bluuebunny/crossref_metadata_2025_split huggingface dataset:
Effect of Hydroxylamine on Respiration and Oxidative Phosphorylation
Authors: WikstrÖM M. K. F. , Saris N.‐E. L. 
Published: March 2005

Rat liver mitochondria were used to study the effect of hydroxylamine on respiration and oxidative phosphorylation with succinate or tetramethyl‐p‐phenylenediamine plus ascorbate as substrate, and ATP phosphohydrolase activities.
With these substrates hydroxylamine inhibited respiration in state 3. Inhibition was released by various uncouplers of oxidative phosphorylation, including gramicidin and calcium ions. Respiration in state 4 was affected much less. Arsenate‐induced respiratory increase was effectively inhibited by hydroxylamine. Succinate oxidase activity in state 3 with ferricyanide as the electron acceptor was not inhibited by hydroxylamine.
Oxidative phosphorylation, with succinate as substrate, was inhibited more than respiration by hydroxylamine, with a concomitant decrease in the P/O ratio. Oxidative phosphorylation during succinate oxidation by ferricyanide was not affected by hydroxylamine.
2,4‐Dinitrophenol‐ and carbonylcyanide p‐trifluoromethoxy phenylhydrazone‐induced ATPase activities were not affected by hydroxylamine. The magnesium‐stimulated ATPase of detergent‐disrupted mitochondria was strongly inhibited but the gramicidin‐induced ATPase of intact mitochondria was only inhibited by about 30%. Under non‐respiring conditions hydroxylamine induced no ATPase by itself.
Our results indicate that the inhibition of oxidative phosphorylation by hydroxylamine is specific for coupling site 3, and in some ways resembles the effects of oligomycin. It is suggested that site specificity exists up to the level of X∼P in the partial reactions of oxidative phosphorylation.
Double Conditional Lethality: Temperature‐Sensitive and Amber Mutations in the Glucosyl Transferase Gene of Bacteriophage T2
Authors: Molholt B. , de Groot B. 
Published: March 2005

The isolation and characterization of two types of conditional lethal mutations in the α‐glucosyl transferase gene of bacteriophage T2 are described.T2gt7 is temperature‐sensitive for glucosyl transferase by the following criteria:

Glucosylation of T2gt7 DNA is temperature‐sensitive in vivo. Compared to T2+ DNA, DNA of the gt7 mutant is two‐thirds glucosylated at 30° and less than 10% glucosylated at 37°.
The glucosyl transferase induced by T2gt7 is temperature‐sensitive in vitro. Catalytic activity of the mutant enzyme is rapidly and irreversibly lost at temperature above 37° both in the presence and absence of substrates.
The gt7 mutation maps in the T2 gene for glucosyl transferase.

T2gt3 is an amber mutation in the T2 gene for glucosyl transferase. DNA of the gt3 mutant is glucosylated in E. coli K704 (supE+) and once glucosylated is not restricted upon growth in E. coli B carrying an amber suppressor. The gt3 mutation also maps in the T2 gene for glucosyl transferase.
On the Mechanism of Uncoupling in Chloroplasts by Ion‐Permeability Inducing Agents
Authors: Karlish S. J. D. , Shavit N. , Avron M. 
Published: March 2005

A general mechanism is presented for uncoupling of chloroplasts by compounds or combination of compounds which increase membrane permeability to both protons and alkali metal cations.Agents which supposedly increase the membrane permeability to protons only, such as 2,4‐dinitrophenol and carbonyl cyanide p‐trifluoromethoxy phenylhydrazone (FCCP) (at low concentrations), did not uncouple chloroplasts. Similarly, the antibiotics valinomycin, and nonactin and the synthetic compound LR6‐125, which apparently increase only the cation permeability of membranes, did not uncouple. However, combinations of dinitrophenol or of low concentrations of FCCP with anyone of the cation permeability inducing agents, led to full uncoupling of the chloroplasts. Studies on the passive permeability properties of the grana membrane, support the assumption that combinations of dinitrophenol or FCCP (at low concentrations), with valino‐mycin or nonactin or LR6‐125, increase the membrane permeability to both proton and cations. High concentrations of FCCP (10 μM) which alone uncoupled chloroplasts, also appeared to increase the permeability of the membrane to both protons and to cations, while low concentrations of FCCP, which did not uncouple alone, sufficed to increase the membrane permeability to protons. It is suggested that the uncoupling produced by the compounds used in this work is of the same type as that induced by the antibiotics, gramicidin and nigericin, both of which were suggested to increase the membrane permeability to both protons and cations.
Kinetics of the Alteration and Modification of DNA‐Dependent RNA‐Polymerase in T4‐Infected E. coli Cells
Authors: Seifert W. , Qasba P. , Walter G. , Palm P. , Schachner M. , Zillig W. 
Published: March 2005

