bluuebunny/crossref_metadata_2025_split huggingface dataset:
Spatial relationship between the nucleotide‐binding site, Lys‐61 and Cys‐374 in actin and a conformational change induced by myosin subfragment‐1 binding
Authors: MIKI Masao , dos REMEDIOS Cristobal G. , BARDEN Julian A. 
Published: March 2005

The spatial relationship between Lys‐61, the nucleotide binding site and Cys‐374 was studied. Lys‐61 was labelled with fluorescein‐5‐isothiocyanate as a resonance energy acceptor, the nucleotide‐binding site was labelled with the fluorescent ATP analogues ɛATP or formycin‐A 5′‐triphosphate (FTP) and Cys‐374 was labelled with 5‐{2‐[(iodoacetyl)amino]ethyl}aminonaphthalene‐1‐sulfonic acid (1,5‐IAEDANS) as a resonance energy donor. The distances between the nucleotide binding site and Lys‐61 or between Lys‐61 and Cys‐374 were calculated to be 3.5 ± 0.3 nm and 4.60 ± 0.03 nm, respectively. (The assumption has been made in calculating these distances that the energy donor and acceptor rotate rapidly relative to the fluorescence lifetime.) On the other hand, when doubly‐labelled actin with 1,5‐IAEDANS at Cys‐374 and FITC at Lys‐61 was polymerized in the presence of a twofold molar excess of phalloidin [Miki, M. (1987) Eur. J. Biochem. 164, 229–235], the fluorescence of 1,5‐IAEDANS bound to actin was quenched significantly. This could be attributed to inter‐monomer energy transfer. The inter‐monomer distance between FITC attached to Lys‐61 in a monomer and 1,5‐IAEDANS attached to Cys‐374 in its nearest‐neighbour monomer in an F‐actin filament was calculated to be 3.34 ± 0.06 nm, assuming that the likely change in the intra‐monomer distance does not change during polymerization by more than 0.4 nm. One possible spatial relationship between Lys‐61, Cys‐374 and the nucleotide binding site in an F‐actin filament is proposed.The effect of myosin subfragment‐1 (S1) binding on the energy transfer efficiency was studied. The fluorescence intensity of AEDANS‐FITC‐actin decreased by 30% upon interaction with S1. The fluorescence intensity of AEDANS‐FITC‐actin polymer in the presence of phalloidin increased by 21% upon interaction with S1. The addition of ATP led to the fluorescence intensity returning to the initial level. Assuming that the change of fluorescence intensity can be attributed to a conformational change in the actin molecule induced by S1 binding, the intra‐monomer distance was reduced by 0.4 nm and the inter‐monomer distance was increased by 0.2 nm.
Characterization of the Na+‐stimulated ATPase of Propionigenium modestum as an enzyme of the F1F0 type
Authors: LAUBINGER Werner , DIMROTH Peter 
Published: March 2005

The ATP‐hydrolyzing activity of Propionigenium modestum was extracted from the membranes with Triton X‐100 or by incubation with EDTA at low ionic strength. The ATPase in the Triton extract was highly sensitive to dicyclohexylcarbodiimide but not to vanadate. These properties are characteristic for enzymes of the F1F0 type. The ATPase was specifically activated by Na+ ions yielding a 15‐fold increase in catalytic activity at 5 mM Na+ concentration. The additional presence of 1% Triton X‐100 caused a further 1.5–fold activation. In the absence of Na+ Triton stimulated the ATPase about 13‐fold. The Triton‐stimulated ATPase was further activated about 1.5–2‐fold by Na+ addition.The ATPase extracted by the low‐ionic‐strength treatment was purified to homogeneity by fractionation with poly(ethylene glycol) and gel chromatography. The enzyme had the characteristic F1‐ATPase subunit structure with Mr values of 58000 (α), 56000 (β), 37600 (γ), 22700 (δ), and 14000 (ɛ). The F1‐ATPase was not stimulated by Na+ ions. The membrane‐bound ATPase was reconstituted from the purified F1 part and F1‐depleted membranes, thus further indicating an F1F0 structure for the ATPase of P. modestum. Upon reconstitution the ATPase recovered its stimulation by Na+ ions, suggesting that the binding site for Na+ is localized on the membrane‐bound F0 part of the enzyme complex.
Construction of a model for the aggregation and cross‐linking region (7S domain) of type IV collagen based upon an evaluation of the primary structure of the α1 and α2 chains in this region
Authors: SIEBOLD Bernhard , QIAN Rui‐qing , GLANVILLE Robert W. , HOFMANN Hans , DEUTZMANN Rainer , KÄHN Klaus 
Published: March 2005

