bluuebunny/crossref_metadata_2025 huggingface dataset:
The Effective Proton Conductance of the Inner Membrane of Mitochondria from Brown Adipose Tissue
Authors: NICHOLLS David G.
Published: March 2005

The nucleotide‐sensitive H+(OH−) conducting pathway of mitochondria from the brown‐adipose tissue of cold‐adapted guinea‐pigs passes an effective proton current which is directly proportional to the proton electrochemical gradient. At 23 °C and pH 7.0 this conductance is 16 nmol H+· min−1· mg−1· mV−1. Addition of 0.2 mM GDP results in a conductance which is linear and low (0.7 nmol H+· min−1· mg−1· mV−1) until ΔH+ exceeds 220 mV. At higher values of ΔH+, which can be attained by glycerol 3‐phosphate oxidation but not palmitoyl‐L‐carnitine plus malate oxidation, the membrane conductance greatly increases, effectively limiting the maximal ΔH+ to 240 mV. High glycerol 3‐phosphate concentrations which have the thermodynamic potential to exceed this value of ΔH+ instead create a greatly increased rate of controlled respiration. The generality and significance of this device to limit ΔH+, and its relation to the nucleotide‐sensitive conductance, are discussed.
α‐D‐Galactosidase from Soybeans Destroying Blood‐Group B Antigens
Authors: HARPAZ Noam , FLOWERS Harold M. , SHARON Nathan
Published: March 2005

α‐D‐Galactosidase was isolated from untoasted soybean meal and purified to homogeneity by affinity chromatography on N‐ɛ‐aminoacaproyl α‐D‐galactopyranosylamine‐Sepharose. The purified enzyme destroyed the B‐specificity of human ovarian cyst B‐glycoprotein with an accompanying increase in H‐specificity, and converted human type‐B erythrocytes to type O.

The enzyme consists primarily of a tetramer, molecular weight 150000 ± 5000 at pH 4.0, and of a monomer, molecular weight 40000 ± 3000 at pH 8.0. Polyacrylamide gel electrophoresis in dodecyl sulfate at pH 7.2 distinguished between two types of monomeric unit of similar molecular weight. N‐terminal alanine was identified as the sole N‐terminal amino acid residue. The enzyme was shown to be devoid of carbohydrate.
Conformational Studies on Histone H3 and Its CNBr Peptides
Authors: MORRIS Gregory , LEWIS Peter N.
Published: March 2005

Histone H3 and H3 peptides 1–120, 1–90, 91–135, 91–120 and 121–135 have been prepared and examined for salt‐induced conformational changes by circular dichroism measurements. It was found that reduced histone H3 and the reduced peptides 1–120, 91–135 and 91–120 exhibit biphasic changes with the formation of α‐helix and β structures. H3 peptide 1–90, on increasing the ionic strength to moderately high levels, monophasically formed appreciable quantities of α‐helix and β structures, while peptide 121–135 remained unfolded under all ionic strengths examined. All the above peptides except 121–135 also aggregate when the ionic strength is raised. The salt‐induced near‐ultraviolet circular dichroic spectra of histone H3 and peptide 1–90 were found to be very similar, suggesting that the conformational changes induced in the peptide 1–90 are essentially the same as those observed for the intact histone. These results support the contention that the polypeptide segments of this histone interact initially by parallel self‐association followed by the formation of even larger aggregates on a longer time scale.
Further Study of Nucleotide‐Binding Site on Chloroplast Coupling Factor 1
Authors: GIRAULT Guy , GALMICHE Jean‐Michel
Published: March 2005

The adenine nucleotide binding sites on spinach chloroplast coupling factor 1 have been investigated by measuring the fluorescence changes of the substrate or of the enzyme as a function of the amount of nucleotide bound to the protein.

The decrease of the fluorescence of ethenoadenine nucleotide and the quenching of the intrinsic fluorescence of the protein has been studied when these nucleotides bind to the factor. The enhancement of the fluorescence of a probe, 4‐nitrobenzofurazan (Nbf), is analyzed when adenine nucleotides bind to the protein. These three spectral changes do not parallel the total amount of nucleotide bound to the coupling factor.

We propose that the first molecule of nucleotide which binds to the factor exhibits, after binding, specific properties and induces structural changes of the protein. Thus the first molecule of ethenoadenine nucleotide bound to the factor has a very low fluorescence yield and brings about a quenching of the intrinsic fluorescence of the enzyme. Furthermore the first molecule of adenine nucleotide which binds to the factor induces a conformation change which brings the Nbf amino groups from an hydrophilic to an hydrophobic surrounding and consequently causes an enhancement of the fluorescence of the N‐Nbf group.

The action of the different nucleotides on the fluorescence intensity of the N‐Nbf group of the factor has been studied.

The properties of the solubilized factor are discussed in terms of intersubunit interactions modified by ligand binding on only one site of one subunit. Properties of the factor in vitro are compared with those of the factor embedded in the chloroplast membrane.
A DNA Polymerase from Ustilago maydis
Authors: YARRANTON Geoffrey T. , BANKS Geoffrey R.
Published: March 2005

The 3′→ 5′ deoxyribonuclease activity associated with an Ustilago maydis DNA polymerase hydrolysed non‐complementary 3′‐primer termini about 12 times more rapidly than complementary termini. An analysis of its substrate specificity suggested that, although it was unable to hydrolyse fully single‐stranded polynucleotides, it could hydrolyse such regions less than about four nucleotides in length covalently bound to a primer molecule which was base‐paired to a complementary template strand. Template‐primer combinations containing complementary or non‐complementary primer termini both supported polynucleotide synthesis, but whereas the former were conserved, the latter were hydrolysed during the reaction thus allowing synthesis to occur. No addition of nucleotides onto a conserved non‐complementary 3′‐primer terminus was detected. The deoxyribonuclease activity therefore fulfilled a proof‐reading function during DNA synthesis in vitro.
Sensitivity of Hemoglobin Thiol Groups within Red Blood Cells of Rat during Oxidation of Glutathione
Authors: KOSOWER Nechama S. , KOPPEL Rela L. , KOSOWER Edward M.
Published: March 2005

