bluuebunny/crossref_metadata_2025 huggingface dataset:
An Investigation of the 16‐S RNA Binding Sites of Ribosomal Proteins S4, S8, S15 and S20 from Escherichia coli
Authors: UNGEWICKELL Ernst , GARRETT Roger , EHRESMANN Chantal , STIEGLER Patrick , FELLNER Peter
Published: March 2005

The RNA binding sites of four 30‐S ribosomal subunit proteins from Escherichia coli, namely S4, S8, S15 and S20 were prepared from reconstituted single protein · 16‐S‐RNA complexes by mild enzymic digestion of non‐protected RNA regions. Oligonucleotide fingerprints of the protected RNA regions were obtained and their positions were located within the 16‐S RNA sequence. They were not completely contiguous regions of RNA; oligonucleotides had been excised from each of them. The binding sites of S4 and S20, and those of S8 and S15 showed overlapping. The specificity of the RNA binding sites was confirmed by a reconstitution method.
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.
The Covalent Structure of Collagen: Amino‐Acid Sequence of the Cyanogen‐Bromide Peptides α1‐CB2, αl‐CB4 and α1‐CBS from Calf‐Skin Collagen
Authors: FIETZEK Peter P. , KÜHN Klaus
Published: March 2005

The amino acid sequence of 120 residues in the N‐terminal region of the α1‐chain of calf skin collagen (comprising the cyanogen‐bromide‐derived peptides α1‐CB2, α1‐CB4 and α1‐CB5) has been determined by automated Edman degradation. The lysyl residue in position 87 is completely hydroxylated, while those in positions 99 and 108 partially hydroxylated. Two substitutions are found with respect to the homologous region of the α1‐chain from rat skin collagen. Positions 101 and 102 of calf skin collagen are occupied by Asp‐Ala, in rat skin collagen by Asn‐Thr. The extensive homology in this region is remarkable and is not found in other regions of the α1 and α2‐chain.
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.
The Specificity of a Neutral Deoxyribonuclease from Cancer pagurus
Authors: SABEUR Georgette , AUBEL‐SADRON Geneviève , BERNARDI Alberto , BERNARDI Giorgio
Published: March 2005

The recently isolated neutral deoxyribonuclease from crab (Cancer pagurus) testes has been characterized in its mode of action and its specificity. The enzyme is a typical endonuclease, forming 5′‐phosphate oligonucleotides of large average size; after extensive digestion of calf thymus DNA over 75% of the fragments have a size larger than pentanucleotides and mononucleotides are absent. As far as specificity is concerned, thymidine is very abundant in the 5′‐penultimate position (∼ 50%) and in the 3′‐terminal position (37%) and almost absent in the 5′‐terminal position (∼ 1%), the values quoted concerning Escherichia coli digests of average size (P̄n) between 50 and 10.
Nuclear‐Magnetic‐Resonance Studies of Carboxypeptidase B
Authors: ZISAPEL Nava , SOKOLOVSKY Mordechai , NAVON Gil
Published: March 2005

Longitudinal and transverse proton relaxation rates of water in solutions of porcine manganese carboxypeptidase B have been measured in the presence of various competitive inhibitors by pulse nuclear magnetic reasonance (NMR) spectrometry. The inhibition constant of Mn‐carboxypeptidase activity by l‐argininic acid and acetyl‐l‐arginine was in agreement with the equilibrium constant obtained by the NMR method, indicating similar and specific binding of the inhibitors to the active site of the manganese enzyme. Titration of the water bound to the metal ion revealed the presence of one water molecule which could be displaced from the sphere of the manganese ion by various inhibitors. The structural features of the inhibitors required for this displacement as well as the mode of interaction is described.
On the pH‐Dependence of the Reaction of Hemoglobin with Carbon Monoxide
Authors: MAY Roland P. , MAYER Adalbert
Published: March 2005

Flash‐photolysis experiments were performed on solutions of carbonmonoxy hemoglobin (human Hb A) as function of pH. The fraction of fast reaction and the amount of photodissociation as produced by a given amount of light quanta has been analyzed in terms of the allosteric model of ligand binding by Monod, Wyman and Changeux. It is shown how the switch‐over point of the allosteric transition from the T to R state is controlled by the protons, which act as allosteric effectors.
Metabolic Instability of Myelin Proteins
Authors: BENUCK Myron , MARKS Neville , HASHIM George A.
Published: March 2005

