bluuebunny/crossref_metadata_2025 huggingface dataset:
Two‐Dimensional Gel Electrophoresis of Ribosomal Proteins from Streptomycin‐Sensitive and Streptomycin‐Resistant Mutants of Chlamydomonas reinhardi
Authors: BRÜGGER Martin , BOSCHETTI Arminio
Published: March 2005

Ribosomal proteins from three mutant strains of Chlamydomonas reinhardi were analysed and compared by one‐dimensional and two‐dimensional gel electrophoresis. One mutant was streptomyin‐sensitive the other two were streptomycin‐resistant, one with a Mendelian the other with a non‐Mendelian pattern of inheritance. In the 30‐S subunits of chloroplast ribosomes approximately 25 proteins are found and in the 50‐S subunits 34 proteins. The 40‐S subunits of cytoplasmic ribosomes contain about 31 proteins and the 60‐S subunits 44 proteins. The molecular weights of most proteins in all subunits are in the range of 10000 to 35000. However, the 60‐S subunits contain in addition a protein of molecular weight 50 000 and the 30‐S subunits show 6–7 bands of molecular weights from 50000 to 83000. The proteins of the cytoplasmic 80‐S ribosomes or of their subunits from all three mutants are electrophoretically identical. The proteins of the 70‐S organellar ribosomes and both of their subunits show distinct differences between the three strains. Our results indicate that organellar ribosomal proteins are in part controlled by nuclear DNA and in part by organellar DNA.
The Pyruvate‐Dehydrogenase Complex from Azotobacter vinelandii
Authors: GRANDE Hans J. , BRESTERS Tjarda W. , DE ABREU Ronney A. , DE KOK Arie , VEEGER Cees
Published: March 2005

Labelling studies with N‐ethylmaleimide show that either in the presence of Mg2+, thiamine pyrophosphate (TPP) and pyruvate or in the presence of NADH the overall activity of the pyruvate dehydrogenase complex from Azotobacter vinelandii is inhibited without much inhibition of the partial reactions. The complex undergoes a conformational change upon incubation with NADH. The inhibition by bromopyruvate is less specific.

Specific incorporation of a fluorescent maleimide derivative was observed on the two transacetylase isoenzymes. Binding studies with a similar spin label analogue show that 3 molecules/FAD are incorporated by incubation of pyruvate, Mg2+ and TPP, whereas 2 molecules/FAD are incorporated via incubation with NADH. The spin label spectra support the idea that in the complex the active centres of the component enzymes are connected by rapid rotation of the lipoyl moiety.

Three acetyl groups are incorporated in the complex by incubation with [2‐14C]pyruvate. Time‐dependent incorporation supports the view that the two transacetylase isoenzymes react in non‐identical ways with the pyruvate dehydrogenase components of the complex. The results show that the complex contains 2 low‐molecular‐weight transacetylase molecules and 4 molecules of the high‐molecular‐weight isoenzyme.

Mn2+‐binding studies show that the complex binds 10 ions, with different affinities. 2 Mn2+ ions are bound with a 20‐fold higher affinity than the remaining 8 Mn2+ ions. The latter 8 ions bind with equal affinities and are thought to reflect binding to the pyruvate dehydrogenase components of the complex.

It is concluded that the complex contains 8 pyruvate dehydrogenase molecules, 4 high‐molecular‐weight transacetylase molecules, 2 low‐molecular‐weight transacetylase molecules and 1 dimeric (2‐FAD‐containing) symmetric molecule of lipoamide dehydrogenase. Evidence comes from pyruvate‐dependent inactivation and labelling studies that the pyruvate dehydrogenase components contain either an –SH group or an S‐S bridge which participates in the hydroxyethyl transfer to the transacetylase components.
The Renaturation of Reduced Polyalanyl‐chymotrypsinogen and Chymotrypsinogen
Authors: ORSINI Gilbert , GOLDBERG Michel E. , SKRZYNIA Cécile
Published: March 2005

Chymotrypsinogen has been successfully renatured in solution, after reduction of its 5 disulfide bonds in 6 M guanidine‐HCl. This has been made possible by the study of the renaturation of a model derivative, polyalanyl‐chymotrypsinogen. The reduced derivative is shown to refold and reoxidize spontaneously, with a 30–40% yield, into molecules which are monomeric and fully susceptible to activation by trypsin.

Chymotrypsinogen can also be renatured but only in the presence of reagents allowing disulfide interchange and of moderate concentrations of guanidine‐HCl or urea. These results illustrate how the kinetic trapping of incorrectly folded molecules by wrong S‐S bonds and aggregation can be overcome, thus allowing the correct refolding of the protein.
Amino‐Acid Sequence of lac Repressor from Escherichia coli
Authors: BEYREUTHER Konrad , ADLER Klaus , FANNING Ellen , MURRAY Carolyn , GEISLER Norbert , KLEMM Alex
Published: March 2005

The lac repressor from Escherichia coli, composed of four identical subunits with a molecular weight of 37 160, was carboxymethylated and fragmented by tryptic digestion and cyanogen bromide treatment. Using ion‐exchange chromatography, gel filtration and preparative thin‐layer electrophoresis and chromatography 29 of the 30 tryptic peptides were isolated in pure form. Direct Edman degradation and the dansyl‐Edman technique were used to determine the sequence of the small tryptic peptides. Special emphasis was put on the sequence determination of the six large tryptic fragments which together account for 177 residues, corresponding to 51% of the repressor subunit with its 347 residues. The large tryptic fragments were analyzed after fragmentation with chymotrypsin, thermolysin and dipeptidyl aminopeptidase I. Thus the sequence of all 30 tryptic peptides could be deduced. The complete sequences of all cyanogen bromide fragments were deduced from peptides obtained by tryptic, chymotryptic and thermolytic digestion of the individual fragments and by automated stepwise Edman degradation of lac repressor and of the large cyanogen bromide fragments. The order of the cyanogen bromide fragments was given by overlapping tryptic peptides. The resulting amino acid composition of the monomer is Asp15, Asn11, Thr18, Ser30, Glu14, Gln27, Pro13, Gly22, Ala44, Cys3, Val33, Met9, Ile17, Leu40, Tyr8, Phe4, Trp2, Lys11, His7, Arg19.

