUKAVISHNIKOV Igor G. , DZHUZENOVA Cholpon S. , TRETIAKOVA Larisa Z. , ARCHAKOV Alexander I. 
Published: March 2005

The relation between microenvironment and the tertiary structure of cytochrome P‐450 LM2 has been investigated. No complete relaxation to the most active state of the native enzyme took place in the case of membrane‐incorporated hemoprotein with three or four intramolecular cross‐links.The spatial organization of the enzyme was predicted to determine the cross‐link location on the hemoprotein surface and membrane‐incorporated parts of the polypeptide chain. It was concluded on the basis of the predicted structure that hemoprotein has an amphipathic structure and, thus, the greater part of molecule is exposed to the water phase. Not more than one NH2‐terminal α helix is able to incorporate into the membrane. The location of this region is believed to control the formation of the catalytically‐active‐conformational state of cytochrome P‐450 LM2.
Sequence of the cysteinyl‐containing peptides of 4‐aminobutyrate aminotransferase
Authors: KIM Young Tae , CHURCHICH Jorge E. 
Published: March 2005

Three cysteine‐containing tryptic peptides were isolated and sequenced from mitochondrial 4‐aminobutyrate aminotransferase using DABIA (4‐dimethylaminoazobenzene‐4‐iodoacetamide) as specific labeling reagent for sulfhydryl groups. The enzyme is a dimer made up of two identical subunits, but four out of the six cysteinyl residues/dimer form disulfide bonds when treated with iodosobenzoate to yield inactive enzyme species.To identify the cysteinyl residues undergoing reversible oxidation/reduction, the S‐DABIA‐labeling patterns of the fully reduced (active) and fully oxidized (inactive) forms of the enzyme were compared. Tryptic digests of the reduced enzyme contained three labeled peptides. If the enzyme was treated with iodosobenzoate prior to reaction with DABIA and tryptic digestion, only one labeled peptide was detected and identified (peptide I), indicating that the two missing cysteinyl‐containing peptides (peptides II, III) have been oxidized.The sulfhydryl groups undergoing oxidation/reduction were found to be intersubunit, based on SDS/polyacrylamide gel electrophoresis results. The loss of catalytic activity of 4‐aminobutyrate aminotransferase by oxidation of sulfhydryl residues is related to constraints imposed at the subunit interface by the insertion of disulfide bonds.