bluuebunny/crossref_metadata_2025 huggingface dataset:
Maleimide and Isomaleimide Pyrrolidine‐Nitroxide Spin Labels
Authors: Barratt Martin D. , Davies Alan P. , Evans Mervyn T. A.
Published: March 2005

2,2,5,5‐Tetramethylpyrrolidine‐1‐oxyl‐3‐maleimide and the corresponding isomaleimide have been synthesized and characterised. The electron‐spin‐resonance spectra exhibited by these labels when bound to poly‐L‐lysine and bovine‐plasma albumin are discussed in terms of their structures and their relative degrees of binding to thiol and amino groups. Contamination of male‐imide spin labels with isomaleimide is shown to be a real possibility. Emphasis is thus laid on the need for careful characterisation of spin labels prior to their use.
Oxygen‐Affinity Studies of Avian Hemoglobins
Authors: Vandecasserie Christian , Schnek Arthur Georges , Léonis José
Published: March 2005

The oxygenation of hemoglobin is under the control of the regulatory function of organic phosphates.

The one in mammals corresponds to 2,3‐bisphosphoglycerate but in birds and turtles appears to be inositol hexakisphosphate.

The latter component decreases the oxygen affinity of the two chicken hemoglobin compounds (the minor and the major) and the lone pigeon hemoglobin component.

The Bohr effects have also been determined for the two hemoglobin samples.

As has already been observed by electrophoresis and by chromatography, pigeon hemoglobin seems to behave more like that of the major chicken component by disclosing analogous log p50 values, i.e. the partial pressure of oxygen corresponding to a 50% load of the hemoglobin.
An Association between the Kinetic and Electrophoretic Properties of Human Purine‐Nucleoside‐Phosphorylase Isozymes
Authors: Turner Bryan M. , Fisher Rachel A. , Harris Harry
Published: March 2005

Human purine‐nucleoside phosphorylase has been studied in extracts of red‐blood cells, cultured fibroblasts and cultured‐lymphocytoid‐cell lines. The enzyme from red cells has been found to contain at least seven electrophoretically distinct isozymes while the enzyme from the cultured cells gives a single electrophoretic band corresponding to the slowest band of the red‐cell enzyme.

Human erythrocytes have been separated into groups of different mean‐ages by densitygradient centrifugation; nucleoside phosphorylase from the youngest cells was found to give only four or five electrophoretic bands of which the slowest was the most intensely staining. There was a progressive increase in number and intensity of the faster bands as the mean age of the red‐cell fraction increased. An age‐related decline in the total activity of the enzyme was also observed.

The Lineweaver‐Burk plot for human‐erythrocyte purine‐nucleoside phosphorylase assayed in a red‐cell haemolysate with inosine as the variable substrate showed a downward curvature at inosine concentrations above about 0.2 mM. When a form of the enzyme giving only a single electrophoretic band was used the Lineweaver‐Burk plots were found to be completely linear. Generation of secondary isozymes in vitro resulted in a change in this linear plot to the curved form shown by the red‐cell enzyme.

It is concluded that the secondary isozymes generated as a result of ageing in vivo or in vitro differ in kinetic behaviour from the primary isozyme. This may account for the observed curvature of the Lineweaver‐Burk plot at high inosine concentrations.
Substrate Properties of Yeast tRNAPhe Oxidized and Reduced at the 3′‐Terminal Ribose
Authors: von der Haar Friedrich , Schlimme Eckhard , Gómez‐Guillen Manuel , Cramer Friedrich
Published: March 2005

Native yeast tRNAPhe and this tRNAPhe with the 3′‐terminal AMP removed were oxidized by NaIO4 and subsequently reduced by NaBH4. The following investigations were undertaken with these substrates: aminoacylation in borate buffer and measurement of the life‐time of the aminoacylated oxidized an reduced tRNA, hydrolysis with snake‐venom phosphodiesterase and pyrophosphorolysis with tRNA nucleotidyl transferase.

Whereas the oxidized and reduced yeast tRNAPhe is a good substrate for the enzymes investigated, the corresponding tRNAPhe with the 3′‐terminal AMP removed is a very poor, if any, substrate in these reactions. From the data obtained and from proton‐magnetic‐resonance investigations with oxidized and reduced AMP and ATP it is concluded that the removal of the C2′—C3′ bond and introduction of two hydrogen atoms instead distorts the original ribose conformation at the 3′‐terminus. Under the assumption that this distortion is different for C75 and A76 the difference in reactivity between the two oxidized and reduced tRNAs can be explained.
Iodine, Hemin and Heminester as Oxidants in a Synthesis of ATP from ADP and Pi Mediated by Thiols and Disulfides
Authors: Bäuerlein Edmund , Klingenfuss Marlies , Wieland Theodor
Published: March 2005

The conversion of oxidation energy into the energy‐rich phosphoryl linkage of ATP has been demonstrated in a system consisting of the tertiary‐butylammonium salts of ADP and Pi in pyridine and utilising several mercapto carboxylic acids or their disulfides as mediators and iodine or hemin or hemindimethyl ester as oxidants.

Iodine with several thiols in 1:1 molar proportions produces relatively poor yields of ATP (1.5–5.4%) which can be considerably increased (10–15%) when the corresponding disulfides are used, whereas a thioether (N‐acetylmethionine) had no effect. This led to the conclusion that disulfides are better substrates and accordingly much higher amounts of ATP were formed from all thiols on using a two‐fold amount of oxidant (up to 25.6%). Taking this into account a mechanism has been formulated in which the formation of a phosphorylating species, RS‐OPO3H2, is assumed to be formed from disulfides.

