bluuebunny/crossref_metadata_2025 huggingface dataset:
Protein Kinase of Bacteriophage T7
Authors: PAI Shou‐Hsiung , RAHMSDORF Hans‐Jobst , PONTA Helmut , HIRSCH‐KAUFFMANN Monica , HERRLICH Peter , SCHWEIGER Manfred
Published: March 2005

Protein kinase, which was isolated from cells infected with T7, is indeed a viral gene product. This is shown by DNA‐dependent synthesis in vitro. The protein kinase transfers phosphate from ATP to seryl or threonyl residues in protein. The enzyme has only a relative requirement for magnesium ions, but is only active at low ionic strength. The best substrate is lysozyme. T7 protein kinase activity is not stimulated by cyclic 3′: 5′‐AMP and/or cyclic 3′: 5′‐GMP. The T7 protein kinase carries – SH groups essential for activity. There is indication that the enzyme phosphorylates itself and causes self inactivation, which may explain the fast disappearance of enzyme activity in vivo. Bacteriophage T3 also induces a protein kinase which is similar to the T7‐induced enzyme in all respects tested.
Stimulation of Tumor‐Cell Respiration by Inhibitors of Pyruvate Kinase
Authors: GOSALVEZ Mario , LÓPEZ‐ALARCÓN Luisa , GARCÍA‐SUAREZ Socorro , MONTALVO Antonio , WEINHOUSE Sidney
Published: March 2005

In a model system consisting of highly coupled rat liver mitochondria respiring in the presence of substrate, pyruvate kinase, phosphoenolpyruvate, ATP, hexokinase and glucose, the increase in the mitochondrial concentration results in a progressive decrease in the activity of pyruvate kinase. These results are in accord with a role of pyruvate kinase as a determinant of glycolytic activity by competing with mitochondrial oxidative phosphorylation for the available ADP.

The addition of adequate amounts of the amino acids, cysteine, alanine and phenylalanine, known as inhibitors of pyruvate kinase, to living Ehrlich ascites tumor cell suspensions results in a stimulation of the respiratory rate and in a decrease of the glycolytic rate of the cells. Concomitant with these changes, there is an accumulation of intracellular phosphoenolpyruvate and ADP, and a decrease in pyruvate kinase as another key enzyme whose properties and activities may be major determinants for the control of glycolysis and the Crabtree and Pasteur effects of tumor cells.
Cytochrome Components of Plant Microsomes
Authors: RICH Peter R. , BENDALL Derek S.
Published: March 2005

The electron transport components of the microsomal fraction of cauliflower buds and mung bean hypocotyls were investigated using split‐beam and dual wavelength spectrophotometry under a variety of reducing conditions.

Cauliflower microsomes were found to contain an ascorbate‐reducible component, termed cytochrome b‐559.5 [E′0=+ 135 ± 20 mV; λmax (reduced minus oxidised) = 559.5, 527 and 429 nm at 23 °C], cytochrome b5 [E'0=−20 ± 20 mV; λmax (reduced minus oxidised) = 556, 526 and 425 nm at 23°C], cytochromes P‐450 and P‐420. On the basis of binding studies with ethyl isocyanide, degradation of cytochrome P‐450 to P‐420, redox potential, aniline binding, and relative rates of reduction by NADPH and NADH, it is suggested that the cytochrome P‐450 system is analogous to that in mammalian microsomes. Other components, reducible only by dithionite, may also be present.

Mung bean microsomes were found to contain an ascorbate‐reducible component, termed cytochrome b‐562 [E′0=+ 120 ± 20 mV; λmax (reduced minus oxidised) = 562, 528, and 430 nm at 23 °C], cytochrome b5, and a low potential component which was reducible only by sodium dithionite. No cytochrome P‐450 or P‐420 could be detected.

A general method of analysis of the cytochromes was developed and applied to the microsomes from a variety of plant sources. The results indicate that large variations, both in type and amount of components, occur between the microsomes from different plant materials.
Determination of Protein‐Ligand Binding Constants at Equilibrium in Biological Samples
Authors: BLONDEAU Jean‐Paul , ROBEL Paul
Published: March 2005

Protein‐ligand complexes can be separated functionally into two classes. “Specific” binding is characterized, in relative terms, by a high affinity for the ligand and a low binding capacity. “Nonspecific” binding is characterized by a low affinity and a very large capacity. The calculation of equilibrium binding constants for any specific protein‐ligand interaction requires the exact determination of the unbound ligand concentration and the specifically bound ligand concentration. These determinations usually require corrections for the contribution of non‐specific binding. The use of two correction terms, kn and f, is proposed: kn is the product of the affinity constant k times the number of binding sites n of the non‐specific components, while f is the fraction of the non‐specific binding included in the experimental estimates of bound ligand. Several theoretical solutions using these terms are proposed for the calculation of specific binding constants. The practical choice of the correction factor may be different when the simultaneous measurement of the affinity constant and maximum number of binding sites, or when only the latter, is desired. In the case of complex binding systems containing more than one specific component, the individual constants can be determined by non‐graphical methods, using computer‐aided iterative statistical calculations. A complete solution is given for a system containing two specific plus non‐specific interactions and actual experiments are reported for steroid hormone‐receptor complexes.
Identification of Chloride‐Binding Sites in Hemoglobin by Nuclear‐Magnetic‐Resonance Quadrupole‐Relaxation Studies of Hemoglobin Digests
Authors: CHIANCONE Emilia , BRUNORI Maurizio , ANTONINI Eraldo , WYMAN Jeffries , NORNE Jan Erik , FORSÉN Sture , BONAVENTURA Joseph
Published: March 2005

