bluuebunny/crossref_metadata_2025 huggingface dataset:
31P‐NMR studies of the freeze‐tolerant larvae of the gall fly, Eurosta solidaginis
Authors: STOREY Kenneth B. , MICELI Michael , BUTLER Keith W. , SMITH Ian C. P. , DESLAURIERS Roxanne
Published: March 2005

31P NMR was applied to an examination of the freeze‐tolerant larvae of the gall fly, Eurosta solidaginis. Resonances from sugar phosphates, inorganic phosphate, adenylates and arginine phosphate were identified. Two peaks of Pi were identified corresponding to intracellular and extracellular Pi. Anoxia produced an expected decrease in peak intensities of ATP and arginine phosphate while the peak of intracellular Pi was enhanced and shifted to indicate intracellular acidification during anoxia. Spectra of whole larvae were monitored over a temperature range from –30° to + 25°C. No abrupt alterations in the spectra were seen at the point of extracellular freezing which occurs at about –8°C but temperature had dramatic effects upon the peak intensities of ATP and arginine phosphate. A reversible increase/decrease in peak intensities, relative to Pi, was observed as temperature was raised/lowered. At 15° and –20°C, the β peak of ATP was 64% and 2% of the peak intensity of Pi while that of arginine phosphate was 78% and 11%, respectively. This temperature effect was not an artifact of instrumentation (as model solutions containing Pi, ATP and arginine phosphate did not show this effect) or a result of changes in the total amounts of these compounds in the cell with temperature. Rather it is apparent that these molecules become restricted in their rotational movement as temperature is lowered perhaps via binding to subcellular components. Changes in the amounts of freely soluble ATP and arginine phosphate with temperature could have important implications for metabolism and its control. Analysis of the effect of temperature on the chemical shift of Pi was also used to determine pH in the intracellular and extracellular compartments. Temperature change had no effect on extracellular (hemolymph) pH which remained constant at 6.1–6.3. Intracellular pH varied with temperature, however, from pH 6.8 at 15°C to pH 7.3 at –12°C with a change, ΔpH/Δθ, of –0.0185°C consistent with alphastat regulation.
Regulation of synthesis of citrate synthase in regreening Euglena gracilis
Authors: CANNONS Andrew C. , MERRETT Michael J.
Published: March 2005

Exposure of dark‐grown Euglena to white or red light, but not blue light, produced a twofold increase in the specific activity of citrate synthase. A 400‐fold purification of mitochondrial citrate synthase (subunit Mr= 44000) was achieved from cells of Euglena gracilis by affinity chromatography on ATP‐activated agarose. Antisera, raised against the homogeneously pure enzyme, were used to demonstrate that the increase in citrate synthase activity on exposure of dark‐grown cells to light resulted from an increase in citrate synthase protein. Anti‐(citrate synthase was used to detect precursor citrate synthase resulting from the translation of total polyadenylated RNA from Euglena in a cell‐free rabbit reticulocyte lysate system. Citrate synthase mRNA was found to be present in cells at all stages of regreening. However, extraction and translation of polyadenylated RNA from free polysomes isolated from dark‐grown and regreening cells demonstrated that appreciable translation of citrate synthase mRNA was only occurring in regreening cells.
Molecular cloning of the gene encoding CDPdiacylglycerol–inositol 3‐phosphatidyl transferase in Saccharomyces cerevisiae
Authors: NIKAWA Jùn‐ichi , YAMASHITA Satoshi
Published: March 2005

The structure gene encoding CDPdiacylglycerol – inositol 3‐phosphatidyltransferase was isolated by complementation in a Saccharomyces cerevisiae mutant after transformation with a comprehensive gene library containing Sau3AI partial restriction fragments of wild‐type yeast DNA inserted into the YEp13 shuttle vector.

Introduction of the cloned plasmid into the mutant restored both growth of cells and CDPdiacylglycerol – inositol 3‐phosphatidyltransferase activity.

A subcloning study indicated that the complementing gene was contained within a 1700‐base‐pair fragment of DNA. Expression of the cloned sequence was independent of its orientation in the vector.

CDPdiacylglycerol – inositol 3‐phosphatidyhransferase activity in the transformant was eight fold higher than that in the wild‐type strain. Although the mutant enzyme had a greatly increased Km for myo‐inositol, the enzyme in the transformant had an apparent Km for myo‐inositol equal to that of the wild‐type enzyme, indicating the presence of the wild‐type structure gene on the recombinant plasmid.

The elevated level of enzyme activity in the transformant did not lead to an increase in the content of phosphatidylinositol. In contrast, supplementation of the culture medium with increasing concentrations of myo‐inositol resulted in an increase in the phosphatidylinositol content. Thus, the availability of myo‐inositol is a critical regulatory factor in yeast phosphatidylinositol synthesis.
Phosphorylation in vivo of red‐muscle pyruvate kinase from the channelled whelk, Busycotypus canaliculatum, in response to anoxic stress
Authors: PLAXTON William C. , STOREY Kenneth B.
Published: March 2005

That red muscle pyruvate kinase from anoxic Busycotypus canaliculatum (PK‐anoxic) is a phosphoprotein was demonstrated by the anoxia‐dependent, in vivo, covalent incorporation of injected [32P]orthophosphate into the enzyme molecule. Specificity in labelling of PK‐anoxic was strongly suggested by: (a) coincidental elution of pyruvate kinase activity and radioactivity following chromatography of purified PK‐anoxic on Sepha‐rose CL‐6B, and (b) comigration of the area containing [32P]phosphate and Coomassie‐Blue‐staining protein following SDS‐polyacrylamide gel electrophoresis of homogenous PK‐anoxic. The [32P]phosphate content of the enzyme was calculated to be 7.3 mol phosphate/mol enzyme (233 kDa, 180 units/mg protein). Evidence for the reversibility of this phosphorylation was provided by the consistent kinetic similarities between purified red muscle pyruvate kinase from aerobic animals (PK‐aerobic) and homogenous, unlabelled, alkaline phosphatase treated PK‐anoxic. Comparison of the electrophoretic mobilities of products derived from acid hydrolysis of purified 32P‐labelled PK‐anoxic with authentic substances suggest the presence of an O‐phospho‐L‐threonine residue in the protein. That this residue plays a probable role in an interconversion mechanism was suggested by the lack of phosphate exchange of homogenous 32P‐labelled PK‐anoxic in the presence of all substrates. A possible role of protein phosphorylation as a mechanism for the overall control of molluscan anaerobic metabolism is suggested.
Mode of action of miconazole on yeasts: inhibition of the mitochondrial ATPase
Authors: PORTILLO Francisco , GANCEDO Carlos
Published: March 2005