The amino acid sequence of the 212‐residues‐long N‐terminal aggregation and cross‐linking region of the α2(IV) chain of human basement membrane collagen is presented. Comparing this with the primary structure of α1(IV)7S [Glanville et al. (1985) Eur. J. Biochem. 152, 213–219] revealed a high degree of similar subdivisions in three functional regions. These are the 21‐residue‐long N‐terminal non‐triple‐helical regions (NH1) containing cysteine and lysine residues which are putative cross‐linking sites, a 117‐residue‐long triple‐helical region (TH1) responsible for the aggregation of four molecules to form the 7S domain and which also possess cross‐linking sites, and finally a 10‐residues‐long non‐triple‐helical region (NH2) which introduces the first of many flexible areas into the triple helical body of the molecule [Hofmann et al. (1984) J. Mol. Biol. 172, 325–343].Computer calculations of interaction scores between parallel and antiparallelly aligned triple‐helical regions (TH1) of the 7S domain allowed the prediction of a detailed model for the structure of the 7S complex which agreed well with models based primarily on electron micrographs of rotary shadowed type IV collagen tetramers. The results indicated that the assembly of the 7S domain is directed by hydrophobic interactions and is self‐limiting to a tetramer. The most favourable chain configuration is α2‐α1‐α1.
Brain pyridoxal kinase dissociation of the dimeric structure and catalytic activity of the monomeric species
Authors: KWOK Francis , SCHOLZ Glen , CHURCHICH Jorge E. 
Published: March 2005

Reversible dissociation of the dimeric structure of brain pyridoxal kinase into subunits was attained by addition of guanidinium · HCl (2 M). The molecular mass of the subunits (40 kDa) was determined by HPLC chromatography.Separation of the processes of refolding and association of the monomeric species was achieved by attaching the protein subunits to a rigid matrix (Affi‐gel 15). The matrix‐bound monomer is catalytically competent. The reaction of the crosslinking reagent 4,4′‐dimaleimidestilbene 2,2′‐disulfonate (DMDS), a derivatized stilbene, with the dimeric structure of pyridoxal kinase resulted in the formation of an oligomeric species of 80 kDa detectable by SDS‐PAGE. The crosslinked subunits exhibit the same catalytic parameters as the native enzyme. The presence of two nucleotide‐binding sites per dimer was determined by fluorimetric titrations using pyridoxyl‐ATP, a strong competitive inhibitor with respect to ATP.The ATP analog binds with a Kd= 5 μM to each nucleotide site of the dimeric enzyme. The mode of binding pyridoxyl‐ATP to the kinase is discussed in reference to a model which assumes the presence of two binding domains per subunit.
Effect of bacteriophage M13 infection on phosphorylation of dnaK protein and other Escherichia coli proteins
Authors: RIEUL Corinne , CORTAY Jean‐Claude , BLEICHER Françoise , COZZONE Alain J. 
Published: March 2005

