bluuebunny/crossref_metadata_2025 huggingface dataset:
Internal pH, ΔpH, and the Kinetics of Electron Transport in Chloroplasts
Authors: Bamberger Elchanan S. , Rottenberg Hagai , Avron Mordhay
Published: March 2005

The relation between the rate of electron transport, ΔpH, and internal pH was studied in lettuce chloroplasts, by following, in parallel, the fluorescence quenching of 9‐aminoacridine and the rate of electron transport. At low external pH (< 8.0) there was an initial burst in the rate of electron transport (R1) followed by a slower steady‐state rate (R2). However, at high external pH (> 8.5) R1 was slower than R2. Nevertheless, when analyzed as a function of internal pH, both R1 and R2 showed the same dependence with a maximal rate observed around internal pH 5.0. Thus, at low external pH, the initial pH is initially around 5.0 and, therefore, R1 is high, but as ΔpH is increased, the internal pH falls below 5.0 and R2 is lower. At high external pH, the internal pH is initially above 5.0 and, therefore, R1 is relatively low. As ΔpH increases the internal pH is lowered toward 5.0 and R2 becomes faster. A similar conversion of the ratio R1/R2 from R1/R2 > 1 to R1/R2 < 1 was also observed at constant external pH by varying the light intensity, or concentration of the uncoupler nigericin.

In the presence of nigericin or at low light intensities, the maximal rate was shifted to higher internal pH values. At internal pH values above 5.0 in the presence of nigericin, the rate of electron transport was inversely proportional to ΔpH. These results indicate that the internal pH is not the only parameter that controls the rate of electron transport. It is shown that when all these diverse results are plotted as a function of an average pH (between the internal and external pH) they show a single optimum. Thus, it is suggested that the rate controlling site is embedded in the thylakoid membrane, and is a function of both the internal and external pH. This membrane pH and the size of ΔpH seem to be the major factors controlling the rate of electron transport in chloroplasts.
The Problem of Cation‐Binding Sites in the Energized Membrane of Intact Mitochondria
Authors: Colonna Raffaele , Massari Stefano , Azzone Giovanni Felice , Ziche Bruno , Veronese Paolo
Published: March 2005

Weak bases, such as the acridine and the aminophenazine dyes, which undergo protonation in the chromophoric group, are actively bound in sonicated fragments. On the other hand strong bases such as the divalent cations, the safranine and the cyanine dyes, which carry a permanent charge, are actively bound in intact mitochondria. In the latter case the process results in stimulation of the respiration, H+ ejection, and a large change in absorbance, which is attributed to a stacking of the dyes. The effects are inhibited or reversed by uncouplers, and insensitive to nigericin.

Four alternatives are considered: accumulation in the inner aqueous space; binding to matrix proteins; binding to the outer membrane surface; and binding to sites in the membrane fabric. The view that the energized membrane contains high affinity sites for cationic dyes is supported by: comparison of the spectra of the membrane‐bound and polyanion‐bound dye; competition with divalent cations; osmotic activity; and dependence of the spectral shift on the protein/dye ratio. The data are in accord with the concept that the mitochondrial membrane acts as a fixed negative‐charge system where electrostatic interactions occur in microenvironments of low polarity.
Interprétation du rôle antilipolytique de la tétracycline
Authors: Counis Raymond , Raulin Jeanine , Koumanov Kamen , Infante Recaredo
Published: March 2005

In order to observe whether or not the lipolytic inhibitory effect due to the injection of tetracycline to rats could be related to the decrease of enzymic reactivity in the membrane, adenylate cyclase activities were determined in adipose cell ghosts.

Basal, noradrenaline and glucagon‐stimulated activities were significantly depressed by added tetracycline (in vitro experiments). It appeared that tetracycline could influence both V (inhibition of catalytic subunit) and the apparent affinity for hormones, as indicated on dose‐response curves. Fluoride‐stimulated adenylate cyclase activity was also affected, with no change in Km. Phosphodiesterase activity was identical whatever the concentration of added tetracycline.

The adenylate cyclase inhibition by added tetracycline was not related to any chelation effect on Mg2+ of this anionic‐antibiotic compound.
Yeast 3‐Phosphoglycerate Kinase
Authors: Roustan Claude , Brevet Annie , Pradel Louise‐Anne , van Thoai Nguyen
Published: March 2005

The mechanism of the transphosphorylation reaction catalyzed by yeast 3‐phosphoglycerate kinase has been studied by means of spectrophotometric investigations.

The binding sites, one for the nucleotide substrates and one for the acceptor, have been characterized. The interaction of nucleotide substrates with the enzyme, which is not metal‐dependent, resulted only in a perturbation of the spectrum of the nucleotide chromophore characterized by hypochromic and red shift effects. The hydrophobicity of this site was estimated by using a reporter group reagent, 2‐(dansylamino)ethyl monophosphate. The interaction of 3‐phosphoglycerate with its corresponding site produced perturbation of a tyrosyl chromophore. Furthermore, spectrophotometric studies evidenced the formation of binary and ternary complexes in accord with a random reaction mechanism proceeding via interconversion of ternary complexes.