35Cl−‐nuclear magnetic resonance (NMR) studies indicate that various digests of human hemoglobin with carboxypeptidase A and B, or a combination of the two, may be used for the identification of chloride binding sites. All the digestion products contain, like hemoglobin itself, at least two classes of binding sites, one of high, the others of low affinity. The pH dependence of the excess linewidth of the 35Cl−‐NMR signal indicates that in the simple digests with either carboxypeptidase A or B, chloride is bound with high affinity at or near His‐β146–Asp‐β94 and at or near Val‐α1–Arg‐α141. The high‐affinity sites show, in the case of the simple digests, a strong oxygen linkage which is lost in the forms digested with both carboxypeptidase A and B; this linkage may thus be correlated to the presence of quaternary conformational changes. Organic phosphates, like inositol hexaphosphate, show competition for some of the high‐affinity chloride binding sites in hemoglobin and in the simple digests. This competition is likewise lost in the doubly digested hemoglobins.
Tertiary Structure of tRNAPhe (Yeast): Kinetics and Electrostatic Repulsion
Authors: URBANKE Claus , RÖMER Roland , MAASS Günter
Published: March 2005

Conditions were established that allowed the observation of the unfolding of the tertiary structure of tRNAPhe (yeast) without the interference of either secondary structure or low salt aberrant structures. Relaxation kinetics of tertiary structure melting show that the reaction proceeds according to a co‐operative all‐or‐none mechanism. The negative activation enthalpy of formation (ΔH‡R=−14 ± 5 kcal/mol, −59 ± 21 kJ/mol) implies a fast pre‐equilibrium preceding the rate‐limiting step. The ionic strength dependence of the corresponding rate constant demonstrates that most of the electrostatic repulsion characteristic of tertiary structure folding is overcome before the rate‐limiting step is reached. On the other hand, most of the stabilizing enthalpy change occurs after the rate‐limiting step. At the usual ionic strength (0.1 M Na±) tertiary structure folding is about 100 times slower than double‐helix formation. Extrapolation of the rate constants to high ionic strengths, however, indicates that the dynamic differences between secondary and tertiary structure are only due to electrostatic repulsion. The stabilization of tertiary structure by alkaline salts is increased by decreasing the cationic radius. Double helices show virtually no dependence on the radius of monovalent cations. This indicates considerable geometric restrictions for the stabilization of tertiary structure.
Heterogeneity of Enterotoxin‐Like Protein Extracted from Spores of Clostridium perfringens Type A
Authors: FRIEBEN William R. , DUNCAN Charles L.
Published: March 2005

Enterotoxin‐like protein was extracted from spores of three enterotoxin‐positive and three enterotoxin‐negative strains of Clostridium perfringens type A by urea/mercaptoethanol, alkaline mercaptoethanol and alkaline dithiothreitol. Disc immunoelectrophoresis demonstrated that three distinct enterotoxin‐like proteins could be extracted. In 7% acrylamide gels, type I, type II, and type III enterotoxin‐like proteins had relative mobilities of 0.52, 0.63, and 0.73 respectively. In contrast to disc immunoelectrophoresis, immunoelectrophoresis in agar gel demonstrated identical electrophoretic properties for the various enterotoxin‐like proteins. Immunoelectrofocusing experiments gave isoelectric points of 4.43, 4.43, 4.36, and 4.52 for purified enterotoxin and type I, type II, and type III enterotoxin‐like proteins respectively. Ferguson plots (i.e., log relative mobility versus acrylamide concentration) yielded nonparallel lines which intersected at a nonsieving concentration of acrylamide indicating that the various species of enterotoxin‐like protein differed in size. Estimation of the molecular weight of purified enterotoxin and the three species of enterotoxin‐like protein was done by comparing the slopes obtained in Ferguson plots with those obtained using proteins of a known molecular weight. Molecular weights of 38000, 36500, 23000, and 14500 were obtained for purified enterotoxin, type I, type II, and type III enterotoxin‐like protein respectively. Collectively, the evidence indicates that fractionation of the different species of enterotoxin‐like protein was due primarily to differences in their size, and that different forms of enterotoxin‐like protein can be extracted from spores of different strains of C. perfringens type A.
d‐Alanyl‐d‐Alanine Carboxypeptidase in the Bacterial Form and L‐Form ofProteus mirabilis
Authors: MARTIN Hans Herbert , MASKOS Christian , BURGER Reinhard
Published: March 2005

Membranes of the bacterial form and the stable and unstable L‐forms ofProteus mirabiliscontain LD and DD‐carboxypeptidase. The DD‐carboxypeptidase is inhibited non‐competitively by penicillin G. The enzyme of the bacterial form is highly penicillin‐sensitive (Ki= 4 × 10−9M penicillin G). Inhibition is only partly reversible by treatment with penicillinase or by dialysis against buffer. In contrast, the DD‐carboxypeptidase of the unstable L‐form, grown in the presence of penicillin, is 175‐fold less penicillin‐sensitive (Ki= 7 × 10−7M penicillin G). Inhibition is completely reversed by penicillinase or dialysis. After inhibition by penicillin and subsequent reactivation the penicillin sensitivity of the bacterial DD‐carboxypeptidase is similar to the sensitivity of the enzyme of the unstable L‐form. The hypothesis is proposed thatP. mirabiliscontains two DD‐carboxypeptidases of different penicillin sensitivity and with different mechanisms of penicillin binding. Peptidoglycan synthesis in the cell walls of the unstable L‐form is probably carried out with the help of only one DD‐carboxypeptidase,viz. the completely reactivatable enzyme with the lower penicillin sensitivity.
Pigeon‐Liver NAD Kinase
Authors: APPS David K.
Published: March 2005

NAD kinase was purified from pigeon liver by an improved procedure which included chromatography on phosphocellulose. The resultant preparation was homogeneous as judged by gel electrophoresis, but electrofocusing gave indications of heterogeneity. The enzyme appeared to be of molecular weight 270000, and to consist of subunits of molecular weight 34000; it may therefore be an octomer. Kinetic studies over a wide range of substrate concentrations revealed departures from Michaelis‐Menten behaviour with the substrate NAD±; these were interpreted tentatively in terms of negative homotropic interactions between identical binding sites, since thermal and chemical inactivation studies revealed no evidence for more than one type of catalytic site. The significance of the kinetics and of the type of inhibition produced by NADPH is discussed in terms of the regulation of NAD kinase activity in vivo.
Solubilization and Characterization of Phosphoprotein Phosphatase(s) from Bovine Corpus‐Luteum Plasma Membranes
Authors: AZHAR Salman , MENON K. M. Jayaram
Published: March 2005

