bluuebunny/crossref_metadata_2025 huggingface dataset:
The Role of Lysine and Leucine Binding on the Catalytical and Structural Properties of Aspartokinase III of Escherichia coli K 12
Authors: RICHAUD Catherine , MAZAT Jean‐Pierre , FELENBOK Béatrice , PATTE Jean‐Claude
Published: March 2005

The lysine‐sensitive aspartokinase of Escherichia coli K12, a 100000‐molecular weight dimer, possesses four binding sites for L‐lysine, its allosteric inhibitor. The lysine binding is cooperative. In the presence of leucine this cooperative effect disappears, that may explain the synergistic inhibition phenomenon observed between these two amino acids. The four sites are non‐equivalent two by two, with a K of, respectively, 8 μM and 100 μM. The tetramerization of the protein observed in the presence of lysine appears not to be related to the inhibition of enzyme activity. Lysine or leucine binding leads to transconformational changes evidenced by difference spectra and modification in – SH titration rates. All the results are consistent with the hypothesis that the lysine‐sensitive aspartokinase belongs to the class V of allosteric enzymes.
Structural Studies of Adenovirus Type‐2 Hexon Protein
Authors: JÖRNVALL Hans , PETTERSSON Ulf , PHILIPSON Lennart
Published: March 2005

The adenovirus type 2 hexon protein produced in excess during infection was carboxymethylated by reduction and alkylation with iodo[2‐14C]acetate in 6 M guanidine hydrochloride. Only one type of polypeptide chain was detected by exclusion chromatography in dissociating buffers. Peptide mapping experiments, sequence analysis of peptides and identification of possible protein terminal residues also suggest that all subunits are similar and probably identical.

Structural analysis of [14C]carboxymethylated hexon and of hexon which was labeled in vivo with [35S]cysteine revealed six unique cysteine derivatives in soluble tryptic peptides. A seventh unique cysteine derivative, only partly carboxymethylated, was present in the tryptic core material and was recovered after chymotryptic digestion. Seven unique cysteine derivatives in the hexon subunit, correspond to a minimum molecular weight of about 100 000 for the subunit. This value is consistent with values obtained from exclusion chromatography in guanidine hydrochloride, from mercurial titration of an accessible thiol group in the intact protein and from peptide mapping experiments.

Peptides corresponding to more than one tenth of the total number of residues in the protein chain were characterized and, with the inclusion of other peptides studied, about one quarter of all residues are accounted for. The results exclude extensive regions of identity in primary structure within the hexon subunits and the partial resistance towards attack by proteolytic enzymes is structurally explained.
Deoxyribosyl Transfer Catalysis with trans‐N‐Deoxyribosylase
Authors: DANZIN Charles , CARDINAUD Robert
Published: March 2005

Kinetic studies were carried out in order to investigate the enzymic mechanism of a 1420‐fold purified purine nucleoside : purine deoxyribosyl transferase fraction from Lactobacillus helveticus. Deoxyinosine and adenine were used as substrates. Initial velocity, product inhibition and isotopic exchange studies are consistent with a ping‐pong bi‐bi mechanism (the first product is released before the second substrate combines with the enzyme). The numerical values of the Michaelis and dissociation constants, maximum velocities and equilibrium constants for the forward and reverse reactions are derived graphically from secondary plots. A comparison with purine nucleoside phosphorylase is discussed.
Intracellular Carboxy‐Terminal Degradation of the αA Chain of α‐Crystallin
Authors: de JONG Wilfried W. , van KLEEF Frans S. M. , BLOEMENDAL Hans
Published: March 2005

Chromatographically and electrophoretically homogenous preparations of αA‐chains of α‐crystallin actually contain two types of chains. Beside the normal αA‐chain, 173 residues in length, a variable proportion of a shortened chain is present, which lacks five residues at the C‐terminal end. This chain, referred to as αA1–168, is found in all mammalian species thus far investigated. The two electrophoretic forms of the αA‐chain, αA1 and αA2, both exhibit the shortened version. The proportion of shortened αA‐chain is very high in early embryonic stages and rapidly declines with increasing age. In older lenses the level of αA1–168 increases from the outer cortex toward the nucleus. It is concluded, on the basis of structural studies, that the αA1–168 chain is derived from the αA‐chain by degradation in vivo. The αA1–168 chain probably represents a first stage in the sequential breakdown of ageing α‐crystallin.
Studies on the Partial Degradation of Polyunsaturated Fatty Acids in Rat‐Liver Mitochondria
Authors: KUNAU Wolf.‐H. , BARTNIK Friedhelm
Published: March 2005

In rat liver some highly unsaturated fatty acids are partially degraded. In contrast to “normal”β‐oxidation some intermediates accumulate in this catabolic pathway. When a docosapolyenoic acid with the first double bond in position 4 is metabolized, two degradation products are to be found. The sequence of reactions has been elucidated. As a first step the docosapolyenoic acid loses its double bond in position 4 while the carbon chain remains unchanged. The enzyme involved has tentatively been called 4‐enoyl‐CoA reductase. Subsequently chain shortening occurs. Using polyenoic acids with different chain lengths and different double bond positions, it has been possible to demonstrate that the double bond positions rather than the chain length determine whether or not an unsaturated fatty acid is partially degraded. The four double bonds in the positions 4, 7, 10 and 13 seem to be the minimal structural requirement for docosapolyenoic acids in order to function as substrates for the 4‐enoyl‐CoA reductase. 4, 7, 10‐docosatrienoic, 4, 7‐docosadienoic, and 4‐docosenoic acid, which have not been detected in nature so far, are not metabolized when incubated with rat liver mitochondria.
Carbon Monoxide Oxidation by Growing Cultures of Clostridium pasteurianum
Authors: FUCHS Georg , SCHNITKER Ursula , THAUER Rudolf K.
Published: March 2005

Growing cultures of Clostridium pasteurianum were found to oxidize carbon monoxide to CO2 at a maximal rate of approximately 15 nmol × min−1× mg protein−1. The dependence of the rate on the carbon monoxide concentration followed simple Michaelis‐Menten kinetics. Half‐maximal velocity was obtained with 3 μM carbon monoxide in the growth medium. The values of V and[S]0.5V determined in vivo are similar to those reported for carbon monoxide oxidation with flavin nucleotides in cell‐free extracts of C. pasteurianum (V= 14 nmol × min−1× mg protein−1; [S]0.5V= 5 μM), indicating that in vivo and in vitro carbon monoxide is oxidized by the same system.