The decrease in the activity of DNA‐dependent RNA polymerase in Escherichia coli after infection with bacteriophage T4 is the consequence of a structural change of the polymerase and not of the appearance of an inhibitor. The kinetics of this change were followed using polymerases highly purified at different times after infection (a) by measuring the specific enzymatic activity with T4‐ and calf thymus DNA as templates and (b) by following the appearance of the modified polymerase‐subunit in disc‐electrophoresis.The results allow one to differentiate between two consecutive processes:

A very rapid alteration which inactivates the RNA polymerase for T4‐DNA, but not for other DNA templates, and which does not require protein synthesis.
A structural modification which depends on protein synthesis and leads to a change in one subunit of the polymerase.

As shown by the use of the T4‐mutants am BL 292 and am N.134 neither gene 55 nor gene 33, both involved in the transcription of late genes, are participating in the modification reaction.
Specificity and Function of Exonuclease II in the Synthesis of Alternating Copolymers by DNA Polymerase
Authors: Lezius A. G. , Metz E. 
Published: March 2005

The poly d(A‐T) dependent synthesis of the analog alternating copolymer poly d(A‐4thioT) (in which thymidine is replaced by 4‐thiothymidine) by DNA polymerase of Escherichia coli is accompanied by hydrolysis of at least one mole of primer d(A‐T) per mole of product d(A‐4thioT) formed. Exonuclease II, the deoxyribonuclease associated with DNA polymerase, is responsible for the hydrolysis of the primer. Since the growing 3′ end of the primer is protected from hydrolysis during the enzymatic polymerisation the hydrolysis must occur at the free 5′ end. The enzymatic synthesis of poly d(A‐4thioT) on a poly d(A‐T) template proceeds in a two phase time course. The ratio of polymerisation versus hydrolysis is close to unity for the first rapid phase of synthesis. The temperature‐dependence of poly d(A‐T) and poly d(A‐4thioT) synthesis, represented by usual Arrhenius plots has a discontinuity at 15° and 25°, respectively. Exonuclease II exhibits a similar discontinuity at 26° under the same conditions. Above 25°, the synthesis of poly d(A‐4thioT) has a smaller apparent activation energy than the poly d(A‐T) synthesis, but the latter is twice to three times as fast. The obligatory hydrolysis of the primer offers a plausible explanation of why the synthesis of poly d(A‐4thioT) depends on the amount of poly d(A‐T) primer in a stoichiometric manner since the former copolymer is not a primer for extensive synthesis of poly d(A‐4thioT). A modified enzyme model for DNA polymerase is suggested, implicating the function of exonuclease II in providing an additional driving force for the unidirectional movement of the enzyme along the template. Emphasis is put on the energy contribution arising from formation of new base pairs during synthesis.The specificity of exonuclease II was also studied using as substrates such polymers which consist of poly d(A‐T) and poly d(A‐4thioT) in one half each. The polymer bearing the d(A‐4thioT)n at the 3′ half is readily hydrolysed by exonuclease II from both the 3′ and 5′ end synchronously at pH 7.2. Upon mercuration of d(A‐4thioT)n with p‐chloromercuribenzoate, preferentially the poly d(A‐T) part is hydrolysed from the 5′ end at a reduced rate. In contrast, no synchronism of nuclease reaction is observed with the polymer bearing d(A‐4thioT)n at the 5′ end. the latter being generally more resistant against hydrolytic attack by the enzyme.
Attempts at Purifying the Galactose Carrier from Galactose‐Induced Baker's Yeast
Authors: Haškovec C. , Kotyk A. 
Published: March 2005

An attempt was made to isolate the protein associated with the formation of the inducible d‐galactose‐transporting system in baker's yeast. Of the several fractions obtained by centrifugation of homogenized yeast cells at different speeds only the fraction sedimenting at 40000 x g and containing a predominance of membranes was found to bind d‐galactose, the dissociation constant being 0.2–0.6 mM. No galactokinase activity was found in this fraction.
The membrane proteins were solubilized using various technique but the amount of sugar bound by the solubilized proteins was lower than that bound by the intact membranes, the most promising appearing to be treatment with 1 M NaI.
Membrane proteins labelled by a double‐labelling technique were solubilized with 1 M NaI and separated on DEAE‐cellulose in a linear gradient of NH4HCO3. A peak of unequal labelling with 14C and 3H was identified both in normal baker's yeast and in a galactokinase‐less mutant and assumed to represent the inducible galactose carrier or a part of its transport system.
Preparation and Properties of Cyanoethylated Insulin
Authors: Bosshard H. R. , Jørgensen K. H. , Humbel R. E. 
Published: March 2005