Plasma membrane fractions I and II isolated from bovine corpus luteum contain phosphoprotein phosphatases. Enzyme activities associated with both membrane fractions showed pH optima in the neutral range and were most active with phosphoprotamine as the exogenous substrate. The enzyme activity was partially inhibited by Co2+, Zn2+ and Fe2+. Dithioerythritol, glutathione (reduced) and 2‐mercaptoethanol stimulated the enzyme activity, whereas N‐ethylmaleimide and N‐phenylmaleimide were inhibitory. Similarly, various cyclic nucleotides and nucleoside triphosphates also inhibited phosphoprotein phosphatase activities. The phosphatase activity was also observed with endogenous phosphorylated membrane proteins as substrate. The endogenous phosphorylation of membranes was rapid and attained a maximal level after 15–20 min of incubation. Initially endogenous dephosphorylation was also very rapid, but did not reach completion. In addition to phosphoprotein phosphatase, membrane preparations also possessed very active cyclic‐AMP‐dependent protein kinase activity. Phosphoprotein phosphatase activity from plasma membranes was solubilized by ionic and nonionic detergents. Optimal solubilization was achieved with 0.1% sodium deoxycholate. Sucrose density gradient centrifugation of deoxycholate‐solubilized fraction I and fraction II membranes resolved phosphoprotein phosphatase activity into two species with apparent sedimentation coefficients of 6.7 S (Mr 130000) and 4.8 S (Mr 90000). Cyclic‐AMP‐stimulated protein kinase activity sedimented as a broad peak with a sedimentation coefficient of 5.5 S (Mr 110000).
Mode of Action of the Macrolide‐Type Antibiotic, Chlorothricin
Authors: SCHINDLER Peter W. , SCRUTTON Michael C.
Published: March 2005

The macrolide‐type antibiotic chlorothricin inhibits pyruvate carboxylases purified from rat liver, chicken liver and Azotobacter vinelandii. Under standard assay conditions the concentration of chlorothricin required for half‐maximal inhibition of oxalacetate synthesis is 0.26 mM (rat liver), 0.12 mM (chicken liver), and 0.5 mM (Azotobacter vinelandii). Inhibition by chlorothricin appears non‐competitive in character when measured as a function of the concentration of the substrates of the pyruvate carboxylase reaction as well as of CoASAc and Mg2+. This pattern of inhibition suggests that this antibiotic interacts at unique sites on chicken and rat liver pyruvate carboxylase which are distinct from both the catalytic and activator sites. Interaction of chlorothricin with the two vertebrate liver pyruvate carboxylases differs from the effect exerted by this antibiotic on pyruvate carboxylase purified from Azotobacter vinelandii. A sigmoidal relationship between initial velocity and inhibitor concentration is observed for the vertebrate enzymes under most conditions whereas a hyperbolic profile characterizes the concentration dependence of inhibition of the Azotobacter vinelandii enzyme by chlorothricin. In the case of rat liver pyruvate carboxylase chlorothricin does not alter the extent of cooperativity in the relationship between initial rate and CoASAc concentration. However, a small but significant increase of the Hill coefficient from a value of 2.7 in the absence of antibiotic to that of 3.3 in the presence of 0.5 mM chlorothricin is observed for chicken liver pyruvate carboxylase. Chlorothricin decreases the rate of inactivation observed when rat liver pyruvate carboxylase is incubated with trinitrobenzenesulfonate and when chicken liver pyruvate carboxylase is incubated at 2° C. The maximal decrease in inactivation observed in the presence of saturating concentrations of antibiotic is 50% for cold inactivation of the chicken liver enzyme and 60% for inactivation of the enzyme from rat liver by trinitrobenzenesulfonate. In both cases a sigmoidal relationship is observed between inactivation rate and chlorothricin concentration. These data as well as the initial rate studies suggest that multiple interacting sites for this antibiotic are present on the vertebrate liver pyruvate carboxylases. The occupancy of these sites appears to cause significant distortion of both the catalytic and the activator sites.
Quaternary Structure of Polynucleotide Phosphorylase from Escherichia coli: Evidence of a Complex between Two Types of Polypeptide Chains
Authors: PORTIER Claude
Published: March 2005

A new form of polynucleotide phosphorylase containing α and β subunits was isolated (form B) by purification without preparative electrophoresis; this form was compared to the enzyme obtained by preparative electrophoresis purification (form A).

The Stokes radius of these two forms are very different: 6.4 nm for form A and 8.7–9.0 nm for form B; on the other hand the sedimentation coefficients are close: 8.9 S and 9.9 S respectively. Such a result suggests that form B is very asymmetric. The apparent molecular weights, calculated from the Stokes radii and from the sedimentation coefficients, are approximately 365000 for form B and 252000 for form A. The latter is homogeneous on polyacrylamide gel, whereas the former yields two components, one of which behaves similarly to form A. Finally, whereas form A is composed of only one type of subunit, α, form B contains two types of chains: α (Mr 86000 ± 5000) and β (Mr 48000 ± 2000) in stoichiometric proportions.

