bluuebunny/crossref_metadata_2025_split huggingface dataset:
A New Concept of the Function of Elongation Factor 1 in Peptide Chain Elongation
Authors: GRASMUK Hans , NOLAN Robert D. , DREWS Jürgen 
Published: March 2005

An entirely new model for the mechanism of elongation factor 1 (EF‐1) function is presented. Experiments, in which mixtures of [3H]EF‐1, ribosomes from Krebs II ascites cells and various additional co‐factors were analyzed by chromatography on Sepharose 6B, show that EF‐1 binds to the ribosome early in the translation process and remains bound on the ribosome during translation. Optimal EF‐1 binding occurs on polynucleotide‐programmed ribosomes carrying a tRNA in their P‐site.On the other hand it was clearly shown that EF‐2 attached at each translocation event and was then released before a new Phe‐tRNA could be bound.
Acetyl‐Coenzyme‐A Carboxylase of Candida lipolytica
Authors: MISHINA Masayoshi , KAMIRYO Tatsuyuki , TANAKA Atsuo , FUKUI Saburo , NUMA Shosaku 
Published: March 2005

The level of acetyl‐coenzyme‐A carboxylase activity in Candida lipolytica undergoes large variations depending upon the carbon source on which the yeast is grown. Cells grown on n‐alkanes or fatty acids exhibit a lower activity level than do cells grown on glucose. Among the n‐alkanes and fatty acids tested, n‐heptadecane, n‐octadecane, oleic acid and linoleic acid reduce the enzyme activity to the lowest levels, which are 16–18% of the activity level in glucose‐grown cells.Immunochemical titrations and Ouchterlony double‐diffusion analysis with specific antibody as well as kinetic studies have indicated that the observed decrease in the level of acetyl‐CoA carboxylase activity is due to a reduction in the cellular content of the enzyme. Furthermore, isotopic leucine incorporation studies with the use of the immunoprecipitation technique have demonstrated that the relative rate of synthesis of the enzyme in oleic‐acid‐grown cells is diminished to 12% of that in glucose‐grown cells. Evidence has also been obtained to support the view that the enzyme in this yeast is not degraded at a rate high enough to contribute to the marked decrease in the cellular content of the enzyme. Thus, it is concluded that the reduction in acetyl‐CoA carboxylase content in fatty‐acid‐grown cells is due to diminished synthesis of the enzyme.
Glucose Effect in tgl Mutant of Escherichia coli K12 Defective in Methyl‐α‐D‐glucoside Transport
Authors: ERLAGAEVA Raisa S. , BOLSHAKOVA Tatyana N. , SHULGINA Marina V. , BOURD Genrich I. , GERSHANOVITCH Vladimir N. 
Published: March 2005

The dependence of the rate of accumulation of methyl‐α‐d‐glucoside on its extracellular concentration was studied in the tgl mutant of Escherichia coli K12, isolated earlier. It has been shown that the kinetics of methyl‐α‐d‐glucoside transport differ sharply from those in wild‐type bacteria.
The β‐galactosidase synthesis in tgl strain is much less sensitive both to permanent and transient glucose catabolite repression. The level of cyclic AMP in mutant cells under the conditions of glucose catabolite repression is several times higher than in the parent strain.
The tgl mutation does not affect the manifestation of catabolite inhibition and inducer exclusion with glucose.
The data obtained are discussed in the light of a hypothesis concerning the existence of two sites, binding and phosphorylating, in the molecule of the glucose (and methyl‐α‐d‐glucoside) specific enzyme II of the phosphoenolpyruvate‐dependent phosphotransferase system. The tgl mutation alters the first site, and the second one is damaged by the gpt mutation.
It is suggested that the products of the tgl and gpt genes are necessary for the manifestation of the phenomena of glucose permanent and transient repression. The effects of catabolite inhibition and inducer exclusion are realized irrespective of the existence or absence of the tgl product.
Anabolic Ornithine Carbamoyltransferase of Pseudomonas
Authors: STALON Victor , LEGRAIN Christiane , WIAME Jean‐Marie 
Published: March 2005

