bluuebunny/crossref_metadata_2025_split huggingface dataset:
The Interaction of Actinomycin C3 and Actinomine with DNA
Authors: ZIPPER Peter , BÜNEMANN Hans 
Published: March 2005

Small‐angle X‐ray scattering was applied to solutions of calf thymus DNA and calf thymus DNA complexed with various amounts of actinomycin C3 or actinomine in phosphate‐saline buffer at pH 6.9 and I= 0.2. From the measurements of DNA in the absence of dye, two cross‐section radii of gyration of Rc, = 0.875 ± 0.015 nm and Rc2, = 0.81 ± 0.02 nm, and a mass per unit length of M/l= 1906 ± 43 daltons/nm resulted. The investigation of DNA complexed with dye revealed a decrease of the cross‐section radii of gyration as compared to those for the DNA in the absence of dye and a relatively low increase of the mass per unit length on binding of actinomycin and a slight decrease of M/l on binding of actinomine.The latter results are interpreted on the basis of a length increase of the DNA double helix by 0.47 ± 0.03 nm per actinomycin molecule and by 0.355 ± 0.03 nm per actinomine molecule bound. The results for Rc, and Rc2 obtained for the various samples of complexed DNA were extra polated to the limiting binding ratio where each dye molecule is associated with a minimum of six nucleotide pairs. According to this extrapolation, the cross‐section radii of gyration of such a complex would amount to (Rc)b= 0.805 ± 0.015 nm and (Rc2)b= 0.76 ± 0.015 nm for the complex with actinomycin, and to (Rc)b= 0.77 ± 0.015 nm and (Rc2)b= 0.75 ± 0.01 nm for the actinomine complex. On the basis of a core and shell model for solvated DNA, these results can be understood as to indicate a decrease of the radial dimensions of both the core and the shell when the dye is bound.The experimental results are compared with theoretical data calculated from the atomic coordinates of the detailed intercalation model for the actinomycin · DNA complex as recently proposed by Sobell and Jain. The model proves to be consistent fairly well with our data on the length increase of the double helix, but it appears to be unable to explain the experimentally observed decrease of Rc2, on binding of dye.
The Hormonal Control of Activity of Skeletal Muscle Phosphorylase Kinase
Authors: COHEN Philip , WATSON David C. , DIXON Gordon H. 
Published: March 2005

Two tryptic phosphopeptides containing the sites on the α and β subunits of phosphorylase kinase which are phosphorylated by protein kinase, dependent on adenosine 3′: 5′‐monophosphate (cyclic AMP), have been isolated and their amino acid sequences have been determined. 32P‐labelled phosphorylase kinase, containing 1.9 mol phosphate per mol enzyme, was digested with an equimolar quantity of trypsin for 2.5 min at pH 7.0, 20°C. This treatment released nearly all the 32P radioactivity associated with the β subunit as trichloroacetic‐acid‐soluble material. Only a small proportion of the 32P radioactivity associated with the α subunit was solubilised, the remainder being removed in the trichloroacetic acid pellet.The β‐subunit tryptic phosphopeptide was completely resolved from traces of the α‐subunit phosphopeptide by gel filtration on Sephadex G‐25. Further purification by peptide mapping separated the phosphopeptide into four components, each derived from the same nine‐amino‐acid segment of the βchain, which was found to possess the sequence:Gln‐Ser‐Gly‐Ser(P)‐Val−IleTyr‐Pro‐Leu‐Lys. The four components were produced by the partial cyclisation of the N‐terminal glutaminyl residue, and by the presence of two alleles for the β subunit in the rabbit population, which led to a valine‐isoleucine ambiguity.The α‐subunit phosphopeptide was liberated from the trichloroacetic acid pellet by redigestion with trypsin. It was the largest component in the digest which remained soluble in 5% trichloroacetic acid, and obtained in a highly purified form by a single filtration on Sephadex G‐50. The peptide comprised 39 amino acids of which nine were serine and three were threonine residues. Only one residue, the serine at position three from the amino terminus, was phosphorylated. The amino‐terminal sequence of the peptide was shown to be: Arg‐Leu‐Ser(P)‐Ile‐Ser‐Thr‐Glu‐Ser‐Glx‐Pro‐Asx‐Gly.The sequences confirm the stoichiometry of the reaction and the absolute specificity of cyclic‐AMP‐dependent protein kinase for just two of the 200 serine residues in the enzyme. These results and an inspection of the rate of phosphorylation of a number of skeletal muscle proteins, including each enzyme of the glycolytic pathway, lead to the conclusion that cyclic‐AMP‐dependent protein kinase is an extremely specific enzyme. The molecular basis of this specificity is discussed.
Soluble and Membrane‐Bound Enzyme‐Active Antigens of Rat‐Liver Lysosomes
Authors: BERZINS Klavs , BLOMBERG Fred , PERLMANN Peter 
Published: March 2005

