bluuebunny/crossref_metadata_2025_split huggingface dataset:
Physicochemical Properties of T4 Polynucleotide Kinase
Authors: LILLEHAUG Johan R. 
Published: March 2005

Some physicochemical properties of T4 polynucleotide kinase (EC 2.7.1.78) have been studied. The enzyme is an oligomer of one polypeptide chain. The molecular weight of the monomer is 33000, as determined from the amino acid analysis. Phenylalanine is the N‐terminal amino acid. Each monomer contains two −SH groups, one exposed and one more buried. Circular dichroic spectra suggest a high content of α‐helical structure, 45 – 55%. Excitation at 280 nm gave a strong emission fluorescence spectrum with a maximum centering at 340 nm.Sedimentation studies suggested the enzymically active form to be a tetramer. High ionic strength (0.1 M KCl), spermine, and the substrates ATP and thymidine 3′‐monophosphate were found to be essential factors in order to stabilize the protein in an oligomeric structure. The association constants for ATP, thymidine 3′‐monphosphate, and Pi were determined fluorimetrically to be 7.9 × 105, 4.8 × 105, and 7.2 × 102 M−1 respectively.
Thyrotropin Stimulation of the Phosphorylation of Serine in the N‐Terminal Region of Thyroid H1 Histones
Authors: LAMY Françoise , LECOCQ Raymond , DUMONT Jaques E. 
Published: March 2005

The analysis of thyroid nuclear proteins by polyacrylamide gel electrophoresis has demonstrated that thyrotropin and dibutyryl adenosine 3′:5′‐monophosphate stimulate specifically the phosphorylation of H1 histones in an intact cell system.This effect does not require new protein synthesis and implicates the phosphorylation of serine residue(s) situated in the N‐terminal part of H1 histones.
An Investigation of Mistranslation in vivo Induced by Streptomycin by an Examination of the Susceptibility of Abnormal Proteins to Degradation
Authors: HEWITT Jane , KOGUT Margot 
Published: March 2005

Proteolysis rates in vivo were measured in Escherichia coli cultures during treatment with dihydrostreptomycin and under various other conditions. Dihydrostreptomycin treatment caused an increase in the proteolysis rate, compared to untreated controls. The proteolytic system in vivo responsible for the elevated proteolysis in the early stages of dihydrostreptomycin treatment, or that during canavanine and puromycin treatment, were not inhibited by addition of phenylmethanesulphonyl fluoride. This agent did inhibit proteolysis rates in cultures whose growth was inhibited by starvation, or had been completely stopped by dihydrostreptomycin. It seems, therefore, that the extremely high proteolysis rates in cultures at this stage of dihydrostreptomycin treatment were due to the action of two protease systems: the one concerned with the breakdown of abnormal proteins, and the other concerned with normal protein turnover and active during a non‐specific decline of growth.The proteolytic rate at complete growth inhibition brought about by dihydrostreptomycin was intermediate between those induced by canavanine and puromycin at the same stage of treatment. This indicated a similar hierarchy in the extent and nature of abnormality in the proteins synthesised under these conditions. The relationship between the abnormality of proteins induced by dihydrostreptomycin and the importance of this in the antibiotic mechanism is discussed.
Cathepsin L
Authors: KIRSCHKE Heidrun , LANGER Jürgen , WIEDERANDERS Bernd , ANSORGE Siegfried , BOHLEY Peter 
Published: March 2005

Cathepsin L was purified from rat liver lysosomes by cell fractionation, osmotic disruption of the lysosomes in the lysosomal mitochondrial pellet, gel filtration of the lysosomal extract and chromatography on CM‐Sephadex.
Cathepsin L is a thiol proteinase and exists in several multiple forms visible on the disc electropherogram. By polyacrylamide‐gel electrophoresis in the presence of sodium dodecyl sulphate, its molecular weight was found to be 23000 – 24000. The isoelectric points of the multiple forms of cathepsin L extended from pH 5.8 – 6.1 ascertained by analytical isoelectric focusing.
Using various protein substrates, cathepsin L was found to be the most active endopeptidase from rat liver lysosomes acting at pH 6 – 7. In contrast to cathepsin B1, its capability of hydrolyzing N‐substituted derivatives of arginine is low and it does not split esters.
Greatest activity is obtained close to pH 5.0 with 70 – 90% of maximal activity at pH 4.0 and pH 6.0 and 30–40% at pH 7.0.
The enzyme is strongly inhibited by leupeptin and the chloromethyl ketone of tosyl‐lysine. Leupeptin acts as a pseudo‐irreversible inhibitor.
The enzyme is stable for several months at slightly acid pH values in the presence of thiol compounds in a deep‐frozen state.
Spectroscopic Studies on Effector‐Induced and Substrate‐Induced Conformation Changes of Phosphofructokinase
Authors: GROSSE Richard , ECKERT Klaus , OTTO Matthias , JACOBASCH Gisela , REPKE Kurt R. H. 
Published: March 2005

