bluuebunny/crossref_metadata_2025 huggingface dataset:
Glyceraldehyde‐3‐Phosphate Dehydrogenase(NADP) from Sinapis alba L.
Authors: CERFF Rüdiger
Published: March 2005

Partially purified glyceraldehyde‐3‐phosphate dehydrogenase (NADP) (EC 1.2.1.13) from Sinapis alba seedlings was filtered on Sephadex G‐200 in the presence of NADP(H) and NAD(H). With NADP(H) the enzyme elutes as a dissociated species (Mr∼ 170 000), whereas with NAD(H) it travels as an aggregate in the void volume (Mr≥ 800 000). The NAD‐specific enzyme (EC 1.2.1.12) elutes with an Mr of about 160 000, regardless of whether NADP(H) or NAD(H) is present.

If the NADP‐dependent enzyme is first filtered with NADP+ on Sephadex G‐200 and then with NAD+ on Sepharose 6B, only part of the enzyme aggregates during the second filtration. The other part (fraction S) travels as a narrow band slightly ahead of the marker cytochrome c. Refiltration of fraction S with NADP+ and with increasing [NADP+]/[NAD+]ratios on Sepharose 6B demonstrates that this transport retardation is NAD‐dependent and counteracted by NADP. The effect is also eliminated by inorganic cations and is not observed on Sephadex G‐200.

Dodecylsulfate/polyacrylamide gel electrophoresis of fraction S before and after affinity chromatography on NADP‐agarose shows that the enzyme is pure after transport retardation on Sepharose 6B. It is active with both NADP and NAD and contains at least three different polypeptide chains with apparent Mr of 39 000, 42 000, and 43 000. The NAD‐specific enzyme is shown to be a tetramer of four 38 000‐Mr subunits.

Kinetic investigations show that the binding of NADP(H) and NAD(H) with glyceraldehyde‐3‐phosphate dehydrogenase (NADP) is mutually exclusive.

The present data indicate that the NADP‐dependent enzyme, a species structurally distinct from the NAD‐specific enzyme, occurs in two different conformations in the presence of NADP(H) and NAD(H) respectively. The existence of a ‘sticky’ NAD conformer is suggested, which interacts with Sepharose 6B and/or aggregates with other molecules in crude or partially purified extracts.
Evidence for a Dimeric Form of Artemia salina Extracellular Hemoglobins with High‐Molecular‐Weight Subunits
Authors: MOENS Luc , KONDO Masatoshi
Published: March 2005

The brine shrimp, Artemia salina, produces at least three chemically and ontogenetically distinct extracellular hemoglobins (Hb‐I, Hb‐II and Hb‐III). The estimated molecular weights of these hemoglobins are 240 000–260 000, containing 14 heme groups based on the iron and heme contents of a major species, Hb‐II, which corresponds to a minimal molecular weight of about 18 000 per heme [Moens, L. and Kondo, M. (1977) Biochem. J. 165, 111–119]. Denaturation of the reduced and alkylated hemoglobins with 8 M guanidine hydrochloride revealed apparently one polypeptide chain having a molecular weight of 126 000. Thus a single native hemoglobin molecule should be composed of two of these high‐molecular‐weight subunits each of which is bound with seven hemes. Upon sodium dodecyl sulfate/polyacryamide gel electrophoresis of either native hemoglobins or isolated subunits it was found that the 126 000‐Mr polypeptide was cleaved specifically into two unequally‐sized fragments of Mr 50 000 and 80 000. Further denaturation of native hemoglobins with urea at pH 2.5 or 11 followed by sodium dodecyl sulfate gel electrophoresis confirmed these results.

The amino acid compositions determined for native Hb‐II and its subunit and fragments are found to be very similar, implying that no specifically localized amino acid sequences are present and that the subunit globin chain could be composed of seven similar repeat units (Mr∼ 18 000) being linked covalently to one another. The amino acid compositions of Hb‐I and Hb‐III showed only minor differences to that of Hb‐II.
Purification and Properties of Monomeric Cytochrome f from Charlock, Sinapis arvensis L.
Authors: GRAY John C.
Published: March 2005

Monomeric cytochrome f has been purified to homogeneity, as judged by polyacrylamide gel electrophoresis, isoelectric focusing and analytical ultracentrifugation, from the leaves of charlock, Sinapis arvensis L. The cytochrome was obtained in an aqueous extract following extraction of leaf lipids with butan‐2‐one, and was subsequently purified by acetone precipitation and chromatography on DEAE‐cellulose, Sephadex G‐100 and hydroxylapatite. The purified cytochrome had absorbance ratios of A422/A280= 7.3 and A554/A280= 1.07 in the reduced form. There was no indication of the presence of an absorbance band at 695 nm in the oxidised form. The cytochrome had a midpoint redox potential of +365 mV and was oxidised very rapidly by parsley plastocyanin. The molecular weight of the cytochrome was approximately 27000 as determined by sedimentation equilibrium and by polyacrylamide gel electrophoresis in the presence of dodecylsulphate. The sedimentation coefficient (S20, w0) of cytochrome f was 2.48 S. The cytochrome had an isoelectric point at pH 5.50 determined by isoelectric focusing in polyacrylamide gels.
A Spectrophotometric Rapid Kinetic Study of Reactions Catalysed by Coenzyme‐B12‐Dependent Ethanolamine Ammonia‐Lyase
Authors: HOLLAWAY Michael R. , WHITE Hugh A. , JOBLIN Keith N. , JOHNSON Alan W. , LAPPERT Michael F. , WALLIS O. Caryl
Published: March 2005

Spectral changes during the transient states and the steady states of the reactions catalysed by the adenosylcobalamin‐dependent ethanolamine ammonia‐lyase with l‐2‐aminopropanol and 2‐aminoethanol as substrates were followed by using a rapid‐wavelength‐scanning, stopped‐flow spectrophotometer (800 spectra per second and each spectrum 340–560 nm).