The sequence of lac repressor shows no similarities with that of other proteins known to bind to DNA or RNA. The N‐terminal 55 residues contain two homologous regions. This part of the sequence which is involved in lac operator binding might have been formed by gene duplication.
A Urinary Pentasaccharide in Bovine Mannosidosis
Authors: LUNDBLAD Arne , NILSSON Bo , NORDÉN Nils E. , SVENSSON Sigfrid , ÖCKERMAN Per‐Arne , JOLLY Robert D.
Published: March 2005

Abnormally high amounts of low molecular weight mannose‐rich carbohydrate material were found in the urine of an Angus calf with mannosidosis. At least five oligosaccharide fractions were detected by paper chromatography. The most abundant compound was purified by gel chromatography, zone electrophoresis, and two consecutive preparative paper chromatographic steps. The yield was 10 mg/liter of urine. From structural studies including nuclear magnetic resonance spectroscopy, optical rotation, sugar analysis, methylation analysis, and partial enzymatic degradation the following structure was deduced: α‐d‐Manp‐(1→6)‐β‐d‐Manp‐(1→4)‐β‐d‐GlcNAcp‐(1→4)‐β‐d‐GlcNAcp‐(1→4)‐d‐GlcNAc. This oligosaccharide is distinct from all the oligosaccharides previously described which are excreted by patients with mannosidosis.
The Hormonal Control of Activity of Skeletal Muscle Phosphorylase Kinase
Authors: YEAMAN Stephen J. , COHEN Philip
Published: March 2005

Phosphorylase kinase was activated 5–10‐fold in vivo by an intravenous injection of adrenalin. Sodium fluoride, an inhibitor of phosphorylase kinase phosphatase, was required to prevent the reversal of this process; the activated and non‐activated forms of the enzyme were indistinguishable by dodecylsulphate gel electrophoresis. This suggested that the activation had resulted from a phosphorylation of the enzyme, and that it was not a consequence of the well known activation by proteolytic cleavage that can be demonstrated in vitro.

Phosphorylase kinase activated in vivo was purified and digested with trypsin, and the two tryptic peptides which contain the serine residues which are phosphorylated in vitro by the action of cyclic‐AMP (adenosine 3′: 5′‐monophosphate) dependent protein kinase, were isolated. It was found that the same nine‐amino‐acid segment of the β chain and the same seven‐amino‐acid segment of the α chain had become phosphorylated in vivo in response to adrenalin, as were phosphorylated in vitro. The degree of phosphorylation of each of the two sites was at least 50%. The data provide direct proof that the activation of phosphorylase kinase which occurs in vivo in response to adrenalin results from a phosphorylation of the enzyme. They also indicate that the novel form of regulation associated with the phosphorylation of the α subunit, the stimulation of protein dephosphorylation by “second site phosphorylation”, can now be regarded as a new form of enzyme control mechanism which operates in vivo.

The regulation of phosphorylase kinase activity was studied in the protein · glycogen complex from skeletal muscle. The enzyme could be rapidly converted to a phosphorylated form in a cyclic‐AMP‐stimulated reaction upon addition of magnesium ions and ATP, but the conversion of phosphorylase b to phosphorylase a in the complex still showed an absolute requirement for calcium ions.

The implications of these findings and major problems in the hormonal control of skeletal muscle glycogenolysis which are not yet resolved, are discussed.
Studies on Choline Permeation through the Plasma Membrane and Its Incorporation into Phosphatidyl Choline of Ehrlich‐Lettré‐Ascites Tumor Cells in vitro
Authors: HAEFFNER Eberhard W.
Published: March 2005

The initial rate of incorporation of 14C or 3H‐labeled choline into Ehrlich‐Lettre ascites cells of the glycogen‐free strain seven days after inoculation was investigated in vitro.

At choline concentrations in the medium between 6 to 30 μM and 100 to 500 μM the choline uptake by the cells followed Michaelis‐Menton kinetics with V values between 31 to 100 and 59 to 500 pmol per minute at a given cell density, and average Q10‐values of 2.1 at the high and of 2.4 at the low choline molarity. The Km‐values increased from 27 μM to 58.8 μM at low and from 0.11 mM to 0.22 mM at high choline concentrations over a temperature range between 15°C and 37°C. Arrhenius plot of the V values gave two lines, one with a transition temperature at 25°C at low and one straight line at high choline concentrations, from which the energy of activation for choline uptake was determined to be 16 kcal/mol.

It is assumed that two systems exist for the choline uptake by the ascites cells. One, operative at low substrate concentrations, which is saturable and probably is to be classified as a carrier‐mediated facilitated diffusion process, can be strongly inhibited by deoxyglucose or 2,4‐dinitrophenol and also by substrate analogues such as chlorocholine or benzoylcholine. Ouabain affects this system to a lesser extent. The other system functioning at high choline concentrations may be a simple diffusion process, which is little inhibited by substrate analogues, ouabain and deoxyglucose; however, it is also inhibited by 2,4‐dinitrophenol and p‐chloromercuribenzoate.

Choline incorporation into the acid‐insoluble material (lecithin) gave linear Michaelis‐Menton kinetics at the low and the high substrate concentration respectively. Km‐values decreased with an increase in temperature at low and increased with rising temperature at high substrate concentrations thus reflecting a close relationship between choline uptake and its metabolism. Labeling of lecithin choline in the various subcellular fractions under the conditions of the functioning of a carrier‐mediated process was in the order: mitochondria (50%) > plasma membranes (25%) > nuclei (14%) > microsomes (9%) > supernatant (1.5%).