The intracellular thiol‐oxidising diazenes, diazenedicarboxylic acid bis‐N,N‐dimethylamide and diazenedicarboxylic acid bis‐N′‐ethylpiperazinide, have been used in the study of red cells. A difference in the consequences of diazene oxidant treatment between the human red cell and rat red cell has been found in respect to the quantity of oxidant needed for glutathione (GSH) oxidation, to the fate of GSH, and to the reactivity of hemoglobin. In the first place, significantly more oxidant is needed for GSH oxidation in the rat red cell than in the human cell. Secondly, in the human cell, all of the GSH is converted to glutathione disulfide (GSSG), from which GSH is regenerated. In the rat cell, GSH disappears without being converted to GSSG, and GSH is not regenerated. Thirdly, a decrease in rat hemoglobin thiol groups, but no change in human hemoglobin, is found.

Sterically unhindered thiol groups in the rat hemoglobin are thought to react with the usual adduct intermediate in GSH oxidation by diazene (formed from RCON=NCOR + GSH →RCON(SG)NHCOR) to produce mixed disulfides, from which GSH is not easily regenerated. The results support the idea that reduction of mixed disulfides of GSH and protein is not carried out directly by GSSG reductase but necessitates thiol transferase and GSH.

The thiol‐oxidising diazenes may be of use in mapping of exposed, reactive thiol groups in proteins.
Identification of 2‐Methyl‐3‐hydroxydecanoic and 2‐Methyl‐3‐hydroxytetradecanoic Acids in the ‘Lipid X’ Fraction of the Bordetella pertussis Endotoxin
Authors: HAEFFNER Nicole , CHABY Richard , SZABÓ Ladislas
Published: March 2005

The ‘lipid X’ fraction, released from the Bordetella pertussis endotoxin upon treatment with trifluoroacetic acid of pH 3 at 50°C, was shown to contain, in addition to 3‐hydroxydecanoic, 3‐hydroxydodecanoic, 3‐hydroxytetradecanoic, and tetradec‐2‐enoic acids, 2‐methyl‐3‐hydroxydecanoic‐, and 2‐methyl‐3‐hydroxytetradecanoic acids. The structure of these was established by gasliquid chromatography/mass spectrometry.
Cooperative, Low‐Molecular‐Weight Dimeric Myoglobins from the Radular Muscle of the Gastropod Mollusc Nassa mutabilis L
Authors: GERACI Giuseppe , SADA Alfonso , CIROTTO Carlo
Published: March 2005

Two chromatographically distinct dimeric myoglobins can be isolated from the radular muscles of the sea‐snail Nassa mutabilis L. The purification procedure is based on ammonium sulfate fractionation of the centrifuged homogenate followed by CM‐cellulose column chromatography and gel filtration through Sephadex G‐100. The total recovery, on a heme basis, is about 50%. The two myoglobins show one band by gel electrophoresis and practically identical physical properties. The isoelectric point is at pH 10.2 ± 0.2; the apparent molecular weight is 28000 ± 1500 by gel filtration and is not dependent on the ligand state of the hemes. Ageing of the myoglobin solutions produces a form showing an apparent molecular weight of about 15 000 ± 500 by gel filtration and abnormal spectrophotometric properties. This form is incapable of reassociation. S20, w= 2.0 ± 0.05 S for the native oxy derivative by velocity of sedimentation, S20, w= 1.25 ± 0.005 S for the molecule denatured in sodium dodecylsulfate. Acrylamide gel electrophoresis in sodium dodecylsulfate, in reducing conditions, indicates a molecular weight of about 13000. About 15 000 g protein/mol heme are found by biuret analysis of the isolated globins. Determinations of the absorption coefficients have been carried out by iron analysis and by spectrophotometric measurements on the CN‐metmyoglobin and on the pyridine‐chromogen derivatives. Oxygen equilibrium curves show that the myoglobin binds cooperatively oxygen with values of the interaction coefficient, n, = 1.5 with no Bohr effect.
Studies on Avian Erythrocyte Metabolism
Authors: ISAACKS Russell , HARKNESS Donald , ADLER Janet , ROTH Sandra , KIM Chang , GOLDMAN Phyllis , SAMPSELL Ronald
Published: March 2005

The erythrocyte count of the adult ostrich (∼ 2.0 × 106/mm3) is about 40% of that of man (∼ 5.0 × 106/mm3) but the red blood cell is approximately three times larger (220 μm3). The total phosphate content per cm3 red blood cell is 37.7 μmol. Inositol tetrakisphosphate (inositol‐P4), a compound not previously reported in the erythrocytes of any species of animal, is the major organic phosphate in erythrocytes of the adult ostrich, accounting for 35 % of the total phosphate. Inositol pentakisphosphate (inositol‐P5), the major phosphorylated compound in the erythrocytes of most mature species of birds, is also present but in amounts approximately half that of inositol‐P4. The effects of adenosine triphosphate, 2,3‐bisphosphoglyceric acid (P2‐glycerate), inositol‐P4, inositolP5, and inositol hexakisphosphate (inositol‐P6) on the oxygen affinity of whole ‘stripped’ hemoglobin, hemoglobin A (major component), and hemoglobin D (minor component) isolated from erythrocytes of the mature ostrich have been determined. All five compounds cause a right shift in the oxygen dissociation curve of each hemoglobin and their relative effectiveness in decreasing the oxygen affinity is ATP < P2‐glycerate < inositol‐P4 < inositol‐P5 < inositol‐P6. No preferential interaction of inositol‐P4 or inositol‐P5 with either hemoglobin A or hemoglobin D was observed. The unique organic phosphate composition of the erythrocyte of the ostrich probably explains the relatively high whole blood oxygen affinity (P50= 24.5 torr) of these birds.
DNA Unwinding Enzyme II of Escherichia coli
Authors: ABDEL‐MONEM Mahmoud , DÜRWALD Hildegard , HOFFMANN‐BERLING Hartmut
Published: March 2005