Carbon monoxide was even removed from the medium by growing cultures of C. pasteurianum at concentrations as low as 1 nM equal to a concentration in the gas phase of approximately 1 part per million. Carbon monoxide was oxidized at a rate of 8 μmol × 1−1× h−1 under these conditions. The biological significance of this finding is discussed.
Antagonism of Insulin and Lipolytic Hormones in the Control of Adenylate‐Cyclase Activity in Fat Cells
Authors: RENNER Rolf , KEMMLER Wolfgang , HEPP K. Dieter
Published: March 2005

The control of adenylate cyclase activity by lipolytic hormones was studied in fat cell “ghosts” in the presence and absence of physiological concentrations of insulin. Insulin was found to inhibit the effect of submaximal concentrations of norepinephrine, adrenocorticotrophin, and glucagon. Addition of the hormone to the assay system leads to an immediate reduction of the linear rate of formation of adenosine 3′:5′‐monophosphate (3′:5′‐AMP). For norepinephrine and adrenocorticotrophin a dose‐response relationship was established which was shifted by insulin in a “pseudocompetitive” fashion. Increasing the concentration of insulin beyond the physiological range resulted in a reversal of its inhibitory effect in the presence of lipolytic hormones. Insulin alone at concentrations between 5 and 5000 μU/ml had no effect, whereas a small stimulatory effect of high insulin concentrations (5–500 mU/ml) could be found in the presence of submaximal concentrations of norepinephrine. No effect of insulin was obtained against several concentrations of NaF. The effect of insulin upon the norepinephrine‐stimulated adenylate cyclase disappeared after trypsin treatment of intact cells prior to preparation of ghosts. Further incubation of intact fat cells after trypsin treatment lead to a recovery of the effect of insulin. Analogous to insulin, nonsuppressible insulin‐like material which is believed to be related to “somatomedin”, was found to inhibit adenylate cyclase activity in ghosts, but this effect was not abolished by trypsin treatment. Ghosts were found to contain 3′:5′‐AMP phosphodiesterase activity which was not influenced by direct addition of insulin as measured with low substrate concentrations or under the conditions of the adenylate cyclase reaction.

It is suggested that insulin at low concentrations interferes with the activation of the adenylate cyclase system by lipolytic hormones, at a step between hormone receptor and catalytic unit. In view of similar characteristics of the antilipolytic effect of insulin in intact lipocytes and the observed modulation of the action of lipolytic hormones in ghosts, a fundamental physiological role of this effect upon the adenylate cyclase system in the mechanism of action of insulin is postulated.
The Amino‐Acid Sequence of the αB2 Chain of Bovine α‐Crystallin
Authors: VAN DER OUDERAA Frans J. , DE JONG Wilfried W. , HILDERINK Ans , BLOEMENDAL Hans
Published: March 2005

Isolated cyanogen bromide fragments of the αB2 chain of α‐crystallin contained 67 and 107 residues, respectively. Part of the sequence of the C‐terminal fragment was elucidated by Edman and Begg sequenator analysis. Sequences of the tryptic peptides were determined by a combination of various Edman degradation techniques. Overlaps of tryptic peptides were established by selective modification of lysine residues and subsequent tryptic cleavage and by isolation of peptides after hydrolysis with chymotrypsin, thermolysin and a staphylococcal protease. The sequence of the αB2 chain comprises 175 residues corresponding to a molecular weight of 20070. This value is somewhat lower than that assumed hitherto. A high degree of homology between the αB2, and the αA2 chain, the major polypeptides, of bovine α‐crystallin was observed.
A Mathematical Model for the Influence of Fructose 6‐Phosphate, ATP, Potassium, Ammonium and Magnesium on the Phosphofructokinase from Rat Erythrocytes
Authors: OTTO Matthias , HEINRICH Reinhart , KÜHN Brigitte , JACOBASCH Gisela
Published: March 2005

The influence of the substrates fructose 6‐phosphate and ATP and the effect of potassium, ammonium and magnesium ions on the phosphofructokinase from rat erythrocytes have been investigated. The results are expressed in terms of the Monod‐Wyman‐Changeux model. Potassium and ammonium ions are equally bound to the active R‐state and the inactive T‐state (type‐I binding site) the allosteric equilibrium between R and T is not affected. Both ions compete for this site, the dissociation constants being 0.2 mM for ammonium and 10 mM for potassium. Ammonium ions additionally occupy a specific site (type‐II binding site, dissociation constant 10 mM) of the R‐state thus shifting the sigmoidal fructose 6‐phosphate saturation curve to the left. Magnesium does not activate the phosphofructokinase. It removes the inhibition by uncomplexed ATP which is strongly bound to the T‐state (dissociation constant 10 μM). In higher concentration magnesium ions inhibit the enzyme. MgATP is equally well bound to both states having therefore no inhibiting influence on the enzyme. The importance of these effects under physiological conditions is discussed.
Regulatory Behavior of Monomeric Enzymes
Authors: MEUNIER Jean‐Claude , BUC Jean , NAVARRO André , RECARD Jacques
Published: March 2005

The wheat‐germ hexokinase L1 is made up of one polypeptide chain only and exhibits one active site for glucose per enzyme molecule as shown by equilibrium dialysis experiments. However, MgATP2− cannot be bound to a significant extent on the free enzyme in the absence of the hexose.