Myelin basic protein, purified from bovine spinal cord, is cleaved by a purified bovine brain acid proteinase to yield three peptide fragments which were purified by gel filtration procedures combined with preparative gel electrophoresis. The purity of the fragments was established by end group analysis, amino acid analysis, polyacrylamide disc gel electrophoresis, and by their encephalitogenic activity. Chemical and biological characterization shows that one peptide, (peptide II), originates from the N‐terminal end of the basic protein and contains residues 1–43; a second peptide, (peptide I), contains residues 44–89 and a third peptide (peptide III). originates from the C‐terminal region with an N‐terminal phenylalanine, residue 90, through arginine, residue 170, of the basic protein. The combined amino acid composition of the three fragments account for the amino acid content of the basic protein. The structure of the three peptides demonstrates that the purified brain acid proteinase selectively cleaves the two phenylalanine‐phenylalanine linkages formed between residues 43–44 and 89–90 of the basic protein molecule. This study shows that cathepsin D could be the endogenous enzyme responsible for the breakdown of basic protein to form smaller encephalitogenic fragments, some of which are known to be present in crude tissue extracts.
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].
Observations Concerning the Sequence of Two Additional Specifically Encapsidated RNA Fragments Originating from the Tobacco‐Mosaic‐Virus Coat‐Protein Cistron
Authors: GUILLEY Hubert , JONARD Gerard , RICHARDS Kenneth Eugene , HIRTH Léon
Published: March 2005

The incubation of 25‐S tobacco mosaic virus (TMV) protein with a mixture of RNA fragments produced by partial T1 RNase digestion of TMV RNA results in the encapsidation of only a few species of RNA. In addition to the most predominant species, fragment 1, whose sequence has been described in the preceding paper, two other species, fragment 41 and fragment 21, are coated by the protein. These two RNA fragments were purified by polyacrylamide gel electrophoresis and subjected to total digestion with pancreatic and T1 RNase. The oligonucleotides were separated by paper electrophoresis and characterized insofar as possible by digestion with the complementary ribonuclease. From the amino acid coding capacity of the oligonucleotides liberated from fragments 41 and 21 by T1 RNase digestion, it appears that these two fragments, like fragment 1, are derived from the coat protein cistron. They are situated immediately prior to fragment 1 and, together with this fragment, constitute a continuous stretch of 232 nucleotides of the cistron which codes for amino acids 53 to 130 of the coat protein. The order of the fragments in the sequence is 21 – 41 – 1. A possible model for the secondary structure of this portion of the sequence is proposed.
The “Hidden Ligand” of the Galactose‐Binding Protein
Authors: SILHAVY Thomas J. , BOOS Winfried
Published: March 2005

Following tryptophan fluorescence of the galactose‐Binding Protein during dissociation of the ligand it has been found that glucose dissociates with a half life of less than 5 s. Similarly, fast dissociation was also observed by following release of radioactively labelled glucose from Sepharose‐coupled galactose‐binding protein upon dilution. Accordingly, a previous claim that the galactose‐binding protein contains glucose as a non‐dissociable “hidden ligand” [G. Richarme and A. Kepes (1974) Eur. J. Biochem. 45, 127–133] has to be reinterpreted.
Studies on the de novo Biosynthesis of NAD in Escherichia coli
Authors: GRIFFITH Gary R. , CHANDLER Jerry LR , GHOLSON Robert K.
Published: March 2005

