bluuebunny/crossref_metadata_2025_split huggingface dataset:
Reconstitution of Lactic Dehydrogenase after Acid Dissociation
Authors: ZETTLMEISSL Gerd , RUDOLPH Rainer , JAENICKE Rainer 
Published: March 2005

Reconstitution of lactic dehydrogenase from pig muscle after dissociation at acidic pH produces native tetramers and inactive aggregates, both of which have been previously described with respect to their structural properties and the kinetics of their formation. The ratio of the two fractions is determined by the relative rates of reassociation and aggregation [G. ZettlmeißI, R. Rudolph and R. Jaenicke (1979) Biochemistry 18, 5567–5571]. The present study is concerned with the influence of residual structural elements within the acid‐denatured monomers on the process of reconstitution.Long incubation at low pH leads to a marked decrease of the rate and yield of reactivation, caused by conformational changes within the partially unfolded monomers. These slow rearrangements are reflected by slight changes of the far‐ultraviolet circular dichroism of the acid‐dissociated monomers, indicating a decrease in helix content from 30 observed immediately after the transfer to low pH, to 25% after completion of the acid transition. The unchanged near‐ultraviolet circular dichroism and fluorescence properties prove local structural alterations to be of lesser importance compared to the changes of the residual backbone folding. Varying the experimental conditions during denaturation proves the slow decrease in the rate and yield of reactivation, as well as the changes in the far‐ultraviolet circular dichroism to be enhanced with incubation time and temperature (0–30°C). The first‐order rate constants of the decrease in the reactivation yield after incubation in 1 M glycine/H3PO4 pH 2.3, 0.1 M sodium phosphate pH 2.3, and 1 M H3PO4 were found to be in the same time range as the respective changes in the far‐ultraviolet circular dichroism.Apart from the similarity of the rate constants, the activation energy for both the acid induced changes of the yield of reactivation and the far‐ultraviolet circular dichroism is found to be identical (80 kJ/mol).The effects of low pH on the reconstitution of lactic dehydrogenase can be eliminated by complete disruption of the structure of the polypeptide chain, e.g. by adding 6 M guanidine · HCl. This proves unambiguously that the above‐mentioned structural rearrangements at acid pH are responsible for the observed decrease of the rate and yield of reactivation.
Interaction of Fluorescein Isothiocyanate with Nucleotide‐Binding Sites of the Ca‐ATPase from Sarcoplasmic Reticulum
Authors: PICK Uri 
Published: March 2005

We have previously shown that fluorescein 5′ isothiocyanate is a potent inhibitor of the Ca‐ATPase from sarcoplasmic reticulum [Pick, U. and Karlish, S. J. D. (1980) Biochim. Biophys. Acta, 626, 255–261] and completely blocks Ca uptake with ATP but not with acetyl phosphate [Pick, U. and Bassilian, S. (1981) FEBS Lett. 123, 127–130]. Here we show that fluorescein isothiocyanate inhibits in parallel all the ATP‐dependent partial reactions: the Ca‐ATPase, ATP‐dependent Ca uptake and phosphorylation by ATP whereas the phosphorylation reaction by inorganic phosphate is resistant to fluorescein isothiocyanate and Ca uptake with acetyl phosphate is only partly inhibited by the modification.The time course of inactivation by fluorescein isothiocyanate is biphasic and the rate of inactivation is markedly increased at alkaline pH. The observations that a lysine‐specific reagent (2,4,6‐trinitrobenzene sulfonic acid) com‐ petes with binding of fluorescein isothiocyanate and the pH dependence of inactivation strongly suggests binding to a lysine E‐amino group.Mg ions accelerate the rate of inactivation at pH 7.5–8.0 and Ca ions slightly inhibit the inactivation and the binding of fluorescein isothiocyanate.The adenine nucleotides ATP and AdoPP [NH]P protected against inactivation by fluorescein isothiocyanate. A kinetic analysis of the initial rates of inactivation indicates competition between ATP and fluorescein isothiocyanate (the apparent dissociation constant KATP= 38 μM) and suggests a common binding site. Ca ions increase the apparent affinity of the non‐hydrolyzable analog AdoPP [NH]P by an order of magnitude but have no appreciable effect on the apparent affinity of ATP. Mg ions decrease the apparent affinity for adenine nucleotides both in the presence and absence of Ca.It is suggested that (a) fluorescein isothiocyanate binds to and modifies the ATP‐binding site of the enzyme. (b) The enzyme has only one ATP‐binding site but the site exists in two different configurations which sequentially appear at the E2 and the El conformations of the enzymes; and correspond to the ‘regulatory’ ATP‐binding site and to the phosphorylation site and (c) the site is blocked by fluorescein isothiocyanate in both configurations.
Proton Nuclear‐Magnetic‐Resonance Spectroscopy of Myosin Subfragment 1 Isoenzymes
Authors: PRINCE H. Paul , TRAYER Hylary R. , HENRY Gillian D. , TRAYER Ian P. , DALGARNO David C. , LEVINE Barry A. , CARY Peter D. , TURNER Christopher 
Published: March 2005

