bluuebunny/crossref_metadata_2025 huggingface dataset:
Characterization and expression of a NADP‐malic enzyme cDNA induced by salt stress from the facultative crassulacean acid metabolism plant, Mesembryanthemum crystallinum
Authors: CUSHMAN John C.
Published: March 2005

The facultative halophyte and crassulacean acid‐metabolism plant, Mesembryanthemum crystallinum shifts from C3 photosynthesis to crassulacean acid metabolism when exposed to high‐salt or drought conditions. To study the molecular basis of this metabolic transition, the expression of NADP+‐dependent malic enzyme (NADP‐ME), which catalyzes the decarboxylation of malate to release pyruvate and CO2, has been investigated. The complete nucleotide sequence of a full‐length cDNA clone was determined and found to contain a single open reading frame encoding a 585‐amino‐acid polypeptide of 64284 Da. The ice plant (M. crystallinum) NADP‐ME shares amino acid identities in the range 72.5–79.0% when compared to other higher‐plant enzymes and is more closely related to C3 rather than C4 forms of the enzyme. Genomic Southern‐blot analysis of ice‐plant DNA indicates that NADP‐ME is encoded by a small gene family. Steady‐state transcript levels increase 8–10‐fold in response to salt stress in the leaves. Transcript levels in roots are extremely low and are unaffected by salt‐stress treatment. Nuclear run‐on experiments, using isolated nuclei from leaf tissue, confirm that the accumulation of NADP‐ME transcripts is, in part, the result of increased transcription of this gene during salt stress.
A missense mutation Pro157Arg in lipoprotein lipase (LPLNijmegen) resulting in loss of catalytic activity
Authors: BRUIN Taco , KASTELEIN John J. P. , VAN DIERMEN Denise E. , MA Yuanhong , HENDERSON Howard E. , STUYT Paul M. J. , STALENHOEF Anton F. H. , STURK Augueste , BRUNZELL John D. , HAYDEN Michael R.
Published: March 2005

Here we report on the molecular defect that leads to a deficiency of lipoprotein lipase (LPL) activity in a proband of Dutch descent. Southern‐blot analysis of the LPL gene from the patient did not reveal any major DNA rearrangements. Sequencing of polymerase‐chain‐reaction‐amplified DNA revealed that the proband is a homozygote for G725C, resulting in a substitution of Pro157 for Arg. This substitution alters a restriction site for PvuII, which allowed rapid identification of the mutant allele in family members. Site‐directed mutagenesis and transient expression of the mutant LPL in COS cells produced an enzymatically inactive protein, establishing the functional significance of this mutation. This naturally occurring mutation which alters the Pro157 adjacent to Asp156 of the proposed catalytic triad, indicates that this region of the protein is indeed crucial for LPL catalytic activity.
Crystal structure of carboxypeptidase T from Thermoactinomyces vulgaris
Authors: TEPLYAKOV Alexei , POLYAKOV Kostya , OBMOLOVA Galya , STROKOPYTOV Boris , KURANOVA Inna , OSTERMAN Andrei , GRISHIN Nikolai , SMULEVITCH Sergei , ZAGNITKO Olga , GALPERINA Olga , MATZ Michael , STEPANOV Valentin
Published: March 2005

The crystal structure of carboxypeptidase T from Thermoactinomyces vulgaris has been determined at 0.235‐nm resolution by X‐ray diffraction. Carboxypeptidase T is a remote homologue of mammalian Zn‐carboxypeptidases. In spite of the low degree of amino acid sequence identity, the three‐dimensional structure of carboxypeptidase T is very similar to that of pancreatic carboxypeptidases A and B. The core of the protein molecule is formed by an eight‐stranded mixed β sheet. The active site is located at the C‐edge of the central (parallel) part of the β sheet. The structural organization of the active centre appears to be essentially the same in the three carboxypeptidases. Amino acid residues directly involved in catalysis and binding of the C‐terminal carboxyl of a substrate are strictly conserved. This suggests that the catalytic mechanism proposed for the pancreatic enzymes is applicable to carboxypeptidase T and to the whole family of Zn‐carboxypeptidases. Comparison of the amino acid replacements at the primary specificity pocket of carboxypeptidases A, B and T provides an explanation of the unusual ‘A + B’ type of specificity of carboxypeptidase T. Four calcium‐binding sites localized in the crystal structure of carboxypeptidase T could account for the high thermostability of the protein.
Non‐linear inhibition curves for tight‐binding inhibitors of dimeric ubiquinol — cytochromecoxidoreductases
Authors: BECHMANN Georg , WEISS Hans , RICH Peter R.
Published: March 2005

Steady‐state electron flow through and electron delivery into isolated dimericbc1complex (ubiquinol–cytochromecoxidoreductase) fromNeurospora crassaand beef heart mitochondria were studied in the presence of increasing concentrations of antimycin A, funiculosin and/or myxothiazol. Parabolic or linear inhibition curves were obtained, depending upon the different quinols and inhibitors that were used. Linear curves occur when the inhibitor directly affects the rate‐determining step. The most reasonable explanation for the parabolic curves is given by a fast intradimeric exchange of the hydrophobic inhibitors antimycin A, funiculosin (rate < 500 s−1) and of myxothiazol (rate > 1 s−1). Using mitochondria from beef heart, the shape of the inhibition curve with antimycin A is parabolic if the quinol – O2oxidoreductase turns over at about 300 s−1, but hyperbolic if the rate is 5 times less. The hyperbolic titration curve may be the result of both intradimeric and an additional interdimeric redistribution (rate ∼ 100 s−1) of inhibitors between enzymes incorporated in a continuous phospholipid membrane. This explanation is supported by experiments with chromatophores obtained fromRhodobacter capsulatus. As recently described [Fernandez‐Velasco, J. & Crofts, A. R. (1992)Biophys. J. 2, A153], cytochromebbecomes fully reoxidized within 1 s after a flash at substoichiometric concentrations of antimycin A. This kinetic of the slow reoxidation can be expressed in terms of the intradimeric and interdimeric redistribution with rate constants of about 10 s−1and 2 × 106M−1s−1, respectively. It seems that rapid inhibitor redistribution may be a widespread phenomenon for hydrophobic inhibitors of enzymes incorporated in lipid membranes.
Lipid vesicles which can bind to protein kinase C and activate the enzyme in the presence of EGTA
Authors: EPAND Richard M. , STAFFORD Alan R. , LESTER David S.
Published: March 2005

