bluuebunny/crossref_metadata_2025_split huggingface dataset:
Perchlorate Binding to Cytochrome c
Authors: ANDERSSON Thomas , ÅNGSTRÖM Jonas , FALK Karl‐Erik , FORSÉN Sture 
Published: March 2005

1. The effects of perchlorate on cytochrome c have been investigated by 1H and 35Cl NMR, electron paramagnetic resonance and optical spectroscopy.2. The pK values for the formation and disappearance of the major alkaline conformation were found to be displaced from 9.3 to 8.3 and from 10.4 to 10.9, respectively. The displacement was dependent on the ClO4− concentration below 0.1 M.3. Competition experiments between perchlorate and chloride show that ClO4− binds both to the neutral and alkaline forms but with a higher affinity for the latter. The appearance of a new binding site in the alkaline form accounts for the markedly enhanced relaxation rate of 35ClO4− in this pH range. Complex formation between cyanide and the alkaline species results in the loss of this binding site, which probably is located close to or within the heme crevice.4. The neutral form of ferricytochrome c also binds perchlorate strongly as evidence by the unique appearance of a high‐spin signal dependent on pH and perchlorate concentration. This signal disappears with the same pK value as the neutral form.The effects of perchlorate on cytochrome c are due to specific binding of this ion.
Cryoenzymic Studies on the Transition‐State Analog Complex Creatine Kinase ‐ ADPMg ‐ Nitrate ‐ Creatine
Authors: TRAVERS Franck , BARMAN Thomas E. 
Published: March 2005

Concomitant with the formation of the transition state analog complex creatine kinase ‐ ADPMg. nitrate ‐ creatine [Milner‐White, E. J. and Watts, D. C.1971 Biochem. J. 122, 727–7401 there results a large difference spectrum. The shape of the spectrum was characteristic of tryptophan exposure.The kinetics of formation and final amplitude of the spectrum were studied at – 15°C using a stopped‐flow apparatus. The results obtained allowed for a plausible reaction pathway for nitrate fixation: the ordered addition of nitrate and creatine to the binary enzyme‐ADPMg complex, a slow protein isomerization and the final addition of a molecule each of nitrate and creatine.The kinetic parameters for the formation of the transition state analog complex were compared with those already known for the overall reaction obtained under the same conditions [Barman, T. E., Brun, A. and Travers, F.1980 Eur. J. Biochem. 110, 397–403], This comparison revealed a striking analogy between the two processes. This suggests that the conformation of creatine kinase in the analog complex is very similar to that in the transition state complex on the catalytic pathway.
Transcriptional and Translational Control of Glucose‐Stimulated (Pro)insulin Biosynthesis
Authors: JAHR Henning , SCHRÖDER Dieter , ZIEGLER Brigitte , ZIEGLER Manfred , ZÜHLKE Hartmut 
Published: March 2005

Islets of Langerhans were isolated from the pancreata of fed or 48‐h‐fasted Wistar rats. The islets were incubated with either [3H]leucine of [3H]uridine. Inhibition of RNA synthesis by actinomycin D or by α‐amanitin for 4 h had no influence on the (pro)insulin biosynthesis of isolated islets of fed rats. The (pro)insulin biosynthesis was not inhibited after two days incubation of islets of fed rats with α‐amanitin either. Incorporation of labelled uridine into total RNA for 3 h was stimulated by glucose in islets of fasted, but not of fed rats. Therefore, it was concluded that transcriptional control does not participate, even for longer periods than believed previously, in acute regulation of (pro)insulin biosynthesis of islets isolated from fed rats. Despite the strong and preferential stimulation of (pro)insulin biosynthesis of islets of fed rats by glucose the radioactivity of the [3H]uridine‐labelled polysomes active in proinsulin synthesis remained unchanged.To interprete these experimental data we suggest that glucose triggers the transformation of a translationally inactive form of pre‐proinsulin mRNA to a translationally active form.
A Switch from Translational Control to Transcriptional Control of Protein Synthesis in Mid‐exponential Growth Phase of Bacterial Cultures
Authors: BAHRAMIAN Mohammad B. , HARTLEY Brian S. 
Published: March 2005