The anabolic ornithine carbamoyltransferase of Pseudomonas appears to be extremely specialized. Unlike the other carbamoyltransferases studied, this enzyme catalyzes the phosphorolytic cleavage of citrulline with a very poor efficiency.The main goal of this paper is to understand what, in the catalytic process, causes this directed functional specialization. On the basis of kinetic data and thermodynamic properties of the reaction, it appears that the reaction mechanism is the same as for ornithine carbamoyltransferases from other sources, that is, of the sequential ordered type, where carbamoylphosphate is the first substrate to be bound and phosphate the last product to be released. In addition to this, and here lies the difference with other ornithine carbamoyltransferases, the anabolic transferase of Pseudomonas forms a binary dead‐end complex with citrulline, leading to inefficient binding of phosphate and citrul‐line to the enzyme. Therefore the phosphorolytic cleavage of citrulline is equally inefficient. It should be mentioned that the affinity of the enzyme for citrulline at its catalytic site is low as compared to other transferases.
Effects of Nucleoside Phosphates and Salts on the Activity of a Heart Phosphoprotein Phosphatase and Its Catalytic Subunit
Authors: LI Heng‐Chun , HSIAO Kwang‐Jen 
Published: March 2005

A phosphoprotein phosphatase (phosphatase B, Mr= 161000) isolated from canine heart extract can be dissociated by ethanol into a catalytic subunit (phosphatase S, Mr= 34800). The dissociation of phosphatase B into phosphatase S is accompanied by an increase in the enzymic activity toward phosphohistone, phosphocasein and phosphorylase a and by a pronounced change in catalytic properties. Phosphatases B and S exhibit different substrate saturation kinetics. pH profile and respond differently to nucleoside phosphates and salts.The increased activity toward phosphohistone accompanying the dissociation of phosphatase B reflects loss of substrate inhibition. Preincubation of either phosphatase B or S with ATP results in an inactivation of its activity and phosphatase S is much more susceptible to ATP inactivation than phosphatase B. ATP and various ionic compounds, however, can greatly stimulate the activity of phosphatase B by interacting with phosphohistone, indicating the modification at the substrate level represents an important regulatory mechanism. By contrast, the activity of phosphatase S is slightly stimulated by relatively low concentrations of salts, and is inhibited by nucleoside phosphates. The present data indicates that the activity of phosphatase B is more sensitive to the modification of the substrate, phosphohistone, by nucleoside phosphates and salts than is phosphatase S. These observations suggest that the regulatory properties of phosphatase B are lost following its dissociation.
Determination of the Chain Stoichiometries from the Number of Reactive Sulfhydryl Groups in the Pyruvate Dehydrogenase Complexes of Azotobacter vinelandii and Escherichia coli
Authors: DE ABREU Ronney A. , DE KOK Arie , DE GRAAF‐HESS Adriana C. , VEEGER Cees 
Published: March 2005