Hemin and (less efficiently) its dimethyl ester used as oxidants also give rise to the formation of ATP in our system, but only under aerobic conditions. Here optimal yields (up to 16.7%) were obtained with thioglycolate or its disulfide as mediators, a thioether (N‐acetylmethionine) being almost ineffective, and no difference in efficiency between thiol acid and disulfide was observed at a hemin: sulfur ratio of 1:1. The yields depend in some way on the hemin: S ratio, lower values giving higher amounts of ATP on oxidation of the mercapto compound, but not of its disulfide. Therefore a reaction mechanism different from that of iodine must be presumed. The formation of the same phosphorylating species as above has been assumed to occur preferently with a thiol and to a lesser extent with a disulfide. The role of oxygen is still rather obscure, for it can not be determined whether it is the terminal electron acceptor or electron carrier. A catalytic participation of hemin could not be demonstrated, perhaps on account of the slow autoxidation rate of the ferrous heme iron.
The Covalently‐Bound Flavin of Hepatic Monoamine Oxidase
Authors: Walker Wolfram H. , Kearney Edna B. , Seng Richard L. , Singer Thomas P.
Published: March 2005

In the previous paper in this series it was shown that a pure flavin pentapeptide isolated from hepatic monoamine oxidase contains 1 mole each of serine and tyrosine and 2 moles of glycine, as well as an additional amino acid which is covalently linked to the 8α carbon of riboflavin. This amino acid has been identified as cysteine and the linkage to the flavin as a thioether on the basis of positive chloroplatinic and negative iodine‐azide tests, performic acid oxidation or reduction with zinc, followed by acid hydrolysis, which yield cysteic acid or cysteine, respectively. The fluorescence properties of the flavin from monoamine oxidase agree with those expected for a thioether and its oxidation products. In regard to optical and electron spin resonance spectra, chemical stability, and fluorescence characteristics the flavin peptide isolated from the enzyme agrees excellently with the properties of synthetic cysteinyl 8α‐riboflavin.
The pH‐Dependence of Rates of Individual Steps in Ficin Catalysis
Authors: Hollaway Michael R. , Antonini Eraldo , Brunori Maurizio
Published: March 2005

The kinetics of the ficin‐catalysed hydrolysis of benzoylglycine p‐nitrophenyl ester have been studied at different pH values with widely varying initial concentrations of enzyme and substrate. The time‐courses of proton release and p‐nitrophenol release were followed.

The failure to observe a lag period for p‐nitrophenol release employing a stopped‐flow spectrophotometer leads to a minimum value for the rate constant for the formation of the E · S complex of 5 × 105 M−1 sec−1. Evidence is presented for the formation of E · S being a pseudo‐equilibrium process.

The results are consistent with a three‐step mechanism involving an acyl‐enzyme intermediate. Time‐courses of reactions with [E0] ∼ [S0] were independent of wavelength in the range 291 to 400 nm so that it is unlikely that an oxazolinone is formed as an intermediate.

The pH‐dependencies have been determined for the dissociation constant of the E · S complex (Ks) as well as for the individual rate constants associated with the catalytic steps (k+2 and k+3).

pH profiles of k+2/Ks implicate ionising groups of pKa values 4.3 and 8.58 in the free enzyme (25 °C, I= 0.1).

The pH dependency of k+2 (measured directly at pH values below neutrality) implicates an ionising group in the catalytic step with pKa∼ 5.8. The change in the value of Ks with pH is in accord with this being identical to the group with pK 4.3 in the free enzyme.

The value of k+3 does not vary significantly between pH 3.9 and 6.6, from which it is deduced that the group of pKa∼ 5.8 in the E · S complex undergoes a downward shift in pKa by at least 2 units on formation of the acyl‐enzyme intermediate. The release of protons concomitant with acyl‐enzyme formation (pH 5 and [E0] ∼ [S0]) is consistent with such a pKa shift.

At neutral pH values the k+3 step is rate limiting in catalysis whereas at pH 3.9 k+2 becomes rate limiting. This change in rate‐limiting step with pH illustrates the danger in the assumption that kcatversus pH profiles for thiol protease action on substrates with good leaving groups are equivalent to k+3versus pH profiles.
Regulatory Properties of the Pyruvate‐Dehydrogenase Complex from Escherichia coli
Authors: Bisswanger Hans , Henning Ulf
Published: March 2005

It is shown that the pyruvate‐dehydrogenase complex from Escherichia coli K12 produces, under certain conditions, positive cooperativity for the substrate pyruvate and negative cooperativity for the inhibitor acetyl‐CoA. The target of the two effectors is the pyruvate dehydrogenase component of the complex. Suggestive evidence indicates that pyruvate cooperativity may mostly be of intramolecular nature (i.e., occurring within individual pyruvate dehydrogenase molecules) although some interactions between such molecules become apparent in the presence of acetyl‐CoA inhibition.

Negative cooperativity exerted by acetyl‐CoA may not be a direct consequence of ligand‐induced conformational changes of or non‐exclusive ligand binding. It was found that a partially inactivated pyruvate dehydrogenase component is activated by acetyl‐CoA rather than inhibited. It has not been possible to demonstrate the occurrence of this activation with the fully active enzyme component; nonetheless, it does not seem unlikely that the negative cooperativity observed is the consequence of acetyl‐CoA both activating and inhibiting (presumably by acting at different sites) pyruvate dehydrogenase catalysis.
Nuclear Dependency of Chloroplast Proteins in Acetabularia
Authors: Apel Klaus , Schweiger Hans‐Georg
Published: March 2005

A membrane protein fraction, insoluble in water, has been prepared from Acetabularia chloroplasts. The greater part of these proteins could be dissolved in a phenol—formic acid—water mixture (2:1:1, v/v/v). The dissolved proteins were subjected to gel electrophoresis. The electrophoretic pattern was reproducible for one species.