Miconazole ([1‐{2‐(2,4 dichlorophenyl)‐2‐(2,4 dichlorophenyl) methoxy]ethyl}‐1H‐imidazole) completely inhibited growth ofSaccharomyces cerevisiaeandCandida albicanson glycerol at 10 μM. 50 μM miconazole was needed to achieve the same effect during growth on glucose. Miconazole inhibited competitively the mitochondrial ATPase ofS. cerevisiaewith aKiof 1 μM. F1activity of the enzyme was not affected. Mutants resistant to miconazole were isolated. The ATPase of these mutants was resistant to 10 μM miconazole. Higher concentrations of miconazole inhibited the ATPase of the plasma membrane. The inhibition of theS. cerevisiaeenzyme was competitive with aKiof 50 μM. The results point to the mitochondrial ATPase as the primary target of miconazole action at least during growth on non‐fermentable carbon sources.
The interaction of skeletal myosin subfragment 1 with the polyanion, heparin
Authors: LABBÉ Jean‐Pierre , BERTRAND Raoul , AUDEMARD Etienne , KASSAB Ridha , WALZTHÖNY Doris , WALLIMANN Theo
Published: March 2005

The association between chymotryptic skeletal muscle myosin subfragment 1 (S1) and the polyanion, heparin, was investigated as an experimental approach in probing the functional importance of the cationic sites on S1 and their involvement in ionic interactions within the myosin head during energy transduction. The direct binding of heparin, used at micromolar concentrations, and its influence on the structural and functional properties of S1 were followed by gel chromatography, electron microscopy, chemical cross‐linking techniques and limited digestion studies.

The limited tryptic digestion of S1 showed that the presence of heparin, as well as of the homopolymer, poly‐(L‐glutamic acid) causes a specific structural change in the 50‐kDa heavy chain region of S1 and accelerates the breakdown of this segment into a 45‐kDa species by a proteolytic cleavage restricted to its COOH‐terminal portion. Under similar experimental conditions, the binding of MgATP and MgADP to S1 led also to the 50‐kDa → 45‐kDa conversion, suggesting that the S1‐nucleotide interactions exhibit some resemblances to the polyanion‐S1 interactions; a distortion of the COOH‐terminal peptide stretch of the 50‐kDa fragment seems to result from the binding of polyanionic ligands to S1. This particular area is adjacent to the actin site containing the 45‐kDa and 20‐kDa segments of the S1 heavy chain. On the other hand, the polyanions as well as nucleotides induced changes in the interface between the heavy chain and the alkali light chains.

Moreover, the binding of heparin to S1 resulted in the self‐association of the enzyme and the production of stable small S1 oligomers, most likely dimers, which were demonstrated by the alteration of the size of the S1 particles examined by electron microscopy and their freezing by chemical cross‐linking agents. These findings are relevant to the recently reported property of skeletal chymotryptic S1 to form dimers under convenient ionic conditions, in particular in the presence of Mg‐nucleotides. The interaction of cationic sites on S1 and possibly on the 50‐kDa region of the heavy chain with polyanions promotes the dimerization of the S1 molecules. The binding of S1 to F‐actin abolished S1 aggregation.
Conformation and domain structure of the non‐histone chromosomal proteins HMG 1 and 2
Authors: CARY Peter D. , TURNER Christopher H. , LEUNG Iris , MAYES Elaine , CRANE‐ROBINSON Colyn
Published: March 2005

The sequence of the 224 residues of HMG 1 suggests it consists of three domains. We have previously proposed [Cary et al. (1980 Eur. J. Biochem. 131, 367–374] that the A and B domains can fold autonomously and that there is also a small N domain. Several proteases are now found to cut at the end of the B domain (at or close to residue 184). It is shown that the A+B‐domain fragment also folds and probably contains all the helix of intact HMG 1. The stability of the B domain is enhanced by the presence of the A domain. The acidic C domain undergoes a coil → helix transition on lowering the pH. Several peptides have been prepared by cleavage at tryptophan. Peptide 57–C‐terminus contains complete B and C domains but does not fold. In the absence of the A domain the C domain is thus able to destabilise the B domain. It is concluded that the stability of the B domain in HMG 1 is due to interaction with the A domain and the C domain has a separate function from the other domains.
van der Waals' induced 13C NMR shifts in crystalline amino acids and peptides
Authors: BREWER Curtis F.
Published: March 2005