Treatment of the cells with p‐chloromercuribenzoate or heat shock at 50°C markedly reduced the choline uptake and concomitantly its conversion into lecithin. Kinetic analysis revealed that the inhibitory effect of p‐chloromercuribenzoate was competitive and that of the heat shock non‐competitive in nature. Further the choline uptake by the cells was found to be the rate‐limiting step, since the rate of choline phosphorylation was determined by the extracellular choline concentration. Pulse chase experiments showed a rapid turnover of the choline moiety with a concomitant increase in activity of the lecithin fraction and little change within the choline phosphate pool.
On the Activity and Regulation of Anaplerotic and Gluconeogenetic Enzymes during the Growth Process of Baker's Yeast
Authors: HAARASILTA Sampsa , OURA Erkki
Published: March 2005

The anaplerotic and gluconeogenetic metabolism of baker's yeast was studied at the enzymatic level during glucose‐ethanol diauxic growth in the presence and absence of aspartate. Of the two possible anaplerotic systems, only the pyruvate carboxylase by‐pass was present during the whole growth process. The second system, the glyoxylate by‐pass (isocitrate lyase as the indicator), like the specific enzymes of the gluconeogenetic metabolism, phosphorenolpyruvate carboxykinase and hexosediphosphatase began to appear only after the glucose had been consumed. The addition of glucose during the growth phase based on ethanol effected a rapid disappearance of phosphorenolpyruvate carboxykinase and hexosediphosphatase activities. The activity of pyruvate carboxylase decreased when the growth medium was supplied with asparate. The presence of aspartate had no effect on the activities of the other enzymes studied.
tRNA Nucleotidyltransferase‐Catalyzed Incorporation of CMP and AMP into RNA‐Bacteriophage Genome Fragments
Authors: PROCHIANTZ Alain , BÉNICOURT Claude , HAENNI Anne‐Lise , CARRÉ Danièle
Published: March 2005

Fragments of bacteriophage RNAs R17, MS2 and Qβ obtained by incubation with commercial snake venom phosphodiesterase become substrates of the Escherichia coli tRNA nucleotidyltransferase. The transferase adds back CMP and AMP in conditions in which it remains highly specific of CCA‐deprived tRNAs. The results suggest that the fragment from the 3′ end of the viral genome and/or possibly one or more internal fragment(s) are recognized by the transferase. These observations might indicate that bacteriophage RNAs contain certain features probably present in all tRNAs and which are recognized by the transferase.
Studies on New Intracellular Proteases in Various Organs of Rat
Authors: KOMINAMI Eiki , BANNO Yoshiko , CHICHIBU Kenji , SHIOTANI Taiichi , HAMAGUCHI Yoshitaka , KATUNUMA Nobuhiko
Published: March 2005

1.The mechanism of proteolysis of ornithine transaminase apoenzyme II by group‐specific protease and the relation between the conformation of ornithine transaminase and its susceptibility to group‐specific protease were studied to elucidate the mode of action of the protease.

2.Differences in the conformations of ornithine transaminase apoenzyme II, molecular weight 67000, and ornithine transaminase holoenzyme, molecular weight 140000, were shown by studies on difference spectra produced by various concentrations of ethylene glycol. Increase of the titratable sulfhydryl groups on resolution of the coenzyme from ornithine transaminase also supports this finding. These results are consistent with the facts that the apoenzyme was sensitive to group‐specific protease, while the holoenzyme was not.

3.Kinetics studies showed that ornithine transaminase apoenzyme II was degraded by limited proteolysis. Reaction of the native enzyme with group‐specific protease resulted in a nick in the enzyme molecule with formation of one homogeneous large product and small peptides. The large product was not degraded further. The large product was indistinguishable from native ornithine transaminase apoenzyme II in various properties including its elution volume on gel filtration, its mobility on disc electrophoresis, its antigenicity, its estimated number of exposed tryptophan residues, and its titratable number of sulfhydryl groups. But unlike the apoenzyme the product did not show tetramerization with coenzyme or catalytic activity, although it retained the ability to bind with coenzyme and had the same number of bound pyridoxal phosphate as the native ornithine transaminase molecule. Thus, native ornithine transaminase apoenzyme I1 was degraded by limited proteolysis. Unfolded enzyme, denatured by 8 M urea, was degraded extensively.

4.The initial step of intracellular proteins degradation is discussed on the basis of these results.
The Mode of Action of Pleuromutilin Derivatives
Authors: HÖGENAUER Gregor
Published: March 2005

Using equilibrium dialysis techniques it could be demonstrated that (a) the pleuromutilin derivative 14‐deoxy‐14[(2‐diethylaminoethyl)‐mercaptoacetoxy] dihydromutilin HCl binds to one site per ribosome specifically, (b) the binding constant is 1.3 × 107 M−1 and (c) chloramphenicol and puromycin compete with binding of the pleuromutilin derivative. Similarly the nucleotides CpA and CpCpA also displace the unsaturated derivative of the above‐mentioned pleuromutilin compound. These findings suggest that the ribosomal binding site for pleuromutilin overlaps with that for chloramphenicol and analogs of the 3′‐terminus of a tRNA, like puromycin or the nucleotides CpA and CpCpA.
A Study of Codon‐Dependent Binding of Aminoacyl‐tRNA with the Ribosomal 30‐S Subparticle of Escherichia coli
Authors: GLUKHOVA Marina A. , SPIRIN Alexander S. , BELITSINA Nadezhda V.
Published: March 2005

Titration of isolated Escherichia coli ribosomal 30‐S particles with [14C]phenylalanyl‐tRNA in the presence of poly(uridy1ic acid) was used for a quantitative assay of codon‐dependent binding of aminoacyl‐tRNA with the small ribosomal subparticle. The technique has allowed the estimation both of the fraction of “active” 30‐S subparticles capable of forming the 30‐S · poly(U) · phenylalanyl‐tRNA complexes and the equilibrium constants of phenylalanyl‐tRNA binding in different media.

