bluuebunny/crossref_metadata_2025_split huggingface dataset:
The Properties of Cytochrome f and P700 in Chloroplasts Suspended in Fluid Media at Sub‐zero Temperatures
Authors: COX Raymond P. 
Published: March 2005

The properties of P700 and cytochrome f have been studied at sub‐zero temperatures in chloroplasts suspended in a medium containing 50% (v/v) ethylene glycol. The dark reduction of these components after a period of illumination provided information about the rate‐limiting step of photosynthetic electron transport under these conditions.
The oxidation of P700 on illumination in the presence of methyl viologen and its subsequent dark reduction can be observed at −35°C. This cycle of reactions could be repeated many times. The rate of reduction was increased by NH4Cl and reduction was inhibited by 3(3,4‐dichloro‐phenyl)‐1,1‐dimethylurea.
The oxidation and reduction of cytochrome f could also be observed under similar conditions. The activation energies for the reduction of cytochrome f and P700 are similar (about 75 kJ mol−1) and the reduction of cytochrome f is also inhibited by dichlorophenyldimethylurea and stimulated by NH4Cl.
The reduction of both cytochrome f and P700 seemed to follow first‐order kinetics, but the t1/2 for the reduction of the cytochrome was at least three times that for the reduction of P700 at the same temperature. It was concluded that the results were only compatible with a model in which the main pathway of electrons from plastoquinone to P700 involved cytochrome f if the equilibrium constant between the cytochrome and P700 was very much less than that expected from their redox potentials.
Enzymic Synthesis of an Aromatic Ring from Acetate Units
Authors: KREUZALER Fritz , HAHLBROCK Klaus 
Published: March 2005

Flavanone synthase was isolated and purified about 300‐fold from fermenter‐grown, light‐induced cell suspension cultures of Petroselinum hortense. The enzyme catalyzed the formation of the flavanone naringenin from p‐coumaroyl‐CoA and malonyl‐CoA. Trapping experiments with an enzyme preparation, which was free of chalcone isomerase activity, revealed that in fact the flavanone and not the isomeric chalcone was the immediate product of the synthase reaction. Thus the enzyme is not a chalcone synthase as previously assumed.No cofactors were required for flavanone synthase activity. The enzyme was strongly inhibited by the two reaction products naringenin and CoASH, by the antibiotic cerulenin, by acetyl‐CoA, and by several compounds reacting with sulfhydryl groups. Optimal enzyme activity was found at pH 8.0, at 30°C, and at an ionic strength of 0.1–0.3 M potassium phosphate. EDTA, Mg2+, Ca2+, or Fe2+ at concentrations of about 0.7 μM did not affect the enzyme activity. Apparent molecular weights of approx. 120000, 50000, and 70000, respectively, were determined for flavanone synthase and two metabolically related enzymes, chalcone isomerase and malonyl‐CoA : flavonoid glycoside malonyl transferase.The partially purified flavanone synthase efficiently catalyzed the formation of malonyl pantetheine from malonyl‐CoA and pantetheine. This malonyl transferase activity, and a general similarity with the condensation steps involved in the mechanisms of fatty acid and 6‐methyl‐salicylic acid synthesis from “acetate units”, are the basis for a hypothetical scheme which is proposed for the sequence of reactions catalyzed by the multifunctional flavanone synthase.
Transport of Messenger RNA into Different Classes of Membrane‐Associated Polyribosomes in Ehrlich‐Ascites‐Tumor Cells
Authors: VAN VENROOIJ Walther J. , GIELKENS Arno L. J. , JANSSEN Albert P. M. , BLOEMENDAL Hans 
Published: March 2005

The membrane‐bound polyribosomes in Ehrlich ascites tumor cells can be separated into a loosely bound and a tightly bound fraction by means of a high salt treatment. Both membrane fractions as well as the free polyribosomes in the supernatant synthesize about the same set of proteins, suggesting a close relationship between these polyribosome fractions in the Ehrlich cell.Relatively high concentrations of cycloheximide do not prevent newly synthesized poly(A)‐containing mRNA from entering the tightly bound polyribosome fraction. Nor had treatment of the cells with puromycin in the presence of cycloheximide, which released about 70% of the nascent chains, any significant effect on the entrance of newly synthesized mRNA into tightly bound polyribosomes.These results suggest that in Ehrlich ascites tumor cells nascent polypeptide chains are not involved in the binding of polyribosomes to membranes.
Ribonucleotide Reductase from Escherichia coli: Demonstration of a Highly Active Form of the Enzyme
Authors: ERIKSSON Staffan 
Published: March 2005