The effects of infection with the filamentous phage M13 on the phosphorylation of Escherichia coli proteins were studied. Phosphorylated proteins were labeled with [32P]orthophosphate and analyzed by the O'Farrell two‐dimensional gel technique and autoradiography.
Phage infection was shown to induce significant changes in the pattern of protein phosphorylation. At least eight different proteins were found to be phosphorylated to a larger extent while seven others were, by contrast, much less labeled than in uninfected bacteria.
Labeling experiments with [35S]methionine demonstrated that these quantitative changes in protein phosphorylation were not connected, in any case, with changes in the amount of protein synthesized. They rather seemed to result from a variation of the phosphorylating capacity of the relevant protein kinase(s).
The individual proteins, whose phosphorylation was affected by phage infection, were characterized by both their molecular mass and isoelectric point. One of them, whose phosphorylation was increased by a factor of 7, was identified as the dnaK protein which is necessary for both cellular and phage DNA replication.
The chemical analysis of the phosphorylated moiety of dnaK protein showed that it was modified exclusively at serine residues during normal growth of cells, and mostly at threonine residues after phage infection. These results were discussed in terms of stimulation of the protein activity by phosphorylation.
Processing of a plant vacuolar protein precursor in vitro
Authors: HATTORI Tsukaho , NAKAMURA Kenzo , ICHIHARA Shigeyuki 
Published: March 2005

A precursor for sporamin A, the storage protein of the tuberous roots of sweet potato deposited in the vacuole, is synthesized on membrane‐bound polysomes and has an extra peptide of 37 amino acids at the N‐terminus of the mature form, which can be divided into an N‐terminal putative signal peptide sequence (residues −37 to −17) and a segment enriched with charged amino acids (residues −16 to −1) [Hattori, T., et al. (1985) Plant Mol. Biol. 5, 313–320]. We examined the in vitro processing of the sporamin A precursor using a messenger RNA derived from a full‐length cDNA by the SP6 transcription system. When the in vitro translation in a wheat germ cell‐free system was carried out in the presence of dog pancreas microsomal membranes, the precursor polypeptide (Mr= 24000) was processed into an intermediate form still larger than the mature polypeptide (Mr= 20 000). The processed intermediate form was also produced by addition of microsomal membranes from sweet potato and potato in the translation reaction, although less efficiently compared to dog membranes. Moreover, Escherichia coli cells expressing sporamin precursor accumulated a polypeptide with the same electrophoretic mobility as the intermediate form produced in vitro.The processing by dog membranes is accompanied by translocation of the polypeptide across the membranes as assayed by resistance to externally added proteases. The N‐terminal amino acid sequencing analysis of [3H]leucine‐labelled intermediate form produced in vitro by dog membranes indicated that co‐translational processing of the sporamin precursor by endoplasmic reticulum membranes removes only the signal peptide segment from the extra peptide, and suggested that the charged segment following the signal peptide is removed post‐translationally during the transport of sporamin into vacuole. The significance of two‐step processing of plant vacuolar protein precursor is discussed in relation to the two‐step processing of precursors for yeast vacuolar proteins and animal lysosomal proteins.
Structure and expression of genes coding for xylan‐degrading enzymes of Bacillus pumilus
Authors: MORIYAMA Hideaki , FUKUSAKI Eiichiro , CABRERA CRESPO Joaquin , SHINMYO Atsuhiko , OKADA Hirosuke 
Published: March 2005

The complete nucleotide sequence of the β‐xylosidase gene (xynB) of Bacillus pumilus IPO and its flanking regions was established. A 1617‐bp open reading frame for β‐xylosidase, a homodimer enzyme, was observed. The amino acid sequence of the N‐terminal region and the molecular mass (62607 Da) of the β‐xylosidase subunit, deduced from the DNA sequence, agreed with the result obtained with the purified enzyme. The Shine‐Dalgarno sequence was found 8 bp upstream of the initiation codon, ATG.The xylanase gene (xynA) of the same strain was 4.6 kbp downstream of the 3′ end of xynB, and its DNA sequence was reported in our previous paper [Fukusaki, E., Panbangred, W., Shinmyo, A. & Okada, H. (1984) FEBS Lett. 171, 197–201]. The results of the Northérn hybridization suggested that the mRNA of xynA and xynB were produced separately. The 5′ and 3′ ends of the xynA and xynB gene were mapped with nuclease S1. The'‐10′ regions for promoter sequences of both genes were similar to the consensus sequence for B. subtilis RNA polymerases, the'‐35′ regions were different from all the known promoters for B. subtilis RNA polymerases.
Fructose 2,6‐bisphosphate in germinating oat seeds
Authors: LARONDELLE Yvan , CORBINEAU Françoise , DETHIER Monique , COME Daniel , HERS Henry‐Géry 
Published: March 2005

