bluuebunny/crossref_metadata_2025_split huggingface dataset:
Kinetics of Thyroglobulin Iodination and of Hormone Synthesis Catalyzed by Thyroid Peroxidase
Authors: DÉME Daniéle , POMMIER Jacques , NUNEZ Jacques 
Published: March 2005

The kinetics of tyrosine iodination and of thyroxine synthesis in thyroglobulin, different reactions catalyzed by the sane enzyme (thyroid peroxidase), have been compared.Thyroxine synthesis always began after a lag period of 3–5 min. This lag was constant whatever the rate of iodination; this rate of iodination was increased either by increasing the concentration of iodide or enzyme or by decreasing the concentration of thyroglobulin. Increasing the rate of iodination resulted in increasing the number of iodine atoms incorporated during the lag period. Thus the lag observed for thyroxine synthesis was constant and did not depend on the fact that free iodide or non‐iodinated tyrosine residues of thyroglobulin were exhausted before thyroxine synthesis occurred. Finally, it appeared that, whatever the explanation of the lag, the enzyme catalyzes thyroid hormone synthesis at a slower rate than iodination.The existence of a lag also allowed us to prepare thyroglobulin samples with different iodine contents but without thyroid hormones. Thus iodination and thyroxine synthesis could be studied independently and the following results were obtained.

Iodotyrosine residues which can couple to form thyroxine are made considerably before coupling occurs.
H2O2 is required for coupling of these hormonogenic residues; thus the coupling reaction requires enzymic oxidation of the iodotyrosine residues.
In addition a strict requirement for iodide was needed for coupling: the requirement was dependent on the concentration of iodide. Thus iodide, a substrate of the iodination reaction, may also have other effects on the activity of thyroid peroxidase.
RNA Sequences Associated with Proteins L1, L9, and L5, L18, L25, in Ribonucleoprotein Fragments Isolated from the 50‐S Subunit of Escherichia coli Ribosomes
Authors: BRANLANT Christiane , KROL Alain , SRIWIDADA Johanes , BRIMACOMBE Richard 
Published: March 2005

32P‐labelled 50‐S subunits from Escherichia coli ribosomes were hydrolysed under conditions known to give rise to two specific ribonucleoprotein fragments, containing proteins L1, L9, and L5, =L 18 and L 25 respectively. RNA corresponding to these ribonucleoproteins was isolated and purified, and the various RNA fragments obtained were subjected to oligonucleotide analysis.The results showed that the RNA associated with proteins L1and L9 was very similar to the RNA found with protein L1 after controlled digestion of 23‐S‐RNA. L1 complexes (described elsewhere); this RNA lies within a region 550–1000 nucleotides from the 3′ terminus of 23‐S RNA. The RNA associated with proteins L5, L18, and L25 consisted predominantly of two species of similar size. One was 5‐S RNA, and the other a fragment of 23‐S RNA, lying within the region 450–1000 nucleotides from the 3′ terminus.
Studies on 3‐Deoxy‐d‐arabinoheptulosonate‐7‐phosphate Synthetase(phe) from Escherichia coli K12
Authors: SIMPSON Richard J. , DAVIDSON Barrie E. 
Published: March 2005

3‐Deoxy‐d‐arabino heptulosonate‐7‐phosphate synthetase(phe) from Escherichia coli K12 has been purified to near homogeneity. The purified enzyme has a specific activity of 67 units mg which is about 1000 times that found in cell‐free extracts of wild type E. coli K12.
The minimum molecular weight of the enzyme was estimated by dodecylsulphate‐gel eleetrophoresis to be 33000. Re‐estimation of the native molecular weight by gel filtration confirmed the previously determined value of 110000.
Amino acid analysis and tryptic fingerprints indicated that the subunits of the enzyme are very similar and possibly identical.
The purified enzyme does not contain Co2+
Studies on 3‐Deoxy‐d‐arabino heptulosonate‐7‐phosphate Synthetase(phe) from Escherichia coli K12
Authors: SIMPPSON Richard J. , DAVIDSON Barne E. 
Published: March 2005