Plots of the reciprocal of the reaction rate versus the reciprocal of glucose concentration exhibit a significant downward curvature, but they are linear versus the reciprocal of MgATP2− concentration. The curvature of the plots with regard to glucose is modified neither by the concentration of MgATP2− nor by that of the product MgADP−. The intercepts and the slopes of the straight lines obtained versus the reciprocal of MgATP2− concentrations give, when replotted against the reciprocal of glucose concentration, a curve concave downwards and a straight line, respectively. The downward curvature of the reciprocal plots, obtained with regard to glucose, is strongly increased by glucose 6‐phosphate.

The whole set of kinetic results along with equilibrium dialysis data strongly suggest that the non‐Michaelian behavior is due to a mnemonical transition of the wheat germ hexokinase L1. The free enzyme very probably exists in two conformation states. The collision of any of these enzymes forms with glucose induces the same new conformation. After the binding of MgATP2−, which is thus the second substrate, and the release of MgADP−, the enzyme appears in one of the initial conformations which is stabilized by glucose 6‐phosphate. That ligand can thus be bound on one only of the two conformation states of the free enzyme.

The view that wheat germ hexokinase L1 is a mnemonical enzyme gains further support from the study of the effect of denaturing agents on the reaction kinetics. Low concentrations of urea or dodecylsulfate “desensitize” the enzyme and completely suppress its non‐Michaelian behavior, which clearly appears as a consequence of a conformational transition.
Involvement of DNA Polymerases I and III in the Replication of Bacteriophage M‐13
Authors: STAUDENBAUER Walter L.
Published: March 2005

M‐13 replication was investigated in Escherichia coli mutants deficient in DNA polymerase I (polA) and DNA polymerase III (dnaEts). The following results were obtained:

M‐13 infection is abortive in both polA− and dnaEts mutants at 42°C.

The conversion of the parental single‐stranded DNA to double‐stranded replicative forms (RF) is only slightly affected by the dnaE mutation. However, no parental double strands are formed in the polA− dnaEts double mutant at the restrictive temperature, indicating a possible involvement of DNA polymerase I in this process.

The replication of the replicative‐form molecules as well as the synthesis of progeny single strands are inhibited by thermal inactivation of the dnaE function implying a requirement of DNA polymerase III in both types of DNA synthesis.

The presence of the polA mutation leads to an accumulation of open‐circular replicative forms. DNA polymerase I might, therefore, also be involved in the filling of gaps in the newly synthesized DNA molecules.
On the Mechanism of Translocation of Pyruvate and Other Monocarboxylic Acids in Rat‐Liver Mitochondria
Authors: PAPA Sergio , PARADIES Giuseppe
Published: March 2005

Pyruvate equilibrates, like other carboxylic acids, across the membrane of rat‐liver mitochondria according to the transmembrane pH difference.

Comparison of the rate of pyruvate, acetoacetate and acetate efflux from mitochondria as well as a study of the effect of mersalyl and N‐ethylmaleimide on this process, show that the diffusion of pyruvic and acetoacetic acid across the mitochondrial membrane is carrier‐mediated. The process might consist of pyruvate (acetoacetate) hydroxyl ion exchange‐diffusion or of pyruvic acid (acetoacetic acid) uniport. On the contrary acetic acid appears to diffuse freely across the membrane.

The initial rate of the net pyruvate uptake by rat liver mitochondria follows saturation kinetics. The increase of the external pH of the medium as well as the addition of acetoacetate inhibit competitively pyruvate uptake. Pyruvate uptake is inhibited by mersalyl and N‐ethylmaleimide.

Mitochondria1 [14C]pyruvate exchanges with unlabelled pyruvate and acetoacetate but not with other monocarboxylates, Pi and citric‐acid cycle intermediates.

The pyruvate‐pyruvate and pyruvate‐acetoacetate exchanges follow saturation kinetics and exhibit the same V but different Km values. Both the exchanges are inhibited by mersalyl, but not by N‐ethylmaleimide.

It is concluded that pyruvate translocation across the mitochondrial membrane is mediated by a specific transport system.
The Inhibition of Proteinases by Human α1‐Antitrypsin
Authors: HERCZ Albert
Published: March 2005

The inhibition of human plasmin by α1‐antitrypsin was time dependent and the rate of inhibition rapidly decreased with decreasing temperature.

The effect of substrates on the inhibitions of trypsin and chymotrypsin was investigated. Theoretically the final degree of inhibition should be the same, whether the substrate is added to the proteinase‐inhibitor system before or after the formation of the complex. The inhibition of chymotrypsin by α1‐antitrypsin and of trypsin by commercial soybean trypsin inhibitor was in line with the prediction, with either casein or synthetic esters as substrates. However, in the trypsin‐α1‐antitrypsin system the degree of inhibition depended on the order in which the reactants were mixed. With both casein and p‐tosyl‐l‐arginine methyl ester as substrates, inhibition was high when trypsin was preincubated with the inhibitor and the substrate was added later, and low or absent when the proteinase was added to the mixture of inhibitor and substrate.

