bluuebunny/crossref_metadata_2025 huggingface dataset:
Light‐Induced Increase in the Activity of Maize Plastid DNA‐Dependent RNA Polymerase
Authors: APEL Klaus , BOGORAD Lawrence
Published: March 2005

Illumination of dark‐grown maize plants induces a rapid rise in DNA‐dependent RNA polymerase activity in plastids. Within 16 h of illumination the enzyme activity in isolated plastids increases 3–4 fold as compared to other dark‐grown plants illuminated for only 2 h. This change in activity is unaccompanied by either a quantitative rise in the amount of chloroplast RNA polymerase or qualitative changes in the purified enzyme. It is suggested that other factors may be present which interact either with the enzyme or the DNA template and that these factors are responsible for the light‐induced enhancement of the RNA polymerase activity in plastids.
Crosslinking with Bifunctional Reagents as a Means for Studying the Symmetry of Oligomeric Proteins
Authors: HAJDU János , BARTHA Ferenc , FRIEDRICH Peter
Published: March 2005

A method based upon the principle that unlike domains of bonding are reflected in different re‐activities and distribution of residues that can be crosslinked, has been elaborated for the determination of symmetry of oligomeric proteins.

The derivation of theoretical curves for the prediction of crosslinking patterns of tetramers produced by reaction with a bifunctional reagent and subsequent sodium‐dodecylsulphate‐gel electrophoretic analysis is presented. Based upon the theory the symmetry properties of a tetramer, to the extent whether it is an isologous or heterologous associationn, can be deduced by a simple calculation.

Crosslinking patterns obtained with rabbit muscle aldolase and pig muscle lactate dehydrogenase after treatment with a series of diimidoesters of increasing chain length are evaluated and shown to be consistent with the expectations for isologous tetramers. From the patterns obtained with the various reagents the distances between lysyl residues located nearest to each other in different subunits in the two proteins could also be determined.
The Importance of Arginine Residues in the Catalytic and Regulatory Functions of Bovine‐Liver Glutamate Dehydrogenase
Authors: PAL Pranab K , COLMAN Roberta F.
Published: March 2005

Bovine liver glutamate dehydrogenase reacts rapidly with 2,3‐butanedione to yield modified enzyme with 29% of its original maximum activity, but no change in its Michaelis constants for substrates and coenzymes. No significant reduction in the inactivation rate is produced by the addition of the allosteric activator ADP or inhibitor GTP, while partial protection against inactivation is provided by the coenzyme NAD+ or substrate 2‐oxoglutarate when added separately. The most marked decrease in the rate of inactivation (about 10‐fold) is provided by the combined addition of NAD+ and 2‐oxoglutarate, suggesting that modification takes place in the region of the active site. Reaction with 2,3‐butanedione also results in loss of the ability of the enzyme to be activated by ADP. Addition of ADP (but not NAD+, 2‐oxoglutarate or GTP) to the incubation mixture protects markedly against the loss of activatability by ADP. It is concluded that 2,3‐butanedione produces two distinguishable effects on glutamate dehydrogenase: a relatively specific modification of the regulatory ADP site and a distinct modification in the active center. Reaction of two arginyl residues per peptide chain appears to be responsible for disruption of the ADP activation property of the enzyme, while alteration of a maximum of five arginyl residues can be related to the reduction of maximum catalytic activity. Electrostatic interactions between the positively charged arginine groups and the negatively charged substrate, coenzyme and allosteric purine nucleotide may be important for the normal function of glutamate dehydrogenase.
Purification of Six Neurotoxins from the Venom of Dendroaspis viridis
Authors: BECHIS Guy , GRANIER Claude , VAN RIETSCHOTEN Jurphaas , JOVER Emmanuel , ROCHAT Hervé , MIRANDA François
Published: March 2005

Six neurotoxins were purified from Dendroaspis viridis venom using gel filtration and equilibrium chromatography. The amino acid sequences of two of these neurotoxins (72 and 73 residues, live disulphide bridges) have been determined using almost exclusively automated Edman degradation. These two sequences are very similar: the only difference lies in the presence of one extra glycine at the C‐terminal end of one of them. There is a good homology with the sequences of toxins now isolated from other Elapidae and Hydrophidae venoms
An Approach to the Antigenic Structure of Arginine Kinase and Creatine Kinase
Authors: BENYAMIN Yves , GUILLOU Yvonne , DESORMEAU‐BEDOT Jean‐Pierre , ROBIN Yvonne
Published: March 2005

The antigenic structure of arginine kinase and creatine kinase has been approached using chemical modifications and enzymatic cleavage.

Mild performic oxidation that, with a restricted number of oxidized amino acid residues, results for both enzymes in a severe decrease of the helical structure and in a large increase of the protein‐solvent interactions, affects differently their antigenic reactivity: when compared with antisera to the homologous native enzymes, arginine kinase and its oxidized derivative cross‐react fully, while creatine kinase and its oxidized derivative cross‐react only about 30%. The persistence of the antigenic reactivity of arginine kinase through drastic structural alterations is confirmed by the high inhibitory capacity (about 80%) of crude tryptic hydrolyzates towards the combination of arginine kinase with its specific antibodies. Tryptic peptides of creatine kinase, obtained in the same conditions, inhibit weakly (about 12 %) the homologous antigen‐antibody interaction.