The protein patterns of A. calyculus and A. mediterranea were composed of 8 peaks common for both species and of at least 3 peaks which were species‐specific.

By means of nuclear transplantation and implantation techniques it was shown that the species‐specific chloroplast‐proteins were nucleus‐dependent and therefore were coded by the nuclear DNA. Six weeks after the operation the patterns of the transplants and implants had changed into that of the nucleus donor species. It was concluded that the mRNA of the nucleus acceptor cell lost its stability under the influence of the implanted or transplanted nucleus.

Both cycloheximide and chloramphenicol inhibited the incorporation of radioactive amino acids into the insoluble chloroplast‐protein fraction.

The electrophoretic pattern of the labelled chloroplast‐proteins consisted of at least 3 peaks. Cycloheximide inhibited the incorporation into peak 3, whereas chloramphenicol affected the incorporation into the two other peaks. Isolated chloroplasts incorporated amino acids only into peaks 1 and 2. Peak 3 was absent in the pattern in vitro.

The protein groups which were involved in the formation of chloroplasts were synthesized either by cytoplasmic 80 S or by 70 S chloroplast ribosomes. It was concluded that the autonomy of the chloroplasts was limited by the influence of the nucleus.
Incorporation of 5‐Iodocytidine into Yeast tRNAPhe with tRNA Nucleotidyl Transferase in vitro
Authors: Sprinzl Mathias , von der Haar Friedrich , Schlimme Eckhard , Sternbach Hans , Cramer Friedrich
Published: March 2005

5‐Iodocytidine (io5C) is incorporated into tRNAPhe‐A73 as well as into tRNAPhe‐A73‐C74 using tRNA nucleotidyl transferase. The tRNAPhe‐A73‐C74‐io5C75‐A76 and tRNAPhe‐A73‐io5C74‐io5C75‐A76, respectively, thus obtained can be aminoacylated by the corresponding synthetase exhibiting the same Km but a slightly lowered V compared to tRNAPhe‐A73‐C74‐C75‐A76. An analytical method is presented which enables quantitative determination of the 3′ terminal nucleosides.
Structural Studies of Alcohol Dehydrogenase from Human Liver
Authors: Jörnvall Hans , Pietruszko Regina
Published: March 2005

Tryptic peptide maps of human liver alcohol dehydrogenase show this protein to be homologous to the corresponding protein of the horse. A subunit molecular weight very close to 40000 is established for the human enzyme.

Sequence analysis of about one quarter of the tryptic peptides of human alcohol dehydrogenase show identical residues to the horse enzyme at about 90% of the positions, which is considered fairly representative of the general resemblance between the two proteins. All amino acid exchanges found are compatible with one‐base mutations in the genetic code.

Two different types of subunits of human liver alcohol dehydrogenase are identified. They are essentially similar but differ at some positions, one of which is No. 43 (valine in one subunit and alanine in the other). Still other subunit types may exist. The known occurrence of isoenzymes of human liver alcohol dehydrogenase may therefore be explained at least in part by subunits of different primary structures.

The amino acid differences between the subunits of the human enzyme are not found at the same positions as those between the two types of subunit of the horse enzyme. The structural differences between subunits from the two species seem greater than between the subunits within either species. Isoenzyme differences may, therefore, have evolved independently in the two species. The suggestion that some of the amino acid exchanges between the horse subunits may be directly involved in the substrate binding is supported.

The isoenzyme and species differences at position 43 is only three residues away from the reactive “active site” cysteine residue. The region around this important residue is thus not kept constant in liver alcohol dehydrogenase during evolution.
The Role of Ribosomal Conformation in Protein Biosynthesis
Authors: Sherman Michael I.
Published: March 2005

Hydrogen‐tritium exchange studies on ribosomes and poly(U)‐directed protein synthesis assays have been carried out at 12°C in the absence and presence of a wide spectrum of streptomycin concentrations. At very low (“catalytic”) streptomycin/ribosome, ratios, the ribosomal hydrogen‐tritium exchange rate is increased; this “loosening” phase is characterized functionally by a slight but reproducible inhibition of the incorportion of phenylalanine, and often of leucine and isoleucine, into polypeptide. At higher streptomycin/ribosome ratios, the exchange rate is decreased. This “tightening” of structure correlates with the onset of miscoding. The “loosening” and “tightening” phases appear to represent different ribosomal conformations with qualitatively different effects upon ribosomal function. This in turn suggests that there is more than one type of binding site for streptomycin on the ribosome, not inconsistent with the results of binding studies. Evidence is also provided suggesting that streptomycin interferes with initiation in the poly(U) system, although ribosomes are not irreversibly inactivated by streptomycin. The bearing of our results upon the phenomenon of phenotypic suppression is discussed.
The Effect of Magnesium‐Ion Concentration on the Translation of Phage‐f2 RNA in a Cell‐Free System of Escherichia coli
Authors: Zagórski Włodzimierz , Filipowicz Witold , Wodnar Aleksandra , Leonowicz Andrzej , Zagórska Ludwika , Szafrański Przemysław
Published: March 2005

The phage‐f2 specific protein synthesis was studied in the cell‐free system of Escherichia coli at 10.6–18.6 mM Mg2+. The products formed under the direction of f2 RNA between 12.6 mM and 18.6 mM Mg2+ were separated by polyacrylamide gel electrophoresis and found to correspond to phage‐specific proteins, i. e. RNA synthetase and the coat protein. The rate of f2‐RNA translation was optimal at 12.6 mM Mg2+. Lowering of Mg2+ concentration from the optimal value to 10.6 mM caused the lack of formation of complete RNA synthetase while the coat protein was still synthesised.