In the inhibition mixtures of plasmin, trypsin and chymotrypsin with α1‐antitrypsin, distinct changes were detected by electrophoresis in polyacrylamide gel. The unreacted inhibitor diminished or disappeared and reaction products were formed at rates corresponding to rates of inhibition.
Cyclic AMP and Catabolite Repression in Yeasts
Authors: SCHLANDERER Gustav , DELLWEG Hanswerner
Published: March 2005

In Schizosaccharomyces pombe an inverse proportionality between glucose concentration and intracellular adenosine 3′:5′‐monophosphate (CAMP) was established. The cAMP levels were in the range of 12 to 785 pmol per g yeast cells dry weight. In continuous cultures a steady‐state concentration of as low as 0.05% glucose is sufficient to cause a decrease in intracellular cAMP content. Lowest levels of CAMP were found in the presence of more than 5% glucose, while highest levels were synthesized by cells grown on glycerol or maltose.

In all strains tested, CAMP is transported into the fermentation broth, both if grown on glucose or cultured on maltose as the sole carbon source.

The synthesis of α‐glucosidase (EC 3.2.1.20) is induced by maltose. Glucose represses the synthesis of invertase (EC 3.2.1.26), NADH‐ferricyanide reductase (EC 1.6.99.3) and both the constitutive and the inducible α‐glucosidase. The possibility of a coordinate synthesis of α‐glucosidases and invertase is indicated by the experimental data but cannot definitely be proved.

Moreover, there exists a relation between the intracellular concentration of cAMP and the activities of invertase, α‐glucosidases and a NADH‐ferricyanide reductase. Under comparable growth conditions a mutant strain partially resistant to catabolite repression synthesizes more cAMP and produces increased activities of the catabolite‐sensitive enzymes tested than the wild‐type strain. Strains classified as catabolite‐sensitive show reduced values of intracellular cAMP and enzyme activities. In contrast to Saccharomyces cerevisiae in Schizosaccharomyces pombe there exists no relation between the intracellular level of cAMP and the activity of alcohol dehydrogenase (EC 1.1.1.1).

We suppose, that in Schizosaccharomyces pombe cAMP is concerned with the regulation of catabolite‐sensitive enzyme synthesis.
Biochemistry and Pharmacology of the Crotoxin Complex
Authors: BREITHAUPT Henning , RÜBSAMEN Klaus , HABERMANN Ernst
Published: March 2005

Crotapotin and the basic crotalus phospholipase A, which are the main components of the crotoxin complex, have been purified to homogeneity with respect to their behaviour on polyacrylamide gel electrophoresis, cellogel electrophoresis, immunoelectrophoresis, isoelectric focusing, and sedimentation equilibrium analysis.

The isoelectric points were found to be 3.4 for crotapotin and 9.7 for the crotalus phospholipase A.

The molecular weight of phospholipase A has been determined by gel filtration in 6 M guanidine · HCl (14500), by ultracentrifugation (15800), and by dodecylsulfate‐gel electrophoresis (15800). Phospholipase A consists of a single peptide chain intramolecularly crosslinked by eight disulfide bridges.

The molecular weight of crotapotin was 8900 as shown by gel filtration in 6 M guanidine · HCl. Sedimentation equilibrium studies in 4 M guanidine · HCl gave a value of 6700. In solutions of lower ionic strength, the Mr, was 12500, indicating that crotapotin can exist as a dimer. In contrast the Mr of reduced and carboxamidomethylated crotapotin was much lower (4000–4500), in gel filtration experiments.

Crotapotin consists of three peptide chains, held together by disulfide bridges. Chain A contains 40 amino acid residues (Mr 4274), chain B 34 residues (Mr 3658) and chain C 14 residues (Mr 1558). A free NH2‐terminal residue (serine) could be detected only for chain A.
Alkaline Denaturation of Ferrihemoglobins A, S and C
Authors: ANUSIEM Alphonso C. I. , BEETLESTONE John G.
Published: March 2005

Various workers have interpreted their data on the rates of alkaline denaturation of ferrihemoglobin either as a consequence of the presence of two distinct species or as a result of two consecutive first order steps involving an intermediate. Our results with a chromatographically purified homogeneous sample indicate that the rate of alkaline denaturation support the latter hypothesis. The rates of denaturation of ferrihemoglobins A, S and C under the same conditions differ, and it is concluded that the amino acid sequence of the ferrihemoprotein plays a significant role in the conformational stability of the molecule.
Interaction between Arginase and l‐Ornithine Carbamoyltransferase in Saccharomyces cerevisiae
Authors: PENNINCKX Michel , SIMON Jean‐Paul , WIAME Jean‐Marie
Published: March 2005

l‐Ornithine carbamoyltransferase and arginase from Saccharomyces cerevisiae have been extensively purified and their quaternary structures have been determined by polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate. Subunit cross‐linking with glutaraldehyde shows that both enzymes have trimeric structures. Ornithine carbamoyltransferase is built up by the association of three subunits of molecular weight 37500; arginase is made of three subunits of 39000. The latter observation has lead us to reinvestigate the quaternary structure of rat liver arginase for which, in spite of an earlier report to the contrary [H. Hirsch‐Kolb and D. Greenberg, J. Biol. Chem. 243, (1968) 6123–6129], evidence of a trimeric structure is also obtained.

The data suggest that the 1‐to‐1 regulatory complex of ornithine carbamoyltransferase and arginase of S. cerevisiae, leading to ornithine carbamoyltransferase inhibition, is an hexamer.
Molecular Forms of Rat‐Liver Arginase. Isolation and Characterization
Authors: TARRAB Rebeca , RODRÍGUEZ Jesús , HUITRÓN Carlos , PALACIOS Rafael , SOBERÓN Guillermo
Published: March 2005

Debate continues over the physical characteristics and even the existence of arginase isoenzymes. This paper gives additional support for such multiplicity and reports differences in physical characteristics among the various forms.

After 2500–5000‐fold purification of rat liver arginase, three molecular forms were separated on carboxymethyl‐cellulose columns and were purified 2500–5000‐fold, 800–1000‐fold and 600–1000‐fold, respectively. The molecular forms have also been identified by chromatography in the supernatant of tissue extracts. The isolation of these molecular forms by affinity chromatography, using Sepharose‐lysine as a competitive inhibitor of arginase, shows only one main form, however.