High‐resolution proton NMR spectrocopy has been used to study the solution structures of the subfragment 1 (S1)isoenzymes (containing either the A1 or A2 light chains) from rabbit skeletal muscle myosin and to investigate their interaction with actin. Superimposed upon broad components, the narrow signals of the S1 spectra are unexpectedly sharp, indicating that domains of varying sidechain mobility occur in the conformation adopted solution. These observations are in agreement with previous studies of the mixed isoenzymes [Highsmith et al. (1979) Biochemistry, 18, 4238 ‐42431]. Peptide amide exchange studies show also that the S1 structure accommodates fluctuations of sufficient amplitude to allow most of the peptide groups to come into contact with the solvent on the time scale‐of the 1H‐NMR experiment. The overall impression is that S1 is a molecule possessing backbone motility well as domains of different sidechain mobility.Close comparison of the Sl(A1) and Sl(A2) spectra indicate that the N‐terminal41 residues of the A1 light chain, rich in lysine, proline and alanine, display a high degree of segmental mobility. The difference spectrum [S1(A1)‐S1(A2)] obtained closely resembles the spectral simulation of the 41‐residue segment. Upon addition of actin, many of the narrow S1 resonances decrease in intensity or progressively disappear altogether, indicative of intermediate‐ slow exchange conditions consistent with the recognised high affinity between the two proteins. These changes are interpreted as an overall modulation in the observed and hence more mobile regions of S1 as has been suggested earlier 1H‐NMR studies referred to above. In particular, the differences noted between S1(A1) and S1(A2) have now largely disappeared in their complexes with actin indicating a marked reduction in the segmental mobility of the N‐terminal region of the light chain in S1(A1). Together with other affinity chromatography results [Winstanley and Trayer (1979) Biochem. Soc. Trans. 7, 703–704], this is good evidence for a direct interaction between this area of Sl(A1) and actin.
Glycoproteins Containing Peanu t‐Agglutinin Receptors from Human‐Peripheral‐ Blood T‐Lymphocyte Plasma Membranes
Authors: FARRAR Graham H. , HOLZ Gisela , UHLENBRUCK Gerhard 
Published: March 2005

A glycoprotein fraction possessing peanut agglutinin receptors has been isolated from detergent extracts of neuraminidase‐treated human peripheral blood T‐lymphocyte plasma membranes with affinity matrices comprising the peanut agglutinin co‐valently immobilised on Sepharose 4B.This fraction could be specifically eluted from affinity columns using buffer solutions supplemented with 0.2 M d‐galactose and was shown, by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate (0.1 %), to contain four major glycoprotein components with apparent molecular weights of 200000, 190000, 110000 and 95000.It is suggested, from the observed reactivity of these glycoproteins with various lectins in double‐diffusion experiments, that they possess both O‐glycosidically and N‐glycosidically linked carbohydrates.
Nucleotide Sequence and Secondary Structure of Citrus Exocortis and Chrysanthemum Stunt Viroid
Authors: GROSS Hans J. , KRUPP Guido , DOMDEY Horst , RABA Manfred , JANK Peter , LOSSOW Christine , ALBERTY Heidemarie , SÄNGER Heinz L. , RAMM Karla 
Published: March 2005

