bluuebunny/crossref_metadata_2025 huggingface dataset:
Modulation in vivo of β-adrenergic-receptor subtypes in rat brown adipose tissue by the thermogenic agonist Ro 16–8714
Authors: Revelli J P , Muzzin P , Giacobino J P
Published: August 2015

The number of beta 3-adrenergic receptors (AR) in plasma membranes from interscapular brown adipose tissue (IBAT) was decreased by 62% in lean Zucker rats treated with the thermogenic beta-adrenergic agonist Ro 16-8714 as compared with controls after 72 h of treatment. The loss of beta 3-AR number was preceded by a 93% decrease in the steady-state level of beta 3-AR mRNA at 30 h. Similar results were obtained in obese (fa/fa) Zucker rats. Ro 16-8714 had no effect on the number of beta 1- and beta 2-ARs in IBAT. This is the first report to demonstrate that the beta 3-AR in IBAT can be specifically down-regulated in vivo by exposure to a thermogenic agonist.
Two distinct transcriptional activities of pea (Pisum sativum) chloroplasts share immunochemically related functional polypeptides
Authors: Lakhani S , Khanna N C , Tewari K K
Published: August 2015

An RNA polymerase activity has been purified from pea (Pisum sativum) chloroplast extracts with a distinct transcriptional specificity for a chloroplast messenger gene. This activity (ms-RNA pol) differs from the pea RNA polymerase preparation reported by Sun, Shapiro, Wu & Tewari [(1986) Plant Mol. Biol. 6, 429-439], which specifically transcribes only the rRNA gene (rb-RNA pol). The specificity of transcription has been assessed by the synthesis in vitro of discrete transcripts of predicted sizes using cloned promoter regions of the chloroplast psbA and 16 S rRNA genes. The ms-RNA pol preparation, with polypeptides ranging in apparent molecular mass from 22 to 180 kDa, correctly initiates transcription from recombinant plasmids containing the psbA promoter and does not support 16 S rRNA promoter-directed transcription. The two activities differ also in their response to Mn2+ ions. To investigate whether the two transcriptional activities share common functional polypeptides, monoclonal antibodies were developed against the rb-RNA pol preparation. Three clones were selected on the basis of their ability to inhibit transcription in vitro of the 16 S rRNA gene by rb-RNA pol. The antibodies from these clones independently recognized three polypeptides with molecular masses of 27, 90 and 95 kDa on immunoblots. Antibodies cross-reacting with the 90 kDa polypeptide completely eliminated the specific retardation of an end-labelled 16 S rRNA promoter fragment in a mobility-shift assay, whereas the antibodies against the 95 kDa polypeptide resulted in the formation of a ternary complex (enzyme-DNA-antibody). The antibodies cross-reacting with the 27 kDa polypeptide, however, did not alter the mobility of the retarded DNA-enzyme complex on the gel. These antibodies also inhibited transcription in vitro of the psbA gene by ms-RNA pol and recognized polypeptides of identical molecular masses in the ms-RNA pol. These results show that the three polypeptides are functional components of the chloroplast transcriptional complex and appear to be involved in the transcription of both rRNA and mRNA genes. Transcriptional specificity is probably conferred by ancillary transcription factor(s) which remain to be identified.
A cortical phosphoprotein (‘PP63’) sensitive to exocytosis triggering in Paramecium cells. Immunolocalization and quenched-flow correlation of time course of dephosphorylation with membrane fusion
Authors: Höhne-Zell B , Knoll G , Riedel-Gras U , Hofer W , Plattner H
Published: August 2015

We had previously shown that a phosphoprotein of 63 kDa (‘PP63’) is rapidly and selectively dephosphorylated during synchronous (less than or equal to 1 s) trichocyst exocytosis in Paramecium cells and then rephosphorylated within less than or equal to 1 min [Zieseniss & Plattner (1985) J. Cell Biol. 101, 2028-2035]. Using a new quenched-flow device, we now find a strict correlation between PP63 dephosphorylation and the process of membrane fusion, both occurring within 80 ms. Uptake of 32P over 90 min, followed by exocytosis and rephosphorylation for 1 min, results in a rather selective phosphorylation of the dephosphorylated form, P63, to PP63. Solubilization by repeated freezing and thawing allows isolations of P63 and PP63. On isoelectric focusing autoradiograms they have pI values of 6.05, 5.95 (major spots), 5.85 and 5.75. All spots are sensitive to alkaline, but not to acidic, hydrolysis (except for the pI-6.05 spot). On two-dimensional-gel autoradiograms the most prominent spot, of pI 5.95, is most extensively de- and re-phosphorylated. This spot, from de- and re-phosphorylated samples, was used to produce monospecific antibodies. A cortical localization of PP63 was revealed by producing Western blots from isolated cell-surface fragments (‘cortices’) and by immunofluorescence labelling. We assume that both P63 and PP63 are attached to cortical structures, e.g. around trichocysts, though they are partly soluble. This localization and the strict correlation of PP63 dephosphorylation with exocytotic membrane fusion suggests a role in fusion regulation.
Clearance from plasma of lymph chylomicrons and chylomicron remnants labelled with 125I-tyramine-cellobiose
Authors: Ly H L , Mortimer B C , Baker E , Redgrave T G
Published: August 2015