Kinetic studies were done for two of the molecular forms isolated, specifically the activation energy (Ea) the energy of denaturatization (Ed), Km, pH and the effect of divalent cations were determined. Significant differences were found for the Ea, between the two molecular forms. The isolated isoenzymes are cationic at pH 5.5 and pH 8.8. However, they show different mobilities in electrophoresis. The molecular weight determination by gel filtration yields a value of 110000 to 115000 for both forms. The use of thin‐layer immunochromatography plates, a combination of molecular weight and immunodiffusion technique, gave only one peak with the same molecular weight as that determined by gel filtration. The immunological studies showed that the isoenzymes have similar antigenic determinants.
Control of Fatty‐Acid Biosynthesis by Long‐Chain Acyl CoAs and by Lipid Membranes
Authors: SUMPER Manfred
Published: March 2005

The possibility of a regulatory role of long‐chain acyl‐CoAs was studied in a system synthesizing palmitoyl‐CoA and stearoyl‐CoA in vitro. This system contained acetyl‐CoA carboxylase (yeast), fatty acid synthetase (yeast) and lipid membranes as acceptors for the long‐chain acyl‐CoA compounds. In this system acetyl‐CoA carboxylase is controlled by long‐chain acyl‐CoAs, whereas this is not true for fatty acid synthetase. The degree of inhibition of acetyl‐CoA carboxylase depends strongly upon the capacity of the lipid membrane to incorporate long‐chain acyl‐CoAs. This capacity is not fixed but is a function of different parameters such as surface charge of the membrane. The effects of inositol deficiency in vivo (overproduction of lipids) and the shift to shorter chain lengths of the fatty acids synthesized under anaerobic conditions are discussed and interpreted.
The Synthesis of Adenine‐Substituted Derivatives of NADP+ and Their Potential as Active Coenzymes and Affinity Adsorbents
Authors: LOWE Christopher R. , MOSBACH Klaus
Published: March 2005

Alkylation of NADP+ with iodoacetic acid at pH 6.5 yielded N1‐carboxymethyl‐NADP+ which, following enzymic reduction, alkaline rearrangement and enzymic reoxidation, gave N6‐carboxy‐methyl‐NADP+. Condensation of these analogues with 1,6‐diaminohexane in the presence of a water‐soluble carbodiimide generated the N1 and N6N‐(6‐aminohexyl)‐acetamide derivatives of NADP+ respectively. The coenzymic activities of the N1 and N6 adenine‐substituted carboxymethyl and N‐(6‐aminohexyl)‐acetamido derivatives of NADP+ were tested with several NADP+‐dependent dehydrogenases. The carboxymethyl derivatives were more active (up to 145%) relative to un‐substituted NADP+ than the corresponding N‐(6‐aminohexyl)‐acetamido compounds (up to 10%).

NADP+ ‐N6‐[N‐(6‐aminohexyl)‐acetamide] was coupled to a soluble dextran and to Sepharose 4B by the cyanogen bromide technique. The dextran‐bound derivative contained 85 μmol NADP+ analogue/g dry dextran and was reduced at approximately 35% the rate of native NADP+ with glucose‐6‐phosphate dehydrogenase. The dextran‐bound NADP+ cycled at a rate of about 30% relative to free NADP+ in a system comprising glucose‐6‐phosphate dehydrogenase and l‐glutamate dehydrogenase. Sepharose‐bound NADP+ proved to be an effective biospecific adsorbent for affinity chromatography and was competent for the resolution of a mixture of bovine albumin, lactate dehydrogenase and glucose‐6‐phosphate dehydrogenase. The latter was eluted almost quantitatively with a pulse of 0.5 mM NADP+.
A New Subgroup of Human L‐Chains of the λ‐Type
Authors: EULITZ Manfred
Published: March 2005

Bence‐Jones protein DEL was isolated from the urine of a patient with multiple myeloma by ammonium sulfate precipitation. Ion‐exchange chromatography provided no further purification of the protein; the ammonium sulfate precipitate consisted of only one protein moiety as tested by polyacrylamide electrophoresis. Protein DEL contains 213 amino acids and has a molecular weight of 22500. Eighteen peptides have been isolated by ion‐exchange chromatography after tryptic digestion of the completely reduced and aminoethylated protein. Sequence studies were performed mainly on these tryptic peptides. In addition, 14 chymotryptic peptides were isolated and characterized to obtain overlapping peptides for the whole V‐region and for some tryptic peptides of the C‐region.

Protein DEL is related to subgroups III and IV of the λ‐chains as indicated by a deletion of three amino acids after position 27, a common pattern of these two subgroups. Furthermore, position one is deleted in protein DEL as in the other proteins of subgroups III and IV but in contrast to subgroups I and II of the λ‐chains. However, marked differences exist between protein DEL and the proteins of subgroups III and IV.

a) The deletion of two amino acids in positions 95 and 96, characteristic of subgroup IV, was not found in protein DEL.

b) 19 of the 27 linked exchanges specific for subgroup IV are substituted in protein DEL. Although the insertion of two amino acids after position 94 is the same as in subgroup III, a high exchange rate was observed for proteins DEL and Sh. Therefore it is concluded that protein DEL does not belong to either subgroups III or IV, but is the first example of a new subgroup of the λ‐chains.
Yeast Phenylalanyl‐tRNA Synthetase
Authors: FASIOLO Franco , REMY Pierre , POUYET Jean , EBEL Jean‐Pierre
Published: March 2005

