bluuebunny/crossref_metadata_2025 huggingface dataset:
Determination of the Kinetic Constants of Glucosephosphate Isomerase by Non‐linear Optimization
Authors: PLESSER Theodor , WURSTER Bernd , HESS Benno
Published: March 2005

The rate constants for the anomerization and isomerization reactions catalysed by glucose‐phosphate isomerase (EC 5.3.1.9) are determined from kinetic measurements by non‐linear optimization technique, employing the simplest reaction pathway with only one enzyme‐substrate complex.
Occurrence of 3‐Oxo‐acyl Groups in the 6,6′‐Diesters of α‐d‐Trehalose
Authors: PUZO Germain , TISSIÉ Gérard , AURELLE Hélène , LACAVE Charlotte , PROMÉ Jean‐Claude
Published: March 2005

The ‘cord factor’ from Corynebacterium diphtheriae was fractionated by thin‐layer chromatography of the trimethylsilyl derivatives into three main components: the true ‘cord factor’ B1 which has been described previously and two compounds, B2 and B3, containing 3‐Oxo‐acyl esters. These products were analyzed by mass spectrometry (field desorption/cationization and electron impact of the trimethylsilyl derivatives) and by chemical degradation.

Compound B3 is a 6,6′‐bis(3‐oxo‐acyl)‐α‐d‐trehalose. The 3‐oxo‐acyl groups are mainly saturated species, branched at C‐2, containing 24, 26, 28, 30 or 32 carbon atoms. The main component of B3 is the 6,6′‐bis(3‐oxo‐2‐tetradecyl‐octadecanoyl)‐α‐d‐trehalose.

Compound B2 is a 6,6′‐mixed ester of α‐d‐trehalose with the preceding 3‐oxo acids and with corynomycolic acids. These latter contain an even number of carbon atoms from 24 to 32 and are a mixture of saturated, mono‐unsaturated and di‐unsaturated species. The main compound of B2 is 6‐(3‐oxo‐2‐tetradecyl‐octadecanoyl)‐6′‐(3‐hydroxy‐2‐tetradecyl‐octadecanoyl)‐α‐d‐trehalose.

The metabolic implications of this structural study, together with results already published, suggest that condensation of the common fatty acids by a Claisen‐like process via the oxo ester pathway may not be the only way for the synthesis of corynomycolic acids.
The Stimulus‐Secretion Coupling of Glucose‐Induced Insulin Release
Authors: SENER Abdullah , MALAISSE Willy J.
Published: March 2005

The combination of Ba2+ and theophylline provoked a dramatic release of insulin from rat pancreatic islets incubated in the absence of extracellular Ca2+. The secretory response was unaffected by poisons of glycolysis; inhibited by epinephrine, organic calcium‐antagonists, respiratory inhibitors, and uncoupling agents; and augmented by glucose, 2‐oxoisocaproate and 2‐oxoiso‐valerate. The latter augmentation occurred even when the nutrients were used at concentrations which did not stimulate insulin release from islets maintained in a normal ionic environment. The release of insulin evoked by the combination of Ba2+ and theophylline was associated with an increase in the rate of oxidation of endogenous nutrients, but a fall in the concentration of both ATP and reduced pyridine nucleotides. The facilitation of insulin release by glucose (3.3 and 16.7 mM) was associated with a dose‐related increase in both the concentration of NAD(P)H and net uptake of 133Ba. The magnitude of the augmenting action of glucose upon insulin release evoked by the combination of Ba2+ and theophylline correlated with the rate of glucose oxidation. These data indicate that insulin release can be stimulated by non‐metabolizable secretagogues, despite a fall in the concentration of both ATP and NAD(P)H. However, the insulinotropic action of glucose, whatever its concentration, may depend on an increased generation of both reducing equivalents and high‐energy phosphate intermediates.
Recognition of tRNATrp by Initiation Factors from Escherichia coli
Authors: LEON Monique , DONDON Jacques , LABOUESSE Julie , GRUNBERG‐MANAGO Marianne , BUCKINGHAM Richard H.
Published: March 2005

Binding of acetyl or formyltryptophanyl‐tRNATrp from Escherichia coli or beef liver to E. coli ribosomes is strongly stimulated by E. coli initiation factors and requires GTP. The N‐acylated tryptophan is puromycin reactive. Polypeptide chain initiation with acetyltryptophan dependent on poly(U,G) has been demonstrated and is highly dependent on added initiation factors. tRNATrp appears, therefore, to share some structural features with tRNAMetf of significance to the process of polypeptide chain initiation.
Catalytic Activity of Aspartate Aminotransferase in the Crystal
Authors: MOZZARELLI Andrea , OTTONELLO Simone , ROSSI Gian Luigi , FASELLA Paolo
Published: March 2005

Natural substrates and analogs rapidly diffuse through crystals of pig heart mitochondrial aspartate aminotransferase and react at the active sites causing spectral changes that can be measured by single‐crystal microspectrophotometry. Dissociation constants for natural substrates and rate constants of transamination for slowly reacting substrates have been determined.

A comparison between the data obtained in the crystal and in solution shows that the crystalline enzyme is catalytically competent and that events occurring in the crystal essentially parallel those occurring in solution, even though minor differences have been detected.
The Role of Chloroplast‐Membrane‐Protein Synthesis in the Circadian Clock
Authors: LEONG Ta‐Yan , SCHWEIGER Hans‐Georg
Published: March 2005

A polypeptide (polypeptide P39), which is presumed to be involved in the photosynthetic circadian rhythm in the green alga Acetabularia, was purified from the EDTA‐insoluble chloroplast membrane fraction by means of preparative dodecylsulfate gel electrophoresis and then partially characterized.