The aims of the present study were to evaluate the metabolism of chylomicrons (CM) and of CM remnants after labelling with radioactive iodine and converting the iodinated CM into remnants in vitro. Lymph CM were radiolabelled with 125I or sham-labelled with 127I by either the ICl procedure or the tyramine-cellobiose (TC) procedure, then injected into rats. The clearance from plasma of the iodinated CM was compared with control non-iodinated lipid-labelled CM. After iodination with ICl, the plasma removal of endogenously labelled CM was significantly different from non-iodinated CM, with increased uptake of CM triacylglycerols by the liver. In contrast, the clearances from plasma and the uptake by organs of radiolabelled lipids of CM iodinated by the TC method (TC-CM) were similar to control CM. About 40% of the label from 125I-TC-CM was insoluble in 50% propan-2-ol, indicating association with CM apolipoprotein B48. Only about 8% of label was lipid soluble, mostly in phosphatidylethanolamine. Radioactivity from 125I-TC-CM injected intravenously in rats was cleared rapidly and by 30 min only 20% remained in plasma, whereas 48% was recovered in the liver. After fractionation of the plasma by density-gradient ultracentrifugation, most label remained associated with d (relative density) less than 1.006 lipoproteins. In intact rats label was also found associated with the low-density and high-density lipoprotein fractions of plasma. When the liver was excluded from circulation, the recovery of label in low-density- and high-density-lipoprotein fractions was greatly decreased. CM remnants were prepared in vivo by injecting 125I-TC-CM into functionally hepatectomized donors and compared with remnants prepared in vitro by incubation with purified bovine milk lipoprotein lipase. Although remnants prepared in vitro cleared from plasma slower than remnants prepared in vivo, the size, lipid composition and apolipoprotein profile on gradient PAGE of the remnants were similar. We conclude that labelling of CM by the TC method avoided the ‘artefactual’ changes in metabolism seen after labelling by the ICl procedure. CM remnants when prepared in vitro using lipoprotein lipase were found to be similar to those prepared in vivo after injection into functionally hepatectomized rats.
Human neutrophil phospholipase D activation by N-formylmethionyl-leucylphenylalanine reveals a two-step process for the control of phosphatidylcholine breakdown and oxidative burst
Authors: Gélas P , Von Tscharner V , Record M , Baggiolini M , Chap H
Published: August 2015

A comparative study of real-time kinetics of respiratory burst, monitored by H2O2-dependent chemiluminescence, and phospholipase D (PLD)-mediated phosphatidylcholine breakdown has been undertaken on human neutrophils stimulated by N-formylmethionyl-leucylphenylalanine in the absence of cytochalasin B. The fungal metabolite 17-hydroxywortmannin (HWT), an inhibitor of NADPH oxidase activation, decreases phosphatidic acid (PA) production by 30% at a concentration of 1 nM. Higher concentrations (10 nM-1 microM) inhibit PA formation maximally by 50% as compared with control. In all cases, the inhibition is delayed by 20-30 s after addition of the agonist. Thus the full PA generation is actually the result of an early (HWT-insensitive) and a late (HWT-sensitive) phosphatidylcholine breakdown. However, under all conditions, alkylacylglycerol remains at the basal level. PLD activity is dependent on Ca2+ influx, but is fully inhibited in cells depleted of Ca2+ with EGTA and Quin 2. The effect of HWT on the respiratory burst was investigated by measuring the kinetics of H2O2-induced chemiluminescence. This method allows to distinguish various phases of superoxide ion production: a lag, an increase in H2O2 formation (early phase), the duration of H2O2 production (late phase) and the termination of the oxidative burst. The lag remains constant for all HWT concentrations. A concentration of 10 nM-HWT, which fully inhibits the HWT-sensitive part of PA production, decreases superoxide ion production with a delay of about 20 s after addition of the agonist. Higher HWT concentrations, which have no additional effect on PLD inhibition, equally affect an early and a late phase of the burst. Thus high doses of HWT have a site of action which decreases the whole burst but does not affect the PLD any more. Therefore HWT and Ca2+ provide evidence for a two-step process for PLD activation. Only the delayed PA generation is functionally linked to a late phase of the oxidative burst.
Substrate specificity of the purine-2′-deoxyribonucleosidase of Crithidia luciliae
Authors: Steenkamp D J , Hälbich T J F
Published: August 2015

The purine-2′-deoxyribonucleosidase of Crithidia luciliae catalyses an efficient deoxyribosyl transfer between a variety of purine bases, benzimidazole and 5,6-dimethylbenzimidazole. Since the deoxyriboside of a deoxyribosyl acceptor is necessarily also a substrate, the trans-N-deoxyribosylase activity of the enzyme allows a study of its specificity to be extended to a large number of purines and purine analogues. Amongst 27 different deoxyribosyl acceptors, only hypoxanthine gave rise to isomeric products. The introduction of methyl groups at appropriate positions in either purine or benzimidazole lowered the Michaelis constant, KB, for deoxyribosyl acceptors: by about 10-fold for 6-methylpurine (KB 351 +/- 87 microM) compared with purine (KB 3.91 +/- 0.8 mM) and by about 10(3)-fold for 5,6-dimethylbenzimidazole (KB 7.0 +/- 0.79 microM) compared with benzimidazole (Km,app. 7.8 +/- 2.4 mM). The maximal rates of deoxyribosyl transfer to different acceptors, on the other hand, varied by only 4.5-fold, and can be ascribed to decreases in the rate of release of the newly formed purine deoxyriboside from the enzyme.
Inducible overproduction of the Aspergillus nidulans pentafunctional AROM protein and the type-I and -II 3-dehydroquinases from Salmonella typhi and Mycobacterium tuberculosis
Authors: Moore J D , Lamb H K , Garbe T , Servos S , Dougan G , Charles I G , Hawkins A R
Published: August 2015