We also observed partial exchange reaction between ATP and ADP. Properties of this exchange reaction have been investigated. The partial reaction points out the existence of a covalent phosphoryl‐enzyme intermediate.
Covalently Bound Flavin in d‐6‐Hydroxynicotine Oxidase from Arthrobacter oxidans
Authors: Brühmüller Margarete , Decker Karl
Published: March 2005

d‐6‐Hydroxynicotine oxidase from Arthrobacter oxidans was previously shown to contain FAD covalently bound to the N‐3 of a histidyl residue of the apoprotein via the 8α‐methylene group of the flavin nucleus. For elucidating the structure around the flavin centre a tryptic‐chymotryptic FAD‐peptide from d‐6‐hydroxynicotine oxidase was isolated and purified. The amino acid sequence of this FAD‐peptide was found to be Ser‐Gly‐Gly‐Asn‐Asn‐Pro‐Asp‐His‐Tyr‐(Gln‐Pro)‐Ala.
The Steady‐State Kinetics of Glyoxalase I from Porcine Erythrocytes
Authors: Mannervik Bengt , Górna‐hall Barbara , Bartfai Tamás
Published: March 2005

The steady‐state kinetics of glyoxalase I from porcine erythrocytes were subjected to a detailed investigation. Three chemical species, methylglyoxal, glutathione, and their hemimercaptal adduct, are in equilibrium and the concentrations of these components were varied over a wide range to evaluate their influence on the velocity of the glyoxalase‐I‐catalyzed reaction. The experimental data were fitted by nonlinear regression methods to a number of alternative mathematical models, corresponding to possible enzymatic mechanisms, and the discrimination between the rival models was based on statistical criteria. The selection of the best model was supported by a procedure of weighting for discrimination and by examination of subsets of experimental data. The best model is interpreted as a random pathway mechanism composed of a two substrate branch involving the hemimercaptal.
Mutants of Escherichia coli K12 Defective in Oxidative Phosphorylation
Authors: Schairer Hans U. , Gruber Dorothee
Published: March 2005

A mutant of Escherichia coli K12 has been isolated with a defect in oxidative phosphorylation. Though the mutation does not affect the activity of the Mg2+, Ca2+ ‐activated ATPase the mutant strain does not show ATP‐dependent transhydrogenase activity. Also, in contrast to the wild‐type strain, active β‐galactoside transport is abolished after inhibition of respiration by cyanide. Mapping of the mutation locus shows that it lies between asn and ilv on the E. coli chromosome.
Reversible Photolysis of the Purple Complex in the Purple Membrane of Halobacterium halobium
Authors: Oesterhelt Dieter , Hess Benno
Published: March 2005

A suspension of the purple membrane from Halobacterium halobium in concentrated salt solutions saturated with diethylether is bleached by light. During this process, the purple complex is converted in to a compound absorbing maximally at 412 nm (412‐nm complex). The purple complex reappears in the dark with a rate constant of 0.04 s−1 at 20°C.

The initial rate of formation of the 412‐nm complex is proportional to the intensity of the bleaching light and a quantum yield of 0.79 is measured. The action spectrum of the photolytic reaction is identical to the absorption spectrum of the purple complex.

The light reaction is temperature independent. The regeneration of the purple complex requires an activation energy of 11.4 kcal × mol−1.

Illumination causes a decrease in the tryptophan fluorescence of the membrane. Regeneration of the purple complex restores the original fluorescence level. The half times of both processes correlate with the half times observed by absorption spectroscopy.

The photochemical reaction is accompanied by release of protons during bleaching and uptake of the same amount of protons during regeneration of the purple complex. The half times of the measured pH changes again correspond to the one observed by absorption spectroscopy. The rapid release of protons upon illumination is temperature independent. For the dark uptake of protons an activation energy of 14 kcal × mol−1 has been determined. The significance of this isolated photochemical system is discussed with respect to a possible function of the purple membrane in vivo.
NADP‐Linked Isocitrate Dehydrogenase from Beef Liver
Authors: Carlier Marie‐France , Pantaloni Dominique
Published: March 2005

NADP‐dependent isocitrate dehydrogenase has been 500‐fold purified from beef liver; the protein is homogeneous in the ultracentrifuge as well as in electrophoresis. From beef liver sub‐cellular fractionation, the enzyme is found to be cytoplasmic. Some physical parameters of the enzyme have been determined. The enzyme consists of two subunits having the same molecular weight of 48000 ± 5000. From equilibrium sedimentation studies, it is shown that the dimer dissociates at low concentration, and that the dimeric form is induced by the substrate metal2+ isocitrate complex; the reassociation is a low phenomenon, which results in a lag time preceding the establishment of the catalytic activity at the steady state; this lag decreases when the enzyme is preincubated with the substrate. A study of the number of –SH groups shows that 2‐SH among 5 in the protomer, are implicated in the catalytic activity and protected by metal2+‐isocitrate against the attack of the classical sulfhydryl reagents.
Synthesis and Processing of Ribosomal RNA in Cultured Plant Cells
Authors: Cox Brian J. , Turnock Geoffrey
Published: March 2005

Four species of rapidly labelled, high‐molecular‐weight RNA have been extracted from partially purified nuclei of cultured sycamore cells. The molecular weights of these RNA species have been tentatively estimated by comparison of their electrophoretic mobilities in polyacrylamide gels with thóse of HeLa 45‐S and 32‐S RNA as 3.4 × 106, 2.4 × 106, 1.4 × 106 and 1.0 × 106. All four species of RNA are methylated and the components with molecular weights of 2.4 × 106, 1.4 × 106 and 1.0 × 106 have a pattern of labelling consistent with their being precursors of ribosomal RNA. Labelling studies with [14C]uridine and [Me‐3H]methionine have indicated that the 2.4 × 106‐Mr molecule is cleaved to give the 1.4 × 106‐Mr molecule and the 1.0 × 106‐Mr molecule which then give rise to 1.3 × 106‐Mr and 0.7 × 106‐Mr ribosomal RNAs respectively. The kinetics of labelling of the 3.4 × 106‐Mr component, however, show that it cannot be an obligatory precursor of the 2.4 × 106‐Mr RNA and the implications of this observation for the process of transcription of the ribosomal genes and the method of cleavage of the transcription products are discussed. The kinetics of labelling of the RNA with [Me‐3H]methionine have indicated that methylation occurs during the synthesis of the largest precursor molecules, as is known to be the case in mammalian tissues but, in contrast to the mammalian system, there also appears to be a significant amount of direct methylation of the 1.4 × 106‐Mr precursor RNA.
Purification et caractérisation partielle des deux malate synthases d'Escherichia coli
Authors: Falmagne Paul , Wiame Jean‐Marie
Published: March 2005

A simple procedure is reported for the purification of the two malate synthases of Escherichia coli involved in the use of acetate (A) and glycolate (G), respectively, as carbon source. Both isoenzymes have been isolated simultaneously and in equally high amounts from a strain constitutive for the glyoxylate cycle, after growth on glycolate. After separation from each other by column chromatography on DEAE‐cellulose, the malate synthases have been purified by heating, ammonium sulfate precipitation and molecular sieve chromatography on a Sephadex G‐200 column, in the constant presence of their specific protectors, glycolate for malate synthase A, and oxalate for malate synthase G.

