bluuebunny/crossref_metadata_2025 huggingface dataset:
Visible, EPR and electron nuclear double‐resonance spectroscopic studies on the two metal‐binding sites of oxovanadium (IV)‐substituted D‐xylose isomerase
Authors: BOGUMIL Ralf , HÜTTERMANN Jürgen , KAPPL Reinhard , STABLER Rainer , SUDFELDT Christoph , WITZEL Herbert
Published: March 2005

The two metal‐binding sites of the D‐xylose isomerase from Streptomyces rubiginosus were studied using VO2+ as a sensor for the ligand environment. Titration of the tetrameric enzyme with VO2+, followed by EPR spectroscopy and inhibition studies, show that the first four VO2+ equivalents occupy, in analogy to Co2, Cd2+ and Pb2+, the binding site B. The visible absorption data and the EPR parameters indicate that a nitrogen ligand is involved in the ligand sphere of the high‐affinity B site. The low‐affinity A site could be studied selectively by blocking the B site with visible and EPR‐silent Cd2+. The visible data and EPR parameters for this site are consistent with a ligand environment composed of oxygen donors without nitrogen ligation. The nitrogen coordination in the high‐affinity site could be demonstrated by electron nuclear double‐resonance (ENDOR) studies of the 4VO2+ enzyme, and was assigned to a histidine ligand. The 14N resonances are interpreted in terms of a quartet with a coupling value of 13.2 MHz. 1H‐ENDOR coupling of 1.7 MHz, exchangeable in D2O, has been assigned to the N–H proton of the histidine. Additional proton ENDOR couplings, which are not exchangeable, are due to protons bound to the carbon atoms of the histidine. For the low‐affinity binding site, a nitrogen coordination could be definitely excluded by the ENDOR measurements. Exchangeable 1H‐ENDOR couplings observed in this sample were assigned to H2O ligands in the vicinity of VO2+. The results closely relate to what is known from X‐ray structure. However, the relative affinities for the two binding sites seem not to be the same for different bivalent cations.

In mixed metal samples with four VO2+ and four Co2+ equivalents, the VO2+ is distributed between both binding sites. Small changes in the complex geometry of the A site, indicated by different EPR features, seem to occur if the B site is occupied by Co2+ or by Cd2+.
Treatment of intact striatal neurones with cholera toxin or 8‐bromoadenosine 3′,5′‐(cyclic)phosphate decreases the ability of pertussis toxin to ADP‐ribosylate the α‐subunits of inhibitory and other guanine‐nucleotide‐binding regulatory proteins, Gi and Go
Authors: MAUS Marion , HOMBURGER Vincent , CORDIER Jocelyne , PANTALONI Colette , BOCKAERT Joël , GLOWINSKI Jacques , PRÉMONT Joël
Published: March 2005

Using primary cultures of striatal neurones from the mouse embryo, we showed that treatment of intact cells with cholera toxin (5 μg/ml, 22 h) decreases the subsequent ADP‐ribosylation of the α subunit of the guaninenucleotide‐binding regulatory protein Go (Goα) and the α subunit of the inhibitory guanine‐nucleotide‐binding regulatory protein (Giα) of adenylate cyclase, which is catalyzed in vitro on neuronal membranes by pertussis toxin. The inhibitory effect of cholera toxin could not only be attributed to an increased production of cAMP in neurones. Treatment of cells with 0.1 μM 8‐bromoadenosine 3′,5′‐(cyclic)phosphate (Br8cAMP) for 16 h, or with 0.1 mM Br8cAMP for 5 min, mimicked the effect of cholera toxin on the ADP‐ribosylation of Goα and Giαin vitro. However, the two agents seem to act through distinct mechanisms. The protein kinase inhibitor 1‐(5‐isoquinolinesulfonyl)‐2‐methylpiperazine prevented the action of Br8cAMP but not that of cholera toxin. In addition, measurements of the pI of the Goα, deduced from immunoblots of two‐dimensional gels performed using a specific antibody directed against Goα, suggest that treatment of neurones with cholera toxin induces ADP‐ribosylation of Goα in intact cells, while Br8cAMP does not.
Expression of three human β‐adrenergic‐receptor subtypes in transfected chinese hamster ovary cells
Authors: TATE Keri M. , BRIEND‐SUTREN Marie‐M. , EMORINE Laurent J. , DELAVIER‐KLUTCHKO Colette , MARULLO Stefano , STROSBERG A. Donny
Published: March 2005

The genes coding for three pharmacologically distinct subtypes of human β‐adrenergic receptors (β1AR, β2AR and β3AR) were transfected for expression in Chinese hamster ovary (CHO) cells. Stable cell lines expressing each receptor were analyzed by ligand binding, adenylate cyclase activation and photoaffinity labeling, and compared to βAR subtypes observed in previously described tissues, primary cultures and transfected cell lines. Each of the three receptor subtypes displayed saturable [125I]iodocyanopindolol‐binding activity. They showed the characteristic rank order of potencies for five agonists, determined by measuring the accumulation of intracellular cAMP. These recombinant cell lines express a homogeneous population of receptors and display the known pharmacological properties of β1AR and β2AR, in human tissues. It is therefore likely that the pattern of ligand binding and adenylate cyclase activation, mediated by the new β3AR in CHO cells, also reflects the yet‐undetermined pharmacological profile in humans.
Functional analysis of the pathogenesis‐related 1a protein gene minimal promoter region
Authors: BEILMANN Andrea , PFITZNER Artur J. P. , GOODMAN Howard M. , PFITZNER Ursula M.
Published: March 2005