The stoichiometries of phenylalanyl‐tRNA‐synthetase · tRNAPhe complexes were found close to 2, as determined by the following methods: zone centrifugation in sucrose gradient, Sephadex filtration, sedimentation boundary technique and fluorescence quenching of Y base of tRNAPhe. The two former techniques allow the isolation of a complex composed of active enzyme (which is only a fraction of total enzyme used) and tRNA; the two last methods take into account the input protein. All the observed stoichiometries are consistent with the existence of two binding sites for tRNAPhe. Under the experimental conditions used (pH 7.0 and 12°C) a dissociation constant of 3 × 10−8 M was calculated from the fluoresence quenching of tRNAPhe by the enzyme.

tRNAPhe protects the enzyme against heat denaturation. A protection constant of 9 × 10−7 M was found at 55°C, pH 7.0. The presence of phenylalanine and ATP did modify the strength of interaction of the enzyme‐tRNA system. They induce, however, a synergistic effect of the protection afforded by tRNAPhe.

The binding of phenylalanine was examined by equilibrium dialysis in presence of tRNAPhe. The stoichiometry obtained indicates two binding sites for phenylalanine. The two sites however do not possess the same affinity: the corresponding Kd values are 6 and 30μM. The presence of more than one active site is also manifested by the curvature of the double‐reciprocal plots of v versus [S] for phenylalanine, when measuring the initial rates of phenylalanyl‐tRNA formation. With regard to the site having the highest affinity, a 20‐fold higher Kd value was found, in the absence of tRNAPhe, when binding of the amino acid was followed by the phase partition technique.
Cholesterol 7 α‐Hydroxylase
Authors: VAN CANTFORT Jacques , GIELEN Jacques
Published: March 2005

70% of the microsome‐bound cholesterol is directly accessible to cholesterol 7α‐hydroxylase in an assay in vitro. After 5 min of incubation this endogenous cholesterol makes a single pool with the exogenously added substrate and modifies its specific radioactivity. Thus, an accurate estimation of the enzymic activity should take the participation of endogenous cholesterol into account.

Cholesterol 7α‐hydroxylase activity is enhanced in vitro by thiol‐containing substances like mercaptoethanol, dithiothreitol, or cysteamine. On the contrary, the enzymic activity is inhibited by heavy cations (Hg2+, Pb2+, Cu2+, Zn2+), or −SH‐blocking agents (mersalic acid, p‐chloro‐mercuribenzoic acid).

Several steroids are potent inhibitors (Ki < Km) of the enzyme, among them pregnenolone and its acetate derivative and the cholesterol closely related 7‐oxocholesterol and 7‐dehydrocholesterol. The cholesterol esters are neither substrates nor inhibitors of cholesterol 7α‐hydroxylase. Only a high concentration (1 mM) of biliary acids or of their glyco or tauro derivatives inhibits cholesterol 7α‐hydroxylase. The quantitatively less important lithocholic acid and deoxycholic acid are the strongest inhibitors; the most common cholic acid does not affect the enzymic activity even at 1 mM.
Preparation and Properties of Vertebrate Smooth‐Muscle Myofibrils and Actomyosin
Authors: SOBIESZEK Apolinary , BREMEL Robert D.
Published: March 2005

A new technique for obtaining a myofibril‐like preparation from vertebrate smooth muscle has been developed. An actomyosin can be readily extracted from these myofibrils at low ionic strength and in yields 20 times as high as previously reported. The protein composition of all preparations has been monitored using dodecylsulfate‐gel electrophoresis. By this method smooth muscle actomyosin showed primarily only the major proteins, myosin, actin and tropomyosin, while the myofibrils contained, additionally, three new proteins not previously described with polypeptide chain weights of 60000, 110000 and 130000. The ATPase activities of both the myofibrils and actomyosin preparations are considerably higher than previously described for vertebrate smooth muscle. They are sensitive to micromolar Ca2+ ion concentrations to the same degree as comparable skeletal and cardiac muscle preparations, even though troponin‐like proteins could not be identified in these smooth muscle preparations. From the latter observation and the presence of Ca2+ ‐sensitivity in tropomyosin‐free actomyosin it is suggested that this calcium sensitivity is, as in some invertebrate muscles, a property of the myosin molecule.
RNA Metabolism in Nuclei Isolated from HeLa Cells
Authors: BUSIELLO Enzo , DI GIROLAMO Mario
Published: March 2005

Nuclei with low cytoplasmic contamination, capable of synthesizing RNA for an extended period of time, were prepared from HeLa cells. Besides elongating RNA chains already initiated in vivo, the nuclear preparation initiates the synthesis of new RNA chains. This was shown by labelling the newly synthesized RNA with [γ‐32P]GTP and by detecting the presence of labelled guanosine tetraphosphate among the alkaline hydrolysis products of synthesized RNA. By synthesizing RNA in the presence of each of the four γ‐32P‐labelled nucleoside triphosphates, it was possible to conclude that RNA chain synthesis starts predominantly with a purine base. Both nucleolar and nucleoplasmic RNAs are made. The nuclear preparation methylates the nucleolar RNA by utilizing S‐adenosyl‐l‐methionine as a methyl‐group donor.
A Pathway of Chitosan Formation in Mucor rouxii
Authors: ARAKI Yoshio , ITO Eiji
Published: March 2005

An enzyme that catalyzes hydrolysis of acetamido groups of chitin derivatives was found in the supernatant fraction of Mucor rouxii.

Partially O‐hydroxyethylated chitin (glycol chitin) was used as a substrate in the purification and characterization of this enzyme. A 140‐fold purification was obtained by means of ammonium sulfate fractionation followed by chromatography on carboxymethylcellulose and DEAE‐cellulose.

