bluuebunny/crossref_metadata_2025_split huggingface dataset:
Two‐Dimensional Polyacrylamide‐Gel Electrophoresis for Purification of Small RNAs Specified by Virulent Coliphages T4, T5, T7 and BF23
Authors: IKEMURA Toshimichi , OZEKI Haruo 
Published: March 2005

RNAs synthesized in Escherichia coli infected with virulent phages T4, T5, T7 and BF23 were labelled with 32PO43− after phage infection. [32P]RNAs of low molecular weight were separated by two‐dimensional polyacrylamide gel electrophoresis, in which electrophoresis was carried out in two dimensions at different concentrations of acrylamide. The fractionated RNAs were characterized by RNA‐fingerprint patterns made after T1 ribonuclease digestion. The two‐dimensional gel of 10%→ 20% acrylamide was suitable for RNA of less than 200 nucleotides, while that of 5%→ 10% was preferred for RNAs of about 150–400 nucleotides.With T4 phage, 16 RNA species were separable on a single slab gel. Among those, 11 were identified as the known RNA species, including eight T4 tRNAs, one tRNA precursor and two non‐tRNA molecules. In the case of T5 and BF23, more than 20 RNA species were separated on a slab gel; 15 or more RNAs were found in the 4‐S RNA region, and several in 5‐S and 6‐S region. The RNA‐fingerprint patterns of many BF23 RNAs were very similar to those of corresponding RNAs of T5. Pseudouridine and ribosylthymidine, minor nucleosides generally present in tRNA, were found in several BF23 4‐S RNAs tested. Possibility of those BF23 4‐S RNAs as tRNAs is discussed. With phage T7, three RNAs were detected, two of which were much smaller than tRNAs.
Translation of mRNA for 3,4‐Dihydroxyphenylalanine Decarboxylase Isolated from Epidermis Tissue of Calliphora vicina R.‐D. in an Heterologous System
Authors: FRAGOULIS Emmanuel G. , SEKERIS Constantin E. 
Published: March 2005

RNA was isolated from the epidermis of Calliphora vicina larvae by phenol–chloroform extraction. The RNA sedimenting in sucrose gradients between 5 and 18 S was submitted to chromatography on oligo(dT)‐cellulose columns. The fraction binding to the oligo(dT) is able to stimulate protein synthesis in a system consisting of mouse liver ribosomal subunits, pH‐5 factors from rat liver and initiation factors from rabbit reticulocytes. Optimal Mg2+ concentration for the translation of insect mRNA is 3.5 mM, that of K+ 76 mM. Initiation factors prepared from epidermis of Calliphora larvae are less efficient in the translation of insect mRNA than initiation factors isolated from reticulocytes. The pH‐5 fraction from epidermis inhibits protein synthesis independent of the source of the mRNA fraction used.One of the proteins synthesized in the reconstituted system under the direction of insect mRNA has been identified as 3,4‐dihydroxyphenylalanine (DOPA) decarboxylase by immunoprecipitation with specific antiserum against DOPA decarboxylase and comigration in dodecylsulphate‐acrylamide electrophoresis with pure DOPA decarboxylase.Both mRNA from white prepupae and from 6–7‐days‐old larvae contain sequences coding for DOPA decarboxylase. However, white prepupae contains 3–4 times more DOPA decarboxylase‐mRNA than 6–7‐days‐old larvae. The content of DOPA decarboxylase mRNA is proportional to the amount of active DOPA decarboxylase molecules present in the animals from which the mRNA was isolated.
Fluorescence Studies on the Interaction between Yeast Seryl‐tRNA Synthetase and Its Substrates
Authors: MAELICKE Alfred , CRAMER Friedrich 
Published: March 2005