1. We present evidence suggesting a sudden switch from translational control to transcriptional control of protein synthesis in mid‐exponential growth of bacterial batch cultures. At a critical cell density a switch from large to small polysomes occurs during a short period of exponential growth. The profile of specific polysomes engaged in synthesis of a constitutive enzyme, ribitol dehydrogenase, changes at the same point but in an opposite way: a linear profile peaking at monosomes changes to a dome‐shaped profile peaking at about 15 ribosomes/mRNA, which persists into late exponential phase despite a gradual reduction in the total polysome population. The switch in the pattern of protein synthesis is exhibited dramatically by changes in the specific activity of ribitol dehydrogenase in cell extracts at different stages of batch culture. In early exponential phase the specific activity of the enzyme is constant, but it begins to rise suddenly, at the same point at which the polysome profiles change, and continues to increase up to the end of exponential phase. This effect is exhibited by the strains of Klebsiella aerogenes taht are inducible for (in the presence of the inducer), consitutive for, or superproducers of ribitol dehydrogenase, and it appears to be unrelated to catabolite repression.2. The above results depend on improved techniques for production of large amounts of bacterial polysomes and the ability to label nascent peptides attached to polysomes very specifically with radioactive antibody to ribitol dehydrogenase. Our success was due to the observation that sodium heparin completely abolishes non‐specific interactions of the antibody with the polysomes.
Factors Affecting the Quaternary Structure of the Allosteric L‐Lactate Dehydrogenase from Lactobacillus casei and Lactobacillus curvatus as Investigated by Hybridization and Ultracentrifugation
Authors: MAYR Ulrich , HENSEL Reinhard , KANDLER Otto 
Published: March 2005

The allosteric L‐lactate dehydrogenases of Lactobacillus curvatus and Lactobacillus casei exist in the tetrameric form (molecular weight about 145000) at pH 5.0–5.5 even in the absence of the effectors Mn2+ and Fru(1,6)P2 (fructose 1,6‐bisphosphate), but undergo reversible dissociation to monomers (molecular weight about 35000) at higher pH values or in the presence of urea.In the range between pH 5.5 (tetrameric state) and pH 7.4 (monomeric state) the L. curvatusL‐lactate dehydrogenase exists in a dissociation‐association equilibrium comprising tetramers, dimers and monomers as indicated by the sc20,w values and the results of hybridization experiments. The simultaneous addition of both effectors [Mn2+ and Fru(1,6)P2] at pH 7.4, however, resulted in the stabilization of the tetrameric form. The addition of Fru(1,6)P2 alone at pH 7.4 had almost no influence on the quarternary structure, whereas the addition of Mn2+, as well as that of NADH, largely prevented dissociation.The L‐lactate dehydrogenase of L. casei showed similar properties, although the enzyme dissociates only at about pH ≧ 7.8. As in the case of the L. curvatus enzyme, Fru(1,6)P2 has no influence on the pH‐dependent dissociation of the L. casei enzyme, whereas Mn2+ stabilizes the tetrameric structure. Reconstitution of a mixture of the two dissociated enzymes results in the formation of all statistically possible, enzymatically active hybrids. No hybridization between the allosteric enzymes from L. casei and L. curvatus and the non‐allosteric ones of Lactobacillus plantarum and Lactobacillus acidophilus was observed.
A High Production Rate of Translatable IgG mRNA Accounts for the Amplified Synthesis of IgG in Myeloma Cells
Authors: WALLACH Michael , LASKOV Reuven 
Published: March 2005

The aim of the present work was to determine whether the accumulation of Ig mRNA in myeloma cells is due to a high rate of production or to a high stability of these molecules. Specific mRNAs for the light and heavy polypeptide chains of IgG were isolated from the murine MPC‐11 myeloma tumor cells by immune precipitation of polysomes which synthesize these chains. It was found that the immune‐precipitated polysomes were enriched 10 –30‐fold in the γ and χ mRNA sequences respectively. In the wheal germ cell‐free system the χ mRNA preparation was translated mainly into three polypeptides of Mr 25000, 18000, and 15000. The method of immune precipitation of polysomes was also used to characterize three variant clones of MPC‐11 myeloma. It was found that little if any γ‐chain polysomes are present in the L‐chain producer and non‐producer clones, while a substantial amount of χ‐chain polysomes was present in the non‐producer clone. This may be due to the presence in the non‐producer cells of the constant region χ‐chain fragment.In order to determine the relative synthesis rate of χ and γ mRNAs, pulse‐labeled polysomes were immune precipitated using antibodies to χ and γ chains. It was found that χ and γ mRNA molecules are produced at a very high relative rate each accounting for 10–15% of the total labeled mRNA after 1 h of labeling. These values are higher than the steady‐state pool size of χ and γ mRNA, which was 5–6%, and indicates that the half‐life of these molecules is not unusually high. It is concluded that the amplified synthesis of immunoglobulin chains in myeloma cells is mainly due to a high rate of production of translatable χ and γ mRNAs.
Stoichiometry of Interaction of Chicken Ovoinhibitor with Pancreatic Trypsin, Chymotrypsin and Elastase I
Authors: GERTLER Arieh , BEN‐VALID Isaac 
Published: March 2005