Co2+ is not a cofactor for 3‐deoxy‐d‐arabinoheptulosonate‐7‐phosphate synthetase(phe).
The following analogues of phosphoenolpyruvate were tested as inhibitors of 3deoxy‐d‐arabinoheptolosonate‐7‐phosphate synthetase(phe): pyruvate, lactate, glycerate, 2‐phosphoglycerate, 3‐methylphosphoenolpyruvate, 3‐ethylphosphoenolpyruvate and 3,3‐dime‐thylphosoenolpyruvate. The results obtained indicate that the binding of phosphoenolpyruvate to the enzyme requires a phosphoryl group on the C‐2 position of the substrate and one free hydrogen atom at the C‐3 position.
The dead‐end inhibition pattern observed with the substrate analogue 2‐phosphoglycerate when either phosphoenolpyruvate or erythrose 4‐phosphate was the variable substrate is inconsistent with a ping‐pong mechanism and indicates that the reaction mechanism for this enzyme must be sequential. The following kinetic constants were determined: Km for phosphoenolpyruvate, 0.08 ± 0.04 mM: Km for erythrose 4‐phosphate, 0.9 ± 0.3 mM; Kis for competitive inhibition by 2‐phosphoglycerate with respect to phosphoenolpyruvate, 1.0 ± 0.1 mM.
The enzvmc was observed to have a bell‐shaped pH profile with a pH optimum of 7.0. The effects of pH on V and V/(Km for phosphoenolpyruvate) indicated that an ionizing group of pKa.8.0–8.1 is involved in the catalytic activity of the enzyme. The pKa of this group is unallected by the binding of phosphoenolpyruvate.
Isolation and Characterization of a Cat 2+‐Binding Polysaccharide Associated with Coccoliths of Emiliania huxleyi (Lohmann) Kamptner
Authors: DE JONG Elisabeth W. , BOSCH Leendert , WESTBROEK Peter 
Published: March 2005

Coccolithophoridae, a group of mostly unicellular algae, possess a cell wall containing calcified plates, called coccoliths. The coccoliths from the species Emiliania huxleyi (Lohmann) Kamptner contain a water‐soluble acid polysaccharide. In this paper we describe the isolation and some characteristic properties of the polysaccharide, in particular its Ca2+ ‐binding capacity.A large‐scale cultivation of the Coccolithophoridae was worked out and a new procedure for isolating coccoliths was developed. The polysaccharide obtained from the coccoliths contained two types of monobasic acid groups in a total amount of 1.8 μmol/mg polysaccharide.One type consisted of weakly acid groups which were identified as uronic acids. The nature of the stronger acid groups remains to be established. The ratio between the respective groups was 1:0.8. Studies with; 45Ca2+ demonstrated that the isolated polysaccharide is capable of binding Ca2+. Equilibrium dialysis revealed that the maximum amount of Ca2+ which can be bound is 0.92 ± 0.05ltmol/mg polysaccharide. Flow‐rate dialysis experiments strongly suggested the presence of two classes of Ca2+‐binding sites differing in affinity for Ca2+. High‐affinity sites (dissociation constant Kd for Ca2+: 2.2 + 1.0 × 10−5 M) were found to be present in amounts (0.38 ± 0.04 μmol/ mg polysaccharide) approximately equivalent to the strongly acid monovalent groups mentioned above (0.8 μmol/mg polysaccharide).Low‐affinity sites (Kd for Ca2+: 111 ± 39 × 10−s M) were estimated at 0.74 ± 0.11 μmol/ mg polysaccharide. Although this figure could be determined less accurately, it is suggested that the uronic acids (1.0 μmol/mg polysaccharide) are identical to the low‐affinity sites. Preferential binding of Ca2+ occurred in a 100‐fold excess of Na+ and Mg2+ as was shown by gel filtration. A 100‐fold excess ofSr2+ inhibited Ca2+ binding to a great extent while no Ca2+ was bound in the presence of an equimolar amount of La3+. The dissociation constants of the high‐affinity sites for Na+, Mg2+, Sr2+ and La3+ (in the presence of Ca2+) were determined with the flow‐rate dialysis technique. They confirm the order of binding preference found with gel filtration.A polysaccharide with similar properties could be isolated from subfossil coccoliths of E. huxieyi (about 1000 years old).The possible role of the polysaccharide as a heterogeneous matrix in coccolith formation is discussed.
Isolation and Characterization of Poly(glycosyl)ceramides (Megaloglycolipids) with A, H and I Blood‐Group Activities
Authors: KOŚCIELAK Jerzy , MILLER‐PODRAZA Halina , KRAUZE Robert , PIASEK Andrzej 
Published: March 2005