The DNA‐stimulated 75000‐Mr ATPase described in the preceding paper is shown to be a further catalytic DNA unwinding principle (DNA unwinding enzyme II) made in Escherichia coli cells (the first being the 180000‐Mr ATPase of the cells: DNA unwinding enzyme I). Unwinding depends, strictly, on the supply of ATP. It occurs only under conditions permitting ATP dephosphorylation and it proceeds as long as enzyme molecules are permitted to enter the enzyme. DNA complex. The enzyme binds specifically to single‐stranded DNA yielding a complex of only limited stability. These results are interpreted in terms of a distributive mode of action of the enzyme. It is argued that chain separation starts near a single‐stranded DNA region and that, forced by continued adsorption of enzyme molecules to the DNA, it develops along the duplex. This mechanism is different from that deduced previously for DNA unwinding enzyme I. Complicated results were obtained using ATPase prepared from rep3 mutant cells.
Small‐Angle X‐Ray Titration Study on the Complex Formation between 5‐S RNA and the L18 Protein of the Escherichia coli 50‐S Ribosome Particle
Authors: ÖSTERBERG Ragnar , GARRETT Roger A.
Published: March 2005

The 5‐S RNA (A) and the L18 protein (B) from Escherichia coli ribosomes form one single AB complex in the concentration ranges supposed to prevail in vivo; at concentrations of L18 higher than 40 mM there is some indication for a minor species, most probably an AB2 species. This is indicated from the X‐ray scattering titration data of the 5‐S RNA/L18 system recorded at 21°C in ribosomal reconstitution buffer. As a result of the 1:1 complex formation, there is a relatively small but defined increase in the radius of gyration from 3.61 to 3.85 nm. This result as well as the experimental scattering curve can be explained by models where it is assumed that the elongated L 18 model is quite far from the electron density centre and where protein L18 interacts with one or both of the minor arms of the supposed Y‐shaped 5‐S RNA molecule.
Preparation of Polypeptide Subunits of Cytochrome Oxidase from Neurospora crassa
Authors: WERNER Sigurd
Published: March 2005

Cytochrome oxidase was purified from Neurospora crassa by ammonium sulfate fractionation in the presence of bile salts. The enzyme preparations contained 10–13 nmol of heme a per mg of protein; no other hemoproteins could be detected. Dodecylsulfate gel electrophoresis resolved the enzyme complex into seven major bands, representing seven polypeptide subunits.

A procedure is described that allows the isolation of these enzyme subunits on a large scale starting from a single batch of oxidase preparation. It involves dissociation of the enzyme complex by dodecylsulfate and subsequent separation of the obtained polypeptides by chromatography in the presence of various dodecylsulfate concentrations. Purification of subunits 3, 4, 5, 6 and 7 was achieved by column chromatography using molecular sieves (Sephadex G‐100, Bio Gel P‐60) and hydroxylapatite. For the purification of subunits 1 and 2 an electrophoretic separation on a preparative polyacrylamide gel was required.

The advantages and disadvantages of the separation procedure of the enzyme polypeptides are discussed. As a special point of interest, the conservation of antigenic determinants of the polypeptide chains during the dodecylsulfate treatment is considered.
Reaction of Yeast Fatty Acid Synthetase with Iodoacetamide
Authors: KRESZE Georg‐Burkhard , STEBER Liesel , OESTERHELT Dieter , LYNEN Feodor
Published: March 2005

When yeast fatty acid synthetase is treated with iodoacetamide at 0°C, three carbamoylmethyl residues are incorporated into the protein under concomitant complete loss of the enzymatic activity [Oesterhelt, D., Bauer, H., Kresze, G.‐B., Steber, L. and Lynen, F. (1977) Eur. J. Biochem. 79, 173–180]. Here we report that, in this reaction, only cysteinyl residues are alkylated. During protein hydrolysis of the modified enzyme, S‐carboxymethyl cysteine is formed which, when treated with performic acid in the presence of a strong acid and chloride ions, is oxidized only partially to yield open‐chain S‐carboxymethyl cysteine sulfone but, for the greater part, is transformed into S‐carboxymethyl cysteine lactam sulfone (3‐carboxy‐5‐oxo‐tetrahydro‐1,4‐thiazine‐1,1‐dioxide), a novel derivative of S‐carboxymethyl cysteine which cannot be detected with ninhydrin. If, on the other hand, the carbamoylmethylated enzyme is treated first with performic acid and hydrolyzed afterwards, a large part of the carbamoylmethyl residues is lost due to decarboxylation of the α‐sulfonyl carboxylic acid S‐carboxymethyl cysteine sulfone.