The aroQ gene of Mycobacterium tuberculosis, encoding a type-II 3-dehydroquinase, and the aroD gene of Salmonella typhi, encoding a type-I 3-dehydroquinase, have been highly overexpressed in Escherichia coli using the powerful trc promoter contained within the expression vector pKK233-2. The M. tuberculosis type-II 3-dehydroquinase has been purified in bulk from overproducing strains of E. coli to greater than 95% homogeneity. The protein is extremely heat-stable, is active as a homododecamer and has the lowest reported Km value of any type-II 3-dehydroquinase. The pentafunctional aromA gene of Aspergillus nidulans has been overexpressed more than 120-fold in an A. nidulans aromA- qutB- double mutant from a truncated quinate-inducible qutE promoter, such that the AROM protein is visible as a significant fraction (approx. 6%) in cell-free crude extracts. The M. tuberculosis aroQ gene has been fused to the same truncated qutE promoter and shown to encode quinate-inducible 3-dehydroquinase activity that allows a qutE- mutant strain of A. nidulans to utilize quinate as sole carbon source.
Structurally and functionally conserved regions of cytochrome P-450 reductase as targets for DNA amplification by the polymerase chain reaction. Cloning and nucleotide sequence of the Schizosaccharomyces pombe cDNA
Authors: Miles J S
Published: August 2015

1. Alignments of the available cytochrome P-450 reductase amino acid sequences, and comparison with the crystal structure of ferredoxin-NADP reductase, indicate that two highly conserved regions are of functional importance. 2. Degenerate oligonucleotide primers, based on these sequences, were used in the polymerase chain reaction to amplify a 309 bp fragment of the cytochrome P-450 reductase gene from Schizosaccharomyces pombe for use as an homologous probe. 3. A 2.6 kb cDNA was cloned from a lambda library, and sequencing revealed an open-reading frame of 2034 bp encoding a protein of M(r) 76774. This protein shares 38-41% identity with other eukaryotic cytochrome P-450 reductases, and 30% identity with that of Bacillus megaterium. 4. Comparison of the N-terminal FMN-binding domain with flavodoxin, and the C-terminal FAD- and NADP-binding domain with ferredoxin-NADP reductase, indicates the presence of several functionally conserved regions. 5. The Sc. pombe cytochrome P-450 reductase gene was shown to contain no introns.
Analysis and elimination of protein perturbation in infrared difference spectra of acyl-chymotrypsin ester carbonyl groups by using 13C isotopic substitution
Authors: White A J , Drabble K , Ward S , Wharton C W
Published: August 2015

I.r. spectroscopy has been applied to the study of hydrogen-bonding of the unique ester carbonyl group of acylchymotrypsins in the oxyanion hole of the enzyme. This catalytic device provides electrophilic stabilization of negative charge in the transition states and tetrahedral intermediates along the reaction pathway. The use of 13C isotope substitution of the ester carbonyl group reinforces the previous observation [White & Wharton (1990) Biochem. J. 270, 627-637] that the ester carbonyl group is significantly polarized in the ground state by hydrogen bonding in the oxyanion hole. I.r. difference spectra of [carbonyl-12C]-minus [carbonyl-13C]-cinnamoyl-chymotrypsin as well as each of these acylenzymes minus free enzyme are reported. These spectra show that the contribution of protein perturbation (i.e. spectral features that arise from the enzyme which is distorted on acylation) in [carbonyl-12C]cinnamoyl-chymotrypsin minus free enzyme spectra is significant. The contribution of the perturbation components of the spectra is pH-dependent and can represent up to 50% of the total absorbance in the spectral region from 1690 to 1740 cm-1. Use of the isotopic difference method has allowed problems associated with protein perturbation to be eliminated. Similar difference spectra are presented for dihydrocinnamoyl-chymotrypsin. In this case the effect of perturbation is very marked and leads to the cancellation of the band assigned to the non-bonded conformation of the acyl group which has previously only been observed at higher pH. The isotopic difference method again proves reliable and shows that the frequency difference previously used to calculate the ground-state electronic strain induced by the oxyanion-hole catalytic device is not affected by the perturbation, although the amplitudes of the spectral features are different. A study of the deacylation of cinnamoyl-chymotrypsin in water and deuterium oxide using both u.v. and i.r. spectroscopies has confirmed that the use of deuterium oxide as solvent has no serious effect on the deacylation behaviour of the enzyme. I.r. bands assigned to nonproductive and productive conformers decline identically during deacylation, which shows that the conformers are in dynamic exchange on the reaction time-scale.
The effect of metal ions on the activity and thermostability of the extracellular proteinase from a thermophilic Bacillus, strain EA.1
Authors: Coolbear T , Whittaker J M , Daniel R M
Published: August 2015

The proteinase from the extremely thermophilic Bacillus strain EA.1 exhibits maximum stability at a pH of approx. 6.5. In the presence of calcium ions the half-life at 95 degrees C of the enzyme at this pH was 17 min, and loss of activity followed first-order decay kinetics. The role of metal ions in the activity and stability of the enzyme was studied using the holoenzyme, the metal-depleted apoenzyme, and a zinc-enriched apoenzyme preparation. Zinc and calcium ions were the preferred bivalent cations for the active site and stabilization site(s) respectively. Stabilization by metal ions was not in itself a highly stringent process, but ions other than calcium which stabilized the enzyme generally had a concomitant inhibitory effect on activity. Inhibition and stabilization of the enzyme by cations were concentration-dependent effects and certain ions activated the apoenzyme but not the holoenzyme. Manganese(II) ions conferred some stability and also activated the enzyme, but in the latter case were not as effective as zinc ions. The results are discussed with reference to the ionic radii, co-ordination number and preferred ligand donors of the ions. Mercury(II) ions severely compromised enzyme activity and stability, and the effects of thiol-reactive agents suggest that thiol groups also have a role in enzyme integrity.
A high-density-lipoprotein receptor appears to mediate the transfer of essential fatty acids from high-density lipoprotein to lymphocytes
Authors: Xu Q , Bühler E , Steinmetz A , Schönitzer D , Böck G , Jürgens G , Wick G
Published: August 2015