Very complex glycosphingolipids with A, H and I blood‐group activities were isolated from human erythrocyte membranes. The membranes were obtained from erythrocytes of blood group A, A2 and O respectively. A general formula for the antigens is:(Fuc)3–4(Gal)n(GlcNAc)n–2(Glc)1(Sphingosine)1(where Fuc is fucose, Gal is galactose, GlcNAc is N‐acetylglucosamine and Glc is glucose) with values of n ranging from 10–27. A‐active preparations contain additionally 2–3 residues of N‐acetyl‐galactosamine. In view of the unusual complexity of these compounds they were designated poly(glycosyl)ceramides (formerly megaloglycolipids). Individual poly(glycosyl)ceramide fractions were isolated from A erythrocytes and were found to differ by about 8 glycosyl residues per molecule forming a series of compounds with 22, 30, 38, 51 and 59 glycosyl residues per mole. Structural studies indicate that the main sequence of poly(glycosyl)ceramides consists of the residues of galactopyranose and 2‐deoxy‐2‐acetamidoglucopyranose substituted at 3 and 4 position respectively. These residues are probably alternating. N‐Acetylglucosamine substituted at 3 position was not found in poly‐(glycosyl)ceramides. Branches of poly(glycosyl)ceramides originate from 3 and 6 position of galacto‐pyranosyl residues. The number of branches is proportional to the degree of molecular complexity. In poly(glycosyl)ceramides isolated from A and A2 erythrocytes the branches are terminated with the following structures GalNAc α 1→3 [Fuc α 1→2] Gal; Fuc α 1→2 Gal and Gal (presumably Gal β 1→4 GlcNAc). In poly(glycosyl)ceramides from A cells the total number of A and H‐active structures per average molecule of 30–35 glycosyl residues amounts to 2.1 and 1.2 respectively while the number of terminal galactose structures is 1.8. For poly(glycosyl)ceramides from A2 erythrocytes the corresponding figures are 0.75, 3.5, and 2.1 respectively.Poly(glycosyl)ceramides from O cells comprise about 3.8 H‐active structures and 1.8 terminal galactopyranosyl residues. In poly(glycosyl)ceramides with high “n” values the number of terminal galactose structures is increased. These fractions display high blood‐group I activity. However, the removal of terminal galactose with β‐galactosidase affects I‐activity only slightly.
A Spin‐Label Study of the Chromaffin Granule Membrane
Authors: MARSH Derek , RADDA George K. , RITCHIE Gillian A. 
Published: March 2005

The structure of the chromaffin granule membrane has been probed using a number of different spin labels. Both the effect of temperature and high levels of calcium have been studied.

The results from three positional isomers of the stearic acid spin label demonstrate that a substantial part of the membrane lipid (that is sensed by the probe) is in a bilayer structure which undergoes a structural transition at 32–36 °C, characterized by an increase in the population of gauche isomers in the lipid chains. A possible mechanism for this transition would be the preferential segregation of cholesterol.
The covalently bound iodoacetamide spin label reveals a transition within the protein component of the membrane or its immediate lipid environment at 32°C. This transition corresponds to an increased degree of motional freedom of the spin label above the transition temperature.
The lipid‐soluble spin label 2,2,6,6‐tetramethyl‐piperidine‐1‐oxyl exhibits a break at 34°C in the temperature‐dependence of its partitioning into the membrane. This could correspond to the onset of a lateral separation in the membrane lipid, again possibly involving a re‐distribution of cholesterol.
Calcium abolishes, diminishes or shifts the transition observed by the spin label and decreases the amplitude of motion of the stearic acid spin labels, again possibly involving a restribution of cholesterol and also lysolecithin.