Substrate‐induced variations in the native fluorescence of seryl‐tRNA synthetase from yeast have been used to evaluate the binding equilibria with its ligands. Binding of l‐serine to the enzyme can be detected by equilibrium dialysis but not by fluorescence quenching. Hence, in contrast to ATP and tRNASer, l‐serine does not induce any fluorescence‐sensitive conformational change of the synthetase. A comparison of the binding constants for ATP (2 × 104 M−1) and seryl adenylate (1 × 106 M−1) indicates that the activation reaction is mainly driven by the higher affinity of seryl adenylate to the enzyme.tRNASer and seryl‐tRNASer though causing rather different maximal fluorescence quenching are bound to the enzyme with similar stoichiometry and association constant. The implications of this result are discussed with respect to the mechanism of the aminoacylation reaction.It is also shown that the enzyme clearly is capable of discriminating l‐serine from other amino acids, ATP from other nucleotidetriphosphates and AMP, and tRNASer from other tRNA species in the binding processes. The spectral properties, the effects of outside quenchers and the fluorescence decay times of the free enzyme and the enzyme‐substrate complexes are discussed with respect to the intercalation hypothesis of Hélène.
Adenosine Deaminase in Chicken‐Egg Yolk and Its Relation to Homologous Enzymes in Liver and Plasma of the Adult Hen
Authors: DE BOECK Stefaan , RYMEN Theo , STOCKX Jan 
Published: March 2005

Chicken egg yolk contains an adenosine deaminase that was investigated after purifying about 500 times. It has a pH optimum at 6.5, a Km of 6.6 × 10−5 mol/l and an approximate molecular weight of 14000; higher molecular forms could not be detected. It was compared with the adenosine deaminases of chicken liver and blood plasma. From this comparison it is evident that this protein has undergone certain changes during the successive events leading to its final structure (secretion by the liver, transport through blood plasma to the oocytes and development of the egg): a common subunit with an approximate molecular weight of 15 000 may be the basis of the physiological diversifications.Substrate specificity of the purified extracts extends to cytidine and guanosine also, although certain observations point to different enzymes being involved. Deoxyadenosine is also deaminated. Cu2+, Zn2+ and Pb2+ are inhibiting and free – SH seems essential for activity.
Quantitative Analysis of Mixed Association between Different Protein Molecules
Authors: IFFLAENDER Uwe , MARKAU Klaus , SUND Horst 
Published: March 2005

The theoretical and experimental analysis of a reversible association‐dissociation equilibrium between different proteins (mixed association) is described. The experiments were performed with glutamate dehydrogenases from beef and rat liver. These enzymes are different, especially with respect to their association behavior.The association constant of rat liver glutamate dehydrogenase has been determined by light‐scattering measurements. Its value (1.3 × 10−4 M−1) is much lower than that of the beef liver enzyme, but the difference in the free association energy is only 30%. Association between these two enzymes is observed, also employing light‐scattering experiments. Theoretical curves for mixed associating systems have been calculated and by comparison with these curves the mixed association constant could be determined. Since the free association energy of the mixed association is very near to the arithmetic mean between the values for the pure enzymes, the association interactions appear to be additive. The model of an open association with a virial coefficient is also true for the rat enzyme and the mixed association. The ultracentrifuge data are also explained by the same model and yield a similar value for the mixed association constant. Differences in the enzyme kinetics are small, but a somewhat reduced lifetime of the ternary complexes with the coenzymes and with substrates or GTP can be concluded for the rat liver enzyme. The circular dichroism measurements indicate no significant difference in the dissociation constants of the nucleotides, but the different amplitudes of the ellipticity indicate small differences in the electrical environment of the active center.
Metabolism of Isolated Kidney Tubules Independent Actions of Catecholamines on Renal Cyclic Adenosine 3′: 5′‐Monophosphate Levels and Gluconeogenesis
Authors: GUDER Walter G. , RUPPRECHT Arnold 
Published: March 2005

