bluuebunny/crossref_metadata_2025 huggingface dataset:
Ganglioside Biosynthesis in Golgi Apparatus of Rat Liver
Authors: YUSUF Harun K. M. , POHLENTZ Gottfried , SCHWARZMANN Günter , SANDHOFF Konrad
Published: March 2005

Golgi vesicles were isolated and purified from rat liver, in which the specific activities of glycosyltransferases (e.g. GM3 : CMP‐NeuAc sialyltransferase, GD3 synthase; GM3 : UDP‐GalNAc galactosaminyltransferase, GM2 synthase) were 50–60‐times enriched relative to microsomes or total homogenate. Synthesis of gangliosides GM2 and GM1 in such Golgi vesicles is, in the absence of any detergents, stimulated 6‐fold and 20‐fold respectively by phosphatidylglycerol. Other phospholipids like phosphatidylethanolamine and phosphatidylserine are also significantly stimulatory. With 50μg Golgi protein and 1 nmol UDP‐GalNAc, optimal stimulation of GM2 synthase was obtained with 20 μg of phosphatidylglycerol and 7.5 nmol of the lipid acceptor GM3. Under the same experimental conditions this stimulation exceds (by about 40%) that obtained with optimal amount (200μg) of the detergent octylglucoside. Phosphatidylglycerol, on the other hand, has virtually no stimulatory activity on the synthesis of ganglioside GD3 either in the presence of Mg2+ or Mn2+, indicating that facilitation by phospholipid of GM3 transport into Golgi vesicles was not the basis of stimulation of GM2 synthesis.

Tunicamycin inhibits the synthesis of gangliosides GM2 and GM1 in isolated Golgi vesicles, but only in the absence of detergents. In the presence of phosphatidylglycerol, GM2 synthesis, for example, was inhibited by 60% by 2 μg tunicamycin and more than 85% by 10 μg tunicamycin, per 50 μg Golgi membrane protein. The inhibition was stronger on GM1 synthesis: 85% with 2.5 μg of the antibiotic. The dependence on phosphatidylglycerol and the degree of inhibition by tunicamycin of the synthetic activities are strictly dependent on the intactness of the Golgi vesicles: both phenomena become increasingly less evident when the vesicles are pelleted, and frozen and thawed several times, and completely disappear when the vesicles are solubilized by detergents or disrupted by ultrasonication. Furthermore, tunicamycin inhibition is reversible by increased concentration of phosphatidylglycerol. All these results indicate tht phosphatidylglycerol does not stimulate, and tunicamycin does not inhibit, the transferases themselves; rather, the two opposing effects might relate to carrier‐mediated transport, e. g. of nucleotide sugars, across Golgi vesicles.
Na+‐Dependent Uptake of 4‐Azidophenylalanine by Pig Intestinal Microvillus Vesicles
Authors: NORÉN Ove , SJÖSTRÖM Hans , LARSEN Per
Published: March 2005

A procedure for the synthesis of tritiated 4‐azidophenylalanine in a radioactively homogeneous form is described. The characteristics of the 4‐azidophenylalanine uptake by pig intestinal microvillar vesicles and its interaction with the neutral amino acid transport have been studied. 4‐Azidophenylalanine is transported into an osmotically sensitive space and the transport is sodium dependent. It is demonstrated that 4‐azidophenylalanine transport is inhibited by l‐alanine and l‐phenylalanine but not by l‐lysine and d‐glucose. Likewise 4‐azidophenylalanine inhibited the initial transport of l‐lysine and d‐glucose was unaffected. Treatment of microvillar vesicles with 4‐azidophenylalanine in combination with photolysis irreversibly inactivated the l‐alanine transport.

Photolabelling in the presence of sodium using tritiated 4‐azidophenylalanine resulted in labelling of 10 peptides, all found in the fraction insoluble in Triton X‐100. Omission of sodium from the reaction mixture or addition of l‐alanine did not specifically abolish labelling in any of the labelled components.
The Influence of Cholesterol Incorporation and Removal on Lipid‐Bilayer Viscosity and Electron Transfer in Rat‐Liver Microsomes
Authors: ARCHAKOV Alexander I. , BORODIN Evgeni A. , DOBRETSOV Gennadi E. , KARASEVICH Elena I. , KARYAKIN Alexander V.
Published: March 2005

The incorporation and removal of cholesterol from rat liver microsomes was used as a methodical approach to investigate the molecular organization of microsomal redox‐chains.

It was shown that the incorporation of cholesterol in microsomes increases and removal of cholesterol decreases lipid bilayer viscosity as indicated from the rate of fluorescent probe‐pyrene eximerisation in cholesterol‐enriched and cholesterol‐depleted microsomes.

The increase of membrane viscosity slows down the initial rates and decreases the rate constants of cytochrome b5 reduction by NAD(P)H, whereas the decrease of membrane viscosity enhances the initial rates and increases the rate constants of these reactions. The rates of cytochrome P450 reduction by reduced pyridine nucleotides do not depend on the viscosity of lipid bilayer. The incorporation and removal of cholesterol from microsomes was not followed by any essential changes in the rates of dimethylaniline N‐demethylation, aniline p‐hydroxylation, p‐nitroanisole O‐demethylation, oxygen consumption, oxidation of NADH and NADPH.