The temperatures of the structural transition agree well with the changes in the enzymic activity of the membrane ATPase and NADH oxidase functions and also with the results from fluorescent probes [Bashford et al., Eur. J. Biochem. 67, 105–114 (1976)]. It is possible that triggering of the transition either by calcium or some other stimulus may play a role in catecholamine release and membrane fusion.
Human Coagulation Factor IX
Authors: SUOMELA Hannu 
Published: March 2005

Human coagulation factor IX was purified by two ion‐exchange chromatographies on DEAE‐Sephadex A‐50, heparin‐Sepharose chromatography, hydroxyapatite chromatography and immuno‐adsorbent technique. Factor IX was homogeneous by ordinary and sodium dodecylsulphate disc electrophoresis, N‐terminal amino acid analyses and ultracentrifugation and by immunological criteria. The following molecular data were observed:

Sedimentation equilibrium indicated a molecular weight of 66 100 and sedimentation velocity gave S20, w= 3.97 S. A partial specific volume of v̄= 0.712 ml/g was calculated from the amino acid and carbohydrate composition.
Sodium dodecylsulphate disc gel electrophoresis suggested a molecular weight of 65 000.
Gel filtration indicated a Stokes radius of 4.08 nm, and ‘a molecular weight’ of 72 000, as well as a diffusion coefficient D20, w= 5.15 × 10−7 cm2 s−1 and a frictional ratio f/f0= 1.54.
Tyrosine was the N‐terminal amino acid. The amino acid composition is described. Factor IX contained approximately 17.5% carbohydrate, which includes 4.7% hexose, 6.8%N‐acetylhexos‐amine and 6% sialic acid.
Microheterogeneity of pure factor IX was demonstrated by isoelectric focusing. The isoelectric points of the major components lay within the range of pH 4.0 to 4.6.
The antibody raised in rabbits against the pure factor IX did not react with the other vitamin‐K‐dependent coagulation factors measured by coagulation factor assays or immunodiffusion in gels.
Microtubular Proteins in Pigeon Erythrocyte Membranes
Authors: ZENNER Hans P. , PFEUFFER Thomas 
Published: March 2005

[3H]Colchicine binds in a concentration and temperature dependent, saturatable and noncooperative manner to cytoplasmic proteins from pigeon erythrocytes: Kd= 3.5 × 10−7 M at 37 °C. Binding of [3H]colchicine at 0 °C and of [3H]lumicolchicine at 37 °C was significantly reduced. Hence microtubular proteins are present in the cytoplasm of pigeon erythrocytes.Antibody against bovine brain tubulin was raised in rabbits and confirmed by immunodiffusion, passive immunohaemolysis and in radioimmunoassay. Pigeon erythrocyte membrane proteins solubilized with 2% sodium cholate competed with 125I‐labelled tubulin in the radioimmunoassay although much higher concentrations of membrane proteins than of purified bovine brain tubulin were required for effective competition. No binding to antibody occurred with boiled solubilized membrane preparations. Similar results were obtained with antitubulin‐dependent passive immunohaemolysis of tubulin‐coated sheep erythrocytes in the presence of complement. The presence of tubulin in membranes was verified by binding intact pigeon erythrocytes to colchicine‐Sepharose beads at 37 °C. Free colchicine (5 mM) or incubation at 0 °C prevented binding. Lumicolchicine‐Sepharose beads did not attach to erythrocytes at 37 °C. Thus pigeon erythrocyte membranes contain microtubular proteins.
Chorismate Mutase/Prephenate Dehydratase from Escherichia coli K12
Authors: GETHING Mary‐Jane H. , DAVIDSON Barrie E. , DOPHEIDE Theo A. A. 
Published: March 2005