Heterogeneity of the ternary complexes formed has been revealed: at least two classes of complexes differing in stability have been observed.

The stability of the 30‐S · poly(U) · phenylalanyl‐tRNA complexes has been shown to decrease with the lowering of the Mg2+ concentration, the increase of K + concentration and the addition of urea. The stability of the complexes increases with the increase of Mg2+ concentration, with the addition of ethanol and decrease of temperature.

It is demonstrated that the fraction of actively binding 30‐S particles also varies in different medium conditions; it decreases with the increase of ionic strength (K +) and with the addition of urea, and increases with the increase of Mg2+ concentration and addition of ethanol.
Structural Studies on the Coat Protein of Alfalfa Mosaic Virus
Authors: VAN BEYNUM Gerard M. A. , KRAAL Barend , DE GRAAF J. Martien , BOSCH Leendert
Published: March 2005

The reduced and carboxymethylated coat protein of alfalfa mosaic virus (AMV 425) was fragmented by means of cyanogen‐bromide cleavage. The tryptic peptides from the protein and its four cyanogen‐bromide fragments were isolated on a preparative scale by combinations of column and paper separation techniques. The tryptic digest of the carboxymethylated protein contained 24 peptides and two free amino acids. All peptides have been characterized by amino acid analyses and end‐group determinations. Together the tryptic peptides account for a total chain length of 228 amino acids. The data are in good agreement with previous reports from this laboratory.
Liver 3‐Phosphoglycerate Kinase
Authors: KULBE Klaus D. , BOJANOVSKI Marina , LAMPRECHT Walther
Published: March 2005

Homogeneous phosphoglycerate kinase from bovine liver possesses a maximum ultraviolet absorption at 278 nm (A1%, 1cm280= 6.7; Amax/Amin= 2.26; ɛ280= 31.5 mM−1× cm−1). The enzyme consists of about 420 amino‐acid residues and is a slightly acidic protein with an isoelectric point of 6.5 as expected from amino‐acid analysis. The most notable features of the chemical composition are two tryptophan, 12 methionine and four half‐cystine residues per enzyme molecule. Although phosphoglycerate kinases from mammalian tissues are partially similar to each other, clear differences in serine, glutamic acid, glycine, cysteine, valine, leucine, tyrosine, tryptophan and arginine contents were found. Fingerprinting and column chromatography of tryptic digests of the S‐carboxymethylated protein confirm the data of amino‐acid analysis.

Liver phosphoglycerate kinase is inactivated when modified with either p‐chloromercuribenzoate or 5,5′‐dithio‐bis(2‐nitrobenzoic acid) (Nbs2). The enzyme has two thiol groups available for reaction with Nbs2 under denaturing conditions, one of which is essential for catalysis. After reduction by NaBH4 four cysteine residues per molecule were determined with Nbs2 suggesting the presence of a disulfide bridge.

Using sedimentation equilibrium studies, the molecular weight was found to be 49600. Gel filtration yielded values of 43000–50000. By analytical dodecylsulfate‐polyacrylamide gel electrophoresis a molecular weight of 45600 was estimated. Inconsistent with these results is the value 37500 obtained by thin‐layer gel chromatography in 6 M guanidine‐HCl. Sedimentation velocity experiments revealed a sedimentation coefficient s20,w, = 3.4 S. The Stokes radius was 2.77 nm, the partial specific volume v̄ 0.747 ml × g−1. The diffusion coefficient was found to be 76.9 μm2× s−1 by analytical gel filtration. From these data a molecular weight of 44000 was calculated. Other physical constants of bovine‐liver phosphoglycerate kinase are: frictional ratio f/f0= 1.18, axial ratio = 3.3, maximal degree of hydration = 0.1 g per g of protein.

Bovine‐liver phosphoglycerate kinase could not be dissociated into smaller subunits by treatments which have caused dissociation of various other proteins (8 M urea, 6 M guanidine‐HCl, dodecyl sulfate, carboxymethylation, maleylation). All experiments strongly support the lack of subunit structure of the enzyme.

Some characteristics of bovine‐liver phosphoglycerate kinase are compared with the corresponding proteins from rabbit muscle, yeast and human erythrocytes.
N‐Acetylglutamate 5‐Phosphotransferase of Pseudomonas aeruginosa
Authors: LEISINGER Thomas , HAAS Dieter
Published: March 2005

Some kinetic properties of N‐acetylglutamate 5‐phosphotransferase (ATP: N‐acetyl‐l‐glutamate 5‐phosphotransferase EC 2.7.2.8) purified approx. 2000‐fold from Pseudomonas aeruginosa have been studied. The enzyme required Mg2+ for activity. Mn2+, Zn2+, Co2+, and Ca2+, in this order, could replace Mg2+ partially. The substrate specificity was narrow: N‐carbamoyl‐l‐glutamate and N‐formyl‐l‐glutamate were phosphorylated, but at a lower rate than N‐acetyl‐l‐glutamate; N‐propionyl‐l‐glutamate was almost inactive as a substrate. dATP, but neither GTP nor ITP, could be used instead of ATP. The enzyme had a broad pH optimum from pH 6.5 to 9. Feedback inhibition by l‐arginine was markedly dependent on pH. Above pH9 no inhibition was observed. l‐Citrulline was three times less potent an inhibitor than l‐arginine.