The interaction of phosphofructokinase with NH4+, AMP, ATP, citrate, MgATP or fructose 6‐phosphate, and in part with their mixtures forming either binary or ternary complexes has been studied by means of ultraviolet difference spectroscopy and circular dichroism spectroscopy in the wavelength range 265 – 300 nm with the aim of characterizing the conformational corollaries of the ligand effects on phosphofructokinase.The positive as well as the negative effectors change phosphofructokinase conformation in different ways, not easily interpretable in terms of one active and one inactive enzyme conformation. The spectroscopic equivalents of phosphofructokinase conformation changes resulting from catalytic activity are similar to those produced by the reaction products. The ligand concentration‐dependent changes of absorption differences in the tryptophyl, tyrosyl and phenylalanyl region parallel each other, i.e. the interactions of the ligands with phosphofructokinase are not confined to specific aromatic side chains, but involve conformation changes of the large domains of the protein. ATP affinity to the enzyme shows temperature‐dependent biphasic changes so that ATP binding appears to be either an entropy‐driven or enthalpy‐driven process. The dissociation constants of the ligands derived from spectroscopic titration of binary complex formation are comparable to those calculated from kinetic experiments. MgATP and fructose 6‐phosphate each alone change phosphofructokinase conformation by binary complex formation in keeping with a random order of reaction sequence.
Isolation of Mutants Conditionally Blocked in the Biosynthesis of the 3‐Deoxy‐d‐manno‐octulosonic‐acid—Lipid‐A Part of Lipopolysaccharides Derived from Salmonella typhimurium
Authors: LEHMANN Volker , RUPPRECHT Eckhard , OSBORN Mary Jane 
Published: March 2005

A procedure is described for the selection of conditional 3‐deoxy‐d‐manno‐octulosonic‐acid—Lipid A mutants which depends on temperature sensitivity for both synthesis of complete lipopolysaccharide and for growth. Using this procedure new types of mutants were isolated which cease growth and accumulate lipid A precursors following a shift to nonpermissive temperatures. All precursor molecules differ in their charge as judged by DEAE‐cellulose chromatography. While they all contain glucosamine, phosphate and 3‐hydroxymyristic acid, they lack detectable 3‐deoxy‐d‐manno‐octulosonic acid (dOclA) as well as the nonhydroxylated fatty acids of the complete lipid A structure.Three mutants proved to be conditionally defective in dOclA metabolism, whereas one seems to be blocked at a relatively early step in lipid A synthesis. The phenotypes of all these mutants appear to be due to single mutations by reversion analysis and by characterization of the temperature‐resistant revertants.Studies of these mutants may shed light on the essential role of the complete dOclA—lipid A part of lipopolysaccharides in membrane function.
The Purification and Properties of 4‐Hydroxyisophthalate Hydroxylase from Pseudomonas putida NCIB 9866
Authors: ELMORSI Elmorsi A. , HOPPER David J. 
Published: March 2005

4‐Hydroxyisophthalate hydroxylase has been purified from cells grown with 2,4‐xylenol and gave a single protein band on polyacrylamide‐gel electrophoresis after a 24‐fold purification.
In the reaction, 4‐hydroxyisophthalate was converted to protocatechuate by an oxidative decarboxylation and the reaction stoichiometry is typical of a monooxygenase with an external electron donor.
The enzyme can function with either NADH (Km 105 μM) or NADPH (Km 71 μM). Besides 4‐hydroxyisophthalate (Km 42 μM), 5‐sulphosalicylate was the only aromatic substrate found for the enzyme.
The enzyme has a molecular weight of about 103000, consisting of two identical polypeptide chains, and contains one mole of FAD per mole of protein. The FAD could not be replaced by FMN in reconstituting active enzyme from native apoenzyme.
The formation of an enzyme‐substrate complex was detected by the change in flavin spectrum when 4‐hydroxyisophthalate was added to the enzyme.
Rapid reduction of the enzyme‐bound FAD by NADPH only occurred in the presence of substrate. No stable flavin semiquinones were observed in the titration with NADPH or during anaerobic photoreduction in the presence of EDTA.
Although a number of substrate analogues inhibit the enzyme no non‐hydroxylatable effectors of the NADPH oxidation were found.
The enzyme was inhibited by sulphydryl‐binding reagents and not protected by substrate. Addition of sulphydryl compounds was essential for stabilization of the enzyme during purification.
Extracts of cells grown with 2,4‐xylenol and 4‐hydroxyisophthalate also contain a separate 4‐hydroxybenzoate hydroxylase.
On the Interrelationship of Prostaglandin Endoperoxide G2 and Cyclic Nucleotides in Platelet Function
Authors: CLAESSON Hans‐Erik , MALMSTEN Curt 
Published: March 2005