During the steady state of the reaction with the ‘poor’ substrate, l‐2‐aminopropanol (kcat∼ 1 s−1), under conditions of V, the spectrum of the coenzyme closely resembled that of cob(II)alamin. With the ‘good’ substrate, 2‐aminoethanol (kcat∼ 140 s−1), under the same conditions, the spectrum corresponded to a mixture of 58% cob(II)alamin and 42% cob(III)alamin. The latter may be either unchanged adenosylcobalamin or a coenzyme derivative with a covalent bond between a substrate carbon and the cobalt atom. The magnitude of a reported kinetic deuterium isotope effect favours the second possibility. There was no evidence for the presence of a cob(I)alamin intermediate.

The transient phases of the reactions were dependent on the order of mixing of reactants. When enzyme was placed in one syringe of the stopped‐flow apparatus and coenzyme and substrate in the other, the cob(II)alamin intermediate formed slowly (first‐order rate constants of about 5 s−1 with l‐2‐aminopropanol as substrate and about 2 s−1 with 2‐aminoethanol). When the enzyme and coenzyme were placed in one syringe and the substrate in the other, the formation of the cob(II)alamin occurred much more rapidly (90 s−1 with l‐2‐aminopropanol and greater than 300 s−1 with 2‐aminoethanol). It is concluded that binding of adenosylcobalamin to the enzyme is followed by a slow change in the conformation of the enzyme molecule to give a catalytically active species (there is no observable change in the spectrum of the coenzyme concommitant with this process). The formation of active holoenzyme is slow on the time scale of subsequent catalytic steps when 2‐aminoethanol is the substrate.

The spectra of the intermediates formed in the reactions with both substrates, the rates of their formation and their lifetimes allowed a detailed kinetic analysis of the reactions in terms of a three‐step mechanism involving binding of substrate, cob(II)alamin formation (k+2 step) and cob(II)alamin breakdown (k+3 step). In catalysis the difference between the substrates is expressed mainly in the k+3 step (240 s−1 and about 1 s−1 respectively for 2‐aminoethanol and l‐2‐aminopropanol, the respective k+2 values being 336 s−1 and 90 s−1).
Purification and Properties of Phage P22 c2 Repressor
Authors: BALLIVET Marc , EISEN Harvey
Published: March 2005

The c2 repressor of phage P22 has been purified to homogeneity. It specifically binds to λimm21 and P22 DNA. Its affinity for the presumed operator mutant P22 virB is reduced. The initial dissociation rates of the complex between c2 repressor and λimm21 DNA are 0.02 min−1 at 0°C, 0.08 min−1 at 20°C and 0.17 min−1 at 32°C. The dissociation rates of complexes formed between the c2 repressor and the λimm21 operators OR, OL and ORvira were measured and compared to the corresponding rates obtained with 21 cI repressor.
Transport of d‐Glucose and 3‐O‐Methyl‐d‐Glucose in the Cyanobacteria Aphanocapsa 6714 and Nostoc strain Mac
Authors: BEAUCLERK Alan A. D. , SMITH Arnold J.
Published: March 2005

The cyanobacterium Aphanocapsa 6714 which grows in the dark on D‐glucose, will take up D‐glucose and the analogue 3‐O‐methyl‐D‐glucose; uptake of each of these compounds was inhibited competitively by the other and by 6‐deoxy‐D‐glucose.

This cyanobacterium accumulated 3‐O‐methyl‐D‐glucose up to 100‐fold relative to the medium but did not modify or metabolize it to a significant degree.

Intracellular 3‐O‐methyl‐D‐glucose was rapidly displaced from Aphanocapsa 6714 by exogenous D‐glucose and 3‐O‐methyl‐D‐glucose.

Although not characterized to the same extent, D‐glucose and 3‐O‐methyl‐D‐glucose uptake by Nostoc strain Mac, another cyanobacterium capable of growth in the dark on D‐glucose, was similar.

Other cyanobacteria that do not grow on D‐glucose take up this compound at much lower rates which were unaffected by analogues of D‐glucose that greatly reduced carbohydrate uptake by Aphanocapsa 6714 and Nostoc strain Mac.

It is therefore proposed that Aphanocapsa 6714 and Nostoc strain Mac possess a mechanism for the active transport of D‐glucose. The absence of this transport mechanism is suggested as the reason why other strains fail to grow in the dark on this substrate. These latter organisms are therefore naturally cryptic with respect to D‐glucose as a growth substrate.
Major Proteins of the Escherichia coli Outer Cell Envelope Membrane. Interaction of Protein II* with Lipopolysaccharide
Authors: SCHWEIZER Margarete , HINDENNACH Ingrid , GARTEN Wolfgang , HENNING Ulf
Published: March 2005

One of the major proteins of the Escherichia coli outer cell envelope membrane, protein II*, is shown to interact specifically with the same membrane's lipolysaccharide. In cell envelopes the protein (Mr∼ 33 000) is partially degraded by trypsin or pronase, and fragments with molecular weights of 24 000 or 19 000, respectively, are left with the outer membrane. The protein and these fragments in the cell envelope can act as receptors for a phage, and, most likely, as receptors in F‐mediated conjugation. Lipopolysaccharide protects isolated protein II* (in solution) in the same way against proteolytic attack, and the tryptic or pronase fragments are identical with those fragments obtained from protease‐digested cell envelopes. All fragments represent the N‐terminal moiety of the protein, and are, in the presence of lipopolysaccharide, active as phage receptors. Protein II* is heat‐modifiable in that its apparent molecular weight upon boiling in dodecylsulfate is 33 000 and without boiling is 28 000. Addition of lipopolysaccharide to the boiled species causes a renaturation to the species of molecular weight 28 000. Total E. coli phospholipid, synthetic dimyristoyl phosphatidylcholine, or the oligosaccharide moiety of lipopolysaccharide do not show any of these effects. However, the lipid part of lipopolysaccharide (lipid A) exhibits essentially the same activity as complete lipopolysaccharide.