The participation of the lysines in the antigenicity of arginine kinase and creatine kinase is suggested by the enhanced inhibitory capacity of the tryptic hydrolyzates when the cleavage is restricted to the arginyl peptide bounds, and was verified for arginine kinase through assays with lysine‐modified derivatives
Torpedo marmorata Acetylcholinesterase; a Comparison with the Electrophorus electricus Enzyme
Authors: RIEGER François , BON Suzanne , MASSOULIÉ Jean , CARTAUD Jean , Brigitte PICARD  , BENDA Philippe
Published: March 2005

Electron microscopy, sequential degradation by hydrolytic enzymes and the physical‐chemical properties of the molecular forms of Torpedo acetylcholinesterase indicate that these molecules are structurally related to each other in the same way as the molecular forms of Electrophorus acetylcholinesterase: all are derived from a complex structure in which three tetrameric groups of subunits are associated with a rod‐like ‘tail’. In aged preparations the catalytic subunits are split into fragments in a manner similar to those of Electrophorus acetylcholinesterase. Immunological cross‐reaction between both enzymes demonstrates the occurence of common antigenic sites. The enzymes from the two sources, however, are different in their molecular weights and susceptibility to hydrolytic enzymes. Also, Torpedo acetylcholinesterase does not precipitate with either isologous or heterologous antibodies.
Molecular Forms of Electrophorus Acetylcholinesterase
Authors: BON Suzanne , HUET Marianne , LEMONNIER Marguerite , RIEGER. François , MASSOULIÉ Jean
Published: March 2005

Molecular weights for the series of six Electrophorus acetylcholinesterase forms have been determined either by the sedimentation‐diffusion equilibrium method or, particularly in the case of the very scarce G′ and G″ forms, from their Stokes radius and sedimentation coefficient values.

Both methods are in excellent agreement. The results provide good evidence for the model previously proposed, G″, G′ and G containing one, two and four subunits, whereas A, C and D possess, in addition to respectively one, two and three tetrameric sets of such subunits, a structural element, the tail. Although the amino acid composition of ‘tailed’ and globular forms did not reveal any significant feature of this element, its mass, about 100000 daltons, could be deduced from a comparison of molecular weights for the two classes of acetylcholinesterase forms. This value is in close agreement with electron microscopic data. The tail is thought to consist of three 30000‐dalton strands.
Efficiency of Oxidative Phosphorylation and Energy Dissipation by H+Ion Recycling in Rat‐Liver Mitochondria Metabolizing Pyruvate
Authors: STUCKI Jörg W.
Published: March 2005

A method was developed for the calculation of metabolic fluxes through individual enzymatic reactions of pyruvate metabolism including the citric acid cycle in rat liver mitochondria incubated at metabolic states between state 4 and state 3. This method is based on the measurement of the specific radioactivities of the products formed from [2‐14C]pyruvate. With this procedure the energy balance of mitochondria incubated in the presence of [2‐14C]pyruvate, ATP, bicarbonate and phosphate at different ATP/ADP ratios in the medium was calculated. The ATP/ADP ratios were maintained at a steady state with creatine kinase plus creatine as a phosphoroyl acceptor. The calculations revealed that by adding increasing concentrations of creatine up to 20 mM the energy dissipated by the mitochondria decreased but showed a local maximum at 13 mM creatine. Omission of bicarbonate from the medium led to a shift of this maximum. When energy dissipation was minimal the overall P/O ratio was maximal. The amount of energy dissipated was paralleled by the magnitude of the pH gradient across the inner membrane. From these results it was concluded that the recycling of H+ions which consists of a passive leakage of H+ions into the matrix and an active extrusion of these ions out of this compartment, is an important energy dissipating process. The H+ion recycling is thus one of the processes which give rise to the state 4 respiration in mitochondria.
Kinetic Investigation of the Staphylococcal Protease‐Catalyzed Hydrolysis of Synthetic Substrates
Authors: HOUMARD Jean
Published: March 2005

In investigating the staphylococcal protease‐catalyzed hydrolysis of N‐tert‐butoxycarbonyl‐l‐glutamate α‐phenyl ester, N‐benzyloxycarbonyl‐l‐glutamate α‐phenyl ester and N‐benzyloxycarbonyl‐l‐glutamate α‐p‐nitroanilide, we obtained kinetic evidence consistent with the formation of an acyl‐enzyme intermediate. We found that addition of a nucleophile, such as methanol, led to the partition of the common acyl‐enzyme intermediate between water and the alcohol. With N‐benzyloxycarbonyl‐l‐glutamate α‐phenyl ester, a specific ester substrate, deacylation was shown to be the rate‐limiting step.

By studying the kcat/Km ratio of these hydrolyses as a function of pH, we have shown that two ionizable groups on the enzyme are essential to the catalytic process. One of these groups has a pK of 6.58 and the other, a pK of 8.25. The assignment of these pK values is discussed in connection with the known features of the serine proteinase reaction mechanism.

In addition, monovalent anions were shown to inhibit staphylococcal protease hydrolyses. They seem to compete with the negative charge of the substrate, thus inhibiting its binding on the enzyme molecule. Finally we compared the kinetic parameters obtained with five proteases isolated from different strains of Staphylococcus aureus.
p‐Nitroanilides of 3‐Carboxypropionyl‐peptides
Authors: KASAFÍREK Evžen , FRIČ Pǐemysl , SLABÝ Jan , MALIŠ František
Published: March 2005

Fourteen 3‐carboxypropionyl‐tripeptide p‐nitroanilides of the general formula 3‐carboxypropionyl‐alanyl‐alanyl‐Y p‐nitroanilide (Y = glycine, norvaline, S‐methylcysteine, valine, norleucine. S‐ethylcysteine, methionine, leucine, isoleucine, phenylalanine, tyrosine, S‐benzylcysteine, Cx‐phenylglycine, and proline) were synthesized and their cleavage by elastase, trypsin, and chymotrypsin (Km, kcat, and kcat/Km) was determined.

The significance of amino acid residues in the position of Y was evaluated firstly with respect to their structure (topographically), and secondly with respect to their free energy (thermodynamically). The alanine residue substrate was cleaved best by elastase, the phenylalanine substrate by chymotrypsin. Trypsin cleaved two substrates only, that is those containing a phenylalanine and a tyrosine residue.