The prostaglandin endoperoxide G2 caused rapid aggregation and release of ADP and [14C]serotonin in human platelets. Since the presence of the ADP phosphorylating system creatine phosphate creatine phosphokinase markedly inhibited the aggregation caused by the endoperoxide, this effect seemed to be mediated mainly by ADP, which is instantaneously released by the endoperoxide.The prostaglandin G2 counteracted the increasing effect of prostaglandin E1 on the adenosine 3′:5′‐monophosphate (cAMP) levels in platelet‐rich plasma. This effect of prostaglandin G2 was only observed when ADP was released by the endoperoxide. This finding indicates that the effect of prostaglandin G2 on the cAMP levels in platelet‐rich plasma is principally mediated by ADP. The rapid release of ADP by prostaglandin G2 and the time courses for the effects of the endoperoxide and ADP on the level of cAMP give further evidence for this hypothesis. ADP also caused primary aggregation in the presence of indomethacin, and prostaglandin synthesis inhibitors did not influence the decreasing effect of ADP on the cAMP levels. N2,O2‐Dibutyrylguanosine 3′:5′‐monophosphate did not influence the aggregation and release‐reaction caused by ADP and no changes of the cGMP levels were observed after addition of prostaglandin G2.
Bacteriorhodopsin‐Mediated Photophosphorylation in Halobacterium halobium
Authors: HARTMANN Rainer , OESTERHELT Dieter 
Published: March 2005

The rate of halobacterial photophosphorylation was found to be a linear function of light intensity over a wide range (between 1 and 20 mW/cm2). At higher light intensities (above 25 mW/cm2) the ATP‐synthesizing system itself limits the maximal rate of photophosphorylation. The optimal external pH range for this type of photophosphorylation is between pH 6.2 and 7.2 external. The photophosphorylation rate is directly proportional to the bacteriorhodopsin content of the cells. The quantum requirement for photophosphorylation was found to be 22 ± 5 photons per ATP molecule synthesized.According to Mitchell's chemiosmotic hypothesis of energy coupling phosphorylation can be driven by a membrane potential or a pH gradient or a combination of both. From the results of experiments with drugs which abolish or reduce either one of the two components we conclude that the major driving force for photophosphorylation above an external pH value of 6.5 is the membrane potential, while at more acidic pH value the pH gradient becomes dominating.We did not observe a correlation between a transient alkalinization of the medium and ATP‐synthesis upon illumination under certain conditions.
Protein Components of Very Low Density Lipoproteins from Hen's Egg Yolk
Authors: BENGTSSON Gunilla , MARKLUND Sven‐Erik , OLIVECRONA Thomas 
Published: March 2005

Egg yolk lipoproteins of very low density were found to contain proteins with cofactor activity for lipoprotein lipase. When delipidated very low density lipoproteins were dissolved in 10 mM HCl and fractionated by gel filtration about two thirds of the protein were in several components with estimated molecular weights of 60000 to more than 170000. The major low‐molecular‐weight proteins were the dimeric and monomeric forms of a previously characterized 9000‐dalton peptide. The cofactor activity was not associated with any of these major proteins.A large‐scale fractionation method was developed by which two protein fractions with cofactor activity for lipoprotein lipase were purified more than thousand‐fold. One fraction had a molecular size of about 9000 daltons and the other had a size of about 5000 daltons. Both these fractions could be further separated on the basis of charge into several fractions with cofactor activity. The cofactor proteins were relatively soluble both at high and at low pH. They retained their cofactor activity after denaturation in guanidinium hydrochloride and after reduction. During the initial steps in the purification of the cofactor proteins another low‐molecular‐weight protein followed the cofactors. It had a single 17500‐dalton peptide chain and was present in four variants, three of which contained carbohydrate.
A Kinetic Study of Calcium Transport by Heart Mitochondria
Authors: NOACK Eike A. , HEINEN Edgar M. 
Published: March 2005