Maximal protein kinase C activity with vesicles of phosphatidic acid and 1,2‐dioleoyl‐sn‐glycerol is observed in the absence of added Ca2+. Addition of phosphatidylcholine to these vesicles restores some calcium dependence of enzyme activity. 1,2‐Dioleoyl‐sn‐glycerol eliminates the Ca2+‐dependence of protein kinase C activity found with phosphatidic acid alone. Phorbol esters do not mimic the action of 1,2‐dioleoyl‐sn‐glycerol in this respect. This suggests that the 1,2‐dioleoyl‐sn‐glycerol effect is a result of changes it causes in the physical properties of the membrane rather than to specific binding to the enzyme. The effect of 1,2‐dioleoyl‐sn‐glycerol on the phosphatidic‐acid‐stimulated protein kinase C activity is dependent on the molar fraction of 1,2‐dioleoyl‐sn‐glycerol used and results in a gradual shift from Ca2+ stimulation at low 1,2‐dioleoyl‐sn‐glycerol concentrations to calcium inhibition at higher concentrations of 1,2‐dioleoyl‐sn‐glycerol. Phosphatidylserine‐stimulated activity is also shown to be largely independent of the calcium concentration at higher molar fractions of 1,2‐dioleoyl‐sn‐glycerol. Thus, with certain lipid compositions, protein kinase C activity becomes independent of the calcium concentration or requires only very low, stoichiometric binding of Ca2+ to high affinity sites on the enzyme.

Protein kinase C can bind to phosphatidic acid vesicles more readily than it can bind to phosphatidylserine vesicles in the absence of calcium. Addition of 1,2‐dioleoyl‐sn‐glycerol to phosphatidylserine vesicles promotes the partitioning of protein kinase C into the membrane in the absence of added Ca2+. There is no isozyme specificity in this binding. These results suggest that a less‐tightly packed headgroup region of the bilayer causes increased insertion of protein kinase C into the membrane. This is a necessary but not sufficient condition for activation of the enzyme in the presence of EGTA.
Effect of diets rich in medium‐chain and long‐chain triglycerides on lipogenic‐enzyme gene expression in liver and adipose tissue of the weaned rat
Authors: FOUFELLE Fabienne , PERDEREAU Dominique , GOUHOT Betty , FERRE Pascal , GIRARD Jean
Published: March 2005

The activity and mRNA concentrations of two lipogenic enzymes, fatty‐acid synthase and acetyl‐CoA carboxylase were measured in the liver and white adipose tissue of rats weaned to a carbohydrate‐rich diet containing either long‐chain or medium‐chain fatty acids, and compared to those of rats weaned on a diet containing less than 1% (total energy) fat (high‐carbohydrate diet). In the liver, the diet containing long‐chain fatty acids inhibited the increase of both lipogenic‐enzyme mRNA concentrations and activities seen at weaning on the high‐carbohydrate diet but did not prevent the decrease in phosphoenolpyruvate carboxykinase mRNA and activity. In contrast, the diet containing medium‐chain fatty acids induced a slower but finally similar increase in lipogenic‐enzyme mRNA concentrations and activities. In adipose tissue, a similar trend was observed, although the inhibitory effect of the diet containing long‐chain fatty acids was considerably less marked than in liver. It is concluded that medium‐chain and long‐chain fatty acids have not the same inhibitory potency of the gene expression of lipogenic enzymes, and that long‐chain fatty acids have a more marked effect in the liver.
In‐vitro studies on the folding characteristics of the Escherichia coli precursor protein prePhoE
Authors: BREUKINK Eefjan , KUSTERS Ron , DE KRUIJFF Ben
Published: March 2005

We characterised the behaviour of the purified precursor protein prePhoE upon dilution from 8 M urea by CD, fluorescence spectroscopy and gel‐filtration techniques. It is demonstrated that prePhoE rapidly adopts β structure, folds and aggregates upon dilution to urea concentrations below 3 M. These processes are paralleled by a loss of translocation competence. Furthermore the interaction of prePhoE with SecB was investigated. SecB is shown to have a very high content of β structure, therefore we propose that precursor recognition by SecB is mediated through β‐β interaction. It is shown that SecB has little effect on the adoption of secondary structure and tertiary folding upon dilution of the precursor from urea. However, SecB prevents the precursor from aggregating by forming a functional and stable complex.
Site‐specific mutagenesis of Escherichia coli asparaginase II
Authors: WEHNER Andreas , HARMS Etti , JENNINGS Michael P. , BEACHAM Ifor R. , DERST Christian , BAST Peter , RÖHM Klaus H.
Published: March 2005

Site‐specific mutagenesis was used to replace the three histidine residues of Escherichia coli asparaginase II (EcA2) with other amino acids. The following enzyme variants were studied: [H87A]EcA2, [H87L]EcA2, [H87K]EcA2, [H183L]EcA2 and [H197L]EcA2. None of the mutations substantially affected the Km for l‐aspartic acid β‐hydroxamate or impaired aspartate binding. The relative activities towards l‐Asn, l‐Gln, and 1‐aspartic acid β‐hydroxamate were reduced to the same extent, with residual activities exceeding 10% of the wild‐type values. These data do not support a number of previous reports suggesting that histidine residues are essential for catalysis.