Isolated kidney cortex tubules from starved rats have been used to study the actions of catecholamines on renal adenosine 3′:5′‐monophosphate (Ado‐3′:5′‐P) levels and gluconeogenesis.In accordance with previous workers, norepinephrine was found to increase glucose formation from lactate and pyruvate and to a smaller degree from malate, succinate, fumarate, glutamate and glutamine. The stimulatory effect of 0.5 μM norepinephrine was additive to that of 0.1 mM Ado‐3′:5′‐P, indicating an Ado‐3′:5′‐P‐independent mechanism of catecholamine action. The effects of parathyroid hormone and oleate on gluconeogenesis were also additive to that of norepinephrine. A comparative study of the actions of different catecholamine derivatives revealed that gluconeogenesis was stimulated in parallel to the α‐adrenergic potency of the hormones, whereas Ado‐3′: 5′‐P levels were increased according to the known β‐stimulatory potency of the agents. Although iso‐proterenol was by far the most effective in raising Ado‐3′: 5′‐P levels, it was without effect on glucose formation from pyruvate, when added at 0.1 μM. At the same concentration, phenylephrine, which had no effect on Ado‐3′:5′‐P levels, was the best stimulator of gluconeogenesis.The α‐receptor blocking agent phentolamine inhibited the stimulatory effect of catecholamines on gluconeogenesis with a 50 times higher potency than propranolol, a,β‐blocking agent. The fact that the stimulatory effect of Ado‐3′:5′‐P was also blocked by propranolol, indicated an unspecific mechanism of action of this substance. The results indicate that the stimulatory effect of catecholamines on renal gluconeogenesis are mediated by an a‐receptor and that they are independent from the stimulation of renal adenyl cyclase by these agents.
Mechanism of Action of Rifamazine, a Member of a New Class of (Dimeric) Rifamycins
Authors: FIETTA Anna M. , SILVESTRI Luigi G. 
Published: March 2005

Rifamazine (AF/RP) a dimeric rifamycin, is active against bacterial DNA‐dependent RNA polymerase and against viral RNA‐dependent DNA polymerase.
Rifamazine is active also against DNA‐dependent RNA polymerase extracted from rifampicin‐resistant mutants of Escherichia coli. It does not interfere with enzyme‐template interaction or with RNA elongation. It blocks initiation.
A comparison is made between the mechanism of action of rifamazine and that of rifampicin, and of AF/013 (octyloxime of 3‐formylrifamycin SV), a C‐class rifamycin. Our results show that the mechanism of action of rifamazine is more similar to that of rifampicin than to that of the octyloxime derivative.
Activity of rifamazine against RNA polymerase from rifampicin‐resistant mutants is thought to be due to binding of the dimer to both the rifamycin‐specific binding site and to a second weak site.
Assay of Collagen‐Galactosyltransferase and Collagen‐Glucosyltransferase Activities and Preliminary Characterization of Enzymic Reactions with Transferases from Chick‐Embryo Cartilage
Authors: MYLLYLÄ Raili , RISTELI Leila , KIVIRIKKO Kari I. 
Published: March 2005

Procedures are described for the assay of collagen galactosyltransferase and collagen glucosyltransferase activities. The methods are based on the transfer of [14C]galactose or [14C]glucose from the corresponding radioactive UDP‐glycoside to hydroxylysyl or galactosylhydroxylysyl residues in calf‐skin gelatin substrate, and on the specific assay of the products of the enzymic reactions. The assay of the products includes precipitation of the protein, and removal of most of the radioactivity present in the free UDP‐glycoside by several washings of the precipitate. The [14C]galactosylhydroxylysine or [14C]glucosylgalactosylhydroxylysine is then liberated by alkaline hydrolysis, and purified further by a modified Dowex chromatography and by paper electrophoresis. The procedure is entirely specific when judged by amino acid analysis, and it is possible to assay a series of 36 samples in 2 days. The sensitivity of the method permits assays in extracts from several tissues, including tissues with low transferase activities, such as liver.Preliminary characterization of the transferases from chick embryo cartilage indicated that the galactosyltransferase did not catalyze the transfer of galactose to free hydroxylysine. Dialyzable peptides were found to act as substrates for both transferases. The Km, for the gelatinized collagen substrate was 150 g/l in the reaction with the galactosyltransferase, and 14 g/1 in that with the glucosyltransferase. The Km, for UDP‐galactose or UDP‐glucose in the reaction with the corresponding transferase was about 30 μM. Both transferases required a metal co‐factor, and manganese was found to be far more effective than any other metal tested. The optimal manganese concentration for both transferases was 10 mM. No stimulation of the glucosyltransferase activity was found with folate, which has recently been reported to stimulate collagen glucosyltransferase from rat kidney.
Glycerol‐3‐Phosphate Shuttle and Its Function in Intermediary Metabolism of Hamster Brown‐Adipose Tissue
Authors: HOUŠTÊK Josef , CANNON Barbara , LINDBERG Olov 
Published: March 2005