When carboxamidomethylated fatty acid synthetase was digested with trypsin at 0°C, two different carbamoylmethyl peptides were found which could be separated by gel filtration. The larger peptide TA was cleaved, during prolongated incubation with trypsin at 30°C, to yield the smaller peptide TB which was purified and its amino acid sequence determined to be Thr‐Pro‐Val‐Gly‐Ala‐Cys(carbamoylmethyl). By comparison of tryptic and peptic peptides from acetylated and carboxamidomethylated synthetase, respectively, it is demonstrated that the same cysteinyl residues are alkylated by iodoacetamide which are the acetyl‐binding sites of the condensing enzyme component, i.e. the peripheral SH‐groups.
Stimulation and Inhibition of Milk (Lipoprotein) Lipase by Proteins from Egg Yolk Lipoproteins
Authors: BENGTSSON Gunilla , OLIVECRONA Thomas
Published: March 2005

In an accompanying paper we describe a method to purify from very low density lipoproteins of egg yolk, two protein fractions (T1 and T2) with cofactor activity for lipoprotein lipases. When added to media containing (bovine milk) lipoprotein lipase and Intralipid (a lecithin‐stabilized emul‐sion of soy bean triacylglycerols) they caused an immediate, several‐fold increase in the rate of lipolysis. It has been considered characteristic for lipoprotein lipases that their activity is strongly suppressed by high concentrations of NaCl. Such inhibition was seen in assay systems with whole serum, lipoproteins, or crude fractions of egg apoproteins as source of cofactor activity. In contrast NaCl caused little or no inhibition in assay systems with no cofactor source added or with T1 or T2 proteins as cofactors, indicating that salt inhibition is not exerted directly on the active site of the enzyme or on its ability to interact productively with cofactor proteins. Addition of some of the other egg apoproteins changed the response to salt of activity stimulated by T1 and T2 proteins so that it became similar to that seen in assay systems with serum or other crude sources of cofactor activity. Thus, these other proteins could inhibit the enzyme activity and this inhibition was strongly potentiated by increasing the salt concentration of the medium. Inhibition at high salt concentration was seen also with hemoglobin and with myoglobin, suggesting that it is a rather nonspecific effect. It occurred immediately on addition of the inhibitory proteins to the medium, it was reversible and was relieved in a competitive manner by increased amounts of lipid substrate or of cofactor proteins.
Ionization Behaviour of Native Apolipoproteins and of Their Complexes with Lecithin
Authors: ROSSENEU Maryvonne , SOETEWEY Frederik , LIEVENS Marie‐José , VERCAEMST Raphél , PEETERS Hubert
Published: March 2005

The ionization behaviour of native apoA‐I protein is compared to that of its complex with synthetic dimyristoyl lecithin in studies using calorimetric, potentiometric and spectrophotometric titration. In the presence of phospholipids, 10 out of the 21 lysines together with 22 acidic residues are masked in the complex. All tyrosines remain accessible to titration below pH 13. The apparent ionization enthalpy of the 11 lysine residues is not affected by the presence of phospholipids. These data are consistent with discrete binding sites located in the apoprotein helical segments as suggested by the model of Segrest et al. [FEBS Lett. 38, 247–253 (1974)]. A tentative localisation of lysine, arginine, aspartic acid and glutamic acid residues directly involved in phospholipid binding is suggested, assuming that such helical regions are involved in apoprotein‐phospholipid association.
Binding of 4‐Methylumbelliferyl N‐Acetyl‐Chitooligosaccharides to Wheat‐Germ Agglutinin
Authors: VAN LANDSCHOOT Anita , LOONTIENS Frank G. , CLEGG Robert M. , SHARON Nathan , DE BRUYNE Clemens K.
Published: March 2005

The binding of 4‐methylumbelliferyl per‐N‐acetyl‐chitooligosaccharides, MeUmbGlcNAc (I), MeUmb(GlcNAc)2 (II) and MeUmb(GlcNAc)3 (III) to wheat germ agglutinin, was studied by equilibrium dialysis, difference absorption spectrometry and extrinsic fluorescence quenching [Privat, J. P., Delmotte, F. & Monsigny, M. (1974) FEBS Lett. 46, 229–232] titration at a fixed wavelength or above 370 nm. The change in optical properties of the MeUmb group upon binding of the glycosides to the agglutinin depends on the length of the carbohydrate chain. An intense and carbohydrate‐specific difference absorption spectrum was observed with I (‐Δɛ at 316 nm equal to 2770 M−1 cm−1), a weaker one with II (‐Δɛ at 316 nm = 1070 M−1 cm−1), but none was observed with III; the fluorescence quenching is 100% for compounds I and II and 44% for compound III. As determined with I, using equilibrium dialysis and difference absorption spectrometry, there are two identical and independent binding sites per subunit. For I the value of the association constant is independent of the experimental method or the saturation range used. The association constants increase with the chain length of the fluorescent oligosaccharides: at 25°C the values (in M−1) are (1.98 ± 0.12)104 for I, (7.5 ± 0.3)104 for II and (1.17 ± 0.60)105 for III; the thermodynamic parameters do not differ greatly: ΔH0 and ΔS0 are ‐8.2 ± 0.4 kcal (–34.3 kJ) mol−1 and –7.8 ± 1.5 cal (–32.6 J) mol−1 K−1 for I, –10.0 ± 0.3 kcal (–41.8 kJ) mol−1 and –11.2 ± 1.0 cal (–46.9J) mol−1 K−1 for II, –8.4 ± 0.3 kcal (–35.1 kJ) mol−1 and –4.8 ± 1.2 cal (–20.1 J) mol−1 K−1 for III. The results, taken together with data from the literature, are interpreted as binding to a region composed of subsites with a protein – MeUmb interaction for I and II amounting to a contribution of the MeUmb group in the free energy of binding, equal to – 2 kcal (– 8.4 kJ) mol−1 for I.
Dependence of tRNA Structure in Solution upon Ionic Condition of the Solvent
Authors: LABUDA Damian , AUGUSTYNIAK Jacek
Published: March 2005