Pathogenesis‐related (PR) proteins are a heterogeneous group of host encoded, low‐molecular‐mass proteins that are induced in plants by various external stimuli, such as pathogen attack or exposure of the plants to certain chemicals. To examine the regulation of these genes, the 5′‐flanking region of the tobacco PR‐1a gene [Pfitzner U. M., Pfitzner, A. J. P. & Goodman, H. M. (1988) Mol. Gen. Genet. 211, 290–295] was joined by a transcriptional fusion to the Escherichia coliβ‐glucuronidase (GUS) gene. Expression of the reporter gene was monitored in transient expression assays as well as in stable transformants. The PR‐1a 5′‐flanking sequences from ‐335, ‐149 or ‐71 to +28 are functional promoter elements in tobacco and carrot protoplasts, as determined by transient expression. These constructs direct correct initiation at the normal transcription‐start site of the PR‐1a gene. The level of gene expression was about twofold less than that obtained with the cauliflower mosaic virus 35S RNA promoter. Regulation of gene expression by acetylsalicylic acid, however, could not be detected in the transient assays. When the same constructs were stably integrated into the tobacco genome, neither constitutive nor induced β‐glucuronidase activity was observed. A comparison of the results from the transient and the stable transfection experiments suggests that expression of the reporter gene may be due to a constitutive transcriptional activity of the PR‐1a 5′‐flanking regions under the conditions of the transient assays and that the PR‐1a promoter may contain at least two functional domains.
Long‐chain‐acyl‐CoA synthetase activities in peroxisomes and microsomes from rat liver
Authors: LAGEWEG Wessel , WANDERS Ronald J. A. , TAGER Joseph M.
Published: March 2005

We have investigated the palmitic acid (C16:0) and cerotic acid (C26:0) activating activities in rat‐liver microsomes and peroxisomes. The activation of the two fatty acids showed similar dependencies on ATP and coenzyme A, reflected in about equal apparent Km values both in microsomes and peroxisomes. In microsomes and peroxisomes similar apparent Km values for palmitic acid were found (15 μM and 22.8 μM, respectively), whereas apparent Km values for cerotic acid were 8.4 μM and 1.0 μM in microsomes and peroxisomes, respectively. The activation of cerotic acid was found to be inhibited to a progressively greater extent by increasing concentrations of 1‐pyrenedecanoic acid (P10) as compared to the activation of palmitic acid, both in microsomes and peroxisomes. The inhibition by P10 of palmitic acid activation and cerotic acid activation was non‐competitive in both organelles. From the observation that P10 activation is not affected by palmitic acid and cerotic acid, we conclude that P10 is activated by a distinct enzyme. Furthermore, our results are in accordance with earlier suggestions that activation of cerotic acid is brought about by an enzyme distinct from the palmitoyl‐CoA synthetase.
Eicosanoid‐mediated increase in glucose and lactate output as well as decrease and redistribution of flow by complement‐activated rat serum in perfused rat liver
Authors: MUSCHOL Waldemar , PÜSCHEL Gerhard P. , HÜLSMANN Martina , JUNGERMANN Kurt
Published: March 2005

Rat serum, in which the complement sytem had been activated by incubation with zymosan, increased the glucose and lactate output, and reduced and redistributed the flow in isolated perfused rat liver clearly more than the control serum. Heat inactivation of the rat serum prior to zymosan incubation abolished this difference. Metabolic and hemodynamic alterations caused by the activated serum were dose dependent. They were almost completely inhibited by the cyclooxygenase inhibitor indomethacin and by the thromboxane antagonist 4‐[2‐(4‐chlorobenzenesulfonamide)‐ethyl]‐benzene‐acetic acid (BM 13505), but clearly less efficiently by the 5′‐lipoxygenase inhibitor nordihydroguaiaretic acid and the leukotriene antagonist N‐{3‐[3‐(4‐acetyl‐3‐hydroxy‐2‐propyl‐phenoxy)‐propoxy]‐4‐chlorine‐6‐methyl‐phenyl}‐1H‐tetrazole‐5‐carboxamide sodium salt (CGP 35949 B). Control serum and to a much larger extent complement‐activated serum, caused an overflow of thromboxane B2 and prostaglandin F2x into the hepatic vein. It is concluded that the activated complement system of rat serum can influence liver metabolism and hemodynamics via release from nonparenchymal liver cells of thromboxane and prostaglandins, the latter of which can in turn act on the parenchymal cells.
cDNA cloning and expression of the mRNA for cytochrome P‐450kd which shows a fatty acid ω‐hydroxylating activity
Authors: YOKOTANI Noboru , KUSUNOSE Emi , SOGAWA Kazuhiro , KAWASHIMA Hidenori , KINOSAKI Masahiko , KUSUNOSE Masamichi , FUJII‐KURIYAMA Yoshiaki
Published: March 2005

We have recently purified three distinct forms of fatty acid ω‐hydroxylase cytochrome P‐450 (P‐450), designated P‐450ka‐1, P‐450ka‐2 and P‐450kd, from rabbit kidney cortex microsomes, and isolated and sequenced cDNA clones corresponding to P‐450ka‐1 and P‐450ka‐2 [Yokotani, N., Bernhardt, R., Sogawa, K., Kusunose, E., Gotoh, M., Kusunose, M. & Fujii‐Kuriyama, Y. (1989) J. Biol. Chem. 264, 21665–21669]. The present paper describes cloning, sequencing and expression of a cDNA for the third fatty acid, ω‐hydroxylase, P‐450kd, from a rabbit kidney cDNA library. The cDNA for P‐450kd encodes a polypeptide of 511 amino acids with sequence similarity of 87% to P‐450ka‐1. Its deduced NH2‐terminal sequence of amino acids 5–24 is in complete agreement with the NH2‐terminal sequence of P‐450kd. The identity of the cDNA was further confirmed by its expression in COS‐7 cells. When 14C‐labeled lauric acid was added to the culture medium of COS‐7 cells transfected with the cDNA, significant amounts of radioactive dodecanedioic acid, together with ω‐ and (ω‐1)‐hydroxylauric acids, were produced. Microsomes prepared from the transfected cells also efficiently catalyzed the ω‐ and (ω‐1)‐hydroxylation of lauric acid without formation of dodecanedioic acid. RNA blot analysis demonstrated that the mRNA for P‐450kd gave a single band at the approximately 2.6‐kb position. The mRNA for P‐450kd was expressed in the liver and kidney, but not in many other tissues examined. Treatment of rabbits with clofibrate resulted in a elevated level of mRNA for P‐450kd in both liver and kidney. Furthermore, the mRNA was remarkably increased in the kidney by the administration of cyclosporin A.
Regulation of S‐adenosylmethionine decarboxylase in L1210 leukemia cells
Authors: AUTELLI Riccardo , STJERNBORG Louise , KHOMUTOV Alex R. , KHOMUTOV Radii M. , PERSSON Lo
Published: March 2005