Quinolinic acid (pyridine 2,3‐dicarboxylic acid) which is an immediate precursor of the pyridine nucleotides, is synthesised from l‐aspartate and dihydroxyacetone phosphate in Escherichia coli. Extracts from certain nadB mutants complement the extracts prepared from all nadA mutants for the enzymic synthesis of quinolinate. Using the complementation assay, the quinolinate synthetase B protein has been purified more than 300‐fold. The quinolinate synthetase B protein exists in all nadA and nadC mutants examined. The quinolinate synthetase A protein was present in all nadC mutants and most (but not all) nadB mutants. The facile separation of the wild‐type quinolinate synthetase A and B proteins out of a nadC mutant suggests that quinolinate synthetase does not exist as a tightly bound complex. The partially purified quinolinate synthetase is inhibited by physiological concentrations of NAD and NADH but not by NADP or NADPH.
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.
Interactions of Heteroaromatic Compounds with Nucleic Acids
Authors: MÜLLER Werner , BÜNEMANN Hans , DATTAGUPTA Nanibhushan
Published: March 2005

This paper presents the results of a systematic study on the effects of substituents on the base and sequence specificity of tricyclic heteroaromatic compounds interacting with DNA by intercalation. All the compounds tested are derived from proflavine and acridine orange analogs with different heteroatoms in the middle ring. Their base and sequence specificities were determined by differential dialysis of the ligand against DNA samples of differing G · C content. The main results indicate that (a) the introduction of a phenyl substituent into one of the two available positions of the middle ring increases or decreases the G · C specificity of the ligand depending on the position where the substitution takes place; (b) compounds of the substitution type of neutral red (2‐methyl‐3‐amino‐7‐dimethyl‐amino‐phenazine) show unexpectedly high G · C specificities and (c) DNA ligands of pronounced sequence specificity for adjacent G · C pairs can be constructed by combining the structural elements of neutral red with an additional phenyl residue in the same molecule.

The further study of compounds related to the phenylated neutral red revealed that the G · C specificity can be improved or destroyed by additional substituents.

The comparison of the G · C specificity and the DNA‐affinity data of the compounds studied leads to the suggestion that the specificity arises mainly from electronic factors which are strongly controlled through steric constraints on possible complex geometries. As a basis for the discussion a possible structure for the DNA complex of the phenylated neutral red is considered in which the extra phenyl ring at N‐5 of the phenazinium system, protrudes into the large groove of the DNA helix while the tricyclic part of the ligand is inserted between the DNA base‐pairs.
The Stimulation of Liver Phosphorylase b by AMP, Fluoride and Sulfate
Authors: STALMANS Willy , HERS Henri‐Géry
Published: March 2005

The activity of liver phosphorylase b from several mammalian species has been studied. The enzyme from rat or mouse has a higher activity than the rabbit enzyme, which is itself more active than pig liver phosphorylase b.

The activity of liver phosphorylase b is influenced by anions and by AMP, and these effects are influenced by pH. Fluoride, which is currently added to the assay mixture of phosphorylase a in crude preparations, is about as active as sulfate as a stimulator of phosphorylase b.

When assayed at pH 6.1 and in the presence of 0.15 M NaF, the activity of rat liver phosphorylase b reaches 25% of that of the a enzyme; if 1 mM AMP is also present, this value rises to 50%.

Methods are described that allow the determination of liver phosphorylase a without interference of b, and the determination of total phosphorylase (a+b) in rat liver.
Non‐Helical Sequences of Rabbit Collagen
Authors: BECKER Udo , FIETZEK Peter P. , TIMPL Rupert , FURTHMAYR Heinz
Published: March 2005

Non‐helical peptide fragments were isolated from rabbit skin collagen after cleavage of α chains with cyanogen bromide and proteases. Determination of their amino acid sequence indicated a length of 9, 16 and 25 amino acid residues for the non‐helical sequences located in the N‐terminal region of α2 and α1 chain and in the C‐terminal region of α1 chain, respectively. The C‐terminal sequence Tyr‐Tyr hitherto considered as the genuine end of collagen α 1 chain is in part of rabbit collagen extended by two residues, alanine and arginine.