Initial velocities of energy‐dependent Ca2+ uptake in cardiac mitochondria were measured with the ethyleneglycol bis(2‐aminoethylether)‐N,N,N′,N′‐tetraacetic acid/ruthenium red quenching technique. Accurate concentrations of free Ca2+ between 1 and 15μM were generated with ATP as a Ca2+ buffer system. Using the method of iterative approximation, the accurate value of the initial velocity of ATP‐driven 45Ca uptake, and the time (t0.5) for half‐maximal uptake can be calculated.The rate of initial Ca2+ uptake increased in response to the rising free Ca2+ concentrations in the medium (in the presence of 0.6–5.0 mM ATP). A maximal rate was obtained between 10 and 15 μM of free Ca2+. For different ATP concentrations (0.6–5.0 mM), the half‐maximal rate of Ca2+ transport (Km) was observed between 4.1 and 7.4 μM Ca2+ (with a mean Km of 5.6 ± 0.6 μ Ca2+; n= 5). Hill plots of the data yield straight lines with an average slope n, of 1.93 ± 0.29, indicating the existence of two binding sites for Ca2+ in the transmembrane transport system. Keeping the free Ca2+ concentration on a constant level (6 μM), the rate of initial Ca2+ uptake also increased continually with rising ATP concentrations (maximum velocity v= 7.1 nmol of Ca2+× mg protein−1× s−1).From energy‐independent calcium‐ion binding studies with cardiac mitochondria, a dissociation constant (Kd) of 26.8 × 10−6M and a total number of external binding sites (n) of 12.6 nmol of Ca2+/mg of protein was obtained.The data are discussed with regard to the physiological role which cardiac mitochondria may play as a reversible storage site during the contraction relaxation cycle of the heart.
Kinetics of Refolding of Completely Reduced Human‐Serum Albumin
Authors: WICHMAN Anders , SVENSON Anders , ANDERSSON Lars‐Olov 
Published: March 2005

The kinetics of refolding of completely reduced human serum albumin has been studied by various methods including immunological techniques. The decrease in thiol content is very rapid in the beginning of the reoxidation process and rather slow in the later stages. Polyacrylamide gel electrophoresis studies show that, in the earlier stages of refolding, the main part of the albumin is present as various oligomers and that a slow conversion to monomer occurs as reoxidation proceeds.Rocket immunoelectrophoresis shows that the completely reduced protein is devoid of native albumin antigenic determinants but that a rapid regain of immunoprecipitability is obtained upon reoxidation. A new ‘consumption’ rocket immunoelectrophoretic method has been used to estimate the total regain of antigenicity.The data obtained indicate that there is a preferential rapid folding to native structure in certain parts of the molecule but that areas with wrong or incomplete foldings exist a considerable time after the initial refolding period.
Change in Location of Ornithine Carbamoyltransferase and Carbamoylphosphate Synthetase among Yeasts in Relation to the Arginase/Ornithine Carbamoyltransferase Regulatory Complex and the Energy Status of the Cells
Authors: URRESTARAZU L. Antonio , VISSERS Stephan , WIAME Jean‐Marie 
Published: March 2005

Partial fractionation of Saccharomyces cerevisiae spheroplast lysate shows that arginase, ornithine carbamoyltransferase and carbamoylphosphate synthetase have the same distribution as the cytosol marker enzyme glucose‐6‐phosphate dehydrogenase. The same holds true in stepwise homo‐genization experiments of spheroplast lysates of S. cerevisiae.This second method applied to Debaryomyces hansenii, Hansenula anomala and Schizosaccharomyces pombe shows, in contrast, that ornithine carbamoyltransferase is particulate in these yeasts. Its solubilization is in line with that of citrate synthase taken as mitochondrial matrix marker, whereas arginase acts like glucose‐6‐phosphate dehydrogenase. In Schizosaccharomyces pombe carbamoylphosphate synthetase is particulate too. Density gradient centrifugation of a particulate fraction of Schizosaccharomyces pombe suggests that ornithine carbamoyltransferase and carbamoylphosphate synthetase are associated with the mitochondria.In Saccharomyces cerevisiae and in some other yeasts [Messenguy & Wiame (1969) FEBS Lett. 3, 47–49; Wiame (1974) Proc. 9th Meet. Fed. Eur. Biochem. Soc., pp. 161–177] arginase binds to ornithine carbamoyltransferase in the presence of arginine and ornithine, thereby inhibiting its activity but leaving arginase activity unmodified. This regulation by reversible stoechiometric interaction between the two enzymes prevents recycling of ornithine to arginine when the latter is used as a source of nitrogen. Results presented here show a correlation between the shift of ornithine carbamoyltransferase and carbamoylphosphate synthetase from mitochondrial to extramitochondrial fraction and the existence of arginase/ornithine carbamoyltransferase regulatory interaction. This regulation and extramitochondrial ornithine carbamoyltransferase are absent from obligate aerobe yeasts and may reflect an adaptation to the fermentative type of energy production.
Enzyme Activities in Different Cell Compartments of the Involuting Rat Myometrium
Authors: GEYER Helmut , MÜLLER Ulrich , AFTING Ernst‐Günter 
Published: March 2005