Ribonucleotide reductase from Escherichia coli consists of two nonidentical subunits, proteins B1 and B2. The activity of the enzyme in crude extracts prepared from mechanically disrupted bacteria is very low. Enzyme activity is stimulated 5 to 10‐fold by addition of an excess of either subunit. Concentrated extracts from cells lysed gently on Cellophane discs (Schaller et al.) contained 10 to 20‐fold higher activity than extracts from mechanically disrupted cells. This activity was not further stimulated by either B1 or B2. The system is suitable for complementation tests for the analysis of temperature‐sensitive mutants affecting the ribonucleotide reductase system.Concentrated high‐spced supernatants from E. coli treated with lysozyme (Wickner et al.) also contained a high ribonucleotide reductase activity, which was stimulated slightly or not at all by addition of B1 and B2. This active form of the enzyme was unstable and could not be purified.The results suggest that the intracellular form of the enzyme consists of a tight complex of proteins B1 and B2, possibly stabilized by other intracellular structures.
Human Luteinizing Hormone
Authors: CLOSSET Jean , VANDALEM Jean‐Louis , HENNEN Georges , LEQUIN Rudy M. 
Published: March 2005

A new procedure is described for the isolation of the α and β chains of the hormone. In this method, the native hormone is incubated in acidic urea and the chains are then separated by ion‐exchange chromatography.The amino‐terminal residue of the α subunit is valine. The carboxy‐terminal end of the α subunit is of variable length. No amino‐terminal residue was detected for the β chain; glycine was found at its carboxy‐terminal end by the selective titration method.The amino acid and carbohydrate compositions of the hormone and both subunits are presented. The β chain contains sialic acid and is devoid of galactosamine in contrast to the β subunits of other species.Contamination of our human lutenizing hormone preparation by other pituitary glycoprotein hormones such as thyroid‐stimulating hormone and follicle‐stimulating hormone amounted to 0.5 and 0.25 percent by weight respectively.Cross‐contamination of the initial α and β subunit preparations was measured by specific radioimmunoassays and amounted to 4.1 and 2 percent by weight respectively.Further extensive purification of these subunit preparations was then performed by means of affinity chromatography using immunosorbants.The final preparations exhibited a residual cross‐contamination amounting to 0.2 and 0.02 percent by weight for the α and β subunits respectively.
Binding Studies of NADPH to NADP‐Specific l‐Glutamate Dehydrogenase from Saccharomyces cerevisiae
Authors: VENARD Renée , JALLON Jean Marc , FOURCADE Anne , IWATSUBO Motohiro 
Published: March 2005

Optical characteristics of enzyme‐reduced coenzyme complexes of yeast NADP‐specific glutamate dehydrogenase have been investigated in the presence and absence of product (l‐glutamate) and in the presence or absence of phosphate.The phosphate effect, pointed out in a previous work, is found again: inorganic phosphate (Pi) destabilizes the binary complex (E · NADPH), the dissociation constant of which is equal to 14 μM, a value much higher than that determined in Tris‐HCl buffer: Kd= 0.9 μM. Concerning the role of phosphate some assumptions are drawn up with respect to a similar behaviour of Pi toward yeast glutamate dehydrogenase and ADP toward the beef liver enzyme.In the same way, l‐glutamate induces a stabilization of the binary complex; this latter effect is unchanged in the presence of phosphate, yet it is less marked than in the case of beef liver glutamate dehydrogenase.Protein fluorescence, nucleotide fluorescence and circular dichroism measurements allowed the determination of three identical and independent NADPH binding sites per hexameric active unit. In analogy with beef liver enzyme, it seems that yeast glutamate dehydrogenase is a good model to study anticooperativity in ligand binding.
Immunologically Active and Structurally Similar Fragments of Protein A from Staphylococcus aureus
Authors: HJELM Hans , SJÖDAHL Jörgen , SJÖQUIST John 
Published: March 2005

To study the active site(s) in protein A, partial tryptic digestions of the protein and of intact Staphylococcus aureus were performed. Fragments which bind to the Fc‐part of human IgG were isolated by affinity chromatography on IgG‐Sepharose 4B and purified by ion‐exchange chromatography on phosphocellulose.From a partial tryptic digest of pure protein A at 30°C, pH 8.2 for 30 min we have isolated and characterized six active fragments with molecular weights ranging from 6000 to 8000. Two active fragments, obtained in high yields by digestion at pH 7.2 of intact protein‐A‐containing bacteria, were shown to be similar to two of the six characterized fragments from the digest of pure protein A.All fragments appeared to have similar amino acid sequences, judged by peptide mapping, specific staining and amino acid analysis; some are very possibly overlapping peptides. Each fragment probably contains only one active site region since all are monovalent in the Fc‐reaction when studied with a hemagglutination technique. The maximal molar yield of active fragments obtained from the digestion of pure protein A accounts for about 210% of the amount of protein A used. Thus protein A, suggested to consist of repeating units, should exhibit at least three similar if not identical active regions.
Similarities and Differences of Five Peroxidases from Turnip and Horseradish
Authors: WELINDER Karen Gjesing , MAZZA Gilbert 
Published: March 2005