Dependence of barley phenylalanine tRNA (tRNAphe) fluorescence intensity at 430 nm upon LiCl, NaCl, KCl, CsCl or NH4Cl concentration was measured in 0.01 M Tris‐HCl, pH 7.5, 0.001 M Na2EDTA solutions. Increase of monovalent cation concentration in the solvent from 0 to 2 M induced about 3‐fold fluorescence intensity enhancement. The fractional fluorescence change was used as a measure of bound ligand concentration. Fluorescence Scatchard plots revealed three classes of monovalent cation binding sites on the tRNA molecule: interacting (strong) and independent (weak and very weak) sites. Calculated from Scatchard plots binding constants (K), for strong and weak binding of monovalent cations (in the case of Na+ binding: Ks= 26 M−1 and Kw= 4.3 M−1 respectively) exhibit linear dependence upon ionic radius (r). Two limiting values obtained from the plot of K versus r: K(max) at r= 0 and r(max) at K= 0, characterize additionally strong and weak monovalent cation binding sites (Ks(max)= 42 M−1Kw(max)= 8.5 M−1, rs(max)= 0.23 nm and rw(max)= 0.22 nm). A model of the relationship between weak Mg2+ binding sites and monovalent cation binding sites as well as of monovalent cations binding to tRNA is proposed.
Comparison of the Properties of Plasma Membrane‐Bound and Mitochondria‐Bound ATPases in the Yeast Schizosaccharomyces pombe
Authors: DELHEZ Jacques , DUFOUR Jean‐Pierre , THINES Denise , GOFFEAU André
Published: March 2005

Two distinct membrane‐bound ATPases of the glucose‐grown yeast Schizosaccharomyces pombe are separated by differential and isopycnic gradient centrifugation. The mitochondrial ATPase, with optimal activity at pH 9.0, equilibrates with cytochrome c oxidase at densities 1.17 and 1.19 g × cm−3 while the non‐mitochondrial ATPase, with optimal activity at pH 6.0 is associated with mannans in 1.21‐g × cm−3 particles. Cytochemical staining for carbohydrates confirms the plasma membrane origin of vesicles found in a subcellular fraction enriched about 10 times in pH‐6.0 ATPase activity.

The following analogies are found between the two purified membrane‐bound ATPases. They hydrolyse preferentially ATP and require magnesium. They exhibit a Km of 3.3 mM for ATP‐Mg at pH 6.0. The two activities are inhibited by ADP, Dio‐9, dicyclohexylcarbodiimide, triethyltin, miconazole, sodium fluoride and p‐hydroxymercuribenzoate.

Nevertheless, the plasma membrane‐bound ATPase is distinct from the mitochondria‐bound ATPase in the following properties. The plasma membrane‐bound ATPase is not sensitive to the antibody dressed against the purified mitochondrial enzyme. Excess of Mg2+ inhibits the mitochondria‐bound ATPase but not the plasma membrane one. The relative specificities for nucleoside triphosphates and cations of the two enzymes are different. In contrast to the mitochondrial enzyme, the plasma membrane‐bound ATPase is not stimulated by carbonate and maleate anions. The plasma membrane‐bound ATPase is not inhibited by the mitochondrial inhibitors oligomycin and venturicidin.
Biosynthesis of Triacylglycerols Containing Short‐Chain Fatty Acids in Lactating Cow Mammary Gland
Authors: MARSHALL Michael O. , KNUDSEN Jens
Published: March 2005

Microsomal 1,2‐diacylglycerol acyltransferase from lactating cow mammary gland incorporated equal molar amounts of microsomal‐bound 1,2‐dipalmitoyl [2‐3H]glycerol and [1‐14C]‐butyrate, [1‐14C]hexanoate or [1‐14C]palmitate from their CoA esters into triacylglycerol. The enzyme could also utilize exogenous 1,2‐diacylglycerols in the presence of ethanol.

The pH optimum of the enzyme was 6.1 and 6.4 with butyryl‐CoA and hexanoyl‐CoA respectively. Values of V were approximately the same (2.7 and 2.4 nmol · min−1· mg−1, respectively), but values of Km were different (34 and 10 μM, respectively) with these two substrates. Mg2+ was not required as cofactor.

The presence of a Mg2+‐dependent phosphatidate phosphatase in the microsomal fraction was demonstrated.

It is proposed that triacylglycerols containing butyric and hexanoic acid are biosynthesized in cow mammary gland by the glycerolphosphate pathway, in which long‐chain 1,2‐diacylglycerols derived from phosphatidic acid are acylated at the sn‐3 position by short‐chain acyl‐CoA esters.
The Role of Iron‐Sulfur Center 2 in Electron Transport and Energy Conservation in the NADH‐Ubiquinone Segment of the Respiratory Chain in Paracoccus denitrificans
Authors: MEIJER Emmo M. , WEVER Ron , STOUTHAMER Adriaan H.
Published: March 2005

The electron paramagnetic resonance spectra at 15 K of reduced membrane particles of Paracoccus denitrificans exhibit resonance signals with g values, line shapes and temperature profile which are similar to the signals of the iron‐sulfur centers observed in the NADH‐ubiquinone segment of mitochondrial respiratory chains. These iron‐sulfur centers are reducible with NADH, NADPH as well as chemically with dithionite.

Sulphate‐limited growth of Paracoccus denitrificans results in the loss of an electron paramagnetic resonance signal (gz∼ 2.05, gy∼gx∼ 1.92) which has properties similar to those of iron‐sulfur center 2 of the NADH dehydrogenase of mitochondrial origin. The loss of this signal is accompanied by a decrease in the NADH oxidase and NADH ferricyanide oxidoreductase activities to respectively 30 and 40% of the values found for succinate‐limited growth conditions. In addition respiration in membrane particles from sulphate‐limited cells loses its sensitivity to rotenone.

Since sulphate‐limited growth of Paracoccus denitrificans induces loss of site I phosphorylation [Arch. Microbiol. (1977) 112, 25–34] these observations suggest a close correlation between site I phosphorylation, rotenone‐sensitivity and the presence of an electron paramagnetic resonance signal with gz∼ 2.05 and gy∼gx∼ 1.92.
Aminopeptidase N from Escherichia coli: Ionizable Active‐Center Groups and Substrate Specificity
Authors: CHAPPELET‐TORDO Danielle , LAZDUNSKI Claude , MURGIER Maryse , LAZDUNSKI Andrée
Published: March 2005

Effects of pH on the Michaelis constant and maximal velocity of hydrolysis of l‐alanine‐4‐nitroanilide by aminopeptidase N have been studied in the range between pH 6.5 and pH 9.5. V does not vary with pH in this range, therefore any ionizable group shown to be involved in the action of aminopeptidase N will likely be affecting binding and not the catalytic step. The pKmvs pH profile suggests that two ionizable groups (pKa= 7.2 and pKa= 8.2) affect binding of this substrate. The pKivs pH profile observed with the competitive inhibitor hexylamine does not show any inflection point in the alkaline region (pH > 7.2), which suggests that the pKa= 8.2 at 37°C corresponds to the pKa of the substrate ammonium group.