From these results we believe that one should assume for form B the existence of a complex between form A and β chains; the role of the latter still remains to be specified.
Inhibition by Carbon Monoxide of the Secondary‐Amine Mono‐oxygenase of Pseudomonas aminovorans and the Photochemical Action Spectrum for Its Reversal
Authors: BROOK David F. , LARGE Peter J.
Published: March 2005

The secondary‐amine mono‐oxygenase (EC 1.14.99.–) of Pseudomonas aminovorans is potently inhibited by carbon monoxide. The degree of inhibition of the purified enzyme was determined by the CO:O2 ratio rather than by the absolute concentration of carbon monoxide. The partition constant (the CO:O2 ratio causing 50% inhibition of activity) was 9.2 × 10−4. The inhibition could be reversed by light, and the extent of reversal was proportional to the light intensity. With monochromatic light of wavelength 417 nm, the light sensitivity, L, was determined to be 2.5 × 108 cm2 min/mol quantum. The photochemical action spectrum for the light reversal of inhibition showed a single maximum of effectiveness at about 420nm. The difference spectrum of the enzyme (reduced by NADH) on bubbling with CO (compared with an NADH‐reduced reference sample) showed a peak at 426 nm. The preparation showed none of the spectral properties of cytochrome P‐450 mono‐oxygenase preparations, and was much more sensitive to carbon monoxide. The enzyme behaves as a typical o‐type cytochrome (i.e. a carbon‐monoxide‐reactive b‐type cytochrome), and in its sensitivity to carbon monoxide as well as in its spectral properties, shows close resemblances to haemoglobin.
Nucleotide Sequences of Escherichia coli 16‐S RNA Associated with Ribosomal Proteins S7, S9, S10, S14 and S19
Authors: YUKI Atsushi , BRIMACOMBE Richard
Published: March 2005

A ribonucleoprotein fragment containing proteins S7, S9, S10, S14, and S19 was isolated in high yield from Escherichia coli 30‐S ribosomal sub‐particles. The same fragment was obtained whether ribosomes from E. coli strain A19 or MRE 600 were used, despite the fact that protein S7 differs widely between the two strains. RNA was extracted from this fragment and fractionated on gels containing 7 M urea, to reveal “hidden breaks”. A well‐defined and reproducible pattern of RNA fragments was obtained, with the main components being approximately 300, 240, 130, 115 and 75 nucleotides in length, respectively. The pattern of RNA fragments obtained was also independent of the strain of E. coli used.

Two‐dimensional fingerprints were made from ribonuclease T1 hydrolysates of these RNA fragments, labelled with 32P, and the oligonucleotides were further analysed by digestion with either ribonuclease A or U2. The data obtained were fitted in detail to the new 16‐S RNA sequence map of Ehresmann et al. (1975). Again no significant differences were observed between the RNA from E. coli A19 or MRE 600. The RNA sequences found lay in the region O′‐D‐E′‐K‐P‐P′‐E‐A of the 16‐S RNA, with a clear excision of several nucleotides in section E′‐K. The total sequence length covered was approximately 430 nucleotides.
The Modification of DNA‐Dependent RNA Polymerase from Escherichia coli by an Alkylating Derivative of Rifamycin SV
Authors: STENDER Walter , STÜTZ Anton A. , SCHEIT Karl Heinz
Published: March 2005

3‐(2‐Bromo[1‐14C]acetamidoethyl)‐thio‐rifamycin SV, abbreviated BrAcNEtS‐Rif, an alkylating derivative of rifamycin SV was synthesized. A four‐fold excess of BrAcNEtS‐Rif inhibited the enzymic activity of RNA polymerase from Escherichia coli to 97%. Incubation of RNA polymerase with Br[14C]AcNEtS‐Rif led to covalent substitution. The reaction of Br[14C]AcNEtS‐Rif with enzyme at a ratio of 1.4:1 and a concentration of 63 nM was found to proceed with a half life of 1 h at 37°C. The enzyme could be protected from reaction with BrAcNEtS‐Rif by either rifampicin or the hybrid [poly(dT)] · [r(Ap)5A]. The modification of holoenzyme by Br[14C]AcNEtS‐Rif in the presence of p‐hydroxymercuribenzene sulfonic acid (pOH‐HgBzSO3H) or 4 M LiCl occurred with faster kinetics and led to a higher degree of substitution. Reaction of Br[14C]AcNEtS‐Rif with RNA polymerase core enzyme caused predominant substitution of subunit β. In the case of RNA polymerase holoenzyme the radioactive substituents were evenly distributed between subunits β and σ. Apparently the topology of the rifamycin binding site of holoenzyme, similarly to core enzyme, precludes attacks of nucleophilic functions from β' and α, but it allows nucleophilic functions from subunits β and σ to react with equal probability on BrAcNEtS‐Rif. In the presence of a 20‐fold excess of pOH‐HgBzSO3H, the modification of holoenzyme was drastically altered. Virtually all substitution took place on subunit β', very little on β and none on subunits σ and α.
A New Mode of Ring Cleavage of 2,3‐Dihydroxybenzoic Acid in Tecoma Stans (L.)
Authors: SHARMA Harmesh Kumar , VAIDYANATHAN Chelakara Sivarama
Published: March 2005

2,3‐Dihydroxybenzoic acid has been shown to be oxidized via the 3‐oxoadipate pathway in the leaves of Tecoma stans. The formation of 2‐carboxy‐cis, cis‐muconic acid, a muconolactone, 3‐oxoadipic acid and carbon dioxide during its metabolism has been demonstrated using an extract of Tecoma leaves. The first reaction of the pathway, viz., the conversion of 2,3‐dihydroxybenzoate to 2‐carboxy‐cis, cis‐muconic acid has been shown to be catalysed by an enzyme designated as 2,3‐dihydroxybenzoate 2,3‐oxygenase. The enzyme has been partially purified and a few of its properties studied. The enzyme is very labile with a half‐life of 3–4 h. It is maximally active with 2,3‐dihydroxybenzoate as the substrate and does not exhibit any activity with catechol, 4‐methyl catechol, 3,4‐dihydroxybenzoic acid, etc. However, 2,3‐dihydroxy‐p‐toluate and 2,3‐dihydroxy‐p‐cumate are also oxidized by the enzyme by about 38% and 28% respectively, compared to 2,3‐dihydroxybenzoate. Sulfhydryl reagents inhibit the enzyme reaction and the inhibition can be prevented by preincubation of the enzyme with the substrate. Substrate also affords protection to the enzyme against thermal inactivation. Sulfhydryl compounds strongly inhibit the reaction and the inhibition cannot be prevented by preincubation of the enzyme with its substrates. Data on the effect of metal ions as well as metal chelating agents suggest that copper is the metal cofactor of the enzyme. Evidence is presented which suggests that iron may not be participating in the overall catalytic mechanism.
Synthesis of Mitochondrial Proteins in an Escherichia coli Cell‐Free System Directed by Yeast Mitochondrial DNA
Authors: SCRAGG Alan H. , THOMAS David Y.
Published: March 2005