Secondary lysosomes were isolated from rat liver and separated into a soluble and a membrane fraction. Plasma membranes and microsomes were also isolated and antisera against the various fractions were prepared in rabbits. Lysosomal content and detergent‐solubilized membrane fractions were analysed in two‐dimensional immunoelectrophoresis (crossed immunoelectrophoresis). The immunoprecipitates were stained by histochemical procedures for different enzyme activities such as phosphatases, non‐specific esterase, arylsulphatase, glycosidases and l‐leucyl‐β‐naphthylamidase.When lysosomal content was tested against its corresponding antiserum, 17 different precipitates could be seen. Most of the enzyme activities tested were shown to reside separately in one or a few precipitates each. In contrast, when the membrane extracts were investigated, a more polymorphic pattern of enzyme‐active precipitates appeared. Thus, when lysosomal membrane extracts were reacted with homologous antiserum 11 precipitates with acid phosphatase activity were obtained. Several of the antigens were electrophoretically different and immunologically non‐identical. As expected from the biology of secondary lysosomes, many of their antigens were also found in microsomes and/or plasma membranes, but several antigens unique for lysosomes were detected concomitantly.Closer analysis of these results indicated that several seemingly identical enzyme‐active proteins occurred both in soluble and membrane‐associated forms. However, while many of the membrane antigens expressed 2–4 different enzyme activities, only one activity was detected in individual precipitates of the lysosomal content. Thus, acid phosphatase activity was found together with esterase activity in three membrane‐associated antigens. The precipitates formed by two of these also stained for arylsulphatase and nucleoside tri‐, di‐ and monophosphatase activities. l‐Leucyl‐β‐naphthylamidase activity was found in one additional acid‐phosphatase‐active precipitate.
Extracellular Enzyme System Utilized by the Fungus Sporotrichum pulverulentum (Chrysosporium lignorum) for the Breakdown of Cellulose
Authors: ERIKSSON Karl‐Erik , PETTERSSON Bert 
Published: March 2005

An exo‐1,4‐β‐glucanase from culture solution of the rot fungus Sporotrichum pulverulentum (formerly called Chrysosporium lignorum) grown on powder cellulose as the sole carbon source has been extensively purified and characterized with respect to some physico‐chemical properties. The purification has been carried out in a five‐step procedure comprising chromatography on DEAE‐Sephadex, gel filtration on polyacrylamide P‐150, activation on a Dowex 2‐X8 anion exchanger, chromatography on Concanavalin A‐Sepharose and chromatography on SP‐Sephadex.The purified enzyme was found to be pure and homogeneous by analytical polyacrylamide electrophoresis, by electrophoresis on dodecylsulphate gels and by analytical isoelectric focusing. A single symmetrical peak was obtained with the free zone electrophoresis method. The purification factor is about 15 and the yield of exo‐1,4‐β‐glucanase activity 7%.After purification, the enzyme showed no viscosity‐decreasing activity towards carboxymethyl‐cellulose solutions.The exo‐1,4‐β‐glucanase was isoelectric at pH 4.3 (4°C).A molecular weight of 48600 was calculated on the basis of a knowledge of the partial specific volume, ultracentrifugation data and the amino acid composition. The enzyme contained no carbohydrate.
Crystalline Adenylate Kinase from Carp Muscle
Authors: NODA Lafayette , SCHULZ Georg E. , Von ZABERN Inge 
Published: March 2005

The concentration of adenylate kinase in carp muscle is about 0.3 mg/g. An improved isolation procedure makes use of a dilute solution of the substrates, ATP and AMP, to elute the enzyme from a phosphocellulose column in overall yields of 60% before crystallization. By the hexokinase–pH‐stat assay the specific activity is 3550 units/mg. The preparation has been found to be essentially homogeneous by dodecylsulfate gel electrophoresis, isoelectrofocusing and gel filtration.
The molecular weight has been determined to be 22000 by several methods. The absorbance of a 1% solution at 280 nm is 6.9 and the isoelectric point by electrofocusing is pH 5.9.
The crystals of carp adenylate kinase have the space group P4122 or P4322.
The amino acid composition has been determined. There is no tryptophan, no cystine. There is one amino acid residue each of cysteine and histidine which are at or close to the catalytic center.
Several peptides derived by tryptic hydrolysis have been isolated and identified with corresponding peptides of porcine adenylate kinase. Consideration is given to histidine and cysteine being a part of the active site.
Multiple Forms of Tryptophanyl‐tRNA Synthetase from Beef Pancreas
Authors: LEMAIRE Geneviève , GROS Claude , EPELY Sylvie , KAMINSKI Marie , LABOUESSE Bernard 
Published: March 2005

