bluuebunny/crossref_metadata_2025_split huggingface dataset:
Similarity of Acetylcholine Receptors of Denervated, Innervated and Embryonic Chicken Muscles
Authors: SUMIKAWA Katumi , BARNARD Eric A. , DOLLY J. Oliver 
Published: March 2005

The native (9‐S) form of the acetylcholine receptor, purified from chick embryonic or adult innervated or denervated chicken muscles, was shown in each case to contain three subunits of Mr about 40000 (predominant), 50000 and 54000. The 40000‐Mr subunit (termed α) appears to exist in two forms; the minor variant form of it, with Mr about 41000, may have a different carbohydrate content. Bromo[3H]acetylcholine, an affinity alkylating reagent for this receptor, labels the 40000‐Mr subunit and the accompanying 41000‐Mr variant. The apparent ratio of the subunits varies with the several methods of detection used (protein staining, radio‐iodination or tritiation) but the α subunit always predominates.The α subunit was separated in urea gel isoelectric focussing; its isoelectric point was identical there for the three receptors from innervated, denervated and embryonic muscles. Their respective peptide maps for the α subunit were identical, and likewise for the 54000‐Mr subunit. All of the evidence obtained shows that the subunit structures of the junctional, extra‐junctional and embryonic chick muscle receptors are identical.Although the α subunit in the receptor from Torpedo electric organ, cat muscle and chicken muscles is distinguishable in all three cases in dodecylsulphate gel electrophoresis, it is affinity‐labelled in each case. The peptide map of this subunit after limited proteolysis is also very similar in all three cases. It is concluded that the α subunits from these three diverse sources are closely related structurally.
Structural Studies on Microvillus Aminopeptidase from Pig Small Intestine
Authors: SVENSSON Birte , SJÖSTRÖM Hans , NORÉN Ove 
Published: March 2005

Two amphiphilic forms of intestinal microvillus aminopeptidase were obtained by purification of the enzyme in the absence and in the presence of aprotinin respectively. By subsequent treatment with trypsin in vitro a hydrophilic form was obtained. These three enzyme preparations all contained three subunits, A, B and C (molecular weights 168000, 118000 and 54000 respectively). The individual subunits were purified by gel filtration on Ultrogel AcA 22 in the presence of sodium dodecyl sulphate. Subunits A and B from aminopeptidase prepared in the presence of aprotinin contained both N‐terminal lysine and alanine, whereas N‐terminal alanine and glycine were found for both of these subunits from the other amphiphilic form. Conversion of amphiphilic aminopeptidase to the hydrophilic form in vitro led only to a change in the N terminus of the B subunit to a valine. N‐terminal serine was found in subunit C from all three forms of the aminopeptidase. The amino acid composition and peptide maps of the isolated subunits from one of the amphiphilic forms are reported. It is suggested that subunit B and C arise by cleavage of subunit A, whereas it seemed unlikely that C originates from B.Brush border membrane vesicles were cross‐linked by dimethyl‐3,3′‐dithiobispropionimidate, and the aminopeptidase subsequently isolated. Analysis of molecular weights and subunit composition of its constituent polypeptide complexes by two‐dimensional polyacrylamide slab gel electrophoresis in the presence of sodium dodecyl sulphate showed that AB, BBC, BCC, BB, BC and CC complexes had been formed. The subunit structures ABC and BBCC were thus proposed in agreement with the maximum molecular weight of the membrane‐bound enzyme previously determined as 330000.
Characterization of the Ca2+ Binding Sites of Calmodulin from Bovine Testis Using 43Ca and 113Cd NMR
Authors: ANDERSSON Thomas , DRAKENBERG Torbjörn , FORSÉN Sture , THULIN Eva 
Published: March 2005

The exchange rates of Ca2+ ions to the two classes of sites on calmodulin have been determined from the temperature dependence of the 43Ca NMR line width. The exchange rates were found to differ by a factor of about 40 at room temperature. The apparent pK value for one of these classes was estimated from the pH dependence of the 43Ca line width.The PK values of the two‐high‐affinity sites were found to differ about 0.1–1.Zn2+ ions were shown to bind to calmodulin and affect the exchange rates of Ca2+ and Cd2+ for all four sites.
Evolutionary Divergence of Chloroplast and Cytosolic Glyceraldehyde‐3‐phosphate Dehydrogenases from Angiosperms
Authors: CERFF Rüdiger 
Published: March 2005