In the neutral pH range the inflection points occur at pH 7.2 both in plots of pKmvs pH and pKivs pH. Thus the corresponding ionizable group should belong to the enzyme. Since the strategic–SH group of aminopeptidase N is protected from the action of N‐ethylmaleimide by hexylamine and other competitive inhibitors, we propose that the value of pKa= 7.2 corresponds to the ionization of this group. Aminopeptidase N is competitively inhibited by l‐amino acids having hydrophobic side‐chains such as leucine, isoleucine, phenylalanine, tyrosine and tryptophan. Blocking of the α‐amino group prevents binding, and blocking of the α‐carboxylic group reduces the inhibitory capacity of the amino acid. Aliphatic amines are also competitive inhibitors; the longer the aliphatic chain, the smaller the Ki, which varies by a factor 20 from butylamine to octylamine. Even though aminopeptidase N is not a metalloenzyme, divalent cations competitively inhibit the hydrolysis of l‐alanine‐4‐nitroanilide. The efficiency of binding is in the order: Cd2+ > Zn2+ > Cu2+ > Co2+ > Mn2+ > Mg2+ > Ca2+ with Cd2+ ions having a 103 higher binding constant than Ca2+ ions.

The Ki values and initial rates of hydrolysis of 15 peptides by aminopeptidase N are reported. The peptides tested included: alanyl‐X, X‐alanine, di, tri and penta‐alanine, and dipeptides devoid of alanine, where X represents any one of several amino acids. The C‐terminal amino acid of alanyl‐X dipeptides contributes mainly to the binding constant, which depends on the hydrophobicity of their side‐chain. This affinity varies by a factor of 28 from glycine to phenylalanine whereas V varies only by a factor of 2. Effects of changes in the NH2‐terminal amino acid in X‐alanine dipeptides and in dipeptides devoid of alanine on the rate of hydrolysis and Ki, was also studied. V values for the enzyme of dipeptides devoid of alanine decreased with Ki of the peptides. For di, tri, and pentaalanine, the increase of the peptide size increases the affinity but not the rate of hydrolysis.
The Denaturation‐Renaturation of Chicken‐Muscle Triosephosphate Isomerase in Guanidinium Chloride
Authors: McVITTIE John D. , ESNOUF M. Peter , PEACOCKE Arthur R.
Published: March 2005

The process of denaturation of the chicken muscle dimeric enzyme triosephosphate isomerase on addition of guanidinium chloride has been studied at pH 7.6, the pH at which the recovery of activity is optimal (100%) on removal of denaturant. Determinations of the sedimentation coefficient, intrinsic viscosity, molecular weight (by sedimentation equilibrium studies) and the absorption coefficient at 280 nm in various concentrations of guanidinium chloride concurred in showing a single, sharp transition at about 0.7 M guanidinium chloride at a protein concentration 1 – 5 mg/ml from the native enzyme to the dissociated, unfolded chains of the monomer. Relative fluorescent intensity measurements revealed a single transition at about 0.4 M guanidinium chloride at enzyme concentrations of about 0.05 mg/ml.

The process of denaturation in different guanidinium chloride concentrations was first order with respect to enzyme and about sixth order with respect to denaturant.

The rate of attainment of equilibrium during the renaturation obeyed second‐order/first‐order reversible kinetics. It was concluded that the rate‐determining step in renaturation at pH 7.6 must be the association of two subunits.
The Crystal Structure of Complexes between Horse Liver Alcohol Dehydrogenase and the Coenzyme Analogues 3‐Iodopyridine‐adenine Dinucleotide and Pyridine‐adenine Dinucleotide
Authors: SAMAMA Jean‐Pierre , ZEPPEZAUER Eila , BIELLMANN Jean‐François , BRÄNDÉN Carl‐Ivar
Published: March 2005

We have studied the binding of the enzymatically active NAD+ analogue, 3‐iodopyridine‐adenine dinucleotide, and the inactive analogue, pyridine‐adenine dinucleotide to the enzyme horse liver alcohol dehydrogenase using X‐ray crystallographic methods. These studies were made under such conditions that crystals of the complexes were isomorphous to apoenzyme crystals. Both analogues bind in the same conformation. The binding of the adenosine moiety is very similar to that of ADP‐ribose or NADH bound to the enzyme. The conformation and mode of binding of the remaining portions of the analogue molecules is, however, quite different. The pyridine ring is not situated in the active‐site pocket as the nicotinamide group in the isomorphous enzyme · NADH · imidazole complex but lies at the surface of the crevice between the two domains of the subunit, approximately 1.5 nm away from the catalytically active zinc atom. Lys‐228 which has been shown to be important for NADH dissociation is in this region of the molecule.
Hormone‐Induced Changes in Pyruvate Kinase
Authors: VAN BERKEL Theo J. C. , KRUIJT Johan K. , KOSTER Johan F.
Published: March 2005

The kinetic properties of pyruvate kinase from isolated hepatocytes of fed rat liver incubated with and without glucagon (1 μM) are compared. Glucagon incubation of hepatocytes decreases the apparent affinities of the enzyme for the substrate phosphoenolpyruvate and the allosteric activator fructose 1,6‐bisphosphate while the apparent Ki values for alanine and ATP are lowered. The Km for the second substrate ADP is not influenced by glucagon incubation. The kinetic curves show intermediate plateaus indicative of the presence of two enzyme forms, an active form (called La) and a less active form (called Lb). It is concluded that the effect of glucagon on the kinetic parameters of pyruvate kinase is caused by a shift of the equilibrium between both enzyme forms to the less active Lb side.