1. The interaction of chicken ovoinhibitor with pancreatic trypsin, chymotrypsin and elastase I was studied in the absence of substrate by affinity chromatography, electrophoresis and gel filtration.2. It was found that ovoinhibitor has, at least, five separate and non‐overlapping binding sites: two for trypsin, two for chymotrypsin and one for elastase. Most likely, all five sites may be occupied simultaneously.3. Electrophoretic studies indicated that two binding sites for trypsin and probably also for chymotrypsin differ in their affinity toward the respective enzyme.
Studies of Liposome Interactions with Rat Thymocytes
Authors: KRAMERS Mary T. C. , PATRICK John , BOTTOMLEY Juliana M. , QUINN Peter J. , CHAPMAN Dennis 
Published: March 2005

1. The consequences of incubating liposomes with rat thymocytes have been studied using liposomes of dipalmitoylphosphatidylcholine and cholesterol or dipalmitoylphosphatidylcholine only.2. Dipalmitoylphosphatidylcholine‐cholesterol liposomes do not bind to the cells and can be removed by washing. An increase in cellular cholesterol is observed. However dipalmitoylphosphatidylcholine liposomes bind rapidly to the cells and cannot be removed by repeated washing. Cholesterol is removed from the cells.3. There are small changes in intracellular cations in the cholesterol‐enriched cells, but no transport studies have been made. Cells depleted of cholesterol lose K+ with little change in intracellular Na+. Na+ influx is increased. The majority of this increase appears to be ouabain‐sensitive, indicating an increase in pump‐mediated Na+ influx, K+ influx is reduced.4. The significance of these results is discussed.
Surface Labeling of Membrane Glycoproteins and Their Drastic Changes during Development of Dictyostelium discoideum
Authors: TODA Katsumi , ONO Ken‐ichi , OCHIAI Hiroshi 
Published: March 2005

Surface glycoproteins on plasma membranes from Dictyostelium discoideum were labeled by sodium inctaperiodate oxidation and sodium bore[3H]hydride reduction. The amount of incorporation of tritium from NaB3H4 reached a platcau after 10 min at a periodate concentration of 20 mM. The density analysis carried out by sucrose density gradient centrifugation showed that the plasma membranes were selectively labeled by this technicue. About 84% of the radioactivity incorporated was released by hydrolysis with 0.25 M H2SO4 for 3 h at 100° C. The released materials were eluted at it bed volume after chromatography using a Sephadex G‐50 column. From the paper chromatographic analysis of the eluate, four radioactive spots were detected. Two of them were glycerol and glyceraldehyde and the other two spots seemed to be oligosaccharides.Using the above method, the plasma membranes from aggregation‐phase cells were labeled four times more than those front growth‐phase cells. The labeled plasma membrane fraction during the aggregation phase was separated into at least 39 distinct glycoprotein bands on a one‐dimensional dodecylsulfate/polyacrylamide gel. Six of the 39 bands increased markedly in density and two new bands appeared. In contrast, four bands decreased in density in the aggregation phase.Using this method, the surface glycoproteins can be analyzed directly by gel electrophoresis of the lystate of labeled whole cells without preparing the plasma membranes. Changes during development of glycoproteins on outer cell surfaces were also confirmed by O'Farrell's method of twodimensional polyacrylamide gel electrophoresis. Utilizing this technique, glycoproteins on plasma membranes of D. discoideum were separated into 63 individual spots; 45 of these 63 spots changed during the early developmental course of D. discoideum. This is in contrast to the slight change in the soluble and membrane proteins during this phase. This fact also suggests that the glycoproteins have important roles in the process of aggregation.
The Quaternary Structure of Bovine α‐Crystallin
Authors: SIEZEN Roland J. , BINDELS Jaques G. , HOENDERS Herman J. 
Published: March 2005