The enzyme releases about 30% of the acetyl groups of glycol chitin, giving a product with a decreased sensitivity to lysozyme. The enzyme also deacetylates chitin and N‐acetylchitooligoses, whereas it is inactive toward bacterial cell wall peptidoglycan, N‐acetylated heparin, a polymer of N‐acetylgalactosamine, di‐N‐acetylchitobiose and monomeric N‐acetylglucosamine derivatives.

This enzyme shows a pH optimum of 5.5. The Km value for glycol chitin is 0.87 g/l or 2.6 mM with respect to monosaccharide residues.

The occurrence of this enzyme accounts for the formation of chitosan in fungi.
Isolation and Partial Characterisation of the Relaxation Protein from Nuclei of Cultured Mouse and Human Cells
Authors: VOSBERG Hans‐Peter , GROSSMAN Lawrence I. , VINOGRAD Jerome
Published: March 2005

A protein, called relaxation protein because of its ability to remove superhelical turns in closed‐circular DNA, has been isolated and partially characterized from the nuclei of LA9 mouse and HeLa cells. The purification was facilitated by an assay method, with PM2 DNA, which used the fluorescence enhancement of the intercalating dye ethidium bromide upon binding to the closed‐circular DNA. The amount of dye bound depends upon the degree of the superhelix density of the DNA. The relaxation products were analysed by the buoyant separation method in CsCl containing ethidium bromide and were shown to be completely relaxed. The purification resulted in a single band in a dodecylsulfate gel electrophoresis with an apparent molecular weight of 37000. The pH optimum is 7.0 and the optimal salt concentration is 0.2 M NaCl. The relaxation protein removes negative as well as positive supercoils, the latter generated by the interaction of ethidium bromide with closed‐circular DNA. Relaxation of positive supercoils results, after removal of the dye, in the formation of molecules with superhelix densities exceeding that of native PM2 DNA (0.054). The highest negative superhelix density observed was −0.098 ± 0.001. The corresponding positive superhelix density has been calculated to be + 0.023.

A nicking—swivelling—closing mechanism is postulated, but nicked intermediates have so far not been demonstrated. The relaxation protein is not inhibited by known mammalian endonuclease I inhibitors, except for denatured DNA, and does not possess a conventional polynucleotide ligase activity. The relaxation activity was found to be predominantly in the nuclei, with only small amounts present in the cytoplasm and mitochondria. The biological function of transient swivels induced by the relaxation protein is not known. However, transient swivels are considered necessary or useful in the replication of closed‐circular DNA or long linear DNA, respectively. Relaxation protein could replace the combined action of an endonuclease and a ligase ahead of the replication fork. Alternatively, transient swivels could be involved in the transcription process.
Amino‐Acid Sequence of the Peptide Segment Liberated during Activation of Prochymosin (Prorennin)
Authors: PEDERSEN Vibeke Barkholt , FOLTMANN Bent
Published: March 2005

By conversion of prochymosin into active chymosin an N‐terminal segment of 42 amino acid residues is liberated. In one activation experiment this segment was recovered in two peptides; in a second experiment the activation segment was cleaved into three peptides. The primary structures of the peptides have been determined. Overlaps between these peptides and between the activation segment and the active enzyme have been obtained from peptides produced by tryptic digestion of denatured prochymosin.

Comparison of the amino acid sequences of the activation segments from bovine prochymosin, bovine pepsinogen and porcine pepsinogen shows considerable homology.
The Structure of Dihydrofolate Reductase
Authors: GLEISNER John M. , BLAKLEY Raymond L.
Published: March 2005

Dihydrofolate reductase from the amethopterin‐resistant mutant (strain A) of Streptococcus faecium var. Durans was reacted with iodo[14C]acetate according to three procedures; (a) in the absence of an inhibitor, (b) in the presence of aminopterin, and (c) in absence of inhibitor, but after treatment with unlabeled iodoacetate in presence of aminopterin. The first and last procedures resulted in the loss of approximately 90% of the catalytic activity, whereas in the presence of aminopterin essentially no activity was lost. Peptides were produced from all three labeled proteins by tryptic digestion after citraconylation of the lysine residues. From the amino acid compositions and partial amino acid sequences of these peptides the position of all modified methionines in the sequence was determined. The extent of labeling at each methionine, in enzyme labeled in the different procedures, indicated that methionines 28 and 50 may be at the binding site for inhibitors and that residue 50 is less accessible to iodoacetate than is residue 28. It is likely that carboxymethylation of residue 28 is responsible for the loss of enzyme activity.
Change of Cytochrome c Structure during Development of the Mouse
Authors: HENNIG Bernd
Published: March 2005

The structure of cytochrome c during mouse development is investigated. For this purpose the amino acid sequence of cytochrome c of the adult mouse had to be determined. The structure of cytochrome c of adult differentiated mouse cells differs in two amino acid residues from the known amino acid sequence of rabbit cytochrome c. No indication of different forms of cytochrome c in the adult differentiated cells was obtained. The structure of cytochrome c from 11.5‐day‐old mouse embryos is identical with that of adult mouse tissues. Since germ cells after meiotic division are the immediate precursors of a new individual, the structure of cytochrome c from sperm‐containing mice testes was investigated. By means of chromatography of the cytochrome c and of peptide maps and amino acid analyses of its tryptic peptides, it is shown that mouse testis contains two isocytochromes c in about equal amount. The structure of one of these two isocytochromes c is identical with the structure of the adult‐type cytochrome c of mouse. The testis‐specific cytochrome c, which is assumed to be located in the sperm cells, differs in 13 of its 104 amino acid residues from the adult‐type cytochrome c. From comparison of the primary and the spatial structures of the adult‐type and the sperm‐type isocytochromes c with the known structures of cytochrome c of more than 65 different species it is concluded that the duplication of the cytochrome c structural gene, causing the existence of the two ontogenetic‐specific isocytochromes c in mouse, has occurred early in the evolution of eucaryotes.
Nuclear Protein‐Kinase Activity in Perfused Rat Liver Stimulated with Dibutyryl‐adenosine Cyclic 3′: 5′‐Monophosphate
Authors: CASTAGNA Monique , PALMER Warren K. , WALSH Donal A.
Published: March 2005