The purity of the isolated polypeptide P39 was confirmed by a further electrophoresis on an analytical dodecylsulfate gel and further elucidated by amino‐terminal analysis which shows that glycine is the only amino‐terminal amino acid of the purified polypeptide material. The molecular weight of the polypeptide P39 was found to be about 39000 on analytical gel electrophoresis and the value was further supported by those obtained from amino acid composition and peptide mapping.

The amino acid composition of polypeptide P39 showed that the proportion of intermediate amino acid groups is high while the proportion of hydrophilic amino acid groups is well balanced by that of hydrophobic amino acid groups, a property characteristic of membrane proteins.
Studies on the Methylation of Cytoplasmic Ribosomal RNA from Cultured Higher Plant Cells
Authors: CECCHINI Jean‐Pierre , MIASSOD Raymond
Published: March 2005

The methylation of cytoplasmic ribosomal RNA of cultured sycamore cells (Acer pseudoplatanus L.) was investigated. Labelled 17‐S and 26‐S rRNA were prepared from cells that had been incubated with either [32P]phosphate, [Me‐3H]methionine or [Me‐14C]methionine. Ion‐exchange resin chromatography of 0.3 M KOH or 1 M HCl hydrolysates and two‐dimensional chromatographic analyses of phosphodiesterase plus phosphatase digests of 17‐S and 26‐S rRNA were performed.

17‐S and 26‐S rRNA contain 49 and 91 methyl groups per molecule, respectively. These values were verified in several ways. The high degree of methylation of sycamore rRNA, particularly for the 26‐S rRNA, contrasts with the situation in all other investigated organisms.

Several methylated bases were identified. 7‐Methylguanine and 5‐methylcytosine both occur in 17‐S and 26‐S rRNA. N6‐Methyladenine and N6,N6‐dimethyladenine are restricted to the 17‐S rRNA while 3‐methyluracil and 1‐methyladenine occur in the 26‐S rRNA. One hypermodified uridine was also tentatively identified in the small rRNA. In 17‐S rRNA, there is one copy of 7‐methylguanine, N6‐methyladenine and hypermodified uridine and two copies of N6,N6‐dimethyl‐adenine. 3‐Methyluracil, 1‐methyladenine and 5‐methylcytosine occur twice, twice and three times, respectively, in 26‐S rRNA. 7‐Methylguanine and 5‐methylcytosine are only in submolar amounts in the 26‐S and 17‐S rRNA, respectively.

There are 40 ± 2 and 83 ± 3 2′‐O‐methylriboses per 17‐S and 26‐S rRNA molecule, respectively. In addition to the four 2′‐O‐methylnucleosides, one 2′‐O‐methylpseudouridine is present in the 17‐S rRNA. Several lines of evidence argue for a non‐random distribution of the methylriboses. In particular, one and seven Nm‐Nm‐Np structures occur in the 17‐S and 26‐S rRNA, respectively.

The data are discussed comparatively with the methylation pattern of Escherichia coli, yeast and HeLa cell rRNA.
Increased Biosynthesis of CoA in the Liver of Rats Treated with Clofibrate
Authors: SKREDE Sverre , HALVORSEN Ola
Published: March 2005

The amounts of CoA in rat liver increased about three fold after treatment with clofibrate. About 10 of total CoA were long‐chain acyl‐CoA in normal rats, and slightly less in clofibrate‐treated animals. The wet weight and protein content of the liver increased by about 25%.

From livers of clofibrate‐treated rats, mitochondrial and lysosomal fractions were obtained in increased amounts by conventional tissue fractionation techniques. The supernatants from such livers had rather high activities of marker enzymes for lysosomes and peroxisomes.

No changes of the specific activities of the two first enzymes in the degradation pathway for CoA, i.e. acid phosphatase and nucleotide pyrophosphatase, were found in the liver homogenate from clofibrate‐treated rats. In the mitochondrial fraction from such livers, both (contaminating) activities were decreased. The degradation of CoA was increased in the supernatant, however, probably because of an increased leakage of acid phosphatase from lysosomes during the fractionation procedure.

The synthesis of CoA from pantothenic acid was estimated by two different methods in vitro: by quantitation of CoA formed and by estimation of the incorporation of [14C]pantothenic acid into CoA. By both methods, CoA synthesis was found to be increased 3–4‐fold in livers from clofibrate‐treated rats.

Dephospho‐CoA kinase was found to have a dual localization, with a minor pool in the mitochondrial fraction. This enzyme was only slightly increased in the clofibrate‐treated animals.

The activity of pantothenate kinase was more than doubled after clofibrate treatment. This is thought to be the most important factor for the increase of CoA. CoA exerted feed‐back inhibition at this step in liver supernatant from normal and clofibrate‐treated rats.
Transcription by Eukaryotic RNA Polymerases A and B of Chromatin Assembled in vitro
Authors: WASYLYK Bohdan , CHAMBON Pierre
Published: March 2005

Chromatin was assembled in vitro from simian virus 40 DNA form I and the calf‐thymus four histones H2A, H2B, H3 and H4. Transcription with calf thymus RNA polymerases A and B (I and II) was greatly inhibited. Nucleosomes were found to inhibit both RNA chain initiation and elongation. The inhibition of elongation could be relieved by increasing ionic strength, suggesting that electrostatic interactions between histone octamer and DNA have to be broken for RNA polymerase to transcribe DNA organised into nucleosomes.
Stereochemistry of Reactions Catalysed by Mammalian‐Brain L‐Glutamate 1‐Carboxy‐lyase and 4‐Aminobutyrate: 2‐Oxoglutarate Aminotransferase
Authors: BOUCLIER Martine , JUNG Michel J. , LIPPERT Bruce
Published: March 2005