Thus the reduction of cytochrome b5 by NADH and NADPH is the diffusion‐dependent reaction in the redoxchains of microsomes only.
Partial Purification and Characterization of mRNA (Guanine‐7‐) Methyltransferase from the Yeast Saccharomyces cerevisiae
Authors: LOCHT Camille , BEAUDART Jean‐Luc , DELCOUR Jean
Published: March 2005

As a tool for the study of the capping‐methylation process of yeast mRNA, we developed a procedure for the purification of the mRNA (guanine‐7‐)methyltransferase using the commercial cap analog guanosine(5′)triphospho(5′)guanosine as a substrate and radioactive S‐adenosylmethionine (AdoMet) as the methyl group donor. The osmotic‐sensitive yeast strain VY 1160 was used as the enzyme source. Little methyltransferase activity was detectable in a crude lysate obtained after osmotic shock. We showed that this was due to the presence of a low‐molecular‐weight inhibitor which could easily be eliminated by Sephadex G‐25 gel filtration. The 10000 ×g supernatant from the crude lysate was submitted to DEAE‐cellulose and DNA‐agarose chromatography. The resulting preparation was enriched about 450‐fold in specific activity. Under standard assay conditions, the incorporation rate remained constant for at least 6 h at 30°C. Transmethylation was not stimulated by KCl nor NaCl. Divalent cations were strong inhibitors. The partially purified enzyme was able to methylate undermethylated poly(A)‐rich mRNA isolated from an AdoMet auxotrophic yeast strain briefly exposed to AdoMet‐free medium.
The Primary Structure of Subunit II of NADH Dehydrogenase from Bovine‐Heart Mitochondria
Authors: von BAHR‐LINDSTRÖM Hedvig , GALANTE Yves M. , PERSSON Magnus , JÖRNVALL Hans
Published: March 2005

Subunit II (with a molecular mass of about 24000 dalton, ∼24 kDa) of NADH dehydrogenase from beef heart mitochondria was [14C]carboxymethylated and cleaved with CNBr and proteolytic enzymes. Sequence analyses of purified fragments suggest that the subunit is composed of a homogeneous polypeptide chain, containing just over 230 residues. The primary structure of this chain was established except for a 14‐residue internal part which was only determined by composition. The amino acid sequence suggests that four cysteine residues are involved in the binding of an iron‐sulfur cluster. The subunit contains no long hydrophobic segment, in contrast to structures often found in membrane proteins, but in agreement with a model where the functional unit of NADH dehydrogenase in the membrane is shielded by other intra‐membrane proteins. The polypeptide has a weak similarity to the iron‐sulfur binding region of ferredoxin and has interesting but possibly insignificant similarities to part of previously compared flavin‐linked enzymes.
The Properties of Citrate Transport in Membrane Vesicles from Bacillus subtilis
Authors: BERGSMA Jack , KONINGS Wil N.
Published: March 2005

The uptake system for citrate is induced in Bacillus subtilis W23 by growth in the presence of citrate and only membrane vesicles isolated from these cells show energy‐dependent citrate uptake. Citrate transport in membrane vesicles is strictly dependent on the presence of divalent cations such as Mg2+, Mn2+, Zn2+, Ba2+, Be2+, Ca2+, Cu2+, Co2+, or Ni2+. The initial rate of citrate transport increases with the divalent cation concentration up to a maximum. The maximum initial rate of citrate uptake is reached with 2 mM Mg2+.

The cations form stable chelates with citrate. The metal citrate complex is the transported solute. This is demonstrated for citrate uptake in the presence of Ca2+. Membrane vesicles from citrate‐grown cells accumulate Ca2+ and citrate only if both solutes are present. Citrate and Ca2+ are accumulated in equimolar quantities. The uptake of Ca2+ but not of citrate is inhibited by Mg2+.

Uptake of the metal‐citrate complex is inhibited by the uncoupler carbonylcyanide p‐trifluoromethoxyphenyl‐hydrazone and in the presence of K+ ions by valinomycin and nigericin. The inhibitory effects correlate with the effects observed on the components of the proton‐motive force, indicating that the proton‐motive force is a driving force for metal‐citrate transport. The number of protons (n) symported with the metal‐citrate complex has been determined under different experimental conditions from the steady state levels of citrate accumulation, the electrical potential and pH gradient. This number varies from 1 at pH 4.7 to 2 at pH 8.0.
Comparative Binding of Ethidium and Three Azido Analogs to Dinucleotides: Affinity and Intercalation Geometry
Authors: LAUGAA Philippe , DELBARRE Alain , LE PECQ Jean‐Bernard , ROQUES Bernard P.
Published: March 2005

Geometrical and thermodynamic information has been obtained from theoretical analysis of both visible and 1H‐NMR spectroscopic binding isotherms of ethidium and three photoactivable derivatives (8‐azido‐ethidium, 3‐azido‐ethidium and 3,8‐diazido‐ethidium) to self‐complementary ribodinucleosides. The following results have been obtained.

Interaction with pyrimidine(3–5′)purine sequences is well accounted for by multicomponent equilibria involving self‐association of the dyes in oligomers, formation of 1:1 and 2:1 (nucleoside:dye) complexes. This model provided evidence for intercalation of all dyes, though with weaker affinity in the case of diazido‐ethidium (2 × 106 M−2 vs 6 × 107 M−2). Moreover 3‐azido‐ethidium was shown to intercalate into cytidylyl(3′–5′)guanosine (CpG) with its phenyl group lying in the major groove of the minihelix. This geometry is inverted with respect to that of all other compounds. It should be emphasized that visible and 1H‐NMR techniques independently provided similar results (intercalation, affinity constants) therefore supporting this stepwise model.

Interaction of all dyes with purine(3′–5′)pyrimidine sequences is not intercalative, even at low temperature (4°C), but is well described by self‐association of the dyes and formation of 1:1 (nucleoside:dye) complexes.