It has been shown previously that a specific high-density lipoprotein (HDL) receptor exists on human lymphocytes that recognizes apoprotein (apo) A1 as its ligand, and may be responsible for utilization of HDL lipids to respond optimally to mitogenic stimulation when cultured in serum-free medium supplemented with HDL. To clarify further the relationship between various HDL lipids used by lymphocytes and HDL receptor activity, the lipid composition of the cells and the regulation of HDL and low-density lipoprotein (LDL) receptors on freshly isolated lymphocytes and mitogen-activated T-blasts after treatment with lipoproteins, liposomes or fatty acids were investigated. Our data show that the linear increase in cell proliferation correlates with the presence of HDL in fatty-acid-free culture medium in the concentration range of HDL receptor saturation. Decreased binding/uptake of dioctadecylindocarbocyanine (DiI)-fluorescence-labelled HDL by freshly isolated lymphocytes was observed in the presence of unlabelled HDL in 4-day culture, whereas T-blast binding/uptake was down-regulated by preincubation not only with HDL but also with LDL. T-blasts pretreated with HDL showed increased cellular contents of phosphatidylcholine, oleic acid (C18:1,n-9) and linoleic acid (C18:2,n-6), which are essential for optimal proliferation of mitogen-stimulated lymphocytes. Furthermore, DiI-HDL binding on lymphocytes was down-regulated by up to 20% (resting T cells) and 50% (T-blasts) when cultured in the presence of apoA1-phosphatidylcholine liposomes (T-blasts only), oleic acid or linoleic acid, but not by stearic acid (C18:0). The results indicate that HDL provide lymphocytes with essential fatty acids, which in turn regulate HDL receptor activity.
Protein kinase A modulates Ca2+- and protein kinase C-dependent amylase release in permeabilized rat pancreatic acini
Authors: O'Sullivan A J , Jamieson J D
Published: August 2015

The role of protein kinase A (PKA) in the release of amylase from permeabilized pancreatic acini was investigated. Addition of cyclic AMP (cAMP) to permeabilized acini resulted in a potentiation of Ca(2+)-dependent amylase release, shifting the Ca2+ dose/response curve leftwards. As with protein kinase C (PKC) activation, this is due to an increase in the time of active discharge. The effect of cAMP was shown to be blocked by two inhibitors of PKA, H89 and the PKI-(5-24)-peptide. At low concentration, cAMP synergizes from phorbol 12-myristate 13-acetate (PMA), while at optimal concentrations cAMP and PMA are additive. PKA and PKC appear to work via similar, but not identical mechanisms.
Determining stability of proteins from guanidinium chloride transition curves
Authors: Ahmad F , Yadav S , Taneja S
Published: August 2015

The guanidinium chloride (GdmCl) denaturation of RNAase A, lysozyme and metmyoglobin was investigated at several pH values by using absorbance measurements at 287, 300 and 409 nm respectively. From these measurements the free-energy change on denaturation, delta Gapp., was calculated, assuming a two-state mechanism, and values of delta Gapp. at zero concentration of the denaturant were measured. For each protein all delta Gapp. values were adjusted to pH 7.00 by using the appropriate relationship between delta Gapp. and pH. Dependence of the adjusted delta Gapp. value on GdmCl concentration increases for metmyoglobin and decreases for the other two proteins as the denaturant concentration decreases. It has been shown that these are expected results if the presence of the acid-denatured state during the GdmCl denaturation of proteins is considered.
Bivalent-metal binding to CheY protein. Effect on protein conformation
Authors: Kar L , Matsumura P , Johnson M E
Published: August 2015

CheY is a 14 kDa cytoplasmic protein that is activated by the transfer of a phosphoryl moiety to Asp-57 from phosphoCheA during signal transduction in bacterial chemotaxis. It has been established that metal ions are necessary for the autophosphorylation of CheA, the transfer of phosphate from phosphoCheA to CheY and the autodephosphorylation of phosphoCheY. In this work, paramagnetic relaxation enhancement has been used in conjunction with one- and two-dimensional n.m.r. to study the interaction of CheY with bivalent metal ions. These studies have led to the discovery of two conformations of the protein in water, corresponding to the metal-free and the metal-bound states. Binding of bivalent cations like Mg2+, Ca2+, Sr2+, Zn2+ and Mn2+ results in a conformational change from the metal-free to the metal-bound state. Preliminary assignments of the aromatic proton resonances are reported. Comparison of phase-sensitive double-quantum-filtered COSY, homonuclear Hartmann-Hahn coherence transfer and nuclear Overhauser enhancement spectra from the metal-bound and metal-free protein indicates that Trp-58, Thr-87 and Tyr-106 are particularly affected by the conformational change involved, and that this change is limited to a small number of residues. In addition, homonuclear Hartmann-Hahn coherence transfer experiments with paramagnetic Mn2+ show significant suppression of cross-peaks associated with Trp-58 and several neighbouring residues. Comparison of the distances estimated using n.m.r. with the CheY crystal structure indicates that the n.m.r. results are consistent with bivalent metal binding at the cluster of aspartic acid residues that includes Asp-13 and Asp-57. These studies also demonstrate the utility of paramagnetic metal-induced relaxation in conjunction with two-dimensional n.m.r. measurements for exploring ligand-binding sites.
Expression of transfected stathmin cDNA reveals novel phosphorylated forms associated with developmental and functional cell regulation
Authors: Doye V , Le Gouvello S , Dobransky T , Chneiweiss H , Beretta L , Sobel A
Published: August 2015