All results taken together strongly suggest that the interaction observed in vitro reflects such an interaction in vivo, and the possibility exists that this interaction is at least partially responsible for the specific cellular localization of the protein. Very likely a number of other major outer membrane proteins also interact specifically with lipopolysaccharide.
The Binding of Calcium and Magnesium to Sarcoplasmic Reticulum Vesicles as Studied by Manganese Electron Paramagnetic Resonance
Authors: KALBITZER Hans Robert , STEHLIK Dietmar , HASSELBACH Wilhelm
Published: March 2005

The binding pattern of the biologically relevant ions calcium and magnesium has been investigated via the binding of the ion‐analogue manganese. The binding parametes of manganese are obtained conveniently by standard electron paramagnetic resonance techniques, the binding of calcium and magnesium is inferred from competition experiments.

The quantitative analysis was carried out with a computer program which was able to treat the competition of three kinds of ions for three classes of independent and one class of cooperative binding sites.

It was possible to correlate specific binding classes with biological functions of the sarcoplasmic reticulum membrane. The magnesium and manganese specific binding class of medium affinity is related to the catalytic function of these ions in the ATP‐splitting. For the first time a manganese and calcium specific class of cooperative binding sites has been observed and it can be related to the inhibition of the calcium transport at high concentrations of manganese or calcium.

From the binding results a model can be developed which allows a full description of the role of the divalent ions and their specific binding sites during calcium transport.
Isolation of 4.5‐Dihydroxyisophthalic Acid, an Inhibitor of Brain Glutamate Decarboxylase, Produced by a Streptomyces Species
Authors: ENDO Akira , KITAHARA Nobuaki , OKA Hidehiko , MIGUCHI‐FUKAZAWA Yumi , TERAHARA Akira
Published: March 2005

A potent inhibitor of brain glutamate decarboxylase (L‐glutamate 1‐carboxy‐lyase, EC 4.1.1.15) was isolated from cultures of a Streptomyces species, and its structure was found to be 4,5‐dihydroxyisophthalic acid. The metabolite inhibited beef brain glutamate decarboxylase 50% at a concentration of 0.12 μg/ml (0.61 μM). The kinetic analysis indicated that 4,5‐dihydroxyisophthalic acid was a competitive inhibitor having a Ki value of 0.18 μM.
Reagents Specific for Cell Surface Components
Authors: PITHA Josef
Published: March 2005

Mercury, diazonium ions and dyes which bind nucleic acids were covalently linked to dextrans using methods that resulted in non‐hydrolyzable reagent‐dextran bonds without impairing the binding abilities of the reagents, i.e. these dextran derivatives reacted with thiols, phenols/imidazoles and nucleic acids respectively. Since these dextran derivatives cannot penetrate into cells and since dextran itself does not bind to cells, these compounds represent reagents specific for the cell surface. They may be used both to evaluate cell surface constituents of intact cells and to affect viable cells via an interaction with those constituents. Mercury‐dextran was found to bind to cells; the amount of mercury thus attached to the cells was about ten times smaller than when an equivalent concentration of free mercury ions was used. Mercury‐dextran, bound to cells after a 30‐min exposure at room temperature, was localized on the surface of these cells, as sodium borohydride reduced this complex giving rise to the intact cells, elementary mercury and free dextran which was released into medium. When cells were constantly exposed to the mercury‐dextran, its toxic effects were comparable to that of free mercury ions. Diazonium‐dextran, which also binds tightly to the cell surface, was also considerably toxic. Dextrans substituted with dyes which bind to nucleic acids were less toxic than the parent dyes themselves; it was shown that the attachment of such a dye to dextran decreased the binding of dye to cells under detection limits.
Properties of the Main Endonuclease Specific for Apurinic Sites of Escherichia coli (Endonuclease VI)
Authors: GOSSARD Francis , VERLY Walter G.
Published: March 2005

The main endonuclease for apurinic sites of Escherichia coli (endonuclease VI) has no action on normal strands, either in double‐stranded or single‐stranded DNA, or on alkylated sites. The enzyme has an optimum pH at 8.5, is inhibited by EDTA and needs Mg2+ for its activity; it has a half‐life of 7 min at 40°C. A purified preparation of endonuclease VI, free of endonuclease II activity, contained exonuclease III; the two activities (endonuclease VI and exonuclease III) copurified and were inactivated with the same half‐lives at 40°C. Endonuclease VI cuts the DNA strands on the 5′ side of the apurinic sites giving a 3′‐OH and a 5′‐phosphate, and exonuclease III, working afterwards, leaves the apurinic site in the DNA molecule; this apurinic site can subsequently be removed by DNA polymerase I. The details of the excision of apurinic sites in vitro from DNA by endonuclease VI/exonuclease III, DNA polymerase I and ligase, are described; it is suggested that exonuclease III works as an antiligase to facilitate the DNA repair.
Blue‐Dextran — Sepharose Affinity Chromatography: Recognition of a Polynucleotide Binding Site of a Protein
Authors: DROCOURT Jean‐Louis , THANG Diec‐Chi , THANG Minh‐Nguy
Published: March 2005

Native Escherichia coli polynucleotide phosphorylase can be retained on blue‐dextran–Sepharose. The bound enzyme cannot be displaced by its mononucleotide substrates such as ADP, UDP, CDP, GDP and IDP, but it is easily eluted by its polymeric substrates. Under identical conditions, lactate dehydrogenase, bound on blue‐dextran–Sepharose, is not eluted by poly(I) but can be specifically displaced by NADH. On the other hand, the trypsinized polynucleotide phosphorylase, known to be an active enzyme which has lost its polynucleotide site, does not bind to the affinity column. The native polynucleotide phosphorylase can also be tightly bound to poly(I)–agarose and displaced from it only by high salt concentration. The trypsinized enzyme is not bound at all on poly(I)–agarose. Moreover, the native enzyme linked on blue‐dextran–Sepharose, remains active indicating a free access of nucleoside diphosphates to the active center. These results taken together show that the dye ligand is not inserted onto the mononucleotide binding site and suggest rather that it binds to the polynucleotide binding region. The implications of this study and the application of blue‐dextran–Sepharose affinity chromatography to other proteins having affinity for nucleic acids are discussed.
Purification and Characterization of Acidic Adenylate Kinase in Porcine Heart
Authors: ITAKURA Takao , WATANABE Kiyotaka , SHIOKAWA Hiroyuki , KUBO Shuichiro
Published: March 2005