The optimum length of the elastolytic substrates was studied in a series of N‐3‐carboxypropionyl‐(Ala)n‐p‐nitroanilides (n= 1, 2, 3, 4, 5), N‐3‐carboxypropionyl‐(Gly)n‐p‐nitroanilides (n= 1, 2, 3), and in p‐nitroanilides of fatty acids with two to seven carbon atoms. Elastase cleaved tri, tetra, and pentapeptides of alanine.

p‐Nitroanilides of the glycine series, as well as p‐nitroanilides of fatty acids were riot cleaved. 3‐Carboxypropionyl‐tetra‐alanine‐p‐nitroanilide was the most suitable substrate so far found for elastase cleavage; it is not cleaved by trypsin nor chymotrypsin. The optimum distance between Y and the terminal anionic carboxyl residue was found to be 1.8 nm in clastolytic substrates.
Some Properties of Phospholipid Exchange Proteins from Rat Liver
Authors: LUMB Roger H. , KLOOSTERMAN Aat D. , WIRTZ Karel W. A. , VAN DEENEN Laurens L. M.
Published: March 2005

The phospholipid exchange proteins of rat liver that catalyze the transfer of phosphatidylcholine and phosphatidylinositol from rat liver microsomes to liposomes, have been purified and characterized.

Two proteins were detected with dual specificities catalyzing the transfer of both phosphatidylinositol and phosphatidylcholine. Both proteins showed a strong preference for phosphatidylinositol transferring 8 to 9 times as much of the microsomal phosphatidylinositol pool as the microsomal phosphatidylcholine pool. The two proteins had iso‐electric points of 5.1 and 5.3 and were purified 300‐fold and 500‐fold, respectively.

A protein that catalyzed specifically the transfer of phosphatidylcholine, was purified 7000‐fold. This protein had an iso‐electric point of 8.4 and a molecular weight of approximately 16000 calculated from Sephadex G‐50 chromatography and sodium dodecylsulfate‐polyacrylamide gel electrophoresis; the amino acid composition was determined. An antiserum against this protein was raised in rabbits. Treatment of a rat liver supernatant fraction with the antiserum immunoglobulin fraction demonstrated that 60 % of the phosphatidylcholine transfer activity is due to this protein.
Daunorubicin Inhibition of DNA‐Dependent RNA Polymerases from Ehrlich Ascites Tumor Cells
Authors: BARTHELEMY‐CLAVEY Véronique , MOLINIER Catherine , AUBEL‐SADRON Geneviève , MARAL René
Published: March 2005

Two different forms of DNA‐dependent RNA polymerase have been solubilized and purified from nuclei of Ehrlich ascites tumor cells. The purification procedure involves ammonium sulfate precipitation and gel filtration on Sephadex G‐25. The separation of A and B activities is achieved by chromatography on DEAE‐cellulose. Nuclei arc prepared from cells, sensitive or resistant to daunorubicin. RNA polymerases A and B have an absolute requirement of divalent cations for activity. Native DNAs are better templates than heat‐denatured DNAs for RNA polymerase A. On the contrary heat‐denatured DNA is more transcribed than the native one by RNA polymerase B. The low level of transcription of total and nucleolar ascites DNAs is due to the DNA, the same results being obtained with ascites and calf thymus RNA polymerases A and B.

The inhibitory action of daunorubicin on RNA polyrnerases A and B from Ehrlich ascites tumor cells has been studied in vitro. The same results are obtained with enzymes extracted from sensitive or resistant cells. Daunorubicin does not inhibit the binding of the RNA polymerases to the DNA template, but prevents the transformation of the DNA daunorubicin RNA‐polymerase unstable complex into the highly stable one. This inactive ternary complex has a dissociation rate faster than the stable complex formed without daunorubicin. The size of the RNA synthesized in the presence or absence of daunorubicin is the same.
Comparison between Strontium and Calcium Uptake by the Fragmented Sarcoplasmic Reticulum
Authors: MERMIER Pierre , HASSELBACH Wilhelm
Published: March 2005

The ATP‐supported uptake of strontium by the fragmented sarcoplasmic reticulum is monophasic and proceeds more rapidly than the fast uptake of calcium. Strontium uptake is not activated by P4. The accumulation of strontium is nearly proportional to the external strontium concentration even in the millimolar range. Internal and external strontium quickly equilibrate. One mole of strontium is stored for every mole of ATP split by the Sr2+ ‐activated ATPase. In the absence of oxalate most of the strontium is taken up with a transport ratio of one. On the opposite, the transport ratio of calcium decreases immediately, especially when ADP is not instantaneously phosphorylated to ATP. In this case, energy, conversion is uncoupled more effectively by the simultaneous action of ADP and free internal calcium, resulting in the interruption of the fast uptake. After depletion of ATP most of the stored strontium is released and the remaining fraction appears to be not exchangeable. Strontium activates the slow uptake of calcium, but reduces the amplitude of the fast uptake. The calcium induced release of strontium, and vice versa, is partial and transient. The strontium activated ATPase does not transport calcium at low ionic calcium concentrations.
An Improved Procedure for Automated Edman Degradation Used for Determination of the N‐Terminal Amino Acid Sequence of Human Transcobalamin I and Human Intrinsic Factor
Authors: THOMSEN Johannes , BUCHER Ditlef , BRUNFELDT Kay , NEXØ Ebba , OLESEN Henrik
Published: March 2005

An improved procedure for automated Edman degradation is presented. Three programs are described, one with double cleavage and two with single cleavage. The programs presented are characterized by a reversed delivery scheme for buffer, and phenyl isothiocyanate, and by reduced cleavage times. The modified procedures applied on automated Edman degradation of the vitamin B12‐binding proteins human transcobalamin I and human intrinsic factor, containing approximately 390 and 350 amino residues respectively, gave the following N‐terminal amino acid sequences:

Human transcobalamin I

Glu‐Ile‐Cys‐Glu‐Val‐Ser‐Glu‐Glu‐Asn‐Tyr‐Ile‐Arg‐Leu‐Lys‐Pro‐Leu‐Leu‐Asn‐Thr‐Met‐Ile‐Gln‐Ser‐Asn‐Tyr‐Asn‐?‐Gly‐

Human intrinsic factor

Ser‐Thr‐Gln‐Thr‐Gln‐Ser‐Ser‐Cys‐Ser‐Val‐Pro‐Ser‐Ala‐Gln‐Glu‐Pro‐Leu‐Val‐Asn‐Gly‐Ile‐Gln‐?‐Leu‐Met‐Glu‐Thr‐

The background accumulation seems to be related not only to the length of the polypeptide chain being degraded, but also to the content of serine (and possibly threonine). A possible N O acyl shift during the cleavage is a tentative explanation. The programs here represented lead to a significant reduction in background compared to conventional programs and allowed considerable prolongation of the degradations.
Epoxides Derived from Various Polycyclic Hydrocarbons as Substrates of Homogeneous and Microsome‐Bound Epoxide Hydratase
Authors: BENTLEY Philip , SCHMASSMANN Hansueli , SIMS Peter , OESCH Franz
Published: March 2005

A general assay for epoxide hydratase using epoxides derived from polycyclic aromatic hydrocarbons as substrates is described. Addition of dimethylsulphoxide to the incubation mixture after incubation allows unreacted epoxide and its phenolic by‐product to be extracted into light petroleum whilst the product dihydrodiol remained in the aqueous phase. The product was then extracted into ethyl acetate and estimated radiochemically. This assay gave low extraction blanks (0.8–3.8%) when six K‐region epoxides of polycyclic hydrocarbons were used, with high recoveries of the corresponding dihydrodiol in the ethyl acetate phase (65–89%). Radiochromatograms demonstrated that all the radioactivity in the ethyl acetate extracts of active incubations above that of boiled enzyme blanks was confined to a single band that always cochromatographed with the authentic trans‐dihydrodiol.

Using this assay, the kinetic parameters of six K‐region epoxides were estimated. In all cases the apparent Km was low (2–5.9 μM). This is about 100‐fold lower than the known apparent Km of epoxide hydratase for styrene oxide, an alkene oxide that is widely used as a substrate for epoxide hydratase. The rate of hydration varied with the substrate. Thus the maximum velocity for hydration of phenanthrene 9,10‐oxide > 7‐methylbenz[a]anthracene 5,6‐oxide:∼ benz[a]anthracene 5,6‐oxide ∼ benzo[a]pyrene 4,5‐oxide > 3‐methylcholanthrene 11,12‐oxide > dibenz[a,h] anthracene 5,6‐oxide. This relationship between the individual epoxides was found in microsomal fractions from both rat skin and rat liver, although the activity was always much lower in skin microsomes. All six arene oxides derived from polycyclic hydrocarbons were substrates for the homogeneous epoxide hydratase that was isolated from rat liver microsomal fractions using styrene oxide, an alkene oxide, as substrate to follow the purification.
The Action Pattern of Amylomaltase from Escherichia coli
Authors: PALMER T. Norman , RYMAN Brenda E. , WHELAN W. J.
Published: March 2005

Amylomaltase, the inducible 4‐α‐glucanotransferase of Escherichia coli strain ML, has been purified to homogeneity. Its specific activity with a commercial maltose substrate was 500 mkat/kg protein (30 μmol glucose formed min−1 mg protein−1). The purified enzyme, dependent on buffer concentration, exists in interconvertible low‐molecular‐weight (apparent molecular weight 71000) and high‐molecular‐weight (apparent molecular weight 370000) forms.

The specificity of amylomaltase has been redefined. Hitherto, the enzyme was thought to be a glucosyltransferase, catalysing the transfer of single glucosyl units, and maltose has been regarded as its most important substrate. Amylomaltase is now shown to exhibit both glucosyl‐transfer and 4‐α‐glucanosyl‐transfer specificity. 4‐α‐Glucanosyl chains containing up to at least nine glucosyl units can be transferred. However, it is concluded that the transfer reaction by which amylomaltase action was originally expressed, does not take place, i.e.

Maltose + maltose? Maltotriose + glucose

and that maltose has a restricted role as a substrate. This may be due to the inability of maltose to function as a donor substrate, serving only as an acceptor substrate. It is confirmed that when a maltodextrin serves as a donor, that portion of the molecule transfered by the enzyme is that containing the nonreducing‐end‐group.

Enzyme action on chromatographically pure maltose is characterized by a lag phase in the time course of glucose release. The lag phase is overcome by addition of ‘priming’ (catalytic) concentrations of maltotriose or higher maltodextrins. An autocatalytic reaction mechanism involving the generation of primer molecules is proposed to explain the action of the enzyme on maltose.

The redefined action pattern of amylomaltase is consistent with the redefined role of the enzyme in the utilization of exogenous and endogenous 1,4‐α‐glucans by E. coli.
Kinetics of Dimerization of the Variable Fragment of the Bence‐Jones Protein Au
Authors: MAEDA Hiroshi , ENGEL Juergen , SCHRAMM Hans J.
Published: March 2005

The dimerization of the variable fragment of the Bence‐Jones protein Au was examined in phosphate buffers at pH 6.8 ‐ 6.9 and ionic strength of 0.1 M or 0.2 M at 200 C. The dimerization constant was about 1 × 105 M−1. The reaction enthalpy was positive and the process was entropy driven. The association and dissociation rate constants were 9×106 M−1 s−1 and 1.5×102 s−1 respectively. Temperature‐jump experiments exhibited the presence of two isomers of the dinier, which are present at equilibrium in a ratio of about 1: 1. Isomerization occurred wich a half‐life of about 0.1 s.
Protein Synthesis in Toluene‐Treated Escherichia coli Exclusive Synthesis of Membrane Proteins
Authors: HALEGOUA Simon , HIRASHIMA Akikazu , SEKIZAWA Jun , INOUYE Masayori
Published: March 2005