A Potent irreversible inhibitor of S‐adenosylmethionine (AdoMet) decarboxylase, S‐(5′ ‐adenosyl)‐methylthio‐2‐aminooxyethane (AdoMeSaoe), was used to study the regulatory control of this key enzyme in the polyamine biosynthetic pathway. Treatment of L1210 cells with the inhibitor completely eradicated the growth‐induced rise in AdoMet decarboxylase activity, resulting in a marked decrease in cellular content of spermidine and spermine. The putrescine content, on the other hand, was greatly elevated. Although no detectable AdoMet decarboxylase activity was found in the L1210 cells after treatment with AdoMeSaoe, the cells contained 50‐fold higher amounts of AdoMet decarboxylase protein, compared to untreated cells during exponential growth. Part of this increase was shown to be due to elevated synthesis of the enzyme. This stimulation appeared to be related to the decrease in cellular spermidine and spermine content, since addition of either one of the polyamines counteracted the rise in AdoMet decarboxylase synthesis. The synthesis rate was determined by immunoprecipitation of labeled enzyme after a short pulse with [35S]methionine. In addition to a protein that co‐migrated with pure rat AdoMet decarboxylase (Mr∼ 32000), the antibody precipitated a somewhat larger labeled protein (Mr∼ 37000) that most likely represents the proenzyme form. Treatment of the L1210 cells with AdoMetSaoe also gave rise to a marked stabilization of the decarboxylase which contributed to the increase in its cellular protein content. Addition of spermidine did not significantly affect this stabilization, whereas the addition of spermine reduced the half‐life of the enzyme to almost that of the control cells.
Conformational fluctuations and protein reactivity
Authors: LAVALETTE Daniel , TETREAU Catherine , BROCHON Jean‐Claude , LIVESEY Alastair
Published: March 2005

When a protein's active site happens to be strongly coupled with the protein structure, the rate constant of the reaction may eventually be modulated by the conformational fluctuations. Evidence for this effect has long been provided by extensive flash photolysis investigations of liganded hemoproteins and more recently of the nonheme respiratory protein hemerythrin in hydro‐organic solvents. Within a given protein conformational substate, an elementary reaction step is characterized by one single free energy barrier and by a first‐order rate constant, k, which changes with temperature according to an Arrhenius law. At physiological temperature and low viscosity, ultrafast conformational relaxation causes efficient averaging of the reaction rates and the protein displays exponential kinetics with and average rate constant 〈k〉. Under sufficiently general conditions, it can be shown that 〈k〉 also follows a simple Arrhenius law with ‘effective’ values of the pre‐exponential factor Aeff and activation enthalpy Heff. It is found that Aeff strongly depends on the overall shape of the rate constant distribution and that Heff actually corresponds to the lower limit of the enthalpy of activation, i.e. the value associated with the highest possible reaction rate.

The underlying distribution of rate constants can be reconstructed from a set of experiments in which the kinetics depart from an exponential, i.e. at low temperature and high viscosity. The most probable distribution of exponentials consistent with the observed kinetics of the geminate recombinations of oxygen with photodissociated hemerythrin has been determined by using a new approach, known as the maximum entropy method. The results are consistent with a single pre‐exponential value and a distributed enthalpy spectrum. As expected, Heff does not coincide either with the most probable nor with the average value of the enthalpy. The most salient findings are that the probability for any protein molecule to have an enthalpy of activation equal to the effective value Heff vanishes and that Aeff differs by nearly three orders of magnitude from the true value Ao.

Biochemical reaction rates are actually average values, since protein reactions are measured under physiological conditions, where conformational relaxation is always fast. Our understanding of the significance of Aeff and Heff is therefore entirely dependent on the knowledge of the distribution function of the rate constants. In particular, enthalpy and entropy terms of similar reactions performed by different proteins cannot be compared as long as the distribution of the rate constants remains unknown.
Class π glutathione S‐transferase from pig lung
Authors: DIRR Heini W. , MANN Karlheinz , HUBER Robert , LADENSTEIN Rudi , REINEMER Peter
Published: March 2005

A cytosolic glutathione S‐transferase from pig lung was purified 210‐fold to apparent homogeneity. The enzyme was classified as a class φ isoenzyme on the basis of its physical and chemical properties. It is homodimeric with a subunit Mr of 23 500, has a pl of 7.2, and shows a high specific activity towards ethacrynic acid. The glutathione analogues, S‐hexylglutathione and glutathione sulfonate, were strong reversible inhibitors. The enzyme's primary structure, established entirely by protein chemical methods, consists of 203 amino acids and is highly similar (82–84% residue identity) to the rat and human class φ isoenzymes. Furthermore, there was no evidence of microheterogeneity or post‐translational modifications. Each subunit contains a highly reactive cysteine residue, the modification of which leads to enzyme inactivation. None of the cysteine residues in the pig enzyme appear to form intramolecular disulfide bonds. Single crystals of the glutathione‐S‐transferase‐glutathione‐sulfonate complex were obtained by the hanging‐drop method of vapour diffusion from poly(ethylene glycol) 4000 solutions. The crystals belong to the orthorhombic space group P212121 with unit cell dimensions of a= 10.125 nm, b= 8.253 nm and c= 5.428 nm and diffract to better than 0.22 nm.
Purification and characterization of a plant peroxisomal Δ2,Δ3‐enoyl‐CoA isomerase acting on 3‐cis‐enoyl‐CoA and 3‐trans‐enoyl‐CoA
Authors: ENGELAND Kurt , KINDL Helmut
Published: March 2005