Brown adipose tissue of the hamster possesses high specific activities of soluble, cytoplasmic NAD‐linked, as well as mitochondrial flavin‐coupled, glycerol‐3‐phosphate dehydrogenases. The ratio of the two enzyme activities is high (close to 1), when compared with other tissues of the hamster.
In the presence of rotenone, NADH is oxidised very poorly by homogenates of brown adipose tissue. A high rate of oxidation is obtained upon further addition of dihydroxyacetone phosphate, which itself is negligible oxidised. When followed fluorimetrically glycerol 3‐phosphate can also be observed to induce NADH oxidation, but only after a significant lag time. Similar results are obtained with isolated mitochondria + high‐speed supernatant. With high‐speed supernatant alone, only dihydroxyacetone phosphate has any effect, whereas with isolated mitochondria neither dihydroxy‐acetone phosphate nor glycerol 3‐phosphate induce any NADH disappearance.
Respiration induced by NADH + dihydroxyacetone phosphate in homogenates equals 56% of the respiration induced by glycerol 3‐phosphate alone.
Respiration induced by NADH + dihydroxyacetone phosphate, as well as that induced by glycerol 3‐phosphate, is inhibited by the same concentrations of inhibitors as are required for inhibition of the mitochondrial dehydrogenase i.e. EDTA, long‐chain unsaturated fatty acids, long‐chain fatty acyl CoA esters.
In isolated brown adipocytes in the presence of rotenone, norepinephrine significantly inhibits respiration induced by glycerol 3‐phosphate.
The results obtained are discussed with respect to the role of glycerol 3‐phosphate as an electron sink for cytosolic reducing equivalents to maintain a low level of extramitochondrial NADH. A means of maintaining a level of glycerol 3‐phosphate adequate for triglyceride synthesis is also considered.
The Reactive Sites of Kunitz Bovine‐Trypsin Inhibitor
Authors: CHAUVET Jacqueline , ACHER Roger 
Published: March 2005

Kunitz bovine trypsin inhibitor gave with α‐chymotrypsin a stoichiometric complex stable at neutral pH. The complex has been characterized by amino acid composition, molecular sieving and zone electrophoresis. Complete dissociation occurred at pH 4.0 as shown by gel filtration. α‐Chymotrypsin was displaced from the complex by trypsin either in solution or by affinity chromatography on trypsin‐Sepharose: α‐chymotrypsin was recovered in the filtrate (yield about 100%) and the inhibitor was eluted from trypsin‐Sepharose with 0.1 M HCl (yield: 83%).Lysine‐15 of the inhibitor was shown to be involved in the interaction between α‐chymotrypsin and the inhibitor. When the complex was maleylated, the maleylated chymotrypsin‐bound inhibitor was displaced by affinity chromatography on trypsin‐Sepharose. The recovered derivative was oxidized, subjected to tryptic hydrolysis and the products separated by peptide mapping and analyzed. The peptides were compared with those obtained with non‐maleylated inhibitor and fully maleylated free inhibitor. In the fully maleylated inhibitor, the four lysyl residues of the molecule were blocked but in the maleylated chymotrypsin‐bound inhibitor, Lys‐15 was unmodified in contrast to Lys‐26, Lys‐41 and Lys‐46; therefore Lys‐15 is shielded by chymotrypsin in the complex.On the other hand, when inhibitor with a selectively reduced carboxamidomethylated Cys‐14–Cys‐38 disulfide bridge was allowed to react with chymotrypsin, cleavage occurred not only at Tyr‐21, Tyr‐35 and Phe‐45 but also at Lys‐15, cleavage not observed in the case of the fully oxidized inhibitor. This result shows that under particular conditions the bond Lys‐15–Ala‐16 can be the substrate for chymotrypsin and the side chain of Lys‐15 can be inserted in the chymotrypsin specificity pocket. Apparently the contact area of inhibitor with chymotrypsin seems to be similar to that with trypsin [J. Chauvet and R. Acher (1967) J. Biol. Chem. 242, 4274–4275].
Substrate Stereochemistry of the Enoyl‐CoA Hydratase Reaction
Authors: WILLADSEN Peter , EGGERER Hermann 
Published: March 2005