Stathmin is a ubiquitous, highly conserved phosphoprotein, which most likely acts as an intracellular relay integrating various transduction pathways triggered by extracellular signals. Two post-translational isoforms (alpha and beta) have been previously identified whose increasingly phosphorylated forms migrate as a set of isoelectric variant spots (molecular mass 19 kDa; pI 6.2-5.6) on two-dimensional electrophoretic gels. In parallel with the phosphorylation of these forms of stathmin, two sets of three proteins migrating with slightly higher apparent molecular masses (21 and 23 kDa respectively) also incorporated radioactive phosphate in response to cell regulation through various transduction pathways. These phosphoproteins, previously referred to as proteins ‘16’ and ‘17’, share several biochemical properties with stathmin and are recognized by antibodies directed to stathmin or to stathmin peptides. Furthermore, when rat stathmin cDNA was transfected into mouse myogenic C2 cells, it directed the expression of protein sets 16 and 17 together with the 19 kDa forms of stathmin, as detected with a species-specific anti-stathmin antiserum. Proteins 16 and 17 are thus novel phosphorylated derivatives of stathmin, encoded by the same cDNA as its previously identified 19 kDa forms. These results increase the known complexity and diversity of stathmin patterns, which may yield the molecular support for its proposed role as a relay integrating various signals which regulate the proliferation, differentiation and functions of cells during development and adult life.
Interaction of GTPase-activating protein with p21ras, measured using a continuous assay for inorganic phosphate release
Authors: Webb M R , Hunter J L
Published: August 2015

The mechanism of GTPase-activating protein (GAP) activation of p21ras GTP hydrolysis has been investigated by measuring the kinetics of release of Pi during the hydrolysis. The measurement uses a continuous spectroscopic assay for Pi, based on a guanosine analogue, 2-amino-6-mercapto-7-methylpurine ribonucleoside, as substrate for purine nucleoside phosphorylase [Webb, M.R. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 4884-4887]. This phosphorolysis gives an absorbance increase at 360 nm, so that when the reaction is coupled to GTP hydrolysis, the change in absorbance gives the total amount of Pi released from the p21ras. The rate of the absorbance increase gives the GTPase activity. This provides a non-radioactive method of determining p21ras concentration and GAP activity. It was used to determine the interaction of GAP with wild-type p21ras and two mutants (Leu-61/Ser-186 and Asp-12), all in the GTP (or guanosine 5′-[beta gamma-imido]triphosphate) form. The Leu-61/Ser-186 mutant binds 10-fold tighter than does the wild-type protein. The Asp-12 mutant binds to GAP with the same affinity as the wild-type protein. A novel GTPase activity was characterized whereby the EDTA-induced nucleotide release and GAP-activated cleavage of bound GTP leads to steady-state turnover of GTP hydrolysis. An assay for GAP is described based on this activity.
Overproduction of the pyruvate dehydrogenase multienzyme complex of Escherichia coli and site-directed substitutions in the E1p and E2p subunits
Authors: Russell G C , Machado R S , Guest J R
Published: August 2015

The aceEF-lpd operon of Escherichia coli encodes the pyruvate dehydrogenase (E1p), dihydrolipoamide acetyltransferase (E2p) and dihydrolipoamide dehydrogenase (E3) subunits of the pyruvate dehydrogenase multienzyme complex (PDH complex). An isopropyl beta-D-thiogalactopyranoside-inducible expression system was developed for amplifying fully lipoylated wild-type and mutant PDH complexes to over 30% of soluble protein. The extent of lipoylation was related to the degree of aeration during amplification. The specific activities of the isolated PDH complexes and the E1p component were 50-75% of the values normally observed for the unamplified complex. This could be due to altered stoichiometries of the overproduced complexes (higher E3 and lower E1p contents) or inactivation of E1p. The chaperonin, GroEL, was identified as a contaminant which copurifies with the complex. Site-directed substitutions of an invariant glycine residue (G231A, G231S and G231M) in the putative thiamine pyrophosphate-binding fold of the E1p component had no effect on the production of high-molecular-mass PDH complexes but their E1p and PDH complex activities were very low or undetectable, indicating that G231 is essential for the structural or catalytic integrity of E1p. A minor correction to the nucleotide sequence, which leads to the insertion of an isoleucine residue immediately after residue 273, was made. Substitution of the conserved histidine and arginine residues (H602 and R603) in the putative active-site motif of the E2p subunit confirmed that H602 of the E. coli E2p is essential, whereas R603 could be replaced without inactivating E2p. Deletions affecting putative secondary structural elements at the boundary of the E2p catalytic domain inhibited catalytic activity without affecting the assembly of the E2p core or its ability to bind E1p, indicating that the latter functions are determined elsewhere in the domain. The results further consolidate the view that chloramphenicol acetyltransferase serves as a useful structural and functional model for the catalytic domain of the lipoate acyltransferases.
Secretion of 72 kDa type IV collagenase/gelatinase by cultured human lipocytes. Analysis of gene expression, protein synthesis and proteinase activity
Authors: Arthur M J P , Stanley A , Iredale J P , Rafferty J A , Hembry R M , Friedman S L
Published: August 2015

The matrix metalloproteinases play an important role in matrix degradation, but there is limited information about this family of enzymes in either normal or diseased human liver. In this study, we have examined the synthesis of a 72 kDa type IV collagenase/gelatinase by human hepatic lipocytes in primary culture. Hepatic lipocytes were isolated from wedges of normal human donor liver by Pronase/collagenase perfusion, purified by density-gradient centrifugation, and established in primary culture on uncoated plastic. By Northern-blot analysis, the total RNA extracted from cultured human lipocytes was found to contain 3.4 kb mRNA for the 72 kDa type IV collagenase/gelatinase. Low levels of expression of this mRNA were observed in freshly isolated lipocytes but expression increased with the duration of lipocyte culture. Using anti-human 72 kDa type IV collagenase/gelatinase IgG, synthesized enzyme was immunolocalized to monensin-treated human lipocyte cultures. De novo synthesis and secretion of 72 kDa type IV collagenase/gelatinase were confirmed by immunoprecipitation of radiolabelled enzyme from medium obtained from [35S]methionine-treated cells. Activity of the secreted enzyme was demonstrated by gelatin-zymography and by degradation of soluble, radiolabelled [14C]gelatin. The enzyme was released both in active and latent pro-enzyme forms and its inhibition profile was that of a metalloproteinase. These studies indicate that cultured human hepatic lipocytes express the gene for the 72 kDa type IV collagenase/gelatinase, and secrete this enzyme, particularly in prolonged primary culture. As this enzyme exhibits degradative activity against basement membrane collagen, its release by activated hepatic lipocytes in the space of Disse could lead to disruption of the normal subendothelial liver matrix. It is suggested that this enzyme may have an important role in human liver injury and fibrosis.
Assay method for monitoring the inhibitory effects of antimetabolites on the activity of inosinate dehydrogenase in intact human CEM lymphocytes
Authors: Balzarini J , De Clercq E
Published: August 2015