A Δ3,Δ2‐enoyl‐CoA isomerase was extracted from fat‐degrading cotyledons of cucumber seedlings. The enzyme is required for the degradation of cis‐unsaturated fatty acids, e.g. linoleic acid being present in the storage tissue of cucumber seedlings in high amounts. The Δ3,Δ2‐enoyl‐CoA isomerase was exclusively localized within peroxisomes. Its purification included chromatography on a hydrophobic matrix, a cation exchanger, and on hydroxylapatite. 17000‐fold purification yielded a protein of apparent homogeneity. The isomerase is a homodimer with a Mr of 50000 and an isoelectric point of pH approximately 8.1. Δ3,Δ2‐Enoyl‐CoA isomerase reversibly catalyzes the conversion of both cis‐3‐enoyl‐CoA and trans‐3‐enoyl‐CoA into trans‐2‐enoyl‐CoA. The isomerase exhibited optimal activity at pH 9 and was active with 3‐enoyl‐CoA species having chain lengths of C6–C12, with cis‐hexenoyl‐CoA being the most effective substrate. The purified enzyme did not show enoyl‐CoA hydratase activity.
Domain structure and interaction within the pentafunctional arom polypeptide
Authors: HAWKINS Alastair R. , SMITH Melanie
Published: March 2005

The AROM locus of Aspergillus nidulans specifies a pentafunctional polypeptide catalysing five consecutive steps leading to the production of 5‐enolpyruvylshikimate 3‐phosphate in the shikimate pathway. Aided by oligonucleotide‐mediated site‐directed mutagenesis, the whole AROM locus and various overlapping subfragments from within it have been fused to the powerful hybrid trc promoter in the Escherichia coli plasmid pKK233–2. Expression of these subfragments in appropriate aro mutants of E. coli has (a) allowed the delineation of functional domains within the arom polypeptide, (b) shown that the arom polypeptide falls in two independently folding and functioning regions, the N‐terminal half specifying 3‐dehydroquinate (DHQ) synthase and EPSP synthase and the C‐terminus specifying shikimate kinase, biosynthetic 3‐dehydroquinase (DHQase) and shikimate dehydrogenase, and (c) strongly suggested an interaction between the DHQ synthase and EPSP synthase domains to stabilise the EPSP synthase activity. In addition an isoenzyme of biosynthetic DHQase, catabolic DHQase, encoded by the QUTE gene of A. nidulans has been transcribed from the trc promoter and upon isopropyl‐thio‐β‐d‐galactoside induction produces up to 20% of the total soluble cell protein.
Identification of the major protein phosphatases in mammalian cardiac muscle which dephosphorylate phospholamban
Authors: MACDOUGALL Lindsay K. , JONES Larry R. , COHEN Philip
Published: March 2005

The protein phosphatases which dephosphorylate native, sarcoplasmic reticulum (SR)‐associated phospholamban were studied in cardiac muscle extracts and in a Triton fraction prepared by detergent extraction of myofibrils, the latter fraction containing 70–80% of the SR‐associated proteins present in the tissue. At physiological concentrations of free Mg2+ (1 mM), protein phosphatase 1 (PP1) accounted for approximately 70% of the total phospholamban phosphatase activity in these fractions towards either Ser‐16 (the residue labelled by cAMP‐dependent protein kinase, PK‐A) or Thr‐17 (the residue phosphorylated by an SR‐associated Ca2+/calmodulindependent protein kinase). Protein phosphatase 2A (PP2A) and protein phosphatase 2C (PP2C) accounted for the remainder of the activity.

A major form of cardiac PP1, present in comparable amounts in both the extract and Triton fraction, was similar, if not identical, to skeletal muscle protein phosphatase 1G (PP1G), which is composed of the PP1 catalytic (C) subunit complexed to a G subunit of approximately 160kDa, responsible for targetting PP1 to both the SR and glycogen particles of skeletal muscle. This conclusion was based on immunoblotting experiments using antibody to the G subunit, ability to bind to glycogen and the release of PP1 activity from glycogen upon incubation with PK‐A and MgATP. PP1 accounted for approximately 90% of the phospholamban (Ser‐16 or Thr‐17) phosphatase activity in the material sedimented by centrifugation at 45000xg, a fraction prepared from cardiac extracts which is enriched in SR membranes. The G subunit in this fraction could be solubilised by Triton X‐100, but not with 0.5 M NaCl or digestion with α‐amylase, indicating that it is bound to membranes and not to glycogen. By analogy with the situation in skeletal muscle, the PK‐A catalysed phosphorylation of the G subunit, with ensuing release of the C subunit from the SR, may prevent PP1 from dephosphorylating SR‐bound substrates and represent one of the mechanisms by which adrenalin increases the phosphorylation of cardiac phospholamban (Ser‐16 and Thr‐17) in vivo.

Hearts left in situ post mortem lose 85–95% of their PP1 activity within 20–30 min. This remarkable disappearance of PP1 may partly explain why the importance of this enzyme in cardiac muscle metabolism has not been recognised previously.
Determination of disulfide bridges in natural and recombinant insect defensin A
Authors: LEPAGE Pierre , BITSCH Francis , ROECKLIN Dominique , KEPPI Elisabeth , DIMARCQ Jean‐Luc , REICHHART Jean‐Marc , HOFFMANN Jules A. , ROITSCH Carolyn , VAN DORSSELAER Alain
Published: March 2005

The primary‐structure comparison of natural insect defensin A from Phormia terranovae and recombinant insect defensin A from Saccharomyces cerevisiae has been accomplished using a combination of Edman degradation and liquid secondary ion mass spectrometry. The natural and recombinant proteins have the same primary structure with identical disulfide‐bond designations ( and ) as determined from the peptides obtained after thermolysin digestion. The combined use of Edman degradation and mass spectrometry allowed the disulfide‐bridge structure to be determined with a total of only 40μg (9.9 nmol) natural peptide. Mass spectrometry provides a rapid means of disulfide‐bridge verification, requiring not more than 20μg recombinant insect defensin A, which is compatible with use in batch analysis.
Staphylococcal phosphoenolpyruvate‐dependent phosphotransferase system
Authors: EISERMANN Reinhard , FISCHER Roland , KESSLER Ursula , NEUBAUER Andrea , HENGSTENBERG Wolfgang
Published: March 2005