Toluene‐treated Escherichia coli K12 cells were shown to be able to synthesize proteins. Protein synthesis in cells treated with toluene in the presence of Mg2+ and at 0 oC, was completely dependent upon the external addition of an energy source, ATP, and was sensitive to antibiotics which are known to inhibit protein synthesis. The toluene‐treated cells were shown to exclusively synthesize membrane proteins.
Snake Venom Toxins
Authors: STRYDOM Daniel J.
Published: March 2005

Twelve low‐molecular‐weight proteins, of which eleven have subcutaneous LD50 values of less than 40 μg/g mouse, were purified from Dendroaspis polylepis polvlepis venom. Ion‐exchange chromatography on Amberlite CG‐50 and ion‐exchange chromatography on carboxymethylcellulose and/or phosphocellulose was used for the purification. The amino‐terminal sequences of these proteins were determined and used to indicate that five groups of low‐molecular‐weight polypeptides are to be found in black mamba venom. Proteins from two of these groups which have low toxicity individually, when used together show synergism, in that their toxicity in combination is greater than the sum of their individual toxicities.
The Binding of Specific Ligands to Adenosine‐Triphosphate Phosphoribosyltransferase
Authors: DALL‐LARSEN Torbjørn , KLUNGSØYR Leiv
Published: March 2005

Ligand binding by adenosine‐triphosphate phosphoribosyltransferase was studied by different methods. 200000 daltons of enzyme bound approximately 3 molecules of histidine cooperatively with a Hill plot slope of 1.23 (half‐maximal binding at 520 μM). AMP increased the affinity of the enzyme for histidine (half‐maximal binding at 80 μM). In the presence of AMP the binding of histidine was strongly cooperative with a Hill plot slope of 2.3. The transferase binds a little more than 3 molecules of AMP per hexamer of enzyme with a dissociation constant of the transferase · AMP complex of approximately 25 μM. ATP was able to displace radioactive AMP from the enzyme only at a concentration ratio of 25 in favour of ATP. The transferase bound 3 molecules of ATP per 200000 daltons to an inhomogeneous population of sites, or by a mechanism of negative cooperativity. The binding of phosphoribosyladenosine triphosphate took place preferably at 1–2 sites per hexamer of enzyme, depending on several factors including the magnesium concentration.
Ferredoxin from a Red Alga, Porphyra umbilicalis
Authors: ANDREW Peter W. , ROGERS Lyndon J. , BOULTER Donald , HASLETT Barry G.
Published: March 2005

A plant‐algal type ferredoxin was isolated from the red alga, Porphyra umbilicalis. In its oxidised form the ferredoxin had absorption maxima at 277, (281), 323, 420 and 462 nm. Two atoms each of non‐haem iron and labile sulphur were present per molecule protein. The midpoint potential of the protein was 400 mV and it effectively mediated electron transport in the NADP‐photoreduction system of barley. The amino acid composition of Porphyra umbilicalis ferredoxin was determined as (Lyso4 His2, Argi, Asx10, Thr8, Ser7 Glx16–17, Pro3, G1y7, A1a8, Cys5, Val6, Meti, Ile5, Leu8, Tyr5, Phe2). The minimum molecular weight of approximately 11000 was confirmed by sedimentationequilibrium studies in the analytical ultracentrifuge. Approaching half of the total amino acid sequence was determined by means of an automatic sequencer.
Nuclear‐Magnetic‐Relaxation Studies of the Interaction of Inhibitor with the Threonine‐Sensitive Aspartokinase of Escherichia coli
Authors: TILAK Anup , WRIGHT Keith , DAMLE Suresh , TAKAHASHI Mark
Published: March 2005

The nature of the feedback inhibition of the bifunctional enzyme, aspartokinase I‐homoserine dehydrogenase I of Escherichia coli was studied using 13C nuclear magnetic resonance (NMR). Since aspartokinase is activated by Mn(II), the interaction of the inhibitor l‐threonine (specifically enriched to 9O%13C in the carboxyl carbon) with the metal‐enzyme complex was studied. Spinlattice (T1) and spin‐spin (T2) relaxation times were determined by the partially relaxed Fourier transform method and line‐width measurements respectively at 20 MHz. The pronounced broadening of the dl‐threonine carboxyl peak in the presence of the Mn(II)‐enzyme complex indicates that an l‐threonine binding site is close to the metal binding site of the kinase active site. The nonidentity of (T1) * and (T2)*m indicates that conditions of fast exchange prevail. The (T1)* (T2)*m ratio was used to estimate a correlation time of 2.0 ns for the dipolar interaction at 25 °C. An estimate for the distance between Mn(II) and the threonine carboxyl carbon of 4.4 Å (0.44 nm) was obtained. This 13C NMR study has thus located one of the two classes of threonine regulatory sites which exist per subunit; the threonine site identified here is at the aspartokinase active site, adjacent to the catalytic metal site.
Substrate Specificity of the Ultraviolet‐Endonuclease from Micrococcus luteus
Authors: TOMILIN Nikolai V. , PAVELTCHUK Elena B. , MOSEVITSKAYA Tatyana V.
Published: March 2005

The ultraviolet‐endonuclease isolated from Micrococcus Iuteus, specific for pyrimidine dimers, is able to attack not only ultraviolet‐irradiated DNA (leading to 3′OH‐5′PO4 single‐strand breaks) but also superhelical covalently‐closed circular DNA of phage λ damaged by heating at 70°C, pH 5.93.

The number of endonuclease‐sensitive defects in the DNA corresponds to the number of alkailabile bonds (apurinic sites) induced by heating. Competition between ultraviolet‐induced lesions and apurinic, sites for ultraviolet‐endonuclease is demonstrated; the affinity of the enzyme for pyrimidine dimers is about three times that for apurinic sites. Both activities of the ultraviolet‐endonuclease are inactivated at 50 °C at the same rate.