This was due to a sharp decrease in rate of polypeptide‐chain elongation, which led to incomplete translation of the longer gene. Our results indicate that, within the studied range of Mg2+ concentration, specific initiation of f2‐RNA translation occurs even at 18.6 mM Mg2+.
The Synthesis of d‐arabino‐Hexulosonic Acid 6‐Phosphate and Its Stability in Acid and Alkaline Medium
Authors: Trigalo François , Szabó Ladislas
Published: March 2005

The chemical synthesis of d‐arabino‐hexulosonica cid 6‐phosphate from d‐gluconic acid 6‐phosphate and a simple synthesis of the latter from d‐glucose 6‐phosphate are described. The phosphorylated keto acid reduces 3 molar equivalents of periodate; its ultraviolet spectrum possesses a broad maximum of low intensity at 280 nm, but none at 260 nm; it is fully active as substrate for specific NAD‐ and NADP‐dependent reductases and a specific decarboxylase. The compound is quite unstable in alkaline medium: it yields, by retro aldol reaction, hydroxy pyruvate and triose phosphate as the main products but some alkali resistent phosphate ester (s) is (are) also formed. In 0.1 N acid at 50 °C the phosphorylated acid is relatively stable; in more drastic conditions strongly reducing substances, probably of the ascorbic acid type, are also produced.
Studies on the Mode of Action of Nalidixic Acid
Authors: Pedrini Antonia M. , Geroldi Diego , Siccardi Antonio , Falaschi Arturo
Published: March 2005

Nalidixic acid, a very specific inhibitor of bacterial DNA synthesis, has been studied for its action on purified enzymes acting on DNA and in subcellular DNA‐synthesizing systems. The drug does not inhibit the following enzymes: DNA polymerase I, endonuclease I, exonuclease I, II and III from Escherichia coli, polynucleotide‐ligase and DNA methyl‐transferase from T4‐ infected E. coli, DNA polymerase from Bacillus subtilis. A significant inhibition of ATP‐dependent DNA synthesis is observed in cells of a strain of E. coli lacking DNA polymerase I after short treatments with toluene (2 min); longer treatments reduce the rate of synthesis and abolish its sensitivity to nalidixic acid. Equally insensitive are the ATP‐dependent DNA synthesizing systems bound to membrane fractions of E. coli and B. subtilis.

The results suggest a mode of action through a still unidentified physiological component of the growing point apparatus, and supply a new criterion of physiological integrity of subcellular DNA‐synthesizing systems, namely their sensitivity to nalidixic acid.
RNA Polymerase from Eukaryotic Cells
Authors: Mondal Hrishikes , Mandal Radha K. , Biswas Birendra B.
Published: March 2005

This paper describes the isolation and purification of two RNA polymerases (CI and CII) and several protein fractions (A, B and C) from the chromosomal acidic proteins of coconut endosperm nuclei. The method involves disruption of isolated nuclei in dilute salt medium, centrifugation to sediment the crude chromatin, solubilization of chromatin in concentrated salt, dialysis of soluble chromatin to dilute salt to precipitate DNA‐histone complex and centrifugation to get the acidic proteins in the supernatant. Further purification involves ammonium sulphate fractionation and chromatography on DEAE‐cellulose and QAE‐Sephadex.

RNA polymerase CI purified through the QAE‐Sephadex step gives a single band on polyacrylamide gel electrophoresis. In gels containing dodecylsulfate, this enzyme shows multiple bands indicating its subunit nature. The pH optima for both RNA polymerases is 8.0. RNA polymerase CI is maximally activated by Mn2+; while CII, by Mg2+. The activities of both the enzymes are stimulated by fraction B. All the fractions except A are substantially free from nucleases. Both RNA polymerases require the addition of DNA for activity. Fraction B is ineffective either with denatured coconut DNA or native λ DNA.
Porcine Carboxypeptidase B
Authors: Sokolovsky Mordechai , Eisenbach Lea
Published: March 2005

Coupling of porcine carboxypeptidase B with diazotized arsanilic acid labels an active‐site tyrosine residue of the enzyme. Tyrosine modification is accompanied by a change in the activities of carboxypeptidase B towards both basic and non‐basic substrates. The major arsanilazotyrosine‐containing peptide, Thr‐Ile‐Tyr‐Pro‐Ala, isolated by affinity chromatography is homologous with the peptide containing tyrosine‐248 from the active center of bovine carboxypeptidase A.

Formation of the arsanilazotyrosine residue generates extrinsic Cotton effects above 300 nm. The dichroic spectrum of the azochromophore constitutes a sensitive probe of conformational changes accompanying the interaction of arsanilazocarboxypeptidase B with inhibitors, as monitored by changes in the circular dichroic spectrum. The changes are consistent with the hypothesis that the tyrosine residue is a component of the active‐site region.
Chemical Modification and Kinetic Studies on Glucarate Hydro‐Lyase from a Species of Pseudomonas A
Authors: Jeffcoat Roger
Published: March 2005

Glucarate hydro‐lyase, obtained from cells of Pseudomonas A grown on glucarate as the sole source of carbon, was purified 40‐fold in 70% yield.

The effects of a variety of chemical modifiers and competitive inhibitors on the enzyme activity were studied.

Enzyme activity was irreversibly lost by incubating the enzyme with p‐chloromercuribenzene sulphonate.

(+)‐Tartrate, a competitive inhibitor of the enzyme, protected the enzyme from inhibition by this reagent but was not effective when the enzyme was inhibited by coupling it to a diazonium salt. Glucarate, unlike (+)‐tartrate, was an effective protecting group against this type of inhibition.