Extracts from 13 different angiosperm species (spinach, mustard, pea, bean, tomato, cucumber, pumpkin, maize, sorghum, rye, wheat, oats, barley) were submitted to electrophoresis under nondenaturing conditions and stained for enzyme activities of cytosolic and chloroplast glyceraldehyde‐3‐phosphate dehydrogenases by a modified tetrazolium test of high sensitivity. Zymograms of the cytosolic enzyme revealed a single band of similar electrophoretic mobility for all but one species, the tomato, which displayed an ordered set of five different bands. In contrast, zymograms of the chloroplast dehydrogenase are highly different, containing between two and five distinct bands of variable electrophoretic mobilities according to the plant species examined. This variability of the native chloroplast enzyme is paralleled by a remarkable interspecific heterogeneity of the enzyme with respect to subunit size and number, as shown by dodecylsulfate electrophoresis of the purified chloroplast enzyme from 11 different angiosperm species.The present data suggest that cytosolic and chloroplast glyceraldehyde‐3‐phosphate dehydrogenases belong to two separate protein families of different evolutionary rate. While the cytosolic enzyme is probably an extremely conservative protein like the corresponding enzymes from animals, yeast and bacteria, the chloroplast enzyme seems to change rather rapidly during evolution.
Oxidase and Oxygenase Function of the Microsomal Cytochrome P450 Monooxygenase System
Authors: KUTHAN Hartmut , ULLRICH Volker 
Published: March 2005

The rates of the NADPH‐dependent formation of superoxide radicals and hydrogen peroxide have been measured in liver microsomes from phenobarbital‐pretreated rats. Correcting a quenching of O2− radicals by microsomes, a stoichiometry of O2− to H2O2 close to 2:1 was obtained. This, and the fact that pseudo‐substrates of microsomal cytochrome P450 like perfluoro‐n‐hexane and perfluorinated cyclohexane did not increase H2O2 formation in a catalase‐inhibited assay, rules out a two‐electron reduced oxygen species as the source of H2C2.The rates of O2− as well as H2O2 generation in the presence of 7‐ethoxycoumarin were equally inhibited by carbon monoxide (75%) and resulted in photochemical action spectra with a maximum reactivation at 450 nm. Using the same conditions the monooxygenation was inhibited to a high degree (83%) but without exogenous substrate the inhibition of H2O2 formation dropped to 55%.It was concluded that most of the O2− originated from the oxycomplex of cytochrome P450 and that substrates can modify the rates of its decomposition and sensitivity to carbon monoxide. No correlation of H2O2 formation or of substrate monooxygenation with the optical substrate binding spectra could be observed. From the pH dependence a proton‐assisted decomposition of oxy‐cytochrome P450 appears likely. H2O2 formation was only slightly decreased at 20 μM dioxygen suggesting that H2O2 formation via cytochrome P450 should also occur in vivo.
Solubilization and Partial Characterization of a Multienzyme Complex of DNA Synthesis from Human Lymphoblastoid Cells
Authors: WICKREMASINGHE R. Gitendra , YAXLEY John C. , HOFFBRAND A. Victor 
Published: March 2005

Gently lysed human lymphoblastoid cells (HPB‐ALL) catalysed incorporation of the distal DNA precursors [3H]thymidine, [3H]dTMP, and [3H]dUMP into DNA. Measurement of the [3H]dTTP, produced during the incorporation reactions, provided kinetic evidence for the channelling of the distal precursors into DNA by a multienzyme complex consisting of precursor‐synthesizing enzymes and of DNA polymerase.
In the presence of the DNA polymerase inhibitor, 1‐β‐d‐arabinofuranosylcytosine triphosphate, incorporation of [3H]dTMP into DNA was abolished. However, there was no accumulation of [3H]dTTP, suggesting that the flow of substrates through the complex was regulated by the activity of DNA polymerase.
Multienzyme complexes were detected by chromatography of a lysate on a column of Sepharose 6B. These complexes retained the ability to channel [3H]thymidine and [3H]dTMP into DNA.
Model for the Interaction of Membrane‐Bound Substrates and Enzymes
Authors: SCHEEL Günther , ACEVEDO Enrique , CONZELMANN Ernst , NEHRKORN Heidi , SANDHOFF Konrad 
Published: March 2005