The ultraviolet‐endonuclease is able to reduce the infectious activity of depurinated λ DNA towards Ca2+‐treated uvr+ and uvr A Escherichia coli cells.
The Transient‐State Kinetics of Two‐Substrate Enzyme Systems Operating by an Ordered Ternary‐Complex Mechanism
Authors: PETTERSSON Gösta
Published: March 2005

Transient‐state kinetic equations, applicable to single‐turnover experiments with limiting amounts of substrate and catalytic‐suicide experiments with substrates in excess to enzyme, have been derived for two‐substrate enzyme reactions occurring by an ordered ternary‐complex mechanism. The relative magnitude and dependence on substrate concentrations of rates and amplitudes for the exponential transients corresponding to this mechanism are quantitatively defined and related to steady‐state kinetic parameters for the reaction mechanism. It is shown that ordered ternary‐complex reactions under standard assay conditions may exhibit a biphasic transient rate behaviour, and the precise conditions under which two transients can be detected are described and discussed.
Primary Structure of α‐Factor Peptides from Saccharomyces cerevisiae
Authors: STÖTZLER Dieter , KILTZ Hans‐Hermann , DUNTZE Wolfgang
Published: March 2005

The amino acid sequences of the mating hormones produced by α‐mating‐type cells of the yeast Saccharomyces cerevisiae (α factor peptides α1, α2, α3 and α4) have been determined. By cleavage of the peptides with thermolysin and pepsin and structural analysis of the fragments the sequence H2N‐Trp‐His‐Trp‐Leu‐Gln‐Leu‐Lys‐Pro‐Gly‐Gln‐Pro‐Met‐Tyr‐COOH could be assigned to α1. α2 lacks the N‐terminal tryptophan residue. α3 and α4 represent the methionine sulfoxides of α1 and α2 respectively.
Essential Role of Monovalent Cations in the Firm Binding of Pyridoxal 5′‐Phosphate to Tryptophanase and β‐Tyrosinase
Authors: TORAYA Tetsuo , NIHIRA Takuya , FUKUI Saburo
Published: March 2005

Roles of inorganic monovalent cations in tryptophanase of Escherichia coli B and β‐tyrosinase of Escherichia intermedia were studied. Like tryptophanase, β‐tyrosinase absolutely required certain monovalent cations for catalytic activity. The cation selectively of β‐tyrosinase was quite similar to that of tryptophanase; K+, NH4+ and Rb+ were the most effective as cofactor, and Li+ and Na+ were essentially inactive. Cs+ was partially active in the β‐tyrosinase reaction but essentially inactive in the tryptophanase reaction. A close relationship was observed between their cofactor activities and crystal ionic radii. It was established in β‐tyrosinase as well as in tryptophanase that K+ is absolutely required for conversion of the apoenzyme‐pyridoxal 5′‐phosphate complex into the functional form. Effects of monovalent cations and substrates on formation and resolution of the holoenzymes of tryptophanase and β‐tyrosinase were investigated by a Sephadex G‐25 gel filtration method. In both cases, highly active monovalent cations facilitated the formation of the tight enzyme‐pyridoxal 5′‐phosphate complex (holoenzyme) and retarded its resolution, and the reverse was the case for the inactive cations. Upon Sephadex G‐25 gel filtration using triethanolamine‐HCI buffer, the dissociation of pyridoxal 5′‐phosphate from holotryptophanase proceeded according to the first‐order kinetics. Affinities of Na+‐tryptophanase for both coenzyme and substrate were found to be much lower than those of the K+‐enzyme. Thus, it can be concluded that certain monovalent cations play an essential role in formation and maintenance of the firm binding of pyridoxal 5′‐phosphate to the apoenzyme leading to the catalytically active holoenzyme.
Structure Determination of a Modified Nucleoside Isolated from Escherichia coli Transfer Ribonucleic Acid
Authors: KASAI Hiroshi , MURAO Katsutoshi , NISHIMURA Susumu , LIEHR Joachim G. , CRAIN Pamela F. , McCLOSKEY James A.
Published: March 2005

A modified nucleoside, N+, was isolated from Escherichia coii tRNA, and its structure was, determined to be N‐[N‐[(9‐β‐D‐ribofuranosylpurin‐6‐yl)carbamoyl]threonyl]‐2‐amido‐2‐hydroxymethylpropane‐1,3‐diol. The structure determination of N+ was based on its ultraviolet absorption, nuclear magnetic resonance spectrum, high and low‐resolution mass spectra, and its properties following chemical degradation. The ultraviolet absorption spectra of N+ closely resembled the spectra of N‐[N‐[(9‐β‐d‐ribofuranosylpurin‐6‐yl)carbamoyl]threonyl]amide, suggesting that N+ is a derivative of the latter. Alkaline and acid hydrolysis resulted in the formation of N‐[N‐(9‐β‐d‐ribofuranosylpurin‐6‐yl)carbamoyl)threonine. Low and high‐resolution mass spectrometry of the trimethylsilyl derivative of N+ indicated the presence of an aminobutanetriol moiety, NH2C4H6(OH)3, attached to the threonine through an amide linkage. The aminobutanetriol structure was determined to be NH2‐C‐(CH2OH)3 by analysis of the nuclear magnetic resonance spectrum of N+, and by direct comparison of the mass spectrum of the side chain with that of an authentic compound, following acid hydrolysis of N+ and gas chromatography/mass spectrometry. It is very likely that the aminobutanetriol moiety is derived from tris(hydroxymethyl)aminomethane, namely Tris buffer, that was added in the medium to suspend E. coli cells. Covalent bonding between tris(hydroxymethyl)‐aminomethane and the threonyl moiety presumably occurs in vivo in E. coli cells, rather than in vitro during isolation of N+ from tRNA.
Basic Charges on Mammalian R bonuclease Molecules and the Ability to Attack Double‐Stranded. RNA
Authors: LIBONATI Massimo , FURIA Adriana , BEINTEMA Jaap J.
Published: March 2005

A ribonuclease recently isolated from the pancreas of the lesser rorqual or pike whale (Ba1aenoptera acutorostrata), definitely more basic than bovine ribonuclease A, degrades very efficiently double‐stranded RNA under conditions where the action of bovine pancreatic ribonuclease is minimal.