Recent reports in the literature of solid‐state 13C nuclear magnetic resonance (NMR) spectra of crystalline L‐alanine [Naito, A., Ganapathy, S., Akasaka, K., and McDonnell, C. A. (1981) J. Chem. Phys. 74, 3190–3197] and L‐leucine [Frey, M. H. and Opella, S. J. (1980) J. Chem. Soc. Chem. Commun., 474–475], recorded with cross‐polarization and magic‐angle spinning (CP‐MAS), show downfield resonance shifts of several parts per million in their side‐chain methyl groups, relative to their resonance positions in aqueous solution. Similar findings are reported here for crystalline aliphatic amino acids and L‐alanine peptides, including tetra(L‐alanine), which show similar, specific downfield shifts in their side‐chain methyl resonances. Coupled with X‐ray crystallographic data of these compounds, and previous gas and solution‐phase 13C NMR studies, the CP‐MAS 13C NMR data indicate that these downfield shifts are a result of van der Waals' interactions. This group have reported similar van der Waals' induced shifts of the same magnitude for 13C resonances of the side‐chain methyl groups of 13C‐enriched tetra(L‐alanine) upon binding to high‐affinity Fab' fragments of heterogeneous sheep anti‐[poly(L‐alanine)] antibodies in aqueous solution(Geller, S., Wei, S. C., Shkuda, G. K., Marcus, D. M., and Brewer, C. F. (1980) Biochemistry 19, 3614–3623]. The above findings show that van der Waals' induced 13C NMR shifts of similar magnitudes can be detected in specific antibody‐hapten complexes and the side chains of crystalline aliphatic amino acids and peptides. The results also indicate that water possesses relatively little attractive van der Waals' interactions with aliphatic molecules.
Precursors of the nerve growth factor γ subunit and renin bind to microtubules
Authors: DICOU Eleni , BRACHET Philippe
Published: March 2005

Translation products of a reticulocyte lysate reaction, programmed with poly(A)‐rich RNAs from the male mouse submaxillary gland, were subjected to affinity chromatography on a tubulin‐Sepharose column. Analysis of the bound proteins in sodium dodecylsulfate/polyacrylamide gels revealed two polypeptides of Mr 27000 and 45 000, that were shown to bind to tubulin in a specific manner. These polypeptides were absent from the translation products coded by poly(A)‐rich RNAs from the female mouse. They were eluted from the tubulin‐Sepharose resin under conditions similar to those employed for the dissociation of immune complexes. The Mr‐27000 and Mr‐45000 proteins were identified by immunoprecipitation with specific antisera as the precursors of the γ subunit of the nerve growth factor (NGF) and renin respectively. These two precursors as well as a third, unidentified polypeptide of Mr 38000, probably unrelated to the β subunit of NGF, bound also to microtubules. The mature form of renin, purified from the submaximillary gland, also displayed an affinity for the microtubules. In contrast, the mature form of the γ subunit of NGF did not bind to the microtubules. The possible involvement of the microtubules (tubulin) in the biosynthesis of these two secretory proteins is discussed.
Nucleotide pyrophosphatase from potato tubers
Authors: BARTKIEWICZ Marcjanna , SIERAKOWSKA Halina , SHUGAR David
Published: March 2005

Purification of potato tuber nucleotide pyrophosphatase (EC 3.6.1.9) has been modified to furnish a rapid and reproducible procedure yielding a preparation purified 1800‐fold and homogeneous in sodium dodecyl sulphate/polyacrylamide gel electrophoresis. The Mr of the enzyme, from gel filtration or sucrose density gradient centrifugation, is 343000 or 346000 respectively; and SDS electrophoresis indicates an Mr for the subunit of 74000. Analytical isoelectrofocusing reveals a broad isoelectric range of pH 8.3–8.7. The enzyme is a glycoprotein.

The purified enzyme exhibits the previously reported activities versus pyrophosphate linkages located at either the 5′‐OH or 3′‐OH of nucleosides, and phosphodiester linkages in: (a) aryl esters of nucleoside 3′‐ and 5′‐phosphates, p‐nitrophenylphosphate and orthophosphate, and (b) nucleoside cyclic 2′,3′‐phosphates. However, the relative rates of activity towards these substrates, and the corresponding V values, differ significantly. The enzyme exhibits additional novel activities, including ability to cleave dinucleoside polyphosphates such as A(5′)p2(5′)A– A(5′)p5(5′)A, and aryl phosphonates. Contrary to previous reports, there is no activity towards nucleoside cyclic 3′,5′‐phosphates.

The present preparation is also devoid of endonucleolytic activity, so that it specifically cleaves m7GMP from the 5′‐terminal m7G(5′)p3(5′)Gm of intact reovirus mRNA.

NAD+ was found to be the most effective inhibitor of enzyme activity versus thymidine 5′‐p‐nitro‐phenylphosphate, with a Ki= 0.1 mM. Kinetic analyses demonstrated competitive inhibition between these two substrates. Both 2′,3′‐cAMP and thymidine 3′‐β‐nitrophenylphosphate inhibit hydrolysis of NAD+ non‐competitively and vice‐versa.
Azidonaphthoyl‐ADP: a specific photolabel for the high‐affinity nucleotide‐binding sites of F1‐ATPase
Authors: LÜBBEN Mathias , LÜCKEN Uwe , WEBER Joachim , SCHÄFER Günter
Published: March 2005

3′‐O‐[5‐azidonaphthoyl]‐ADP has been synthesized as a photoreactive analog to 3′‐O‐naphthoyl(1)‐ADP which is known to bind to the high‐affinity nucleotide sites of mitochondrial F1‐ATPase, considered to be the catalytic sites. The photolabel in (he dark acts as a ligand to F1‐ATPase and as a competitive inhibitor with K1= 11 μM. Binding to the enzyme is accompanied by a quench of endogenous protein fluorescence leveling off at an occupancy or 1 mol/mol F1, whereas the total number of reversible sites accessible to the analog is 3 mol/mol F1 as measured by isotope studies. Covalent insertion by near ultraviolet activation of the probe yields labeling of both α and β polypeptides of F1: it is accompanied by corresponding removal of reversible high‐affinity sites for ADP or naphthoyl‐ADP and by an inhibition of the enzyme; total inactivation occurs at a covalent occupancy of 2 mol/mol F1. This is the maximum number of sites accessible to covalent modification by the label; one reversible site is still available in the totally inactivated enzyme. This observation is discussed in terms of a stochastic model requiring a minimum of two interacting catalytic domains out of three in order to commence catalysis.
Interaction of calmodulin with muscle phosphofructokinase
Authors: MAYR Georg W.
Published: March 2005

The hysteretic calmodulin‐induced inactivation of muscle phosphofructokinase and the calmodulin‐mediated reactivation are essentially dependent on environmental conditions. The interplay of calmodulin during these reactions and at allosteric conditions with Mg. ATP, fructose 6‐phosphate, adenosine 5′‐[β, γ‐imido]triphosphate and with the allosteric effectors AMP, ADP, fructose 1,6‐bisphosphate, fructose 2,6‐bisphosphate and glucose 1,6‐bisphosphate was studied by two techniques, (a) A two‐step technique with a preincubation of enzyme, calmodulin and effectors in close to physiological concentrations before dilution into an optimal activity assay. It reveals aggregation and slowly reversible conformation changes. (b) A direct assay of dilute enzyme at allosteric conditions.