When dormant oat seeds were imbibed at the non‐permissive temperature of 30°C, the concentration of phosphoenolpyruvate and of glycerate 3‐phosphate, which are two inhibitors of phosphofructokinase 2, increased almost linearly during 30 h. By contrast, these metabolites increased only after a lag period of about 10 h in nondormant seeds imbibed at the same temperature. As a consequence of this, the concentration of the C3 derivatives remained always remarkably lower in non‐dormant than in dormant seeds. Accordingly, the concentration of fructose 2,6‐bisphosphate, which increased similarly in the two types of seeds during the first 8 h after the start of imbibition, then reached a plateau in dormant seeds but continued to increase for another 8 h in non‐dormant seeds, reaching a maximal value a few hours before the beginning of radicle protrusion. When the dormant seeds were imbibed at the permissive temperature of 10°C, the evolution of all metabolites was slowed down but behaved like that of non‐dormant seeds imbibed at 30°C. Experiments in which the dormant seeds were submitted to a jump from 10°C to 30°C and vice versa, always provoked reverse changes in the concentration of the C3 derivatives and of fructose 2,6‐bisphosphate, the latter being increased in all conditions that allowed germination. Dormant seeds were also allowed to germinate at 30°C by imbibition during 24 h in the presence of 3% ethanol. Again, this permissive treatment caused an arrest in the accumulation of C3 derivatives and an increase in fructose 2,6‐bisphosphate.Another, apparently unrelated, biochemical difference between dormant and non‐dormant oat seeds was their inorganic pyrophosphate content, which was approximately five‐fold higher in non‐dormant than in dormant seeds. This difference was observed before and persisted during imbibition as long as measurement could be made and was not affected by the temperature jumps or by ethanol. In contrast to the phosphoric esters under investigation, pyrophosphate was not preferentially located in the embryo.
Trout sperm motility
Authors: CHRISTEN Richard , GATTI Jean‐Luc , BILLARD Roland 
Published: March 2005

For freshwater fish the motile period of sperm is extremely brief, even after a dilution in isotonic media. This result is in contrast to most other animals (ranging from invertebrates to mammals), in which sperm are generally motile for at least several hours.We have analyzed the reasons for the brevity of this movement by studying the relationships between the metabolism of trout sperm and the activation of their motility upon dilution. Sperm motility was not initiated when the dilution medium contained an elevated concentration of potassium (20–40 mM), but dilution in an isotonic solution of sodium chloride triggered an immediate activation of motility, and sperm swam vigorously. Motility of sperm decreased rapidly and 15 s after dilution sperm were moving slowly in small circles. Sperm became abruptly immotile at 20–30 s and flagella straightened. When millimolar concentrations of Ca2+ were also present in the dilution medium, movement did not not stop abruptly, flagella kept beating and stopped only after 1–2 min.When sperm remained immotile they retained a high concentration of ATP. The activation of motility induced a rapid decrease of ATP. In the absence of calcium, and after the cessation of motility, ATP increased slowly back to its original concentration. In the presence of millimolar concentrations of calcium the concentration of ATP decreased to a very low level and remained low thereafter.The progressive decrease of the flagellar beat frequency, that had been observed during the period of trout sperm movement, might be related to the rapid exhaustion of intraflagellar ATP. Motility could be reinduced in sperm that had recovered high concentrations of ATP, demonstrating the functional integrity of the motile apparatus even after flagellar arrest.In conclusion we suggest that the maximum duration of trout sperm motility, at most 2 min (as a consequence of a depletion of ATP during the movement), is due to a low mitochondrial oxidative phosphorylation capacity.
The high‐resolution X‐ray crystal structure of the complex formed between subtilisin Carlsberg and eglin c, an elastase inhibitor from the leech Hirudo medicinalis
Authors: BODE Wolfram , PAPAMOKOS Evangelos , MUSIL Djordje 
Published: March 2005