The histidine‐containing protein (HPr) of the bacterial phosphoenolpyruvate‐dependent phosphotransferase system (PTS) was isolated from Staphylococcus carnosus and purified to homogeneity. The protein sequence was determined by Edman degradation of peptides obtained by proteolytic digestion with proteases V8, trypsin and chemical cleavage with BrCN. Furthermore, immunological screening of a chromosomal S. carnosus DNA gene library in pUC19 vector enabled us to isolate S. carnosus HPr‐expressing colonies. The nucleotide sequence of this ptsH gene and its flanking regions was determined by the dideoxy‐chain‐termination technique. Upstream, the 264‐bp open reading frame of the ptsH gene is flanked by a putative S. carnosus promoter structure and a putative ptsI gene downstream suggesting that ptsH gene is the first gene in the PTS operon of S. carnosus. Comparison of the amino acid sequence of S. carnosus HPr with the HPr sequence of Staphylococcus aureus (derived from peptide sequencing) showed a high degree of similarity.
The histidines of the iron‐uptake regulation protein, Fur
Authors: SAITO Takeshi , DULY Dominique , WILLIAMS Robert J. P.
Published: March 2005

There are 12 histidine residues/molecule in the iron‐uptake regulation protein (Fur). Here we examine their pH dependence using proton nuclear magnetic resonance spectroscopy. The histidines have widely spread acid dissociation constants but we can not offer a simple explanation for their complicated behaviour.
Enzymatic properties of phosphatidylinositol‐glycan‐specific phospholipase C from rat liver and phosphatidylinositol‐glycan‐specific phospholipase D from rat serum
Authors: STIEGER Susi , DIEM Susanna , JAKOB Andrea , BRODBECK Urs
Published: March 2005

Using anchored acetylcholinesterase from bovine erythrocytes and phosphatidylinositol‐glycan (PtdIns‐glycan) as substrate, we found PtdIns‐glycan‐anchor‐degrading activity in rat liver and serum. The hepatic enzyme was only soluble in detergents, whereas the serum enzyme occurs as soluble, slightly amphiphilic protein. Using 3‐trifluoromethyl‐3‐(m‐[125I]iodophenyl)diazirine‐labelled acetylcholinesterase as substrate, we showed that the hepatic anchor‐degrading enzyme had a cleavage specificity of a phospholipase C, whereas the serum enzyme was a phospholipase D. Both enzymes exhibited maximal activity in slightly acidic conditions and at low ionic strength. They had a high affinity for the PtdIns‐glycan anchor of the substrate (Km= 0.1 μM and 0.16 μM, respectively). Both hepatic PtdIns‐glycan‐specific phospholipase C and serum PtdIns‐glycan‐specific phospholipase D gave a large increase in activity between 0.1–10 μM Ca2+, indicating that PtdIns‐glycan‐specific phospholipases are only marginally active at physiological intracellular Ca2+ concentrations. The enzymes were inhibited by heavy metal chelating agents such as 1, 10‐phenanthroline and 2,2′‐bipyridyl but not by the corresponding Fe2+ complexes or non‐chelating analogues, indicating that they both require a heavy metal ion for the expression of catalytic activity in addition to Ca2+. Another interesting property of PtdIns‐glycan‐specific phospholipases is their inactivation by bicarbonate and cyanate. The inactivation was time‐ and pH‐dependent and could be reversed by dialysis. These observations are in agreement with a covalent modification of the enzymes by carbamoylation.
Site‐directed chemical conversion of serine to cysteine in penicillin acylase from Escherichia coli ATCC 11105
Authors: SLADE Andrew , HORROCKS A. Janet , LINDSAY Christopher D. , DUNBAR Bryan , VIRDEN Richard
Published: March 2005

Penicillin acylase (EC 3.5.1.11) was completely inactivated with equimolar phenylmethane [35S]sulphonyl fluoride (PhMe35SO2F); the stability of the sulphonyl group in the modified protein was determined by measurement of the radioactivity in ultrafiltrates. In 8 M urea, the rate of loss of the sulphonyl group was similar to that observed in PhMeSO2F‐inactivated chymotrypsin [Gold, A. M. & Fahrney, D. (1964) Biochemistry 3, 783–791]. Incubation of the PhMeSO2F‐inactivated acylase with 0.7 M potassium thioacetate yielded an acetylthiol enzyme which was subsequently converted to a thiol‐enzyme during incubation with 10 mM 6‐aminopenicillanic acid. 4‐Pyridyl‐ethylcysteine was released by acid hydrolysis after reaction of the thiol‐protein with 4‐vinylpyridine. The rates of reaction of thiol‐penicillin acylase with iodoacetic acid and 2,2′‐dipyridyl disulphide were consistent with the presence of an incompletely accessible cysteinyl sidechain. After carboxymethylating the thiol‐enzyme with iodo[2‐3H]acetic acid, the label was shown by SDS/PAGE and sequencing analysis to be associated exclusively with the β‐chain NH2‐terminal residue, indicating conversion of Ser290 to S‐carboxymethyl‐cysteine. Near‐ultraviolet CD spectra showed the conformation of thiol‐penicillin acylase to be indistinguishable from that of the native protein but the catalytic activity was less than 0.02% of that of the normal enzyme. The possibility that Ser290 acts as a nucleophile in catalysis is discussed.
Binding of α‐ and βγ‐subunits of G0 to β1‐adrenoceptor in sealed unilamellar lipid vesicles
Authors: KURSTJENS Nicol P. , FRÖHLICH Margareta , DEES Christian , CANTRILL Richard C. , HEKMAN Mirko , HELMREICH Ernst J. M.
Published: March 2005

First, we describe a preparation of sealed unilamellar lipid vesicles. When this preparation was subjected to sucrose density gradient centrifugation, two rather uniform fractions emerged, one consisting of lighter lipid‐rich vesicles with average diameters ranging over 150–200 nm (fraction I), the other consisting of heavier vesicles with average diameters ranging over 30–70 nm (fraction II). When the lipid mixture containing dimyristoylglycero‐phosphocholine, cholesterol, dipalmitoylglycerophosphoserine and dipalmitoyglycerophosphoethanolamine at molar ratios of 54:35:10:1 was reconstituted with α‐ and γ‐subunits of G0‐proteins purified to homogeneity from bovine brain, the lipid‐rich lighter vesicle fraction I took up these subunits nearly exclusively. Whereas, when a β1‐adrenoceptor preparation purified from turkey erythrocyte membranes was reconstituted, it was found nearly completely in the smaller heavier vesicle fraction II where it was incorporated inside‐out. On co‐reconstitution of either αo or alone with β1‐adrenoceptors, some of these subunits appear together with β1‐adrenoceptors in the small vesicle fraction II, but much more αo was bound to the receptor in the presence of βγ‐subunits.