The purified preparations so obtained appeared to be homogeneous on the basis of analytical ultracentrifugation and moving‐boundary electrophoresis.

The sedimentation coefficient at infinite dilution was 4.6 S for malate synthase A and 5.2 S for malate synthase G; their electrophoretic mobilities at pH 8 were 4.3 X 10−5 cm2 V−1 s−1 and 4.1 × 10−5 cm2 V−1 s−1, respectively.

The apparent molecular weight of malate synthase A, estimated by Archibald's method, was 54000; that of malate synthase G, estimated by light scattering, was 52000.

Threonine and serine have been found, by Sanger's method, to be N‐terminal residues of malate synthases A and G, respectively.
Effect of Tightly Bound Divalent Cation on the Equilibria between G‐Actin‐Bound and Free ATP
Authors: Strzelecka‐Golaszewska Hanna
Published: March 2005

The effects of various divalent cations on the rate of exchange of G‐actin‐bound ATP and on the rate of dissociation of ATP from G‐actin under conditions of a continuous removal of free ATP from the protein solution by Dowex‐1 were investigated. The order of inhibitory effects of equal concentrations of various cations on the ATP‐exchange rate did not parallel the order of affinities of the cations to the high affinity binding site in actin, inconsistent with what was expected on the basis of earlier findings. A kinetic analysis of the exchange of ATP in G‐actin preparations containing either Ca2+ or Mg2+ as the bound cation in the presence of various concentrations of the respective free cation revealed that the rate constants governing the equilibria between free and bound ATP in G‐actin solutions are markedly influenced by the kind of the G‐actin‐bound cation. The dependence of the rate of release of the bound ATP during incubation with Dowex‐1 on the kind of the bound cation (Ca2+, Mg2+ or Mn2+) led to the same conclusion. It is suggested that the conformation of the ATP binding site depends upon the cation bound at the high affinity site in actin.
Biosynthesis of the Crystal Protein of Bacillus thuringiensis var. tolworth
Authors: Herbert Barry N. , Gould Hannah J.
Published: March 2005

Our previous work showed that the crystal protein of Bacillus thuringiensis var. tolworth, a highly potent toxin for Lepidopteran larvae consists of two polypeptide components of molecular weights 55000 (“component A”) and 120000 (“component B”) in the molar ratio 2:1. In this study we demonstrate by several criteria that A and B are synthesized de novo, synchronously, for a predetermined and relatively brief period, during the 24‐h growth cycle in culture, about 17 h after innoculation of the culture, midway between the cessation of growth and cell lysis. This conclusion is supported by the results of the following types of experiments: 1. pulse‐labelling of cultures of B. thuringiensis with radioactive amino acids and subsequent analysis of the radioactivity in the separated crystal protein components; 2. antigenic analysis of protein isolated from cultures at different times, both by precipitin reactions and immunodiffusion in Ouchterlony plates, using antiserum against purified crystal protein and 3. bioassay of the toxicity of similar protein preparations to the larvae of Pieris brassicae.
Detection of α‐Glycosidic Bonds in the Ganglioside GM1 by Stereospecific Enzymatic Degradation
Authors: Werries Eckhard , Buddecke Eckhart
Published: March 2005

The monosialosyl‐N‐tetraglycosyl‐ceramide (GM1) isolated from ox brain and submitted to a stepwise degradation by specific α‐ and β‐glycosidases isolated and purified from ox spleen.

By the sequential action of β‐galactosidase and β‐N‐acetylhexosaminidase, 100% of the terminal galactose and the N‐acetylgalactosamine residues were removed, while the corresponding α‐glucosidases were completely ineffective. When the sialic acid was removed from the remaining sialated dihexosyl ceramide, the internal galactose residue becomes suspectible again to β‐galactosidase which removed up to 89%, but α‐galactosidase also hydrolyzed 2% of the internal galactose residues. A subsequent action of α‐glucosidase hydrolyzed 3% whereas β‐galactosidase released 47% of the remaining glucose moiety.

The results reported prove a β‐configuration of the glycosidic‐linked terminal galactose and of the penultimate N‐acetylgalactosamine moiety of ganglioside GM1 throughout. A β‐configurated glycosidic linkage is also predominant for the internal galactose and the glucose residues, but the presence of a small percentage of α‐glycosidic bonds must be concluded from their susceptibility to α‐galactosidase and α‐glucosidase, respectively.
Bacterial‐Protein Synthesis
Authors: Burns Donald J. W. , Cundliffe Eric
Published: March 2005

Bacterial protoplasts, treated with fusidic acid, have been used to examine the modes of action of various other antibiotics in vivo. This system, in which ribosomal function is severely impeded, is particularly useful for studying the actions of drugs which do not inhibit protein synthesis potently in vivo. One such drug is spectinomycin which is here shown to be an inhibitor of trans‐location; similar results were obtained with lincomycin and celesticetin. Althiomycin, conversely, was found to inhibit the peptidyl transferase reaction. Supplementary evidence for these conclusions was obtained by examining the effects of the drugs upon the isolated peptidyl transferase reaction in vivo and by studying their effects upon polyribosome stability. We conclude that lincomycin and celesticetin inhibit translocation only on those ribosomes which bear nascent peptides not exceeding a critical, but short, chain length.
A New Group of Chromatin‐Associated Proteins with a High Content of Acidic and Basic Amino Acids
Authors: Goodwin Graham H. , Sanders Clive , Johns Ernest W.
Published: March 2005