Regarding the reversible DNA intercalation process, these studies show that 8‐azido‐ethidium is the only photoactivatable derivative which behaves exactly as ethidium. Therefore 8‐azido‐ethidium can be used as a covalent probe to investigate the DNA‐related cytotoxic effects of ethidium.
A Monoclonal Antibody Specific for the Amino Terminal Cleavage Site of Procollagen Type I
Authors: FOELLMER Harald G. , KAWAHARA Kazuko , MADRI Joseph A. , FURTHMAYR Heinz , TIMPL Rupert , TUDERMAN Leena
Published: March 2005

A monoclonal mouse IgG1 antibody was produced against the aminopropetide of dermatosparactic sheep procollagen type I by using the hybridoma technique. Radioimmunoassays demonstrated an apparent affinity constant of 1081 · mol−1. The antibody reacted with a 19‐amino‐acid‐long sequence spanning the procollagen N‐proteinase cleavage site with stronger binding to structures contributed by the aminopropeptide. The antibody showed strong cross‐reactions with similar antigens of bovine, human or chick origin but failed to react with the aminopropetide of procollagen type III. Incubation of chick or sheep procollagen type I with stoichiometric amounts of antibody blocked the release from procollagen molecules of the aminopropeptide by procollagen N‐proteinase. Thus, this antibody seems useful for studying various biological problems encountered in the conversion of procollagen.
Perturbation of the Structure and Function of a Membranous Ca2+ ‐ATPase by Non‐solubilizing Concentrations of a Non‐ionic Detergent
Authors: ANDERSEN Jens P. , LE MAIRE Mare , KRAGH‐HANSEN Ulrich , CHAMPEIL Philippe , MØLLER Jesper V.
Published: March 2005

The present study characterizes the effect of octa(ethyleneglycol)‐monododecylether (C12E8) on Ca2+ ‐ATPase membranes, prepared from sarcoplasmic reticulum (SR). At low concentrations C12E8 is incorporated into the membrane (≤0.2g/g protein), without any solubilization or appreciable morphological changes of freezefracture replica. Binding studies of C12E8 to ATPase membranes and SR lipid liposomes suggest that the major part of the detergent interacts with lipid. Solubilization of ATPase membranes occurs at a free concentration of C12E8 close to the critical micellar concentration (c.m.c); at low temperatures (2°C) phospholipid is extracted somewhat more easily than ATPase. Electron‐spin resonance (ESR) spectra of appropriate spin labels, in‐corporated into ATPase membranes, show that C12E8 strongly increases the fluidity of the lipid phase and the rotational diffusion of ATPase in the membrane. The effect of C12E8 on the ESR spectra is indistinguishable from that produced by a rise in temperature. Incorporation of C12E8 alters the functional properties of Ca2+ ‐ATPase in a characteristic way: V is decreased and the modulatory effect of high ATP concentrations is reduced, in contrast to what occurs by a rise of temperature. The intrinsic fluorescence of the protein is increased, especially in the absence of Ca2+, suggesting that C12E8 modified in particular the E* form (Ca2+ ‐depleted conformation) of the enzyme. Furthermore, stopped‐flow data indicate that C12E8 strongly activates the E* to E transition, which may account for the effect of the detergent on ATP modulation during steady‐state ATP hydrolysis. It is concluded that C12E8 perturbs ATPase turnover by direct interaction with the enzyme, rather than by an indirect effect exerted via a change in the lipid phase or protein aggregation.
The Lipopolysaccharide from Pseudomonas aeruginosa NCTC 8505
Authors: TAHARA Yasutaka , WILKINSON Stephen G.
Published: March 2005

Structural studies have been carried out on the O‐specific fraction from the lipopolysaccharide of Pseudomonas aeruginosa NCTC 8505, Habs serotype 03. The O‐specific polysaccharide has a tetrasaccharide repeating‐unit containing residues of l‐rhamnose (Rha), 2‐acetamido‐2‐deoxy‐d‐glucose (GlcNAc), 2‐acetamido‐2‐deoxy‐l‐galacturonic acid (GalNAcA), and 2,4‐diacetamido‐2,4,6‐trideoxy‐d‐glucose (BacNAc2). The following structure has been assigned to the repeating‐unit:

→3)Rhap(β1→6)GlcpNAc(α1→4) GalpNAcA(α1→3)BacpNAc2(α1→.

The parent lipopolysaccharide is a mixture of S, R, and SR species, and its high phosphorus content is partly due to the presence of triphosphate residues, as found for other lipopolysaccharides from P. aeruginosa. In addition to phosphorus, heptose, a 3‐deoxyoctulosonic acid, and amide‐bound alanine, the core oligosaccharide contains glucose, rhamnose, and galactosamine (molar proportions 3:1:1). The rhamnose and part of the glucose are present as unsubstituted pyranoside residues: other glucose residues are 6‐substituted.
Metabolic Resistance: The Protection of Enzymes against Drugs which are Tight‐Binding Inhibitors by the Accumulation of Substrate
Authors: CHRISTOPHERSON Richard I. , DUGGLEBY Ronald G.
Published: March 2005

Blockade of a metabolic pathway by interaction of a drug with a particular ‘target enzyme’ results in depletion of essential end‐products of the pathway and accumulation of intermediates prior to the blockade. Metabolic resistance to a particular drug can arise if the substrate of the inhibited enzyme accumulates to levels sufficiently high to compete effectively with the inhibitor, leading to restoration of full activity of the metabolic pathway after a transitory delay.

Such resistance has recently been demonstrated in vitro for the interaction of the tight‐binding inhibitor N‐phosphonacetyl‐l‐aspartate (PAcAsp) with the aspartate transcarbamoylase activity of the trifunctional protein which initiates pyrimidine biosynthesis in mammals [Christopherson, R. I. and Jones, M. E. (1980) J. Biol. Chem. 255, 11381–11395]. Carbamoyl phosphate, the product of the carbamoyl phosphate synthetase activity of this trifunctional protein, accumulates to a sufficiently high concentration that the inhibitory effect of PAcAsp is effectively abolished. We have developed a theoretical model for metabolic resistance which quantitatively accounts for these experimental data. This model has been used to simulate the interaction between the following potential or proven anti‐cancer drugs and their target enzyme, under conditions similar to those which would occur in vivo: PAcAsp with aspartate transcarbamoylase; various OMP analogues [the 5′‐monophosphates of 6‐azauridine, pyrazofurin and 1‐(β‐d‐ribofuranosyl)‐barbituric acid] with OMP decarboxylase; 5‐fluorodeoxyUMP with thymidylate synthase; methotrexate with dihydrofolate reductase; and deoxycoformycin with adenosine deaminase.
Some Properties of a Partially Purified Inhibitor(s) of Protein Synthesis from Rat‐Liver Mitochondria
Authors: WU Joseph M. , MOSCA Joseph , SUHADOLNIK Robert J. , IBRAHIM Nader G.
Published: March 2005