The roles of the modified amino acid residues are discussed.
Light‐Absorption Studies on Neutral Flavin Radicals
Authors: Müller Franz , Brüstlein Martin , Hemmerich Peter , Massey Vincent , Walker Wolfram H.
Published: March 2005

Quantitative light absorption data of the neutral free flavosemiquinone are reported. It is shown that the visible range of the spectrum of the radical is drastically dependent on the solvent. The spectra of the neutral blue flavosemiquinone are compared with those of the blue flavoprotein radicals and conclusions are drawn concerning the environment of the protein‐bound flavosemiquinone.

The stabilization of the neutral blue flavosemiquinone through N(5)‐alkylation and/or π‐complex formation with aromatic compounds is described.

The light absorption characteristics of a tautomeric neutral, but red‐coloured, flavosemiquinone are described. The results are compared with those obtained from the free anionic and protein‐bound red flavosemiquinones. The two types of radicals cannot be distinguished from each other by electron spin resonance spectrometry but may be recognized by means of light absorption spectrophotometry. The possible biological significance of the neutral red flavosemiquinone is discussed. The optical properties of this radical are compared with those of free and protein‐bound anionic flavosemiquinones.
La D‐altronate: NAD‐oxydoréductase d'Escherichia coli K12 Purification, proprié0tés et individualité
Authors: Portalier Raymond C. , Stoeber François R.
Published: March 2005

D‐Altronate: NAD‐oxidoreductase of Escherichia coli K12 has been purified. The purification consists of four steps: nucleic acids precipitation with protamine sulfate; ammonium sulfate precipitation; ion exchange chromatography on DEAE‐cellulose and Sephadex G‐150 gel filtration. The overall procedure results in a 53‐fold purification with a yield of 60%.

Altronate oxidoreductase is an inducible enzyme: galacturonate, tagaturonate, glucuronate and fructuronate are good inducers. The activity remains stable at 4 °C or at —20 °C for several months.

Optimal activity is obtained at pH 6.3 for tagaturonate reduction, and at pH 8.9 for altronate oxidation. Altronate oxidoreductase does not require any cofactor, except NADH.

The apparent Michaelis constants have been determined: for tagaturonate, the Km is 350 μM; for NADH, 60 μM; for altronate, 90 μM, and for NAD+, 110 μM. Moreover, altronate oxidoreductase is active with NADPH as coenzyme and the apparent Km value for NADPH is 400 μM. Other carbohydrates tested are unsuitable as substrates. Many other carbohydrates tested do not prove to be inhibitors.

The value for the energy of activation is 38 kJ/mol (9 kcal/mol); the Q10 is 1.5. The results of heat inactivation of altronate oxidoreductase (58.5 °C) indicate homogeneous enzyme activity in crude extract. Thermal inactivations also indicate that the same enzyme, altronate oxidoreductase, is induced by galacturonate and glucuronate, and catalyzes the direct reactions with NADH or NADPH. Finally, it has been confirmed by heat inactivation that there is no interference between specific assays of altronate and mannonate oxidoreductases.

Compounds such as NADH, NADPH, altronate and mannonate protect enzymatic activity from degradation by heat, whereas NAD+, tagaturonate and fructuronate do not. NADH, in particular, is effective as a stabilizer; this ability is proportional to its concentration.

Enzyme inhibition with para‐chloromercuribenzoate is non‐competitive and the apparent K1 is 1 mM. para‐Chloromercuribenzoate also inactivates altronate oxidoreductase indicating homogeneous activity and specificity of the assay method in crude extracts.

Chromatographic properties of the enzyme are identical whether the inducer is galacturonate or glucuronate.

Using paper chromatography, the reaction product of enzyme action on D‐tagaturonate is identified as D‐altronate.
Purification and Characterization of Pyruvate‐Dehydrogenase Phosphatase from Pig‐Heart Muscle
Authors: Siess Elmar A. , Wieland Otto H.
Published: March 2005

Pyruvate‐dehydrogenase phosphatase was extensively purified from pig heart muscle.

The molecular weight was determined to be in the range between 92000 and 95000. By dodecylsulfate‐gel electrophoresis there was no indication for a subunit structure of the protein.

The phosphatase is dependent on Mg2+ or Mn2+. Half‐maximal activity was obtained, in histidine buffer, at 2.5 mM MgCl2 and 1.8 mM MnCl2, respectively.

Fluoride at a concentration of 0.6 mM inhibited phosphatase activity by 50% in the presence of Mg2+, but not of Mn2+. The enzyme was not affected by alkylating thiol‐reagents.

Studies with sequestering agents suggest that pyruvate‐dehydrogenase phosphatase contains a metal which, besides Mg2+ or Mn2+, is essential for its function.
Interaction of Rose Bengal with Apo‐Hemoproteins
Authors: Coulson Andrew F. W. , Yonetani Takashi
Published: March 2005

The interaction of “ose bengal with apo‐hemoproteins was found to give rise to a large absorption change near 550 nm. This was used to study the binding of the dye to apo‐cytochrome c peroxidase, apo‐myoglobin, apo‐horseradish peroxidase and apo‐hemoglobin. In all cases there was about one tight and a number of weak binding sites per heme‐binding site, and the binding was impaired in the presence of stoichiometric quantities of heme.

Rose bengal was found to be an effective photo‐oxidation sensitizer for apo‐cytochrome c peroxidase. Kinetic evidence was obtained consistent with the possibility that binding of the dye to the protein is an important step in the photo‐oxidation process. Heme was found to protect the enzyme from loss of enzymic activity caused by photo‐oxidation. Photo‐oxidation does not simply destroy the binding of heme to apo‐cytochrome c peroxidase, but produces changes in the spectrum of the bound heme. There is a loss of ability of recombined enzyme to form an enzyme substrate complex in the presence of stoichiometric quantities of hydrogen peroxide, which apparently causes the overall loss of enzymic activity. Photo‐oxidation was accompanied by loss of one tryptophan and two histidine residues per mole of protein, but apparently only one of the latter is essential to the enzymic activity.