The complete nucleotide sequence of citrus exocortis viroid (CEV, propagated in Gymura) and chrysanthemum stunt viroid (CSV, propagated in Cineraria) has been established, using labelling in vitro and direct RNA sequencing methods and a new screening procedure for the rapid selection of suitable RNA fragments from limited digests.The covalently closed circular single‐stranded viroid RNAs consist of 371 (CEV) and 354 (CSV) nucleotides, respectively. As previously shown for potato spindle tuber viroid (PSTV, 359 nucleotides), CEV and CSV also contain a long polypurine sequence. Maximal base‐pairing of the established CEV and CSV sequences results in an extended rod‐like secondary structure similar to that previously established for PSTV and as predicted from detailed physicochemical studies of all these viroids. Although the three viroid species sequenced to date differ in size and nucleotide sequence, there is 60–73% homology between them. As PSTV, CEV and CSV also contain conserved complementary sequences which are separated from each other in the native secondary structure. We postulate that the resulting ‘secondary’ hairpins, being formed and observed in vitro during the complex process of thermal denaturation of viroid RNA, must have a vital, although yet unknown, function in vivo. The possible origin and function of viroids are discussed on the basis of the characteristic structural features and of a considerable homology with Ula RNA found for a region highly conserved in the three viroids.
Glutathione Reductase from Human Erythrocytes
Authors: KRAUTH‐SIEGEL R. Luise , SALEH Mansoor , UNTUCHT‐GRAU Renate , SCHIRMER R. Heiner , BLATTERSPIEL Rosi , SCHILTZ Emile 
Published: March 2005

Sequence analysis of the NADPH domain (residues 158 – 293) and of the interface domain (365 – 478) was based on 12 CNBr fragments, which were isolated using ion‐exchange chromatography and paper methods. Fragments with more than 15 residues were digested further with trypsin and chymotrypsin. The isolated peptides were sequenced by automated solid‐phase Edman degradation. All sequenced peptides were ordered and overlapped by computerized comparisons with a complete sequence guessed from the electron density map of the protein. In the case of short CNBr fragments, this alignment was confirmed by the sequence analysis of protein fragments resulting from incomplete CNBr cleavage.
In the NADPH domain, residue 197, which is involved in an induced‐fit mechanism, was identified as a tyrosine. The structure of the NADPH domain is probably homologous with the NAD domain of lipoamide dehydrogenase and with the FAD domain of several proteins, but not with NADPH domains of known chainfold in other proteins.
The paper completes the sequence analysis of glutathione reductase so that the enzyme is now known in atomic detail. The numbering scheme of the chemically determined sequence will be used henceforth in crystallo‐ graphic studies also. As inferred from the sequence data each of the two identical chains contains 478 amino acid residues, the composition being Cys10, Asp21, Asn17, Thr31, Ser31, Glu29, Gln11, Pro24, Gly43, Ala42, Val44, Met15, He29, Leu34, Tyr13, Phe14, Lys34, His16, Argl7, and Trp3. From these data an Mr of 2 × 51600 was calculated for the FAD‐free apoenzyme and an Mr of 2 × 52400 for the holoenzyme.
Evidence for the Existence of Actomyosin ATPase in the Rat Pancreas
Authors: VANDERMEERS André , VANDERMEERS‐PIRET Marie‐Claire , HEPBURN Anne , RATHE Jean , CHRISTOPHE Jean 
Published: March 2005

In a crude extract of rat pancreas, myosin was associated with a protein having the same electrophoretic mobility as actin. This myosin was purified after dissociation of the actomyosin complex with KI‐ATP. On sodium dodecyl‐sulfate/acrylamide gel electrophoresis, the isolated pancreatic myosin showed a major component of approxi‐ mately 200 kDa, and two smaller components with apparent molecular weight of 22 and 15 kDa, respectively.This purified myosin exhibited high ATPase activity in the presence of K++ EDTA or Ca2+ and very little activity in the presence of Mg2+. (K++ EDTA)‐ATPase activity showed one pH optimum at 8.0, while Ca2+‐ATPase activity showed two pH optima at 6.0 and 9.0, respectively. (K++ EDTA)‐stimulated enzyme activity was specific for ATP whereas Ca2+‐stimulated activity showed low specificity for nucleoside triphosphates.
Incorporation of Deoxyribonucleosides into DNA of Coryneform Bacteria and the Relevance of Deoxyribonucleoside Kinases
Authors: AULING Georg , PRELLE Hans , DIEKMANN Hans 
Published: March 2005