Comparison of the kinetic parameters of pyruvate kinase of a total homogenate from a fed and a 48‐h‐starved rat indicates that starvation causes a decrease of the apparent affinities of the enzyme for phosphoenolpyruvate and fructose 1,6‐bisphosphate while the apparent Ki values for alanine and ATP are lowered. The Km for ADP is not influenced by starvation. This indicates that a change in kinetic properties of pyruvate kinase also occurs in the intact animal stressing its physiological importance. The change in kinetic parameters introduced by glucagon incubation of hepatocytes and starvation of the intact animal are similar and identical to the changes observed on in vitro phosphorylation of the enzyme purified from rat liver [Ekman et al. (1976) Biochim. Biophys. Acta, 429, 374–382] or from pig liver [Ljungström et al. (1976) Eur. J. Biochem. 68, 497–506]. This indicates that the La and Lb form of pyruvate kinase might represent the dephosphorylated and phosphorylated forms of the enzyme respectively.

The glucagon inhibited enzyme is partially reactivated in vitro by incubation at 37°C. This reactivation is promoted by the presence of 10mM Mg2+. The reactivation in vitro can be blocked completely by 20 mM KF. These properties of reactivation are most consistent with a protein‐phosphatase‐mediated process. The reactivation is inhibited by a high Ca2+ concentration (2.6 mM).

Incubation in vitro of a liver homogenate of a fed and starved rat with 10 mM Mg2++ 0.5 mM ATP in the presence of F− shows that starvation increases the pyruvate kinase inactivation reaction presumably due to protein kinase activity.

It is concluded that hormonal regulation of pyruvate kinase activity is exerted both by changing the degree of (de)phosphorylation of the enzyme and by changing the concentration of the allosteric activator fructose 1,6‐bisphosphate which depends, at least in part, on the hormonal control of the phosphofructokinase–fructose‐1,6‐bisphosphatase cycle.
Microheterogeneity in Lipid A Demonstrated by a New Intermediate in the Biosynthesis of 3‐Deoxy‐d‐manno‐octulosonic‐acid–Lipid A
Authors: LEHMANN Volker , RUPPRECHT Eckhard
Published: March 2005

A previous report [Lehmann, Rupprecht & Osborn (1977) Eur. J. Biochem. 76, 41–49] described the characterisation of a new class of conditional lethal mutants which are temperature sensitive both in synthesis of 3‐deoxy‐d‐manno‐octulosonic acid 8‐phosphate and in growth. It was found that these mutants synthesize at restrictive temperatures lipid A intermediates consisting of glucosamine disaccharides carrying four 3‐hydroxymyristic acids and two unsubstituted phosphate groups (acidic precursor) [Lehmann (1977) Eur. J. Biochem. 75, 257–266]. The present paper describes the discovery of a new lipid A intermediate (neutral precursor) which is accumulated in the same mutant under slightly modified cultivation conditions. It differs from the acidic precursor by the presence of 4‐aminoarabinose, phosphorylethanolamine and polyamines and exhibits, like the complete lipid A structure of lipopolysaccharides, a considerable degree of intrinsic heterogeneity [Mühlradt, Wray & Lehmann (1977) Eur. J. Biochem. 81, 193–203].

Application of different degradation pathways combined with 31P nuclear magnetic resonance spectroscopy showed that in the neutral precursor 4‐aminoarabinose is linked to the ester‐bound 4‐phosphate while phosphorylethanolamine is linked to the 1‐phosphate. The presence of polyamines such as putrescine, spermidine, cadaverine and lysine suggest that some species of the neutral precursor fraction are crosslinked by ionic interaction. Evidence will be presented that the neutral precursor, like the acidic one, is an intermediate in the 3‐deoxy‐d‐manno‐octulosonic acid–lipid A synthesis. Furthermore the findings altogether support a notion in which the acidic precursor could serve as acceptor for the transfer of 4‐aminoarabinose, phosphorylethanolamine and polyamines to the glucosamine backbone structure.
The Inhibition of Ribosomal Translocation by Viomycin
Authors: MODOLFLL Juan , VÁZQUEZ David
Published: March 2005

The basic peptide antibiotic viomycin rapidly inhibits polypeptide chain elongation when added to purified endogenous Escherichia coli polysomes actively engaged in polypeptide synthesis. After inhibition is established, the polysomal nascent peptide chains react only slowly with puromycin, while the chains of uninhibited polysomes or tetracycline‐blocked polysomes react very rapidly. Since viomycin does not inhibit the peptidyl transferase reaction, these results indicate that the antibiotic confines peptidyl‐tRNA to the ribosomal acceptor site and, consequently, that it blocks polypeptide chain elongation by inhibiting ribosomal translocation.

Viomycin at concentrations strongly inhibitory for translocation (10 μM) does not impair the elongation‐factor (EF)‐G‐plus‐ribosome‐dependent GTP hydrolysis, the formation of GDP · EF‐G · ribosome · fusidic‐acid complex and guanyl‐5′‐yl‐methylene‐diphosphonate · EF‐G · ribosome complex, or the non‐enzymic binding of AcPhe‐tRNA to the ribosomal donor site. In contrast, viomycin strongly inhibits the non‐enzymic translocation and the release of AcPhe‐tRNA from the ribosomal acceptor site promoted by depletion of NH4+ ions. It is suggested that viomycin inhibits the movement of peptidyl‐tRNA associated with translocation by interfering with its exit from the ribosomal acceptor site. Viomycin at high concentrations (10–100 μM) partially inhibits the EF‐G‐plus‐ribosome‐dependent GTP hydrolysis and the turnover of the GDP · EF‐G · ribosome · fusidic‐acid complex. These effects, however, are probably unrelated to the inhibition of ribosomal translocation.