A new group of chromatin‐associated proteins having a high content of acidic and basic amino acids have been isolated from the 0.35 M NaCl‐extractable proteins from calf thymus. This new group, designated “high‐mobility group” proteins have been partially separated and some interesting new proteins identified. One such protein contains over 55% acidic and basic amino acids.
Acyl‐Donor Specificities of Partially Purified 1‐Acylglycerophosphate Acyltransferase, 2‐Acylglycerophosphate Acyltransferase and 1‐Acylglycerophosphorylcholine Acyltransferase from Rat‐Liver Microsomes
Authors: Yamashita Satoshi , Hosaka Kohei , Numa Shosaku
Published: March 2005

1. A partially purified preparation containing 1‐acylglycerophosphate acyltransferase, 2‐acylglycerophosphate acyltransferase and 1‐acylglycerophosphorylcholine acyltransferase was obtained from rat liver microsomes which were resolved with a nonionic detergent, Triton X‐100, in glycine buffer pH 8.6. The purification procedure involves molecular sieve chromatography and sucrose density gradient centrifugation.

2. The partially purified 1‐acylglycerophosphate acyltransferase exhibits a significant specificity for mono‐ and dienoic fatty acyl‐CoA thioesters. The effectiveness of the various acyl‐donors examined is as follows: oleyl‐CoA > linoleyl‐CoA ∼ palmitoleyl‐CoA > palmityl‐CoA ∼ myristyl‐CoA; arachidonyl‐CoA, stearyl‐CoA and lauryl‐CoA are almost ineffective. The newly formed ester bond is sensitive to the action of phospholipase A2.

3. The partially purified 2‐acylglycerophosphate acyltransferase shows a predominant selectivity for saturated acyl‐CoA thioesters. Stearyl‐CoA is the most effective acyl‐donor, and palmityl‐CoA is utilized fairly efficiently.

4. The partially purified 1‐acylglycerophosphorylcholine acyltransferase is specific for arachidonyl‐CoA.

5. These findings, together with our previous studies on the acyl‐donor specificity of glycerophosphate acyltransferase, indicate that the asymmetric fatty acid distribution found in naturally occurring glycerolipids results mostly from the specific at the step of phosphatidic acid synthesis and partly from the deacylation‐reacylation cycle operating after the synthesis de novo of phospholipids.
Influence of Membrane Lipids on the Regulatory Properties of UDP‐Glucuronyltransferase
Authors: Zakim David , Goldenberg Jovita , Vessey Donald A.
Published: March 2005

The maximal potential activity of UDP‐glucuronyltransferase is constrained by the structure of the phospholipid environment in intact microsomal membranes. This constraint can be relieved by treatment of microsomes with phospholipase A. As shown by the data in this paper, however, relief of constraint is associated with a loss of specificity in the binding of UDP‐sugars at the UDP‐glucuronic acid site of UDP‐glucuronyltransferase. As a result; several UDP‐sugars which have no effect on the activity of the untreated enzyme act as inhibitors of the unconstrained, phospholipase A‐treated form of UDP‐glucuronyltransferase. In addition to the loss of specificity of substrate binding, the phospholipase‐A‐treated form of UDP‐glucuronyltransfease cannot be activated by UDP‐N‐acetylglucosamine, which is a positive K‐type of allosteric effector for the untreated form of the enzyme. UDP‐N‐acetylglucosamine, in fact, is an inhibitor of the phospholipase‐A‐treated from of the enzyme. In contrast to the effects of other UDP‐sugars, inhibition by UDP‐N‐acetylglucosamine seems to result from the binding of UDP‐N‐acetylglucosamine at an allosteric site rather than the active site. Inhibition of the phospholipase‐A‐treated form of UDP‐glucuronyltransferase by UDP‐N‐acetylglucosamine and other UDP‐sugars is additive.
Interaction of Mixed‐Function Oxidation with Biosynthetic Processes
Authors: Scholz Roland , Hansen Werner , Thurman Ronald G.
Published: March 2005

Gluconeogenesis from lactate was studied in perfused livers from phenobarbital‐pretreated rats fasted for 24 h. Maximal rates of glucose production obtained with lactate concentrations of more than 2 mM were suppressed to about 50% following the addition of aminopyrine, a substrate for NADPH‐utilizing mixed‐function oxidations. Submaximal rates (i.e. with 0.5–2 mM lactate) or gluconeogenesis from dihydroxyacetone were only slightly affected by aminopyrine, whereas no inhibition was observed at low gluconeogenic rates. The data are consistent with the following hypothesis. In the presence of active NADPH‐utilizing processes (such as mixed‐function oxidation of aminopyrine) a futile cycle involving malic enzyme occurs in the pathway of gluconeogenesis which is compensated for by an increased flux through the pyruvate carboxylase reaction. Thus, gluconeogenesis is suppressed when maximal activity of pyruvate carboxylase is reached. It is assumed that the observed inhibition of gluconeogenesis is not specific for mixed‐function oxidation, but may be an example for a more general mechanism of metabolic interdependences involving the extramitochondrial NADPH pool.
Synthesis of Lens Protein in vitro
Authors: Strous Ger J. A. M. , van Westreenen Hanske , Bloemendal Hans
Published: March 2005

N‐terminal acetylation of the lens protein α‐crystallin has been studied in a lens cell‐free system. This system is capable of synthesizing de novoα‐crystallin, a protein with acetylmethionine as N‐terminus. When [35S]Met‐tRNAfMet is used as radioactive precursor only the α‐crystallin chains are labeled in the N‐terminal position. This phenomenon enables the study of the process of acetylation. Newly synthesized polypeptides could be separated according to chain length. Electrophoretic analysis of thermolytic peptides revealed that acetylation of the N‐terminus occurs while the chain is still on the ribosome.