Cytoplasmic and nuclear protein kinase activities from perfused rat liver have been studied in response to dibutyryl‐adenosine cyclic 3′:5′‐monophosphate added at a concentration that stimulates hepatic gluconeogenesis (100 μM). Total nuclear protein kinase, as assayed using a mixed histone fraction as phosphate acceptor, is increased by 5‐fold within 8 min of the addition of cyclic nucleotide to the perfusate. In contrast the total cytoplasmic protein kinase activity is decreased to 50% of the control value. The protein substrate specificity of the protein kinase that is present in the nucleus in response to dibutyryl‐adenosine cyclic 3′:5′‐monophosphate stimulation is similar to that of cytoplasmic, adenosine cyclic 3′:5′‐monophosphate‐dependent, protein kinase but is distinct from that of the enzyme(s) present in control nuclei. The predominant species of protein kinase from stimulated nuclei has a sedimentation constant of 3.9 S. This value is identical to that of the catalytic subunit of cytoplasmic adenosine 3′:5′‐monophosphate‐dependent protein kinase. These data suggest that some of the effects of adenosine 3′:5′‐monophosphate on nuclear events may be mediated through its interaction with the inactive protein kinase holoenzyme in the cytoplasm and the subsequent redistribution of the active catalytic subunits generated by this interaction.
Chicken Antichromatin Antibodies: Specificity to Different Chromatin Fractions
Authors: ZARDI Luciano
Published: March 2005

Specific complement‐fixing and precipitin antibodies were induced in chicken to chromatin from WI‐38 human diploid fibroblast. Chicken antibodies were chosen because they present two major advantages with respect to rabbit antibodies: (a) the optimal NaCl concentration for chicken precipitin is 1.5 M, which is also an optimal solvent for chromatin proteins that are insoluble at the lower salt concentration required by rabbit precipitin; (b) chicken antichromatin antibodies require an antigen concentration much lower with respect to rabbit antibodies for saturation at the complement‐fixation reaction.

These antibodies are species specific and they react at the complement‐fixation and precipitin assay not only with the whole chromatin but also with dissociated proteins, albeit at a higher concentration of both antigen and antibodies. The specificity of these antibodies to different fractions of chromosomal proteins and of the chromatin after washing with a solution at a different sodium chloride concentration has been investigated.
Fingerprinting Studies of the Maturation of Ribosomal RNA in Mammalian Cells
Authors: GALIBERT Francis , ELADARI Marthe‐Elisabeth , TIOLLAIS Pierre
Published: March 2005

32P‐labelled ribosomal RNA of L 5178 Y cells (a mouse cell line) was digested with T1 ribonuclease and fingerprinted by electrophoresis at pH 3.5 on cellulose acetate and homochromatography on DEAE‐cellulose thin‐layer plate. From this, it can be concluded that 18‐S and 28‐S RNA have different and characteristic fingerprints and that the number, the size and the frequency of the large T1 oligonucleotides demonstrate that the guanylic residues are randomly interspaced along the molecule.

Using a double‐labelling technique with 32P‐labelled 45‐S RNA and 14C‐labelled 18‐S RNA or 28‐S RNA, long T1 oligonucleotides of the 45‐S RNA can be divided into three classes: (a) those which are lost during the transition, (b) those which are present in the 18‐S RNA and (c) those which are present in the 28‐S RNA. These results provide direct evidence for the existence of one common precursor for the two mature ribosomal RNAs.

The comparison of the fingerprints of T1‐ribonuclease‐digested 47‐S, 45‐S and 41‐S RNA shows that the 47‐S and 41‐S RNA have a characteristic ribosomal pattern. Finally the size, the number and the mobility of the oligonucleotides present in the different RNA precursors but absent from the mature RNA demonstrate that the non‐conserved RNA pieces do not have a monotonous and repetitive sequence.
Interaction of Wheat‐Germ Agglutinin with Bacterial Cells and Cell‐Wall Polymers
Authors: LOTAN Reuben , SHARON Nathan , MIRELMAN David
Published: March 2005

Wheat germ agglutinin was found to agglutinate cells of Escherichia coli PAT 84, Micrococcus luteus, Staphylococcus aureus H, and of S. aureus 52A5, but not cells of S. aureus 52A2.

Interaction of wheat germ agglutinin with a soluble linear peptidoglycan secreted by Micrococcus luteus and with the teichoic acid of S. aureus H was demonstrated by agar gel diffusion, quantitative precipitation and inhibition of hemagglutination of trypsinized rabbit erythrocytes. No interaction could be demonstrated with the teichoic acid from a phage‐resistant mutant (S. aureus 52A2) which lacks N‐acetyl‐d‐glucosamine residues.

All interactions were specifically inhibited by low concentrations of chitotriose (GlcNAcβ1 → 4GlcNAcβ1 → 4GlcNAc) and the bacterial cell wall tetrasaccharide, GlcNAcβ1 → 4MurNAcβ1 → 4GlcNAcβ1 → 4MurNAc. Hemagglutination‐inhibition experiments showed that the linear peptidoglycan and the teichoic acid of S. aureus H were several thousand times more potent inhibitors of wheat germ agglutinin than was N‐acetyl‐d‐glucosamine.

Comparison of the efficiency of different saccharides in inhibition of hemagglutination and precipitation of polymers by wheat germ agglutinin, strongly suggests that secondary, non‐specific interactions contribute to the binding of the lectin to the polymers.