Deamination of 4‐aminobutyrate by mammalian or bacterial 4‐aminobutyrate aminotransferases involves the abstraction of the pro‐S hydrogen on C‐4 of 4‐aminobutyrate. Decarboxylation of L‐glutamate by rat brain glutamate decarboxylase occurs with retention of configuration. Inhibition of this enzyme by (S)‐4‐aminohex‐5‐ynoic acid involves the abstraction of the proton at C‐4 of the inhibitor. On the basis of this finding, we postulate the existence of an abnormal reaction of glutamate decarboxylase in which the proton at C‐4 of (S)‐4‐aminohex‐5‐ynoic acid is removed in a manner similar to the one which normally occurs in enzymatic transaminations of L‐amino acids. This reaction is presumably facilitated by the acetylenic group adjacent to the eliminated proton.
Deuterium Isotope Effects on Papain Acylation
Authors: POLGÁR László
Published: March 2005

The experimental data presented in this paper comprise kinetic deuterium isotope effects on acylation of papain with various substrates when conducted in H2O and 2H2O. With alkyl esters of N‐acylamino acids there is no or very little isotope effect, whereas with N‐acylamino acid amides the ratio kH2O/k2H2O is less than 1, i.e. there is an inverse isotope effect. Similarly, alkylation of papain with methyl bromoacetate exhibits no kinetic isotope effect, whereas for the analogous alkylation with bromoacetamide an inverse isotope effect is observed.

It is concluded that (a) general base catalysis does not occur in the acylation of papain and (b) kinetic deuterium isotope effects can be affected substantially by interaction between the substrate leaving group and the enzyme, which has not been considered in previous mechanistic investigations.
The Amino Acid Sequence of Mouse Pancreatic Ribonuclease
Authors: LENSTRA Johannes A. , BEINTEMA Jaap J.
Published: March 2005

The complete amino acid sequence of mouse pancreatic ribonuclease has been determined by analysis of tryptic, chymotryptic, thermolytic and CNBr peptides and by automatic sequence analysis of the intact protein.

The sequence of mouse RNase differs in 20–30% of the positions from other RNase sequences. Three unique or nearly unique substitutions were found, viz. Gly‐68 → Arg‐68, Arg‐85 → His‐85 and Ser‐123 → Thr‐123. All these three residues might be involved in interactions with substrate molecules.

A most parsimonious tree of the myomorph rodent RNase shows that after the divergence of rat and mouse, the ribonuclease of rat accumulated substitutions at a rate 2.5–4.3 times as high as the rates in other branches of the tree and 23 times as high as the average rate in the Bovidae ribonuclease evolution. These extreme fluctuations in substitution rate are difficult to reconcile with the hypothesis of the evolutionary clock. The high evolution rate of rat ribonuclease is thought to be caused by positive selection, leading to new functional properties of the enzyme.
Acidic Ribosomal Proteins from Eukaryotic Cells
Authors: SÁNCHEZ‐MADRID Francisco , REYES Ramón , CONDE Pedro , BALLESTA Juan P. G.
Published: March 2005

Precipitation of Saccharomyces cerevisiae ribosomes by ethanol under experimental conditions that do not release the ribosomal proteins can affect the activity of the particles. In the presence of 0.4 M NH4Cl and 50% ethanol only the most acidic proteins from yeast and rat liver ribosomes are released. At 1 M NH4Cl two more non‐acidic proteins are lost from the ribosomes. The release of the acidic proteins causes a small inactivation of the polymerizing activity of the particles, additional to that caused by the precipitation itself. The elongation‐factor‐2‐dependent GTP hydrolysis of the ribosomes is, however, more affected by the loss of acidic proteins. These proteins can stimulate the GTPase but not the polymerising activity when added back to the treated particles. Eukaryotic proteins cannot be sustituted for bacterial acidic proteins L7 and L12. We have not detected immunological cross‐reaction between acidic proteins from Escherichia coli and those from yeast, Artemia salina and rat liver or between acidic proteins from these eukaryotic ribosomes among themselves.
Glyceraldehyde‐3‐phosphate Dehydrogenase of Scenedesmus obliquus
Authors: WOODROW Sandra , O'BRIEN Michael J. , EASTERBY John S. , POWLS Roy
Published: March 2005

Incubation of the hexadecameric glyceraldehyde‐3‐phosphate dehydrogenase of Scenedesmus obliquus with dithiothreitol causes a transient increase in NADPH‐dependent activity. When NADPH is also incorporated in the incubation buffer, the induced NADPH‐dependent activity is stabilised. In the presence of NADPH, the rate of induction of NADPH‐dependent activity is directly proportional to dithiothreitol concentration. This is consistent with either a dithiothreitol‐mediated reduction of disulphide bonds or dithiothreitol‐promoted disulphide exchange in the enzyme without prior formation of a non‐covalent complex between enzyme and reductant. A kinetic analysis of the transient induction of the NADPH‐dependent activity by dithiothreitol alone shows that the activation is thiol‐dependent with a second‐order rate coefficient of 4.3 M−1 min−1. In contrast, the ensuing in‐activation is spontaneous and does not involve thiol. It proceeds with a first‐order rate coefficient of 0.032 min−1, corresponding to a half‐time of 22 min. It is concluded that dissociation of the hexadecameric enzyme to the tetramer is promoted by dithiothreitol whereas further dissociation to an inactive dimer results purely from the inherent instability of the tetramer in the absence of NADPH. The rate of induction of NADPH‐dependent activity by dithiothreitol increased hyperbolically with respect to NADPH concentration, implying that NADPH has more than a stabilising role in the activation process. These observations are discussed in relation to the photoreductive process thought to occur in vivo.
Differential Binding of Myosin Subfragment One Species to Immobilized ADP, and Actin: the Influence of the Alkali Light Chains
Authors: WINSTANLEY Monica A. , SMALL David A. P. , TRAYER Ian P.
Published: March 2005

Isolated myosin head regions (subfragments 1) have been prepared from a number of muscle types, and studied by affinity chromatography upon immobilized ADP and actin matrices.