We have previously described an inactive inhibitor of protein synthesis from rat liver mitochondria and its activation by brief incubation with N‐ethylmaleimide [Wu, J. M. and Ibrahim, N. G. (1980) FEBS Lett. 119, 25–28]. To study the mode of action of the mitochondrial translational inhibitor (MTI), the relative distribution of monosomes and polysomes in rabbit reticulocyte lysates has been analysed by sucrose gradient centrifugation. These studies show that MTI causes a significant decrease in the amount of polysomes with marginal effect on the polysome profile. Under identical experimental conditions, addition of partially purified heme‐regulated inhibitor results in a complete disaggregation of polysomes. Studies with micrococcal‐nuclease‐treated rabbit reticulocyte lysates suggest that the primary target of MTI is the inactivation of globin mRNA with relatively little effect on other components of the translational machinery. The inhibitor also degrades poly(U), vesicular stomatis virus mRNA, reovirus mRNA, but appears to be inactive against poly(A), Escherichia coli 16S rRNA, HeLa cell rRNA or chick embryo DNA. Chromatography of MTI on heparin‐agarose results in the resolution of at least two inhibitory activities. The first inhibitory activity (eluted with 250 mM KCl) can be reversed (50–70%) with high concentrations of glucose 6‐phosphate or MgGTP (0.7 mM or 3.3 mM) whereas the second inhibitory activity can only be partially reversed with poly(A)‐rich RNA.
The complete amino acid sequence of oncomodulin — a parvalbumin‐like calcium‐binding protein from Morris hepatoma 5123tc
Authors: MACMANUS John P. , WATSON David C. , YAGUCHI Makato
Published: March 2005

Oncomodulin is an acidic calcium‐binding protein of 108 amino acids which has been detected only in tumors. The complete amino acid sequence of rat oncomodulin has been established, and the N‐terminus shown to be N‐acetylserine. Homology of the oncomodulin sequence with members of the troponin C superfamily of calcium‐binding proteins was demonstrated, and putative calcium‐binding domains were located. A further degree of homology was established between oncomodulin and the parvalbumins, including rat parvalbumin. Oncomodulin was therefore considered to be a parvalbumin‐like calcium‐binding protein.
Covalent structure of chicken pepsinogen
Authors: BAUDYŠ Miroslav , KOSTKA Vladimir
Published: March 2005

Chicken pepsinogen is a glycoprotein consisting of a single polypeptide chain and containing the following 367 amino acid residues: Asp23, Asn16, Thr26, Ser41, Glu14, Gln11, Pro18, Gly31, Ala17, Cys7, Val25, Met9, Ile23, Leu28, Tyr22, Phe20, His8, Lys17, Arg7, Trp4. The Mr‐value of the protein is 42074. This value includes the carbohydrate moiety of the protein, i.e. Man3, (GlcNAc)7, (‐SO3H)5. The primary fragmentation of the molecule was effected by limited trypsinolysis at arginine residues after preceding modification of the lysines with citraconic anhydride. All eight peptides expected in theory were obtained and their size, amino acid composition, and N‐terminal amino acid sequence were characterized. To elucidate the amino acid sequence of these large fragments the latter were subjected to secondary cleavage by CNBr, trypsin (after removal of the protecting groups from the lysines), the proteinase from Staphylococcus aureus V8 strain, α‐chymotrypsin, hydroxylamine, or dilute acid; the resulting peptides were isolated by gel permeation and ion‐exchange chromatography and by the fingerprint techniques. Overlaps at sites of the arginine residues were obtained in an earlier study [BaudyŠ, M. & Kostka, V. (1982) Collect. Czech. Chem. Commun. 47, 2814–2832]. Chicken pepsinogen shows the highest degree of homology with the primary structures of pepsinogens A. The internal homologies are apparent in the neighborhood of the two active aspartic acid residues. We have assigned tentatively chicken pepsinogen to the group of pepsinogens A (EC 3.4.23.1); this assignment is a result both of our sequence studies and of an investigation of the kinetic characteristics of the enzyme.
Forskolin regulation of liver membrane adenylyl cyclase
Authors: BIRNBAUMER Lutz , STENGEL Dominique , DESMIER Martine , HANOUNE Jacques
Published: March 2005

The effects of forskolin on rat liver plasma membrane adenylyl cyclase were studied. The diterpene stimulated the Vmax of the enzyme system with apparent Km values of 3–5 μM. Stimulations were marked both in the absence (20‐fold over control) as well as in the presence of various stimulators such as GTP, GuoPP[NH]P, NaF alone or in combination with glucagon. Except with GTP, where stimulations of activities by forskolin and the nucleotide were synergistic (more than additive), stimulations of combinations of GuoPP[NH]P, NaF or glucagon with forskolin were additive.

Forskolin did not alter significantly the apparent Km values of the enzyme for MgATP or MnATP or the apparent Ka values (concentrations giving stimulations that are 50% of maximum) for Mg or Mn ions, GTP, GuoPP[NH]P or NaF. Forskolin caused a decrease in the concentration of glucagon required for half‐maximal stimulation from 5 μM to 1.5 μM. Except for this effect on the Ka for the glucagon, the only kinetic parameter altered was the Vmax under all conditions tested.

Although proteolysis stimulated liver membrane adenylyl cyclase under control conditions, it did not enhance forskolin‐stimulated activities. More extensive proteolysis, which resulted in decreased activities in the absence of forskolin, also resulted in reduced forskolin‐stimulated activities.