In order to obtain basic knowledge of the salvage pathways for DNA synthesis, the ability of Brevibacterium ammoniagenes ATCC 6872 and Micrococcus luteus ATCC 15932 for incorporation of nucleobases and nucleo–sides was investigated. Only adenine and uracil are incorporated by B. ammoniagenes, whereas M. luteus additionally can utilize deoxyadenosine and, less efficiently, thymidine.In M. luteus, the demonstration of deoxyadenosine kinase and thymidine kinase explains the incorporation data. Uptake of thymidine is of short duration because of rapid breakdown of exogenously supplied thymidine to thymine. At a 540–fold excess pyrimidine deoxyribonucleosides inhibit 14C incorporation from thymidine nearly totally and purine deoxyribonucleosides cut by half the uptake rate, probably by interfering with transport of thymidine. However, as no cessation of thymidine incorporation occurs at these concentrations of purine deoxyribonucleosides, incorporation is finally enhanced. During the initial period of this reduced uptake considerable protection of thymidine from breakdown to thymine is provided by deoxyguanosine, but not by deoxyadenosine. At a 108–fold excess there is actually no inhibition of thymidine uptake by deoxyguanosine and only an insignificant impairment by deoxyadenosine resulting in an ultimate enhancement of 14C incorporation up to 20%of the exogenously supplied thymidine.As there is no salvage pathway for thymidine in B. ammoniagenes due to the absence of thymidine kinase, labelling with adenine and hydrolyzing of the ‘contaminated’ RNA fraction with 1 M KOH is recommended for measurements of overall DNA synthesis in this strain.
The Role of Biotin and Sodium in the Decarboxylation of Oxaloacetate by the Membrane‐Bound Oxaloacetate Decarboxylase from Klebsiella aerogenes
Authors: DIMROTH Peter 
Published: March 2005

The biotin‐containing oxaloacetate decarboxylase from Klebsiella aerogenes catalyzed the Na+‐dependent decarboxylation of oxaloacetate to pyruvate and bicarbonate (or CO2) but not the reversal of this reaction, not even in the presence of an oxaloacetate trapping system.The enzyme catalyzed an avidin‐sensitive isotopic exchange between [1‐14C]pyruvate and oxaloacetate, which indicated the intermediate formation of a carboxybiotin enzyme. Sodium ions were not required for this partial reaction, but promoted the second partial reaction, the decarboxylation of the carboxybiotin enzyme, thus accounting for the Na+ requirement of the overall reaction. Therefore, the 14CO2‐enzyme which was formed upon incubation of the decarboxylase with [4‐14C]oxaloacetate, could only be isolated if Na+ ions were excluded. Preincubation of the decarboxylase with avidin also prevented its labelling with 14CO2.The isolated 14CO2‐labelled oxaloacetate decarboxylase revealed the following properties. It was slowly decarboxylated at neutral pH and rapidly upon acidification. The 14CO2 residues of the 14CO2‐enzyme could be transferred to pyruvate yielding [4‐14C]oxaloacetate. In the presence of Na+ this 14CO2 transfer was repressed by the simultaneous decarboxylation of the 14CO2‐enzyme. However, Na+ alone was insufficient as a cofactor for the decarboxylation of the isolated 14CO2‐enzyme, since this required pyruvate in addition to Na+. It is therefore concluded that the decarboxylation of oxaloacetate proceeds over a CO2‐enzyme‐pyruvate complex and that free CO2‐enzyme is an abortive reaction intermediate.The activation energy of the enzymic decarboxylation of oxaloacetate changed with temperature and was about 113 kJ below 11 °C, 60 kJ between 11 °C and 31 °C and 36 kJ between 31–45 °C.
The Generation of an Electrochemical Gradient of Sodium Ions upon Decarboxylation of Oxaloacetate by the Membrane–Bound and Na+–Activated Oxaloacetate Decarboxylase from Klebsiella aerogenes
Authors: DIMROTH Peter 
Published: March 2005

Inverted membrane vesicles from Klebsiella arragenes containing the Na+‐activated oxaloacctate decarboxylase accumulated 22Na+ in response to the decarboxylation of oxaloacetate. Complexation of the biotin prosthetic group of the decarboxylase with avidin completely prevented the oxaloacetate‐dependent Na+ uptake, whereas avidin saturated with biotin was without effect.The rapid accumulation of 22Na+ inside the vesicles following oxaloacetate addition was succeeded by a slower efflux of 22Na+ after exhaustion of oxaloacetate. Other substrates, e.g. d‐lactate, were unable to replace oxaloacetate in accumulating 22Na+ to a comparable extent, with the exception of (S)‐malate, which acted through its conversion to oxaloacetate. The amount of Na+ which was transported into the vesicles by a limited amount of oxaloacetate critically depended on the rate of oxaloacetate decarboxylation. This rate was retarded by using a low temperature (0 °C), by inhibiting the enzyme with oxalatc or by using oxaloacetate‐generating systems, where the activity of the enzyme catalyzing oxaloacetate formation was rate‐determining. With these retarded systems of oxaloacetate decarboxylation the efficiency of the Na+ transport was significantly increased. Under optimized conditions Na+could be concentrated inside the inverted vesicles eight‐times higher than in the incubation medium.The Na+‐carrying ionophores nigericin and trinactin effectively prevented the oxaloacetate‐dependent Na+ accumulation in a K+‐free incubation medium. In the presence of valinomycin and K+, however, the amount of Na+ which was taken up by the vesicles following oxaloacetate addition increased by a factor of two, which indicated limitation of Na+ uptake by electrical parameters. The generation of a membrane potential was determined from the distribution of the lipophilic anion [14C]SCN‐between interior and exterior vesicular spaces. The oxaloacetate‐decarboxylase‐driven Na+ transport into inverted vesicles at 0°C generated a membrane potential of 65 mV, an Na+ gradient equivalent to 49 mV and a total driving force of 114 mV. The proton carrier carbonylcyanide‐p‐trifluoromethoxy phenylhydrazone had essentially no effect on the oxaloacetate‐de‐carboxylase‐driven Na+ uptake, indicating that thc gcneration of a proton gradicnt is not involved. It is concluded from these results that oxaloacetate decarboxylase acts as a primary active Na+ pump which generates an electrochemical gradient of Na+ ions.
Uridylate Trapping Induced by the C‐2‐Modified d‐Glucose Analogs Glucosone, Fluoroglucose, and Glucosamine
Authors: HOLSTEGE Axel , SCHULZ‐HOLSTEGE Christa , HENNINGER Hanspeter , REIFFEN Karl A. , SCHNEIDER Friedhelm , KEPPLER Dietrich O. R. 
Published: March 2005