The binding of viomycin to AcPhe‐tRNA · ribosome · poly(U) complexes is readily reversible, since the antibiotic can be removed by filtration through molecular sieves.
Studies on the Role and Mode of Operation of the Very‐Lysine‐Rich Histones in Eukaryote Chromatin
Authors: RATTLE Henry W. E. , LANGAN Thomas A. , DANBY Shirley E. , BRADBURY E. Morton
Published: March 2005

270‐MHz proton magnetic resonance has been used to study the effect of phosphorylation of histone H1 in vitro on the structure of isolated H1 molecules and on the interaction of H1 with DNA. Phosphorylation at serine‐105, which is located in the globular region of H1, was found to reduce the enthalpy of structure formation from 24 ± 2 kcal mol−1 (100 ± 8 kJ mol−1) to 13 ± 2 kcal mol−1 (55 ± 8 kJ mol−1). Phosphorylation at either or both of serine‐37 and serine‐105 was found to reduce the strength of binding of the histone to DNA considerably at some ionic strengths.
Determination of the Solution Conformation of Dephospho Coenzyme A by Nuclear‐Magnetic‐Resonance Spectroscopy with Lanthanide Probes
Authors: FAZAKERLEY G. Victor , LINDER Peter W. , REID David G.
Published: March 2005

The aqueous solution conformation of the 1 : 1 complex of dephospho CoA bound to a lanthanide ion has been determined by examination of the dipolar shift and induced relaxation at pH 6.4. The experimental data are shown to arise from the presence of both 1 : 1 and 1 : 2 metal : ligand complexes and a graphical method is described to divide the experimental data into information corresponding to each of the two species. The formation constants are also derived. For the 1 : 1 complex the ribose is found in a 2E conformation with the adenine base predominantly anti. A small contribution from a syn conformation is evident. The pantoinic acid fragment of the chain is folded back towards the pyrophosphate while the remainder of the chain is extended.
Regulation of Synthesis of a Brain‐Specific Protein in Monolayer Cultures of Clonal Rat Glial Cells
Authors: LABOURDETTE Gerard , MAHONY James B. , BROWN Ian R. , MARKS Alexander
Published: March 2005

C6 cells were grown in monolayer culture under conditions permitting continued exponential cell division after attainment of a density at which extensive intercellular contacts were formed. An increase in the relative synthesis of S100 protein coincided with the time of formation of extensive intercellular contacts and preceded the onset of the stationary phase of growth by three generations. These observations suggested that the induction of S100 protein synthesis was mediated by cell contact and not by an arrest of cellular growth. The mechanism of this induction was first studied in a homologous non‐initiating cell‐free protein‐synthesizing system from C6 cells, using fixed amounts of free amino acids or fully charged rat liver aminoacyl‐tRNA as a source of precursors for protein synthesis. Real synthesis of total soluble proteins decreased as the cells progressed from logarithmic to stationary growth while synthesis of S100 protein increased during this period. The capacity of poly(A)+ RNA from logarithmic and stationary cultures to direct the synthesis of S100 protein was estimated in a cell‐free protein‐synthesizing system derived from wheat embryos. Increased synthesis of S100 protein in stationary cultures was directly correlated with an increase in translatable S100 protein mRNA.
Purified Human Somatomedin A and Rat Multiplication Stimulating Activity
Authors: RECHLER Matthew M. , FRYKLUND Linda , NISSLEY S. Peter , HALL Kerstin , PODSKALNY Judith M. , SKOTTNER Anna , MOSES Alan C.
Published: March 2005

We have compared the mitogenic activity and reactivity in receptor binding assays of somatomedin A and multiplication‐stimulating activity (MSA), two acid‐soluble polypeptides of molecular weight 7–10 000 that possess weak intrinsic insulin‐like metabolic activity. Somatomedin A was purified to homogeneity from human plasma, and MSA was purified to homogeneity from the conditioned culture media of a cell line, BRL 3A, derived from rat liver. The two peptides stimulated DNA synthesis in chick embryo fibroblasts 3–4‐fold and have superimposable dose‐response curves. Addition of either peptide to chick embryo fibroblasts plated in serum‐free medium increased the cell number by 150% in 4 days, compared with an increase of 25% in control cultures receiving no additions. The two peptides also stimulated DNA synthesis in human fibroblasts in culture 3–4‐fold.

Specific receptors for MSA and/or somatomedin A could be demonstrated in intact cells or membranes from chick embryo fibroblasts, human fibroblasts, human placenta, rat liver, and the BRL 3A2 cell line, a subclone of the line that produces MSA. Unlabeled MSA and somatomedin A inhibited the binding of 125I‐labeled MSA and 125I‐labeled somatomedin A to each of these receptors with comparable potency. In chick embryo fibroblasts, human fibroblasts, and human placental membranes, the binding of both radioactive ligands also was inhibited by insulin, consistent with the interpretation that 125I‐labeled MSA and 125I‐labeled somatomedin A were binding to the same receptor. By contrast, in the BRL 3A2 cell line, insulin inhibited the binding of 125I‐labeled somatomedin A, but not the binding of 125I‐labeled MSA, suggesting that the two labeled peptides were binding to different receptors in this cell line. Moreover, 125I‐labeled MSA, but not 125I‐labeled somatomedin A, bound specifically to rat liver plasma membranes. These results indicate that human somatomedin A and rat MSA are closely related, but not identical, peptides. They have similar mitogenic activity for cultured fibroblasts, and cross‐react with specific cell receptors with comparable potency. The two radioactively labeled peptides, however, can be distinguished by certain cell surface receptors for somatomedin‐like peptides.