A specimen of stereospecifically 2‐tritiated 3‐hydroxybutyric acid was prepared by hydroboration of ethyl crotonate. It was assumed that the hydroboration reaction took asyncourse and hence that (2R,3S) + (2S,3R)‐3‐hydroxy[2‐3H1]butyric acid was formed after oxidation and hydrolysis.3RS‐3‐Hydroxy[2‐3H2]butyric acid, symmetrically tritiated at C‐2, was prepared by isotopic exchange of ethyl acetoacetate in tritiated water, followed by reduction and hydrolysis.The 3R‐enantiomers of the acids listed under paragraphs (1) and (2) were destroyed enzymically by use of 3R‐specific 3‐hydroxybutyrate dehydrogenase and the residual 3S‐enantiomers were isolated.The resulting specimens of 2R,3S‐3‐hydroxy[2‐3H1]butyric acid and 3S‐3‐hydroxy[2‐3H2]‐butyric acid were converted chemically to the acyl‐CoA derivatives. These were incubated with enoyl‐CoA hydratase.In the presence of the enoyl‐CoA hydratase symmetrically labelled 3S‐3‐hydroxy[2‐3H2]butyryl‐CoA lost nearly 50% of its tritium label; 2R,3S‐3‐hydroxy[2‐3H1]butyryl‐CoA lost about 78%.It was concluded that the elimination of the elements of water from 3‐hydroxybutyryl‐CoA on the hydratase occurs stereospecifically withsyngeometry.
Substrate Stereochemistry of the Acetyl‐CoA Acetyltransferase Reaction
Authors: WILLADSEN Peter , EGGERER Hermann 
Published: March 2005