The enzyme showed Michaelis‐Menten kinetics, even at low concentration of the second substrate. The apparent Km was 2 mM for N‐acetyl‐l‐glutamate (at 10 mM ATP) and approx. 3 mM for ATP (at 40 mM N‐acetyl‐l‐glutamate). In the presence of l‐arginine the rate‐concentration curves for N‐acetyl‐l‐glutamate became sigmoidal, while no cooperativity was detected for ATP.

A method was developed allowing the determination of N‐acetyl‐l‐glutamate in the nanomolar range by means of purified enzyme.
Hemachatus haemachatus (Ringhals) Venom. Purification, Some Properties and Amino‐Acid Sequence of Phospholipase A (Fraction DE‐I)
Authors: JOUBERT François J.
Published: March 2005

Two isoenzymes of phospholipase A (fraction DE‐I and DE‐II) were isolated from the venom of Hemachatus haemuchatus (Ringhals) by gel filtration on Sephadex G‐50 and by ion‐exchange chromatography on DEAE‐cellulose. Fraction DE‐I was homogeneous by various physicochemical criteria. The enzyme comprises 119 amino acid residues and is cross‐linked by seven disulphide bridges. The values of the molecular weight of fraction DE‐I from gel filtration in the presence of calcium ions, also by the dodecylsulphate gel method and calculated from the amino acid composition were close to 13 500. The complete amino acid sequence of phospholipase A (fraction DE‐I) was elucidated. The reduced and S‐carboxymethylated enzyme was digested with trypsin, chymotrypsin, thermolysin and subtilisin and the peptides purified by DEAE‐cellulose chromatography, gel filtration and chromatography or electrophoresis on paper. The amino acid sequences of the intact enzyme and the pure peptides were determined by the Edman procedure, either through the use of the automatic sequencer or by manual manipulation. The chymotryptic digest provided the necessary overlapping peptides for aligning the tryptic peptides. The amino acid sequence of Hemachatus haemachatus phospholipase A (fraction DE‐I) shows a high degree of homology with the three isoenzymes of phospholipase A from Naja melanoleuca and is also homologous with the phospholipase A from Bitis gabonica and porcine pancreas.
Iron‐Sulfur Components of Succinate Dehydrogenase: Stoichiometry and Kinetic Behavior in Activated Preparations
Authors: BEINERT Helmut , ACKRELL Brian A. C. , KEARNEY Edna B. , SINGER Thomas P.
Published: March 2005

Extensively or completely activated preparations of beef heart succinate dehydrogenase have been investigated by electron paramagnetic resonance (EPR) techniques at 6 to 97 K. Reductive titrations with dithionite and rapid kinetic studies were performed with various types of soluble and membrane‐bound preparations of the enzyme. The following components were detected and their behavior analyzed: a free radical, presumably arising from the covalently bound flavin on reduction, two iron‐sulfur centers of the ferredoxin type, the signals of which appear on reduction, and a high‐potential iron‐sulfur component, detectable in the oxidized state. The high‐potential component was only detected in complex II and inner‐membrane preparations. This component and one of the ferredoxin‐type centers were present in amounts close to stoichiometric with the flavin and were reduced by substrate. The other ferredoxin‐type center was present in amounts between 0.1 and 0.5 times that of the flavin and was reduced only by dithionite. Of the components reduced by succinate. however, only a fraction (up to 50% of the high‐potential iron‐sulfur center and 40–60% of the ferredoxin‐type iron‐sulfur center) was reduced within the turnover time of the enzymes. In complex II not more than about 10% of the flavin appeared in the semiquinone form at any time. Soluble, purified preparations behaved similarly except that the high‐potential component was nearly or completely absent and extensive accumulation of the free radical occurred (up to 70 to 80% of the flavin) in titration and kinetic experiments. No significant difference was observed between the rates of semiquinone formation and the reduction of the ferredoxin‐type or high‐potential centers by the substrate. Also no qualitative differences in the properties studied in this work became apparent between preparations containing 4 or 8 iron atoms, respectively.
Deficiency of Joining of Okazaki‐Type Fragments in Absence of Cellular Protein Synthesis
Authors: WERNER Dieter , MAIER Gernot
Published: March 2005

The dependence of integration of newly formed DNA chains (< 10 S) into larger DNA on concomitant protein synthesis was studied in a special cellular system. Exponentially growing Ehrlich ascites tumor cells in vivo show decreasing rates and finally complete cessation of protein and DNA synthesis upon transfer into an isotonic but non‐nutritive environment (Hanks' balanced salt solution). Both protein and DNA synthesis is stimulated in these cells for a period of 30 min when they are placed into fresh Hanks' balanced salt solution; however, stimulation of protein synthesis is completely prevented in Hanks' balanced salt solution containing cycloheximide. This system allowed us to investigate the formation and fate of newly formed DNA chains (< 10 S) in dependence of protein synthesis. Analysis of DNA produced in [3H]thymidine pulses showed that DNA chains smaller than 18 S were still formed during the phase of totally delayed protein synthesis and in the presence of cycloheximide, but they were not converted into DNA molecules sedimenting faster than 18 S under these conditions. Stimulation of protein synthesis for a period of 30 min allowed the short DNA pieces to be chased into larger DNA 30 min post stimulation of protein synthesis. The results clearly indicate that DNA chain growth, by sealing of DNA chains smaller than 18 S, is strongly dependent on concomitant protein synthesis. Direct chain elongation by addition of new deoxyribonucleotides is less dependent on concomitant cellular protein synthesis.
Quantitative Binding of Antibiotics to Ribosomes from a Yeast Mutant Altered on the Peptidyl‐Transferase Center
Authors: JIMENEZ Antonio , VAZQUEZ David
Published: March 2005

Quantitative binding studies of [G‐3H]anisomycin and [acetyl‐14C]trichodermin to sensitive and resistant 80‐S ribosomes from yeasts are described in this work. A single mutation, most probably affecting the ribosome peptidyl transferase centre, appears to have pleiotropic effects on the ribosome leading to resistance to trichodermin and anisomycin and to an increased sensitivity to sparsomycin. Resistance to trichodermin is due to a reduced affinity of ribosomes from the mutant for the antibiotic. Ribosomes from the sensitive strain (Y166) bind [acetyl‐14C]trichodermin with a dissociation constant of 0.99 μM while those from the resistant one (TR1) bind [acetyl‐14C]trichodermin with a dissociation constant of 15.4 μM. Similar results are obtained when the binding of [acetyl‐14C]‐trichodermin to Y166 and TR1 60‐S subunits is studied.