Dominating in the interplay of calmodulin with metabolic effectors is the competitive‐like action of calmodulin on Mg · ATP binding to the regulatory sites of the enzyme. At high enzyme concentrations in the absence of hexose phosphates, i.e. at noncatalytic conditions calmodulin counteracts the stabilization of the highly active tetrameric form caused by Mg · ATP. In the allosteric assay it counteracts the ATP‐induced allosteric inhibition. In both cases calmodulin acts synergistic with AMP and ADP. To a miner degree calmodulin also counteracts the stabilization of the tetrameric form caused by fructose 6‐phosphate and hexose bisphosphate, now however antagonistically to AMP and ADP. By the demonstrated interactions the enzyme can be slowly and hysteretically shifted between an active tetrameric and an inactive dimeric state under control of metabolic conditions and of Ca2+ and calmodulin. Resting conditions will inactivate and high contractile activity reactivate available enzyme.
Measurements of electron spin resonance with the pyruvate dehydrogenase complex from Escherichia coli
Authors: SCHRENK Dieter F. , BISSWANGER Hans
Published: March 2005

Binding of the feedback inhibitor acetyl‐coenzyme A to the pyruvate dehydrogenase complex from Escherichia coli was studied by electron spin resonance spectroscopy with the spin‐labelled acetyl‐CoA analogue 3‐carboxy‐2,2,5,5‐tetramethylpyrrolidine‐1‐oxyl‐CoA‐thioester. The spin‐labelled compound binds to the pyruvate dehydrogenase component of the enzyme complex and this binding can be reversed by acetyl‐CoA, while CoA has no effect. AMP and fructose 1, 6‐bisphosphate, which are both activators of the pyruvate dehydrogenase complex, exhibit a partial competition with the spin‐labelled acetyl‐CoA analogue and it could be shown that both activators act essentially by reversion of the feedback inhibition of acetyl‐CoA. The binding site for these activators seems to overlap with the acetyl‐CoA binding site, possibly by a common phosphate attachment point. No competition for binding to the feedback inhibition site exists with pyruvate, thiamine diphosphate, magnesium ions and with the fluorescent chromophore 8‐anilino‐1‐naphthalene sulfonic acid. Thus, the feedback inhibition site proves to be a true allosteric regulatory site, which appears to be completely separate from the catalytic site on the pyruvate dehydrogenase component.

The spin‐labelled acetyl‐CoA analogue binds also to the product binding site of acetyl‐CoA on the dihydrolipoamide acetyltransferase component of the pyruvate dehydrogenase complex. Two binding sites per polypeptide chain with identical affinities on this enzyme component were found and the binding of the analogue can be inhibited by acetyl‐CoA as well as by CoA.
Synthesis of early vaccinia‐virus‐specific enzymes under conditions of immediate early gene expression
Authors: ZÖLLER Barbara , KREMPIEN Ursula , JUNGWIRTH Christoph
Published: March 2005

Cycloheximide reversal experiments in chick embryo fibroblasts and mouse L‐929 cells indicate that the poxvirus‐induced enzymes DNA polymerase and ‘alkaline’ DNase are immediate early gene products of the virus. In contrast to the vaccinia‐WR‐coded enzyme under conditions of immediate early gene expression the cowpox‐virus‐induced DNA polymerase is made only in very small amounts. The studies are consistent with the notion that all poxvirus‐specific early proteins may be immediate early viral gene products.
Permissive action of thyroid hormones in the cAMP‐mediated induction of phosphoenol pyruvate carboxykinase in hepatocytes in culture
Authors: SÜSSMUTH Werner , HÖPPNER Wolfgang , SEITZ Hans J. , LUDA Dagmar , HARNEIT Angelika
Published: March 2005

Thyroid hormones are known to stimulate in vivo the synthesis of several liver proteins. In order to determine their role in the regulation of important hepatic proteins at the cell level, the effect of T3 on the induction of the glucoregulatory enzyme phosphoenol pyruvate carboxykinase (PEPck) was investigated in cultured hepatocytes. The cells were isolated from adult hypothyroid rats and exposed to a chemically defined medium, devoid of serum supplement and hormone‐free over a period of 48 h.

(a) Addition of T3 to the medium had only a minor effect on PEPck induction, (b) Dibutyryladenosine 3′,5′‐monophosphate (Bt2cAMP) or epinephrine provoked significant increase in PEPck synthesis within 4–6h. (c) Simultaneous addition of T3 was found significantly to promote the Bt2cAMP‐mediated enzyme induction, (d) Physiological concentrations of T3 (10 pM to 1 nM) were found to be effective in enhancing this cAMP‐mediated signal, (e) Exposure of the cells to T3 for only 90 min, 16 h prior to addition of Bt2cAMP was nearly as effective as continuous exposure to T3 in enhancing the cAMP‐effect on PEPck synthesis, (f) In contrast, no effect of T3 on the Bt2cAMP‐induced tyrosine‐aminotransferase activity could be detected.