The stability of the native quaternary structure of bovine α‐crystallin was studied, by sedimentation analysis and electron microscopy, as a function of pH (7–11), ionic strength (0.01–0.5), temperature (6–60° C) and calcium ion concentration (0 and 10 mM).Three successive transitions are distinguished at 20° C. Firstly, a slow transconformation step, which is independent of pH, ionic strength or calcium ions. Secondly, an irreversible primary dissociation step, favoured by increasing pH above 8 and/or a lower ionic strength, with formation of ‘alkali‐modified α‐crystallin′, which is spherically shaped like the native protein but has a smaller average diameter, sedimentation coefficient and molecular weight. Thirdly, with further increase of pH above 9, a rapidly reversible dissociation of alkali‐modified α‐crystallin characterized by a single reaction boundary in sedimentation velocity analysis. In the presence of calcium ions the quaternary structure is stabilized to the extent that no dissociation is observed up to at least pH 10.3.Upon increase of temperature, at pH 7.3, a slow irreversible dissociation and swelling run parallel until a limit is reached around 37° C with formation of ‘temperature‐modified α‐crystallin′, which is indistinguishable from the native protein by electron microscopy, but has a higher relative viscosity and lower sedimentation coefficient and molecular weight. Calcium ions have little or no effect on this transition. Above 37° C a reversal of this transition or aggregation is indicated.These findings, together with previous structural data of microheterogeneity, reassociation from urea, and aging of α‐crystallin in vivo, are incorporated into a hypothetical scheme of transitions, based on a three‐layer model for the quaternary structure.
Inhibition of Cholesterol Synthesis in Rat Liver by Physiological Doses of Taurocholate
Authors: WEIS Elmar Eugen , BARTH Christian Arthur 
Published: March 2005

This report deals with the controversial problem whether bile acids exert a direct inhibitory effect on the rate of hepatic cholesterol synthesis. For this purpose rats have been provided with an ‘extracorporeal bile duct′, an experimental model which makes it possible to initiate a bile fistula two weeks after last surgery. In the animals that had recovered completely from operative trauma, a 6‐h infusion of 33.4 μmol sodium taurocholate × (100 g body weight)‐1× h−1, prevented the threefold rise of hepatic cholesterol synthesis following bile diversion.The rate of cholesterol synthesis was monitored by incorporation of [14C] acetate in vitro.There was no difference whether the animals were infused from 4–10 h (dark/light transition) or from 16–22 h (light/dark transition) besides the fact that taurocholate caused a more extensive inhibition during the light‐dark transition.A parallel kind of response was observed for the activity of hepatic 3‐hydroxy‐3‐methylglutaryl‐CoA rcductase (EC 1.1.1.34), the regulatory enzyme of cholesterol synthesis.A twofold rise of hepatic fatty acid synthesis was observed following bile diversion which was not seen in case of simultaneous bile salt infusion.It is concluded that bile salts exert direct feedback inhibition of hepatic cholesterol synthesis and that opposite results reported by other investigators are probably due to the infusion of too low doses of taurocholate.
Membrane Mutants: A Yeast Mutant with a Lesion in Phosphatidylserine Biosynthesis
Authors: KOVÁČ Ladistav , GBELSKÁ Iveta , POLIACHOVÁ Vlasta , ŠUBÍK Július , KOVÁČvÁ Vlasta 
Published: March 2005

A single‐gene nuclear choline‐requiring mutant of Saccharomyces cerevisiae was studied. Choline as a growth supplement to synthetic media could be substituted by low concentrations of d imethylethanolamine, monomethylethanolamine or ethanol amine. DL‐Serine also supported growth, but only at high concentrations: on a molar basis it was approximately one hundred times less effective than choline. When cultured in unsupplemented medium the mutant cells soon ceased to grow. The growth‐arrested cells contained less than one fifth of the phosphatidylethanolamine present in wild‐type cells and only traces of phosphatidylserine. The relative content of the two phospholipid species was raised by growing the mutant cells in the presence of choline of the other supplements but still remained lower than in wild‐type cells.The mutant cells depleted of phosphatidylethanolamine and phosphatidylserine had greatly diminished ability to fuse with other cells in mating and their protoplasts showed increased resistance to hypotonic lysis. Respiration was not substantially affected by the deficit of the two phospholipid species in the mutant.In cell‐free preparations, the affinity of the phosphatidylserine synthesizing system for serine was found to be almost two orders of magnitude lower in the mutant than in the wild‐type. The impairment of phosphatidylserine synthesis accounts for growth requirement and the abnormal phospholipid composition of the mutant cells.
Aldrin Epoxidation Catalyzed by Purified Rat‐Liver Cytochromes P‐450 and P‐448 High Selectivity for Cytochrome P‐450
Authors: WOLFF Thomas , GREIM Helmut , HUANG Mou‐Tuan , MIWA Gerald T. , LU Anthony Y. H. 
Published: March 2005