The mutant TR1 is also resistant to anisomycin. Although trichodermin and anisomycin bind to the ribosome at mutually exclusive sites, the higher affinity binding of [G‐3H]anisomycin that is responsible for the inhibition of the peptidyl transferase center is practically identical for Y166 and TR1 ribosomes. Therefore, the mutation in the ribosome leading to resistance to trichodermin and anisomycin decreases the affinity for trichodermin but not for anisomycin.

Trichodermin, trichothecin and fusarenon X inhibit the binding of [G‐3H]anisomycin to TR1 ribosomes to a lower extent than to Y166 ribosomes, suggesting that the resistance of TR1 ribosomes to the effects of trichothecin and fusarenon X is caused by a decrease in the affinity of the ribosomes for these drugs, as was seen with trichodermin. On the other hand, verrucarin A inhibits [G‐3H]anisomycin binding to Y166 and TR1 ribosomes to a similar extent and therefore its affinity for the ribosome does not appear to be affected by the mutation leading to resistance.

Trichothecin, trichodermin and fusarenon X appear to have a common binding site on the 60‐S ribosomal subunits, which overlaps or is closely linked to the binding sites of anisomycin and verrucarin A.
Biogenesis of Mitochondrial Membranes in Neurospora crassa
Authors: HAWLEY Emerson S. , GREENAWALT John W.
Published: March 2005

The conidia of Neurospora crassa entered logarithmic growth after a 1‐h lag period at 30°C. Although [14C]leucine is incorporated quickly early in growth, cellular protein data indicated that no net protein synthesis occurred until after 2 h of growth. Neurospora is known to produce ethanol during germination even though respiratory enzymes are present. Also, Neurospora mitochondria isolated from cells less than 3‐h old are uncoupled. Since oxygen uptake increased during germination, was largely cyanide‐sensitive, and reached a maximum at 3 h, it is hypothesized that during early germination the uncoupled electron transport chain merely functions to dispose of reducing equivalents generated by substrate level ATP production.

The rate of protein synthesis in vitro by mitochondria isolated from 0–8‐h‐old cells increased as did cell age. Mitochondrial protein synthesis in vivo, assayed in the presence of 100 μg cycloheximide/ml, increased from low levels in the conidia to peak levels at 3–4 h of age and then slowly decreased. The rate of mitochondrial protein synthesis in vivo was linear for at least 90 min in 0–4‐h‐old cells, but declined after 15 min of incorporation in 6 and 8‐h‐old cells.

The products of mitochondrial protein synthesis in vivo were analyzed with dodecylsulfate gel electrophoresis and autoradiography. Early in germination 80% of the synthesis was of two small proteins (molecular weights 7200 and 9000). At 8 h 85% of the radioactivity was in 10 larger proteins (12200 to 80000). Within the high‐molecular‐weight class, proteins of between 12000 and 21500 molecular weight were preferentially labelled early in germination, whereas after 8 h of growth proteins of 27500 to 80000 molecular weight were preferentially labelled. It is hypothesized that the 7200 and 9000‐molecular‐weight products of mitochondrial protein synthesis combine with other proteins to form the larger proteins found later in growth. The availability of these other proteins in cells of different ages could affect the rate of mitochondrial protein synthesis in vivo.
Biochemistry of Development in Insects
Authors: MUNICIO Angel M. , ODRIOZOLA José Maria , PÉREZ‐ALBARSANZ Miguel A.
Published: March 2005

To study the different metabolic behaviour of various stages of development of the insect Ceratitis capitata, the incorporation of labelled decanoic, lauric, myristic, palmitic, stearic, oleic, and linoleic acids into triacylglycerols by insect homogenates was investigated. The time‐course of incorporation of labelled fatty acids was firstly studied by using oleic acid; it showed that after 10 min of incubation the levels of radioactivity incorporated into triacylglycerols and those remaining in the free fatty acids were practically unchanged.

All labelled fatty acids were efficiently incorporated by larval homogenates; however, most of the radioactivity remained as free fatty acids in the presence of pharate adult homogenates, palmitic, and stearic acids being the most scarcely incorporated by this stage of development of the insect.

Plots of triacylglycerol and free fatty acid radioactivities versus the stage of development defined a crossing‐zone in coincidence with the larval‐pupal apolysis. This metabolic difference between larval and pharate adult homogenates could not be explained through differences in the acyl‐CoA synthetase activity of the insect; this enzyme activity was notably higher in pharate adult homogenates than in the larval homogenates whatever would be the nature of the fatty acid.

[14C]Triolein was scarcely hydrolyzed by both larval and pharate adult homogenates.

Double‐label experiments were carried out by incorporating either [3H]oleic acid or [3H]‐palmitic acid and [14C]glycerol 3‐phosphate by larval and pharate adult homogenates at different incubation intervals. Diacylglycerols, triacylglycerols, and phosphoglycerides were isolated and the 14C/3H molar ratio calculated. Results suggest the existence of a different acyltransferase activity in the different stages of development of the insect.
Intermediates in the Interconversion of Acid and Alkaline Ferrihemoglobin
Authors: CHANCE Britton , RUMEN Nevenka M.
Published: March 2005

The acid‐alkaline pH jump in suspension of crystalline sheep hemoglobin has been studied in the range 5.95 to 8.94. Crystals suspended in 3.8 M Cs2SO4 show a rapid optical transition of half‐time equal to or less than 2 ms.