Porcine heart adenylate kinase can be separated into acidic and basic components by electrofocusing, and the latter is identical with the skeletal muscle adenylate kinase. The acidic adenylate kinase was purified approximately 3600‐fold from porcine heart extract in an overall yield of 59% by DEAE‐cellulose chromatography, substrate elution from a column of phosphocellulose, affinity chromatography on a column of blue dextran‐Sepharose 4B, and gel filtration. The purified acidic enzyme had a specific activity of 1100 units per mg of enzyme, and was found to be a homogeneous protein in all the procedures used, i.e. disc and sodium dodecylsulfate electrophoresis, and gel filtration. However, electrophoretic variants were observed on isoelectrofocusing and this variance may depend on the formation of protein‐ampholine complexes. The purified acidic enzyme had a molecular weight of 26 900 and an optimum pH of 5.8 in the forward reaction (ATP + AMP → 2ADP). The amino acid composition of the acidic enzyme was found to be 22 aspartic acid or asparagine, 13 threonine, 14 serine, 29 glutamic acid or glutamine, 17 proline, 17 glycine; 22 alanine, 4 half‐cystine, 13 valine, 8 methionine, 13 isoleucine, 24 leucine, 4 tyrosine, 7 phenylalanine, 5 histidine, 19 lysine, 14 arginine and no tryptophan, totaling 245 residues. The enzyme had two – SS – bonds but no free – SH, and was not inhibited at all by 5,5′‐dithio‐bis(2‐nitrobenzoic acid). From these results, it may be concluded that the acidic adenylate kinase of porcine heart is similar to liver mitochondrial adenylate kinase.
Properties of 3‐(3,4‐Dichlorophenyl)‐1,1‐dimethylurea and Other Inhibitors of the Cytochromebc1Segment of the Mitochondrial Respiratory Chain inSaccharomyces cerevisiae
Authors: CONVENT Bernard , BRIQUET Michel
Published: March 2005

The properties of 3‐(3,4‐dichlorophenyl)1,1‐dimethylurea (diuron) are compared to those of antimycin,n‐heptyl‐hydroxyquinoline‐N‐oxide (HpHOQnO), mucidin and funiculosin in submitochondrial particles ofSaccharomyces cerevisiae.

Diuron inhibits the transport of electrons between cytochromesbandc1of the respiratory chain ofS. cerevisiaeas do the other inhibitors.

Kinetics of inhibition of NADH oxidase are non‐sigmoidal for the inhibitors of low affinities: diuron and HpHOQnO.

Mucidin and funiculosin exhibit high apparent affinities similar to that of antimycin. However the sigmoidicity of the kinetics of inhibition is not apparent for these two inhibitors.

Diuron and funiculosin do not induce the ‘red‐shift’ of cytochromeb562.

In contrast to beef heart, HpHOQnO does induce the ‘red‐shift’ of cytochromeb562inS. cerevisiae.

Diuron induces the ‘extra‐reduction’ of cytochromeb.

Mucidin induces a ‘red‐shift’ but yields a very limited ‘extra‐reduction’ of cytochromeb.

Our results demonstrate that the inhibition of respiration of thebc1segment of the respiratory chain are not necessarily related to the ‘red‐shift’ and the ‘extra‐reduction’ of the cytochrome(s)b. They raise the possibility of the existence in yeast mitochondria of two distinct inhibitor‐binding sites, each of which would be able to control the transfer of electrons between cytochromesbandc1; in addition, one site would be responsible for the ‘red‐shift’ and the other would control the ‘extra‐reduction’ of cytochrome(s)b.
Energy Metabolism of Various Substrates and the 2,3‐Bisphosphoglycerate Bypass in Human Erythrocytes
Authors: QUADFLIEG Karl‐Hugo , BRAND Karl
Published: March 2005

Using different experimental conditions and applying various substrates, a wide range of metabolic rates could be observed in incubations with human erythrocytes.

Lower rates of substrate utilization resulted in a decrease of the ATP and, more pronounced, of the 2,3‐bisphosphoglycerate concentration while carbon utilization rates beyond 14 μmol C ml packed cells in 120 min yielded constant levels of ATP and 2,3‐bisphosphoglycerate indicating that steady state had been achieved.

It can be concluded, that a carbon utilization rate of 14 μmol C/ml cells in 120 min is able to cover the energy requirement of the red cells under steady state conditions which could be calculated to amount to 3.6 μmol ATP/ml cells in 120 min at 37°C.

The contribution of the 2,3‐bisphosphoglycerate bypass to carbon metabolism could be calculated to range between 15–25% at metabolic rates below 14 μmol C/ml cells in 120 min (presteady state). When steady state has been achieved, the share of this bypass increases up to 61% with increasing metabolic rates.
Some Properties of Sialyltransferase in Plasma and Lymphocytes of Patients with Chronic Lymphocytic Leukemia
Authors: KESSEL David , CHOU Ta‐Hsu , ALLEN Janet
Published: March 2005

Some properties of sialyltransferase activity in plasma and lymphocytes from patients with chronic lymphocytic leukemia were compared. Three distinct enzyme fractions were identified in plasma: (1) cation independent, irreversibly bound to agarose; (2) cation dependent, weakly bound to agarose; (3) strongly bound to agarose, lost upon dialysis. Lowering of the peripheral lymphocyte count by leukapheresis markedly decreased the level of serum sialyltransferase, suggesting the circulating lymphocyte is a source of the serum enzyme.