Rabbit collagen may differ considerably in its non‐helical sequences from other vertebrate col‐lagens, particularly in the C‐terminal part. Some but not all of these differences are clustered in areas occupied by antigenic determinants which are recognized in the antibody response of rabbits to rat or calf collagen. On the other hand, a high homology to rabbit collagen, e.g. in the N‐terminal region of rat collagen α1 chain or calf collagen α2 chain, probably prevents immunological recognition by the rabbit. The degree of foreignness alone, however, may not necessarily determine whether a particular non‐helical area is able to express immunogenic activity.
The Mitochondrial Genome of Euglena gracilis
Authors: FONTY Godeleine , BERNARDI Giorgio , CROUSE Edwin J. , STUTZ Erhard
Published: March 2005

Mitochondrial DNA from Euglena gracilis has been investigated in its chemical and physical properties. Its G + C content is equal to 25%; its buoyant density in a CsCl density gradient (1.690 g/cm3) is higher, by 5 mg/cm3, than expected for a bacterial DNA having the same base composition. The buoyant densities of denatured and renatured DNA are higher than that of native DNA by 10–12 mg/cm3 and 6 mg/cm3, respectively. The melting temperature, Tm, is 77°C in standard saline citrate; the first derivative of the melting curve shows a striking multimodality. Degradation of the DNA by micrococcal nuclease indicates that about 40% of the DNA is formed by stretches lower than 10% in G + C. In all its properties the mitochondrial DNA from Euglena gracilis is strikingly similar to that of Saccharomyces cerevisiae.
Reassociation Kinetics of Deoxyribonucleic Acid in Antigen‐Stimulated Mouse‐Spleen Cells
Authors: MOAV Neomi , FRENSDORFF Asher , MOAV Boaz
Published: March 2005

In order to directly compare the complexity of the genome of lymphoid cells which have been antigenically stimulated, with that of non‐immunized and non‐lymphoid cells, DNA was pulse labeled and extracted from BALB/c mouse spleen cells at various time intervals after antigenic stimulation in vivo; the reassociation rates of these newly synthesized DNA preparations were compared with those of the total mouse spleen DNA, obtained from the same sources and at the same times.

DNA labeled for 60 min at 43, 53, or 72 h after antigenic restimulation, reassociated faster than the corresponding total DNA. On the other hand, the reassociation profile of DNA, labeled for 60 min during the first 24 h after restimulation did not differ from that of the total DNA extracted at the same time. When labeled thymidine was available for incorporation at a constant concentration over a period of 24 h, reassociation patterns of labeled DNA were identical to those of the corresponding total DNA at all times after restimulation. Newly synthesized nuclear DNA exhibited reassociation profiles identical to those of the corresponding total nuclear DNA at all times tested. Also, no differences between the reassociation rates of nuclear and total cellular DNA were observed.

It was concluded that antigenic stimulation does not induce a major amplification of genes in the stimulated cells, and that the rapidly reassociating DNA species described represent extranuclear (cytoplasmic) DNA.
The Biosynthesis of Basement‐Membrane Collagen by Isolated Rat Glomeruli
Authors: GRANT Michael E. , HARWOOD Richard , WILLIAMS Isabel F.
Published: March 2005

A technique is described for the rapid isolation of highly purified preparations of viable glomeruli from rat kidney cortex. The synthesis of protein as judged by the incorporation of [14C]proline into non‐diffusible material was shown to be linear for up to 6 h. The synthesis of collagen, measured as non‐diffusible 4‐hydroxy[14C]proline, was also linear over this period but represented only a small proportion of total protein synthesis. Similar studies conducted in vivo confirmed that collagen synthesis accounted for less than 5% of total protein synthesis in glomeruli.

When isolated glomeruli were incubated with [14C]proline, it was found that approximately 16% of the hydroxyproline present in the collagenous component occurred as the 3‐isomer. When glomeruli were incubated with [14C]lysine over 90% of the hydroxy[14C]lysine synthesised was glyco‐sylated and most of the glycosylated hydroxy[14C]lysine was present as glucosyl‐galactosyl‐hydroxy‐[14C]lysine.