Four isoperoxidases of turnip root and isoperoxidase C of horseradish root were digested with trypsin. and their peptide maps, prepared by high‐voltage paper electrophoresis, were compared. All five tryptic digests were completely soluble at pH 8. The maps were developed with a variety of general and specific reagents: ninhydrin, histidine, tyrosine, tryptophan and arginine reagents. Cystine peptides and cysteic acid derivatives have also been characterized. All detected half‐cystine residues seemed engaged in disulfide bridges. For each individual peroxidase the number of specifically staining peptides agreed very well with the amino acid composition.The two most acidic peroxidases of turnip, P1 and P2, only differ significantly in one peptide. The P2 gene is tentatively proposed to have developed from the P1 gene by a single base mutation, changing an asparagine residue to a lysine residue. A less acidic turnip peroxidase, P3, is distinct, although related to peroxidases P1 and P2. Horseradish isoperoxidase C also belongs to this group which appears to be closely related in the amino acid sequences around four disulfide bridges. Peroxidase P7 differs from this group, at least around two of its disulfide bridges, and therefore, may differ from the other four in parts of its three dimensional structure.Sequences of particular importance to peroxidase function must be present in all peroxidases. From the peptide mapping studies we only find two highly homologous sequences present in all five examined peroxidases. Both contain histidine. This finding corroborates previous suggestions of two histidine sequences near the peroxidase heme prosthetic group.The rules applied in relating peptides of different proteins are outlined, and the sources of errors in mapping of glycoproteins of high carbohydrate content (about 20%) are discussed in detail.
The Interaction of Fusidic Acid with Peptidyl‐Transfer‐Ribonucleic‐Acid · Ribosome Complexes
Authors: SAN MILLAN María José , VAZQUEZ David , MODOLELI Juan 
Published: March 2005

The inhibitory action of fusidic acid on peptide‐chain elongation was studied with systems in vitro directed by either polyuridylic acid or endogenous messenger (Escherichia coli polysomes washed with 1 M NH4Cl) or R 17 RNA, and supplemented with either crude or purified elongation factors. In all cases strong inhibition of synthesis required high concentrations of the antibiotic (approx. 1 mM), while a similar inhibition of the EF‐G‐plus‐ribosome‐dependent GTP hydrolysis required between 10 and 100 times less antibiotic. Since most of the GTP hydrolysis observed was presumably due to free ribosomes (without aminoacyl‐tRNA or peptidyl‐tRNA), fusidic acid seemed to interact far more easily with these ribosomes than with ribosomes engaged in peptide‐chain elongation.The role of the GDP · EF‐G · ribosome · fusidic acid complex in the inhibition of polypeptide synthesis was assessed by measuring formation of this complex on polysomes engaged in peptide‐chain elongation. Using purified elongation factors the complex formed on only 25–35% of ribosomes, as measured either by retention of [3H]GDP or by hydrolysis of [3H, γ‐32P]GTP. In contrast, with crude factors (S100 extract) it formed on more than 70% of ribosomes. The results are compatible with the postulated role of the complex in polypeptide synthesis inhibition (blockade of the ribosomal acceptor site and subsequent inhibition of aminoacyl‐tRNA binding) and indicate that formation of the complex takes place by overriding the control that prevents interaction of EF‐G when the donor site is occupied by peptidyl‐tRNA.In the polyuridylic‐acid‐directed system for synthesis of oligophenylalanine the antibiotic inhibits every round of peptide elongation, including dipeptide formation, to roughly the same extent.
Analysis of the Pancreatic‐Ribonuclease‐Digestion Products of Alfalfa‐Mosaic‐Virus Ribonucleic Acid
Authors: PINCK Lothaire , FAUQUET Claude 
Published: March 2005

