bluuebunny/crossref_metadata_2025_split huggingface dataset:
Effects of Formamide on the Polymerization and Depolymerization of Muscle Actin
Authors: ZECHEL Kasper 
Published: March 2005

Formamide was found to interfere with the polymerization of electrophoretically pure rabbit skeletal muscle G‐actin to F‐actin in vitro. It decreased the rate as well as the extent of polymerization. However, this influence was dependent on the way the polymerization reaction was intiated. If polymerization of G‐actin was induced by 2 mM MgCl2, formamide inhibited the rate and extent of the polymerization much less than if the polymerization was induced by either 2 mM CaCl2 or 0.1 M KCl. The critical protein concentration was increased when formamide was present. This effect was small in the presence of MgCl2 but an approximately tenfold increase in the critical value was observed for the CaCl2‐induced or KCl‐induced polymerization.Depolymerization of F‐actin by molar amounts of formamide was faster and proceeded further when the polymer had been formed in the presence of KCl or CaCl2 than when it had been formed in the presence of MgCl2. It is concluded that Mg2+ stabilizes the F‐actin structure readering it more resistant than either Ca2+ or K+ against the destabilizing action of formamide.
Formation of Magnesium‐Phosphoenzyme and Magnesium‐Calcium‐Phosphoenzyme in the Phosphorylation of Adenosine Triphosphatase by Orthophosphate in Sarcoplasmic Reticulum
Authors: SUKO Josef , PLANK Brigitte , PREIS Peter , KOLASSA Norbert , HELLMANN Gertrude , CONCA Walter 
Published: March 2005

The aim of the present study was to test simple reaction sequences which describe calcium‐independent plus calcium‐dependent phosphorylation of sarcoplasmic reticulum transport ATPase by orthophosphate including the function of magnesium in phosphoenzyme formation. The reaction schemes considered were based on the reaction sequence for calcium‐independent phosphorylation proposed previously; namely that the transport enzyme (E) forms a ternary complex (Mg. E. P), by random binding of free magnesium and free orthophosphate, which is in equilibrium with the magnesium‐phosphoenzyme (Mg.E‐P).Phosphorylation, performed at pH 7.0 20°C and a constant free orthophosphate concentration using sarco‐plasmic reticulum vesicles either unloaded or loaded passively with calcium in the presence of 5 mM or 40 mM CaCl2. resulted in a gradual decrease in the apparent magnesium half‐saturation constant and an increase in maximum phosphoprotein formation with increasing calciumloads. When phosphorylation of sarcoplasmicreticulum vesicles preloaded in the presence of 5 mM CaCl2 was performed at a constant free magnesium concentration, a decrease in the apparent otthophosphate half‐saturation constant and an increase in maximum phosphoprotein formation was observed as compared with vesicles from which calcium inside has been removed by ionophore X‐537A plus EGTA treatment; however, both parameters remained unchanged by increasing free magnesium from 20 mM to 30 mM. When phosphorylation of sarcoplasmic reticulum vesicles passively loaded with calcium in the presence of 40 mM CaCl2, at which the saturation of the low‐affinity calcium binding sites of the ATPase is presumably near maximum, was performed at increasing concentrations of free orthophosphate, there was a parallel shift of phosphoprotein formation as a function of free magnesium and vice versa, with no change in the maximum phosphoenzyme formation.Comparison of the experimental data with the pattern of phosphoprotein formation predicted from model equations for various theoretical possible reaction sequences suggests that phosphoenzyme formation from orthophosphate possesses the following features. Firstly, calcium present at the sarcoplasmic reticulum membrane binds to the free enzyme and in sequential order to E. Mg P1 or Mg E‐P or to both, but neither to E. Mg nor to E‐ P1 Secondly, calcium‐independent and calcium‐dependent phosphoproteins are magnesium‐phosphoenzymes. Calcium‐dependent phosphoenzyme is a magnesium‐calcium‐enzyme phosphate complex with 1 magnesium, 2 calciums and 1 orthophosphate (the last covalently) bound to the enzyme [Mg. E‐P. (Cal) 2], and not a ‘calcium‐phosphoprotein’ without bound magnesium.
Fragments of Ribosomal Protein S1 and Its Mutant Form m1‐S1
Authors: SUBRAMANIAN Alap R. , RIENHARDT Peter , KIMURA Makoto , SURYANARAYANA Tangirala 
Published: March 2005