The observations reported are novel and surprising in several respects: firstly, they suggest that βγ‐subunits can bind to the non‐activated β1‐receptor where they may serve as an anchor for α‐subunits. Secondly, the binding of αo‐ and βγ‐subunits to the β1‐adrenoceptors enhances the basal GTPase activity of αo. Thirdly, since the binding domains of the β1‐adrenoceptor for G‐proteins were facing outwards in our sealed vesicle preparations, it follows that interactions of G‐proteins with the β‐receptor can occur at the aqueous membrane interface as was postulated originally by M. Chabre [Trends Biochem. Sci. 12, 213–215 (1987)] for the transducin‐rhodopsin interactions. Finally, the binding of Go‐subunits from bovine brain to a β1‐adrenoceptor from turkey erythrocytes was not expected, since these polypeptides are not likely to be physiological partners.
Studies on the molecular recognition of synthetic methyl β‐lactoside analogs by ricin, a cytotoxic plant lectin
Authors: RIVERA‐SAGREDO Alfonso , SOLIS Dolores , DIAZ‐MAURIÑO Teresa , JIMENEZ‐BARBERO Jesús , MARTIN‐LOMAS Manuel
Published: March 2005

The binding of methyl β‐lactoside and of all possible monodeoxy derivatives of methyl β‐lactoside to the galactose‐specific highly cytotoxin lectin ricin, has been investigated. The distribution of low‐energy conformers of the disaccharide structures has been first determined using molecular‐mechanics calculations and high‐resolution NMR spectroscopy. The nuclear Overhauser enhancements and specific deshieldings observed are in agreement with a similar distribution of low‐energy conformers for all studied compounds which may be described by a major conformer defined by φ (H1′‐C1′‐O1′‐C4) and Ψ (C1′‐O1′‐C4‐H4) torsion angles of 49° and 5°, respectively, with contribution of conformers with angles φ/Ψ 24°/– 59°, 22°/– 32° and 6°/– 44°. Assuming that the disaccharides bind to the lectin in these preferred conformations, the apparent dissociation constants for the ricin‐disaccharide complexes have been interpreted in terms of specific polar and nonpolar interactions. In agreement with X‐ray data, the hydroxyl groups at positions 3, 4 and 6 of the β‐d‐galactopyranose moiety appear as key polar groups in the interaction with ricin. These results are in contrast to previous results which have established that position 6 is not involved in lectin binding. An important nonpolar interaction involving position 3 of the β‐d‐glucopyranose moiety, seems to be operative. The distribution of low‐energy conformers of these disaccharide structures permits this interaction to take place with the hydroxyl group at this position intramolecularly bonded, thus rendering this region of the molecule more lipophylic in character for acceptance into nonpolar regions of the combining site.
Sensitive titration microcalorimetric study of the binding of Salmonella O‐antigenic oligosaccharides by a monoclonal antibody
Authors: SIGURSKJOLD Bent W. , ALTMAN Eleonora , BUNDLE David R.
Published: March 2005

The binding of several oligosaccharide haptens by a monoclonal antibody, Se155–4, specific for Salmonella serogroup B O‐antigen was studied by titration microcalorimetry. In the software developed by Wiseman et al. [Wiseman, T., Williston, S. & Brandts, J. F. (1989) Anal. Biochem. 17, 131–137] the number of binding sites/macromolecule is one of the optional regression parameters in the non‐linear least‐squares analysis of the calorimetric data. Instead, an approach was adopted in which the concentration of binding sites was treated as a regression parameter, obviating the requirement for precise values of antibody absorption coefficients and minimizing effects due to partially inactive antibody preparations. Furthermore, performing the least‐squares analysis in two steps, first using a differential heat mode and then an integral heat mode, was shown to yield the most accurate results. The technique gave accurate results using not more than 1–2 μmol ligand and less than 7 mg antibody. Haptens 2–5 were oligomers of the O‐antigenic repeating unit varying in chain length by 2–5 repeating units and a trisaccharide glycoside 1, which filled the binding site. The latter hapten exhibited a favourable entropy contribution to binding (ΔG°=− 31 kJ · mol−1; ΔH°=−21 kJ · mol−1 and −TΔS°−10 kJ · mol−1), while all four oligomers 2–5 showed a constant binding energy ΔG°=·33 kJ · mol−1, composed of increasingly stronger enthalpy forces compensated by an increasingly unfavourable entropy contribution. These observations are compared with results from enzyme immunoassays and a high‐resolution crystal structure for the dodecasaccharide 3 bound to the Fab derived from Se155–4.
Glycolysis at the climacteric of bananas
Authors: BALL Kathryn L. , GREEN John H. , REES Tom ap
Published: March 2005