‘Uncoupling’ of the guanine‐nucleotide‐binding regulatory component, that mediates guanine nucleotide stimulation by addition of 30 mM MnCl2, did not result in ‘uncoupling’ of forskolin stimulation. The data indicate that the diterpene forskolin stimulates adenylyl cyclase activity by a novel mechanism that differs from that by which NaF or guanylyl nucleotides affect this membrane‐bound system and that the diterpene should be a useful tool with which to explore as yet unrecognized modes of regulation of cyclic AMP production.
Fourier‐transform infrared spectroscopy applied to rhodopsin
Authors: SIEBERT Friedrich , MÄNTELE Werner , GERWERT Klaus
Published: March 2005

By measuring the rhodopsin–bathorhodopsin, isorhodopsin–bathorhodopsin, rhodopsin–isorhodopsin and rhodopsin–meta‐II difference spectra with the method of Fourier‐transform infrared spectroscopy we have identified the C=N stretching vibration of the protonated retinylidene Schiff base of rhodopsin, isorhodopsin and bathorhodopsin. In contrast to resonance Raman spectroscopy additional strong bands were observed between 1700cm−1 and 1620cm−1. Most of them depend on the isomeric state of the chromophore. The origin of these bands will be discussed. In the fingerprint region isorhodopsin and bathorhodopsin are quite similar but no similarities with infrared spectra of model compounds of any isomeric composition are observed. Therefore, no conclusions on the isomeric state of the retinal in bathorhodopsin can be drawn. We provide evidence for the modification of one or two carboxylic group(s) during the rhodopsin–bathorhodopsin and isorhodopsin–bathorhodopsin transition.
The stoichiometry of proton translocation through H+‐ATPase of rat‐liver mitochondria
Authors: SHOLTZ Khristophor F. , GORSKAYA Inna A. , KOTELNIKOVA Alla V.
Published: March 2005

The ratio of the number of protons transported directly by H+‐ATPase of intact mitochondria and that of the hydrolyzed ATP (the proton translocation quotient) is determined. A special kinetic method which makes possible determination of ATPase and H+‐translocase activities in the same experiment has been used to estimate the proton translocation quotient for mitochondrial H+‐ATPase. The quotient is found to be, on average, equal to 3.31 mol/mol.

It is shown that the protonophore 3,5‐di‐tert‐butyl‐4‐hydroxybenzylidenemalononitrile can greatly decrease the proton translocation quotient. This supports our assumption concerning a labile coupling between H+‐translocase and ATPase in mitochondria [I. A. Gorskaya, K. F. Sholtz, S. A. Moreva, A. V. Kotelnikova (1979) Biochemistry (Engl. Transl. Biokhimiya) 44, 765–770]. A decrease in the translocation quotient in the presence of the protonophore is likely to be due to the ability of this uncoupler to take back the translocated protons into the mitochondrial matrix before their release into the medium.

An electrostatic model of the molecular mechanism of H+‐translocase and ATPase coupling in the H+‐ATPase complex is discussed. The model is in agreement with the results obtained: transport of more than two protons per each hydrolyzed ATP molecule, and variable efficiency of the process.
Galactokinase of Vicia faba seeds
Authors: DEY Prakash M.
Published: March 2005

Galactokinase (EC 2.7.1.6) from the dormant seeds of Vicia faba was purified approximately 1300‐fold with an 18% recovery through an eight‐step procedure. The preparation showed the presence of only minor contaminations as judged by disc‐gel electrophoresis. The native enzyme displayed a molecular weight of approximately 60 000 (determined by Sephadex G‐100 gel‐filtration) and the subunit value was 30 000. The isoelectric point of the enzyme was 5.3 and the amino acid analysis showed high percentage of acidic amino acids. The pH optimum of the enzyme was 7.3 at 25°C. The relative activity for phosphorylating various monosaccharides followed the order, d‐galactose >2‐deoxy‐d‐galactose>d‐galactosamine; d‐fucose, l‐arabinose, l‐galactose and d‐glucose were not phosphorylated. Whereas ATP acted as an efficient phosphate donor, ADP, GTP and UTP were unable to act in this capacity. The Km and the V values of the substrates were determined. The metal ion requirement for the enzymic activity followed the order, Mg2+ > Co2+ > Mn2+ > Ni2+ > Ca2+. The enzymic reaction was inhibited by heavy metal ions and sulphydryl reagents indicating the participation of ‐SH group(s) in enzymic catalysis. Product inhibition was observed; galactose 1‐phosphate and ADP were competitive and non‐competitive inhibitors, respectively. Seed germination showed an increase in galactokinase level up to 24 h followed by a rapid decrease. The level of raffinose and stachyose decreased continually. The galactokinase level was found to be sufficiently high to phosphorylate the liberated galactose. No free galactose was observed at any stage of germination.
Coupling between fatty acid binding and sulfhydryl oxidation in bovine serum albumin
Authors: TAKABAYASHI Kenji , IMADA Teruaki , SAITO Yuji , INADA Yuji
Published: March 2005

Albumin with and without a free sulfhydryl group (mercapt‐albumin and nonmercapt‐albumin) has low and high amounts of fatty acids, respectively. It was found that this difference was brought about by the following two mechanisms: (a) the binding of fatty acids increases the rate of oxidation of the sulfhydryl group, (b) the oxidation of the sulfhydryl group of the protein enhances the binding of fatty acids.

The incubation of the protein with fatty acids enhanced reversibly the reaction rate of the free sulfhydryl group with 5,5′‐dithiobis(2‐nitrobenzoic acid). This effect of fatty acids depends on the kind of fatty acid added. Among saturated long‐chain fatty acids tested, the shortest fatty acid, lauric (dodecanoic) acid, had the strongest effect. Oleic (cis‐9‐octadecenoic) acid, which has a long chain but also one cis‐double bond, however, was as effective as lauric acid in enhancing the reactivity of the sulfhydryl group on the protein. Analyses with palmityl‐Sepharose and equilibrium partitioning of palmitic acid between heptane and aqueous protein solutions have shown that nonmercapt‐albumin has higher affinity to the fatty acid than mercapt‐albumin.