Three different forms of tryptophanyl‐tRNA synthetase can be isolated from pancreatic extracts. Structural, immunological and catalytic properties of these various forms have been compared. The native enzyme is a dimeric molecule of molecular weight 108000. Two other forms, of molecular weight 85000 and 82000, are composed of two polypeptide chains identical with the carboxyl terminal region of the native subunits. These molecules are supposed to derive from the original protein by removal, from the amino‐terminal part of each subunit, of a fragment of 11000 to 13000 molecular weight. Such removal modifies the shape and the stability of the molecule and decreases its specific activity.The origin of the derived forms is attributed to proteolysis. In fact, limited proteolysis of purified tryptophanyl‐tRNA synthetase, in its native form, by elastase, results in the formation of an active compound, similar to one of the tryptophanyl‐tRNA synthetase derived forms. Furthermore, incubation with “elastolytic fractions” prepared from pancreatic extracts presenting a particularly high level of proteolytic activity produces the same cleavage in tryptophanyl‐tRNA synthetase polypeptide chain.
On the Mechanism of Action of Isocitrate Lyase
Authors: DIMROTH Peter , MAYER Klaus , EGGERER Hermann 
Published: March 2005

The enzymes citrate lyase and isocitrate lyase catalyse similar reactions in the cleavage of citrate to acetate plus oxaloacetate and of isocitrate to succinate plus glyoxylate, respectively.
Nevertheless, the mechanism of action of each enzyme appears to be different from each other. Citrate lyase is an acyl carrier protein‐containing enzyme complex whereas isocitrate lyase is not. The active form of citrate lyase is an acetyl‐S‐enzyme but that of isocitrate lyase is not a corresponding succinyl‐S‐enzyme.
In contrast to citrate lyase, the isocitrate enzyme is not inhibited by hydroxylamine nor does it acquire label if treated with appropriately labelled radioactive substrate.
Isotopic exchange experiments performed in H218 with isocitrate as a substrate produced no labelling in the product succinate. This was shown by mass‐spectrometric analysis.
The conclusion drawn from these results is that no activation of succinate takes place on the enzyme through transient formation of succinic anhydride or a covalently‐linked succinyl‐enzyme, derived from this anhydride
An Affinity‐Column Procedure Using Poly(l‐proline) for the Purification of Prolyl Hydroxylase
Authors: TUDERMAN Leena , KUUTTI Eeva‐Riitta , KIVIRIKKO Kari I. 
Published: March 2005

An affinity column procedure is reported for purifying prolyl hydroxylase. The procedure is based on the affinity of the enzyme for its competitive polypeptide inhibitor, and involves affinity chromatography in a column containing poly(l‐proline) of molecular weight 30000 linked to agarose, and the elution of the enzyme from the column with poly(l‐proline) of molecular weight 5700. The enzyme is finally separated from this polyproline by gel filtration.The procedure was employed for purifying prolyl hydroxylase from an ammonium sulphate fraction of chick embryo extract. The recovery of enzyme activity varied in ten enzyme preparations from 50 to 82%, and the purified preparations synthesized from 59.3 to 91.5 pmol hydroxyproline per mg enzyme per h at 37°C with a saturating concentration of (Pro‐Pro‐Gly)5 as substrate. The enzyme was pure when examined by polyacrylamide gel electrophoresis as a native protein or in the presence of sodium dodecyl sulphate, and the amino acid composition of the enzyme agreed with that reported previously with only minor exceptions. The molecular weight of the enzyme measured by equilibrium in an analytical ultracentrifuge was 240000, indicating that the enzyme had been isolated in the tetramer form.
Studies on New Intracellular Proteases in Various Organs of Rat
Authors: KATUNUMA Nobuhiko , KOMINAMI Eiki , KOBAYASHI Keiko , BANNO Yoshiko , SUZUKI Kunio , CHICHIBU Kenji , HAMAGUCHI Yoshitaka , KATSUNUMA Tsunehiko 
Published: March 2005