This work was carried out to investigate the relative roles of phosphofructokinase and pyrophosphate—fructose‐6‐phosphate 1 ‐phosphotransferase during the increased glycolysis at the climacteric in ripening bananas (Musa cavendishii Lamb ex Paxton). Fruit were ripened in the dark in a continuous stream of air in the absence of ethylene. CO2 production, the contents of glucose 6‐phosphate, fructose 6‐phosphate, fructose 1, 6‐bisphosphate, phosphoeno/pyruvate and PPi; and the maximum catalytic activities of pyrophosphate—fructose‐6‐phosphate 1‐phosphotransferase, 6‐phosphofructokinase, pyruvate kinase and phosphoenolpyruvate carboxylase were measured over a 12‐day period that included the climacteric. Cytosolic fructose‐1, 6‐bisphosphatase could not be detected in extracts of climacteric fruit. The peak of CO2 production was preceded by a threefold rise in phosphofructokinase, and accompanied by falls in fructose 6‐phosphate and glucose 6‐phosphate, and a rise in fructose 1, 6‐bisphosphate. No change in pyrophosphate—fructose‐6‐phosphate 1‐phosphotransferase or pyrophosphate was found. It is argued that phosphofructokinase is primarily responsible for the increased entry of fructose 6‐phosphate into glycolysis at the climacteric.
Phosphorylation of keratin intermediate filaments by protein kinase C, by calmodulin‐dependent protein kinase and by cAMP‐dependent protein kinase
Authors: YANO Takeo , TOKUI Toshiya , NISHI Yoshimi , NISHIZAWA Kimiko , SHIBATA Masao , KIKUCHI Kunimi , TSUIKI Shigeru , YAMAUCHI Takashi , INAGAKI Masaki
Published: March 2005

Keratins, constituent proteins of intermediate filaments of epithelial cells, are phosphoproteins containing phosphoserine and phosphothreonine. We examined the in vitro phosphorylation of keratin filaments by cAMP‐dependent protein kinase, protein kinase C and Ca2+/calmodulin‐dependent protein kinase II. When rat liver keratin filaments reconstituted by type I keratin 18 (molecular mass 47 kDa; acidic type) and type II keratin 8 (molecular mass 55 kDa; basic type) in a 1:1 ratio were used as substrates, all the protein kinases phosphorylated both of the constituent proteins to a significant rate and extent, and disassembly of the keratin filament structure occurred. Kinetic analysis suggested that all these protein kinases preferentially phosphorylate keratin 8, compared to keratin 18. The amino acid residues of keratins 8 and 18 phosphorylated by cAMP‐dependent protein kinase or protein kinase C were almost exclusively serine, while those phosphorylated by Ca2+/calmodulin‐dependent protein kinase II were serine and threonine. Peptide mapping analysis indicated that these protein kinases phosphorylate keratins 8 and 18 in a different manner. These observations gave the way for in vivo studies of the role of phosphorylation in the reorganization of keratin filaments.
Post‐translational processing of the amino terminus affects actin function
Authors: HENNESSEY Emma S. , DRUMMOND Douglas R. , SPARROW John C.
Published: March 2005

We have studied the importance of N‐terminal processing for normal actin function using the Drosophila Act88F actin gene transcribed and translated in vitro. Despite having different charges as determined by two‐dimensional (2D) gel electrophoresis, Act88F expressed in vivo and in vitro in rabbit reticulocyte lysate bind to DNase I with equal affinity and are able to copolymerise with bulk rabbit actin equally well. Using peptide mapping and thin‐layer electrophoresis we have shown that bestatin ([3‐amino‐2‐hydroxy‐4‐phenyl‐butanoyl]‐l‐leucine), an inhibitor of aminopeptidases, can inhibit actin N‐terminal processing in rabbit reticulocyte lysate. Although processed and unprocessed actins translated in vitro are able to bind to DNase I equally well, unprocessed actins are less able to copolymerise with bulk actins. This effect is more pronounced when bulk rabbit actin is used but is still seen with bulk Lethocerus actin. Also, the unprocessed actins reduce the polymerisation of the processed actin translated in vitro with the bulk rabbit actin. This suggests that individual actins do interact, even in non‐polymerising conditions. The reduced ability of unprocessed actin to polymerise shows that correct post‐translational modification of the N terminus is required for normal actin function.
Pseudomonas 3β‐hydroxysteroid dehydrogenase
Authors: YIN Shih‐Jiun , VAGELOPOULOS Nikolaos , LUNDQUIST Gunilla , JÖRNVALL Hans
Published: March 2005

The 3β‐hydroxysteroid dehydrogenase of Pseudomonas testosteroni commercially available was purified by an FPLC step and submitted to sequence determination by peptide analysis. The structure obtained reveals a 253‐residue polypeptide chain, with an N‐terminal, free α‐amino group, and a low cysteine content. Comparisons with other hydroxysteroid dehydrogenases recently characterized reveal distant similarities with prokaryotic and, to some extent, also eukaryotic forms of separate specificities. Residue identities with a Streptomyces 20β‐hydroxysteroid dehydrogenase are 35% and distributed over the entire molecule, whereas residue identities with the mammalian 17β‐hydroxysteroid dehydrogenase only constitute 20%, and are essentially limited to the N‐terminal and central parts, Nevertheless, all these enzymes exhibit a conserved tyrosine residue (position 151 in the present enzyme) noted as possibly having a functional role in some members of this protein family. Combined, the results establish the prokaryotic 3β‐hydroxysteroid dehydrogenase as belonging to the family of short‐chain alcohol dehydrogenases, reveal that the hydroxysteroid dehydrogenases are no more closely related than dehydrogenases with other enzyme activities within the family (e.g. glucose, ribitol, hydroxyprostaglandin dehydrogenases), show several of the mammalian hydroxysteroid dehydrogenases to have subunits of longer size with different patterns of similarity than those of the prokaryotic family members characterized, and define important segments of the coenzyme‐binding region for this enzyme group.
Purification and characterization of NADPH‐dependent 2‐oxoaldehyde reductase from porcine liver
Authors: LIANG Zhi‐Qun , HAYASE Fumitaka , KATO Hiromichi
Published: March 2005

An enzyme which catalyzes the reduction of 3‐deoxyglucosone to 3‐deoxyfructose and methylglyoxal to acetol, was isolated and purified from porcine liver. 2‐Oxoaldehyde compounds were found to be especially good substrates and monocarbonyl compounds were poor substrates for this reductase. The optimum pH of the enzyme activity was 6.5. The Km for 3‐deoxyglucosone and methylglyoxal were 2.1 mM and 3.3 mM, respectively. The enzyme consisted of a single polypeptide chain with a molecular mass of 38 kDa. The activity of the enzyme was completely inhibited by p‐chloromercuribenzoate. The enzyme inhibited the advanced stage of the Maillard reaction.
Carboxypeptidase yscS: gene structure and function of the vacuolar enzyme
Authors: SPORMANN Dirk O. , HEIM Jutta , WOLF Dieter H.
Published: March 2005