It is concluded that the role of T3 on the cAMP‐elicited PEPck synthesis is selective, rapid, primarily permissive and significant within the physiological range of the hormone. In addition, these data indicate that such hepatocyte cultures prepared from hypothyroid rats are ideally suited to the analysis of the regulatory role of thyroid hormones on specific gene expression.
The primary structure of the cytotoxin restrictocin
Authors: LÓPEZ‐OTÍN Carlos , BARBER Domingo , FERNÁNDEZ‐LUNA Jose Luis , SORIANO Fernando , MÉNDEZ Enrique
Published: March 2005

The complete amino acid sequence of the single polypeptide chain of cytotoxin restrictocin has been determined. Its structure was established by automated Edman degradation of the intact molecule reduced and [14C]carboxymethylated and of fragments obtained by chemical cleavage of the protein with cyanogen bromide and BNPS‐skatole and by enzymatic cleavage of the polypeptide chain with trypsin. The molecule consists of 149 amino acid residues with a calculated relative molecular mass of 16836. The protein presents two disulfide bridges, one between cysteine residues at positions 5 and 147 and the other one formed by cysteine residues at positions 75 and 131. The amino acid sequence of restrictocin shows a high degree of homology (86%) with that of the cytotoxin named α‐sarcin.
31P NMR spin‐transfer in the phosphoglyceromutase reaction
Authors: ROBINSON Gae , CHAPMAN Bogdan E. , KUCHEL Philip W.
Published: March 2005

The rate of exchange of phosphoryl groups between 2‐ and 3‐phosphoglycerate catalysed by (a) high concentrations (∼ 5.0 mg protein ml−1) of rabbit muscle phosphoglyceromutase and (b) lysed human erythrocytes was measured using saturation and inversion transfer techniques with 31P‐NMR spectroscopy. This is the first reported application of these techniques to a study of this particular enzymic reaction either in vitro or in situ in a cell cytosol. Selective irradiation of resonances was achieved by the DANTE pulse sequence which had not previously been used for spin‐transfer studies. New equilibrium exchange theory was developed for the simplest model of a two‐reactant enzyme‐catalysed reaction and this was used to calculate turnover rates for the enzymes. There was a close similarity between the turnover rates calculated from the spin‐transfer data obtained from the systems in vitro and in situ and those obtained by conventional enzymic assays, at low enzyme concentrations. This suggested an absence of any homogeneous enzyme‐enzyme interactions which modify the kinetics at high protein concentrations either in lysates or in the system in vitro.
Primary structure of Vicia angustifolia proteinase inhibitor
Authors: SHIMOKAWA Yukiko , KUROMIZU Kenji , ARAKI Tomiko , OHATA Junko , ABE Okitoshi
Published: March 2005

The complete amino acid sequence (72 amino acid residues) of a double‐headed proteinase inhibitor from seeds of Vicia angustifolia L. var. segetalis Koch has been determined and compared with those of other double‐headed inhibitors of known structure. Sequencing was performed by conventional methods with the aid of the fragments produced by reduction and S‐carboxymethylation of the enzymatically modified inhibitors, and also using tryptic and chymotryptic peptides.

The positions of the 14 half‐cystine residues agreed among all the reported primary structures of the legume double‐headed inhibitors. However, V. angustifolia inhibitor possessed extensive amino acid differences compared to the others. The phylogenetic relationship among these inhibitors was established using the unweighted pair‐group method and revealed that the V. angustifolia inhibitor and the peanut inhibitor B‐III had diverged at a relatively earlier stage compared to the other inhibitors.
Alterations in intracellular calcium compartmentation following inhibition of calcium efflux from isolated hepatocytes
Authors: BELLOMO Giorgio , NICOTERA Pierluigi , ORRENIUS Sten
Published: March 2005

Addition of ATP to the incubation medium of freshly isolated rat hepatocytes causes a marked inhibition of the efflux of Ca2+ from the cells, and its accumulation in intracellular compartments. After an initial rise in cytosolic free Ca2+ concentration, as indicated by the activation of phosphorylase, Ca2+ is preferentially sequestered in the mitochondria, without any apparent contribution by the endoplasmic reticulum. Impairment of mitochondrial Ca2+ homeostasis by pyridine nucleotide oxidation associated with tert‐butyl hydroperoxide metabolism, prevents the ATP‐dependent cellular Ca2+ accumulation and causes a release of Ca2+ from the hepatocytes into the medium. Conversely, maintenance of the mitochondrial pyridine nucleotides in a more reduced state, e. g. in presence of 3‐hydroxybutyrate in the medium, prevents this hydroperoxide‐induced release of intracellular Ca2+. Under conditions of impaired mitochondrial Ca2+ sequestration, there appears to be a redistribution of a minor fraction of the intracellular Ca2+ from the mitochondria to the endoplasmic reticulum.

Our results provide additional evidence for the critical involvement of the plasma membrane Ca2+‐extruding system in the physiological regulation of the cytosolic free Ca2+ concentration in hepatocytes, and suggest that the mitochondria play a more important role than the endoplasmic reticulum in the regulation of the cytosolic free Ca2+ level when the plasma membrane Ca2+ pump is inhibited.
Assignment of the disulphide bonds in the sweet‐tasting protein thaumatin I
Authors: VAN DER WEL Henk , IYENGAR Ramanuya B. , VAN BROUWERSHAVEN Johan , VAN WASSENAAR Pieter D. , BEL Wim J. , VAN DER OUDERAA Frans J. G.
Published: March 2005

The disulphide linkages of the 16 half‐cystine residues in the sweet‐tasting protein thaumatin have been investigated by enzymatic hydrolysis of the intact molecule. The peptides obtained after proteolytic cleavage with trypsin and pepsin, and in one case with chymotrypsin have been purified by gel filtration, high‐performance liquid chromatography and peptide mapping by paper high‐voltage electrophoresis in one direction and paper chromatography in the second dimension. Disulphide bonds appeared to be formed by cysteine residues in positions 9‐204, 56‐66, 71‐77, 121‐193, 126‐177, 134‐149, 145‐158 and 159‐164. The labile disulphide bond responsible for the enzymatic properties of the sweet tasting protein thaumatin appeared to be between Cys‐145 and Cys‐158.
Primary structure of the ram (Ovis aries) protamine
Authors: SAUTIERE Pierre , BELAICHE Denise , MARTINAGE Arlette , LOIR Maurice
Published: March 2005