The enzyme solubilized by detergent from lymphocytes showed a substantially lower Km for CMP‐sialic acid than did the serum enzyme, was less sensitive to several inhibitors, was not irreversible bound to Agarose, and had a substantial cation requirement. The enzyme solubilized from the lymphocyte therefore generally resembles fraction 2 of serum.
Ionic Strength Dependence of the Oxidation of Iodide and Ferrocyanide by Compound I of Horseradish Peroxidase
Authors: STEINER Håkan , DUNFORD H. Brian
Published: March 2005

The kinetics of the oxidation of iodide and ferrocyanide by compound I of horseradish peroxidase have been studied at 25°C as a function of ionic strength and pH. The ionic strength dependencies of the second‐order rate constants are tested with an extended form of the Debye‐Hückel equation for the activity coefficients of the reacting species. For the reaction of iodide with compound I it is shown that the pH variation of the rate constant at zero ionic strength is caused mainly by titrating a catalytically important acid group and not mainly by the varying charge of the protein. The ferrocyanide reaction rate with compound I does not correlate with enzyme net charge, at all pH values. The influence of electrostatic interactions on reaction rates is discussed.
H+ and Cation Movements Associated with ADP, ATP Transport in Mitochondria
Authors: WULF Rainer , KALTSTEIN Andrej , KLINGENBERG Martin
Published: March 2005

H+ and K+ movement associated with the mitochondrial ADP‐ATP exchange have been measured by glass electrode recordings in order to investigate the energy control of the ADP‐ATP exchange. All measurements are taken in the presence of oligomycin and inhibitors of electron transport in order to exclude other types of H+ transfer.

ATP exchange against endogenous ADP as induced by addition of ATP gives only a small H+ uptake which is partially suppressed in a KCl medium. H+ uptake is increased strongly on addition of uncoupler. On addition of valinomycin, K+ is taken up instead of H+. With carbonylcyanide p‐trifluoromethoxyphenylhydrazone (FCCP) and valinomycin respectively, equivalent amounts of H+ and K+ are taken up over a wide range of protein and ATP concentration. The ATP dependence gives a ratio H+ per ATP added = 0.38 and K+ per ATP added = 0.27. Saturation with ATP is reached at about 14 μmol ATP/g protein for H+ uptake and 20 μmol ATP/g protein for K+ uptake.

The FCCP‐facilitated, ATP‐induced H+ uptake increases with endogenous content of ADP, giving a ratio H+/ADP = 0.67. Kinetics of the H+ uptake and ATP‐induced nucleotide exchange are closely correlated to about 0.62 H+/nucleotide released. The temperature dependence of the H+ uptake rate measured between 4 and 11 °C gives an activation energy = 33 kcal (138 kJ), similar to that found for the exchange rate.

In the exchange of ADP against endogenous ATP as started by addition of ADP, H+ are released in the absence of FCCP. In the presence of FCCP, H+ release is only marked when endogenous nucleotides are preloaded with external ATP. This H+ release is decreased by Pi and enhanced by N‐ethylmaleimide, blocking Pi transfer. It varies according to endogenous ATP content reaching the ratio H+ released/endogenous ATP = 0.3 to 0.5.

These data have been interpreted to indicate that the ATPe‐ADPi exchange is largely electrical and the opposite ADPe‐ATPi exchange is only between 50 to 70% electrical. The implications for energy transfer are discussed.
Quantification of Myosin Heavy‐Chain mRNA during Myogenesis
Authors: ROBBINS Jeffrey , HEYWOOD Stuart M.
Published: March 2005

Complementary DNA (cDNA) was synthesized from a 26‐S poly(A)‐containing RNA fraction consisting predominately of myosin mRNA, and used to quantitate the amounts of myosin mRNA during development. RNA · cDNAmyosin hybridizations were performed with RNA isolated from tertiary cultures of chick fibroblasts, and the proportion of myosin RNA determined. The levels of myosin RNA present in primary chick myoblast cultures undergoing development were also analyzed by RNA · cDNAmyosin hybridizations. Correlation of these values with the rate of myosin heavy chain synthesis indicates that myosin mRNA is subject to post‐transcriptional or translational control.
Collagen Synthesis and Secretion by Rat Incisor Odontoblasts in Organ Culture
Authors: MUNKSGAARD E. Christian , RHODES Marsha , MAYNE Richard , BUTLER William T.
Published: March 2005

An organ culture system for studying dentinogenesis has been developed. The system consists of odontoblasts still attached to predentin and dentin and cultured in chemically defined media, supplemented with 20% horse serum and 10% chick embryo extract or 10% fetal calf serum. When incubated for 24 h with [14C]proline, the odontoblast cultures formed [14C]hydroxyproline‐containing protein which was located in cells, media and predentin, showing that these cells both synthesize and secrete collagen. The relative quantities of incorporated [14C]proline and [14C]hydroxyproline showed that collagen produced was particularly enriched in the media and predentin fractions. After extraction of collagen in the presence of pepsin, the collagen chains chromatographed as only α chains on molecular‐sieve chromatography. Thus, type III collagen does not appear to be synthesized by odontoblasts as it would chromatograph as a γ component consisting of three disulfide‐bonded α1(III) chains. Analysis of the denatured collagen by CM‐cellulose chromatography showed that α1(I) and α2 chains were present in a ratio of 5–6:1 instead of 2:1 expected for type I collagen. CNBr peptide analysis established that only α1(I) and α2 chains were made by the odontoblasts. The excess α1(I) chains were found as type I trimer, a molecular species consisting of three α1(I) chains in a triple helix. This was shown by separation of type I collagen and type I trimer by differential salt precipitation. At this time it is not known if the type I trimer is a normal product of odontoblasts in vivo or whether it is only produced by these cells in culture.
Physico‐Chemical Properties of Phosphatidylglycerol in Membranes of Acholeplasma laidlawii
Authors: BEVERS Edouard M. , OP DEN KAMP Jos A. F. , VAN DEENEN Laurens L. M.
Published: March 2005

Membranes isolated from Acholeplasma laidlawii B grown on palmitic acid, elaidic acid, oleic acid or linoleic acid have been treated with excess phospholipase A2 at different temperatures. Hydrolysis of phosphatidylglycerol only occurs at temperatures above the onset of the gel to liquid‐crystalline phase transition of the lipids as measured by differential scanning calorimetry. The extent of phosphatidylglycerol hydrolysis as a function of the temperature reflects the phase transition of this phospholipid in membranes of A. laidlawii.