The gene encoding carboxypeptidase yscS in Saccharomyces cerevisiae, CPS1, was cloned by complementation of the cps1‐3 mutation. The cloned CPS1 gene, which again enabled a leucine auxotrophic cps1‐3 mutant to grow on the modified dipeptide Cbz‐Gly‐Leu (Cbz, benzyloxycarbonyl) as sole leucine source, was sequenced and found to consist of an open reading frame of 1728 bp encoding a protein of 576 amino acids. The putative protein contains a hydrophobic stretch of 20 amino acids and a putative signal sequence cleavage site. Five putative N‐glycosylation sites are also in the protein sequence. This data is consistent with the previous finding of carboxypeptidase yscS being a vacuolar peptidase. Chromosomal disruption of the CPS1 gene completely abolishes carboxypeptidase yscS activity. This protein is yet another member of the peptidases in S. cerevisiae involved in nitrogen metabolism.
Mouse ornithine decarboxylase is phosphorylated by casein kinase‐II at a predominant single location (serine 303)
Authors: ROSENBERG‐HASSON Yael , STRUMPF Dan , KAHANA Chaim
Published: March 2005

Ornithine decarboxylase (ODC), a key enzyme in the biosynthetic pathway of polyamines in mammalian cells is characterized by an extremely short half‐life and by a rapid induction following stimulation with growth‐promoting agents. Inspection of its deduced amino acid sequence revealed the presence of sequences that may serve as targets for phosphorylation by casein kinase II (CK‐II). In the present study we demonstrate that ODC serves as a substrate for phosphorylation by CK‐II in vitro and that it is phosphorylated in intact mammalian cells. One‐dimensional phosphopeptide analysis demonstrated that all the phosphopeptides generated by V8 protease digestion of in vivo phosphorylated ODC correspond to the major phosphopeptides of ODC phosphorylated in vitro by CK‐II. Phosphopeptide analysis of wild‐type ODC and of a mutant in which serine 303 was converted to alanine demonstrated that the latter lacks the phosphopeptides that correspond to those detected in ODC phosphorylated in vivo. In addition, no incorporation of phosphate into the alanine 303 mutant was observed when it was expressed in transfected cos cells. Based on these observations, we conclude that in mammalian cells serine 303 is the major (if not the only) phosphorylated residue of ODC and that CK‐II or another cellular kinase with very similar sequence specificity is responsible for manifestation of this modification. The unphosphorylated alanine 303 mutant retained enzymatic activity, which decayed at a similar rate to that of the wild‐type enzyme. We therefore conclude that phosphorylation is not essential for maintaining enzymatic activity or regulating ODC turnover.
Lipid interaction of diphtheria toxin and mutants
Authors: DEMEL Rudy , SCHIAVO Giampietro , KRUIJFF Ben de , MONTECUCCO Cesare
Published: March 2005

To study the structural change of diphtheria toxin (DT) induced by low pH and its influence on the interaction with membrane lipids, protein and lipid monolayers were formed and characterized. DT at neutral and acidic pH forms stable monolayers, whose surface‐pressure‐increase curves allow an estimation of the apparent molecular area of 29.5 nm2/molecule at pH 7.4 (corresponding to a radius of 3.06 nm) and 34.5 nm2/molecule at pH 5.0 (corresponding to a radius of 3.32 nm).

DT at pH 7.4 does not insert into phospholipid monolayers, while at pH 5.0 it penetrates into the lipid layer with a portion of apparent molecular area of 21.0 nm2/molecule (corresponding to a radius of 2.6 nm).

The low‐pH driven lipid interaction of the toxin is favoured by the presence of acidic phospholipids, without an apparent requirement for a particular class of negative lipids. The DT mutants crm 45 and crm 197 are capable of hydrophobic interaction already at neutral pH and cause an increase of surface pressure with a further increase upon acidification.
Structural and biological properties of human recombinant myeloperoxidase produced by Chinese hamster ovary cell lines
Authors: MOGUILEVSKY Nicole , GARCIA‐QUINTANA Lida , JACQUET Alain , TOURNAY Christophe , FABRY Luc , PIÉRARD Laurent , BOLLEN Alex
Published: March 2005

The cDNA encoding human myeloperoxidase carries three ATG codons in frame; 144, 111 and 66 bp upstream from the proprotein DNA sequence. In order to determine the most efficient signal sequence, three cDNA modules starting at each of the ATG were cloned into an eucaryotic expression vector and stably in Chinese hamster ovary cell lines. In all three cases, recombinant human myeloperoxidase (recMPO) was secreted into the culture medium of transfected cells, indicating that each of the signal peptides functions efficiently. One of the recombinant cell lines, which was amplified using methotrexate, overexpresses enzymatically active recMPO up to 6 μg · ml−1· day−1. The recombinant product was purified by a combination of ion‐exchange and metalchelate chromatography, and characterized in terms of molecular mass, amino‐terminal amino acid analysis, glycosylation, physicochemical properties and biological activity. The data show that recMPO is secreted essentially as a monomeric, heme‐containing, single‐chain precursor of 84 kDa which exhibits peroxidase activity. Aminoterminal analysis indicated that cleavage of the signal peptide occurs between amino acids 48 and 49. In addition, recMPO appeared to be glycosylated up to the last stage of sialylation, to an extent similar to that of the natural enzyme. Specific activity measurements as well as stability data, in various pH, temperature, ionic strength and reducing conditions, indicated that the recombinant single‐chain enzyme behaves essentially in the same way as the natural two‐chain molecule. Finally, recMPO was shown to exert potent cytotoxicity towards Escherichia coli when provided with its physiological substrates, i. e. hydrogen peroxide and chloride ions.