The optimum conditions for transcription in vitro of yeast mtDNA into biologically relevant RNA by Escherichia coli RNA polymerase holoenzyme and yeast mitochondrial RNA polymerase was found to critically depend on salt concentration. RNA was transcribed (at 0.25 M KCl concentration) from high‐molecular‐weight mtDNA which was then translated in an E. coli (S‐30) cell‐free protein synthesising system. Efficient translation of mitochondrial RNA was achieved using conditions which had also been determined to be optimal in other systems. Identification of the polypeptides produced in the translation system was made using antiserum raised against mitochondrial membranes. Electrophoresis of the completely dissociated antigen‐antibody complexes using dodecylsulphate‐polyacrylamide gels revealed that the system in vitro produced polypeptides of similar molecular weight to those synthesised in vivo by cycloheximide‐inhibited whole cells.
Metabolism of Aromatic Compounds by Fungi
Authors: THATCHER David R. , CAIN Ronald B.
Published: March 2005

A preliminary investigation of the kinetic properties of 3‐carboxy‐cis, cis‐muconate cyclase (EC 5.5.1.5) has been performed. The initial velocity of the reaction was shown to be proportional to the concentration of the enzyme in the assay system adopted and the apparent Km was found to be 57 μM at pH 6.0 and 30 °C but at concentrations exceeding 70 μM, substrate inhibition was apparent. At pH 6.0 the Ki for the substrate was 0.45 mM. Plots of V and Km against pH showed inflexions at pH 5.3 and pH 6.4. The enzyme was inhibited by a variety of inorganic anions and by a number of dicarboxylic and tricarboxylic acids. The degree of inhibition exerted by these acids was found to be proportional to the proximity of their carboxyl groups, the cis configuration being a more effective inhibitor than the trans configuration. As inhibition was competitive in each case, the presence of an anion‐sensitive substrate‐binding site has been postulated. The cis‐cis, cis‐trans and trans‐trans isomers of muconate, 3‐chloromuconate and 3‐carboxy‐cis‐trans‐muconate, close analogues of natural substrate but not attacked by the enzyme, were also found to be competitive inhibitors. The variation in pKi with pH was determined in the case of cis,cis‐muconate and cis‐aconitate, both of which gave curves suggesting the importance of a group with a pKa of approximately 6.4 responsible for increasing the inhibition of the enzyme. Modification by ethoxyformic anhydride and the kinetics of Rose‐Bengal‐sensitized photo‐oxidation suggested the participation of a histidine residue in the catalytic reaction. These results are discussed in the light of recent work on enzymes catalysing analogous reactions; a likely reaction mechanism has been proposed.
The Amino‐Acid Sequence of the Most Acidic Major Parvalbumin from Frog Muscle
Authors: CAPONY Jean‐Paul , PINA Concepción , PECHÈRE Jean‐François , DEMAILLE Jacques
Published: March 2005

The primary structure of the most acidic (pI= 4.50) of the two major parvalbumins from frog (Rana esculenta) has been determined from a study of its trypsic peptides and of overlapping peptides generated by limited trypsic digestion, chymotrypsic digestion and N‐bromosuccinimide cleavage of the protein.

The amino acid sequence so obtained is considered in comparison with those known for other parvalbumins and for rabbit troponin‐C.
Fluorescent Labelling of Escherichia coli Ribosomal Sulfhydryl Groups
Authors: PERRIN Martine , POCHON François
Published: March 2005

The reactivity of the sulfhydryl groups of Escherichia coli ribosome has been investigated using a fluorescent label. Under denaturing conditions, all the – SH groups can be titrated. In the native form of the ribosome, six and less than one labels can be respectively conjugated to the 30‐S and 50‐S subparticles without loss of activity for poly(U)‐dependent polyphenylalanine synthesis. The most reactive thiol groups belong to proteins S1, S12, S18, S21. The binding of mRNA plus tRNA to the 70‐S ribosomes affects the spectroscopic properties of the labels showing that conformational changes are induced by these interactions. Furthermore, the treatment of these complexes by the labelling agent demonstrates that the – SH group belonging to protein S1 is partially protected where‐as other thiol groups located on the 50‐S subparticle become reactive.
Kinetics of Interaction between the Exocellular DD‐Carboxypeptidase‐Transpeptidase from Streptomyces R61 and β‐Lactam Antibiotics
Authors: FRÈRE Jean‐Marie , GHUYSEN Jean‐Marie , IWATSUBO Motohiro
Published: March 2005

The simplest model for the interaction between the exocellular DD‐carboxypeptidase‐trans‐peptidase from Streptomyces R61 and β‐lactam antibiotics involves the three following steps. (a) the formation of a reversible equimolar enzyme · antibiotic complex; (b) the irreversible transformation of this complex into a modified enzyme · antibiotic complex; and (c) the breakdown of this latter complex and the concomitant release of a regenerated enzyme and a modified antibiotic molecule. The dissociation constant for step 1 and the rate constants for steps 2 and 3 were measured with various β‐lactam antibiotics. With an antibiotic such as benzylpenicillin, which behaves as a good ‘substrate’, steps 1 and 2 occur at enzymic velocities, whereas step 3 occurs at a very low velocity and hence is responsible for the low efficiency of the overall process.
Cellulases from Sporocytophaga myxococcoides
Authors: OSMUNDSVAG Kristian , GOKSØYR Jostein
Published: March 2005