The amino acid sequence of the protamine isolated from mature sperm nuclei of the ram (Ovis aries) has been established from automated sequence analysis of the S‐carboxymethylated protamine. Ram and bull protamines differ only by two point changes and the deletion in bull protamine of the tripeptide Cys39‐Arg‐Arg41. In mammalian protamines the central region (residues 13–36) consisting mainly of arginine clusters appears to be conserved whereas the N‐terminal and C‐terminal regions are more variable.
Characterisation of size variants of type I DNA topoisomerase isolated from calf thymus
Authors: SCHMITT Bertram , BUHRE Ursula , VOSBERG Hans‐Peter
Published: March 2005

Calf thymus DNA topoisomerase I, which belongs to the eukaryotic type I topoisomerases, is in a typical preparation purified as a set of five major polypeptides with Mr between 70000 and 100000. At least four of these proteins have binding affinity for DNA as was shown by incubating them with radioactive single‐stranded DNA after separation in dodecylsulfate polyacrylamide gels and blotting onto nitrocellulose filters. That these polypeptides have DNA relaxing activity was directly demonstrated with protein extracted from single bands of dodecylsulfate/polyacrylamide gels. We consider the 100000–Mr protein to be the native enzyme. The smaller components are catalytically active fragments of the native topoisomerase most probably arising from limited proteolysis either within the nucleus or during the purification of the enzyme. In two‐dimensional non‐equilibrium pH‐gradient electrophoresis gels the topoisomerase size variants exhibit apparent pI values between 8.1 and 8.3, with small but distinct differences between the components. The calf thymus topoisomerase I, upon binding to phage fd‐ DNA, protects a stretch of 15–25 nucleotides againt digestion with DNase I.
Studies on the nature of transition‐metal‐ion‐mediated binding of triazine dyes to enzymes
Authors: HUGHES Peter , SHERWOOD Roger F. , LOWE Christopher R.
Published: March 2005

Several reactive azoic dichlorotriazinyl dyes specifically and irreversibly inactivate the folate‐degrading enzyme carboxypeptidase G‐2 at a site competitive with the enzyme substrates methotrexate (4‐amino‐N10‐methylfolic acid) and p‐aminobenzoyl‐l‐glutamate. Although the less reactive monochlorotriazinyl dye, Procion red H‐8BN, is unable to inactivate the enzyme, it is capable of marked inhibition of inactivation by dichlorotriazinyl dyes in the presence of Zn2+. Zinc ions and, to a lesser extent other first row transition metal ions, significantly enhance the affinity of Procion red H‐8BN and its analogues Procion red MX‐8B and Procion red MX‐2B, for carboxypeptidase G‐2. It is proposed that this effect is mediated through the formation of a specific tetracoordinate Zn2+ complex between the azo linkage and adjacent sulphonate and hydroxyl functions of the dye and an appropriate ligand on the protein.

Carboxypeptidase G‐2 quantitatively inactivated with the dichlorotriazinyl dye, Procion red MX‐8B, contains approximately 1 mol dye/mol subunit of Mr 42000. Proteolytic cleavage of the labelled enzyme and resolution of the peptides by reverse‐phase high‐performance liquid chromatography yields a principal red peptide which on amino acid sequence analysis results in the identification of the dye binding domain. The affinity label, Procion red MX‐8B, is believed to be attached to the hydroxylic side chain of Thr‐279.
Na+‐dependent regulation of extramitochondrial Ca2+ by rat‐liver mitochondria
Authors: NEDERGAARD Jan
Published: March 2005

The presence and significance of Na+‐induced Ca2+ release from rat liver mitochondria was investigated by the arsenazo technique.

Under the experimental conditions used, the mitochondria, as expected, avidly extracted Ca2+ from the medium. However, when the uptake pathway was blocked with ruthenium red, only a small rate of ‘basal’ release of Ca2+ was seen (0.3 nmol Ca2+× min−1× mg−1), in marked contrast to earlier reports on a rapid loss of sequestered Ca2+ from rat liver mitochondria.

The addition of Na+ in ‘cytosolic’ levels (20 mM) led to an increase in the release rate by about 1 nmol Ca2+× min−1× mg−1. This effect was specific for Na+.

The significance of this Na+‐induced Ca2+ release, in relation to the Ca2+ uptake mechanism, was investigated (in the absence of uptake inhibitors) by following the change in the extramitochondrial Ca2+ steady‐state level (set point) induced by Na+. A five‐fold increase in this level, from less than 0.2 μM to more than 1 μM, was induced by less than 20 mM Na+. The presence of K+ increased the sensitivity of the Ca2+ homeostat to Na+. The effect of Na+ on the extramitochondrial level was equally well observed in an K+/organic‐anion buffer as in a sucrose buffer.

Liver mitochondria incubated under these circumstances actively counteracted a Ca2+ or EGTA challenge by taking up or releasing Ca2+, so that the initial level, as well as the Na+‐controlled level, was regained.