It has been shown that the two alkali light chain isoenzymes of subfragment 1 specific for a given skeletal muscle can be fractionated; therefore, these procedures provide essentially simple and rapid methods for the preparation of myosin head populations which are homogeneous with regard to their light chain content, and readily amenable to further study.

Fractionation of slow twitch skeletal subfragment 1 species upon immobilized actin extends previous findings with fast twitch species [Winstanley, Trayer and Trayer (1977) FEBS Lett.77, 239–2421. In each case the heads containing the less anodic alkali light chain bind more tightly to the actin matrix. These results are supported by kinetic data which show differences only in the apparent Kmfor actin between the two acto‐subfragment 1 MgATPase. Comparison between the subfragment 1 species from different muscle types indicates a correlation between the strength of the interaction between subfragment 1 and immobilized actin and the myosin ATPase activity and contractile properties of the muscle. Tighter actin binding is associated with the low‐ATPase‐activity myosins characteristic of slow twitch skeletal and cardiac muscles.

A possible relationship is suggested between the amino acid composition of the alkali light chain present, and the actin binding properties of the myosin head.
Structure of Pyridine Nucleotide Transhydrogenase from Azotobacter vinelandii
Authors: VOORDOUW Gerrit , VEEGER Cees , VAN BREEMEN Jan F. L. , VAN BRUGGEN Ernst F. J.
Published: March 2005

1. Pyridine nucleotide transhydrogenase of Azotobacter vinelandii purified by affinity chromatography consists of a mixture of polydisperse rods at neutral pH. No other structures are seen by electron microscopy.

2. At high pH (8.5–9.0) the rods depolymerize. Complete depolymerization can be achieved in 0.1 M Tris‐Cl pH 9.0. The depolymerized enzyme has a molecular weight of 421000 (sedimentation equilibrium), its sedimentation coefficient 520,w= 15 S and its Stokes' radius Rs= 7 nm. Since gel electrophoresis in the presence of sodium dodecyl sulphate shows that transhydrogenase consists of a single polypeptide chain of molecular weight (54 ± 2) × 103 it follows that the depolymerized enzyme has an octameric quaternary structure. We propose that this octamer serves as the functional monomeric unit (‘unimer’) from which the polymeric form of transhydrogenase is constructed.

3. Gel filtration and sucrose gradient centrifugation studies of cell‐free extracts from A. vinelandii show the unimer to be the predominant active species.
Mouse‐Liver Glutathione Reductase
Authors: LÓPEZ‐BAREA Juan , LEE Chi‐Yu
Published: March 2005

Glutathione reductase from the liver of DBA/2J mice was purified to homogeneity by means of ammonium sulfate fractionation and two subsequent affinity chromatography steps using 8‐(6‐aminohexyl)‐amino‐2′‐phospho‐adenosine diphosphoribose and N6‐(6‐aminohexyl)‐adenosine 2′,5′‐bisphosphate‐Sepharose columns. A facile procedure for the synthesis of 8‐(6‐aminohexyl)‐amino‐2′‐phospho‐adenosine diphosphoribose is also presented. The purified enzyme exhibits a specific activity of 158 U/mg and an A280/A460 of 6.8. It was shown to be a dimer of Mr, 105000 with a Stokes radius of 4.18 nm and an isoelectric point of 6.46. Amino acid composition revealed some similarity between the mouse and the human enzyme. Antibodies against mouse glutathione reductase were raised in rabbits and exhibited high specificity. The catalytic properties of mouse liver glutathione reductase have been studied under a variety of experimental conditions. As with the same enzyme from other sources, the kinetic data are consistent with a ‘branched’ mechanism.

The enzyme was stabilized against thermal inactivation at 80°C by GSSG and less markedly by NADP+ and GSH, but not by NADPH or FAD. Incubation of mouse glutathione reductase in the presence of NADPH or NADH, but not NADP+ or NAD+, produced an almost complete inactivation. The inactivation by NADPH was time, pH and concentration dependent. Oxidized glutathione protected the enzyme against inactivation, which could also be reversed by GSSG or other electron acceptors. The enzyme remained in the inactive state even after eliminating the excess NADPH. The inactive enzyme showed the same molecular weight as the active glutathione reductase. The spectral properties of the inactive enzyme have also been studied. It is proposed that auto‐inactivation of glutathione reductase by NADPH and the protection as well as reactivation by GSSG play in vivo an important regulatory role.
Purification and Characterization of a Protein Factor that Reverses the Inhibition of Protein Synthesis by the Heme‐Regulated Translational Inhibitor in Rabbit Reticulocyte Lysates
Authors: AMESZ Hans , GOUMANS Hans , HAUBRICH‐MORREE Thea , VOORMA Harry O. , BENNE Rob
Published: March 2005

We have purified and partially characterized a supernatant factor which reverses the effect of the heme‐regulated translational inhibitor on protein synthesis in rabbit reticulocyte lysates. The anti‐inhibitor restores protein synthesis activity in heme‐deficient lysates (and in lysates to which the inhibitor has been added) to the level observed in the presence of heme.

The factor has no effect on the phosphorylation of eIF‐2 by the inhibitor nor on any reaction carried out with purified initiation factors.

The anti‐inhibitor probably consists of three subunits with molecular weights of 81000, 60000 and 41000. The factor is isolated from the postribosomal supernatant of rabbit reticulocytes both free and complexed to eIF‐2. A possible mechanism of action is discussed.
Protein · Nucleic‐Acid Reaction Kinetics
Authors: GIACOMONI Paolo U.
Published: March 2005

This paper presents methods developed in order to analyze experimental results concerning the binding of Escherichia coli DNA‐dependent RNA polymerase to DNA at high and at low DNA concentrations, using the filter retention assay.