It is concluded that the N‐terminal acetylation takes place after chain initiation and before completion of the polypeptide chain.
Inhibition of Enzymes which Interact with Citrate by (—)Hydroxycitrate and 1,2,3,‐Tricarboxybenzene
Authors: Cheema‐Dhadli Surinder , Halperin Mitchell L. , Leznoff Clifford C.
Published: March 2005

The effects of two citrate analogues, (—)hydroxycitrate and 1,2,3‐tricarboxybenzene, were studied using rat liver enzymes which interact with citrate.

The most pronounced effect of 1,2,3‐tricarboxybenzene was inhibition of acetyl‐CoA carboxylase (Ki 20 μM). It also inhibited the mitochondrial citrate transporter (50% inhibition at 3 mM), but was not a substrate for this transporter.

ATP‐citrate lyase was markedly inhibited by both free (—)hydroxycitrate (Ki 8 μM) and (—)hydroxycitrate lactone (Ki 50 to 100 μM). Acetyl‐CoA carboxylase was activated by both the forms of (—)hydroxycitrate (Ka 0.7 mM and 1.6 mM, respectively). (—)Hydroxycitrate is a substrate for the mitochondrial citrate transporter, but its rate of transport is less than 10% of that of citrate.

Other citrate metabolizing enzymes also were inhibited by 1,2,3‐tricarboxybenzene and (—)hydroxycitrate but much higher concentrations were required.
Replication of Bacteriophage M‐13
Authors: Schneck Peter K. , Staudenbauer Walter L. , Hofschneider Peter Hans
Published: March 2005

Nalidixic acid, a specific inhibitor of bacterial DNA synthesis, has no effect on the conversion of M‐13 parental or progeny single strands into double‐stranded replicative form molecules. However, the double strand to double strand replication of the replicative forms as well as the synthesis of single‐stranded M‐13 DNA are completely inhibited. As in the case of M‐13, the formation of the parental replicative form of ΨX 174 is also insensitive to nalidixic acid. It is concluded that nalidixic acid (or some metabolic product thereof) interferes with DNA replication only if a double‐stranded template is involved, but has no effect on the synthesis of a complementary strand on various single‐stranded templates.
Poly(Adenosine Diphosphoribose) Polymerase in Mammalian Nuclei
Authors: Stone Peter R. , Shall Sydney
Published: March 2005

1. The ability of isolated LS‐cell nuclei to incorporate [adenine‐3H]‐NAD into acid‐insoluble products is demonstrated.

2. The incorporation has a pH optimum at 8.5 and an apparent temperature optimum at 25 °C.

3. The optimum Mg2+ concentration is dependent upon the NAD concentration; at 1.5 μM NAD it is 2 mM; at 1.5 mM NAD it is 3 mM; and at 9 mM NAD it is above 20 mM Mg2+ Mg2+ can be replaced by Ca2+.

4. Dithiothreitol or mercaptoethanol, up to 10 mM, enhances the incorporation of NAD.

5. With the optimal conditions the incorporation of NAD (V) is 128 nmol NAD×5 min−1×mg DNA−1 and the Km is 1.47 ± 0.18 mM. Nicotinamide inhibits the incorporation competitively with a Ki of 14.3 ± 1 μM and thymidine inhibits competitively with a Ki of 32.5 μM.

6. The incorporation is not affected by added DNA or poly(U). It is not affected by ribonuclease but is inhibited by about 50% on treatment with deoxyribonuclease.

7. The enzyme system is unstable and the decay is temperature‐dependent; there is little decay at 0 °C but above this temperature the rate of decay follows an Arrhenius relationship. The initial rates of enzyme activity also show a temperature dependence with a linear Arrhenius plot. The temperature optimum at 25 °C is the arithmetic result of these two opposing processes.
Adenylate‐Cyclase Activity of Rat‐Liver Plasma Membranes
Authors: Leray Françoise , Chambaut Anne‐Marie , Perrenoud Marie‐Lise , Hanoune Jacques
Published: March 2005

The adenylate‐cyclase activity from a rat‐liver plasma‐membrane preparation purified according to the Neville's procedure was increased 10 to 12‐fold by glucagon, but the addition of epinephrine, at any concentration, failed to activate the enzyme. When the membranes were isolated from adrenalectomized animals, the responses of the enzyme to glucagon and NaF were enhanced and mainly the adenylate‐cyclase activity became sensitive to epinephrine with an optimal effect at 0.05 mM epinephrine. Intraperitoneal administration of cortisol for 2 days to the adrenalectomized animals reversed the effect of the hormonal deprivation. But addition of cortisol in vitro was without effect.

The use of liver plasma membranes from adrenalectomized rats is therefore proposed as a tool to study the adenylate‐cyclase activation by both glucagon and epinephrine. The epinephrine‐sensitive adenylate‐cyclase activity was completely inhibited by propranolol but not by phentolamine, thus confirming that the metabolic effect of epinephrine is linked to a β‐like receptor. Epinephrine and glucagon effects upon adenylate‐cyclase activity were both inhibited by concentration of calcium above 0.01 mM but differed with respect to the Mg‐ATP concentration. The two hormonally stimulated adenylate‐cyclase activities were found to be additive.