Spectroscopic characterization of the modified enzymes allowed the unequivocal assignment of the histidine resonances in 1H‐NMR spectra of asparaginase II. A histidine signal previously shown to disappear upon aspartate binding is due to His183, not to the highly conserved His87. The fact that [H183L]EcA2 has normal activity but greatly reduced stability in the presence of urea suggests that His183 is important for the stabilization of the native asparaginase tetramer. 1H‐NMR and fluorescence spectroscopy indicate that His87 is located in the interior of the protein, possibly adjacent to the active site.
H2‐forming methylenetetrahydromethanopterin dehydrogenase, a novel type of hydrogenase without iron‐sulfur clusters in methanogenic archaea
Authors: ZIRNGIBL Carmen , VAN DONGEN Walter , SCHWÖRER Beatrix , VON BÜNAU Rudolf , RICHTER Monika , KLEIN Albrecht , THAUER Rudolf K.
Published: March 2005

A novel hydrogenase has recently been found in methanogenic archaea. It catalyzes the reversible dehydrogenation of methylenetetrahydromethanopterin (CH2─H4MPT) to methenyltetrahydromethanopterin (CH═H4MPT+) and H2 and was therefore named H2‐forming methylenetetrahydromethanopterin dehydrogenase. The hydrogenase, which is composed of only one polypeptide with an apparent molecular mass of 43 kDa, does not mediate the reduction of viologen dyes with either H2 or CH2─H4MPT. We report here that the purified enzyme from Methanobacterium thermoautotrophicum exhibits the following other unique properties: (a) the colorless protein with a specific activity of 2000 U/mg (Vmax) did not contain iron‐sulfur clusters, nickel, or flavins; (b) the activity was not inhibited by carbon monoxide, acetylene, nitrite, cyanide, or azide; (c) the enzyme did not catalyze an isotopic exchange between 3H2 and 1H+; (d) the enzyme catalyzed the reduction of CH═H4MPT+ with 3H2 generating [methylene‐3H]CH2─H4MPT; and (e) the primary structure contained at most four conserved cysteines as revealed by a comparison of the DNA‐deduced amino acid sequence of the proteins from M. thermoautotrophicum and Methanopyrus kandleri. None of the four cysteines were closely spaced as would be indicative for a (NiFe) hydrogenase or a ferredoxintype iron‐sulfur protein.

Properties of the H2‐forming methylenetetrahydromethanopterin dehydrogenase from Methanobacterium wolfei are also described indicating that the enzyme from this methanogenic archaeon is very similar to the enzyme from M. thermoautotrophicum with respect both to molecular and catalytic properties.
Differentiation‐inducing‐factor dechlorinase, a novel cytosolic dechlorinating enzyme from Dictyostelium discoideum
Authors: NAYLER Oliver , INSALL Robert , KAY Robert R.
Published: March 2005

Differentiation‐inducing‐factor 1 (DIF‐1) is a dichlorinated alkyl phenone {1‐[(3,5‐dichloro‐2,6‐dihydroxy‐4‐methoxy)phenyl]hexan‐1‐one} from Dictyostelium discoideum, that induces amoebae to differentiate into stalk cells. It was shown previously that DIF‐1 is rapidly metabolized into a series of more polar compounds by living cells [Traynor, D. & Kay, R. R. (1991) J. Biol. Chem. 266, 5291–5297]. The first step in DIF‐1 metabolism is the formation of DIF metabolite 1 (now known to be DIF‐3) by a monodechlorination. We report here the discovery of the enzyme activity catalyzing this dechlorination. A very sensitive enzyme assay was developed, using [3H]DIF‐1 and a TLC system to separate DIF‐1 from the product, DIF‐3. DIF‐1 3(5)‐dechlorinase is present in the high‐speed supernatant of cell lysates, and uses glutathione, at physiological concentrations, as cofactor. Kinetic measurements indicate a Km for DIF‐1 of about 70 nM. The enzyme activity is inhibited by DIF‐2 (the pentan‐1‐one analogue of DIF‐1), with a median inhibitor concentration (IC50) of 1 μM, and DIF‐3 (IC50= 5 μM), which presumably act as substrates, but other compounds structurally related to DIF‐1 were much less effective. Aurothioglucose, an inhibitor of selenocysteine enzymes, inhibited DIF‐1 3(5)‐dechlorinase with IC50= 100 nM.

DIF‐1 3(5)‐dechlorinase activity is developmentally regulated. It is essentially absent from growing cells and increases at the end of aggregation to reach a first peak of activity at the first finger stage, with a further rise at culmination.
Interaction of initiation factors with the cap structure of chimaeric mRNA containing the 5′‐untranslated regions of Semliki Forest virus RNA is related to translational efficiency
Authors: BERBEN‐BLOEMHEUVEL Gerda , KASPERAITIS Marcellé A. M. , van HEUGTEN Han , THOMAS Adri A. M. , van STEEG Harry , VOORMA Harry O.
Published: March 2005

Chimaeric chloramphenicol acetyltransferase (CAT) mRNA, containing the leader sequences of genomic 42S RNA and subgenomic 26S RNA of Semliki Forest virus (SFV) were synthesized by in‐vitro transcription. These transcripts were translated with different efficiencies, as the authentic mRNA in SFV‐infected cells. Therefore, they can be used as model mRNA species to study the mechanism underlying SFV‐directed shut off of host protein synthesis. The interaction of translation initiation factors with the 5′ cap structure was studied. Transcripts prepared in vitro using T7 RNA polymrase were capped and methylated posttranscriptionally with [32P]‐GTP and S‐adenosyl‐l‐methionine to yield cap‐labelled mRNA species. Irradiation with ultraviolet light of 26S CAT and 42S CAT transcripts, together with crude rabbit reticulocyte initiation factors, resulted in the capspecific cross‐linking of eukaryotic initiation factors (eIF) eIF‐4E and eIF‐4B. The relative binding efficiency of these two factors to the cap structure of the various transcripts was, however, markedly different; the cap structure present in 26S CAT mRNA interacted efficiently with cap‐binding proteins, whereas the cap structure of 42S CAT mRNA hardly bound to these proteins. Comparable results were obtained under competitive conditions. Data are presented that the secondary structure close to the 5′ cap structure determines the efficiency of recognition of the mRNA by these initiation factors. Using a chemical cross‐linking assay, it was demonstrated that eIF‐4F, and also eIF‐4E, differentially interacted with the cap structure of the various transcripts. The data are discussed with respect to the possible mechanisms involved in SFV‐induced shut off of host cell protein synthesis.
Splicing of the platelet‐derived‐growth‐factor A‐chain mRNA in human malignant mesothelioma cell lines and regulation of its expression
Authors: LANGERAK Anthonie W. , DIRKS Ron P. H. , VERSNEL Marjan A.
Published: March 2005