Two extracellular cellulases active on carboxymethylcellulose have been isolated from the culture supernatant of Sporocytophaga myxococcoides by a series of gel‐filtration and ion‐exchange chromatography steps. Cellulase II, being present in highest amount, had a molecular weight determined by gel electrophoresis of 52000, pl was 4.75 and a relatively broad pH optimum (5.5–7.5). Cellulase I had a molecular weight of 46000. pl was 7.5 and the pH optimum 6.5–7.5. Both cellulases had a very low carbohydrate content, possibly present as impurities. They had fairly similar amino acid compositions. The specific activity of cellulase I was about 6 times higher than that of cellulase II. Both cellulases acted as endoglucanases. A cell‐associated cellulase, present in amounts corresponding to about 10% of total activity, was partly purified. It showed similarities with cellulase II.
Acetoin Degradation in Bacillus subtilis by Direct Oxidative Cleavage
Authors: LÓPEZ Juan M. , THOMAS Brigitte , REHBEIN Hartmut
Published: March 2005

Acetate and acetaldehyde can be detected as products of the oxidative dissimilation of acetoin in Bacillus subttilis extracts. They arise as the result of the direct cleavage of acetoin without a previous oxidation to diacetyl. This can be deduced from the following observations: (a) no diacetyl was detected in acetoin dissimilation experiments in vitro and (b) methylacetoin, an acetoin analogue which can not be oxidized to the diketone, also undergoes oxidative splitting, yielding acetone and acetate.

The splitting reaction requires thiamine pyrophosphate as a cofactor, suggesting that the oxidative step occurs, as known for similar reactions, by the electron transfer from hydroxyethylthiamine pyrophosphate to a proper acceptor, which in vitro can be replaced by dichlorophenolindophenol.

In vivo the final product of the oxidation of hydroxyethylthiamine pyrophosphate is activated acetate. A mutant which lacks acetoin‐cleaving activity can not reutilize the acetoin accumulated after growth in glucose. This corroborates the actual importance of the cleavage reaction for acetoin dissimilation. The enzyme diacetylmethylcarbinol synthase, thought to be responsible for the formation of diacetylmethylcarbinol from diacetyl, probably is identical to the enzyme catalyzing the cleavage of acetoin.
Preferential Digestion of (A + T)‐Rich Stretches of Yeast Mitochondrial DNA in Isolated Mitochondria
Authors: ZEMAN Leos J. , LUSENA Charles V.
Published: March 2005

Yeast mitochondrial DNA labelled in vitro by incubation of isolated mitochondria with DNA precursors exhibits skewed profiles on isopycnic CsCl gradients. The skew is not due to nuclear DNA nor to single‐stranded mitochondrial DNA in the product labelled in vitro. Simultaneous labelling with [3H]dTTP and [14C]dCTP in vitro indicates a gradient of base composition in the DNA labelled in vitro. Thus, selective degradation of mitochondrial DNA occurs during incubation, converting large molecules having the mean density of mitochondrial DNA into smaller molecules of higher mean density and with higher G:T ratio. Similarly skewed distributions can also be produced by incubation of mitochondrial DNA labelled in vivo with the yeast mitochondrial fraction or with micrococcal endonuclease, an enzyme known to selectively hydrolyse (A + T)‐rich regions of DNA.
Evidence for a Halothane‐Dependent Cyclic Flux of Calcium in Rat‐Liver Mitochondria
Authors: GRIST Elizabeth M. , BAUM Harold
Published: March 2005

The previously reported (Hall et al., Biochem. Soc. Trans. 1973) halothane‐dependent, calcium‐induced loss of respiratory control in rat liver mitochondria is relatively specific to calcium; the effect of strontium ions is much smaller, and comparable additions of potassium salts have no effect on mitochondrial respiration on succinate in the presence of halothane.

The calcium‐dependent loss of respiratory control can be prevented, or reversed, respectively, by the prior or subsequent addition of agents that either chelate extramitochondrial Ca2+ or inhibit calcium accumulation, or that inhibit the efflux of accumulated calcium.

These results suggest that the halothane‐dependent, calcium‐induced loss of respiratory control is due to a cyclic flux of calcium uptake and release.
Adenine Nucleosides in Solution: Circular Dichroism Studies and Base Conformation
Authors: FOLLMANN Hartmut , KUNTZ Irmtrud , ZACHARIAS Wolfgang
Published: March 2005

Adenosine, AMP, S‐adenosylhomocysteine, S‐adenosylmethionine, aristeromycin and 25 other synthetic adenosine analogs modified in the 4′ or 5′ positions show certain groups of different circular dichroism (CD) spectra. Both positive and negative Cotton effects can occur in the long‐wavelength part (250–270 nm) of the spectra. Molar ellipticities [θ] range from – 6000 (in adenosine 5′‐carboxylate) to + 4000 deg. cm2 dmol‐1 (in 5′‐deoxy‐5′iodoadenosine), including some compounds with small, polar 5′‐substituents in which low‐intensity bands are found in signed pairs. Most of these adenosine derivatives that have the same adenine chromophore and a ribofuranose moiety unsubstituted in the 2′ and 3′ positions prefer an anti‐conformation of the adenine base, as evidenced by proton magnetic resonance spectroscopy. In the majority of cases, electronic perturbations of the chromophore or major alterations of the assymmetric sugar residue can be excluded as sources of the CD variations. Therefore a correlation of the long‐wavelength CD bands with the glycosyl torsion angle φCN is suggested, where the gauche, gauche/anti combination which is typical of AMP in the crystal and in solution (φCN∼− 40°, [θ] negative) is one reference point and a region for φCN= 0° ([θ] positive) is assigned to compounds with space‐filling substituents such as S‐adenosylmethionine. Both negative and positive Cotton effects can be associated with the anti conformation range. Within this series, the base conformation of novel nucleoside structures could be predicted from CD measurements. The CD spectrum gives no indication, however, of whether a certain torsion angle is the result of a rigid structure (as in AMP) or the average value of a molecule with high rotational freedom (as in 5′‐deoxyadenosine). The conformations of aristeromycin and 4′‐thioadenosine are discussed in relation to adenosine, and a structure‐determining effect of the 4′ bridge atom is noted.
Influence of Various Factors on the Recognition Specificity of tRNAs by Yeast Valyl‐tRNA Synthetase
Authors: BONNET Jacques , EBEL Jean‐Pierre
Published: March 2005