The interference of nucleotide metabolism induced by d‐arabino‐hexos‐2‐ulose (glucosone), 2‐deoxy‐2‐fluoro‐d‐glucose (fluoroglucose) and, for comparison, 2‐amino‐2‐deoxy‐d‐glucose (glucosamine) was studied in suspension cultures of TA3 mammary tumor cells. High‐performance liquid chromatography, [14C]uridine labeling and enzymatic analyses revealed that glucosone is metabolized to the UDP derivative leading to a diversion of [14C]uridylate from UTP into UDP‐glucosone. The accumulation of UDP‐glucosone, that was measured in TA3 as well as AS‐30D hepatoma cells, was associated with a depletion of UTP pools to less than 15% of control and a reduction of CTP contents to one third. UTP and CTP deficiency could be rapidly rcversed by addition of uridine to the cell suspension.Fluoroglucose acted both as a uridylate‐trapping and as a guanylate‐trapping hexose analog lowering the cellular contcnts of UTP and GTP to 5% and 27% of control, respectively. The lowering of GTP was accompanied by an increase in total acid‐soluble guanine nucleotides and a diversion of 84% of the [14C]guanylate into GDP‐fluoroglucose.As compared to fluoroglucose and glucosone, glucosamine was less effective in TA3 cells in decreasing the content of UTP. The uridylate‐trapping effect of glucosamine was in part compensated by the most active expansion of the acid‐soluble uridylate pool; this reflects an enhanced rate of dc novo pyrimidine synthesis that could be suppressed by the inhibitor 6‐azauridine.As a side effect probably resulting from phosphate trapping, glucosone, fluoroglucose, and glucosamine depressed the levels of ATP and of total adenine 5′‐nucleotides to a similar though moderate extent.The depletion of UTP, induced by these C‐2‐modified d‐glucose analogs, and additionally of GTP in the case of fluoroglucose, represent a metabolic lesion that may explain their growth inhibitory and cytotoxic effects.
Fragmentation and Modification of r‐Carrageenan and Characterisation of the Polysaccharide Order‐Disorder Transition in Solution
Authors: REES David A. , WILLIAMSON Frank B. , FRANGOU Sarah A. , MORRIS Edwin R. 
Published: March 2005

A procedure is described in detail by which the algal polysaccharide, r‐carrageenan, can be modified and fragmented to products of more regular structure and shorter chain length. Since most chains of this product can form uninterrupted helices they undergo the disorder‐order transition without the gelation that is a feature of the behaviour of the natural polymer.The disorder‐order transition becomes much sharper and occurs at higher temperaturcs with increasing chain length. The sign and magnitude of the accompanying shift in optical rotation can be estimated satisfactorily from the solid‐state geometry of the helix using our semi‐empirical method for correlating optical rotation with the conformations at glycosidic linkages. In solutions of the helical form, but not the coil form, cationic dyes such as methylene blue or acridine orange show induced optical activity and a changed visible spectrum which we attribute to binding and stacking on the helix. This also causcs a shift in the temperature of the helix‐coil transition. We discuss the possibility that the tendency of dyes to stack could drive polysaccharides into conformations that they would not otherwise adopt, with implications for their use as ‘conformational probes’.