Platelet‐derived‐growth‐factor (PDGF) A‐chain transcripts differing in the presence or absence of an alternative exon‐derived sequence have been described. In some publications, the presence of PDGF A‐chain transcripts with this exon‐6‐derived sequence was suggested to be tumour specific. However, in this paper it was shown by reverse‐transcription polymerase‐chain‐reaction (PCR) analysis that both normal mesothelial cells and malignant mesothelioma cell lines predominantly express the PDGF A‐chain transcript without the exon‐6‐derived sequence. This sequence encodes a cell‐retention signal, which means that the PDGF A‐chain protein is most likely to be secreted by both cell types. In cultured normal mesothelial cells, the secreted PDGF A‐chain protein might be involved in autocrine growth stimulation via PDGF α receptors. However, human malignant mesothelioma cell lines only possess PDGF β receptors. If this also holds true in vivo, the PDGF A‐chain protein produced and secreted by malignant mesothelial cells might have a paracrine function.

In a previous paper, we described elevated expression of the PDGF A‐chain transcript in human malignant mesothelioma cell lines, compared to normal mesothelial cells. In this paper, the possible reason for this elevation was studied. First, alterations at the genomic level were considered, but cytogenetic and Southern‐blot analysis revealed neither consistent chromosomal aberrations, amplification nor structural rearrangement of the PDGF A‐chain gene in the malignant cells. Possible differences in transcription rate of the PDGF A‐chain gene, and stability of the transcript between normal and malignant cells, were therefore studied. The presence of a protein‐synthesis inhibitor, cycloheximide, in the culture medium did not significantly influence the PDGF A‐chain mRNA level in normal mesothelial and malignant mesothelioma cell lines. Furthermore, nuclear run‐off analysis showed that nuclear PDGF A‐chain mRNA levels varied in both cell types to the same extent as the levels observed in Northern blots. Taken together, this suggests that increased transcription is the most probable mechanism for the elevated mRNA level of the PDGF A‐chain gene in human malignant mesothelioma cell lines.
The interaction of acetate and formate with cobalt carbonic anhydrase
Authors: BERTINI Ivano , LUCHINAT Claudio , PIERATTELLI Roberta , VILA Alejandro J.
Published: March 2005

The interaction of formate and acetate ions with cobalt‐substituted carbonic anhydrase (CA) has been investigated through 13C‐NMR and one‐dimensional and two‐dimensional 1H‐NMR spectroscopy. 13C data on formate are consistent with a regularly coordinated ligand, as previously proposed for the acetate anion [Bertini, I., Luchinat, C. & Scozzafava, A. (1977) J. Chem. Soc. Dalton Trans., 1962–1965]. 1H‐NOE experiments on both anions give evidence of through‐space interactions between ligand protons and protein protons. The latter are assigned to specific residues in the active cavity through nuclear Overhauser effect spectroscopy (NOESY) experiments. The 13C‐derived and 1H‐derived constrains allow reliable docking of these ligands in the active‐site cavity. The resulting geometries are similar to one another and consistent with five‐coordinated structures around the metal ion, as previously proposed from electronic spectroscopy [Bertini, I., Canti, G., Luchinat, C. & Scozzafava, A. (1978) J. Am. Chem. Soc. 100, 4873–4877].

The results are discussed in light of the current debate on anion binding to metal ions in carbonic anhydrase [Lindahl, M., Svensson, A. & Liljas, A. (1992) Proteins, in the press]; Bertini, I., Luchinat, C., Pierattelli, R. & Vila, A. J. (1992) Inorg. Chem., in the press; Banci, L. & Merz, K. (1992) unpublished results] and, in particular, of the proposed long Zn‐O distance found in the recent X‐ray results on the formate adduct [Hakanson, K., Carlsson, M., Svensson, A. & Liljas, A. (1992) J. Mol. Biol., in the press].
Binding of the competitive inhibitor dCDP to ribonucleoside‐diphosphate reductase from Escherichia coli studied by 1H NMR
Authors: ALLARD Peter , KUPRIN Sergei , SHEN Binghua , EHRENBERG Anders
Published: March 2005

Ribonucleoside‐diphosphate reductase (EC 1.17.4.1) from Escherichia coli consists of two nonidentical subunits, proteins R1 and R2. The binding of the product dCDP to protein R1 and to the holoenzyme R1R2 has been studied by means of 1H‐NMR spectroscopy. In presence of the effector dTTP at 25°C, dCDP was found to be in rapid exchange between the binding sites and the solvent which results in a broadening of the dCDP resonances. When both proteins R1 and R2 are present, so that the complex R1R2 is formed, a smaller broadening is observed than with protein R1 alone. No further linewidth decrease was observed when the [R2]/[R1] ratio exceeded 1. The binding constant of dCDP to R1 or R1R2 is the same, Kd= 0.9 mM. The smaller broadening of the dCDP resonances observed with the complex R1R2 as compared with R1 may be explained by the combination of two effects: (a) the overall tumbling time of the protein will increase when going from R1 to R1R2, which will cause the broadening to increase correspondingly, and (b) a twofold decrease of the number of binding sites in rapid exchange, which will decrease the broadening by a factor of 0.5. The effect of R2 without iron (apoR2) is reduced compared with native R2, probably because of some denatured proteins, while a C‐terminal peptide from R2 did not cause any narrowing at all.
Plasma‐membrane‐independent pool of the α subunit of the stimulatory guanine‐nucleotide‐binding regulatory protein in a low‐density‐membrane fraction of S49 lymphoma cells
Authors: SVOBODA Petr , KVAPIL Petr , INSEL Paul A. , RANSNÄS Lennart A.
Published: March 2005