The pyruvate dehydrogenase complex from Azotobacter vinelandii incubated with 0.05–0.7 mM [2‐14C]pyruvate, magnesium chloride and thiamine pyrophosphate under anaerobic conditions at 0°C, incorporates four [14C]acetyl groups per mole FAD which are bound to the high‐molecular‐weight lipoyl transacetylase. With 10 mM pyruvate, the low‐molecular‐weight lipoyl transacetylase is also labelled; to this enzyme 3–4 [14C]acetyl groups are bound per mole FAD. This enzyme is not labelled when pyruvate is used in concentrations lower than 0.7 mM. The complex of A. vinelandii pre‐labelled with non‐radioactive N‐ethylmaleimide did not incorporate label onto low‐molecular‐weight lipoyl transacetylase; a maximum of four acetyl groups are bound to the high‐molecular weight lipoyl transacetylase.Maximum incorporation of four [14C]acetyl groups per mole FAD is found, within five seconds, on the lipoyl transacetylase component when the Escherichia coli complex is labelled under anaerobic conditions, also in the presence of 10 mM pyruvate.In both complexes all incorporated acetyl groups are bound to sulfhydryl groups since they can be completely removed with hydroxylamine, by performic acid oxidation and by reaction with coenzyme A and arsenite. Under aerobic conditions a slow deacetylation is observed, which is blocked in the presence of N‐ethylmaleimide.The multi‐enzyme complex from A. vinelandii pre‐labelled with non‐radioactive N‐ethylmaleimide and then labelled with N‐ethyl [2,3‐14C]maleimide in the presence of pyruvate, magnesium chloride and thiamine pyrophosphate incorporates under anaerobic conditions at 0°C, even at 10 mM pyruvate, a maximum of four N‐ethyl[14C]maleimide groups per mole of FAD. The label is almost exclusively bound to the high‐molecular‐weight lipoyl transacetylase of the A. vinelandii complex.The E. coli complex binds only 2–3 N‐ethyl[14C]maleimide groups per mole of FAD under these conditions. Direct labelling and correction for labelling without pyruvate yields 4–5 N‐ethyl‐[14C]maleimide per mole FAD.The low‐molecular‐weight lipoyl transacetylase can be removed by chromatography of the complex from A. vinelandii on a blue‐dextran–Sepharose 4B column [De Abreu et al. (1977) FEBS Lett. 82, 89–92]. The complex eluted from the column, consisting of the other three enzyme components, binds a maximum of four [14C]acetyl groups per mole of FAD, even with 10 mM pyruvate.It is concluded that the lipoyl/FAD ratio is four in the complexes as isolated by us from both sources. The interpretation of this ratio with respect to the stoichiometries of the different enzyme components will be discussed.
Metabolic Stability of the Two Forms of Initiation Factor IF‐3 in Escherichia coli during the Growth Cycle
Authors: MINKS Michael A. , SURYANARAYANA Tangirala , SUBRAMANIAN Alap R. 
Published: March 2005

Possible alteration in the ratio of the long and short forms of initiation factor IF‐3 (FEBS Lett. 79, 264–275, 1977) during the growth cycle of Escherichia coli was examined. The ratio was found to remain unchanged between the exponential and stationary growth phases. Contrary to an earlier report (Eur. J. Biochem. 29, 319–325, 1972), the total amount of IF‐3 relative to the ribosome content in stationary phase cells was essentially the same as in midlogarithmic phase cells. The activity of IF‐3, assayed after its separation from other initiation factors by chromatography, was also the same in extracts from midlogarithmic and stationary phase cells. The data show that in Escherichia coli the ratio of IF‐3/ribosome is maintained constant.The ribosomes themselves have been shown to retain virtually full activity in vitro during this transition indicating that growth‐cycle‐dependent biochemical modifications of the ribosome do not affect its protein synthetic capacity per se.
The Specificity of Ribonucleoside Triphosphate Reductase
Authors: LUDWIG Walter , FOLLMANN Hartmut 
Published: March 2005