Alfalfa mosaic virus (AMV) genome consists of three pieces of RNA (24‐S, 20‐S and 17‐S RNA). For infectivity these three RNAs and the coat protein are required. In the absence of coat protein, infectivity is obtained by adding the 12‐S RNA also normally present in the virus. This 12‐S RNA represents the message for coat protein. Thus a redundancy of the gene for coat protein exists between 12‐S RNA and one of the other RNAs. Sequence analysis of the oligonucleotides resulting from pancreatic ribonuclease digestion of the AMV RNAs indicates that the nucleotide sequence of 12‐S RNA occurs in 17‐S RNA.Analysis of the pancreatic ribonuclease digestion products of the two larger alfalfa mosaic virus RNAs (20‐S and 24‐S RNA) shows some oligonucleotides containing seven, eight and nine nucle‐otides with the same structure present in both RNAs. The possibility of a limited nucleotide sequence homology between these two RNAs is discussed. The comparison of the RNase digestion products of 20‐S and 24‐S RNA with those of 12‐S or 17‐S RNA revealed no homologous oligonucleotides, thus the origin of 12‐S RNA appears to be 17‐S RNA.
β‐Glucosidases from Cicer arietinum L.
Authors: HÖSEL Wolfgang , BARZ Wolfgang 
Published: March 2005

β‐Glucosidases specific for isoflavone 7‐O‐glucosides have been isolated from garbanzo plants, Cicer arietinum L.These aryl‐β‐glucohydrolases occur in the different organs of the plant as multiple molecular forms. The major isoenzymes of the roots, the leaves and the hypocotyl were purified to electrophoretic homogeneity. When subjected to isoelectric focussing in polyacrylamide gels the electrophoretically homogeneous glucohydrolases were found to consist of one or two major and several minor enzymically active molecular species.In roots the β‐glucohydrolase isoenzymes constitute a considerable portion of the extractable protein, so that purification to an electrophoretically homogeneous form is easily attainable.All β‐glucosidases analyzed possess molecular weights in the range of 125000 (ultracentrifugation) to 135000 (Sephadex G‐200) and contain two subunits of molecular weight near 68000. The pH optimum for enzymic activity is 7–7.5 with a second optimum of 4.5–5. The isoelectric points of the various species range between pH 5.9 and 7.1. Staining for glycoprotein was positive.Kinetic analysis demonstrated a pronounced specificity of the enzymes for aromatic substrates with glucose as the sugar moiety. α‐Glucosides as well as disaccharides were not hydrolyzed at all. Isoflavone 7‐O‐glucosides are the most favoured substrates with a Km of 2 × 10−5 M, while the Km with aromatic glucosides (i.e. salicin, 4‐nitrophenyl glucoside) are 100 times larger. In addition the β‐glucosidases show a pronounced specificity for glucose in the 7‐position of the flavonoid nucleus. Using isoflavone aglycones as substrates glucose transferase activity was also demonstrable.The β‐glucohydrolase activity is strongly inhibited by Hg2+. This inhibition is partially reversible and preferentially influences the Km values of the enzymes compared to to V. Ag+ glucono‐1,5‐lactone, ethyleneglycol monomethyl ether and glycerol are only weakly inhibitory, while glucose, p‐chloromercuribenzoate and Cu2+ are without effect.
Poly(A) Synthesis in T2L Phage‐Infected Escherichia coli
Authors: WUNDERLI Werner , STAEHELIN Matthys , WEHRLI Walter , HÜTTER Ralf 
Published: March 2005

In crude extracts of T2L phage‐infected Escherichia coli cells an enzyme activity was found that produced poly(A) from ATP as substrate.Purification of the extract led to the isolation of two enzymes, a polynucleotide phosphorylase and an ATPase. The polynucleotide phosphorylase possessed the same properties as the well‐known enzyme from uninfected cells and its molecular weight was about 265 000. The ATPase was purified to over 90% purity; its molecular weight was estimated to be about 165000 with three subunits of 55000. The characterization of this enzyme showed that it was different from any ATPase known so far. Mg2+ cannot be replaced by Ca2+, as it can from the membrane‐bound ATPases. The only product yielded by the enzyme was ADP; it was very specific for ATP, other ribonucleotide triphosphates being practically unaffected. The rate of ATP splitting was found to be very high, the turnover number being 2.51 × 104 min‐1 at 37°C. Even at 0°C the enzyme was still active. The optimal assay conditions for ATPase turned out to be very similar to those of polynucleotide phosphorylase. Thus the combination of the two enzymes very efficiently produced poly(A) from ATP. In this combination the polynucleotide phosphorylase was the rate‐limiting enzyme, since its turnover number was about 40 times lower than that of the ATPase. The evaluation of a variety of properties of the poly(A)‐synthesizing constituent found in the crude extracts led us to conclude that this activity arises from the combined action of ATPase and polynucleotide phosphorylase, and is not due to a poly(A) polymerase.
Kinetic Investigation of the α‐Chymotrypsin‐Catalyzed Hydrolysis of Peptide‐Ester Substrates
Authors: BIZZOZERO Spartaco A. , BAUMANN Werner K. , DUTLER Hans 
Published: March 2005