Acrylonitrile has been shown to react not only with α‐ and ɛ‐amino groups, but also with imidazole‐nitrogens in beef insulin.
Experimental methods are described for the preparation and analysis of insulin derivatives with cyanoethylated amino groups and various amounts of cyanoethylated histidine residues.
Cyanoethylated derivatives of insulin can be separated from insulin by electrophoresis.
Cyanoethylated insulin represents a mixture of different molecular species as shown by ion exchange chromatography.
Residual histidine and carboxyethyl‐histidine residues were determined in acid hydrolysates of the isolated peptides B 1–7 and B 8–19. The amounts of blocked histidine B 5 were found to be greater than those of histidine B 10 by a factor of 2.7–3.6.
Under the applied experimental conditions immunological properties of cyanoethylated insulin with a residual histidine content of 50% are similar to those of the native hormone.
Depending on the degree of cyanoethylation, cyanoethylated insulin has a biological activity of 50–20% of that of insulin.
Evidence is presented for the important role of histidine residues for biological activity of insulin. The data presented suggest that histidine B 5 be particularly important.
An Improved Method for Isolation and Identification of Hydroxylated Testosterone Metabolites as Trimethylsilyl Ethers from Biological Material
Authors: Demisch K. , Staib W. 
Published: March 2005

The chromatographic behaviour of 21 testosterone metabolites and their derivatives hydroxylated at various positions is investigated on thin layer plates and on SE‐30 3.8% and XE‐60 1% columns during gas‐liquid chromatography.Distinct retention times of these compounds and their derivatives can give good evidence of identification. An improved method for the isolation and identification of hydroxylated testosterone metabolites from biological material is reported.
The Reduction of Flavins by Borohydride: 3,4‐Dihydroflavin
Authors: Müller F. , Massey V. , Heizmann C. , Hemmerich P. , Lhoste J.‐M. , Gould D. C. 
Published: March 2005

The reduction of flavins by BH4− is slow, but strongly catalyzed by light. The main product of the reaction is 1,5‐dihydroflavin, i. e. normal leucoflavin, which is rapidly reoxidized upon admission of air. Under continuous aeration, however, a second reaction, irreversible by oxygen in the dark, becomes dominant yielding a tetrahydroflavin which, upon exhaustive reaction of BH4−, is autoxidized to give a new dihydroflavin of structure I. This compound is stable toward further oxidation in the crystalline state and in solution with organic solvents. Compound I has to be considered as a formal 3,4‐dihydroflavin. It is photooxidizable to give starting flavin, reacts very slowly with S2O4= compared to flavoquinone (II), but is easily reduced by EDTA in the presence of light. The compound was also synthesized by an unequivocal route and found identical with the product obtained by BH4−‐reduction of lumiflavin. The light absorption spectrum of I shows absorption maxima at about 405, 320, 270, and 220 nm and is practically identical with that of the BH4−‐reduction product of the flavoproteins d‐ and l‐amino acid oxidase. The BH4−‐reduction products of the amino acid oxidases are catalytically active. From this it follows that position 4 of the flavocoenzyme is not involved in enzymatic dehydrogenation.The first photo‐excited triplet state of I has been investigated by optical and electron paramagnetic resonance spectroscopy. The structure of I was elucidated by means of infrared and nuclear magnetic resonance spectroscopy.
On the Problem of an Enzymatic Regulation of DNA Synthesis in Chlorella
Authors: Wanka F. , Poels C. L. M. 
Published: March 2005

Uridine kinase and thymidylate kinase were investigated in Chlorella cultures synchronized by alternating periods of 16 hours light and 8 hours darkness. The enzymes were activated and stabilized by mercaptoethanol and glutathione.Both enzyme activities increased periodically between the 8th and 18th hour after beginning of the light period. The increase was blocked in the presence of 15 μM actidione. When the light was turned off at the 12th hour the enzyme accumulation ceased after another 90 min.DNA synthesis took place between the 10th and 18th hour and subsequently ceased completely, although high dTMP kinase activities were still observed. The results do not support the hypothesis that DNA synthesis in Chlorella is regulated by thymidylate kinase.
The Sedimentation Properties of the Intestinal α‐Glucosidases of Normal Human Subjects and of Patients with Sucrose Intolerance
Authors: Eggermont E. , Hers H. G. 
Published: March 2005