A new simplified procedure for the purification of chorismate mutase/prephenate dehydratase, based on affinity chromatography on Sepharosyl‐phenylalanine, has been developed. The method utilizes the effect of NaCl on the binding properties of the enzyme.NaCl inhibits both the mutase and dehydratase activities of the enzyme. In each case this inhibition is cooperative indicating homotropic interactions between NaCl binding sites on the enzyme. In addition NaCl induces homotropic cooperative effects between chorismate binding sites and between prephenate binding sites. NaCl also increases the sensitivity of the enzyme to inhibition by phenylalanine.
A pH‐Dependent Interaction between Histones H2A and H2B Involving Secondary and Tertiary Folding
Authors: MOSS Tom , CARY Peter D. , ABERCROMBIE Barry D. , CRANE‐ROBINSON Colyn , BRADBURY E. Morton 
Published: March 2005

It has been shown by high‐resolution proton magnetic resonance (PMR) spectroscopy and circular dichroism (CD) that an H2A/H2B histone complex exists after salt extraction of these histones from chromatin and that this complex can be fully renatured from both urea‐denatured acid‐extracted and from urea‐denatured salt‐extracted histones. The histone complex is shown to involve specific secondary and tertiary structure. Formation of this complex is observed to be critically dependent on pH, occurring at and above pH 5. It cannot be induced below pH 5 by increase in ionic strength.From CD spectra the H2A/H2B complex is shown to contain about 37%α helix but no β structure, the latter being confirmed by infrared spectroscopy in the 6‐μm region. The PMR spectra show that the structured region includes most of the aromatic residues of both histones, at least two histidine residues of H2B and probably histidines 31 and 82 of histone H2A.The secondary structure of histones H2A and H2B is predicted using the Chou and Fasman procedure and comparisons are made between the predictions for histones of different species. These results in conjunction with the experimental evidence lead to the conclusion that at least residues 31–95 of H2A and residues 37–114 of H2B, i.e. the more apolar regions of the molecules, are involved in the tertiary structure of the H2A/H2B complex.
Characterization and Site of Action of a Soluble Protein that Stimulates Peptide‐Bond Synthesis
Authors: GLICK Bernard R. , GANOZA M. Clelia 
Published: March 2005

A recently identified soluble protein, named EF‐P, stimulates peptide bond synthesis from ribosomal‐bound N‐formylmethionyl‐tRNA and the aminoacyl‐tRNA analog, puromycin. Using this model of peptide bond formation we have purified this activity approximately 100‐fold from ribosome‐free extracts of Escherichia coli.In order to study the mechanism by which the EF‐P factor stimulates peptide bond formation, we examined and compared the requirements and site of action of the spontaneous and the EF‐P‐mediated synthesis of peptide bonds. We find that ‘enzymic’ peptide bond synthesis (+EF‐P) is characterized by relatively broad temperature and NH4Cl optima, a sharp Mg2+ optimum at 12 mM, and an apparent pKa of approximately 8.5. The characteristics of enzymic peptide bond synthesis closely resemble those reported for native peptidyl‐puromycin formation rather than other models of peptide synthesis.Factor EF‐P requires both 30‐S and 50‐S subunits for activity. The 30‐S particle is inactive by itself and may function in the reaction merely to bind the fMet‐tRNA substrate. Both the peptidyl transferase and the EF‐P binding site may be part of the 50‐S subunit.Unlike all other propagation factors, EF‐P does not require the 50‐S ribosomal proteins L7 and L12 and may therefore occupy a different ribosomal site.
Symmetry and Asymmetry of the Pyruvate Dehydrogenase Complexes from Azotobacter vinelandii and Escherichia coli as Reflected by Fluorescence and Spin‐Label Studies
Authors: GRANDE Hans J. , VAN TELGEN Hendrik J. , VEEGER Cees 
Published: March 2005