Specific proteases which inactivate the apo‐proteins of many pyridoxal enzymes were found in skeletal muscle, liver and small intestine of rats. The protease from these three organs were purified and their properties were compared.
The purified proteases from liver and skeletal muscle appeared homogeneous on acrylamide gel electrophoresis. Two different proteases were separated from small intestine. A homogeneous, crystalline enzyme was obtained from the muscle layer while enzyme from the mucosa was partially purified.
They showed substrate specificity for pyridoxal enzymes. Their pH optima were in an alkaline region. They showed activity with the substrate of chymotrypsin, N‐acetyl‐l‐tyrosine ethyl ester, but not with that of trypsin, p‐toluenesulfonyl‐l‐arginine ethyl ester. They were inhibited by pyridoxal phosphate or pyridoxamine phosphate and seryl residues were involved in their active center.
The four enzymes differed in the following characters: (a) molecular weights; (b) patterns of elution from a CM‐Sephadex column; (c) rates of inactivation of substrate enzymes; (d) rates of cleavage of N‐acetyl‐l‐tyrosine ethyl ester; (e) reactivities with antiserum against the enzyme from the muscle layer of small intestine; (f) specific activities.
The amino acid composition and effect of chemical modifications of the crystalline enzyme from the muscle layer of small intestine were examined to elucidate its active sites and mode of action. Serine and histidine residues were found to be essential for protease activity. A tyrosine residue was also necessary for activity. Modifications of its sulfhydryl group, amino residues and carboxyl group had no effect on its activity.
Studies on New Intracellular Proteases in Various Organs of Rat
Authors: BANNO Yoshiko , SHIOTANI Taiichi , TOWATARI Takae , YOSHIKAWA Daisuke , KATUNUMA Nobuhiko , KATSUNUMA Tsunehiko , AFTING Ernst‐Günter 
Published: March 2005

To study the role of group‐specific protease in enzyme degradation, alternation of its activity under various physiological conditions was examined.
Studies on the distribution of group‐specific protease in various organs of rats showed high activity in skeletal muscle and the muscle layer of small intestine, and rather low activity in liver. The activity varied in different muscles, but red muscle tended to have higher activity than white muscle. Activity was much lower in the muscles of the stomach and colon than in those of the small intestine.
Group‐specific protease in skeletal muscle increased under various dietary conditions (starvation, protein‐free diet or high protein diet), but the activities in the muscle layer of the small intestine and liver were not greatly influenced by dietary conditions. None of the hormones tested (i.e. hydrocortisone, glucagon, insulin, growth hormone and estrogen) influenced the activity of group‐specific protease in liver.
The level of group‐specific protease in skeletal muscle was increased markedly fifteen days after denervation, with a reciprocal decrease in the level of muscle phosphorylase, which is a good substrate of the protease.
Liver protease activity appeared in the late suckling period. The activity in skeletal muscle was high at the time of birth and attained the adult level 3 weeks after birth. The activity in the muscle layer of the small intestine did not change after birth. Thus the mechanisms for evoking these three specific proteases during development are apparently different. The activity of liver protease began to decrease approximately 12 h after partial hepatectomy and reached a minimum after about 72 h. Recovery of the protease activity was very slow and activity had not returned to the normal value 7 days after the operation. This observation seems to be consistent with the fact that there is little or no protease activity in liver in the neonatal period.
Dynamic Control on the Rate of the Reduction of the b Type Cytochromes in Submitochondrial Particles
Authors: EISENBACH Michael , GUTMAN Menachem 
Published: March 2005