Species analysis of the phosphatidylglycerol before and after treatment with phospholipase A2 was carried out by double labeling techniques and fatty acid analysis. Phosphatidylglycerol isolated from cells grown on palmitic acid, predominantly consists of two different species: dipalmitoyl phosphatidylglycerol and monopalmitoyl phosphatidylglycerol in which only one of the two acyl chains is palmitic acid. The physical behaviour of both species in these membranes, as measured with phospholipase A2, resembles that of a monotectic system: at 25°C there is almost complete hydrolysis of the monopalmitoyl species, while all of the dipalmitoyl species, which is still in the highly ordered gel state, remains unhydrolyzed. This implies that the miscibility between lipids in the fluid and ordered domains is restricted.

When cells are grown on elaidic acid also two phosphatidylglycerol species are present, i.e. dielaidoyl phosphatidylglycerol and phosphatidylglycerol with one acyl chain different from elaidic acid (monoelaidoyl phosphatidylglycerol). Irrespective of the incubation temperature, there is little or no preferential hydrolysis of one of the phosphatidylglycerol species, as would be expected for a cocrystallizing system. In oleic‐acid‐enriched membranes the lipids exist in the liquid‐crystalline state in the temperature range from 0–40 °C. Information can be obtained only about a pool of phosphatidylglycerol which is protected against phospholipase attack at low temperature. This pool which comprises about 30–40% of the total phosphatidylglycerol mainly consists of monooleoyl phosphatidylglycerol.
Glyceroglucolipids of the Human Saliva
Authors: SLOMIANY Bronislaw L. , SLOMIANY Amalia , GLASS George B. J.
Published: March 2005

Seven individual glycolipids (I–VII) have been isolated from the lipid extract of human saliva. All glycolipids contained glucose, glyceryl ethers and fatty acids, and differed from each other primarily with respect to the number of glucose residues. In addition, glycolipid V contained also the sulfate ester group. The structures of these glycolipids were identified by partial acid and alkaline hydrolysis, oxidation with periodate and chromium trioxide and methylation studies, as: Glc(α1 → 3)‐diglyceride (glycolipid I), Glc(α1 → 6)Glc(α1 → 3)‐diglyceride (glycolipids II and III), Glc(α1 → 6)Glc(α1 → 6)Glc(α1 → 3)‐diglyceride (glycolipid IV), SO3H‐6Glc(α1 → 6)Glc(α1 → 6)‐Glc(α1 → 3)‐diglyceride (glycolipid V), Glc(α1 → 6)Glc(α1 → 6)Glc(α1 → 6)Glc(α1 → 6)Glc(α1 → 6)Glc(α1 → 3)‐diglyceride (glycolipid VI) and Glc(α1 → 6)Glc(α1 → 6)Glc(α1 → 6)Glc(α1 → 6)Glc(α1 → 6)Glc(α1 → 6)Glc(α1 → 6)Glc(α1 → 6)Glc(α1 → 3)‐diglyceride (glycolipid VII). Diglyceride portion of these compounds consists of 1‐O‐alkyl‐2‐O‐acyl‐glycerol with the docosanoate and glyceryl‐monodocosyl being the predominant acyl and alkyl components.
Inhibition of Yeast Phosphatidic‐Acid Synthesis by Free Fatty Acids
Authors: MORIKAWA Minoru , YAMASHITA Satoshi
Published: March 2005

Particulate preparations obtained from cells of yeast Saccharomyces sake have been shown to possess glycerolphosphate acyltransferase and 1‐acylglycerolphosphate acyltransferase and 1‐acylglycerolphosphate acyltransferase activities. Glycerolphosphate acyltransferase exhibits a high specificity for saturated and monoenoic fatty acyl‐CoA thioesters. When palmitoyl‐CoA is employed as sole acyl group donor, the major lipid product is lysophosphatidic acid. 1‐Acylglycerolphosphate acyltransferase of this yeast species has a rather strict specificity for monoenoic fatty acyl‐CoA thioesters as acyl donor.

These two acyltransferases are strongly inhibited in vitro by low concentrations of free fatty acids. 1‐Acylglycerolphosphate acyltransferase is much more susceptible to fatty acid inhibition than glycerolphosphate acyltransferase. The inhibition is dependent not only on the concentration of fatty acid, but also on the length of exposure to fatty acid. Both saturated and unsaturated fatty acids inhibit the acyltransferase activities. The inhibitory effects of fatty acids cannot be ascribed to a nonspecific surfactant action of fatty acids. The present results support the view that free fatty acid serves as a regulator of glycerolipid synthesis.
Purification and Properties of an Elongation Factor Functionally Analogous to Bacterial Elongation Factor Ts from Embryos of Artemia salina
Authors: SLOBIN Lawrence I. , MÖLLER Wim
Published: March 2005

A protein factor which is functionally analogous to bacterial EF‐Ts was purified from developing embryos of the brine shrimp Artemia salina. The factor, designated as eEF‐Ts, has properties which are similar to EF‐1β from pig liver. Specifically eEF‐Ts in our assay conditions stimulates EF‐1L(eEF‐Tu)‐dependent binding of aminoacyl‐tRNA to ribosomes and eEF‐Tu and EF‐2 dependent poly‐phenylalanine synthesis 2.5–3‐fold. In addition eEF‐Ts is an efficient catalyst of a guanine nucleotide exchange reaction involving eEF‐Tu · GDP complexes and free GTP. We suggest that the latter activity explains the stimulation by eEF‐Ts of eEF‐Tu‐dependent functions.

eEF‐Ts consists of a single polypeptide chain of Mr 26 000, as judged by electrophoresis on acrylamide gels containing dodecylsulfate. However, the native enzyme consists largely of high‐molecular‐weight aggregates with sizes ranging from about 100 000 to greater than 200 000. Stable complexes of EF‐Tu and eEF‐Ts were formed by combining the two factors and these complexes were capable of binding GTP at low temperature (0–2 °C). Antibodies against the heavy form of elongation factor 1 cross‐reacted strongly with purified eEF‐Ts confirming our previous observation that EF‐1 in Artemia consists of eEF‐Tu · eEF‐Ts complexes. The relationship between EF‐1 and eEF‐Tu and eEF‐Ts is discussed.
Photochemical Modifications of the Tryptophan Residues of Wheat‐Germ Agglutinin in the Presence of Trichloroethanol
Authors: PRIVAT Jean‐Paul , CHARLIER Michel
Published: March 2005

Trichloroethanol is an efficient quencher of indole fluorescence of model compounds and proteins [Eftink, M. R. and Ghiron, C. A. (1976) J. Phys. Chem. 80, 486–493]. At low quencher concentrations, the quenching follows the classical Stern‐Volmer law. Bimolecular rate constants calculated from measured quenching constants and lifetimes are equal to 6 × 109 M−1 s−1 and 1.2 × 109 M−1 s−1 for N‐acetyltryptophanamide and wheat germ agglutinin, respectively.