Escherichia coli ribosomal protein S1 and its mutant, shorter, form‐S1 were cleaved at internal methionyl residues to yield, respectively, six and five gragments of M ranging from 1000 to 24000. Methods are described to isolate the fragments in pure form. Four of the fragments (designated F2a, F2b, F3 and F4) contain between 86 and 215 amino acids and are therefore as large as other robosomal proteins. Fragments F2a, derived of S1 [S. Giorginis and A.R. Subramanian (1980) J. Mol. biol. 141, 393‐408]. Here we show that the RNA binding domain of S1 is essentially contained of S1, whose activity is modified by ligand binding, was localized in F2b, a fragment with little RNA binding capacity.The charaterisitic RNAbinding domain and a weak riosome binding domain of S1 have previously been localized in the large trypsin‐resistent core S1‐F1 [T. Suryanarayana and A.R. Subramanian (1979) J. Mol. Biol. 127, 41–54]. Together these date indicate that two of the key functional domains S1 are located in two regions of the molecule separated by an open, exposed segment. The present study also revealed that the nonreactive ‐SH group of S1 becomes reactive m1‐S1 by the loss of remote C‐terminal region in the latter.
Cholesterol 7α‐Hydroxylase of Rat Liver
Authors: OZASA Shigeru , BOYD George S. 
Published: March 2005

Cytochrome P‐450 was prepared from the liver microsomes of cholestryamine‐fed rats by solubilisation with the non‐ionic detergent Nonidet P42 followed by chromatography on a DEAE‐cellulose column and on hydroxyapatite. NADPH‐cytochrome P‐450 reductase was preapred by a technique of affinity column chromatography using 2′,5′ ADP Sepharose.
The activity of cholesterol 7α‐hydroxylase was measured in a reconstituted system of microsomal mixed function oxidase containing cytochrome P‐450 and NADPH‐cytochrome P‐450 reductase from rat liver plus cholesterol and NADPH. Endogenous cholesterol was largely depleted from the enzyme preparations by the treatment of the microsomes with cold n‐butanol/acetone.
The reconstituted system of mixed‐function oxidase catalysed a highly effective and specific 7α‐hydroxylation of cholesterol. The reconstituted system showed higher activity of cholesterol 7α‐hydroxylase than was observed in native liver microsomes. The reconstituted system had an absolute requirement for cytochrome P‐450, NADPH‐cytochrome P‐450 reductase and NADPH.
The apparent Km for cholesterol in the reconstituted system was 15 μm and the V was 1.4 nmol 7α‐hydroxycholesterol formed min−1 (nmol cytochrome P‐450)−1
The reconstituted system also catalysed the 7α‐hydroxylation of taurodeoxycholic acid, the 7α‐hydroxylation of 26‐norcholesterol and to a limited degree the 12α‐hydroxylation reactions and the 12α‐hydroxylation reaction was significantly less than the ability of the system to 7α‐hydroxylate cholesterol.
Regulation of Acetoacetyl‐CoA in Isolated Perfused Rat Hearts
Authors: MENAHAN Lawrence A , HRON W. Theodore 
Published: March 2005

The role of ketone bodies and long‐chain fatty acids in regulating myocardial content of acetyl‐CoA was examined in isolated perfused hearts from fed rats. Addition of 10 mM acetoacetate to a perfusion medium containing 5.5mM glucose as exogenous sustrate resulted in a sevenfold increase of cardiac acetoacetyl‐CoA content. Inclusion of 0.75–0.94 mM palmitate in the perfusate did not augment the basal level of acetoacetyl‐CoA nor did it influence the already elevated content of the metabolic intermediate shown to exist in the presence of acetoacetate alone.In addition to the observed increase in myocardial acetoacetyl‐CoA, a decrease in free carnitine in hearts perfused with acetoacetate was accompanied by elevated ratios of both acetylcarnitine/carnitine and long‐chain faty acylcarnitine/carnitine. Addition of palmitate in the presence of acetoacetate resulted in a further diminution of myocardial free carnitine leading to a further elevation of the long chain fatty acylcarnitine/carnitine ratio when compared to hearts perfused with the ketone body alone. The changes in myocardial CoA and their intermediates in hearts perfused with the ketone body alone. or in combination with palmitate indicate that ketone bodies can divert fatty acids away from oxidation and toward esterification as triacylglycerols. Even though the interrelationships between ketones and myocardial fatty acid metaolism have not been fully characterized, a role for ketone bodies in promoting the increase in myocardial acetoacetyl‐CoA found in diabetic ketoacidosis has been clearly defined.
Limited Proteolysis of Porine‐Muscle Latic Dehydrogenase by Thermolysin during Reconstitution Yields Dimers
Authors: GIRG Rainer , RUDOLPH Rainer , JAENICKE Rainer 
Published: March 2005