As the ammonia concentration is increased in the Cs2SO4‐suspended crystals, a second optical transition is observed as a pseudo‐first‐order reaction, with a rate constant of between 10 and 15 s−1.

The alkaline‐acid pH jump proceeds through a very rapid shift of the alkaline‐acid equilibrium and is followed by a first‐order dissociation constant between 9 and 12 s−1. The dissociation of the ammonia is biphasic, and the ratio between the fast and slow phases is 9.
The Control of the Adenosine Triphosphatase of Rhodospirillum rubrum Chromatophores by Divalent Cations and the Membrane High Energy State
Authors: EDWARDS Patricia A. , JACKSON J. Barry
Published: March 2005

The rate of ATP hydrolysis, catalysed by Rhodospirillum rubrum chromatophores is accelerated by low concentrations and inhibited by high concentrations of uncoupling agent.

The inhibition at high concentrations of uncoupling agent is potentiated by the presence of free magnesium ions. At low uncoupler concentrations magnesium has no effect on the rate of ATP hydrolysis.

Inhibition of ATP hydrolysis by high concentrations of uncoupling agent and free magnesium ions is reversed by illumination. Illumination has less effect at low magnesium concentrations.

Free calcium ions inhibit ATP hydrolysis independently of the coupled state of the membrane.

Under coupled conditions, magnesium ions can overcome the inhibition induced by calcium. The two ions compete for the same site on the enzyme.

Inhibition by free magnesium in highly uncoupled chromatophores and inhibition by free calcium are both non‐competitive with respect to the divalent cation‐ATP substrate.

These data are consistent with a model in which divalent cations can bind to a site on the enzyme which is distinct from the substrate site. The regulation of the enzyme activity by the high energy state of the membrane is dependent on the occupant of this site.
A DNA Polymerase from Ustilago maydis
Authors: BANKS Geoffrey R. , YARRANTON Geoffrey T.
Published: March 2005

The polymerase and deoxyribonuclease activities of the purified Ustilago maydis DNA polymerase coeluted from a hydroxyapatite column, cosedimented in sucrose gradients in both the absence and presence of salt, possessed similar thermolabilities and reaction requirements. These observations suggest that both activities are associated with the same enzyme and that the deoxyribonuclease activity is not a contaminant. The initial rate of degradation of native 3′‐end‐group‐labelled DNA was similar to that of a heat‐denatured substrate, but the final extent was greater for the former. The enzyme exhibits a high specificity for degradation of DNA in a 3′→5′ direction. The degradation of a DNA template was inhibited by the presence of the deoxyribonucleoside triphosphates necessary for simultaneous DNA synthesis, but not that of the newly synthesised DNA. About 50%, 29% and 13% of the purine, cytosine and thymine deoxyribonucleotide residues incorporated by the enzyme into DNA respectively, were subsequently excised when monitored by the resulting conversion of the triphosphate substrates to free monophosphate. The majority of the purine deoxyribonucleoside monophosphates appear after the synthetic phase of the reaction has ceased. In many respects, therefore, the deoxyribonuclease activity of the U. maydis DNA polymerase is similar to the bacteriophage T4‐induced enzyme.
Binding of Secretory Component to Human Immunoglobulin M
Authors: LINDH Erik , BJÖRK Ingemar
Published: March 2005

The binding of human free secretory component to immoglobulin M (IgM) has been studied in vitro as a model for the formation of complexes between the two proteins in vivo. Three IgM myelomas and normal serum IgM were found to bind secretory component in amounts from 0.8 to 2.0 mol per mol of IgM. This variation in binding was not related to a corresponding variation of the J‐chain content of the immunoglobulins, but more likely it was due to varying amounts of unspecifically bound serum proteins blocking the attachment of secretory component. In contrast to complexes with immunoglobulin A (IgA), the binding of secretory component to IgM appeared to be solely of a noncovalent nature, as all secretory component was released from complexes with IgM during gel chromatography in 6 M guanidine hydrochloride. Studies with tryptic fragments of one of the IgM myelomas indicated that the binding site for secretory component is located on the (Fc)5μ part of the IgM pentamer. Finally, only minimal conformational changes were found to accompany complex formation between secretory component and IgM, analogous to what has earlier been reported for the attachment of the secretory component to IgA.
Inhibition by Triphenyltin Chloride of a Tightly‐Bound Membrane Component Involved in Photophosphorylation
Authors: GOULD J. Michael
Published: March 2005

At very low concentrations (< 1 μM) triphenyltin chloride inhibits ATP formation and coupled electron transport in isolated spinach chloroplasts. Basal (−Pi) and uncoupled electron transport are not affected by triphenyltin. The membrane‐bound ATP ⇌ Pi exchange and Mg2+‐dependent ATPase activities of chloroplasts are also completely sensitive to triphenyltin, although the Ca2+‐ dependent and Mg2+‐dependent ATPase activities of the isolated coupling factor protein are insensitive to triphenyltin. The light‐driven proton pump in chloroplasts is stimulated (up to 60 %) by low levels of triphenyltin. Indeed, the amount of triphenyltin necessary to inhibit ATP formation or stimulate proton uptake is dependent upon the amount of chloroplasts present in the reaction mixture, with an apparent stoichiometry of 2 — 2.5 triphenyltin molecules/100 chlorophyll molecules at 50: < inhibition of ATP formation and half‐maximal stimulation of proton uptake. Chloroplasts partially stripped of coupling factor by an EDTA wash are no longer able to accumulate protons in the light. However, low levels of triphenyltin can effectively restore this ability. The amount of triphenyltin required for the restoration of net proton uptake is also dependent upon the amount of chloroplasts, with a stoichiometry of 4 — 5 triphenyltin molecules/100 chlorophyll molecules at 50% reconstitution.