Ribonucleotide reductase of Lactobacillus leichmanii, a monomeric enzyme requiring 5′‐deoxyadenosylcobalamin (coenzyme B‐12), catalyzes reduction of all common ribonucleoside 5′‐triphosphates but its substrate and effector specificity is not precisely known. Structure variations in the base, as in ATP analogs with N6‐dimethyladenine, unsubstituted purine, or even benzimidazole, are tolerated but lead to greatly reduced reaction rates. These substrates respond to the same effector nucleotide (dGTP) as ATP. br8ATP is completely inactive as a consequence of its abnormal molecular (syn) conformation. XTP is not reduced at pH 7.8 but reduction is observed at lower pH values, indicating that the species with an extra negative charge in the base (pK= 5.7) is inactive but uncharged xanthine results in substrate activity. dTTP stimulates reduction of XTP (and also of ITP).The uncommon deoxyribonucleoside triphosphates dUTP, dITP, and dXTP act as allosteric effectors of ribonucleotide reduction like the common ones but they usually produce intermediate, or less specific types of stimulation, or weak inhibition. The same is true for br8dATP the activity of which is explained by a certain conformational flexibility of the aglycone. A complete range of deoxyribonucleotide effects from strong activation to inhibition of enzyme activity is thus observed, indicating that the apparently specific activation pattern among natural ribonucleotide/deoxyribonucleotide couples is in fact part of the protein's general capacity to interact with nucleotides. Inspection of the substituent structures has led us to postulate certain amino acid side chains, among them a carboxylate, and hydrogen bonding interactions at the substrate and effector sites. A two‐sided induced‐fit mechanism is suggested to explain the unusually dynamic allosteric behaviour of Lactobacillus reductase.The activity of modified ribonucleotides and deoxyribonucleotides to promote enzyme‐catalyzed hydrogen exchange between 5′‐[5′‐3H]deoxyadenosylcobalamin and water has been tested. ATP analogs and XTP are active, but in contrast to all other deoxyribonucleotides known so far the effectors dXTP and br8dATP are not. In agreement with our earlier studies of sugar‐modified nucleotides, these results support the existence of two different deoxyribonucleotide sites for stimulation of substrate binding and for modulation of coenzyme action.Because enzymic reduction of modified ribonucleotides by Lactobacillus reductase (and also by other reductases) is very slow these findings provide a rationale as to why such compounds (e.g. biosynthetic intermediates or phosphorylated antibiotics) will not interfere with DNA biosynthesis under normal conditions.
The Submicrosomal Localization of 3‐Hydroxy‐3‐Methylglutaryl‐Coenzyme‐A Reductase, Cholesterol 7α‐Hydroxylase and Cholesterol in Rat Liver
Authors: MITROPOULOS Konstantinos A. , VENKATESAN Soundararajan , BALASUBRAMANIAM Santhirasegaram , PETERS Timothy J. 
Published: March 2005

In order to determine the submicrosomal distribution of cholesterol 7α‐hydroxylase, 3‐hydroxy‐3‐methylglutaryl‐coenzyme‐A reductase and of free cholesterol, the microsomal fraction and the digitonin‐treated microsomal preparation of rat liver were subjected to analytical isopycnic centrifugation on sucrose density gradients.Cholesterol 7α‐hydroxylase, its substrate and hydroxymethylglutaryl‐CoA reductase are present in similar membranes originating from the endoplasmic reticulum with low density of ribosomes. Smooth membranes originating from Golgi apparatus and plasma membranes do not contain appreciable amounts of cholesterol 7α‐hydroxylase and of hydroxymethylglutaryl‐CoA reductase, whilst they contain a considerable portion of the free cholesterol present in microsomal preparations.The addition of trace amounts of [14C]cholesterol to untreated and digitonin‐treated microsomal preparations resulted in heterogeneous labelling of cholesterol as seen from the variation of the specific radioactivity of free cholesterol in the microsomal subfractions. Both with untreated and with digitonin‐treated microsomes the specific radioactivity of cholesterol was minimal in the fractions of maximal contamination with plasma membranes and in those of maximal contamination with smooth membranes from Golgi apparatus. Therefore, the [14C]cholesterol added to the microsomal preparations did not label the plasma membrane and the Golgi membrane cholesterol pools as heavily as it labelled other pools of microsomal cholesterol. The ‘substrate pool’ for cholesterol 7α‐hydroxylase was heavily labelled by [14C]cholesterol added to untreated or digitonin‐treated microsomes and, since the specific radioactivity of 7α‐hydroxycholesterol obtained with various microsomal subfractions from the same preparation was very similar, the ‘substrate pool’ was uniformly labelled.
Homoserine Dehydrogenase of Rhodospirillum rubrum
Authors: EPSTEIN Carol Cherkis , DATTA Prasanta 
Published: March 2005