The effect of epinephrine upon adenylate‐cyclase activity was more sensitive to stimulation by low concentration of GTP and other nucleotides than the effect of glucagon. Addition of insulin in vitro at any concentration failed to alter the adenylate‐cyclase activity regardless of the concentration of magnesium, the presence of glucose or GTP.
Sex‐Dependent Differences in Derangements of Biliary‐Steroid‐Excretion Patterns Produced by Cyanoketone in Rats
Authors: Begue Rene‐Jean , Goldman Allen S. , Gustafsson Jan‐Åke , Gustafsson Sven A.
Published: March 2005

Intramuscular injections of 25 mg/kg body weight of 2α‐cyano‐4,4,17α‐trimethyl‐5‐androsten‐17β‐ol‐3‐one (cyanoketone) produces and immediate cessation of excretion of practically all corticosteroid metabolites (3α, 11β,15α,21‐tetrahydroxy‐5α‐pregnan‐20‐one and 3α‐ and 3β,11β, 21‐trihydroxy‐5α‐pregnan‐20‐one) in bile from female rats. Instead, large amounts of 3β‐hydroxy‐5‐pregnen‐20‐one, 3β, 16α‐dihydroxy‐5‐pregnen‐20‐one and 5‐pregnene‐3β,20α‐diol are excreted. The total daily excretion of steroids in bile from treated female animals is 2580 ± 580 μg compared to 850 μg in a female rat injected with solvent only. When ovariectomized rats are treated with cyanoketone, the principal biliary metabolites are 3β‐hydroxy‐5‐pregnen‐20‐one and 3β,16α‐dihydroxy‐5‐pregnen‐20‐one and the total daily excretion of steroids measured is 1186 ± 197 μg indicating that ovaries contribute about 50% of the total output of 3β‐hydroxy‐Δ5‐steroids in treated, intact female rats. As late as 28 days after a single injection of cyanoketone 3β‐hydroxy‐Δ5‐steroids (44 μg/24 h) are excreted in bile and the total excretion of corticosteroid metabolites is still low (72 μg/24 h) at this time. 130 days after a single dose, no 3β‐hydroxy‐Δ5‐steroids can be identified in bile but the total corticosteroid excretion is still less than 2 standard deviations below values of untreated intact animals (315 μg/24 h). Thus, in females cyanoketone has both an immediate enzyme‐inhibiting effect reflected by rapid onset of action after administration and, in addition, a long‐term depressing action on enzyme biosynthesis manifested by low total steroid excretion values measured at least as long as 130 days after injection of inhibitor.

Administration of cyanoketone to male rats gives rise to a small biliary excretion of 3β‐hydroxy‐Δ5‐steroids (3β,16α‐dihydroxy‐5‐pregnen‐20‐one, 5‐pregnene‐3β,17α,20α‐triol and 5‐pregnene‐3β,20α‐diol) and a decrease in corticosteroid (mainly 3β,11β,21‐trihydroxy‐5α‐pregnan‐20‐one) excretion. Corticosteroid output reaches normal values about 24 h after injection of inhibitor. Testectomy eliminates biliary 3β‐hydroxy‐Δ5‐steroids. These findings indicate that cyanoketone produces biliary 3β‐hydroxy‐Δ5‐steroid mainly of testicular origin. Moreover, it appears that the 3β‐hydroxy‐Δ5‐steroid oxidoreductase system in the adrenals and testes of the male rat is inhibited by cyanoketone considerably less efficiently and for a shorter duration than that in the adrenals and ovaries of the female.
Purification and Properties of Ribose‐Phosphate Isomerase from Candida utilis
Authors: Domagk Götz F. , Doering K. Michael , Chilla Reinhard
Published: March 2005

The purification of ribose‐5‐phosphate isomerase from Candida utilis is described. The procedure used extends over six steps to a 27% yield of a homogeneous protein. The following properties have been elucidated: the enzyme has a molecular weight of 105000 and will dissociate into four subunits upon the addition of sodium dodecylsulfate. The specific activity is about 350 international units per mg protein. The pH optimum, Km for ribose‐5‐phosphate, amino acid composition and isoelectric point have been determined. The isomerase is extremely sensitive to organic mercurials.
Sequence of Events in Initiation of Protein Synthesis
Authors: Benne Rob , Ebes Frieda , Voorma Harry O.
Published: March 2005

Initiation factors recycle during the assembly of a 70‐S initiation complex. At early stages the factors are present, but they are absent in the mature 70‐S initiation complex.

Experiments described in this paper deal with the sequence of events and the contribution of each factor in the assembly process. Initiation factors IF‐1 and IF‐2 bind to 30‐S subunits independent of the presence of the other components involved in initiation. The fate of IF‐2 during 70‐S initiation complex formation has been studied in a system devoid of IF‐1 and in a system in which GTP is replaced by 5′‐guanylylmethylenediphosphonate (Guo‐5′‐P2‐CH2‐P). In both cases it is found that IF‐2 is not released from the 70‐S initiation complex. This leads to the inhibition of the elongation factor EF‐Tu dependent binding of the second aminoacyl‐tRNA, coded for by the messenger. The data are interpreted by assuming partly overlapping binding sites for EF‐Tu and IF‐2.
Phenylalanyl‐tRNA Synthetase from Escherichia coli K10
Authors: Kosakowski Heinz M. , Holler Eggehard
Published: March 2005

A synergistic coupling was observed for ligands binding to the specific sites for l‐phenylalanine and ATP. Dissociation constants were measured by the use of fluorescence titration and equilibrium dialysis techniques. It is found that l‐phenylalanine, l‐phenylalanine methylester, and phenylethylamine bind with an increased affinity when ATP, ADP, AMP or adenosine were present. With l‐phenylalanine, synergistic binding was only observed for adenosine; ATP has probably no effect. Results of competition experiments, the stoichiometry of complex formation and the magnitude of dissociation constants indicate that synergistic coupling involves the binding sites for ATP and amino acid at the active site.
Studies on Lysostaphin
Authors: Iversen Ole‐Jan , Grov Arne
Published: March 2005