Reassociation of lactic dehydrogenase from porcine skeletal muscle at 10°C was studied by limited proteolysis during reconstitution, applying thermolysin as proteolytic enzyme. Proteolysis was achieved by short incubation of the reassociating enzyme with the protease in a ratio lactic dehydrogenase: thermolysin = 10:1 (0.1 M sodium phosphate buffer pH 7.6 plus 1mM dithioerythritol).Thermolysin was found to satisfy all the oligatory requirements for proteolytic studies of reconstituting enzymes:(a) the protease is essentially inactive towards native lactic dehydrogenase;(b) it splits intermediates of reconstitution at a high rate; (c) it can be inactivated instantaneously y the addition of 10 mM EDTA.As demonstrated by gel chromatography, thermolysin digestion stailizes dimers of porcine muscle lactic dehydrogenase as intermediates of reconstitution. These ‘dimers’ consist of virtually intact polypeptide chains of molecular weight about 35000, apart from nicked subunits with molecular weights of 18000 and 12000, as determined y sodium dodecyl sulfate/polyacrylamide gel electrophoresis.Anallyzing the kinetics of reassociation by thermolysin digestion at various times during reconstitution, and susequent gel filtration, the dimer → tetramer transition is found to be rate‐limiting for both reassociation and reactivation. This result corroborates a previously proposed model for the reconstitution of lactic dehydrogenase, based on either cross‐linking experiments [R. Hermann et al. (1979) Nature (Lond.) 277, 243‐245], or the reassembly of dimeric intermediates of dissociation [R. Jaenike et al. Eur. J. Biochem. 114, 525–531].
A Role in vivo for Penicillin‐Binding Protein‐4 of Staphylococcus aureus
Authors: WYKE Anne W. , WARD J.Barrie , HAYES Michael V. , CURTIS Nigel A.C. 
Published: March 2005

The degree of cross‐linking of the peotidoglycan of Staphylococcus aureus H and mutants lacking penicillin‐binding proteins 1 and 4 was studies. No major changes were ovserved in organisms lacking protein 1 whereas oloss of protein 4 was accompanied by a marked reduction in the degree of cross‐linked and the absence of a membrane‐bound ‘model’ transpeptidase activity. A smimlar effect was achieved when cultures of the staphylococci were treated with the β‐lactam antibiotic cefoxitin. At low concentrations (0.05μg ml‐1) cefoxition shows highest affinity for protein 4 to which it appears to bind irreversibly. Treatment of the mutant lacking protein 4 with this concentration of the antibiotic did not affect the degree of cross‐linkage.The possibility that the decrease in cross‐linkage was a consequence of DD‐carboxypeptidase activity on peptidoglycan precursors was investigated. Although both S. aureus H and the mutants possessed such activity it was insensitive to benzylpenicillin and cefoxition and the role of this enzyme(s) in peptidoglycan biosynthesis remains unknown.We conclude that in vivo protein 4 acts as a transpeptidase involved in the secondary cross‐linking of peptidoglycan and this activity is necessary to achieve the high degree of cross‐linkage observed in the peptidoglycan of staphylococci.
The Protochlorophyllide Holochrome of Barley (Hordeum Vulgare L.)
Authors: APEL Klaus 
Published: March 2005

During the illumination of dark‐grown barley plants light induces a rapid decrease of a translatable mRNA which codes for a polypeptide of Mr44000. This component was identified as a precursor of the NADPH: protochlorophyllide oxidoreductase. The precursor has an Mrlarger than the authentic protein by approximately 8000.The light‐induced change in the level of translatable mRNA can be induced by a 15‐s red‐light pulse followed by 5 h of darkness. The red‐light effect is reversed by a subsequent far‐red‐light treatment. It is concluded that the light‐induced decline of translatable mRNA for the NADPH: protochlorophyllide oxidoreductase is controlled by phytochrome. The significance of this finding for present concepts of light‐dependent control of chloroplast development and chlorophyll synthesis is discussed.
Induction of Polymerization of Purified Tubulin by Sulfonate Buffers
Authors: WAXMAN Phyllis G. , DEL CAMPO Anthony A. , LOWE Michael C. , HAMEL Ernest 
Published: March 2005