The basic hypotheses, under which the mathematical expressions for describing the kinetics of binding are derived, are as follows. (a) At low DNA concentration: equivalence and independence of the specific binding sites; first‐order dependence of the binding reaction on both DNA and protein concentration. (b) At high DNA concentration: equivalence and independence of the nonspecific binding sites; no direct transfer or one‐dimensional sliding of the protein along the DNA.

Comparison between theoretical predictions and experimental results at high DNA concentration will alow one to determine the relative value of the rates of binding of RNA polymerase to different promoters (between 1 and 2 in T5 DNA).

Binding experiments performed at low DNA concentration are reported in this paper: these results and the analysis which is reported allow one to determine the value of the rate constant of formation of non‐filterable complexes for the system fd DNA (replicative form) · RNA‐polymerase (ka= 3.3 × 108 M−1 s−1 in 0.1 M NaCl, 0.01 M MgCl2).
Reconstitution of Active 30‐S Ribosomal Subunits in vitro Using Heat‐Denatured 16‐S rRNA
Authors: BARRITAULT Denis , GUERIN Marie France , HAYES Donal H.
Published: March 2005

30‐S ribosomal subunits which have been reconstituted using heat‐denatured 16‐S rRNA can participate in the synthesis of lysosyme in vitro. Therefore all the information contributed by 16‐S rRNA to the reconstitution process is carried in the primary sequence of this RNA. The specific protein‐synthesizing activity of 30‐S subunits reconstituted from 30‐S subunit proteins and heat‐denatured 16‐S rRNA is about one third of that observed if unheated 16‐S rRNA is used and is comparable to the activity of 30‐S particles isolated after dissociation of 70‐S ribosomes in the presence of 0.1 mM Mg2+.
Biosynthesis of a Blood‐Group‐I Determinant Reacting with Anti‐I Ma Serum (Group 1)
Authors: SCHENKEL‐BRUNNER Helmut , KABAT Elvin A. , LIAO Jerry
Published: March 2005

A blood‐group‐I determinant reacting specifically with anti‐I Ma serum (group 1) has been produced biosynthetically by the action of a β‐galactosyl transferase isolated from human milk on a precursor glycoprotein produced by formic acid hydrolysis and β‐D‐galactosidase action on blood‐group H substance prepared from hog gastric mucosa.
Flavanone Synthase from Petroselinum hortense
Authors: KREUZALER Fritz , RAGG Hermann , HELLER Werner , TESCH Rudolf , WITT Irene , Dietrich HAMMER  , HAHLBROCK Klaus
Published: March 2005

Flavanone synthase from irradiated cell suspension cultures of parsley was purified to apparent homogeneity. Molecular weights of about 77000 for the enzyme and about 42000 for the subunits were determined respectively by sedimentation‐equilibrium measurements and disc‐gel electrophoresis in the presence of dodecyl sulfate. A specific antiserum was prepared for the enzyme and was used in an assay for flavanone synthase mRNA activity in partially purified RNA preparations. The apparent molecular size of flavanone synthase mRNA was estimated by sucrose gradient centrifugation and gel electrophoresis under partially denaturing conditions. Values of about 17 S and Mr= 0.62 × 106 were obtained. The fractionation patterns suggested that flavanone synthase mRNA was homogeneous in size. All together, the results support the idea that the enzyme is composed of two subunits which are probably identical.

Amino acid analysis and a microbial assay were carried out to test the possible occurrence of cysteamine, β‐alanine, and pantothenate in the enzyme. The results were negative, indicating the absence of pantetheine or a similar residue. The possible similarity in mechanism between flavanone synthase and 3‐oxoacyl‐(acyl carrier protein) synthase is discussed.
Conversion of Type II Procollagen to Collagen
Authors: UITTO Jouni , ALLAN Ruth E. , POLAK Kathy L.
Published: March 2005

Type II procollagen, the precursor form of a genetically distinct type of collagen present in hyaline cartilages, has previously been shown to contain extension peptides both at the amino‐terminal and carboxy‐terminal ends of the molecule. Here, the synthesis of type II procollagen was studied by pulse‐labeling chick embryo sterna with [14C]proline for 10 min, and the incorporation of radioactivity was then inhibited by the addition of cycloheximide and unlabeled proline. At the end of a 10‐min pulse, most of the 14C‐labeled protein was [14C]procollagen. If the incubation was continued for 30–120 min after the inhibition of the protein synthesis, an increasing fraction of [14C]procollagen was converted to [14C]collagen, as monitored by polyacrylamide slab gel electrophoresis in dodecylsulfate. Two intermediate forms containing either the amino‐terminal or carboxy‐terminal extension peptides were also detected. In further studies the site of the conversion process was examined by comparing the rate of conversion with the time required for secretion of the procollagen molecules out of the cells. Comparison of the time of conversion with the time of secretion, as estimated in intact cartilage by radioautographic techniques, or determined as secretion of hydroxy[14C]proline by matrix‐free cartilage cells incubated under identical conditions, indicated that the secretion started significantly earlier than the conversion. Also, the conversion of the [14C]procollagen secreted by the matrix‐free cartilage cells continued in the medium after separation of the cells by centrifugation. Furthermore, incubation of intact cartilages with α,α'‐dipyridyl, L‐azetidine‐2‐carboxylic acid, cis‐4‐hydroxy‐L‐proline, or colchicine, agents which, by independent mechanisms, delay the secretion of procollagen, also inhibited the conversion. The results suggest that the conversion of type II procollagen to collagen occurs in the extracellular space, the amino‐terminal and carboxy‐terminal extensions beind removed without a preferential sequence.
Isolation of Plasmid‐Protein Complexes from Escherichia coli
Authors: BUSBY Stephen , KOLB Annie , BUC Henri
Published: March 2005