Triclinic crystals of the complex formed by eglin with subtilisin Carlsberg were analyzed by X‐ray diffraction. The crystal and molecular structure of this complex was determined with data that extended to 0.12‐nm resolution by a combination of Patterson search methods and isomorphous replacement techniques. Its structure was refined to a crystallographic R value of 0.178 (1.0–0.12 nm) using an energy‐restraint least‐squares procedure. The complete subtilisin molecule could be traced without ambiguity in the refined electron density. The eglin component, from which an amino‐terminal segment is cleaved off, is only defined from Lys8I (i.e. the lysine residue 8 of the inhibitor) onwards. Per unit cell, 436 fixed solvent molecules and 2 calcium ions were located.In spite of 84 amino acid replacements and one deletion, subtilisin Carlsberg exhibits a very similar polypeptide fold to subtilisin BPN. The root‐mean‐square deviations of all α‐carbon atoms (excluding those at the deletion (1971) Biochem. Biophys. Res. Commun. 45, 337–344] and subtilisin Novo [Drenth, J., Hol, W. G. J., Jansonius, J. N. & Kockoek, R. (1972) Eur. J. Biochem. 25, 177–181] are 0.077 nm and 0.103 nm. Most of these deviations result from global shifts rather than changes of the local geometry. The single‐residue deletion at position 56 affects only the surrounding conformation. Two sites of high electron density and close distances to surrounding oxygen ligands have been found in the Carlsberg enzyme which are probably occupied by calcium ions.Eglin consists of a twisted four‐stranded β‐sheet flanked by an α‐helix and by an exposed proteinase binding loop on opposite sides. Around the reactive site, Leu45I‐Asp46I, this loop is mainly stabilized by electrostatic/hydrogen bond interactions with the side chains of two arginine residues which project from the hydrophobic core [Bode, W., Papamokos, E., Musil, D., Seemüller, W. & Fritz, H. (1986) EMBO J. 5, 813–818]. The reactivesite loop conformation resembles that found in other ‘small’ proteinase inhibitors. The scissile peptide bond is not cleaved but its carbonyl group is slightly distorted from planar geometry. Most of the intermolecular contacts are contributed by the nine residues of the reactive‐site loop Gly40I–Arg48I. The hydrophobic side chains of Pro42I (P4), Leu45I (P1) and Leu47I (P2′), which are exposed to solvent in the free inhibitor, are only partially buried in the complex and are still in van der Waals contact with several solvent molecules.
pH‐dependent interconvertible allosteric forms of murine melanoma tyrosinase
Authors: DEVI Chintamaneni Chaya , RAMAIAH Abburi , TRIPATHI Ram Kumar 
Published: March 2005