Interactions of both purified tubulin and microtubule protein (tubulin plus associated proteins) with two commonly used sulfonate buffers were examined. 1,4‐Piperazineethanesulfonate(Pipes) and 4‐morpholineethane‐sulfonate (Mes) at high concentrations induce the polymerization of purified tubulin in reactions requiring only buffer, tubulin and GTP. While both reactions were temperature‐dependent, cold‐reversible and inhibited by GDP, colchicine or Ca2+, there were significant differences between them. Substantially lower tubulin and buffer concentrations were required for Pipes‐induced polymerization; and turbidity was much more intense in the Pipes‐induced than in the Mes‐induced reaction at the same protein concentration. Electron microscopy demonstrated that for the most part typical smooth‐walled microtubules were formed in Mes, while aberrant forms were the predominant structures formed in Pipes.When the polymerization of microtubule protein was examined as a function of buffer concentration, biphasic patterns were observed with both Pipes and Mes: polymerization occurred at both and high, but not intermediate, buffer concentrations. The turbidity observed at high concentrations of Pipes greatly exceeded that at low concentrations. With Mes, equivalent turbidity developed at both high and low buffer concentrations. Although associated proteins copolymerized with tubulin at low buffer concentrations, they were excluded from the polymerized material at high buffer concentrations.Pipes and Mes were compared to sodium phosphate, Tris/HCl and imidazole/HCl buffers at 0.1 M is several polymerization systems using both purified tubulin and microtubule protein. The sulfonate buffers were invariably associated with more vigorous reactions than the other buffers.
On the Rolw of the Carbohydrate Side Chains of Human Plasminogen in Its Interaction with α2‐Antiplasmin and Fibrin
Authors: LIJNEN H. Roger , VAN HOEF Berthe , COLLEN Désiré 
Published: March 2005

The Dissociation constants (Kd) for the interaction between different plasminogen derivatives and α2‐antiplasmin were determined as the concentrations of ligand that decrease the rate of the reaction between plamin and α2‐antiplasminby 50%. The Kd of Lys‐plasminogen (residues 77–790) type I (with a glucosamine‐based oligosaccharide chain attached to Asn‐288) and type II (with an unsubstituted Asn‐288) was found to be 1.9 μM and 0.2 μM respectively. The isolated plasminogen type I fragments containg the high‐affinity lysine‐binding site (representing residues 79–337 with glucosamine‐based carbohydrate on Asn‐288 or residues 79–353 with glucosamine‐based carbohydrate on Asn‐288 and galactosamine‐based carbohydrate on Thr‐345) both had a dissociation constant of 1.3 μM. The isolated plasminogen type II fragments containg the high‐affinity lysine‐binding site (residues 79–337 without carbohydrate or residues 79–353 with galactosamine‐based carbohydrate on Thr‐345) both had a dissociation constant of 0.24μM. These findings indicate that the difference in Kd is related to the presence of an oligosaccharide side chain on Asn‐288 but is unrelated to the presence of carbohydrate on Thr‐345. The dissociation constants of type I and type II Glu‐plasminogen (residues 1–790) were 4.9 μM and 2.9 μM. These higher values and smaller differences between type I and type II can probably be explained by the presence of the NH2‐terminal preactivation peptide (residues 1–76) which interacts with the high‐affinity lysine‐binding site in Glu‐plasminogen.Using 125I‐labelled plasminogen derivatives, direct binding studies to fibrin were performed. This revealed that the binding of the purified fragments obtained from type II plasminogen was 7‐times higher than that of fragments obtained from type I plasminogen. For both Glu‐plasminogen and Lys‐plasminogen no difference in extent of binding was observed for type I and type II.These finding indicate that the presence of a carbohydrate chain on Asn‐288 to both α2‐antiplasmin and fibrin. This decrease was still observed for the interaction between Lys‐plasminogen and α2‐antiplasmin, but not for the extent of binding to fibrin, suggesting that other interactions than via the high‐affinity lysine‐binding site might contribute in the binding of plasminogen to fibrin.
Origin and Characterization of Short DNA Chains in Escherichia coli
Authors: SIEGMANN Deborah W. , WERNER Rudolf 
Published: March 2005