A procedure is described for the isolation of complexes between pMB9 plasmids and protein from Escherichia coli which are stable during centrifugation on sucrose gradients and are not destroyed in the presence of competitor DNA. The proteins in these complexes have been analysed by dodecyl sulphate/polyacrylamide gel electrophoresis. Only 10 polypeptide species are found in significant quantities, many of which are bound to both the plasmid and host DNA. We have also detected the presence of one protein which binds to a specific DNA sequence inserted in the plasmid.
Isolation of Mitochondrial Succinate: Ubiquinone Reductase, Cytochrome c Reductase and Cytochrome c Oxidase from Neurospora crassa Using Nonionic Detergent
Authors: WEISS Hanns , KOLB Helmut J.
Published: March 2005

The electron transfer complexes, succinate: ubiquinone reductase, ubiquinone: cytochrome c reductase, and cytochrome c:O2 oxidase were isolated from the mitochondrial membranes of Neurospora crassa by the following steps. Modification of the contents of the complexes in mitochondria by growing cells on chloramphenicol; solubilisation of the complexes by Triton X‐100; affinity chromatography on immobilized cytochrome c and ion exchange and gel chromatography. Ubiquinone reductase was obtained in a monomeric form (Mr∼ 130000) consisting of a flavin subunit (Mr 72000) an iron‐sulfur subunit (Mr 28000) and a cytochrome b subunit (Mr probably 14000). Cytochrome c reductase was obtained in a dimeric form (Mr∼ 550000), the monomeric unit comprising two cytochromes b (Mr each 30000), a cytochrome c1 (Mr 31000), an iron‐sulfur subunit (Mr 25000), and six subunits without known prosthetic groups (Mr 9000, 11000, 14000, 45000, 45000, and 52000). Cytochrome c oxidase was also isolated in a dimeric form (Mr∼ 320000) comprising two copies each of seven subunits (Mr 9000, 12000, 14000, 18000, 21000, 29000, and 40000). The complexes were essentially free of phospholipid. Each bound one micelle of Triton X‐100 (Mr∼ 90000). After isolation, the bound Triton X‐100 could be replaced by other non‐ionic detergents such as: alkylphenyl polyoxyethylene ethers, alkyl polyoxyethylene ethers and acyl polyoxyethylene sorbitan esters.
Enzymology of Ubiquinone‐Utilizing Electron Transfer Complexes in Nonionic Detergent
Authors: WEISS Hanns , WINGFIELD Paul
Published: March 2005

The enzymology of isolated succinate: ubiquinone reductase and ubiquinone: cytochrome c reductase in nonionic detergents (alkyl polyoxyethylene derivatives) was studied.

In the membrane the two multiprotein complexes and their hydrophobic substrates ubiquinone and dihydroubiquinone, are embedded in a common lipid bilayer. In detergent solutions the complexes are each inserted into micelles. Detergent micelles also serve as a solvent for the complexes hydrophobic substrates. As a consequence the isolated complexes are in a discontinuous phase with respect to their hydrophobic substrates and with respect to each other.

Three types of assays were used. Firstly, single enzyme assays in which the hydrophobic substrates had to transfer from free micelles to the complex‐bound micelles in order for enzymic reactions to occur. Secondly, assays in which the enzymic reactions were coupled to auxiliary non‐enzymic reactions which rapidly converted the hydrophobic products back into substrates within the complex‐bound micelle. Dichloroindophenol was used for the oxidation of dihydroubiquinone and dihydroduroquinone for the reduction of ubiquinone. Thirdly, assays in which the succinate: ubiquinone reductase reaction was coupled with the ubiquinone: cytochrome c reductase reaction.

With the first type of assay, the kinetics of the substrate transfer reaction was dependent upon the type of detergent. In detergents with small polyoxyethylene head groups the transfer reactions were rate‐limiting, and in detergents with large polyoxyethylene head groups the transfer reactions were fast and the enzymic reactions were rate‐limiting. With the second type of assay, the substrate concentration within the complex‐bound micelle was controlled by the equilibrium position of the substrate transfer reaction. Half‐maximal activity was obtained when the hydrophobic substrate concentration was half the total micelle concentration. This indicated that the equilibrium constant of the substrate transfer reaction was equal to one. With the third type of assay, detergent micelles containing the hydrophobic product/substrate couple functioned as substrate carriers between the two complexes. The reaction rates depended on the kinetics of the substrate transfer reaction. Direct complex‐complex interaction did not occur.
A Molecular Order in the Synthesis and Degradation of Glycogen in the Liver
Authors: DEVOS Pierre , HERS Henri‐Géry
Published: March 2005

When fasted rats or mice were refed, liver glycogen was synthesised at a linear rate of approximately 1%/h for a period of about 6 h. The administration of a radioactive precursor, usually [14C]galactose, allowed pulse labelling of the glycogen formed at various times after the initiation of refeeding. Soon after the administration of the label, the radioactive glucosyl units were preferentially located on the outer chains of glycogen. An almost equal distribution between the inner and outer chains was attained after a time which increased with increasing amounts of preexisting glycogen. Later on, the radioactive molecules were not further enlarged although the mass of glycogen increased threefold.