These results imply that the binding of fatty acids and the oxidation of sulfhydryl groups of the protein are intimately coupled.
Elementary steps in the reaction of the pyruvate dehydrogenase complex from pig heart
Authors: SÜMEGI Balázs , ALKONYI István
Published: March 2005

In the progress curve of the reaction of the pyruvate dehydrogenase complex, a lag phase was observed when the concentration of thiamin diphosphate was lower than usual (about 0.2–1 mM) in the enzyme assay. The length of the lag phase was dependent on thiamin diphosphate concentration, ranging from 0.2 min to 2 min as the thiamin diphosphate concentration varied from 800 nM to 22 nM. The lag phase was also observed in the elementary steps catalyzed by the pyruvate dehydrogenase component. A Km value of 107 nM was found for thiamin diphosphate with respect to the steady‐state reaction rate following the lag phase.

The pre‐steady‐state kinetic data indicate that the resulting lag phase was the consequence of a slow holoenzyme formation from apoenzyme and thiamin diphosphate. The thiamin diphosphate can bind to the pyruvate dehydrogenase complex in the absence of pyruvate, but the presence of 2 mM pyruvate increases the rate constant of binding from 1.4 × 104 M−1s−1 to 1.3 × 105 M−1s−1 and decreases the rate constant of dissociation from 2.3 × 10−2s−1 to 4.1 × 10−3s−1. On the other hand, the effect of pyruvate on the thiamin diphosphate binding revealed the existence of a thiamin‐diphosphate‐independent pyruvate‐binding site in the pyruvate dehydrogenase complex. Direct evidence was also obtained with fluorescence techniques for the existence of this binding site and the dissociation constant of pyruvate was found to be 0.38 mM. On the basis of these data we have proposed a random mechanism for the binding of pyruvate and thiamin diphosphate to the complex.

Binding of substrates to the enzyme complex caused an increase in the fluorescence of the dansylaziridine‐labelled pyruvate dehydrogenase complex, showing that binding of substrates to the complex is accompanied by structural changes.
The function of Nα‐acetylation of the eye‐lens crystallins
Authors: DRIESSEN Huub P. C. , RAMAEKERS Frans C. S. , VREE EGBERTS Wilma T. M. , DODEMONT Huub J. , de JONG Wilfried W. , TESSER Godefridus I. , BLOEMENDAL Hans
Published: March 2005

The putative protective role of the Nα‐acetyl group of proteins has been investigated. Synthetic, non‐acetylated Nterminal tetrapeptides of the αA2‐ and γII‐crystallin chains are good substrates for leucine aminopeptidase, while the acetylated ones are completely resistant. In the native, non‐acetylated, γ‐crystallin the N terminus is not degraded by leucine aminopeptidase. Newly synthesized αA2‐crystallin, in which the normally occurring N‐terminal acetylation has been prevented during cell‐free translation, is virtually resistant against degradation by leucine aminopeptidase. Only at extreme enzyme‐substrate ratios the N‐terminal methionine is removed. Although the Nα‐acetyl group by its very nature protects against this exopeptidase, we conclude that the group is not essential for this purpose in the native crystallins.
Phosphorolytic activity of Escherichia coli glycyl‐tRNA synthetase towards its cognate aminoacyl adenylate detected by 31P‐NMR spectroscopy and thin‐layer chromatography
Authors: LED Jens J. , SWITON Werner K. , JENSEN Kaj F.
Published: March 2005

The catalytic activity of highly purified Escherichia coli glycyl‐tRNA synthetase has been studied by 31P‐NMR spectroscopy and thin‐layer chromatography on poly(ethyleneimine)‐cellulose. It was found that this synthetase, besides the activation of its cognate amino acid and the syntheses of adenosine(5′)tetraphospho(5′)adenosine (Ap4A) and adenosine(5′)triphospho(5′)adenosine (Ap3A), also catalyzes the formation of ADP from inorganic phosphate and the enzyme‐bound glycyl adenylate. Accordingly it was shown that E. coli glycyl‐tRNA synthetase, in the presence of inorganic phosphate, glycine, and Mg2+ ions, catalyzes the synthesis of ADP from three different substrates which all lead to enzyme‐bound glycyl adenylate, that is, ATP, adenosine 5′‐[β,γ‐methylene]triphosphate and Ap4A. It was furthermore demonstrated that the only pathway by which a synthetase‐catalyzed degradation of Ap4A can occur is through the reaction between inorganic phosphate and the enzyme‐bound glycyl adenylate, synthesized from Ap4A. Likewise a 20‐fold increase of the phosphorolytic activity of the investigated synthetase was observed when Mg2+ was replaced by Mn2+.

Besides establishing the phosphorolytic activity of the applied enzyme, the study also showed that the preparation catalyzes a glycine‐independent transfer of the γ‐phosphate group from ATP to nucleoside 5′‐diphosphates.

The importance of the observed reaction between inorganic phosphate and enzyme‐bound aminoacyl adenylate in relation to the remaining catalytic activities of aminoacyl‐tRNA synthetases is discussed, as well as the biological significance of the reaction.
The calmodulin‐dependent glycogen synthase kinase from rabbit skeletal muscle
Authors: WOODGETT James R. , DAVISON Michael T. , COHEN Philip
Published: March 2005

A calmodulin‐dependent glycogen synthase kinase distinct from phosphorylase kinase has been purified ∼ 5000‐fold from rabbit skeletal muscle by a procedure involving fractionation with ammonium sulphate (0–33 %), and chromatographies on phosphocellulose, calmodulin‐Sepharose and DEAE‐Sepharose. 0.75 mg of protein was obtained from 5000 g of muscle within 4 days, corresponding to a yield of ∼ 3 %. The Km for glycogen synthase was 3.0 μM and the V 1.6–2.0 μmol min−1 mg−1.

The purified enzyme showed a major protein staining band (Mr 58 000) and a minor component (Mr 54 000) when examined by dodecyl sulphate polyacrylamide gel electrophoresis. The molecular weight of the native enzyme was determined to be 696 000 by sedimentation equilibrium centrifugation, indicating a dodecameric structure. Electron microscopy suggested that the 12 subunits were arranged as two hexameric rings stacked one upon the other.