A detailed physicochemical characterization of purified homoserine dehydrogenase of Rhodospirillum rubrum is presented. The enzyme has a molecular weight of 110 000 and consists of two subunits of identical molecular weight of 55 000. Depending on the ionic strength and protein concentration it is possible for the native enzyme to dimerize to produce an enzymatically active species of molecular weight 220 000.Titrations of the native and detergent‐treated enzyme with a variety of sulfhydryl reagents show 2 mol free–SH groups per 110 000 g, one of which is buried in the protein interior. l‐Threonine and/or high concentrations of salt can expose the buried–SH group, and this–SH group is essential for the catalytic activity of the enzyme. Two independent lines of evidence show that extensive polymerization of the enzyme caused by l‐threonine and/or high concentrations of salt does not involve the formation of intermolecular disulfide bonds.
Fluorescence Energy‐Transfer Studies on the Pyruvate Dehydrogenase Complex Isolated from Azotobacter vinelandii
Authors: SCOUTEN William H. , DE GRAAF‐HESS Adriana C. , DE KOK Arie , GRANDE Hans J. , VISSER Antonie J. W. G. , VEEGER Cees 
Published: March 2005

Fluorescence energy transfer has been employed to estimate the minimum distance between each of the active sites of the 4 component enzymes of the pyruvate dehydrogenase multienzyme complex from Azotobacter vinelandii. No energy transfer was seen between thiochrome diphosphate, bound to the pyruvate decarboxylase active site, and the FAD of the lipoamide dehydrogenase active site. Likewise, several fluorescent sulfhydryl labels, which were specifically bound to the lipoyl moiety of lipoyl transacetylase, showed no energy transfer to either the flavin or thiochrome diphosphate. These observations suggest that all the active centers of the complex are quite far apart (≧40 nm), at least during some stages of catalysis. These results do not preclude the possibility that the distances change during catalysis.Several of the fluorescent probes used possessed multiple fluorescent lifetimes, as shown by determination of lifetime averages by both phase and modulation measurements on a phase fluorimeter. These lifetimes are shown to result from multiple factors, not necessarily related to multiple protein conformations.
Reactions of Antibodies against Ferredoxin, Ferredoxin‐NADP+ Reductase and Plastocyanin with Spinach Chloroplasts
Authors: BÖHME Herbert 
Published: March 2005

Purified antisera against ferredoxin, ferredoxin‐NADP+ reductase and plastocyanin agglutinated osmotically shocked and washed spinach chloroplasts, prepared according to standard procedures. The monomeric antibody (immunoglobulin G fraction) of the reductase antiserum agglutinated chloroplasts specifically and directly, indicating that protruding structures (for example, the coupling factor) do not act as steric hindrances as has been suggested.With ferredoxin antiserum, the presence of a pentameric antibody (immunoglobulin M fraction) was obligatory to observe a positive agglutination reaction. Immunoglobulin G only inhibited ferredoxin‐dependent reactions, like NADP+ ‐photoreduction, but did not cause agglutination. Ferredoxin seems to be located in depressions of the membrane, possibly caused by a partial release of this protein in shocked chloroplasts.Similar results were obtained with purified immunoglobulins from a plastocyanin antiserum. Again the immunoglobulin G fraction inhibited electron transport reactions catalyzed by plastocyanin, whereas immunoglobulin M showed a positive agglutination, but had no influence on electron transport. It is concluded that ferredoxin, ferredoxin‐NADP+ reductase and plastocyanin are peripheral electron transport components, located at the outer thylakoid membrane.
Studies on the Purification and Properties of a 6.8‐S DNA Polymerase Activity Found in Calf‐Thymus DNA Polymerase‐α Fraction
Authors: HESSLEWOOD Ian P. , HOLMES Andrew M. , WAKELING William F. , JOHNSTON Irving R. 
Published: March 2005