The degradation of the glycogen that had been labelled at various times after initiation of refeeding was induced either in vivo in anesthetized rats, or in vitro in isolated hepatocytes and in a cell‐free system made up of the enzyme‐glycogen complex as isolated by concanavalin A. Under all of these conditions, the radioactive units that were incorporated last were liberated first and vice versa. This ordered degradation could not be explained by the initial removal of outer chains. In the concanavalin‐A‐bound glycogen, the ordered degradation was much less apparent when the preparation had been submitted to high‐speed centrifugation, vigorous homogenization, ultrasonic treatment or exposure to detergents.
The DNA Components of the Chicken Genome
Authors: CORTADAS Jordi , OLOFSSON Birgitta , MEUNIER‐ROTIVAL Michèle , MACAYA Gabriel , BERNARDI Giorgio
Published: March 2005

The organization of the chicken genome was investigated by centrifuging chicken DNA (Mr= 57 × 106) in preparative Cs2SO4/Ag+ and Cs2SO4/BAMD density gradients [BAMD = 3,6‐bis(acetato‐mercurimethyl)dioxane]. An analysis by CsCl density gradient of the DNA fractions obtained from the preparative experiments revealed that 88% of the genome is made up of four DNA components, characterized by buoyant densities of 1.699, 1.7025, 1.7045 and 1.708 g/cm3, and representing 39%, 25%, 15%, and 9%, respectively, of the total DNA. The remaining 12% of the genome is formed by seven minor and/or satellite components. The distribution of the ovalbumin gene in a Cs2CO4/BAMD density gradient, as tested with a cloned cDNA probe, coincides with the distribution of the 1.7025‐g/cm3 component. This shows that the DNA regions flanking the ovalbumin gene are homogeneous in base composition over long distances and that the gene is located on a DNA segment belonging to the 1.7025‐g/cm3 component.
Isolation and Purification of a Neurodepressing Hormone from the Eyestalk of Procambarus bouvieri (Ortmann)
Authors: HUBERMAN Alberto , ARECHIGA Hugo , CIMET Avivah , DE LA ROSA Jorge , ARAMBURO Carlos
Published: March 2005

1. A neurodepressing hormone has been isolated and purified to homogeneity from aqueous extracts of 2000 eyestalks of the Mexican crayfish Procambarus bouvieri (Ortmann).

2. Purification was achieved by gel filtration on Sephadex G‐25 and G‐15, and preparative paper electrophoresis at four pH values (1.8, 3.6, 6.0 and 10.0).

3. The recovery of hormone activity was 85% and the specific activity was 4.0 × 104 times that of the starting material.

4. The hormone is a thermostable peptide of approximately 1200 molecular weight and composed of neutral amino acids.

5. No N‐terminal group could be found. From its electrophoretic behavior it is concluded that the C‐terminal group is also blocked.
Protein Synthesis in Imbibing Wheat Embryos
Authors: CUMING Andrew C. , LANE Byron G.
Published: March 2005

Polypeptides synthesized by imbibing wheat embryos have been compared with those made by cell‐free extracts programmed with bulk poly(A)‐rich RNA from dry wheat embryos. Newly synthesized polypeptides, labeled with [35S]methionine, were resolved by one‐dimensional and two‐dimensional electrophoresis and then records of the separations were prepared by fluorography. When programmed by bulk poly(A)‐rich RNA from dry wheat embryos, a nuclease‐treated rabbit reticulocyte lysate synthesizes an array of polypeptides which is broadly similar to that formed when a wheat germ extract is programmed with the same RNA. Polypeptides made in both homologous and heterologous cell‐free systems, under the direction of bulk poly(A)‐rich RNA from dry wheat embryos, are broadly similar to those formed during early (0–40 min) imbibition of dry wheat embryos. As imbibition progresses beyond 40 min, there are profound changes in the one‐dimensional and two‐dimensional electrophoretic distributions of newly made polypeptides present in the 23000 ×g supernatant fraction of cell‐free homogenates; characteristically, low‐molecular‐weight and basic polypeptides comprise a diminishing proportion of the total polypeptides as imbibition progresses beyond 40 min. Ribosomal proteins are conspicuous among the proteins formed during early imbibition and especially prominent among the products formed when homologous cell‐free polypeptide synthesis is programmed by bulk poly(A)‐rich RNA from dry wheat embryos.
Cell‐Free Synthesis of the Mitochondrial ADP/ATP Carrier Protein of Neurospora crassa
Authors: ZIMMERMAN Richard , PALUCH Ulrich , SPRINZL Matthias , NEUPERT Walter
Published: March 2005

ADP/ATP carrier protein was synthesized in heterologous cell‐free systems programmed with Neurospora poly(A)‐containing RNA and homologous cell‐free systems from Neurospora. The apparent molecular weight of the product obtained in vitro was the same as that of the authentic mitochondrial protein. The primary translation product obtained in reticulocyte lysates starts with formylmethionine when formylated initiator methionyl‐tRNA (fMet‐tRNAfMet) was present. The product synthesized in vitro was released from the ribosomes into the postribosomal supernatant.

The evidence presented indicates that the ADP/ATP carrier is synthesized as a polypeptide with the same molecular weight as the mature monomeric protein and does not carry an additional sequence.
The Primary Structure of the Ovine β‐Caseins
Authors: RICHARDSON Barry C. , MERCIER Jean‐Claude
Published: March 2005

Ovine whole casein contains 2 multiphosphorylated β‐casein components designated as β1 and β2‐caseins. The complete sequence of β1‐casein and the partial sequence of β2‐casein have been determined from the intact proteins and from the peptides isolated from cyanogen bromide and tryptic digests. The ovine β1 and β2‐caseins have the same polypeptide chain and appear to differ only in that they contain 6 and 5 phosphates respectively. The amino acid composition of ovine β‐casein can be written as: Asp4, Asn4, Thr10, ThrP1, Ser9, SerP5, Glu19, Gln21, Pro34, Gly5, Ala4, Val21, Met6, IIe9, Leu22, Tyr3, Phe9, Trp1, Lys12, His5, Arg3. Compared to bovine β‐casein A2, which is made up of 209 residues, ovine β1‐casein has a deletion of 2 residues (either Pro‐179–Tyr‐180 or Tyr‐180–Pro‐181) and 20 largely conservative amino acid substitutions. Although 20% of the substitutions involve proline residues, the proline contents of ovine β1 and bovine β2‐caseins are very similar, around 16%. The average hydrophobicity, calculated according to Bigelow, is 5.51 kJ/residue, which is similar to that calculated for bovine β‐casein A2.