Murine melanoma melanosomal tyrosinase, solubilised at pH 6.8 and 1% Igepal, exhibits a lag in cresolase activity which increases with increasing concentration of tyrosine.
The enzyme, solubilised at pH 5.0 and assayed at pH 5.0, does not exhibit lag even at inhibitory concentrations of tyrosine while the same enzyme when assayed at pH 6.8 exhibits characteristic lag.
When the enzyme was solubilised from a melanosomal fraction with detergent/water without any buffer, significant linear activity for 2 h was seen at an inhibitory concentration of tyrosine, indicating for the first time the presence of a form of tyrosinase without lag and inhibition by excess tyrosine.
Exposure of the enzyme solubilised in buffer/detergent at pH 6.8 to rapid decrease in pH to 5.0 or 4.7 makes the enzyme remain irreversibly in the form without characteristic lag, even at an inhibitory concentration of tyrosine and at pH 6.8.
These results may be interpreted as follows. The enzyme at pH 6.8 exists in the E form with an allosteric site for tyrosine. Decrease of the pH of the enzyme solution from 6.8 to 5.0 or 4.7 by dialysis results in the reversible protonation of the enzyme, which no longer binds tyrosine at its allosteric site and consequently inhibition by excess tyrosine and lag were not observed at acidic pH. However, if the enzyme was rapidly brought to pH 5.0 from 6.8 it remains irreversibly in the protonated form even at pH 6.8.
Ascorbic acid acts as an effective reductant for the hydroxylation of tyrosine by tyrosinase, while 3,4‐dihydroxyphenylalanine is both an effective reductant and counteracts the inhibition by tyrosine at pH 6.8.
Lysosomal and non‐lysosomal peptidyl hydrolases of the bloodstream forms of Trypanosoma brucei brucei
Authors: LONSDALE‐ECCLES John D. , GRAB Dennis J. 
Published: March 2005

African trypanosomes have thiol‐dependent proteolytic activity that resembles some of the cathepsin‐like activity found in mammalian lysosomes [Lonsdale‐Eccles, J. D. & Mpimbaza, G. W. N. (1986) Eur. J. Biochem. 155, 469–473]. Here we show that this activity is found in lysosome‐like organelles which we have isolated (density = 1.082 g/cm3 in Percoll) from bloodstream forms of Trypanosoma brucei brucei. They are approximately 250 nm in diameter, are bounded by a single limiting membrane, and contain acid phosphatase. The predominant proteolytic and peptidolytic activity of these organelles has a pH optimum about 6.0, exhibits latency, and has the characteristics of mammalian cathepsin L (and possibly cathepsin H) with respect to its hydrolysis of small fluorogenic peptidyl substrates such as benzyloxycarbonyl‐phenylalanyl‐arginyl‐7‐amido‐4‐methylcoumarin. This substrate appears to be a good marker for trypanosomal lysosomes. The cathepsin‐L‐like activity is inhibited by the thiol‐protease inhibitors, E‐64, cystatin, leupeptin and mercurial compounds.The proteolytic activity of the lysosome‐like fraction is observed as a single band of activity with an approximate molecular mass of 27 kDa when measured after electrophoresis in the fibrinogen‐containing sodium dodecyl sulphate/polyacrylamide gels. The addition of mammalian serum to this purified fraction, or to whole trypansosome homogenates, results in the appearance of additional bands of activity, with a concomitant increase in the total observed proteolytic activity. The serum of some species of animal (e.g. goat and guinea pig) appear to lack the ability to generate this new and increased activity, while rat, rabbit, human and bovine sera exhibit varying capacities to generate the new activity, the cow being the most effective. The apparent molecular masses of the new bands of activity are different for each mammalian species, suggesting that the activator is a species‐specific molecule or class of molecules.We also show that Trypansosoma brucei contains soluble peptidolytic activity with an alkaline pH optimum. It is inhibited by the serine‐protease inhibitor diisopropylfluorophosphate, but not by inhibitors such as phenylmethylsulphonyl fluoride, α1‐antitrypsin, or aprotinin. Nor is it inhibited by the thiol‐protease‐specific inhibitors E‐64 or cystatin, although it is susceptible to inhibition by tosyllysylchloromethane, leupeptin, HgCl2 and p‐chloromercuribenzoate. This enzymic activity has a preference for arginyl residues in the primary binding site (the P1 position), as also does the activity from the lysosomes. However, the subsite specificity of the soluble enzymic activity is distinct from that of the lysosomes. The soluble enzyme(s) prefers small neutral molecules in the P2 and P3 subsites whereas the lysosomal enzyme(s) prefer hydrophobic molecules in these positions.