On the contrary, a ribonuclease from the pancreas of reindeer, definitely less basic than bovine ribonuclease A, is also, significantly less active than the bovine enzyme at degrading the doublehelical RNA.

Two ribonucleases (from the pancreas of guinea pig, the A component, and red deer), with a net charge and a number of basic amino acids similar to those of bovine pancreatic ribonuclease, show an activity on double‐stranded RNA which is indistinguishable from that of bovine ribonuclease A.

Finally, a ribonuclease from the pancreas of rat, having a net charge equal to that of bovine ribonuclease A, but three basic residues more than the latter enzyme, shows a modest activity towards the double‐helical RNA.

These results are compatible with the idea that the action on secondary structures of RNA by ribonucleases non‐specific for double‐stranded RNA may be related to the number (and possibly the position) of basic charges on the protein molecules.
The Sodium‐Induced Efflux of Calcium from Heart Mitochondria
Authors: CROMPTON Martin , CAPANO Michela , CARAFOLI Ernesto
Published: March 2005

The interrelations between the transport of Na+ and Ca2+ in heart mitochondria have been investigated. Na+ induces a partial release of Ca2+ from heart mitochondria; in the presence of ruthenium red the release of Ca2+ by Na+ is complete. The rate of Ca2+ release appears to be a function of [Na+]3 and is stimulated by energy‐linked respiration. Ca2+ efflux is also induced by Li+ although Na+ is the preferred cation. The data are consistent with the existence of a system able to catalyze an exchange between Na+ (or Li+) and Ca2+ across the inner membrane of heart mitochondria.
Lysosomal Cathepsin B1: Partial Characterization in Rat Preputial Gland and Recompartmentation in Response to Estradiol‐17β
Authors: SZEGO Clara M. , SEELER Barbara J. , SMITH Robert E.
Published: March 2005

Analysis of the properties of the predominant acid proteinase of rat preputial gland has been carried out on partially purified extracts of lysosomes isolated from this organ, using a fluorometric assay and the synthetic substrate, carbobenzyloxy‐alanyl‐arginyl‐arginyl‐4‐methoxy‐β‐naphthylamide. By means of exclusion chromatography on Sephadex G‐200, a molecular weight of ∼ 25000 was estimated. The enzymic activity exhibited a pH optimum of 6.2. Calcium ion was required for full activity within a narrow range of concentrations, beyond which inhibition was noted. Substrate specificity of the enzyme toward arginyl and lysyl residues and an absolute requirement for sulfhydryl activation, coupled with the capacity to inactivate aldolase, strongly suggested cathepsin B1‐like character. This inference was supported by relative efficiencies of several classes of inhibitors. Powerful constraint of activity was exhibited by the known cathepsin‐B1 inhibitors, leupeptin and antipain, while pepstatin, a recognized antagonist of cathepsin D, was without influence. Neither lima bean trypsin inhibitor nor phenylmethylsulfonyl fluoride was active. However, substantial inhibition was seen with tosyl‐l‐lysyl‐chloromethyl ketone, iodoacetate, and ovomucoid. Collectively, these data strongly implicate cathepsin B1 as the predominant lysosomal proteinase of rat preputialgland. In extension of previous work with selected lysosomal hydrolases, estradiol‐17β, but not the inactive 17α‐epimer, elicited, within 2 min of its administration, a marked reduction in structural latency of cathepsin B1 in lysosome‐enriched fractions isolated from the preputial gland of the ovariectomized rat, without evoking concomitant alteration in the total activities in the corresponding unfractionated homogenates. Moreover, administration of the active hormone led to the rapid appearance of this enzymic activity, foreign to the nucleus in the controls, in ultrapurified nuclei isolated froth this organ. The potential significance of such recompartmentation in the subsequent secondary responses initiated by the hormone, including mitogenesis, is discussed.
The Amino‐Acid Sequence of a Fragment of Human‐Serum Albumin Containing Two of Its Antigenic Determinants
Authors: WALKER John E.
Published: March 2005

The amino acid sequence of a fragment of human serum albumin (inhibitor), which contains two of the antigenic determinants of this protein, is described. It contains 90 amino acid residues and is derived from the carboxyl‐terminal region of human serum albumin.

From this sequence the sequence of a smaller fragment (F1), which has previously been shown to contain one antigenic determinant, has been deduced.
Evidence for the Existence of Two Interconvertible Forms of the Phosphofructokinase Dimer from Escherichia coli K‐12
Authors: EWINGS Ken N. , DOELLE Host W.
Published: March 2005

The isolation by affinity chromatography of a low‐molecular‐weight form of phosphofructokinase from Escherichia coli K‐12 has resulted in an improved 2050‐fold purification. The enzyme was readily separable from the higher‐molecular‐weight form by Sepharose 6B fractionation. The enzyme was found to have a molecular weight of 65000 ± 6500 with a subunit molecular weight of 35000 ± 3500. The enzyme exists in either of two interconvertible forms dependent on the presence or absence of the positive of ectors ADP, fructose 6‐phosphate and ATP. A regulatory mechanism is postulated for this enzyme involving hysteric conformational changes; its importance in the mechanism of the Pasteur effect is discussed.
CO2 Reduction to Formate by NADH Catalysed by Formate Dehydrogenase from Pseudomonas oxalaticus
Authors: RUSCHING Ulrich , MÜLLER Ulrich , WILLNOW Peter , HÖPNER Thomas
Published: March 2005

The direct reduction of CO2 to formate is catalysed by formate: NAD oxidoreductase in the presence of substrate amounts of NADH Proof for this reaction is supplied by the detection of a CO2‐dependent NADH oxidation, and by the identification of [14C]carbonate.