The maltase‐isomaltase, maltase‐sucrase and glucoamylase of the human intestinal mucosa have been solubilized by Triton X‐100 and submitted to centrifugation in a mannitol gradient. Under these conditions, the three enzymes sediment together in a 10 S fraction; with one normal mucosa out of 10 which were investigated a supplementary 13 S peak of both glucoamylase and maltase‐isomaltase was observed.Several experimental conditions, like treatment of the extracts at 45° or solubilization of the α‐glucosidases by papain in the absence of potassium ions, induced an important inactivation of the maltase‐isomaltase and deep alterations in the sedimentation profile of the 3 glucosidases: the glucoamylase and the residual maltase‐isomaltase were distributed in 3 fractions with 13 S, 10 S and 6–7 S sedimentation coefficient, whereas the maltase‐sucrase was mostly present as a 7 S structure. In sucrose intolerance, the maltase‐sucrase is absent and the activities of the maltase‐isomaltase and glucoamylase are significantly reduced. The residual enzymes were ‐recovered as 13 S and 6 S structures instead of the normal 10 S species.These observations allow the conclusion that the three α‐glucosidases are naturally linked to each other in the normal mucosa and that the alteration of one of them induces a structural rearrangement of the 3 enzymes. The absence of maltase‐sucrase in patients with congenital intolerance to sucrose can be regarded as the result of a single mutation of the corresponding structural gene and as the cause of a reorganization of the two other α‐glucosidases; this reorganization can explain a partial loss of the enzymatic activity.
On the Redox Potentials of Ubiquinone and Cytochrome b in the Respiratory Chain
Authors: Urban P. F. , Klingenberg M. 
Published: March 2005

The standard redox potential of ubiquinone in submitochondrial particles is measured by equilibration with the succinate/fumarate system to about 65 mV at pH 7, with the redox ratio, n= 2. The pH dependence is ‐60 mV/Δ pH. The lower value measured for bound ubiquinone is discussed in comparison with the value found for isolated ubiquinone (104 to 112 mV, pH 7) determined in ethanol‐HCl.In parallel experiments the standard redox potential of cytochrome b was measured to 72.5mV at pH 7. The pH dependence is ΔE/Δ pH = 0 at pH < 6.8 and ‐ 60 mV ΔHp at pH > 6.8.
Conditions in vitro which Affect Respiratory Control and Capacity for Respiration‐Linked Phosphorylation in Brown Adipose Tissue Mitochondria
Authors: Grav H. J. , Pedersen J. I. , Christiansen E. N. 
Published: March 2005

Oxidative phosphorylation in brown adipose tissue mitochondria is greatly influenced by variations in the in vitro environment. Factors found to be of particular importance were the concentrations of serum albumin, of magnesium and EDTA, and the osmolality and the pH of the medium. The maximal P/O ratios obtained in an isoosmolar medium at pH 6.3, containing 2% serum albumin, 7.5 mM MgCl2, and 7.5 mM EDTA with pyruvate‐malate as substrate, and using the glucose‐hexokinase trapping system, were 2.8 for newborn guinea‐pigs and 2.6 for unweaned rats.Under otherwise equal conditions the P/O ratios obtained with 4‐week‐old guinea‐pigs were higher than those with the newborn.EDTA was a requirement for demonstration of respiratory control in brown adipose tissue mitochondria. Respiration with pyruvate‐malate or succinate (+ rotenone), and stimulated by inorganic phosphate and ADP, was found to be sensitive to oligomycin and to carbonyl cyanide phenylhydrazone. However, the small extent of stimulation by the phosphate acceptor system, the incomplete inhibition by oligomycin, and the fact that the respiratory rate after exhaustion of added ADP was higher than that prior to the addition of ADP, suggest that brown adipose tissue mitochondria are loosely coupled.The measured respiratory control ratios all increased from the newborn to the 3‐week‐old guinea‐pigs.Mitochondria from newborn and 6‐day‐old guinea‐pigs exhibited the phenomenon of “reversed acceptor control”. Thus, in the absence of added inorganic phosphate, ADP inhibited the respiration with pyruvate‐malate or with succinate. The reversed ADP control was abolished by carbonyl cyanide phenylhydrazone, but was not suppressed by treatment of the mitochondria with oligomycin.Inorganic phosphate affects respiration in a composite manner. With all of the preparations tested, inorganic phosphate stimulated respiration whether exogenous ADP was present or not. In the absence of added ADP, the respiration initially stimulated by inorganic phosphate entered a subsequent inhibitory phase.The implications of these findings to the control of brown adipose tissue thermogenesis are discussed.
Mutations Affecting the Repressibility of Arginine Biosynthetic Enzymes in Sacchromyces cerevisiae
Authors: Bechet J. , Grenson M. , Wiame J. M. 
Published: March 2005