One of the major problems in the study of DNA replication involves the presence of numerous short, non‐nascent DNA chains in the cell. Such chains often contaminate nascent DNA preparations, making accurate analysis of the replicative DNA difficult. This complication can be avoided by the use of hydroxyapatite chromatography. Previous results from this laboratory had shown that short nascent DNA preparation which containsonly nascent DNA and is essentially free of non‐nascent chains. 5′‐Terminal labeling patterns and molecular weight distributions show that only nascent DNA chains form the DNA‐protein complex and can therefore be separated from non‐nasent chains if the isolation is performed under non‐denaturing conditions. The protein involved in the complex appears to be rather specific for DNA chains in the replication fork since short chains resulting from the excision of dUMP immediately after replication also form the DNA‐protein complex.Using this isolation technique, 5′‐terminal nucleotides of nascent DNA was pulse–labeled in several ways, purified by hydroxyapatite chromatography, and labeled at the 5′ end with [32] phosphate. Two‐dimensional thin‐layer chromatography of the 32P‐labeled nucleotides, obtained after enzymatic hydrolysis, revealed that all four deoxyribonucleotides are present at the 5′ end in approximately equal amounts. In most cases, this 5′‐terminal nucleotide distribution does not vary significantly, even when the conditions of pulse‐labeling are changed. Only if the cells are allowed to run out of thymine is a variation in the relative amounts of 5′‐end nucleotides. In this case, the amount of dTMP at the 5′ end decreases signigicantly, refleting the low thymine levels and indicating that the cell can compensate for this deficiency by utilizing other available nucleotides. From these results, it appears that DNA chain initiation is essentially random with respect to the nucleotide used in the initiation process.
The Effect of Inhibition of (ADP‐ribose)n Biosynthesis on DNA Repair Assayed by the Nucleoid Technique
Authors: DURKACZ Barbara W. , IRWIN Judy , SHALL Sidney 
Published: March 2005

DNA damage and repair was assayed by the loss and restoration of DNA supercoiling in nucleoids. This technique was used to assess the effects of inhibition of (ADP‐ribose)n biosynthesis by 3‐aminobenzamide on the capacity to repair DNA of mouse leukaemia L1210 cells following damage by γ‐irradiation and by the mono‐ functional alkylating agent, dimethyl sulphate. 3‐Aminobenzamide partially inhibits restoration of supercoiling following γ‐irradiation and dimethyl sulphate treatment, but inhibits neither the enzymic incision events leading to breaks in the DNA nor the repair synthesis. This inhibition of repair can be observed at very low doses of damaging agents. These observations confirm and extend the evidence that (ADP‐ribose)n biosynthesis is required for efficient cellular recovery from DNA damage. In particular, the nucleoid technique permits the demonstration that 3‐aminobenzamide inhibits DNA repair after γ‐radiation; it is not possible to draw this unequivocal conclusion with the data from alkaline sucrose gradients because this technique is too insensitive.1‐β‐D‐Arabinofuranosylcytosine, which inhibits seiniconservative DNA replication, also retards repair, and this effect can be reversed by the addition of deoxycytidine. The inhibitors of DNA excision repair, arabinosyl‐ cytosine, hydroxyurea or 3‐aminobenzoamide increase the steady‐state number of DNA breaks. Thus, they can be used to enhance even further the sensitivity of the nucleoid assay of repair.
The Cell‐Free Protein Synthesis System from the ‘Slime’ Mutant of Neurospora crassa
Authors: SZCZIŃ‐SKORUPA Elzȩbieta , FILIPOWICZ Witold , PASZEWSKI Andrzej 
Published: March 2005

A simple procedure for preparation of a cell‐free protein synthesis system (23000 × g supernatant) from protoplast‐like ‘slime’ mutant of Neurospora crassa is described. A variety of messenger RNAs of viral and cellular origin could be efficiently and faithfully translated in this system into proteins with M, as large as 180000.The importance of the 7‐methylguanosine cap for mRNA translation in the Neurospora system was studied in detail using the cap analogs and chemically decapped messengers. As in the case of reticulocyte lysate or wheat germ extract, the extent of m7G requirement for mRNA translation in a fungal extract strongly depended on translation conditions such as incubation temperature or concentration of potassium ions, mRNA and 23000 × g supernatant protein.