Using filtration through nitrocellulose membranes we found that complexes between yeast valyl‐tRNA synthetase can easily be detected at low pH and ionic strength with the cognate tRNAVal, but also with several non‐cognate tRNAs (tRNAPhe, tRNATyr, tRNAMet and tRNAAsp). We show here that the amino acid linked to the tRNA has no detectable effect on these interactions.

The influence of various factors on the discrimination by the enzyme between the cognate and the non‐cognate tRNAs has been studied. An increase in pH or ionic strength leads to a decrease in the same ratio of the affinity constants between the enzyme and the cognate as well as the noncognate tRNA. The addition of organic solvents has little effect on these constants either in the cognate or in the non‐cognate systems; the addition of substrates of the aminoacylation reaction has not effect on the ratio between the constants. This similar behaviour suggests that at least part of the specific or non‐specific interactions must be identical.

On the contrary, magnesium between 1 mM and 50 mM increases the specificity of recognition, showing the importance of slight conformational changes in the tRNA molecule to the specificity of interaction.
Molecular Changes Associated with Proteolysis of Bovine Factor V by Thrombin
Authors: KANDALL Carol L. , ROSENBERG Robert , COLMAN Robert W.
Published: March 2005

Native factor V contains two major polypeptide chains, h and I. The molecular weights determined by gel electrophoresis in the presence of sodium dodecylsulfate and dithiothreitol (125000 and 73000) are in reasonable agreement with those obtained by gel filtration in 5 M guanidine‐HCI (125000 and 64000). Exposure of factor V to thrombin results in cleavage of the heavier chain to an altered form with a molecular weight of 87000. The other fragment of this proteolytic reaction appears to be a carbohydrate‐rich piece, which migrates abnormally slowly on gel electrophoresis conducted under denaturing and reducing conditions. Both proteolytic cleavage products remain associated with the light chain during the purification of factor V. The 87000‐Mr fragment is present in samples of factor V which are isolated by immunoprecipitation of blood obtained from a single animal by venous catheter. This finding suggests that some proteolysis may occur in vivo. In contrast, the molecular weight of the light chain is unaltered after thrombin proteolysis of either purified factor V or thrombin‐treated plasma.
The Mannosylation of Dolichol‐Diphosphate Oligosaccharides in Relation to the Formation of Oligosaccharides and Glycoproteins in Pig‐Liver Endoplasmic Reticulum
Authors: OLIVER Gerard J. A. , HARRISON Jeffrey , HEMMING Frank W.
Published: March 2005

In the chain‐lengthening of the oligosaccharide chains of endogenous dolichol‐diphosphate oligosaccharides (Dol‐P‐P‐oligosaccharides) by a pig liver microsomal preparation dolichol‐monophosphate mannose (Dol‐P‐Man) was a more efficient donor of mannose (a maximum of 35% transferred by 5 min) than was GDP‐Man (reaching 16% by 45 min). The effects of an excess of GDP, an excess of GDP‐Man, a lack of Mn2+ and an excess of EDTA showed that the transfer from GDP‐Man was via Dol‐P‐Man. The evidence also indicated the presence of two pools of Dol‐P‐Man one of which was difficult to extract and which was possibly closely associated with the Dol‐P‐P‐oligosaccharides and the appropriate transferase.

After 1 h of incubation transfer of 14C to ‘insoluble polymer’ from GDP‐[14C]Man, Dol‐P‐[14C]Man and Dol‐P‐P‐[14C]oligosaccharides reached approximately 3%, 3% and 13% respectively, of that available. The result of adding excess unlabelled GDP‐Man to an incubation with GDP‐[14C]Man in progress confirmed the sequence GDP‐Man → Dol‐P‐Man → Dol‐P‐P‐oligosaccharide → insoluble polymer.

Solubilisation with sodium dodecyl sulphate of the radioactive ‘insoluble polymer’ followed by gel chromatography showed the presence of radioactive glycoprotein and oligosaccharide when either GDP‐[14C]Man or Dol‐P‐P‐[14C]oligosaccharide was used as donor. The proportion of oligosaccharide formed rose sharply when excess EDTA was present and GDP‐[14C]Man was the donor. Under these conditions the oligosaccharide contained 5–6 units and all of the radioactivity could be released by α‐mannosidase. The glycoprotein was susceptible to proteolysis.
Structure and Synthesis of a Lipid‐Containing Bacteriophage
Authors: SCHÄFER Rolf , HUBER Ursula , FRANKLIN Richard M. , SEELIG Joachim
Published: March 2005

The nucleocapsid proteins of bacteriophage PM2 and the inner lamella of the lipid bilayer, containing most of the phosphatidylethanolamine residues, were selectively cross‐linked in the presence of 0.1–0.5% glutaraldehyde, 5 mM dimethylsuberimidate, or 0.05% toluene 2,4‐diisocyanate. The biological activity (p.f.u.) of PM2 modified by these reagents decreased 106‐fold in all cases. The spike and coat proteins were selectively cross‐linked in the presence of 7.5 mM N,N′‐p‐phenylenedimaleimide. The biological activity of virus modified by this reagent was unaffected. The electron paramagnetic resonance spectra of fatty acid spin labels incorporated into native and chemically modified viral membranes were qualitatively similar but show quantitative differences. Fixation with glutaraldehyde increased the rigidity of the membrane while Triton X‐100 induced a more flexible structure. There was no change in the electron paramagnetic resonance spectrum of virus treated with N,N′‐p‐phenylenedimaleimide, however.
Interaction between Arginase and l‐Ornithine Carbamoyltransferase in Saccharomyces cerevisiae
Authors: PENNINCKX Michel
Published: March 2005