Following incubation with Mg‐ATP and Ca2+‐calmodulin, the purified protein kinase underwent an ‘autophosphorylation reaction’. The reaction reached a plateau when ∼ 5 mol of phosphate had been incorporated per 58 000‐Mr subunit. Both the 58 000‐Mr and 54 000‐Mr species were phosphorylated to a similar extent. Autophosphorylation did not affect the catalytic activity.

The calmodulin‐dependent protein kinase initially phosphorylated glycogen synthase at site‐2, followed by a slower phosphorylation of site‐1 b. The protein kinase also phosphorylated smooth muscle myosin light chains, histone H1, acetyl‐CoA carboxylase and ATP‐citrate lyase. These findings suggest that the calmodulin‐dependent glycogen synthase kinase may be a enzyme of broad specificity in vivo.

Glycogen synthase kinase‐4 is an enzyme that resembles the calmodulin‐dependent glycogen synthase kinase in phosphorylating glycogen synthase (at site‐2), but not glycogen phosphorylase. Glycogen synthase kinase‐4 was unable to phosphorylate any of the other proteins phosphorylated by the calmodulin‐dependent glycogen synthase kinase, nor could it phosphorylate site 1 b of glycogen synthase. The results demonstrate that glycogen synthase kinase‐4 is not a proteolytic fragment of the calmodulin‐dependent glycogen synthase kinase, that has lost its ability to be regulated by Ca2+‐calmodulin.
ATP‐AMP phosphotransferase from Paracoccus denitrificans
Authors: YEH Shing‐shing , TOMASSELLI Alfredo G. , NODA Lafayette H.
Published: March 2005

Adenylate kinase has been extracted from Paracoccus denitrificans by toluene treatment and purified 370‐fold (overall yield of 44%, 554 U/mg, Mr 22 000, pI 4.7; pH optimum 7.5–8.0) by using successive column chromatography on DEAE‐cellulose DE‐52, Matrex‐Blue A and Sephadex G‐75. The enzyme was homogeneous by dodecylsulfate gel electrophoresis and constancy of specific activity across the single peak found in Sephadex G‐75 chromatography. Crystals in the form of heavy needles have been obtained in poly(ethylene glycol)‐400. The enzyme is specific for adenine (deoxyadenine) nucleotides with Km values for ATP, ADP and AMP of 340 μm, 980 μM and 93 μM respectively and dissociation constants, for ATP, MgATP and AMP of 7.3 μM, 7.1 μM and 6.9 μM respectively. The enzyme was noted to have no disulfide bond, two free sulfhydryl groups and a total of 208 residues. Aboe 50 mM KCl the enzyme activity drops off gradually. The enzyme is stable at neutral pH and below a temperature of 44°C. Adenylate kinase from P. denitrificans resembles adenylate kinases from other sources with respect to such properties as the dependence on specific divalent cations, maximal activities at a ratio of Mg2+ :ATP of about 1:1 and the ratio of Mg2+ :ADP of 1:2, in having MgATP (or MgADP) binding site and another substrate binding site for AMP (or ADP), and in requiring an intact histidine and one or more arginine residues for enzymatic activity.

Adenylate kinase from P. denitrificans appears to resemble the so‐called muscle‐type (cytoplasmic) rather than the mitochondrial‐type adenylate kinase by comparisons of the following properties: low molecular weight, having — SH groups and no disulfide bonds, pH‐optimum of 8.0 for formation of ADP, positive cross‐reaction by ELISA test and in requiring a low concentration of Ap5A to inhibit 95% activity.
Isolation and characterization of the three fractions (DE‐I, DE‐II and DE‐III) of rat‐liver Z‐protein and the complete primary structure of DE‐II
Authors: TAKAHASHI Kuni , ODANI Shoji , ONO Teruo
Published: March 2005

Three fractions (DE‐I, DE‐II and DE‐III) of Z‐protein (fatty acid binding protein) have been isolated from rat liver cytosol by DEAE‐cellulose chromatography and characterized. They had the same molecular weight of 14000 and essentially identical amino acid composition. However, compositions of endogenous fatty acids were found to differ strikingly from one another. Long‐chain fatty acids detected in DE‐II were palmitic, stearic, oleic, linoleic and arachidonic acids. In contrast to DE‐II, DE‐III contained mainly arachidonic acid. Molar ratios of endogenous long‐chain fatty acids to both DE‐II and DE‐III were estimated to be around 1.0. Unlike the latter two fractions, DE‐I was virtually lipid‐free.

Analyses of the three fractions by polyacrylamide gel electrophoresis, electrofocusing and DEAE‐cellulose chromatography before and after delipidation suggested that the difference between DE‐I and DE‐II was in part due to fatty acids bound to DE‐II. In contrast, DE‐III appeared to be somewhat different from these forms in its protein structure, though tryptic peptide mappings of the three fractions did not reveal clear differences among them.

Analysis of the primary structure was made on the most abundant fraction, DE‐II, to investigate the relationship among the three fractions and to other proteins. The protein was a single chain consisting of 127 amino acid residues and had a mostly acetylated NH2 terminus and a free sulfhydryl group. The complete sequence of Z‐protein showed striking homology to cellular retinoid binding proteins and peripheral nerve myelin P2 protein, which indicated the presence of a new family of cellular lipid‐binding proteins diverged from a common ancestor. A possible intragenic duplication of Z‐protein was also suggested.
Actions of glucagon on flux rates in perfused rat liver
Authors: KIMMIG Rainer , MAUCH Teri Jo , KERZL Willibald , SCHWABE Ursula , SCHOLZ Roland
Published: March 2005

Changes in the rates of glucose, lactate and pyruvate production following infusion of glucagon were studied in isolated livers from fed or fasted rats perfused with non‐recirculating Krebs‐Henseleit bicarbonate buffer.