Microsomes and synaptosomes isolated from calf brain contain a sialidase which cleaves ganglioside substrates. The hydrolysis of [3H]ganglioside GD1a by the membrane‐bound enzyme has been studied under various conditions.The reaction rate decreased with increasing ionic strength in the incubation mixture, and was progressively enhanced by increasing concentrations of the primary alcohols n‐pentanol to n‐octanol. This stimulation correlates quantitatively with an increase in membrane ‘fluidity’ caused by these alcohols as measured by fluorescence depolarization employing l,6‐diphenyl‐1,3,5‐hexatriene as probe. The dependence of the reaction rate on the amount of enzyme in the incubation mixture was linear only with water‐soluble substrates but not with the lipophilic ganglioside substrate. Evidence is presented that lipophilic substrate and enzyme interact mainly within the plane of the membrane presumably by lateral diffusion. Taking this into consideration Michaelis‐Menten theory was modified accordingly.As predicted, apparent Km values increased linearly with the amount of membrane‐bound enzyme added and decreased with the concentration of n‐hexanol in the incubation mixture. In the presence of varying n‐hexanol concentrations the apparent Km‐value decreased with increasing membrane ‘fluidity’, as measured by fluorescence depolarization of l,6‐diphenyl‐1,3,5‐hexatriene. On the other hand, as expected, V values were not affected by membrane ‘fluidity’ and increased linearly with the amount of membrane protein.
Dissociation and Assembly of Pyridine Nucleotide Transhydrogenase from Azotobacter vinelandii
Authors: VOORDOUW Gerrit , de HAARD Hans , TIMMERMANS Johannus A. M. , VEEGER Cees , ZABEL Pim 
Published: March 2005

Thermal inactivation studies of pyridine nucleotide transhydrogenase (55°C, pH 7.5, ionic strength 0.1 M) show a biphasic loss of enzyme activity with time. Approximately half of the activity is lost in both the rapid and the slow phase of the inactivation reaction.With electron microscopy it is found that the quaternary structure of transhydrogenase does not change during thermal inactivation. Using Naja naja venom phosphodiesterase to monitor the release of FAD it is shown that dissociation of FAD and activity loss are proportional at every degree of inactivation. The biphasic loss of enzyme activity thus indicates non‐equivalent positions for the subunits (Mr 54000, 1 FAD per subunit) in the transhydrogenase polymer.The process of thermal inactivation is greatly accelerated when the enzyme‐bound flavin is reduced by addition of NADH. The apoenzyme, obtained by gel filtration of transhydrogenase in the presence of NADH at 37°C, can be partly (10–15%) reactivated by addition of excess FAD. The dissociation of FAD under non‐reducing conditions at 55°C is irreversible.In a guanidine hydrochloride denaturing solvent (cGuHCl > 2 M, 25°C, pH 7.5) transhydrogenase dissociates into subunits. These are highly unfolded as judged by hydrodynamic criteria. Retransfer of the enzyme to conditions stabilizing the native conformation (cGuHcl < 0.3 M) leads to reactivation provided that FAD is also present in the reactivation mixture. The yield of the reactivation is low, 3–8% of the theoretical maximum, and the process is slow. Delaying the time of addition of FAD to the reactivation mixture causes a decrease in reactivation yield. The results indicate that FAD is inserted in an intermediate stage in the assembly path leading to active transhydrogenase.Polyacrylamide gel electrophoresis in combination with the Western blotting technique shows the presence of a covalent dimer in purified transhydrogenase preparations, which is not dissociated by treatment with dodecylsulphate and 2‐mercaptoethanol. Since a dimer or other form of high relative molecular mass is not detected in cell extracts it follows that such forms do not play a role in the assembly of the complex quaternary structure of the enzyme in vivo.
Dynamic Properties of Microtubules at Steady State in the Presence of Taxol
Authors: CAPLOW Michael , ZEEBERG Barry 
Published: March 2005

The dynamic properties of steady‐state microtubules in the presence of the antitumor drug taxol and GTP, but in the absence of microtubule‐associated proteins have been studied. The molecular rate constants for the loss or gain of subunits at steady state was found to be dramatically decreased as compared with that for microtubules formed in the presence of GTP and microtubule‐associated proteins but in the absence of taxol [Zeeberg, B., Reid, R., and Caplow, M. (1980) J. Biol. Chem. 255, 9891–98991. In light of this change it was surprising to find that the degrees of directionality for subunit flux into the microtubule at steady state are nearly identical within 1.5% of each other) in the two systems. One mechanism to account for this would be for taxol to cause a nearly identical decrease in the rate constants for subunit dissociation at both ends of the microtubule, with no effect on the rate constants for subunit addition.Similar results have previously been found in studies with an endogenous effector of the microtubule steady state, a protein kinase [Jameson, J. L. and Caplow, M. (1981) Proc. Natl Acad. Sci. USA, 78, 3413–34171. In this case it was found that phosphorylation of microtubule‐associated proteins altered the molecular rate constants for tubulin subunit addition and dissociation, but had no effect on the degree of directionality for subunit flux. It will be of interest to determine whether other exogenous or endogenous effectors also act in a manner such as to leave the directionality unaltered.
Photoelectric and Spectral Responses of Bacteriorhodopsin Modified by Carbodiimide and Amine Derivatives
Authors: OVCHINNIKOV Yuri A. , ABDULAEV Nazhmutdin G. , DERGACHEV Alexander E. , DRACHEV Alexander L. , DRACHEV Lei A. , KAULEN Andrey D. , KHITRINA Lyubov V. , LAZAROVA Zvetana P. , SKULACHEV Vladimir P. 
Published: March 2005