A rapid and convenient method has been developed to monitor the inhibition of inosinate (IMP) dehydrogenase by antimetabolites in intact human CEM lymphocytes. This method is based on the determination of 3H release from [2,8-3H]hypoxanthine ([2,8-3H]Hx) or [2,8-3H]inosine ([2,8-3H]Ino). The validity of this procedure was assessed by evaluating IMP dehydrogenase inhibition in intact CEM cells by the well-known IMP dehydrogenase inhibitors ribavirin, mycophenolic acid and tiazofurin. As reference materials, several compounds that are targeted at other enzymes in de novo purine nucleotide anabolism (i.e. hadacidine, acivicin) or catabolism (i.e. 8-aminoguanosine, allopurinol) were evaluated. There was a strong correlation between the inhibitory effects of the IMP dehydrogenase inhibitors (ribavirin, mycophenolic acid, tiazofurin) on 3H release from [2,8-3H]Hx and [2,8-3H]Ino in intact CEM cells and their ability to decrease intracellular GTP pool levels. The other compounds (hadacidine, acivicin, 8-aminoguanosine, allopurinol) had no marked effect on 3H release from [2,8-3H]Hx. Using this method, we demonstrated that the novel ribavirin analogue, 5-ethynyl-1-beta-D-ribofuranosylimidazole-4-carboxamide, is a potent inhibitor of IMP dehydrogenase in intact cells.
Real-time study of the urea cycle using 15N n.m.r. in the isolated perfused rat liver
Authors: Geissler A , Kanamori K , Ross B D
Published: August 2015

1. Isolated rat liver was perfused with 10 mM-15NH4Cl, 5 mM-lactate and 1 mM-ornithine, or with 3 mM-[15N]alanine and 1 mM-ornithine, in haemoglobin-free medium. The liver was physiologically stable for over 3 h and synthesized urea at the rate of 1.15 mumol.min-1.g of liver-1 (15NH4(+)-perfused) or 0.41 mumol.min-1.g-1 ([15N]alanine-perfused). 2. The perfused liver was continuously monitored by 15N n.m.r. spectroscopy at 20.27 MHz for 15N. Well-resolved 15N resonances of precursors and intermediates of the urea cycle, present at tissue concentrations of 0.2-3.0 mumol/g, were observed from the intact liver in 5-40 min of acquisition. Key metabolites in liver extract and the final perfusion medium were analysed by n.m.r. and by biochemical assays to determine fractional 15N enrichment and the total 15N recovery. 3. In 15NH4(+)-perfused liver (n = 6), 15N incorporation into glutamate and alanine (1.0-1.3 mumol/g), as well as progressive formation of [15N2]urea, was observed during the first 2 h of perfusion. In the second and third hour, hepatic concentrations of [omega-15N]citrulline and [omega, omega'-15N]argininosuccinate increased to n.m.r.-detectable levels (0.3-0.9 mumol/g). The [15N]aspartate pool was large in the absence of added ornithine, but on its addition was rapidly incorporated into argininosuccinate (n = 3). 4. In [15N]alanine-perfused liver, major metabolites were [15N]glutamate, [gamma-15N]glutamine and [15N]urea. Urea-cycle intermediates were undetectable. 5. The results suggest that, in intact liver provided with excess ammonia, low concentrations of cytosolic argininosuccinate synthetase and argininosuccinate lyase limited the rate of metabolite flux in the urea cycle. By contrast, in alanine-perfused liver at a physiological rate of urea synthesis, mitochondrial carbamoylphosphate synthetase was rate-limiting. 6. The potential utility of 15N n.m.r. for study of metabolite channelling through urea-cycle enzymes in intact liver is discussed.
Characterization and purification of a novel dATP-binding protein in eukaryotes
Authors: Dalton A , Hornby D P , Langston S P , Blackburn G M
Published: August 2015

We characterized and purified an acidic dATP-binding protein, which, in its active form, resides in the nuclear fraction of a range of cells from mammals (including pig liver) and baker's yeast (Saccharomyces cerevisiae). This protein exhibits a high degree of specificity for the deoxy form of the naturally occurring nucleoside triphosphates and shows a marked preference for the purine deoxynucleoside triphosphates dATP and dGTP. The protein cleaves the terminal phosphate of dATP and appears to retain the dADP moiety of the nucleotide in a reaction that is resistant to both SDS and 8 M-urea. Fractionation of the nuclear preparation followed by non-denaturing PAGE and SDS/PAGE electrophoresis was sufficient to produce pure protein. The occurrence of this activity in all nuclei tested suggests that it plays an important role in nuclear metabolism. The specificity of the enzyme for deoxynucleoside triphosphates further suggests a role for this enzyme in DNA replication or repair, but the acidity of the protein argues against a direct interaction with DNA, and, indeed, the catalytic activity is not modulated by the inclusion of DNA in a variety of physical forms.
1H-and 13C-n.m.r. spectroscopy of 2-oxo-3-deoxy-d-glycero-d- galactononulosonic acid-containing oligosaccharide-alditols bearing Lewis X, Lewis Y and A-Lewis Y determinants isolated from the jelly coat of Pleurodeles waltl eggs
Authors: Strecker G , Wieruszeski J M , Michalski J C , Montreuil J
Published: August 2015