A specimen of symmetrically tritiated 3S‐3‐hydroxy[2‐3H2]butyryl‐CoA was prepared from acetoacetyl‐CoA by incubation in tritiated water, followed by enzymic reduction using 3S‐specific 3‐hydroxyacyl‐CoA dehydrogenase. In addition, a specimen of dideuterated, racemic 3RS‐3‐hydroxy‐[2‐2H2]butyryl‐CoA was prepared chemically from 3RS‐3‐hydroxy[2‐2H2]butyric acid.These acyl‐CoA derivatives were incubated respectively with deuterium oxide and tritiated water in presence of enoyl‐CoA hydratase. Stereospecifically tritiated compounds, respectively 2S,3S‐3‐hydroxy[2‐2H1,3H1]butyryl‐CoA and 2R,3S‐3‐hydroxy[2‐2H1,3H1]butyryl‐CoA (plus 3R‐diastereomer) were formed and isolated.3S‐3‐Hydroxy[2‐3H2]butyryl‐CoA and the 3S‐[2‐3H2,3‐14C]‐labelled material were also prepared as described in the preceding paper. The latter substrate was used to establish conditions in which little loss of tritium would occur (found: about 7%) on cleavage to acetyl‐CoA in the presence of 3‐hydroxyacyl‐CoA dehydrogenase and acetyl‐CoA acetyltransferase. Little loss of tritium indicates a small degree of racemization of the stereospecifically 2‐tritiated 3‐hydroxyacyl‐CoA derivatives at the level of transiently formed acetoacetyl‐CoA and acetyl‐CoA. This ensures high optical purity of the chiral acetates generated from the stereospecifically tritiated hydroxybutyryl‐CoA specimens listed in paragraph (2).These two acyl‐CoA derivatives and the symmetrically tritiated 3S‐3‐hydroxy[2‐3H2]butyryl‐CoA listed in paragraph (3) were converted to the acetates enzymically in the sequence hydroxy‐butyryl‐CoA → acetoacetyl‐CoA → acetyl‐CoA → acetate. The isolated acetates were assayed for chirality by conversion to the malates and fumarates as usual.The malate formed from 3S‐3‐hydroxy[2‐3H2]butyryl‐CoA on incubation with fumarase lost nearly 50%, that derived from 2R3S‐3‐hydroxy[2‐2H1,3H1]butyryl‐CoA (plus 3R‐diastereomer) retained about 26% and that derived from 2S,3S‐3‐hydroxy[2‐2H1,3H1]butyryl‐CoA retained about 78% of its total tritium content.It was concluded that the detachment of acetyl‐CoA from acetoacetyl‐CoA on acetyl‐CoA acetyltransferase occurs with inversion of configuration at the methylene group which becomes methyl.
Lysophospholipids in Pig Pancreatic Zymogen Granules in Relation to Exocytosis
Authors: RUTTEN Willem J. , DE PONT Jan Joep H. H. M. , BONTING Sjoerd L. , DAEMEN Frans J. M. 
Published: March 2005

A hypothesis to explain the stimulatory role of cyclic AMP (adenosine 3′:5′‐monophosphate) in pancreatic enzyme secretion has been tested. In this hypothesis cyclic AMP would activate a phospholipase activity, which would lead to a locally increased lysophospholipid formation, resulting in a fusion between the zymogen granule membrane and the apical plasma membrane.Cyclic AMP added to isolated pig pancreatic zymogen granules leads to an increased lysis of these granules, but the slowness of this effect makes its physiological significance dubious. In pancreatic homogenates or zymogen granules no stimulating effect of cyclic AMP on lipase or phospholipase activity could be demonstrated.Isolated zymogen granules have a high lysophospholipid content (27% of total phospholipids), consisting of the 1‐acyl and 2‐acyl forms of lysophosphatidylcholine and lysophosphatidylethanolamine. Experiments with radioactive phosphatidylcholine indicate that the lysophospholipids are due to the action of endogenous (phospho)lipases during the isolation procedure.It is concluded that these experiments do not lend support to the above hypothesis for the mechanism of action of cyclic AMP in pancreatic enzyme secretion.
Interactions of Heteroaromatic Compounds with Nucleic Acids
Authors: MÜLLER Werner , CROTHERS Donald M. 
Published: March 2005

We have examined the origins of base specificity in intercalating ligands by studying the interaction with DNA of a series of proflavine and acridine orange analogs differing in the heteroatoms present in the chromophore. Base specificity was determined by differential dialysis of the dye against DNA samples of differing G · C content. We find that G · C specificity increases as the visible absorbance band of the chromophore moves to longer wavelength, implying a relation between specificity and polarizability of the chromophore. This can be rationalized by recognizing that the G · C pair is more polar than A · T, and should therefore interact more favorably with an easily polarized ring system. We find in addition that dimethylation of the chromophore amino groups increases specificity which we discuss in terms of steric and coupled steric‐electronic contributions.Our results also bear on the origin of G · C specificity in binding actinomycin to DNA. Some of the compounds studied are as G · C specific as actinomycin, yet they lack hydrogen‐bonding functions as plausible determinants of specificity. This observation gives new life to the hypothesis that the specificity of actinomycin is determined primarily by preferential interaction of the chromophore with a G · C pair.