The inhibition of ornithine carbamoyltransferase by arginase in Saccharomyces cerevisiae, which is under the control or arginine and ornithine, involves a regulatory site for arginine on the arginase distinct from its catalytic site. This regulatory site is responsible for the reinforcement effect of arginine on the inhibition of ornithine carbamoyltransferase by arginase. The binding site of ornithine carbamoyltransferase on arginase is also shown by our analysis.
Envelope‐Bound N‐Acetylmuramyl‐l‐alanine Amidase of Escherichia coli K 12
Authors: VAN HEIJENOORT Jean , PARQUET Claudine , FLOURET Bernard , VAN HEIJENOORT Yveline
Published: March 2005

N‐Acetylmuramyl‐l‐alanine amidase activity was detected in Escherichia coli K12 by using N‐acetylmuramyl‐L‐alanyl‐γ‐D‐glutamyl‐(L)‐meso‐[3H] diaminopimelic acid as a radioactive substrate. This activity cleaves the amide bond between the residues of N‐acetylmuramyl acid and L‐alanine. It was readily obtained in a soluble form either by mechanical disruption of the cells or by spheroplast formation. In the latter case the release of most of the activity into the sucrose medium seems to indicate that it is either periplasmic or associated with the outer membrane of the envelope of E. coli K12. The enzyme was purified to near homogeneity. A molecular weight of 39000 was determined by gel filtration and confirmed by polyacrylamide gel electrophoresis. Further characterisation of this N‐acetylmuramyl‐L‐alanine amidase activity was carried out by investigating several of its properties.
Limited Proteolysis of Yeast Phosphofructokinase by Subtilisin
Authors: TAUCHER Martina , KOPPERSCHLÄGER Gerhard , HOFMANN Eberhard
Published: March 2005

Yeast phosphofructokinase having a molecular weight of 750000–800000 (20 S) has been subjected to limited proteolysis by subtilisin and yeast proteases. Two steps of proteolytic degradation could be distinguished: in the first step, which is accompanied by an increase in molecular activity, the subunits α and β (Mr 120000) are converted to α′ and β′ (Mr approximately 90000), and in the second step, accompanied by a decrease in enzyme activity, α′ is converted to α″ (Mr 80000) and two further fragments having Mr 45000 and 35000 become detectable. In the course of this conversion the sedimentation value of the undissociated enzyme drops from 20 S to about 17 S. The two substrates fructose 6‐phosphate and ATP exhibit characteristic protective effects on enzyme activity and on subunit degradation. Whereas the first step is not strongly influenced by the substrates, fructose, 6‐phosphate inhibits significantly the degradation of α′ and β′, whereas ATP prevents only degradation of β′. When in presence of ATP α′ is degraded to α″, the quaternary structure of the 17‐S enzyme is no longer stable and a dissociation of this molecule occurs to a 12‐S form which is enzymically active and ATP‐sensitive and in which the ratio of α″ to β′ is one‐to‐one.
Transport of Pyrimidine Nucleosides in Cells of Escherichia coli K 12
Authors: MYGIND Bente , MUNCH‐PETERSEN Agnete
Published: March 2005

The transport of pyrimidine nucleosides into cells of Escherichia coli has been investigated in mutant strains which cannot metabolize these nucleosides. Such cells transport and concentrate pyrimidine nucleosides several hundred‐fold.

The transport is inhibited by energy poisons and by sulfhydryl reagents.

Pyrimidine nucleosides compete mutually for transport. Adenosine is also a strong competitor while guanosine and inosine are weak competitors.

The rate of pyrimidine nucleoside transport is shown to be under control of the cytR and deoR gene products, which are also known to regulate the synthesis of nucleoside‐catabolizing enzymes. The transport system is repressed by growth on glucose, as is the synthesis of the enzymes.
Immunochemical Studies on Tyrosinase Induction in Neurospora
Authors: KATAN Talma , GALUN Esra , ARNON Ruth
Published: March 2005

An immunoassay for tyrosinase, using the modified bacteriophage technique, was developed: Tyrosinase of Neurospora was conjugated to bacteriophage T4 using glutaraldehyde as a cross‐linking agent. The conjugated phage that survived the coupling process could be inactivated by antiserum raised in rabbits against pure tyrosinase, but not by normal serum. This inactivation was specifically inhibited by pure Neurospora tyrosinase, and the degree of inhibition was proportional to the concentration of tyrosinase within the range of 30 – 150 ng/ml. Crude mycelial extracts possessing tyrosinase activity could similarly inhibit the inactivation of the conjugated phage by the antiserum. To evaluate the tyrosinase content of crude extracts, their inhibitory capacity was compared to that of known amounts of pure tyrosinase, and the amounts thus calculated agreed with those predicted from an enzymatic assay.

The tyrosinase‐bacteriophage immunoassay was used for the quantitation of tyrosinase‐antigen in crude extracts of Neurospora cultures that had been induced to form tyrosinase by the addition of ethionine. Enzymatic activity appeared after a lag of several hours, increased for 2 – 3 days and then declined. Immunological assays of these cultures showed: (a) serologically reactive protein started to accumulate upon culture starvation and was evident during the lag period; (b) specific activity (units per mg antigen) was constant throughout induction; (c) at the phase of decrease in mycelial enzyme content, increasing amounts of serologically reactive protein were detected in the medium, indicating that some enzyme was eventually excreted. These results show that the lag is not a qualitatively distinct period, and support the previously forwarded notion that tyrosinase is synthesized de novo upon induction.