Aldrin epoxidation was studied in monooxygenase systems reconstituted from purified rat livermicrosotnal cytochrome P‐450 or P‐448, NADPH‐cytochrome c reductase, dilauroylphosphatidylcholine and sodium Cholate. Cytochrome P‐450, purified from hepatic microsomes of phenobarbital‐treated rats, exhibited a high rate of dieldrin formation. The low enzyme activity observedin the absence of the lipid and sodium cholate was increased threefold by addition of dilauroyl‐phosphatidylcholine and was further stimulated twofold by addition of sodium cholate. The apparentKm for aldrin in the complete system was 7 ± 2 μM. SKF 525‐A, at a concentration of 250 μM, inhibited aldrin epoxidation by 65% whereas 7,8‐benzoflavone had no inhibitory effect at concentrations up to 250 μM. Addition of ethanol markedly increased epoxidase activity. The increase was three fold in the presence of 5% ethanol.When cytochrome P‐448 purified from hepatic microsomes of 3‐methylcholanthrene‐treated rats was used, a very low rate of epoxidation was observed which was less than 3% of the activity mediated by cytochrome P‐450 under similar assay conditions. Enzyme activity was independent of the lipid factor dilauroylphosphatidylcholine. The apparent Km for aldrin was 27 ± 7 μM. The modifiers of monooxygenase reactions, 7,8‐benzoflavone, SKF 525‐A and ethanol, inhibited the activity mediated by cytochrome P‐448. The I50 was 0.05, 0.2 and 800 mM, respectively.These results indicate that aldrin is a highly selective substrate for cytochrome P‐450 species present in microsomes of Phenobarbital‐treated animals and is a poor substrate for cytochrome P‐448. The two forms of aldrin epoxidase can be characterised by their turnover number, their apparent K, and their sensitivity to modifiers, like 7,8‐benzoflavone and ethanol.
Phosphorylase Kinase from Rabbit Skeletal Muscle Identification of the Calmodulin‐Binding Subunits
Authors: PICTON Colin , KLEE Claude B. , COHEN Philip 
Published: March 2005

Phosphorylase kinase has the structure (αβγδ)4 where the δ‐subunit is identical to the calciumbinding protein termed calmodulin [Shenolikar et al. 1979) Eur. J. Biochem. 100, 329–337].The δ‐subunit was tightly bound to phosphorylase kinase in the absence of calcium ions, and its rate of exchange with [14C]calmodulin was only 15% per week. The δ‐subunit remained associated with phophorylase kinase in the presence of 8 M urea provided that calcium ions were present and this property enabled electrophoretic techniques to be used which demonstrated that the δ‐subunit was associated with the γ‐subunit. This finding was confirmed by cross‐linking experiments with dimethylsuberimidate which resulted in the formation of a γδ complex.Phosphorylase kinase was shown to bind one additional molecule of calmodulin per αβγδ unit, termed the δ‐subunit. Glycerol gradient centrifugation in the presence of [14C]calmodulin indicated that the interaction of the δ‐subunit with phosphorylase kinase only occurred in the presence of calcium ions, and that the Kd value was near 0.01 μM. This was similar to the concentration of δ‐subunit which produced half‐maximal activation. The δ‐subunit did not remain associated with phosphorylase kinase in the presence of 8 M urea, either in the presence or absence of calcium ions. The very slow exchange between the δ‐subunit and [14C]calmodulin, and the calcium‐dependcnt binding of the δ‐subunit allowed cross‐linking experiments to be used which demonstrated that the δ‐subunit was bound to both the α and β subunits. This result was supported by the finding that selective proteolysis of either the δ‐subunit, or the α and β subunits, decreased or abolished the ability of phosphorylasc kinase to bind to calmodulin‐Sepharose.The roles of the different subunits in the regulation of phosphorylase kinase activity are discussed.