We report that compartmentalisation of the stimulatory guanine‐nucleotide‐binding regulatory protein (Gs) exists in S49 lymphoma cells. In addition to the previously reported cytosolic form of the α subunit of Gs (Gsα) [Ransnäs, L. A., Svoboda P., Jasper, J. R. & Insel, P. A. (1989) Proc. Natl Acad. Sci. USA 86, 7900–7903], three membrane‐bound forms of Gsα were identified through ratezonal centrifugation in sucrose density gradients, Gsα‐specific anti‐peptide serum and an adenylate cyclase complementation assay. The sedimentation profile of the first pool of Gsα in the high‐density portion of the gradient (1.13–1.16 g/cm3) is identical with that of β‐adrenergic‐receptor binding, Na/K‐ATPase and adenylate cyclase activity, and may therefore be identified as plasma‐membrane fragments. The second pool, which was recovered in the middle portion of the gradient (1.09–1.11 g/cm3), contains a much lower total amount of Gsα and correlates with the endoplasmic reticulum (microsomal) enzyme markers, NADPH–cytochrome‐c reductase and glucose‐6‐phosphatase.

The identity of the third pool of Gsα located at the top of the gradient (1.06–1.08 g/cm3), is unknown. The Golgi apparatus marker, UDPgalactose:N‐acetylglucosamine glycosyltransferase, was partially recovered in this area; however, this enzyme was also present in the high‐density portion of the gradient. Complete absence of specific adenylate cyclase and Na/K‐ATPase activity indicates that this low‐density (light) membrane form of Gsα is distinct from any plasma‐membrane fragments. Furthermore, sedimentation at 100000 ×g proves its particulate (membrane) character. The light membrane form of Gsα subunit is functionally active in an adenylate cyclase complementation assay using cyc− membranes devoid of Gsα.

Overall, our data indicates that a substantial portion of Gsα is localized in membrane pools other than plasma membrane.
Affinity purification of cytochrome c reductase from potato mitochondria
Authors: BRAUN Hans‐Peter , SCHMITZ Udo K.
Published: March 2005

Ubiquinol‐cytochrome‐c oxidoreductase has been isolated from potato (Solanum tuberosum L.) mitochondria by cytochrome‐c affinity chromatography and gel‐filtration chromatography. The procedure, which up to now only proved applicable to Neurospora, yields a highly pure and active protein complex in monodisperse state. The molecular mass of the purified complex is about 650 kDa, indicating that potato cytochrome c reductase occurs as a dimer. Upon reconstitution into phospholipid membranes, the dimeric enzyme catalyzes electron transfer from a synthetic ubiquinol to equine cytochrome c with a turnover number of 50 s−1. The activity is inhibited by antimycin A and myxothiazol. A myxothiazol‐insensitive and antimycin‐sensitive transhydrogenation reaction, with a turnover number of 16 s−1, can be demonstrated as well. The protein complex consists of ten subunits, most of which have molecular masses similar to those of the nine‐subunit fungal enzyme. Individual subunits were identified immunologically and spectral properties of b and c cytochromes were monitored. Interestingly, an additional ‘core’ polypeptide which is not present in other cytochrome bc1 complexes forms part of the enzyme from potato. Antibodies raised against individual polypeptides reveal that the core proteins are clearly immuno‐distinguishable. The additional subunit may perform a specific function and contribute to the high molecular mass which exceeds those reported for other cytochrome‐c‐reductase dimers.
Stoichiometry of interaction between interferon γ and its receptor
Authors: FOUNTOULAKIS Michael , ZULAUF Martin , LUSTIG Ariel , GAROTTA Gianni
Published: March 2005

The biological response of interferon γ is mediated by binding to a specific cell‐surface receptor. We investigated the stoichiometry of this binding using soluble receptors produced in prokaryotic and eukaryotic expression systems comprising the extracellular ligand‐binding domain of the native protein. The ligand‐receptor complexes were analyzed by cross‐linking, chromatography, analytical ultracentrifugation and laser‐light scattering. Cross‐linking and chromatography showed that the stoichiometry of the interaction between ligand and receptor depends on the molar ratios of the two components mixed. All approaches confirmed that mixtures of ligand‐receptor complexes are formed with one interferon‐γ dimer bound by one or two receptors. The soluble receptor produced in Escherichia coli mainly showed a ligand/receptor stoichiometry of 1:1, while the receptors produced in eukaryotic cells showed a stoichiometry of binding of 1:2. This apparent discrepancy is most likely due to the conformational heterogeneity of the Escherichia‐coli‐derived protein.
Isolation of tocopherol‐binding proteins from the cytosol of smooth muscle A7r5 cells
Authors: NAŁȨCZ Katarzyna Anna , NAŁȨCZ Maciej Jan , AZZI Angelo
Published: March 2005

Cultured smooth muscle A7r5 cells were able to take up α‐tocopherol (32 ± 1.2 nmol/mg protein) the largest part of which (60%) was present in the cytosolic fraction. Using a tocopherol‐based affinity chromatography and α‐, β‐, γ‐, and δ‐tocopherols as eluants, three polypeptides of molecular masses 81, 58 and 31 kDa were eluted. This preparation had α‐[3H]tocopherol binding capability. The 58‐kDa polypeptide could also be eluted by chromanol and the 81‐kDa polypeptide could be eluted also by phytol. The 81‐kDa polypeptide had the unique P‐E‐E‐d‐Q‐X‐Q‐Y N‐terminal sequence.
Synovial protein kinase C and its apparent insensitivity to interleukin‐1
Authors: HULKOWER Keren I. , SAGI‐EISENBERG Ronit , TRAUB Linton M. , GEORGESCU Helga I. , EVANS Christopher H.
Published: March 2005