It was concluded that liver mitochondria should be considered Na+ ‐sensitive, that the capacity of the Na+ ‐induced efflux pathway was of sufficient magnitude to enable it to influence the extramitochondrial Ca2+ level biochemically and probably also physiologically, and that the mitochondria have the potential to act as active, Na+ ‐dependent regulators of extramitochondrial (‘cytosolic’) Ca2+. It is suggested that changes of cytosolic Na+ could be a mediator between certain hormonal signals (notably α1‐adrenergic) and changes in this extramitochondrial (‘cytosolic’) Ca2+ steady state level.
Differentiation of algal chemoreceptors
Authors: BOLAND Wilhelm , JAENICKE Lothar , MÜLLER Dieter G. , PETERS Akira
Published: March 2005

Gamete encounter in the reproduction of brown algae is supported by lures produced by the female cells. These signals are low‐molecular‐mass, volatile, olefinic hydrocarbons. Specific receptors on the motile male gametes recognize the pheromones at threshold concentrations as low as 6.5 pmol/1. The pheromones are possibly bound by dispersion and dipolar interactions. Tight fitting has to compensate for these weak contact forces. This causes strong chiral discrimination (relative affinities to enantiomeric lures 1:100). Structurally related messengers with identical substitution patterns and overall space filling are mutually distinguished in several species by means of altered topologies in their receptor mould. Responses to systematically varied synthetic bis‐alkenyl‐cyclopentene pheromones showed that the sensors of Syringoderma and Cutleria androgametes consist of a mosaic of attachment sites individually adapted to specific structural elements in the messenger molecule. They may be permutated to afford optimal binding. An alternative strategy: distinct reading of electron density combined with variation of sensitivity, brings about ‘fidelity’ in signal transmission by the alkylated cycloheptadiene pheromones in two species of Desmarestia and Ectocarpus. Inhibition of chemotaxis by ectocarpene is observed in Ectocarpus males in the presence of desmarestene, the lure of Desmarestia, and related parapheromones. This indicates complex interactions between different species of algae sharing a common habitat, in their defense of living space.
The refined 2.2‐Å (0.22‐nm) X‐ray crystal structure of the ternary complex formed by bovine trypsinogen, valine‐valine and the Arg15 analogue of bovine pancreatic trypsin inhibitor
Authors: BODE Wolfram , WALTER Jochen , HUBER Robert , WENZEL Herbert R. , TSCHESCHE Harald
Published: March 2005

Large orthorhombic crystals of the complex formed by bovine trypsinogen and a semisynthetic homologous bovine pancreatic trypsin inhibitor with the reactive‐site lysine residue replaced by an arginine residue ([Arg15]PTI) have been obtained which are isomorphous with the crystals of PTI‐trypsinogen [Bode, W., Schwager, P. and Huber, R. (1978) J. Mol. Biol. 118, 99–112]. The X‐ray crystal structure of the ternary complex of trypsinogen‐[Arg15]PTI with the dipeptide Val‐Val has been determined by X‐ray data to 2.2‐Å (0.22‐nm) resolution by means of difference Fourier methods and has been crystallographically refined to a final R‐value of 0.17.

Replacement of the reactive‐site Lys15 by an arginine residue is accompanied in the complex by small movements of polar side groups of trypsin and enclosed solvent molecules within the specificity pocket. Only solvent molecule 414 OH which mediates the hydrogen bond interactions between Lys15 NZ and Asp189carboxylate is expelled, thus allowing the bulkier guanidyl group to approach this carboxylate. The dipeptide Val‐Val binds in the packet accepting the Ile‐Val N‐terminus in trypsin. The cavity left by the CD‐methyl group of He16 upon replacement by a valine residue is only partially filled by slight rearrangements of neighbouring peptide side chains. Part of the positive free energy change observed upon replacement of Ile‐Val may allow for the maintenance of this cavity.
Macromolecular syntheses in the cell cycle mutant cdc25 of budding yeast
Authors: MARTEGANI Enzo , VANONI Marco , BARONI Maurizio
Published: March 2005

A major control point of the cell cycle in Saccharomyces cerevisiae is a G1 event called ‘start’. At start a yeast cell integrates external and internal signals and decides to progress toward mitosis or to choose alternative pathways such as sporulation, conjugation etc.

cdc25 is a class II temperature‐sensitive start mutant that blocks at restrictive temperature in G1 as round unbudded cells.

The arrest of the cell cycle appears to be independent of the carbon and nitrogen sources, and the cell wall of cdc25‐arrested cells shows changes similar to those found in cells undergoing entry in to the stationary phase.

After a shift to 36 °C the increase in cell number of cdc25 cultures is gradually inhibited. The nuclear division cycle appears to be inhibited immediately after the shift and the percentage of budded cells decreases, while cytoplasmic growth, monitored either as increase of adsorbance at 450 nm or as protein accumulation, continues for many hours leading to a progressive increase of mean cell volume and mean protein content per cell.

The stable RNA accumulation instead is immediately inhibited and this is partially due to a 50% inhibition of ribosomal RNA synthesis, while the rate of synthesis of ds‐killer RNA is relatively unaffected.

These data suggest that the CDC25 gene product could be a part of a mechanism that leads yeast cells to choose between the progression towards DNA replication and cell division or to enter into the stationary phase. This mechanism appears to turn off both rRNA accumulation and cell‐cycle progression and to activate differentiative pathways in response to environmental restriction.
The structure of the polysaccharide moiety of Rhodopseudomonas sphaeroides ATCC 17023 lipopolysaccharide
Authors: SALIMATH Paramahans V. , THARANATHAN Rudrapatnam N. , WECKESSER Jürgen , MAYER Hubert
Published: March 2005

The chemical structure of the polysaccharide moiety of the lipopolysaccharide of Rhodopseudomonas sphaeroides ATCC 17023 was established. Mild acetic acid hydrolysis of isolated lipopolysaccharide, followed by preparative high‐voltage paper electrophoresis afforded three oligosaccharides. They were characterized by chemical and physicochemical studies to be: GlcA(α1→4)dOclA8P, Thr(6′) GlcA(α1→4)GlcA and GlcA(α1→4)dOclA, where GlcA is d‐glucuronic acid and dOclA is 3‐deoxy‐d‐manno‐octulosonic acid. Carboxyl‐reduction of the lipopolysaccharide followed by acid hydrolysis gave a trisaccharide: GlcA(α1→4)Glc(α1→4)G1c, showing the presence of three residues of glucuronic acids in the O‐specific chain and indicating that only two of them are reducible by NaBH4. The linkage between the polysaccharide and lipid A was shown to be through a single 1,4‐linked residue of dOclA attached by a 2,6′‐linkage to the lipid A moiety.
Nucleoside diphosphate kinase from brain
Authors: HUITOREL Philippe , SIMON Colette , PANTALONI Dominique
Published: March 2005

Tubulin strictly requires GTP for its polymerization. Nevertheless, microtubule assembly can be observed in the presence of ATP as the only nucleotide triphosphate, due to the nucleoside diphosphate kinase (NDP kinase) present in microtubule preparations, and which phosphorylates the GDP into GTP.