Upon ultraviolet irradiation in the presence of trichloroethanol, transformation of fluorescent trypophan occurs, leading to a fluorescent photoproduct. This can be easily used as a method for the quantitative determination of fluorescent tryptophan residues in proteins. In good agreement with previous results, two fluorescent tryptophan residues per polypeptide chain are found in wheat germ agglutinin. Concomitantly with the photochemical reactions, the hemagglutinating protein activity and its affinity constant towards chitin oligomers are reduced. A probable location of tryptophan residues in the binding sites of wheat germ agglutinin is proposed.
Association of DNA Polymerase with Nucleosomes from Mammalian Cell Chromatin
Authors: SCHLAEGER Ernst‐Jürgen , VAN TELGEN Hendrik‐Jan , KLEMPNAUER Karl‐Heinz , KNIPPERS Rolf
Published: March 2005

More than half of the DNA polymerase β in mouse ascites cell chromatin was found to be associated with monomeric nucleosomal particles (produced by micrococcal nuclease treatment of chromatin). Almost all nuclear DNA polymerase activity in lymphocytes was found to be associated with nucleosomes. The nucleosome‐associated enzyme was mainly DNA polymerase β in chromatin from resting and mainly DNA polymerase α in chromatin from concanavalin‐A‐stimulated lymphocytes.
Inhibition of Eukaryotic Ribosomal Function by the Sesquiterpenoid Antibiotic Fusarenon‐X
Authors: CARTER Christopher John , CANNON Michael
Published: March 2005

Detailed studies have been carried out on the sesquiterpenoid antibiotic fusarenon‐X to determine its precise mode of action as an inhibitor of eukaryotic protein synthesis.

In reticulocyte cell‐free systems low concentrations of fusarenon‐X (2 μg/ml) cause extensive ‘run off’ of polyribosomes and parallel studies on time courses of protein synthesis indicate that the drug inhibits initiation. As the concentration of fusarenon‐X is increased, progressively less ‘run off’ is observed but this effect is coupled with a progressive rise in the level of inhibition of protein synthesis. Inhibition becomes complete at fusarenon‐X concentrations approaching 100 μg/ml and under these conditions polyribosomes are stabilized.

The effects of the drug were studied on the puromycin‐induced release of nascent peptides from sucrose‐washed polyribosomes, an assay for peptidyl transferase activity. Fusarenon‐X did not inhibit the reaction when present at a concentration of 8 μg/ml but did so progressively as the drug concentration was increased above this level.

An assay system has been developed whereby initiation complexes, involving ribosomes, [35S]Met‐tRNAf and poly(A,U,G), can be formed in reticulocyte lysates. This system is sensitive to low levels of puromycin (10 μg/ml) and can be used to assay the formation of the ‘first’ peptide bond in protein synthesis. All concentrations of fusarenon‐X tested inhibited the puromycin reaction in this system, the degree of inhibition increasing progressively with increasing concentrations of fusarenon‐X.

It is concluded that the ribosomal target site for fusarenon‐X is the peptidyl transferase catalytic centre but that the ability of the drug to bind to this centre on polyribosomes (carrying long nascent polypeptide chains) is critically dependent upon the concentration of fusarenon‐X employed.
Sulfate‐Mediated Affinity Chromatography on NADP+ ‐Sepharose of Glutamate Dehydrogenase from Halophilic Bacteria and of Glucose‐6‐Phosphate Dehydrogenase from Escherichia coli
Authors: LEICHT Wolfgang
Published: March 2005

An improved synthesis of the 8‐(6‐aminohexyl)amino derivative of NADP+ is described for use in affinity chromatography. The binding of glutamate dehydrogenase isolated from halobacterium of the Dead Sea on a column of Sepharose linked to this NADP+ derivative could be drastically enhanced by addition of sulfate (1M) and provided a tool for partially purifying the enzyme from a crude extract. A similar finding is reported for glucose‐6‐phosphate dehydrogenase in crude extracts of Escherichia coli. The effects are shown to be biospecific, suggesting that the strength of the interaction between protein and immobilized coenzymes is a function of the sulfate concentration.
The Activities in vitro of dd‐Carboxypeptidase and LD‐Carboxypeptidase of Gaffkya homari during Biosynthesis of Peptidoglycan
Authors: HAMMES Walter P. , SEIDEL Heinz
Published: March 2005