The size of the basement membrane collagen synthesised by the isolated glomeruli was estimated by treating the 14C‐labelled protein with mercaptoethanol and sodium dodecyl sulphate and then chromatographing the 14C‐labelled protein on an agarose column equilibrated and eluted with buffer containing 0.1% (w/v) sodium dodecyl sulphate. The initial form of [14C]collagen synthesised was found to consist of polypeptide chains which had molecular weights of approximately 140000 and which were shown to be distinctly larger than the polypeptide chains from embryonic chick tendon procollagen. Also when glomeruli were labelled with [14C]proline for 2 h and chased with unlabelled proline for 4 h there was a time‐dependent conversion of the initially synthesised collagen moiety to collagen polypeptide chains which co‐chromatograph with tendon pro‐α chains (molecular weight approx. 120000).
The Amino‐Acid Sequence of Leghemoglobin Component a from Phaseolus vulgaris (Kidney Bean)
Authors: LEHTOVAARA Päivi , ELLFOLK Nils
Published: March 2005

Leghemoglobin component a from Phaseolus vulgaris (kidney bean) was digested with trypsin; 15 tryptic peptides and free lysine were purified and the amino acid sequences of the peptides deter mined.

The internal order of the tryptic peptides was determined by the bridge peptides obtained from the thermolytic digest and the dilute acid hydrolyzate of kidney bean leghemoglobin a; 12 thermolytic peptides and two acid hydrolysis peptides were purified and the sequences were partially or completely determined.

The complete amino acid sequence of kidney bean leghemoglobin a is compared to that of leghemoglobin a from soybean (Glycine max) and to some animal globins. As regards sequence, the kidney bean globin has 79% identity with the soybean globin and 21% identity with human hemoglobin γ‐chain. Seven of the 14 amino acid residues common to most globins are found in the kidney bean globin. Trp‐15 and Tyr‐145 are evolutionarily conserved in this globin, which confirms the concept of a common origin of animal and plant globins.
Electrodialysis of Lipopolysaccharides and Their Conversion to Uniform Salt Forms
Authors: GALANOS Chris , LÜDERITZ Otto
Published: March 2005

Ions of low molecular weight like metal cations and basic amines are present in lipopolysaccharides regardless of the isolation procedure employed. They are present in salt form with the acidic groups of the molecule and, partly, bound by chelation. Electrodialysis which removed a large proportion of these basic materials led to acidic lipopolysaccharides often with reduced solubility. Electro‐dialyzed lipopolysaccharides could be rendered soluble by neutralizing with alkali or with a basic amine. Depending on the base employed for neutralization preparations were obtained which showed in water distinct differences in solubility, viscosity and opalescence. These differences were related to differences in the sedimentation coefficients of the various salt forms. Neutralization with tri‐ethylamine led in all cases to highly soluble preparations with low sedimentation coefficients, while, on the other hand, neutralization with Mg(OH)2 led in most cases to insoluble preparations. The acidic lipopolysaccharides obtained by electrodialysis deteriorate on storing in a freeze‐dried form. On heating in distilled water autohydrolysis occurs and free lipid A is liberated. The lipid A which is so far known as a water‐insoluble material showed increased solubility when prepared from electro‐dialyzed lipopolysaccharides.
Purification of Rheumatoid Synovial Collagenase and Its Action on Soluble and Insoluble Collagen
Authors: WOOLLEY David E. , GLANVILLE Robert W. , CROSSLEY Malcolm J. , EVANSON John M.
Published: March 2005

The neutral collagenase released into the culture medium by explants of rheumatoid synovial tissue has been purified by ultrafiltration and column chromatography, utilising Sephadex G‐200, Sephadex QAE A‐50 and Sephadex G‐100 superfine.

The final collagenase preparation had a specific activity against thermally reconstituted collagen fibril of 312 μg collagen degraded min−1 mg enzyme protein−1, representing more than a 1000‐fold increase over that of the active culture medium.

Electrophoresis in polyacrylamide disc‐gels with and without sodium dodecyl sulphate showed the enzyme to migrate as a single protein band. Elution experiments from polyacrylamide gels and chromatography columns have provided no evidence for the existence of more than one collagenase.

The molecular weight of the enzyme, as determined by dodecylsulphate‐polyacrylamide gel electrophoresis, was 33000.

Data obtained from studies with the ion‐exchange resin and from gel electrophoresis in acid and alkaline buffer systems suggested a basically charged enzyme.

It did not hydrolyse the synthetic collagen peptide Pz‐Pro‐Leu‐Gly‐Pro‐d‐Arg and non‐specific protease activity was absent.