The bacteriolytic agent lysostaphin has been isolated from the culture supernatant of Staphylococcus epidermidis, NRRL B‐2628 (K‐6‐W1) on the ion‐exchange resin Amberlite IRC‐50. A salt gradient released three enzyme specificities which were further purified to apparent homogeneity. These enzymes, belonging to three different classes, were endo‐β‐N‐ acetylglucosaminidase, endopeptidase (glycinase) and N‐acetylmuramyl‐l‐alanine amidase. In contrast to the first two enzymes, the third one was not found to be bacteriolytic per se.
The Wall Peptidoglycans of Neisseria perflava, Moraxella glucidolytica, Pseudomonas alcaligenes and Proteus vulgaris Strain P18
Authors: Martin Jean‐Pierre , Fleck Jacqueline , Mock Michèle , Ghuysen Jean‐Marie
Published: March 2005

The primary structure of the wall peptidoglycans of Neisseria perflava, Moraxella glucidolytica and Pseudomonas alcaligenes is similar to those of the Enterobacteriaceae Proteus vulgaris and Escherichia coli, suggesting that the peptidoglycans of all gram‐negative bacteria contain meso‐diaminopimelic acid and belong to the same chemotype I. Differences are observed with regard to the thickness of the peptidoglycan layer in the cell envelopes, the extent of peptide crosslinking and the occurrence of N,O‐diacetylmuramic acid residues.
Products of Mitochondrial Protein Synthesis in Neurospora crassa
Authors: Sebald Walter , Machleidt Werner , Otto Joachim
Published: March 2005

Cytochrome oxidase isolated from Neurospora crassa was resolved into seven protein components by electrophoresis in polyacrylamide gels containing sodium dodecylsulfate. The apparent molecular weights were determined to be 41000, 28500, 21000, 16000, 14000, 11500 and 10000 for the components 1,2,3,4,5,6, and 7, respectively. The components 1,2 and 3 are synthesized on mitochondrial ribosomes as shown by the incorporation of radioactive amino acids in the presence of cycloheximide.

Amino‐acid analysis of the isolated components 1, 2 and 3 revealed a high content of apolar amino acids and a low content of basic amino acids compared to an average amino‐acid composition of components 4–7.

Components 1, 2 and 3 contribute 27.9%, 18% and 14.2% to the whole cytochrome oxidase protein. This was calculated from the contributions of the single components to the total leucine content of the enzyme and the leucine contents (nmol leucine per mg protein) of the single components as determined by amino‐acid analysis. Equimolar relations of the components 1, 2 and 3 are found by dividing the amounts of protein by their apparent molecular weights. A stoichiometry of 1:1:1 results assuming a minimal molecular weight of 150000 for the whole cytochrome oxidase protein.

On the basis of the heme a content a molecular weight of about 70000 per heme group was determined, using an absorption coefficient Δɛ605 (reduced minus oxidized) of 12 mM−1 cm−1. It is concluded that the smallest structural unit of cytochrome oxidase contains two heme groups.
The Covalent Structure of Collagen
Authors: Rexrodt Friedrich W. , Hopper Kelvin E. , Fietzek Peter P. , Kühn Klaus
Published: March 2005

Preliminary to detailed amino acid sequence analysis of the peptide α1‐CB7 from calf skin collagen, the chymotryptic, tryptic and thermolytic peptides were isolated and their positions in the intact chain determined. The collagenase derived peptides α1(78), α1(125) and α1(78)‐CBN were also used as source of peptides and aided considerably the positioning of smaller fragments. All peptides isolated were placed in correct order as found subsequently in the more detailed sequence analysis.
Composition of the 3′‐Phosphate Penultimate Nucleotides Released from Calf‐Thymus DNA by Spleen Acid DNAase
Authors: Devillers‐Thiery Anne , Ehrlich Stanislav D. , Bernardi Giorgio
Published: March 2005

The composition of the 3′‐phosphate penultimate nucleotide liberated by spleen acid DNAase was determined using two different experimental approaches involving the use of pancreatic DNAase, in the presence of Mn2+, to degrade the oligonucleotide released by the former enzyme. The results obtained using both approaches indicate that the 3′‐phosphate penultimate nucleotide is highly specific. Since previous work had already shown that the 3′‐phosphate terminal and the 5′‐hydroxy terminal and penultimate nucleotides also have compositions which very significantly deviate from those expected on the basis of a non‐specific attack, the conclusion is drawn that spleen DNAase recognizes sequences at least four nucleotides long.
Interaction of Haemoglobin with Ions
Authors: Berger Hartmut , Jänig Gerd‐Rüdiger , Gerber Gerhard , Ruckpaul Klaus , Rapoport Samuel M.
Published: March 2005

Under simulated intracellular conditions (pH 7.2, 37 °C, 130 mM KCl, 20 mM NaCl and haemoglobin concentrations > 1.5 mM) the conditional association constants for the binding of ATP and 2,3‐bisphosphoglycerate (P2‐glycerate) to deoxygenated and oxygenated haemoglobin (Hb and HbO2, respectively) and the influence of Mg2+ on the binding of phosphocompounds were determined by means of a cation‐sensitive electrode and ultrafiltration.

The K′ass for the binding of ATP to Hb is 2.6 mM−1 and 0.36 mM−1 for HbO2. P2‐glycerate is bound to Hb with K′ass of 5 mM−1 and to HbO2 with a K′ass of 0.25 mM−1. The anions compete with each other in binding. Mg2+ is not bound to haemoglobin but it reduces the binding of the phosphocompounds by complexing with them. It decreases the shift of the oxygen binding curve produced by ATP. MgATP is weakly bound to Hb with a K′ass of 0.14 mM−′ and to HbO2 with a K′ass of 0.039 mM−1. Binding of the Mg ·P2‐glycerate complex to haemoglobin was not established.

The calculated values for the dependence of p50 (the partial pressure of oxygen at half‐saturation of haemoglobin) on the free concentration of P2‐glycerate, based on the K′ass of Hb and HbO2 complexes, agree well with the experimental data on erythrolysates and intact human red blood cells. The K′ass are furthermore an appropriate basis for the estimation of the intraerythrocatic state of Mg2+ and the metabolites.
Interaction of Haemoglobin with Ions
Authors: Gerber Gerhard , Berger Hartmut , Jänig Gerd‐Rüdiger , Rapoport Samuel M.
Published: March 2005

The intracellular distribution of ATP, 2,3‐bisphosphoglycerate(P2‐glycerate) and Mg2+ was calculated for the oxygenated and deoxygenated human erythrocyte for the normal range and that of pathophysiological variations based on the association constants of the relevant complexes.