Lapine synovial fibroblasts produce prostaglandin E2 (PGE2) and neutral metalloproteinases in response to phorbol 12‐myristate 13‐acetate (PMA), human recombinant interleukin‐1 (hrIL‐1) and, in an autocrine fashion, in response to partially purified preparations of their own cytokines known as cell‐activating factors (CAF). Here we have examined the possible role of protein kinase C (PKC) in these responses. Whereas the 80‐kDa substrate for PKC could not be detected in synovial fibroblasts, these cells contained a 35‐kDa protein which fulfilled the criteria for qualifying as a specific substrate of PKC. Translocation assays based upon phosphorylation of the 35‐kDa protein and Western blotting techniques allowed the movement of PKC from the cytosolic to the particulate fraction in response to PMA and CAF to be detected but not in response to 4α‐PMA or hrIL‐1. Inhibitors of PKC suppressed synovial activation by PMA, partially blocked activation by CAF but had no effect on activation by hrIL‐1. There thus appear to be PKC‐dependent and PKC‐independent routes to synovial cell activation. Our data suggest that IL‐1 uses the latter, while CAF contains cytokines which utilize both routes.
Regulatory protein phosphorylation of phosphoenolpyruvate carboxylase in the facultative crassulacean‐acid‐metabolism plant
Authors: BAUR Bernhard , DIETZ Karl‐Josef , WINTER Klaus
Published: March 2005

Phosphoenolpyruvate PyrP carboxylase (PyrPC) and PyrPC kinase were copurified from dark‐adapted leaves of the common ice plant Mesembryanthemum crystallinum L. with crassulacean‐acid metabolism (CAM). Purification by (NH4)2SO4 fractionation, chromatography on Fractogel‐DEAE and hydroxylapatite resulted in a PyrPC preparation with a specific activity of 23–25 U/mg protein and a protein kinase activity of 255 μmol Pi· mol−1 PyrPC· s−1. After in vitro phosphorylation, the most prominently phosphorylated polypeptide was identified as PyrPC by immunoblotting and sequencing. Phosphorylation of PyrPC in vitro by incubation with 400 μM MgATP decreased its sensitivity towards malate. When purified in the absence of the protease inhibitor chymostatin, PyrPC lost an N‐terminal sequence of 128 amino acids. Although the carboxylation reaction was unaffected, the truncated PyrPC could neither be phosphorylated in vitro nor inhibited by malate. This result and data obtained by limited proteolysis concur with the hypothesis [Jiao, J.‐A. & Chollet, R. (1989) Arch. Biochem. Biophys. 283, 300–305] that Ser11 is the phosphorylation site of the CAM PyrPC of M. crystallinum. At pH 7.0, the Km for ATP of the protein kinase was 25 μM; phosphorylation of PyrPC was maximal after 30 min at pH 7.0. The kinase showed also activity with histone III‐S but not with dephosphorylated casein. It was inhibited by malate. The results show, that reversible protein phosphorylation is an important factor in the regulation of PyrPC in the facultative CAM plant M. crystallinum, similar to C4 and constitutive CAM plants.
Purification, characterization and gene structure of (1→3)‐β‐glucanase isoenzyme GIII from barley (Hordeum vulgare)
Authors: WANG Jun , XU Peilin , FINCHER Geoffrey B.
Published: March 2005

A new member of the barley (1→3)‐β‐glucan glucanohydrolase family of enzymes has been purified from extracts of germinated grain and young seedlings by fractional precipitation with ammonium sulphate, ion‐exchange chromatography, chromatofocussing and gel‐filtration chromatography. The enzyme, which has been designated (1→3)‐β‐glucanase isoenzyme GIII, is a basic protein with an apparent molecular mass of 32 000 Da. Oligosaccharide products released by the enzyme during hydrolysis of the (1→3)‐β‐glucan, laminarin, indicate that the enzyme is an endohydrolase. A 2349‐bp fragment of barley genomic DNA has been isolated and identified as the gene encoding the (1→3)‐β‐glucanase isoenzyme GIII. The open reading frame encoding the isoenzyme is interrupted by a single intron of 180 bp that splits a codon in the putative signalpeptide region. Northern‐blot analyses with gene‐specific probes indicate that the (1→3)‐β‐glucanase isoenzyme GIII mRNA accumulates in developing leaves; no mRNA transcripts were detected in the aleurone or scutellum of germinated grain, or in mature vegetative tissues. Although plant (1→3)‐β‐glucanases are generally classified as ‘pathogenesis‐related’ proteins, the physiological function of the barley (1→3)‐β‐glucanase isoenzyme GIII is unclear.
A gene expressed preferentially in the globular stage of somatic embryogenesis encodes elongation‐factor 1α in carrot
Authors: KAWAHARA Ryoichi , SUNABORI Satoshi , FUKUDA Hiroo , KOMAMINE Atsushi
Published: March 2005

We have isolated cDNA of genes that are preferentially expressed during somatic embryogenesis of carrot (Daucus carota L.) by differential screening of globular embryos and cells that are dividing in an unorganized manner. As a result of Northern‐blot analysis, one of the genes identified in this way, which we refer to as CEM1, was found to be expressed at high levels in somatic embryos at the globular and heart‐shaped stages. In‐situ hybridization using globular embryos revealed that the mRNA transcribed from CEM1 was located preferentially in the spherical region of the globular embryo. A homology search using the amino acid sequence deduced from the nucleotide sequence of the CEM1 cDNA revealed that CEM1 encodes the eukaryotic translational elongation‐factor 1α.
The binding of mutant actins to profilin, ATP and DNase I
Authors: DRUMMOND Douglas R. , HENNESSEY Emma S. , SPARROW John C.
Published: March 2005

Twenty‐five mutations were created in the Drosophila melanogaster Act88F actin gene by in vitro mutagenesis and the mutant actins expressed in vitro. The affinity of the mutant actins for ATP, profilin and DNase I was determined. They were also tested for conformational changes by non‐denaturing gel electrophoresis. Mutations at positions 364 (highly conserved) and 366 (invariant) caused changes in conformation, reduced ATP binding and increased profilin binding. At position 362 (invariant) only the conservative change from tyrosine to phenylalanine had no effect; other changes at this position affected conformation, ATP and profilin binding. Although only glycine or serine occur naturally at position 368, changes to threonine or glutamine had no effect on the actin. The mutant in which Asp363 was replaced by His and that in which Glu364 was replaced by Lys decreased DNase I binding, yet neither amino acid occurs in the DNase I binding site. Likewise several mutations affect ATP and profilin binding but are distant from the binding sites.