Photoelectric activity and photocycle kinetics of bacteriorhodopsin successively treated with a carbodiimide and several amine derivatives have been studied. Both spectral and electrical responses were initiated by a 15‐ns laser flash inducing a single turnover of bacteriorhodopsin. Photocycle kinetics were measured in a suspension of purple sheets. Photoelectric potential generation was monitored by a voltmeter, using a phospholipid‐impregnated collodion film covered on one side with purple sheets or proteoliposomes.It is shown that in bacteriorhodopsin treated with a carbodiimide derivative and ethylenediamine or methyl‐o‐arginine, significant acceleration of the millisecond phase of the photoelectric response is observed, whereas the kinetics of the microsecond phase remain unchanged. This effect is accompanied by an increase in the fast (τ 2 ms) component of the 412‐nm intermediate decay. Methylamine‐modified bacteriorhodopsin shows no such changes in electric and spectral kinetics.Treatment with ethylenediamine or arginine, rather than methylamine, desensibilizes, completely or partially, photoelectric response to inhibition by cationic effectors such as La3+ or by increase in the H+ concentration. At the same time modification sensibilizes this response to the inhibiting effect of an anion, citrate.Dark adaptation of bacteriorhodopsin modified by ethylenediamine or arginine slows down both microsecond and millisecond electrogeniac phases, whereas that of untreated or methylamine modified bacteriorhodopsin does not.Proteoliposomes of ethylenediamine‐modified bacteriorhodopsin generate a photoelectric potential of opposite direction (interior negative) as compared with those of unmodified or methylamine‐modified bacteriorhodopsin.It is concluded that the modifications by ethylenediamine and arginine change the decomposition of the 412‐nm intermediate in such a fashion that a portion of this intermediate decays faster. This results in accelerating those electrogenic phases which are associated with the 412‐nm intermediate decay. Such an effect may be due to replacement of negatively charged groups in the bacteriorhodopsin polypeptide chain by positively charged ones.Different methods for measurement of changes in the bacteriorhodopsin activity are discussed. The conclusion is made that the analysis of the kinetics of photoelectric potential generation in the purple sheet‐collodion film system is a sensitive probe for changes in bacteriorhodopsin activity.
Relationship between Membrane Integrity and the Inhibition of Host Translation in Virus‐Infected Mammalian Cells
Authors: LACAL Juan Carlos , CARRASCO Luis 
Published: March 2005

The inhibition of host protein synthesis after picornavirus infection, and its relationship to the modification of membrane permeability has been studied. In encephalomyocarditis (EMC)‐virus‐infected L cells, cellular protein synthesis is reduced from the third hour after infection, at which time an inhibition in 86Rb+ uptake by the infected cell was observed. The bulk of viral protein synthesis takes place in a cell in which a profound modification in the ionic concentration has occurred. This is in agreement with the idea that optimal viral translation requires a higher concentration of monovalent ions as compared to cellular protein synthesis.A parallel measurement of (a) protein synthesis, (b) 86Rb+ content, (c) membrane potential and (d) modification of permeability to hygromycin B in EMC‐virus‐infected and poliovirus‐infected HeLa cells, indicates that in EMC‐virus‐infected cells a correlation exists between membrane alterations and the inhibition of host protein synthesis. This correlation was not apparent in poliovirus‐infected cells, suggesting that in this system other factors, different from monovalent ion alterations, are involved in the shut‐off. However, in this system, the synthesis of viral proteins also takes place when the cell membrane has been modified and permeability to ions is altered.The analysis of the shut‐off of protein synthesis and permeability modification in different cell lines infected with EMC virus or poliovirus indicates that the kinetic pattern of the shut‐off is variable and depends on the cell line used and the multiplicity of infection used in each experiment.The ATP content of picornavirus‐infected cells was examined throughout infection. A decrease of this nucleotide in the infected cells was apparent from the third hour post‐infection and this factor could perhaps contribute to the inhibition of viral protein synthesis that occurs late in infection. Another factor that could influence this inhibition is the transport of amino acids. For instance, the transport of glycine decreased from the beginning of the infection in EMC‐virus‐infected cells and a leakage of methionine was also observed late in infection. These modifications in membrane permeability could contribute to the development of the cytopathic effect observed in virus‐infected cells.
The Binding of NADH and NADPH to Bovine‐Liver Glutamate Dehydrogenase
Authors: DELABAR Jean M. , MARTIN Stephen R. , BAYLEY Peter M. 
Published: March 2005