Fluorescence‐lifetime measurements of FAD bound to lipoamide dehydrogenase from Azotobacter vinelandii and Escherichia coli were performed. It is shown from these results that the two FAD groups in the isolated dimeric enzyme, as well as in the enzyme in the intact complex of E. coli, are in non‐equivalent surroundings. This contrasts with the near equivalence of the FAD groups of both the enzyme and complex isolated from A. vinelandii. Reduction of the complex with Mg2+, thiamine pyrophosphate and pyruvate or with NADH enables the attachment of a maleimide analogue specifically to the lipoyl moieties of the transacetylase(s). Spin label [N‐(1‐oxyl‐2,2,5,5‐tetramethyl‐3‐pyrrolidinyl)maleimide] introduced in such a way proves the existence of at least two different micro‐environments around the lipoyl moieties in complex isolated from A. vinelandii. Electron paramagnetic resonance spectra of the specifically spin‐labelled complexes from E. coli and A. vinelandii, when dissolved in tricine [N‐tris(hydroxymethyl)‐methylglycine] buffer, show interactions of at least two electron spins with each other, which indicate that the lipoyl moieties are rather close together.Fluorescent label [N‐(1‐anilinonaphthyl‐4)maleimide] is specifically attached to the lipoyl moiety of the high‐Mr transacetylase of the freshly isolated complex from A. vinelandii. From the large differences in the apparent lifetimes τp and τm, as detected by phase fluorimetry, it is shown that this fluorescent label is distributed in different micro‐environments. The differences observed in energy transfer between fluorescent label, attached to the lipoyl moiety of the high‐Mr transacetylase, indicate different conformations of the complex from A. vinelandii. Upon introduction of the label after reduction with NADH a much larger energy transfer, thus a shorter distance, is observed between the label and FAD than when reduction is performed with Mg2+, thiamine pyrophosphate and pyruvate. A similar conformation dependence upon reduction is found for the pyruvate dehydrogenase complex from E. coli. It is thus proposed that the transacetylase of E. coli and the high‐Mr transacetylase of A. vinelandii are both non‐symmetrically distributed within the complex.
Biochemistry of Development in Insects
Authors: MEGIAS Alicia , MUNICIO Angel M. , PEREZ‐ALBARSANZ Miguel A. 
Published: March 2005

A different biochemical behaviour has been previously shown during several stages of the development of the insect Ceratitis capitata; thus, the acyl transferase activity has a different participation in the pathways of synthesis of triacylglycerols and phosphoglycerides by either larval or pharate adult stages of development. The results reported here are concerned with the behaviour of mitochondria and microsomes from larvae and pharate adults for the synthesis of triacylglycerols and phosphoglycerides. In all preparations of either mitochondrial or microsomal fractions the optimum protein amount, previously determined, has been used.
The highest incorporation of [14C]palmitate into triacylglycerols and phosphoglycerides was achieved by the microsomal fraction from the larval stage of development of the insect. However, mitochondrial fractions from pharate adults utilized higher levels of [14C]palmitate than the microsomal preparation. These differences were not correlated with the palmitoyl‐CoA synthetase activity.
Glycerol 3‐phosphate influenced the fatty acid incorporation in a different manner depending on the stage of development of the insect. Increasing concentrations of glycerol 3‐phosphate stimulated the synthesis of triacylglycerols by microsomal and mitochondrial preparations. This effect was not exhibited by the subcellular preparations of pharate adults.
Double‐label experiments using [14C]glycerol 3‐phosphate and [3H]palmitate showed qualitative differences in the synthesis of triacylglycerols by mitochondrial preparations from either larvae or pharate adults. Incorporation of the labelled fatty acids to endogenous triglycerides was preferentially shown by the mitochondrial larval preparations. The pathways of synthesis of triacylglycerols and phosphoglycerides by microsomal preparations showed only quantitative differences depending on the stage of development of the insect.
Acylation of 14C‐labelled lyso‐phosphatidylcholine and 14C‐labelled lyso‐phosphatidylethanolamine by [3H]olate was assayed in the presence of mitochondrial and microsomal preparations from the different stages of development of the insect. Microsomal fractions showed an efficient acyl transferase activity mainly when lyso‐phosphatidyl‐ethanolamine was used as substrate.