A number of peptide‐ester substrates of the general structure Ac‐Lxn‐.Lx2‐Lx1‐OMe have been synthesized and their α‐chymotrypsin‐catalyzed hydrolysis studied. The kinetic analysis involved varying the concentration of substrate and methanol product, and measuring rates along the entire progression curve.For the dipeptide esters Ac‐Lx2‐Lx1‐OMe and the amino‐acid derivatives Ac‐Lx1‐OMe the following constants could be determined: the dissociation constant of the enzyme‐substrate complex, KEA, both rate constants of the acylation step, k23 and k32, and the forward rate constant of the de‐acylation step, k31. For the tripeptide ester Ac‐Ala‐Ala‐Tyr‐OMe it appears that the rate constant for the dissociation of the enzyme‐substrate complex, k21, is smaller than the rate constant for acylation, k23. Thus, for this substrate only the association and dissociation rate constants k12 and k21 could be determined and the values of k23, k32 and k31 only indirectly estimated. The influence of structural changes in the peptide moiety of the substrates on reactivity has been established by comparing the rate constants of appropriate pairs of substrates. It was found that the substrate reactivity, as measured by k23/KEA, increases with the number and strength of the secondary interactions in a manner consistent with the binding scheme which has been proposed on the basis of crystallographic studies. The effect of a particular interaction on k23 and on KEA is dependent on the nature of the other interactions. However, the effect on k23/KEA appears to be independent of the presence of the other interactions and therefore characteristic of that particular interaction. The results for these substrates are compared with those found previously for a series of peptide substrates of the structure Ac‐Lxn‐.‐Lx2‐.‐Lx1‐Gly‐NH2 which have the same acyl moiety as the peptide esters studied in this work.
Non‐histone Chromosomal Proteins
Authors: BLÜTHMANN Horst , MROZEK Stanislav , GIERER Alfred 
Published: March 2005

We describe a method for fractionation of chromatin components by selective dissociation with salt in buffers containing 5 M urea in combination with chromatography on hydroxyapatite at 4 °C. This results in two histone and four non‐histone fractions which are recovered in high yield and with minimal proteolytic contamination.Template capacity measurements of the isolated chromatins and pre‐saturation competition hybridization experiments support the idea that a group of non‐histone proteins activate the transcription of specific DNA sequences which were not transcribed from purified DNA to the same extent.In reconstitution experiments a non‐histone protein fraction, NH4, prepared from lymphocyte chromatin by hydroxyapatite chromatography is shown to cause transcription in vitro of lymphocyte‐specific RNA sequences. A subfraction with a molecular weight of 30000 comprising 40% of the NH4 fraction protein is characteristic for this tissue and not found in liver chromatin.
The Influence of Insulin on Glucose Permeability and Metabolism of Human Granulocytes
Authors: LEROUX Jean‐Paul , MARCHAND Jean‐Claude , HONG TUAN HA Régine , CARTIER Pierre 
Published: March 2005

Viable human polymorphonuclear leukocytes isolated from peripheral blood were incubated for 1 h at 37 °C with variable concentrations of insulin in a saline medium buffered at pH 7.4. The hormone increased glucose consumption by about 40% without influencing the permeability of the membranes to glucose, whose uptake followed a passive diffusion process. The measurement of intermediates localized activation of glycolysis by insulin, down to 0.36 nM, at the phosphofructokinase step. However, the spectrophotometric measurement showed no activation of phosphofructokinase after preincubation with insulin of either intact granulocytes or crude or ultracentrifuged homogenates. The level of cyclic AMP, which is known to activate phosphofructokinase, was not modified by insulin; cyclic GMP did not activate the enzyme in the granulocyte extracts: neither of the two nucleotides can therefore be considered as a direct messenger of the action of insulin on phosphofructokinase.An important fraction of the extra glucose consumed under the influence of insulin was recovered as neither glycogen nor lactate, nor was it oxidized in the Krebs cycle. It might be assumed to have been converted into glycerolipids. However, insulin produced no detectable accumulation of tri‐glycerides and activated neither the pentose phosphate pathway nor oxidative decarboxylation of pyruvate. The fate of the extra glucose consumed under the influence of insulin therefore remains questionable.