We conclude that, although actin has a highly conserved amino acid sequence, individual amino acids can have variable tolerance for substitutions. Also amino acid changes can exert significant effects on the binding of ligands to distant parts of the actin structure.
NMR study of the solution conformation of actinomycin D
Authors: YU Chin , TSENG Yu‐Yu
Published: March 2005

The solution conformation of actinomycin D, the Gram‐positive antibiotic and DNA‐binding drug, has been determined by 1H‐NMR in deuterated dimethyl sulfoxide. The structure determination is based on the experimental data set of NOE restraints. Four structures were obtained from the distance geometry and restrained molecular dynamics calculation. The resultant structures satisfy the experimental restraints very well. These structures are found to be compatible with the X‐ray crystal structures.
Characterization of a Xenopus laevis skin peptidylglycine α‐hydroxylating monooxygenase expressed in insect‐cell culture
Authors: SHIMOI Hiroko , KAWAHARA Takashi , SUZUKI Kenji , IWASAKI Yasuno , JENG Arco Y. , NISHIKAWA Yoshiki
Published: March 2005

The C‐terminal amide structure of peptide hormones and neurotransmitters is synthesized via a two‐step reaction catalyzed by peptidylgiycine α‐hydroxylating monooxygenase (PHM) and peptidylhydroxyglycine N‐C lyase. A Xenopus laevis PHM expressed in insect‐cell culture by the baculovirus‐expression‐vector system was purified to homogeneity and characterized. Using a newly established assay system for PHM, the kinetic features of this enzyme were investigated. As expected, the enzyme required copper ions, L‐ascorbate and molecular oxygen for turnover. Salts like KI and KCl, and catalase stabilized the enzyme in the presence of L‐ascorbate. The optimum pH value for the enzyme reaction was around six when Mes buffer was used and around seven when phosphate buffer was used under the same assay condition. Below pH 6, acetate, iodide and chloride ions activated the reaction. The kinetic analysis is consistent with a ping‐pong mechanism with respect to peptide and L‐ascorbate, and the peptide showed substrate inhibition. The substrate specificity of the enzyme at the penultimate position was examined by competitive assay using tripeptides with glycine at the C‐termini and the inhibitory potency of these peptides in descending order was methionine > aromatic > non‐polar amino acids.
Isolation and structural characterization of a mono‐sulfated isoglobotetraosylceramide, the first sulfoglycosphingolipid of the isoglobo‐series, from rat kidney
Authors: TADANO‐ARITOMI Keiko , KASAMA Takeshi , HANDA Shizuo , ISHIZUKA Ineo
Published: March 2005

A novel sulfoglycosphingolipid based on the isoglobo‐series core structure was isolated from rat kidney and purified by column chromatographies with DEAE‐Sephadex and silica beads. The structure was characterized by solvolysis, compositional analysis, proton NMR spectroscopy, Fourier‐transform infrared spectroscopy, methylation analysis and liquid secondary ion mass spectrometry (LSIMS). The characteristic fragment ions for a sulfate and a sulfated N‐acetylhexosamine were observed in LSIMS spectra. The two‐dimensional chemical‐shift‐correlated spectroscopy (COSY) and nuclear Overhauser enhancement spectroscopy experiments evidenced the presence of a 3‐O‐sulfated N‐acetylgalactosamine and a Galα1‐3Gal structure in the molecule. The major ceramide consisted of 4‐hydroxysphinganine linked to a C24 nonhydroxy fatty acid, deduced from both compositional analysis and LSIMS. From the above results, the following structure was established for this glycolipid: HSO3‐3GalNAcβ1‐3Galα1‐3Galβ1‐4Glcβ1‐1Cer, isoglobotetraosylceramide (iGb4Cer) IV3‐sulfate. Rat kidney also contained globotetraosylceramide (Gb4Cer) IV3‐sulfate which has a carbohydrate core identical to that from human kidney. The yields of iGb4Cer IV3‐sulfate and Gb4Cer IV3‐sulfate were 0.27 and 0.07 nmol/g wet tissue, respectively.
Spectroscopic studies on metal distribution in Co(II)/Zn(II) mixed‐metal clusters in rabbit liver metallothionein 2
Authors: POUNTNEY Dean L. , VAŠÁK Milan
Published: March 2005

Metal selectivity of metal‐thiolate clusters in rabbit liver metallothionein (MT) 2 has been studied by examining the metal distribution of two similarly sized divalent metal ions, cobalt and zinc, which have different thiolate affinity. The forms of mixed‐metal cluster species in (Co/Zn)7‐MT generated with different ratios of both metal ions offered to the metal‐free protein were investigated using EPR, ultraviolet/visible absorption and MCD spectroscopy. The results demonstrated that the distribution of these metals between the two metal‐thiolate clusters is not random. Thus, the EPR absorption intensities of the bound Co(II) ions in the Zn‐cluster matrix increased linearly up to a ratio of Co(II)/ Zn(II) equivalents of 3:4, with the final EPR intensity of three non‐interacting Co(II)‐binding sites. This EPR behaviour is consistent with a binding scheme in which one Co(II) ion occupies a metal‐binding site within the three‐metal cluster and the remaining two Co(II) ions occupy two distinctly separate sites in the four‐metal cluster. With four or more Co(II) ions in the cluster matrix, magnetic coupling between adjacent, sulphur‐bridged Co(II) ions was observed. In previous studies on mixed‐metal clusters in MT formed with Co(II)/Cd(II), Zn(II)/Cd(II) and Cd(II)/Fe(II), changes in the respective cluster volumes were shown to be a significant factor dictating the widely differring metal distributions in these systems. Based on the results of the current study, it is suggested that both the sizes of the two metal ions and their relative affinities towards the cysteine‐thiolate ligands are important in the formation of mixed‐metal clusters in MT.
Purification and paritial characterization of a thermostable trithionate hydrolase from the acidophilic sulphur oxidizer Thiobacillus acidophilus
Authors: MEULENBERG Rogier , PRONK Jack T. , FRANK Johannes , HAZEU Wim , BOS Piet , KUENEN J. Gijs
Published: March 2005

Cell‐free extracts of Thiobacillus acidophilus catalysed the quantitative conversion of trithionate (S3O62−) to thiosulphate and sulphate. A continuous assay for quantification of experimental results was based on the difference in absorbance between trithionate and thiosulphate at 220 nm.