The cluster of 4 phosphorylated serine residues and the highly charged nature of the amino terminal region observed for bovine β‐casein are conserved in the ovine β‐caseins. The substitution from IIe‐12 (bovine) to Thr‐12 (ovine) results in a new phosphorylation site, according to the phosphorylation code proposed for caseins. This site is only partially phosphorylated hence the occurrence of both β1 and β2‐caseins in ovine milk.
Apolipoprotein A‐IV: a Protein Occurring in Human Mesenteric Lymph Chylomicrons and Free in Plasma
Authors: UTERMANN Gerd , BEISIEGEL Ulrike
Published: March 2005

1. Human mesenteric lymph chylomicrons were isolated from chylous ascites fluid by ultracentrifugation and agarose/gel chromatography and their apoprotein composition was analysed by dodecylsulfate/polyacrylamide gel electrophoresis, analytical isoelectric focusing and immunochemically. Major components of mesenteric lymph chylomicrons were apoprotein A‐I, proteins of Mr < 15000 including the C‐group apoproteins and a protein of Mr 46000. Minor components were apoprotein E and a protein of Mr∼ 200000 (B‐like protein). This apoprotein composition was qualitatively identical with that of chylomicrons from intestinal lymph of the rat, but was distinctly different from plasma chylomicrons of humans with fasting chylomicronaemia.

2. The protein of Mr∼ 46000 has been isolated by preparative dodecylsulfate/polyacrylamide gel electrophoresis from human and rat lymph chylomicrons and was compared to a protein of identical Mr present in rat high‐density lipoproteins (apolipoprotein A‐IV) and in the ϱ < 1.006 g/ml serum lipoprotein fraction of individual humans with alimentary hypertriglyceridaemia. In both species the 46000‐Mr proteins isolated from lymph and serum were identical according to amino acid composition and isoelectric point in 6 M urea. The human proteins from both sources were also immunologically identical. The similarities in the molecular properties of the human apolipoprotein and rat apolipoprotein A‐IV indicate that these proteins are homologous.

3. Plasma levels of human apolipoprotein A‐IV determined by electroimmunodiffusion were 14.15 ± 3.66 mg/100 ml (n= 59), but > 90% of the protein was unassociated with the major lipoprotein fractions.

It is concluded, that apolipoprotein A‐IV is a main protein component of human lymph chylomicrons, that is removed from the particles in the plasma compartment.
Interaction between Prealbumin and Retinol‐Binding Protein Studied by Affinity Chromatography, Gel Filtration and Two‐Phase Partition
Authors: FEX Goran , ALBERTSSON Per‐Åke , HANSSON Barbro
Published: March 2005

The interaction between prealbumin and apo or holo retinol‐binding proteins has been studied by affinity chromatography, gel filtration and two‐phase partition. At physiological ionic strength apo and holo retinol‐binding protein form 1:1 molar complexes with prealbumin. Mean dissociation constants for the prealbumin complex with apo retinol‐binding protein and holo retinol‐binding protein with all‐trans retinol, retinoic acid, retinal and retinyl acetate were calculated from the partition data as 0.33 ± 0.11 × 10‐6 M and 0.075 ± 0.015 × 10‐6 M respectively (mean ± S.E.M.). The difference was statistically significant. Quantitative data on the amount of retinol, retinol‐binding protein and prealbumin in plasma and urine were in good agreement with the ratio of the dissociation constants for the complexes of apo and holo retinol‐binding proteins with prealbumin as determined in the partition experiment. The magnitude of the dissociation constants was compatible with previously published data on the turnover of retinol‐binding protein.
Studies on the Actin Activation of Myosin Subfragment‐1 Isoenzymes and the Role of Myosin Light Chains
Authors: WAGNER Paul D. , SLATER Cecilia S. , POPE Brian , WEEDS Alan G.
Published: March 2005

The actin‐activated ATPase activity of myosin subfragment 1 (S‐1) isoenzymes, separated on the basis of their alkali light chain (A1 and A2) content, has been analysed under a number of different conditions. Previous experiments on the effects of increasing actin concentrations on the ATPase have shown that the maximum turnover rate of ATP by S‐1 (A2) was about twice that of S‐1 (A1), and the Km for actin was also considerably larger for this isoenzyme. Here we show that these kinetic differences are maintained when regulated actin (actin + tropomyosin + troponin) is used in place of purified F‐actin. However, while increasing the ionic strength of the solution from 6 mM to 46 mM KCl has little effect on V for S‐1 (A2), the corresponding value for S‐1 (A1) increases to approximately equal that of S‐1 (A2), suggesting that the two isoenzymes are controlled by the same rate‐limiting process in the steady state under these conditions. The value of Km also increases with increasing ionic strength for the two isoenzymes, but that for S‐1 (A1) increases more rapidly and approaches that for S‐1 (A2). These experiments were carried out at at fixed S‐1 concentration, but the maximum rate of actin activation of the S‐1 ATPase can also be obtained from experiments where the actin concentration is fixed and the S‐1 concentration varied. Under these conditions in 6 mM KCl, the maximum rate of ATP hydrolysis expressed per mole of actin at infinite S‐1 concentration is the same for both S‐1 (A1) and S‐1 (A2), and this value numerically equals the maximum rate of hydrolysis of ATP by S‐1 (A2) expressed at infinite actin concentration. These results suggest that the rate‐limiting step in the ATPase cycle for S‐1 (A2) is the release of products from an actin · S‐1 · ADP · Pi complex, while for S‐1 (A1), where the value of V at infinite actin concentration is considerably lower, a different process is rate limiting, and this process may occur before actin reassociation with the S‐1 · ADP · pi complex, using the Lymn‐Taylor kinetic model. Thus increasing the ionic strength appears to change the rate‐limiting process in the steady‐state hydrolysis of ATP by actoS‐1 (A1). These experiments are discussed in terms of the role of the alkali light chains in the myosin ATPase and muscle contraction.