We have purified this enzyme from pig brain to homogeneity, and shown that its relative mass is close to 100000 in its native state, and 17000 under denaturing conditions. Therefore it is probably a hexamer, as previously shown for the enzyme from other sources, and also presents a microheterogeneity, with the major isoforms between pI 5.0 and 6.0. The enzyme is transiently phosphorylated during catalysis, as expected within a ping‐pong bi‐bi mechanism.

The effect of the NDP kinase on pure tubulin polymerization was studied: in the presence of NDP kinase, the lag time observed in the kinetics of microtubule assembly was shorter and the final extent of assembly was unchanged. The effect of the enzyme was observed at enzyme concentrations 900‐fold lower than tubulin concentration, which shows that the NDP kinase acts catalytically.

Kinetic data show that the catalytic effect of the NDP kinase is faster than the rate of nucleotide exchange on tubulin under the same conditons. This result demonstrates that the tubulin‐GDP complex itself is a substrate for the enzyme, which may indicate that the GDP bound to tubulin at the E site is exposed on the surface of dimeric tubulin.
A novel form of the polypeptide PHI isolated in high yield from bovine upper intestine
Authors: CARLQUIST Mats , KAISER Rudolf , TATEMOTO Kazuhiko , JÖRNVALL Hans , MUTT Viktor
Published: March 2005

A novel form of the polypeptide termed PHI (peptide HI with N‐terminal histidine and C‐terminal isoleucine amide) has been isolated from bovine upper intestine. This bovine peptide was obtained in a 40 times higher yield than the corresponding polypeptide isolated from porcine intestine. Bovine PHI is, like porcine PHI, composed of 27 amino acid residues. The complete amino acid sequence of the bovine peptide is His‐Ala‐Asp‐Gly‐Val‐Phe‐Thr‐Ser‐Asp‐Tyr‐Ser‐Arg‐Leu‐Leu‐Gly‐Gln‐Leu‐Ser‐Ala‐Lys‐Lys‐Tyr‐Leu‐Glu‐Ser‐Leu‐Ile‐NH2. This sequence differs from porcine PHI at position 10 and from human PHI at positions 10, 12 and 27. The amino acid residue exchange between porcine and bovine PHI makes the latter more similar to the vasoactive intestinal polypeptide, (VIP), gastric inhibitory polypeptide (GIP), glucagon and the growth‐hormone‐releasing factor (GRF).
Study of the conversion of GDP‐mannose into GDP‐fucose in Nereids: a biochemical marker of oocyte maturation
Authors: BULET Philippe , HOFLACK Bernard , PORCHET Maurice , VERBERT André
Published: March 2005

Homogenates of Perinereis cultrifera oocytes were found to transform GDP‐d‐mannose into another sugar nucleotide. Ultraviolet absorption spectra, chromatographic behaviour, gas‐liquid chromatography coupled to mass spectrometry analysis revealed that GDP‐d‐mannose had been converted into GDP‐l‐fucose. This conversion is a multi‐step reaction as proved by the involvement of two intermediates identified as GDP‐4‐oxo‐6‐deoxy‐d‐mannose and GDP‐4‐oxo‐6‐deoxy‐l‐galactose, this latter being reduced by NADPH to give GDP‐l‐fucose. It is shown that the enzymatic activities responsible for the conversion of GDP‐d‐mannose into GDP‐l‐fucose is recovered only in oocytes and is not present in the other coelomic cells (i.e. coelomocytes). More interesting is the fact that maximum‐activity is recovered at a well defined stage of the hormone‐controlled oogenesis. Thus, this enzymatic system appears as a biochemical marker of the oocyte maturation in P. cultrifera.
A new proposal for the primary and secondary structure of the glycan moiety of pseudomurein
Authors: LEPS Bernd , LABISCHINSKI Harald , BARNICKEL Gerhard , BRADACZEK Hans , GIESBRECHT Peter
Published: March 2005

Using the method of conformational energy calculations, favoured conformations of a pseudomurein sugar strand built up from β1,3‐linked N‐acetyl‐d‐glucosamine and N‐acetyl‐l‐talosaminuronic acid were obtained. Such a completely β1,3‐linked polysaccharide primary structure, although contrasting with the originally proposed alternating β1,3‐α1,3‐linked structure [König, H., Kandler, O., Jensen, M. and Rietschel, E. Th. (1983) Hoppe‐Seyler's Z. Physiol. Chem. 364, 627–636] would be in agreement with all experimental data hitherto known. Starting from an analysis of favoured conformations of the monosaccharide building blocks and those obtained for. disaccharide parts, the favoured helical conformations of the complete polysaccharide chains could be explored. Our completely β1,3‐linked chain could adopt two types of conformation: extended and hollow; the latter was discarded as unsuitable for cell wall assembly. The extended conformation type was shown to exhibit a remarkable similarity if compared to the secondary structures accessible to murein‐type polysaccharide chains. In contrast to the conformations accessible for the β1,3‐α1,3‐linked primary structure, the new hypothetical pseudomurein structure was found to be more extended, possessed a flexible peptide attachment site at every second sugar residue and led to an orientation of consecutive peptide attachment sites analogous to the data known for murein‐type chains. From the two possibilities compatible with the experimental data available, the completely β1,3‐linked sugar strand structure could well be realized in the native pseudomurein network of methanobacteria. In this case the hypothesis for a similar three‐dimensional architecture for murein and pseudomurein would be supported.