The binding of NADH and NADPH to bovine liver glutamate dehydrogenase has been studied using circular dichroism (CD) and fluorescence spectroscopy, under comparable conditions of enzyme concentration. Spectroscopic titrations have been analysed using non‐linear least‐squares techniques to determine dissociation constants and parameters for the different chromophoric portions of the bound coenzymes, according to different models for the binding equilibria.
For NADPH, enhancement of dihydronicotinamide fluorescence and positive CD at 340 nm reflect binding of the coenzyme at the active site (site I) via the dihydronicotinamide moiety with a dissociation constant KI of 50 μM; negative CD at 260 nm reflects the binding of a second molecule of coenzyme at the non‐active site (site II) via the adenine moiety with a KII of 650 μM.
For NADH, the same spectroscopic assignment is made; however, site II affinity is much greater (KII 20 μM) and exceeds the affinity at the active site (KI 50 μM).
In no case was spectroscopic evidence obtained with either coenzyme to support the concept of non‐identical active site binding as three sites IA and three sites IB per hexamer.
Coenzyme affinities are approximately twofold higher in triethanolamine buffer (0.05 + 0.1 M KCl, pH 7.6) compared with phosphate buffer (0.1 M at pH 7.0).
ADP enhances the CD of bound NADPH at 340 nm by up to twofold; phosphate ion also enhances the CD and binds with a Kd of 60–80 mM. GTP causes strong negative CD at 340 nm but only for the NADPH bound at site II. These results are consistent with independent binding sites for ADP (site II) and GTP (site III); phosphate ion is apparently able to affect certain properties of all three sites.
(1,3)‐β‐d‐Glucan Synthase from Budding and Filamentous Cultures of the Dimorphic Fungus Candida albicans
Authors: ORLEAN Peter A. B. 
Published: March 2005

UDPglucose: (1,3)‐β‐d‐glucan 3‐β‐d‐glucosyltransferase (EC 2.4.1.34) was obtained as a particulate fraction from cell‐free extracts prepared after mechanical breakage of cells of a strain of the dimorphic fungus Candida albicans. The properties of this glucosyltransferase were investigated. Budding and filamentous cultures of C. albicans were grown after dilution of a stationary phase inoculum of yeast cells into fresh medium at 30°C and 40°C respectively and the specific activities of (1,3)‐β‐d‐glucan synthase, obtained from budding and filamentous cultures harvested during the first 3 h of their growth, were compared.

UDPglucose was the only glucosyl donor in the reaction (assayed by following the incorporation of radioactivity from UDP[14C]glucose into polymer) and the radioactive product was exclusively β‐(1,3)‐glucan.
Glycogen synthase activity was not detected.
(1,3)‐β‐d‐Glucan synthase activity was enhanced by ATP and GTP.
A threefold increase in the specific activity of the glucosyltransferase was obtained when cell breakage, and subsequent steps in the preparation of the enzyme, were carried out in the presence of 25 μM GTP. A Km value for UDPglucose of 1.5–1.9 mM was obtained for the glucosyltransferase prepared in the presence or absence of GTP.
The glucosyltransferase reaction was not affected by ADPglucose, CDPglucose, GDPglucose or glucose 6‐phosphate, but was competitively inhibited by TDPglucose. GDPglucose was not a glucosyl donor under the present conditions.
There was no evidence that the product was N‐glycosidically linked to protein, since the reaction was neither enhanced in the presence of UDP‐N‐acetylglucosamine, nor inhibited by tunicamycin.
The specific activity of the glucosyltransferase from 3‐h‐old filamentous cultures was about 1.5‐times higher than that from 3‐h‐old budding cultures.
The specific activities of (1,3)‐β‐d‐glucan synthase prepared from budding and filamentous cultures of C. albicans during their first 90 min of growth were similar.