In the presence of antimycin and KCN the reduction of cytochrome b in phosphorylating submitochondrial particles followed a biphasic first‐order kinetics. The transition from the first, rapid phase to the second, slow phase occurred while the reduction of cytochromes c + c1 and a through or around the antimycin block was still linear with time. Thus, the phase transition was due to a fall‐off in the rate of cytochrome b reduction.
The biphasic reduction of cytochrome b was observed over a wide temperature range (0 – 30°C), with succinate of NADH as electron donors and with phosphorylating particles or coupled rat‐heart mitochondria. With rat‐heart mitochondria the same biphasic reduction was observed in the presence of either carbonyl cyanide p‐trifluoromethoxyphenylhydrazone or oligomycin.
In both the rapid and the slow phases, the rate of reduction of cytochrome b‐561 was equal to that of b‐565. Thus both cytochromes b‐561 and b‐565 were affected by the mechanism which determined the reduction‐rate. Furthermore, each of these cytochromes could be reduced individually with rate constants typical of the slow phase.
The proportion of rapidly reduced to slowly reduced cytochrome b was independent of the degree of its reducibility and could be controlled by the experimental conditions. When antimycin was used as the only inhibitor, 96% of the b‐type cytochromes were reduced in the rapid phase. If the c and a‐type cytochromes were first reduced by ascorbate and tetramethyl‐p‐phenylenediamine in the presence of KCN and antimycin, all the b‐type cytochromes were fully reduced at the slow‐rate.
With succinate, the rate of the rapid phase depended on the activation level of the succinic‐dehydrogenase. The rate constant of the second phase was unaffected by the succinic dehydrogenase activity, if the preparation was more than 20% active. Furthermore, the rate constant of the slow reduction was the same with succinate, NADH, or even with durohydroquinone (which reacted directly with cytochromes b).
It is suggested that cytochrome b can exist in two forms: kinetically active or sluggish. The active form is rapidly reduced by the endogenous quinone (QH2) or durohydroquinone. The rate of the reduction of the active form by succinate or NADH is probably determined by the rate of the reduction of Q by the dehydrogenases. The second form of cytochrome b is characterized by its sluggish reduction by QH2 or durohydroquinone.
It is proposed that the transformation from the active to the sluggish form is induced by the reduction of a controlling group, named Y, located on the oxygen side of the antimycin inhibition site. When Y is oxidized, cytochrome b is in its active form, and when Y is reduced, cytochrome b is in its sluggish form. The nature of this kinetic control and a comparison with the mechanism controlling the reducibility of cytochrome b are discussed.
Lens Glycoproteins: Biosynthesis in Cultured Epithelial Cells of Bovine Lens
Authors: HUGHES R. Colin , LAURENT Maryvonne , LONCHAMPT Marie‐Odile , COURTOIS Yves 
Published: March 2005

Radioactivity from [3H]glucosamine is rapidly incorporated into cellular fractions of lens epithelial cells cultured in vitro. The incorporated isotope appears largely in glycoproteins of the cell surface that are exposed to trypsin and are released into a soluble form by proteolysis of intact cells. Glycoproteins are also secreted by cultured cells and can be recovered in the culture fluids. Sodium dodecylsulphate – polyacrylamide gel electrophoresis shows that a range of glycoproteins with apparent molecular weights from approximately 14000 to 120000 are present. The relationships of these glycoproteins to collagen and the non‐collagenous glycoproteins of lens basement membranes are discussed.
A plasma membrane fraction obtained from non‐trypsinised lens epithelial cells contains one major glycoprotein of apparent molecular weight 120000. A major non‐glycosylated polypeptide of molecular weight about 38000 detectable by Bloemendal et al.(1972) in plasma membranes of differentiated lens fibre cells was not prominent in lens epithelial cell membranes.
Examination of lens basement membranes extracted in various ways failed to reveal major glycoproteins of low molecular weight. Higher molecular weight glycoproteins, some of them related to collagen, were present.
Purification and Studies on Some Properties of the 4‐Aminobutyrate: 2‐Oxoglutarate Transaminase from Rat Brain
Authors: MAÎTRE Michel , CIESIELSKI Lucien , CASH Christopher , MANDEL Paul 
Published: March 2005

Using various chromatographic procedures, 4‐aminobutyrate: 2‐oxoglutarate transaminase from rat brain has been purified 2400 times with respect to the initial brain homogenate. The purified protein, which has a specific activity of 10 μmol × min−1× mg−1, gave a single band by acrylamide gel electrophoresis and isoelectric focusing. It has a molecular weight of 105000 ± 5000 and an isoelectric point of 6.8 In the presence of 0.1% sodium dodecylsulphate, a single protein band is seen on polyacrylamide gel, corresponding to a molecular weight of 57000 ± 5000. N‐terminal analysis reveals two chains with the same N‐terminal amino acid, thus the enzyme may be considered as a dimer consisting of two identical subunits. The pH optimum for enzyme activity is 8.5Studies of the enzymic reaction show that the general mechanism is of the ping‐pong bi‐bi model. The Km for 2‐oxoglutarate at saturating 4‐aminobutyrate concentration is 5.5 mM and V is 22 μmol × min−1× mg−1 whereas the km for 4‐aminobutyrate extrapolated to saturating 2‐oxoglutarate concentration is 4 mM. 2‐Oxoglutarate competitively inhibits the enzyme with respect to 4‐aminobutyrate, with a Ki of 1.8 × 10−4 M. The same phenomenon is seen for the reverse reaction where the Ki is 6.6 × 10−4 M for succinic semi‐aldehyde.