The enzyme‐catalysed CO2 reduction by NADH attains the equilibrium predicted by thermodynamic consideration a state which is also reached from the formate side. The Michaelis constant for CO2 is about 40 mM indicating the low affinity of the enzyme for this substrate. The corresponding value for formate is 0.1 mM. Under the special conditions employed the enzyme catalyses the formate oxidation about 30 times faster than the CO2 reduction. That CO2 and not HCO3 is the active species in the reduction was shown by comparing the pH dependency of the velocities of the forward and back reactions and by observing the kinetics of CO2 reduction during the simultaneous attainment of the CO2‐HCO−3 equilibrium.
Efficient Purification and Molecular Properties of Spinach Chloroplast Fructose 1,6‐Bisphosphatase
Authors: ZIMMERMANN Gerhard , KELLY Grahame J. , LATZKO Erwin
Published: March 2005

A relatively straightforward procedure has been developed for the purification of chloroplast fructose bisphosphatase from spinach leaves to apparent homogeneity and with 80% yield. The molecular weight of the enzyme was about 160000. Chloroplast fructosebisphosphatase consists of four possibly identical subunits and, at pH 8.8, easily dissociates into equal halves with lowered activity. Sigmoid saturation curves with Hill coefficients between 3.0 and 3.7 were obtained for fructose 1,6‐bisphosphate and Mg2+. Incubation of the enzyme with 20 mM dithiothreitol slowly altered the response to pH from no activity measured at pH 7.5 and full activity at pH 8.8 to equal activity at each of these pH values; at the same time the number of freely available sulphydryl groups increased from four to twelve per molecule. These properties are considered in the context of the observed activation of this enzyme following illumination of chloroplasts.
Separation and Characterization of Two Phosphorylase Phosphatase Inhibitors from Rabbit Skeletal Muscle
Authors: HUANG Freesia L. , GLINSMANN Walter H.
Published: March 2005

Two heat‐stable and trypsin‐labile inhibitors of phosphorylase phosphatase, designated Inhibitor‐1 and inhibitor‐2, were partially purified from extracts of rabbit skeletal muscle by heating and column chromtoagraphy using DEAF‐cellulose and Bio‐gel P‐60. Inhibitor‐1 exists in an active phosphorylated form and an inactive dephosphorylated form The interconversion of phosphorylated inhibitor‐1 and dephosphorylated inhibitor‐1 is mediated by protein kinase dependent on adenosine 3: 5 ‐monophosphate (cyclic AMP) and a Mn2+‐stimulated phosphoprotein phosphatase. Inhibitory activity of inhibitor‐2 is not influenced by treatment with either the kinase or the Mn2+ stimudated phosphatase. The molecular weights of inhibitor‐1 and inhibitor‐2 estimated by sodium dodecylsulfate‐polyacrylamide gel electrophoresis are 26000 and 33000 respectively. Both inhibitor‐1 and inhibitor‐2 inhibit phosphorylase phosphatase by a mechanism which appears to be non‐competitive with respect to the substrate phosphorylase a. Inhibitor fractions at early stages of purification also inhibit cyclic‐AMP‐dependent histone phosphorylation, but this kinase inhibitory activity resides with a protein moiety which is separable from inhibitor‐I and inhibitor‐2.
Metabolic Regulation of Steroidogenesis in Isolated Adrenal Cells of Rat
Authors: SHARMA Rameshwar K. , AHMED Nahed K. , SHANKER Gouri
Published: March 2005

The data presented with the isolated adrenal cells, in the present study, show that adrenocorticotropin in the physiological concentration range stimulates the synthesis of guanosine 3′:5′‐monophosphate (cyclic GMP), protein kinase activity, and steroidogenesis in a concentration‐dependent manner without detectable rise in the levels of adenosine 3′:5′‐monophosphate (cyclic AMP). Millimolar concentrations of cyclic AMP and cyclic GMP, which stimulate corticosterone synthesis, also activate kinase activity and steroidogenesis in a sigmoid concentration‐response manner. The process of phosphorylation activated by corticotropin, cyclic AMP and cyclic GMP is not inhibited by eyeloheximide or actinomyin D. It is therefore proposed that the hormonal responses mediated by cyclic GMP and cyclic AMP are via the protein kinase enzymatic steps, and the inhibitory effect of' cycloheximide and actinomycin D in corticotropin‐stimulated steroidogenesis follows this step. In conjunction with our previous observations that the biosynthetic steps from (20S)‐20‐hydroxycholesterol to corticosterone are neither inhibited by cycloheximide nor affected by cyclic GMP, it is inferred that the rate‐limiting step of adrenal steroidogenesis is the transformation of cholesterol to (20S)‐20‐hydroxycholesterol and this very step is regulated by cyclic GMP and cyclic AMP.

Of further significance are the findings that micromolar concentrations of cyclic AMP and cyclic GMP, which do not stimulate steroidogenesis, effectively stimulate protein kinase activity in a concentration‐dependent manner. It is therefore concluded that all cyclic‐nucleotide‐dependent protein kinase activities of the cell are not necessarily related to steroidogenesis.
The Binding Site of Protein L1 on 23‐S Ribosomal RNA from Escherichia coli
Authors: BRANLANT Christiane , Viatscheslav KOROBKO  , EBEL Jean‐Pierre
Published: March 2005

The almost complete sequence of a region of' the 23‐S ribosomal RNA from Escherichia Coli, 175 nucleotides in length, located between the 550th and the 1000th nucleotide at the 3′ end of 23‐S RNA and containing the binding site of protein L1, is given. Possible models of secondary structure arc proposed