A method is described for isolating mutants of Saccharomyces cerevisiae which have lost repressibility by exogenous arginine for ornithine transcarbamylase.Besides permeability mutants, three complementary classes of mutations were found: argRI, argRII and argRIII which are recessive and define three loci. No evidence for a linkage between any of these three loci or with the gene coding for ornithine transcarbamylase has been obtained.Strains bearing mutations at either of these loci cannot be distinguished on a phenotype basis: after growth on minimal medium, the l‐ornithine carbamoyl transferase activity is twice that of the wild type strain; the mutations modify neither the growth rate nor the permeability to arginine.The mutations might affect the structure of an hetero‐polypeptidic aporepressor.The level of ornithine transcarbamylase in diploids is proportional to the number of argF+ genes in regulated as well as in non‐regulated cells.
The Subunit Structure of Phenylalanyl‐tRNA Synthetase of Escherichia coli
Authors: Kosakowski M. H. J. E. , Böck A. 
Published: March 2005

Fingerprints of tryptic digests of phenylalanyl‐tRNA synthetase of Escherichia coli NP 3 suggest that the smallest identical subunit is one fourth of the size of the native protein (mol. wt. 181000). Determination of the subunit size by gel electrophoresis reveals a molecular weight of 43000 ± 2000. By the criteria employed the enzyme therefore seems to be composed of four identical subunits.Phenylalanyl‐tRNA synthetase which has been denatured by treatment with 8 M urea is able to reform active enzyme indistinguishable by its size from the native protein. Again, a size of about 45000 was determined for the subunits active in the association reaction.
Chemical Structure of Tubular and Glomerular Basement Membranes of Human Kidney
Authors: Mahieu P. , Winand R. J. 
Published: March 2005

A method involving the use of sieves and differential centrifugation for the preparation and separation of basement membranes from human renal tubules and glomeruli is described. Electron microscopic examination of the material obtained shows the glomerular membrane as a single dense structure, while the tubular membrane is composed of two dense layers separated by a space filled with loose fibrils exhibiting the same periodicity as collagen. The carbohydrate composition of both types of membranes is the same but tubular membranes have a lower content of some specific sugars. Differences also exist in amino acid composition. The content in hydroxylysine is higher in the glomerular membrane while the content in hydroxyproline is higher in the tubular membrane.Gel electrophoresis after solubilization of the material demonstrates the presence of two, may be three, major proteins.
Degradation of Phenols by Intact Cells and Cell‐Free Preparations of Trichosporon cutaneum
Authors: Neujahr Halina Y. , Varga Janos M. 
Published: March 2005

A yeast strain, enriched and isolated from soil using phenol as major carbon source, was identified as Trichosporon cutaneum. After growth on phenol washed cell suspensions of the organism oxidized phenol and some phenol derivatives without delay, others after varying lag periods.Cell‐free preparations from the yeast strain were capable of hydroxylating various phenols as judged by a substrate‐ and NADPH‐dependent oxygen uptake. Certain properties and the cofactor requirement of this hydroxylating system were investigated. Diphenols were hydroxylated at rates considerably higher than monophenols. Mono‐halogen substituted phenols were hydroxylated at rates comparable to that of phenol hydroxylation. The relative hydroxylating activities with respect to different phenol derivatives were markedly changed after 3–5 weeks' storage, thus indicating the presence of several hydroxylating enzymes. The cell‐free preparations also contained the enzyme catechol 1,2‐oxygenase.Results of studies with intact cells and cell‐free preparations indicated that not only phenol but also resorcinol, quinol and catechol were further hydroxylated prior to ring fission.Catechol when supplied as substrate of catechol 1,2‐oxygenase preparations, was oxidized by the “ortho type” of ring fission. The enzyme involved has broad substrate specificity.