The collagenase attacked undenatured collagen in solution at 25°C resulting in a 58% loss of viscosity and producing the two characteristic products TCA (3/4) and TCB (1/4).

At 37°C and pH 8.0 both reconstituted collagen fibrils and gelatin were degraded to peptides of less than 10000 molecular weight.

As judged by the release of soluble hydroxyproline peptides and electron microscopic appearances the enzyme degraded human insoluble collagens derived from tendon and soft juxta‐articular tissues although rates of attack were less than with reconstituted fibrils.

The data suggests that pure rheumatoid synovial collagenase at 37°C and neutral pH can degrade gelatin, reconstituted fibrils and insoluble collagens without the intervention of non‐specific proteases.

The different susceptibilities of various collagenous substrates to collagenase attack are discussed.
High‐Molecular‐Weight DNA Polymerases from Mouse Myeloma
Authors: HACHMANN Henning J. , LEZIUS Axel G.
Published: March 2005

Cytoplasmic (high‐molecular‐weight) DNA polymerase was partially purified from mouse myeloma. Upon chromatography on DEAE‐Sephadex, following fractionation on phosphocellulose, the enzyme was resolved into three species named CI, CII, and CIII. The species CI and CII have equal sedimentation coefficients (10.5 S) in sucrose gradients without salt. In the presence of 125 mM ammonium sulfate the sedimentation coefficients are reduced to 8.6 S. The species CIII shows sedimentation coefficients of 5.7 S and 5.2 S without salt and in the presence of 125 mM ammonium sulfate, respectively. This species is assumed to be an artifact arising from either CI or to a minor extent from CII. The optima for pH, KCI and Mg2+ concentration, and the extent of inhibition by N‐ethylmaleimide are the same. However, the enzymes differ in their responses to Mn2+ (substituting for Mg2+), and to addition of ethanol, dimethylsulfoxide, and various phospholipids in the assay mixture. The enzymes prefer poly [d(A‐T) · d(A‐T)] or partially degraded (activated) DNA as template rather than double‐stranded or single‐stranded DNA. The activity on activated DNA relative to that on poly[d(A‐T) · d(A‐T)] was found to be 93, 66, and 29% for DNA polymerases CI, CTI, and CIII, respectively.
Thermal and Solvent Perturbation as Probes of Conformational Heterogeneity of Haptoglobin 1‐1
Authors: de FORESTA Béatrice , LAVIALLE Françoise , ALFSEN Annette
Published: March 2005

The shifts which occur in the ultraviolet spectra of haptoglobin 1‐1 (Hp) and a conformational isomer with lower affinity for hemoglobin (Hp*), in response to temperature and solvent changes, have been measured by difference spectra. The thermal difference spectra provide evidence for a different participation of tryptophyl residues in the conformational stability of Hp and Hp*. Solvent perturbations have allowed an estimation to be made of the number of surface chromophores in the native Hp and Hp*.

These results are compatible with the existence of two Hp conformational isomers with different free energy, separated by a high‐potential barrier.
Multiple Forms of Human Glutathione S‐Transferase and Their Affinity for Bilirubin
Authors: KAMISAKA Kazuaki , HABIG William H. , KETLEY Jeanne N. , ARIAS Irwin M. , JAKOBY William B.
Published: March 2005

The initial enzymic step in mercapturic acid formation is catalyzed by glutathione S‐transferase. Several species of this enzyme, designated as transferases α. β, γ, δ and e an the basis of increasing isoelectric points, were isolated from human liver. Evidence is presented that each of the purified species is homogeneous with respect to sodium dodecylsulfate‐gel electrophoresis. Transferases α, β and ɛ each appear as a single band an gel electrofocusing: transferases α and δ are present as two and three bands, respectively, with each band catalytically active. Amino acid analysis indicated the five transferases to be either very closely related or identical in this respect.

All enzyme species have a molecular weight of about 48 500 and consist of two apparently identical subunits. The spectrum of substrates is the same for each although the enzymes differ slightly in specific activity. As is the case for the rat liver enzymes, each of the human transferases binds bilirubin although this compound is not a substrate