It was concluded that rose bengal can act as a specific modifying agent for the heme site in apo‐hemo‐proteins, and that this specificity arises from specificity of binding to the heme site. It is suggested that specificity of binding may provide a general basis for the observation of specific destruction of functionally interesting residues in photo‐oxidation.
Studies on the Metabolic Effects Induced in the Rat by a High‐Fat Diet
Authors: Bringolf Michel , Zaragoza Nelly , Rivier Denis , Felber Jean‐Pierre
Published: March 2005

Rats were fed from weaning during 3–4 weeks a high fat carbohydrate‐poor diet. Control groups received a diet in which fat was completely replaced by starch in the same caloric proportion.

The effect of this high‐fat diet on the metabolism of pyruvate in diaphragm in vitro was investigated. Glycogen breakdown and glyceride content of the tissue were also measured.

Fat diet was shown to induce a reduction in 14CO2 production from labelled pyruvate and in the pyruvate uptake. Acetate oxidation was found to be unchanged after fat feeding. This gives evidence for an inhibition of the pyruvate dehydrogenase reaction in diaphragm from fat‐fed rats.

The breakdown of glycogen during either an incubation in vitro or a 24‐h fast was significantly reduced in diaphragm from fat‐fed rats.

Diaphragm glycerides were found to be increased four‐fold by fat diet. Examination by light‐microscopy of muscle sections have shown that glycerides were present in the muscle fibre and not in adipose tissue cells in the preparation.

Inhibition of the oxidation of fatty acids by 2‐bromostearate was shown to reverse completely the impairment of pyruvate metabolism and to stimulate glycolysis. In diaphragm from carbohydrate‐fed rats 2‐bromostearate induced no significant effect on carbohydrate metabolism. Similar results were obtained with glucose as substrate.

Rats were fed the high‐fat diet and then the high‐carbohydrate one for a few days. This change of diet was shown to induce a rapid reversal of the inhibition of glucose oxidation in diaphragm in vitro.

These results indicate that the impairment of carbohydrate metabolism in diaphragm from fat‐fed rats is probably due to an increased oxidation of fatty acids as observed in muscle from alloxan‐diabetic or starved rats rather than to enzymatic adaptations.
The Respiratory‐Chain NADPH Dehydrogenase ofAzotobacter vinelandii
Authors: Ackrell Brian A. C. , Erickson Sandra K. , Jones Colin W.
Published: March 2005

NADPH dehydrogenase activity ofAzotobacter vinelandiirespiratory membranes is inhibited by adenine nucleotides (pure competitive inhibition; AMP>ADP>ATP) and by NAD+(partially competitive, allosteric inhibition). No allosteric activators, other than NADPH, were found for this enzyme.

The dehydrogenase exhibits kinetic properties compatible with the presence of a catalytic site, which binds one molecule of NADPH (or one molecule of adenine nucleotide), and an apparently acid‐sensitive, site (or sites) which binds one molecule of either NADPH (positive homotropic effector) or NAD+(negative heterotropic effector).

These results suggest that in the presence of NAD+or low energy charge, NADPH may be converted to NADHviathe NADPHNAD+transhydrogenase, and thence oxidisedviathe more efficiently phosphorylating NADH dehydrogenase.

The prime function of the NADPH dehydrogenase is probably to oxidise the excess NADPH which might be expected to accumulate when nitrogen‐fixation is switched off (e.g.under highly aerobic conditions) and thus to contribute to the phenomenon of nitrogenase protection by enhanced respiration and also to the maintenance of the desired intracellular NADPH/NADP+ratio.
Ribonuclease T1 Spectrophotometric Investigations of the Interaction of the Enzyme with Substrate Analogues
Authors: Epinatjeff Christa , Pongs Olaf
Published: March 2005

Spectral changes on interaction of ribonuclease T1 with a number of substrate analogues have been measured. For guanosine and guanosine monophosphates these changes are the same as those produced by protonation of the guanine chromophore. It is concluded that the guanine ring of the substrate analogues is protonated by ribonuclease T1 in position N‐7. Evidence is presented for a similar interaction of the enzyme with the purine ring of adenosine monophosphate.

From the magnitude of the spectral changes binding constants have been determined. They decrease in the order guanosine 2′‐phosphate > guanosine 3′‐phosphate > guanosine 5′‐phosphate > guanosine. The temperature dependence of the binding constants has been measured and the changes in enthalpy, free energy and entropy, which occur upon the RNAase T1‐ligand interaction, have been calculated.

The pH dependencies of the different ligand · enzyme complex formations revealed several pKa values (2.5,3.7,6.5,8.5). The pKa values of 2.5 and 6.5 have been assigned to protonations of the ligand. The pKa values of 3.7 and 8.5 have been assigned to specific groups in the active site of ribonuclease T1, which are involved in the recognition of the guanine base. From the pH dependencies of the guanosine monophosphate/enzyme interaction it is concluded that only the monoanionic form of guanosine monophosphate can interact with the enzyme.

The nature of binding of guanosine monophosphate to ribonuclease T1 is discussed. It is proposed that the positions N‐1 and N‐7 of the guanine base are recognized by a basic and an acid group in the active site of ribonuclease T1 and that the monoanionic phosphate moiety interacts with a basic group of the enzyme.
Effect of Degradation of Rat‐Liver Ribosomal RNA on the Structure of the Ribosomal Subunits
Authors: Hüvös Piroska , Gaál Ödön , Vereczkey László
Published: March 2005

The effect of pancreatic ribonuclease on the sedimentation properties of rat liver ribosomal RNA and subunits was studied. When undissociated ribosomes were treated with ribonuclease both the smaller and larger subunits were found to retain original sedimentation properties in spite of the fact, that they contained no more intact 18‐S or 28‐S RNA, respectively.