The synthesis in vitro of peptidoglycan from the precursor UDP‐N‐acetylmuramyl‐peptide, i.e. UDP‐MurNAc‐Ala1‐dGlu2(Lys3‐dAla4‐dAla5) where the indices refer to the position of an amino acid residue within the peptide subunit, radioactively labelled in either position 3, 4 or 5 and from UDP‐N‐acetylglucosamine was studied with a wall‐membrane enzyme preparation from Gaffkya homari. The peptide subunit was partially degraded by the action of dd‐carboxypeptidase(s) and ld‐carboxypeptidase(s), resulting in the formation of polymer containing pentapeptides, tetrapeptides and tripeptides. In the wall‐bound peptidoglycan the percentage distribution of the respective peptides is 48:23:29 whereas in the soluble peptidoglycan it is 82:12:6. In the absence of concomitant peptidoglycan synthesis, i.e. in the absence of UDP‐N‐acetylglucosamine or in the presence of moenomycin, the release of d‐alanine from position 5 was only 22% of the corresponding release with concomitant peptidoglycan synthesis and, most striking, virtually no d‐alanine was released from position 4 of the peptide subunit. When UDP‐MurNAc‐tetrapeptide was used as a substrate, the degradation of the peptide subunit was catalyzed by ld‐carboxypeptidase exclusively; thus, the polymer contained tripeptides besides non‐degraded tetrapeptides. The percentage distribution of the two oligopeptides in the wall‐bound and in the soluble peptidoglycan were 37:63 and 18:82, respectively. In the absence of concomitant peptidoglycan synthesis, no d‐alanine was released from UDP‐MurNAc‐tetrapeptide. Both dd‐carboxypeptidase and ld‐carboxypeptidase act on soluble peptidoglycan with low efficiency whereas they do not act on the wall‐bound polymer. It must be assumed that the true substrate of both carboxypeptidases is the nascent peptidoglycan. This intermediate is not the soluble peptidoglycan since pulse‐chase experiments showed that this product formed in vitro is only a very poor substrate for synthesis of wall‐bound peptidoglycan.
Studies on the Conformational Properties of the High‐Mobility‐Group Chromosomal Protein HMG 17 and Its Interaction with DNA
Authors: ABERCROMBIE Barry D. , KNEALE Geoffrey G. , CRANE‐ROBINSON Colyn , BRADBURY E. Morton , GOODWIN Graham H. , WALKER John M. , JOHNS Ernest W.
Published: March 2005

The conformation of the non‐histone chromatin protein, HMG 17, has been studied using circular dichroism, infrared and nuclear magnetic resonance spectroscopies, and by small‐angle scattering. The results show that in free solution this protein has little or no secondary or tertiary structure in contrast to the other high‐mobility‐group proteins, HMG 1 and 2, which exhibit highly ordered structures. Protein HMG 17 binds to calf thymus DNA in an ionic‐dependent manner, precipitating the DNA at high protein/DNA ratio. The nuclear magnetic resonance data suggest that the principle DNA‐binding segment of HMG 17 is that between about residues 15 and 40.
Restriction Enzyme Analysis of Satellite DNA Components from the Bovine Genome
Authors: KOPECKA Helena , MACAYA Gabriel , CORTADAS Jordi , THIÉRY Jean‐Paul , BERNARDI Giorgio
Published: March 2005

A restriction enzyme analysis was performed on satellite DNA components, isolated, as described in the preceding paper, from the bovine genome by a combination of Cs2SO4/BAMD and Cs2SO4/Ag+ density gradient centrifugation. Such an analysis has led to the unambiguous identification of eight satellite DNA components and to new information on their repeat units; this indicates that identical repeat lengths are shared by them, a fact strongly suggesting a common origin.
Thermotropic Changes in the Surface Structure of Lipoprotein B from Human‐Plasma Low‐Density Lipoproteins
Authors: LAGGNER Peter , KOSTNER Gerhard M.
Published: March 2005

The lipoprotein B fraction of human‐plasma low‐density lipoproteins was labeled with (a) spin‐label analogues of stearic acid I(m/n), the N‐oxyl‐4′,4′‐dimethyloxazolidine derivatives of 5‐oxo‐, 12‐oxo‐, and 16‐oxostearic acid, I(12/3), I(5/10), and I(1/14), respectively, and (b) a spin‐label analog of maleimide (3‐maleimido‐2,2,5,5‐tetramethyl‐1‐pyrrolidinyloxyl) bound covalently to the protein moiety. Electron spin resonance spectra were measured in the temperature range from 4 °C to 50 °C. The evaluation of the spectra yielded the following results.

The stearic acid spin labels I(m/n) show a reversible transition in their motional freedom at temperatures between 25 and 30 °C. This effect is stronger for labels I(1/14) and I(5/10) than for I(12/3). The polarity of their environment decreases in a sigmoidal way between 15 and 40 °C for labels I(12/3) and I(5/10). No polarity changes are observed with label I(1/14).

Below the transition the motional and polarity characteristics of I(m/n) are dominated by lipidprotein interaction, whereas above 30 °C the spectral parameters approach the situation of a model system of phosphatidylcholine‐cholesterol.

The protein‐bound spin label shows two spectral components arising from weakly and tightly immobilized binding sites, the ratio of the former to the latter increasing strongly between 25 and 30 °C concomitantly with a decrease in the separation of the two signals. The combined results indicate reversible changes in the fine structure involving both protein and polar lipids at the surface of lipoprotein B which correlate to the thermotropic transition of the apolar lipids in the particle core.
Valylation of the Two RNA Components of Turnip‐Yellow Mosaic Virus and Specificity of the tRNA Aminoacylation Reaction
Authors: GIEGÉ Richard , BRIAND Jean‐Paul , MENGUAL Raymond , EBEL Jean‐Pierre , HIRTH Léon
Published: March 2005

A comparative study of the aminoacylation of the two RNA components of turnip yellow mosaic virus, of yeast tRNAVal, tRNAfMet and of tRNAPhe by purified yeast valyl‐tRNA synthetase is reported. Aminoacylations were performed in the presence of pure yeast tRNA nucleotidyl‐transferase, since 85% of the viral RNA molecules lacked the 3′‐adenosine. We find that aminoacylation of the viral RNAs, like tRNA aminoacylation, reflects an equilibrium between the acylation and deacylation reactions. The kinetic parameters of TYM virus RNA valylation resemble the values found for tRNAVal valylation; in particular, there is a strong affinity between the viral RNA and valyl‐tRNA synthetase and the rate constant for TYM virus RNA valylation is only slightly lower than that for tRNAVal. This result contrasts with the reduced rates observed in tRNA mischarging, and suggests that the viral RNA could be easily aminoacylated in vivo. Considering the fact that the 3′‐terminal sequence of TYM virus RNA has only a few points of resemblance to a tRNA sequence, we propose that there are some structural motifs found in both tRNAVal and TYM virus RNA which are brought in a similar spatial arrangement recognized by valyl‐tRNA synthetase.