The data indicate that about 20% of ATP is bound both in the oxygenated and deoxygenated cells, while 39 and 73% of P2‐glycerate is bound under these conditions. An increase of the free Mg2+ concentration from 0.7 to 1.1 mM is produced by complete deoxygenation of haemoglobin.

The calculations would indicate that during deoxygenation of haemoglobin the hexokinase reacts to the increased concentration of the activator Mg2+ and the decline of the inhibitor P2‐glycerate with an elevation of its activity which corresponds to experimental data on intact erythrocytes.

The P2‐glycerate formation rate in deoxygenated cells is stimulated about 2.5 times in comparison to oxygenated cells as estimated from the free concentration of P2‐glycerate and the kinetic constants of bisphosphoglycerate mutase.

An assessment of the possible influence of other anions including bicarbonate shows that the distribution of the species of ATP, P2‐glycerate and Mg2+ are changed by less than 20%.

Together with the data presented in the accompanying paper, the results given here indicate that the constants and estimates are approximately valid for intracellular conditions.
Comparison of Escherichia coli and T3 RNA Polymerases
Authors: Küpper Hans A. , McAllister William T. , Bautz Ekkehard K. F.
Published: March 2005

The sensitivities of the Escherichia coli and bacteriophage T3 RNA polymerases to various compounds have been investigated in vitro. Neither enzyme is sensitive to the drug α‐amanitin. The E. coli polymerase is inhibited by an exotoxin from Bacillus thuringiensis; the phage enzyme is not. Although both enzymes are inhibited by actinomycin D and distamycin A, there is a differential effect: the T3 enzyme is more sensitive to distamycin A than the E. coli enzyme, and less sensitive to actinomycin D. While distamycin A preferentially inhibits chain initiation by both enzymes, actinomycin D affects mainly chain elongation. The rifamycin derivative AF/013 was found to selectively inhibit chain initiation by the T3 RNA polymerase and this drug was used to characterize a transient initiation complex observed when the T3 RNA polymerase is incubated with GTP alone as a substrate.
Purification and Properties of Human Acrosin
Authors: Gilboa Eli , Elkana Yehudit , Rigbi Meir
Published: March 2005

The 145‐fold purification of a proteolytic enzyme from human spermatozoa by chromatography on SP‐Sephadex and Sephadex G‐100 is described. The enzyme possesses a molecular weight of 76000 ± 4000, exists as a monomer in solution and consists of a single polypeptide chain. As judged by its inhibition spectrum, substrate specificity and pH‐activity curve it is a trypsinlike enzyme. When initial velocity was measured against substrate concentration, a slight cooperative effect was observed. The enzyme is stable at pH 3.2 and labile at pH 8.1. At this pH, Mn2+, Mg2+ or Ca2+ ions have no effect on its activity or stability. It is identified as human acrosin.
Molecular Properties of Lactic Dehydrogenase under the Conditions of the Enzymatic Test
Authors: Bartholmes Peter , Durchschlag Helmut , Jaenicke Rainer
Published: March 2005

In order to investigate the correlation of enzymatic activity and subunit structure of lactic dehydrogenase from pig heart, the molecular weight, activity and conformation of the enzyme were determined under conditions of the enzymatic test, chosing optimum conditions of the medium regarding subunit dissociation or hybridization and biological function.

At concentrations of 10mg/ml to 0.1 μg/ml no dissociation is to be observed. A population analysis on the basis of high‐speed equilibrium ultracentrifugation does not show significant fractions of other species besides the tetramer. At c < 0.1 μg/ml both activity transport and gelfiltration prove dissociation to the dimer to occur which is accompanied by deactivation. The first‐order rate constant of the dissociation reaction is of the order of 0.002 min−1.

In the presence of the coenzyme, reassociation and reactivation take place, the binding of NADH leading to a tightening of the quaternary structure (Δscorr= 0.17 S). Nicotinamide which does not influence the activity of the native enzyme inhibits reassociation, while ATP does not show any appreciable effect on the dissociation reaction.

The lack of enzymatic activity of the dimer in the presence of nicotinamide and the parallel occurrence of subunit dissociation and deactivation at c < 0.1 μg/ml confirm previous observations implying enzymatic activity to require the intact quaternary structure of the enzyme.
The Size of the Substrate‐Binding Site of an Aspergillus niger Extracellular Endopolygalacturonase
Authors: Rexová‐Benková Ľubomíra
Published: March 2005

The action pattern and kinetics of an Aspergillus niger extracellular endopolygalacturonase were studied with oligogalacturoic acids (digalacturonic acid through hexagalacturonic acid) and their derivatives in which the terminal aldehyde group was reduced. The rate of hydrolysis catalyzed by the enzyme decreases with the shortening of oligogalacturonide chain length; digalacturonic acid is not hydrolyzed. With tetragalacturonic acid one productive complex is formed resulting in (1 + 3) cleavage. Two and three modes of cleavage occur with pentagalacturonic acid and hexagalacturonic acid, respectively. The enzyme is competitively inhibited by trigalacturonic acid whereas digalacturonic acid does not affect the activity. Reduced pentagalacturonic acid forms one productive complex. Reducing trigalacturonic acid and nonreducing digalacturonic acid are the products of the reaction. Lower reduced oligogalacturonic acids are not degraded by the enzyme.

The action pattern and k2 values obtained with the oligouronides suggest that the binding site of the endopolygalacturonase contains four subsites and that the catalytic groups of the enzyme are located in the proximity of the first bond, counting from the reducing end, of the substrate segment bound in the complex.