Treatment of ribosomes with EDTA caused different changes in the ribonuclease sensitivity of the two subunits: the smaller subunit became more sensitive towards nucleolytic degradation than the larger one. On the other hand, addition of EDTA to the ribosomes enhanced the ribonuclease sensitivity of 28‐S RNA to a greater extent than that of 18‐S RNA.
Reversible Chemical Modification of the Tryptophan Residues of Thioredoxin from Escherichia coli B
Authors: Holmgren Arne
Published: March 2005

Treatment of thioredoxin from Escherichia coli with formic acid saturated with HCl by the method of Previero et al. (1967), resulted in a redshift of the ultraviolet spectrum of the protein consistent with chemical modification of both tryptophans to 1‐formyl‐tryptophanyl residues. Concomitantly with the formylation the protein lost its redox activity in the enzymatic reactions with thioredoxin reductase and ribonucleotide reductase. Incubation of thioredoxin in formic acid alone had no effect on the spectrum or biological properties of the protein.

Gel chromatographic experiments on Sephadex G‐50 and G‐75 showed that the formylated thioredoxin was eluted well apart from native thioredoxin and existed in the form of high molecular weight aggregates. This indicated that major conformational changes of thioredoxin accompanied the conversion of the hydrophobic tryptophan residues into the more hydrophilic 1‐formyl‐tryptophans.

Treatment of 1‐formyl‐tryptophanyl thioredoxin at pH 9.5 in 6 M guanidine hydrochloride for 16 h resulted in almost complete regeneration of the ultraviolet spectrum of native thioredoxin. Also regenerated was more than 50% of thioredoxin activity in the reaction with thioredoxin reluctase. The results of this reversible chemical modification strongly indicate that the two tryptophan residues play a major role in maintaining the biologically active conformation of thioredoxin from E. coli.
Effects of Aldosterone and Other Steroids in vivo on Rat‐Kidney Cortex
Authors: Trachewsky Daniel , Majumdar Adhip. P. Nandi , Congote L. Fernando
Published: March 2005

An investigation of the effects of aldosterone, deoxycorticosterone, 9α‐fluorocortisol, and 17β‐estradiol administration, respectively, to adrenalectomized male rats on the polypeptide synthesis by and poly(U)‐directed binding of phenylalanyl‐tRNA to isolated renal cortical ribosomes has revealed several interesting facts. As well as causing an increase in the ability of the ribosomes to synthesize polyphenylalanine in the presence of poly(U), the three mineralcorticoids also increased the poly(U)‐directed binding of phenylalanyl‐tRNA to the ribosomes. 17β‐Estradiol treatment, however, caused a decrease in the ability of the ribosomes to incorporate phenylalanine into polyphenylalanine when compared to the untreated controls. There were also no significant differences in the amount of poly(U)‐directed binding of phenylalanyl‐tRNA to the ribosomes of the estrogen‐treated rats when compared to the untreated animals. Consequently, it was concluded that all four steroids had induced some alterations in the properties of the renal cortical ribosomes.

The effect of aldosterone and 17β‐estradiol on the thiol content of the nuclear proteins from rat kidney cortex showed an interesting correlation with the translational effects of both steroids. Thus aldosterone increased, and 17β‐estradiol slightly decreased, the thiol content of these proteins, measured as the incorporation of radioactive iodoacetate in whole nuclei in vitro. Under the experimental conditions used, the reaction was especially concentrated in discrete protein fractions of molecular weights of about 14000 and 42000. The effect of the hormones on the thiol content was not specifically concentrated in any of these protein fractions.
Kinetic Study of the Tricarboxylate Carrier in Rat Liver Mitochondria
Authors: Palmieri Ferdinando , Stipani Italo , Quagliariello Ernesto , Klingenberg Martin
Published: March 2005

The kinetics of citrate uptake by malate‐loaded mitochondria were measured using the inhibitor stop method and analysed for possible carrier mechanisms.

The citrate exchange is found to follow a first order reaction with a constant k of 1.18 min−1 and the corresponding rate of citrate uptake of 13.4 μmol × min−1× g protein−1 (at 0.5 mM citrate and 9 °C). The half time is 34 sec.

The temperature dependence of citrate exchange was measured in the range between 0 and 14 °C. An activation energy of 20.1 kcal and a Q10 of 3.6 can be calculated from the Arrhenius plot.

The concentration dependence of citrate exchange reveals hyperbolic saturation characteristics. The Km and V values for the rate of citrate uptake are 0.12 ± 0.01 mM and 22.5 ± 1.8 μmol citrate × min−1× g protein−1 respectively at 9 °C in 11 experiments.

The rate of citrate uptake has a pH optimum of about 7. The inhibition by higher pH is competitive with citrate. At pH lower than 7, V is decreased, indicating that the citrate transporting system is inactivated by acid pH.

The rate of citrate exchange is inhibited in a competitive manner by cis‐aconitate, threo‐Ds‐isocitrate, 1,2,3‐propanetricarboxylate, 1,2,3‐benzenetricarboxylate, citrate and propylcitrate. Other tricarboxylates, however, such as trans‐aconitate and 1,3,5‐pentanetricarboxylate have no effect on citrate exchange, even when added in large excess.

Succinate, malonate, oxaloacetate and oxomalonate inhibit the rate of citrate uptake. In contrast, glutarate, adipate, pimelate, 2‐oxoglutarate, aspartate and glutamate do not. Maleate, but not the trans isomer fumarate, also inhibits citrate uptake. The inhibition of citrate uptake is greater with malate than with succinate.

The rate of citrate uptake is also inhibited by the nonpenetrant anions phenylsuccinate, butylmalonate, benzylmalonate and pentylmalonate, previously thought to be specific inhibitors of the dicarboxylate carrier.