Three acidic oligosaccharide-alditols carrying Lewis X, Lewis Y and A-Lewis Y determinants were isolated from the jelly coat of Pleurodeles waltl eggs. These compounds possess the following structures. Gal beta 1-4(Fuc alpha 1-3)GlcNAc beta 1-3[2-oxo-3-deoxy-D- glycero-D-galactononulosonic acid (KDN)alpha 2-6] GalNAc-ol; Fuc alpha 1-2Gal beta 1-4(Fuc alpha 1-3)GlcNAc beta 1-3(KDN alpha 2-6) GalNAc-ol and Fuc alpha 1-2(GalNAc alpha 1-3)Gal beta 1-4(Fuc alpha 1-3)GlcNAc beta 1-3(KDN alpha 2-6)GalNAc-ol. The complete 1H-n.m.r.-spectrum assignment for the three compounds and the 13C-n.m.r. analysis of the A-Lewis Y determinant-containing heptasaccharide are reported.
Mitogen-induced tyrosine phosphorylation of 41 kDa and 43 kDa proteins. Potential role in integrating multiple mitogenic signalling pathways
Authors: Kohno M , Chatani Y , Tanaka E , Hattori A , Nishizawa N
Published: August 2015

We have examined the possible involvement of pertussis toxin (PT)-sensitive GTP-binding protein and protein kinase C (PKC) in mitogen-induced tyrosine phosphorylation of the 41 kDa and 43 kDa cytosol proteins using PT-pretreated (inactivation of PT-sensitive GTP-binding protein) or phorbol 12-myristate 13-acetate (PMA)-pretreated (depletion of PKC) mouse fibroblasts. The effects of the inactivation of PT-sensitive GTP-binding protein and the depletion of PKC on mitogen-stimulated tyrosine phosphorylation of the proteins were similar and varied significantly and systematically in response to growth factors. The important finding was that such inhibitory effects of PT-sensitive GTP-binding protein inactivation and PKC depletion on protein tyrosine phosphorylation induced by each mitogen always correlated well with their inhibitory effects on each mitogen-stimulated DNA synthesis. Although the extent of platelet-derived-growth-factor-induced phosphorylation of the proteins was decreased to approx. 50% in PT- and PMA-pretreated cells compared with native cells, protein phosphorylation itself was not affected and occurred at identical sites on each protein in native, PT- and PMA-pretreated cells. These results suggest that: (1) 41 kDa and 43 kDa proteins are located downstream of PT-sensitive GTP-binding protein and PKC in the mitogenic signalling pathways of growth factors, (2) protein phosphorylation occurs via a cascade of events which includes the activation of the receptor tyrosine kinases, PKC and other unidentified kinase(s) which directly participate(s) in the phosphorylation of the 41 kDa and 43 kDa proteins, and (3) their phosphorylation may play an important role in integrating multiple mitogenic signalling pathways.
Brain nitric oxide synthase is a haemoprotein
Authors: Klatt P , Schmidt K , Mayer B
Published: August 2015

Brain nitric oxide (NO) synthase showed pyridine haemochrome spectra typical of ferroprotoporphyrin IX-containing enzymes. The haem content of purified NO synthase was in the range 0.7-0.9 mol/mol of 160 kDa subunit. In the presence of CO, NO, KCN and miconazole, the L-citrulline-forming activity of NO synthase was markedly diminished, demonstrating that enzyme-bound haem is involved in enzymic NO synthesis.
Protein kinase C phosphorylation of cardiac troponin T decreases Ca2+-dependent actomyosin MgATPase activity and troponin T binding to tropomyosin-F-actin complex
Authors: Noland T A , Kuo J F
Published: August 2015

Effects of phosphorylation of bovine cardiac troponin T (TnT) by protein kinase C on the Ca(2+)-stimulated MgATPase activity of reconstituted actomyosin complex and the binding of TnT to tropomyosin(Tm)-F-actin were investigated. The Ca(2+)-stimulated MgATPase of actomyosin containing phosphorylated TnT (1.8 mol of P/mol), compared with that containing unphosphorylated TnT, was decreased by up to 48%. Phosphorylation of TnT also decreased (up to 48%) its maximum binding to Tm-F-actin, which was accompanied by a decrease (up to 3.5-fold) in its apparent binding affinity. The findings indicate that the effects of phosphorylated TnT in decreasing actomyosin MgATPase might be secondary to its decreased interactions with the other components of the thin filament, representing a new mechanism underlying the negative inotropic responses of various cardiac preparations to protein kinase C-activating phorbol esters.
The binding of amide substrate analogues to phospholipase A2. Studies by 13C-nuclear-magnetic-resonance and infrared spectroscopy
Authors: Slaich P K , Primrose W U , Robinson D H , Wharton C W , White A J , Drabble K , Roberts G C
Published: August 2015