Evidence was presented that under these experimental conditions, the release of lactate plus pyruvate into the perfusate represents 80–90% of the total glycolytic flux; oxidation of pyruvate derived from glucose and/or glycogen accounts for the remaining 10–20%, whereas recycling of pyruvate to glucose is negligible.

In the presence of glucagon, rates of lactate plus pyruvate production were diminished to less than 30% in the fed state (glycogen as substrate) and to less than 10% in the fasted state (glucose as substrate). Rates of pyruvate oxidation were unchanged. Although recycling of pyruvate to glucose was enhanced, it could account for not more than 20% of the decrease in lactate plus pyruvate production. The data indicate a strong inhibition of the substrate flux through glycolysis.

The glucagon concentration for half‐maximal inhibition of glycolysis was 0.2 nM, independent of the substrate (glucose or glycogen) and the nutritional state. The effective concentrations were within the physiological range of glucagon concentrations reported for portal venous blood.

Transient state analyses indicated that the inhibition of glycolysis precedes the stimulation of glycogenolysis. When after a delay glycogenolysis was accelerated, it was followed by a transient stimulation of glycolysis. The stimulatory component in the glucagon effect on glycolysis was diminished in glycogen‐depleted livers and when glucose was the main substrate. The coordinated control of glycolysis and glycogenolysis by glucagon and the interaction of the two processes in the transient state are discussed.
Chemical structure of the lipid A component of the lipopolysaccharide from a Proteus mirabilis Re‐mutant
Authors: SIDORCZYK Zygmunt , ZÄHRINGER Ulrich , RIETSCHEL Ernst Th.
Published: March 2005

The chemical structure of the lipid A component from the lipopolysaccharide of a Proteus mirabilis Re‐mutant (strain R45) was analysed. It consists of a β(1–6)‐linked d‐glucosamine disaccharide which carries two phosphate groups, one being ester‐linked to position 4′ of the nonreducing glucosaminyl residue and the other being bound to the glycosidic hydroxyl group of the reducing glucosaminyl residue. The ester‐bound phosphate group is quantitatively substituted by a 4‐amino‐4‐deoxy‐l‐arabinopyranosyl residue, the glycosidic phosphoryl group appears to be unsubstituted. Two available hydroxyl groups of the disaccharadide (probably at positions 3 and 3) are acylated by approximately 1 mol each of (R)‐3‐tetradecanoyloxytetradecanoic and (R)‐3‐hydroxytetradecanoic acid/mol. The amino group of the nonreducing glucosaminyl residue carries (R)‐3‐tetradecanoyloxytetradecanoic and that of the reducing residue (R)‐3‐hydroxytetradecanoic acid. In addition smaller amounts of (R)‐3‐hexadecanoyloxytetradecanoic acid are present in amide linkage.

The attachment site of the oligosaccharide portion to lipid A was also investigated. It was found that the hydroxyl group at position 6′ of the nonreducing glucosaminyl residue carries 3‐deoxy‐d‐manno‐octulosonic acid. This indicates that the saccharide portion in this Proteus lipopolysaccharide is linked to lipid A via the primary hydroxyl group in position 6′.
Mode of action towards oligopeptides and proteins of hydrolase H, a high‐molecular‐weight aminoendopeptidase from rabbit skeletal muscle
Authors: NISHIMURA Toshihide , OKITANI Akihiro , KATAKAI Ryoichi , KATO Hiromichi
Published: March 2005

The mode of action towards oligopeptides and proteins of hydrolase H purified from rabbit skeletal muscle was studied. The presence of protamine or α‐N‐benzoylarginine p‐nitroanilide (an endopeptidase substrate) changed both the Km and V values of the enzyme towards Leu‐β‐naphthylamide (an aminopeptidase substrate). This indicates that the binding site for an endopeptidase substrate is different from that for an aminopeptidase substrate. Hydrolase H as an aminopeptidase displayed broad specificity. The enzyme hydrolyzed various dipeptides readily except the dipeptides containing Pro or an amino acid with a hydrophobic β‐branched chain at the NH2 terminus. Pro and Val at the NH2 terminus of tripeptides were also difficult to release, whereas Ile and Val of tetrapeptides were easily released in contrast with those of dipeptides. The longer the peptide chain of Glyn (n= 2, 3,4), the more susceptible was it to hydrolase H.

Hydrolase H behaved as an endopeptidase only towards protamine among the proteins tested. The other proteins, casein, bovine serum albumin, myofibrils, troponin, hemoglobin, sarcoplasmic proteins, and myoglobin were probably attacked only by the aminopeptidase activity of the enzyme.
Oxidation of glutathione by the superoxide radical to the disulfide and the sulfonate yielding singlet oxygen
Authors: WEFERS Heribert , SIES Helmut
Published: March 2005

The reaction of superoxide with reduced glutathione (GSH) was studied with two O72‐producing systems: xanthine oxidase using xanthine or acetaldehyde as substrates and, secondly, quinol autoxidation.

The capability of GSH to quench superoxide radicals was detected by lowered O72‐mediated cytochrome c3+ reduction. The formation of the oxidation products, glutathione disulfide (GSSG) and glutathione sulfonate (the latter at levels of about 6–15% compared to GSSG), was dependent on the O72‐production and was inhibited by superoxide dismutase. The presence of GSH together with an O72‐producing system led to an extra uptake of oxygen, which was also depressed by superoxide dismutase. The observed O2 uptake was accounted for by the formation of GSSG and GSO−3 from GSH; the data are in accordance with a mechanism involving thiyl radicals.

Low‐level chemiluminescence measurement indicated the formation of excited oxygen species. The intensity of photoemission was dependent on the GSH concentration and on the O−·2 production rate. Chemiluminescence was inhibited by superoxide dismutase and also by glutathione peroxidase, but not by catalase or OH· quenchers. Spectral analysis and the effects of 1,4‐diazabicyclo[2.2.2]octane and sodium azide indicated the contribution of singlet molecular oxygen to the light emission. It is suggested that singlet oxygen results from an intermediate oxygen addition product such as a glutathione peroxysulphenyl radical.