The inhibition of the rate of citrate uptake by dicarboxylates and their analogues is found to be competitive. The affinity for dicarboxylates is lower than for tricarboxylates (K1 is 0.7 mM for malate).

Phosphoenolpyruvate strongly inhibits the rate of citrate uptake, while P1 and pyruvate have only a slight effect. The inhibition by phosphoenolpyruvate is shown to be competitive (K1 is 0.11 mM).

It is concluded that the tricarboxylate carrier has a single binding site for tricarboxylates, phosphoenolpyruvate and dicarboxylates. The implications of these findings are discussed.
Isolation of Antitransaminase Antibodies with Selective Reactivity against Transaminase or Its Tryptic Hydrolysate
Authors: Katsiris Katherine D. , Dimitropoulos Constantin G. , Patramanis Iphigenia M. , Pavlatos Mary P. , Evangelopoulos Athanasios E.
Published: March 2005

Fractionation of antitransaminase or anti‐whole‐tryptic transaminase hydrolysate antibodies was accomplished by using solid conjugates of the enzyme or its whole‐tryptic hydrolysate with albumin. By mixing in different orders, the solid complexes with antiserums prepared after immunization of experimental animals with transaminase or its hydrolysate, four antibody fractions (A, B, C and D) were isolated.

The antibody reactivity of the isolated fractions was tested by micro‐complement fixation, inhibition of the catalytic activity of the enzyme and haemagglutination. It was found that fractions B and C exhibited more reactivity with the intact transaminase molecule, while A and D reacted more specifically with the various fragments of the enzyme hydrolysate.

After fractionation of the proteolytic hydrolysate through a Sephadex G‐25 column, it was found that high molecular weight peptides were more reactive against the antibody fractions A and D, which reacted specifically with the hydrolysate. On the contrary, low molecular weight peptides reacted more intensively with the antibody fractions B and C, specific for the intact transaminase molecule.

Heterogeneity of the isolated antibody fractions, as well as of the total population of antibodies against transaminase or its whole tryptic hydrolysate, has been demonstrated by analytical acrylamide disc electrophoresis and isoelectric focussing.
The Amino‐Acid Compositions of CNBr Fragment C1 from Antihapten Antibodies
Authors: Friedenson Bernard , Takeda Yasushi , Roholt Oliver A. , Pressman David
Published: March 2005

Most rabbit antibodies contain a methionine residue about 240 residues from the NH2‐terminus of the heavy chain sequence. Cleavage of heavy chains by CNBr at this methionine leads to the formation of a fragment, C1, which contains the variable portion of the H‐chain but is slightly longer than the Fd fragment. Published amino acid compositions of fractions from different antibodies, all taken to represent fragment C1, showed marked similarity. In this work it is shown that the primary reason for this similarity is that previous methods of isolating C1, by gel filtration using 6 M urea or 1 M acetic acid as solvent, led to the isolation of aggregates contaminated with an extensive section of the constant region. Six M guanidine · HCl prevented the formation of these aggregates and gel filtration in this solvent yielded a C1 fragment with an amino acid composition quite different from that of the contaminated aggregate. The amino acid compositions of the uncontaminated C1 fragments from antihapten antibodies from individual rabbits showed large differences. The differences were apparent because of the restricted heterogeneity of the antibodies being compared. Amino acid compositions of C1 fragments from γ‐globulins from individual uninjected rabbits showed appreciably less variation because, unlike the antibodies, the γ‐globulins were complex mixtures.
Regulation of Rat‐Liver Nucleotidase Activity Involving Deoxyribonucleic‐Acid Components as Allosteric Effectors
Authors: Fritzson Per
Published: March 2005

The activity of a highly purified nucleotidase from rat liver cytosol which splits certain 3′‐ and 5′‐nucleotides, was studied in the presence of each of 36 different nucleic acid constituents including 14C‐labeled and chemically modified nucleotides. It was found that the compounds either inhibited or had no effect on dephosphorylation of thymidine 5′‐phosphate, which was used as substrate for measuring 5′‐nucleotidase activity. On the other hand, the 3′‐nucleotidase activity, which was measured with uridine 3′‐phosphate as substrate, was stimulated 2.6 times by deoxyguanosine and, furthermore, by deoxyinosine, thymidine, deoxyuridine and inosine in decreasing order of effectiveness. Experiments with various phosphorylated derivatives of thymidine indicated that a 5′‐phosphoryl group increases the stimulating effect of the nucleoside whereas a 3′‐phosphoryl substitution reduces its ability to activate the enzyme. Di‐ and triphosphates were less stimulatory than the monophosphate. The results are interpreted to indicate that the same catalytic site is responsible for the hydrolysis of the 3′‐ and 5′‐nucleotides and that the enzyme possesses a regulatory site, topographically different from the catalytic site, at which the deoxyribonucleic acid constituents act as stimulators of enzyme activity.
DNA Methylases of Escherichia coli K12
Authors: Bouché Jean‐Pierre , Dubert Jean‐Marie
Published: March 2005

The overall DNA methylating activity of Escherichia coli K 12 has been purified about 1500‐fold. This procedure involves the preservation of high ionic strength during successive steps to keep the activity fairly stable. Both adenine and cytosine activities are purified in these conditions although their ratio varies greatly. Thermodenaturation and sedimentation studies on the most purified fraction show that the adenine and cytosine methylating enzymes are unrelated single protein species.

The sedimentation profile of the most purified activity is different from that observed with crude extracts or partially purified fractions. When heat inactivated, these fractions added to the purified enzyme restore the activity profile which they showed when native. Thus changes in sedimentation properties are due to a factor present in these fractions and lost in the course of the purification.