On the basis of this and other evidence it is concluded that triphenyltin chloride inhibits phosphorylation, ATP + Pi exchange and membrane‐bound ATPase activities in chloroplasts by specifically blocking the transport of protons through a membrane‐bound carrier or channel located in a hydrophobic region of the membrane at or near the functional binding site for the coupling factor.
The Interdependence of Magnesium with Spermidine and Phosphoenolpyruvate in an Enzyme‐Synthesizing System in vitro
Authors: FUCHS Eckart
Published: March 2005

The DNA‐dependent syntheses of different enzymes of the bacteriophages T3 and T7 were studied in an Escherichia coli system in vitro with respect to the optimal Mg2+ concentration and its interdependence with substituting (e.g. spermidine) and complexing agents (e.g. phosphoenolpyruvate). The following results were obtained.

The optimal conditions for the syntheses of the different enzymes were not identical. The optima for RNA polymerase synthesis were 8 mM Mg2+, 10 mM P‐pyruvate and 3 mM spermidine: for S‐adenosyl‐L‐methionine cleaving enzyme synthesis, 6 mM Mg2+, 6 mM P‐pyruvate and 3 mM spermidine; and for lysozyme synthesis, 13‐18 mM Mg2+, 28 mM P‐pyruvate and 3‐0 mM spermidine.

The optimal conditions for the synthesis of analog enzymes (RNA polymerases and lysozymes) from the two templates were identical within experimental error.

Mg2+ and spermidine substituted for each other in relation to the number of their charges. 4. The apparent complexing of one Mg2+ molecule required the addition of 3‐5 P‐pyruvate molecules.

Under the optimal conditions the enzyme‐synthesizing activity was higher by more than a factor of 10 compared to previously described systems.
An Enzyme Complex for the Dehydrogenation of Phytoene in Phycomyces
Authors: ARAGÓN Carlos M. G. , MURILLO Francisco J. , de la GUARDIA Manuel D. , CERDÆ‐OLMEDO Enrique
Published: March 2005

Phycomyces strain C5, carrying mutation carB10, accumulates phytoene instead of β‐carotene. Heterokaryons containing C5 nuclei and different other nuclei carrying the wild type carB allele accumulate significant amounts of phytofluene, ζ‐carotene and neurosporene. From quantitative analyses of carotenes and nuclear proportions in the heterokaryons we conclude that four copies of the carB gene product, assembled in an enzyme complex, act sequentially in the conversion of phytoene to lycopene.
Studies on the Environment of Protein S7 within the 30‐S Subunit of Escherichia coli Ribosomes
Authors: RINKS Jutta , YUKI Atsushi , BRIMACOMBE Richard
Published: March 2005

Analyses are described of three types of ribosomal fragment, all derived from the well‐characterized ribonucleoprotein species consisting of proteins S7, S9, S 10, S14 and S19, together with RNA from sections O′‐D‐E′‐K‐P‐P′‐E‐A of the 16‐S sequence.

When 30‐S subunits were hydrolysed with ribonuclease T1 in the presence of deoxycholate in addition to the components previously described, a fragment could be isolated which contained only proteins S 7 and S19, together with minor amounts of S13 or S 14. Oligonucleotide analysis of this fragment showed that it contained RNA from sections O′‐E′ and P‐A of the 16‐S RNA, but that section K (from the middle of this area) was missing.

It has previously been shown that when 30‐S subunits are irradiated with ultraviolet light, protein S7 is the primary target of cross‐linking of protein to RNA. By making use of this reaction, ribonucleoprotein fragments were isolated from irradiated 30‐S subunits, the unbound proteins were removed, and RNA fragments containing covalently linked protein S7 were identified. It was possible to demonstrate that the site of cross‐linking lies within the O′‐A region of the 16‐S RNA, and more precise experiments showed that this site almost certainly is in the P‐A region.

When the parent five‐protein ribonucleoprotein fragment (above) was deproteinized under very mild conditions, RNA complexes could be isolated which consisted of non‐contiguous sequences, but which migrated as a single species into a polyacrylamide gel. Analysis of one of these complexes showed that it contained sequences from sections O′‐E′ and P‐A, but that section K was missing (cf. first paragraph above). This demonstrated that these two separate regions of RNA interact within the 30‐S subunit, independently of the presence of protein.
Globin mRNA Species Containing Poly(A) Segments of Different Lengths
Authors: NUDEL Uri , SOREQ Hermona , LITTAUER Uriel Z. , MARBAIX Gerard , HUEZ Georges , LECLERCQ Madeleine , HUBERT Evelyne , CHANTRENNE Hubert
Published: March 2005

Rabbit globin mRNA species containing poly(A) segments of different lengths were prepared by partial phosphorolysis of mRNA with Escherichia coli polynucleotide phosphorylase. By varying the salt concentration and the time of incubation of the phosphorolysis mixture, as well as performing oligo(dT)‐cellulose chromatography at 22 °C and at 4 °C, globin mRNA preparations containing poly(A) segments of approximately 122, 95, 68, 39, 32, 21, and 16 adenylate residues were obtained. It was found that the functional stability of the mRNA species containing 32 or more adenylate residues after injection into Xenopus laevis oocytes equaled that of the native globin mRNA. On the other hand, the functional stability of mRNA containing an average number of 21 adenylate residues was about 30% of the native mRNA, while that of mRNA containing 16 adenylate residues was as low as nolv(A)‐free globin mRNA.