The heterogeneity of calf thymus DNA polymerase‐α has been further investigated. In particular, an enzyme (enzyme D) which exhibits higher activity on poly(dA) · (dT)10 (A:T = 20:1) compared with that on activated DNA, has been further purified and its properties compared with two other activities of the DNA polymerase‐α fraction (enzymes A1 and C) which do not show a preference for poly(dA) · (dT)10 over activated DNA.As with A1 and C, enzyme D was shown to have many of the characteristic properties of DNA polymerase‐α in that it is an acidic protein as judged by its binding to DEAE‐cellulose, has a molecular weight of about 140000, does not use a poly(A) · (dT)10 template‐initiator complex and is inhibited by N‐ethylmaleimide. It exhibits anomalous gel filtration behaviour on Sepharose 6B and it binds relatively weakly to DNA‐cellulose compared with DNA polymerase‐β. The extreme sensitivity of enzyme D to inhibition by N‐ethylmaleimide distinguishes it from A1 and C, as does its elution position from a DEAE‐cellulose column. On the other hand enzymes C and D are readily inactivated by heating at 45 °C unlike enzyme A1.The possible interrelationships of the multiple activities of calf thymus DNA polymerase‐α are discussed.
Purification and Properties of Phosphorprotein Phosphatases with Different Substrate and Divalent Cation Specificities from Canine Heart
Authors: LI Heng‐Chun , HSIAO Kwang‐Jen , CHAN Wanda W. S. 
Published: March 2005

Three phosphoprotein phosphatases have been isolated from canine heart by a procedure involving treatment of the enzyme preparation with 80% ethanol at room temperature. They are a divalent‐cation‐independent, nonspecific enzyme (Mr= 35000) referred to as phosphatase S, an Mg2+ ‐activated enzyme (Mr= 49300) with activity predominately toward phosphocasein and an Mn2+ ‐activated enzyme (Mr= 61000) with activity predominately toward phosphohistone. Phosphatase S has been purified over 10000‐fold and the two divalent‐cation‐dependent enzymes have been purified over 1000‐fold from the crude extract. The purified preparation of phosphatase S showed a single broad protein stain band which coincided with the enzymic activities when examined with polyacrylamide gel electrophoresis. Sodium dodecyl sulfate gel electrophoresis of the enzyme preparation also showed a single protein band corresponding to a molecular weight of 35000. Phosphatase S exhibited a broad specificity and was about equally active toward phosphorylase a, phosphorylated histone, casein and cyclic‐AMP‐dependent protein kinase. Phosphatase S showed an optimum between pH 6.5–8 and was inhibited by ATP, other nucleoside phosphates, Pi, PPi and NaF. The Km values of phosphatase S for phosphorylase a, phosphorylated histone, and casein are 4, 27, and 16 μM, respectively. Evidence indicates that phosphatase S is a catalytic subunit of several nonspecific, divalent‐cation‐independent enzymes reported previously [Biochim. Biophys. Acta, 483, 337–347 (1977)].The present findings indicate that at least three basic catalytic entities of phosphorprotein phosphatase can be isolated from canine heart extract. These phosphatases which differ from one another in substrate specificity and divalent cation dependence may play different roles in the regulations of protein dephosphorylation in the cell.
Porcine Follitropin
Authors: CLOSSET Jean , MAGHUIN‐ROGISTER Guy , HENNEN Georges , STROSBERG A. Donny 
Published: March 2005

The amino acid séquence of the porcine β subunit has been established by studies of peptides isolated after tryptic, thermolytic and staphylococcal protease treatments of the reduced and carboxymethylated chain. The primary structure of the amino‐terminal region of the molecule has been solved by automatic sequencing of the reduced and tritium‐labeled carboxymethylated subunit.The amino acid sequence of porcine follitropin β subunit differs from that of its human counterpart by several amino acid replacements, deletion or addition of one or several residues. The porcine chain appears shorter at both its amino and carboxy‐terminal ends. The chemical evolution of follitropin is briefly considered and compared to these of thyrotropin and lutropin.