(R)-(2-dodecanamidoisohexyl)phosphocholine (DAHPC), labelled with 13C at the amide carbonyl group, has been synthesized and its binding to bovine pancreatic phospholipase A2 (PLA2) studied by n.m.r. and i.r. spectroscopy. Two-dimensional 1H-n.m.r. spectra show that, in the presence of Ca2+, DAHPC binds to the active site of the enzyme in a similar manner to other phospholipid amide substrate analogues. The environment of the labelled carbonyl group has been investigated by a combination of 13C n.m.r. and difference-Fourier-transform i.r. spectroscopy. The carbonyl resonance shifts 3 p.p.m. downfield on the binding of DAHPC to PLA2. The carbonyl absorption frequency decreases by 14-18 cm-1, accompanied by a marked sharpening of the absorption band. These results indicate that the carbonyl bond undergoes significant polarization in the enzyme-ligand complex, facilitated by the enzyme-bound Ca2+ ion. This suggests that ground-state strain is likely to promote catalysis in the case of substrate binding. Simple calculations, based on the i.r. data, indicate that the carbonyl bond is weakened by 5-9 kJ.mol-1. This is the first report of observation of the amide vibration of a bound ligand against the strong background of protein amide vibrations.
Induction of nitric oxide synthase in L929 cells by tumour-necrosis factor α is prevented by inhibitors of poly(ADP-ribose) polymerase
Authors: Hauschildt S , Scheipers P , Bessler W G , Mülsch A
Published: August 2015

The fibroblast cell line L929 contains a constitutively expressed NO synthase (EC 1.14.29.-) activity, which can be increased about 10-fold by tumour-necrosis factor alpha (TNF-alpha). Activities of the constitutive and the inducible enzymes are tetrahydrobiopterin-independent and can be inhibited by L-NG-nitroarginine. Induction of NO synthase by TNF-alpha was prevented by inhibitors of poly(ADP-ribose) polymerase, namely nicotinamide, 3-methoxybenzamide and 3-aminobenzamide. TNF-alpha did not lead to an increase in ADP-ribosyltransferase activity nor to a change in the pattern of ADP-ribosylated proteins. The inhibitors were only active during the first 4-5 h after exposure to TNF-alpha and they were found to suppress synthesis of protein, DNA and RNA. These data suggest that the inhibitors prevent induction of NO synthase by interference with RNA and protein synthesis. It is not yet known which reactions of these biosynthetic processes are affected by the inhibitors.
Identification of multifunctional ATP-citrate lyase kinase as the α-isoform of glycogen synthase kinase-3
Authors: Hughes K , Ramakrishna S , Benjamin W B , Woodgett J R
Published: August 2015

Multifunctional ATP-citrate lyase kinase (ACLK) exhibits several properties that are similar to glycogen-synthase kinase-3 (GSK-3). The molecular cloning of two distinct mammalian GSK-3 cDNAs and a Drosophila melanogaster (fruitfly) homologue, zeste-white3sgg, has established the existence of a GSK-3 subfamily. A multifunctional protein kinase first identified as an ACLK has recently been shown to exhibit several similarities to the alpha- and beta-forms of GSK-3. Here we have used immunological and biochemical analyses to directly compare these enzymes. Thus purified preparations of ACLK isolated from brain and liver preferentially cross-react with anti-GSK-3 alpha antisera and phosphorylate previously defined substrates of GSK-3 at identical sites. Conversely, both alpha- and beta-forms of GSK-3 phosphorylated ATP-citrate lyase at the same site(s) targeted by ACLK. These, and other similarities, demonstrate ACLK to be identical with, or highly related to, GSK-3 alpha, the implications of which are discussed.
Effects of insulin and phorbol esters on subcellular distribution of protein kinase C isoforms in rat adipocytes
Authors: Farese R V , Standaert M L , Francois A J , Ways K , Arnold T P , Hernandez H , Cooper D R
Published: August 2015

Effects of insulin and phorbol esters on subcellular distribution of protein kinase C (PKC) isoforms were examined in rat adipocytes. Both agonists provoked rapid decreases in cytosolic, and/or increases in membrane, immunoreactive PKC-alpha, PKC-beta, PKC-gamma, and PKC-epsilon. Effects of phorbol esters on PKC-alpha redistribution to the plasma membrane, however, were much greater than those of insulin. In contrast, insulin, but not phorbol esters, stimulated the translocation of PKC-beta to the plasma membrane, and provoked changes in PKC-zeta redistribution. Neither agonist altered subcellular distribution of PKC-delta, which was detected only in membrane fractions. Our findings indicate that insulin and phorbol esters have overlapping and distinctly different effects on the subcellular redistribution of specific PKC isoforms.
Purification and characterization of a phosphatidylinositol 3-kinase complex from bovine brain by using phosphopeptide affinity columns
Authors: Fry M J , Panayotou G , Dhand R , Ruiz-Larrea F , Gout I , Nguyen O , Courtneidge S A , Waterfield M D
Published: August 2015

Specific phosphorylated tyrosine residues in the kinase insert region of the human platelet-derived-growth-factor beta-receptor mediate the formation of multienzyme complexes with this receptor. When phosphorylated, tyrosine residue 751 within the kinase insert region mediates binding of PtdIns 3-kinase to this receptor. A 17-amino-acid peptide containing this tyrosine residue was synthesized, phosphorylated by using epidermal-growth-factor receptor and then coupled to an Actigel matrix. The tyrosine-751 phosphopeptide column is used here as a final affinity step in the purification of the PtdIns 3-kinase from bovine brain to apparent homogeneity. The active resin-bound PtdIns 3-kinase is composed of two polypeptides, p110 and p85, which are elutable with SDS-containing buffers and detectable by silver staining of polyacrylamide gels. The 85 kDa protein is shown to be identical with the recently cloned p85 alpha. Phosphotyrosine is demonstrated to be an essential part of the structure required for binding of both of these proteins and PtdIns 3-kinase activity to this peptide. The active PtdIns 3-kinase complex from bovine brain, but not recombinant p85 subunits, shows specificity for binding to phosphopeptides containing a YXXM consensus sequence. Neither PtdIns 3-kinase activity, nor the complex of p85 and 110 kDa proteins, binds to several other phosphopeptide affinity columns lacking this sequence motif. The selectivity of binding of baculovirus-expressed free p85 alpha subunit of bovine brain PtdIns 3-kinase, the closely related protein p85 beta and purified bovine brain PtdIns 3-kinase to these and other phosphopeptide columns is examined.