Trithionate hydrolase was purified to near homogeneity from cell‐free extracts of T. acidophilus. The molecular masses of the native enzyme and the subunit were 99 kDa (gel filtration) and 34 kDa (SDS/PAGE). The purified enzyme has a pH optimum of 3.5–4.5 and a temperature optimum of 70°C. Enzyme activity was stimulated by sulphate. The stimulation of the enzyme activity by sulphate was half maximal at a concentration of 0.23 M. The Km for trithionate is 70 μM at 30°C and 270 μM at 70°C. Enzyme activity was lost after 36 days at 0°C, 27 days at 70°C; but after 97 days at 30°C, 40% of the initial activity was still present. The enzyme activity was inhibited by mercury chloride, N‐ethylmaleimide, thiosulphate and tetrathionate. Tetrathionate S4O62− was not hydrolysed by trithionate hydrolase.
Substrate specificity of small‐intestinal lactase
Authors: RIVERA‐SAGREDO Alfonso , CAÑADA F. Javier , NIETO Ofelia , JIMENEZ‐BARBERO Jesús , MARTIN‐LOMAS Manuel
Published: March 2005

Lactase‐phlorizin hydrolase is a disaccharidase present in the small intestine of mammals. This enzyme has two active sites, one being responsible for the hydrolysis of lactose. Lactase activity is thought to be selective towards glycosides with a hydrophilic aglycon. In this work, we report a systematic study on the importance of each hydroxyl group in the substrate molecule for lactase activity. For this purpose, all of the monodeoxy derivatives of methyl β‐lactoside and other lactose analogues are studied as lactase substrates. With respect to the galactose moiety, it is shown here that HO‐3′ and HO‐2′ are necessary for hydrolysis of the substrates by lactase. Using these chemically modified substrates, it has been confirmed that lactase does not behave as a typical β‐galactosidase, since it does not show an absolute selectivity with respect to substitution and stereochemistry at C4′ in the galactose moiety of the substrate. However, the glucose moiety, in particular the HO‐6, appears to be important for substrate hydrolysis, although none of the hydroxyl groups seemed to be essential. In order to differentiate both activities of the enzyme, a new assay for the phlorizin‐hydrolase activity has also been developed.
Genomic organization and DNA sequences of human 17β‐hydroxysteroid dehydrogenase genes and flanking regions
Authors: PELTOKETO Hellevi , ISOMAA Veli , VIHKO Reijo
Published: March 2005

Genomic 17β‐hydroxysteroid‐dehydrogenase (17‐HSD) clones were isolated from a human leucocyte genomic library using cDNA encoding human placental 17‐HSD as a probe. The overlapping fragments spanned more than 21 kbp containing the duplications, 6.2 kbp of each, as well as 7 kbp upstream and 1.6 kbp downstream from the duplicated sequences. 17 complete and eight partial Alu elements were clustered in this area, covering about 30% of the region, including the borders of the duplications. Each duplication contained a 17‐HSD gene and a conserved region of 1.56 kbp with 98% intercopy similarity. The exon structure of the 17‐HSD gene II corresponded to the known cDNA species, but both genes contained a possible promoter region with TATA, GC and inverse CAAT boxes. The 5′ flanking regions contained sequences similar to the consensus sequences of cis‐acting elements, defined as regulators of 17‐HSD gene expression. These putative sequences included estrogen and progesterone/glucocorticoid‐response elements and a cyclic‐AMP regulatory element.
Characteristics of energy‐linked proton translocation in liposome reconstituted bovine cytochrome bc1 complex
Authors: COCCO Tiziana , LORUSSO Michele , DI PAOLA Marco , MINUTO Michele , PAPA Sergio
Published: March 2005

A study is presented on the H+/e− stoichiometry for proton translocation by the isolated cytochrome bc1 complex under level‐flow and steady‐state conditions. An experimental procedure was used which allows the determination of pure vectorial proton translocation in both conditions in a single experiment. The results obtained indicate an H+/e− ratio of 1 at level‐flow and 0.3 at steady‐state. The ratios appear to be independent of the rate of electron transfer through the complex. Making use of pyranine‐entrapped bc1 vesicles, a respiration‐dependent steady‐state ΔpH value of 0.4 was determined in the presence of valinomycin. This value could be either decreased by subsaturating concentrations of the uncoupler carbonyl cyanide m‐chlorophenylhydrazone (CCCP) or increased by introducing bovine serum albumin in the assay mixture. The steady‐state H+/e− ratio appeared to be in linear inverse correlation with the ΔpH. This indicates that ΔpH exerts a control on the proton pump of the bc1 complex at the steady state.

The effect of valinomycin‐mediated potassium‐diffusion potential on electron‐transfer and proton‐translocation activities is also shown. The experiments presented show that the H+/e− ratio is unaffected, both at level flow and steady state, by an imposed diffusion potential up to around 100 mV. At higher potential values the level‐flow H+/e− ratio slightly decreased. Measurements as a function of imposed membrane potential of the rate of electron transfer at level flow and of the rate of the pre‐steady‐state reduction of b and c1 cytochromes in the complex indicate activation of electron transfer at potential values of 40–50 mV. This activation appears, however, to involve a rate‐limiting step which remains normally coupled to proton translocation.