Marker enzymes for various intracellular fractions were measured in rat uterus myometrium during the post‐partum involution. Following a lag phase of 48 h after delivery, the activities of the cytoplasmic enzymes aldolase, glucose‐6‐phosphate dehydrogenase and creatine kinase, decreased at equal rates. The plasma membrane marker 5′‐nucleotidase, again after a lag phase of 48 h, showed a decrease in activity which was parallel to that of the cytoplasmic enzymes when allowance was made for the geometry of the cell.The mitochondrial glutamate dehydrogenase and succinate dehydrogenase activities decreased in parallel together after a lag phase of 72 h. The significantly longer lag phase of the mitochondrial enzymes can be explained in terms of the importance of the mitochondria as a source of the energy which is necessary for intracellular protein degradation.The activity of the lysosomal acid phosphatase showed no significant decrease until the involution of the tissue was essentially complete.The activities of cytosol, mitochondria and plasma membrane enzymes all fell during involution to levels below those measured in non‐pregnant control animals. This can be explained by the absence of estrogenic stimulation of the uterus during the post‐partum period, which results from suppression of ovarian function by lactation.
The Mechanism by which Heparin Stimulates Transcription in Isolated Rat Liver Nuclei
Authors: COUPAR Barbara E. H. , CHESTERTON C. James 
Published: March 2005

The polyanion heparin, whilst inhibiting free RNA polymerase, stimulates the rate of RNA synthesis in eucaryotic nuclei; it has been suggested that this process involves either the inactivation of R Nase or the activation of blocked transcription complexes. These hypotheses have been investigated in isolated rat liver nuclei at 22 °C. We confirm that heparin inhibits free form A and B RNA polymerases but has little effect on the transcribing enzymes. The sensitivity of these enzymes to α‐amanitin is unchanged by the agent and the stimulation is dependent on the presence of (NH4)2SO4 and Mn2+ ions. The size of the RNA product was analysed on denaturing formamide sucrose gradients. It shows only a small increase when heparin is included in the incubation. In addition, no significant size changes are observed if the incubation is continued in the presence of actinomycin D and α‐amanitin which together inhibit 97% of all RNA synthesis. These results show that inactivation of R Nase cannot be an explanation of the stimulatory effect in this system.Polynucleotide elongation rates and numbers of transcribing RNA polymerase molecules were determined by alkaline degradation of RNA labelled from [3H]UTP during 1‐min incubations. The [3H]UMP and [3H]uridine, derived from internal nucleotides and 3′‐terminal nucleosides respectively, were separated by thin‐layer chromatography and their tritium content measured. From this data the above parameters can be calculated. We show that the activity of R Nase and phosphatase do not significantly affect the data and that levels of endogenous UTP are negligible. Data from eight separate experiments show that isolated rat liver nuclei contain 25–35×103 transcribing RNA polymerase molecules per diploid genome comprising 18–20×103 form A and 5–15×103 form B. Form C was not detected. Addition of heparin has no effect on these values. Polyribonucleotide elongation rates in nuclei are approximately 0.15 and 0.4 nucleotide per second for form A and B respectively. In the presence of heparin the latter rate is increased by 2–4‐fold but that of the form A enzyme remains essentially unchanged. Similar results were obtained using [3H]GTP in place of [3H]UTP indicating that polyribonucleotide termination was random. Our findings show that heparin exerts its stimulatory effect by increasing the form‐B‐mediated polyribonucleotide elongation rate. It does not activate previously inactive RNA polymerase molecules.