bluuebunny/crossref_metadata_2025 huggingface dataset:
Molecular cloning, sequencing and expression of the cDNA of the mitochondrial form of phosphoenolpyruvate carboxykinase from human liver
Authors: MODARESSI Said , CHRIST Bruno , BRATKE Jutta , ZAHN Stefan , HEISE Tilman , JUNGERMANN Kurt
Published: August 2015

In human liver, phosphoenolpyruvate carboxykinase (PCK; EC 4.1.1.32) is about equally distributed between cytosol and mitochondria in contrast with rat liver in which it is essentially a cytosolic enzyme. Recently, the isolation of the gene and cDNA of the human cytosolic enzyme has been reported [Ting, Burgess, Chamberlain, Keith, Falls and Meisler (1993) Genomics 16, 698–706; Stoffel, Xiang, Espinosa, Cox, Le Beau and Bell (1993) Hum. Mol. Genet. 2, 1–4]. It was the goal of this investigation to isolate the cDNA of the human mitochondrial form of hepatic PCK. A human liver cDNA library was screened with a rat cytosolic PCK cDNA probe comprising sequences from exons 2 to 9. A cDNA clone was isolated which had overall a 68% DNA sequence and a 70% deduced amino acid sequence identity with the human cytosolic PCK cDNA. Without the flanking 270 bases (=90 amino acids) each at the 5´ and 3´ end, the sequence identity was 73% on the DNA and 78% on the amino acid level. The isolated cDNA had an open reading frame of 1920 bp; it was 54 bp (equivalent to 18 amino acids) longer than that of human or rat cytosolic PCK cDNA. The isolated cDNA was cloned into the eukaryotic expression vector pcDNAI and transfected into human embryonal kidney cells HEK293; PCK activity was increased by 3-fold in the mitochondria, which normally contain 70% of total PCK activity, but not in the cytosol. The isolated cDNA was also transfected into cultured rat hepatocytes; again, PCK activity was enhanced by about 40-fold in the mitochondria, which normally possess only 10% of total PCK activity, but not in the cytosol. In the rat hepatocytes only the endogenous cytosolic PCK and not the transfected mitochondrial PCK was induced 3-fold with glucagon. Comparison of the amino acid sequences deduced from the isolated cDNA with human and rat cytosolic PCK showed that the additional 18 amino acids were located at the N-terminus of the protein and probably constitute a mitochondrial targeting signal. Northern-blot analyses revealed the human mitochondrial PCK mRNA to be 2.25 kb long, about 0.6 kb shorter than the mRNA of the cytosolic PCK. Primer extension experiments showed that the 5´-untranslated region of mitochondrial PCK mRNA was 134 nucleotides in length.
Multiple cis-trans conformers of the prolactin receptor proline-rich motif (PRM) peptide detected by reverse-phase HPLC, CD and NMR spectroscopy
Authors: O'NEAL Kevin D. , CHARI Mohan V. , McDONALD Charles H. , COOK Richard G. , YU-LEE Li-yuan , MORRISETT Joel D. , SHEARER T. William
Published: August 2015

An eight-amino-acid synthetic peptide (Ile1-Phe2-Pro3-Pro4-Val5-Pro6-Gly7-Pro8) corresponding to the conserved proline-rich motif (PRM) of the intracellular domain of the prolactin receptor (PRL-R) was studied by one- and two-dimensional (1D and 2D) proton NMR spectroscopy in water and DMSO in order to characterize its conformational dynamics. The purified PRL-R PRM peptide eluted as two partially resolved peaks in equilibrium on reverse-phase HPLC (RP-HPLC) at 20 °C with a ratio of 60:40. At 30 °C, the two peaks coalesced into a single peak. The two RP-HPLC peaks correspond to two peptide conformers resulting from the slow cis–trans isomerization of one of the four proline amide bonds. Although the peptide has only three amide (NH) protons, its 1D NMR spectrum in water contains approximately 15 discernible NH region peaks, providing evidence for multiple conformers. The amide resonances were assigned on the basis of 2D-COSY spectra, chemical shift values, resonance splitting patterns and temperature coefficients. The cis:trans ratio for each proline in water, calculated from integrated intensities and/or peak heights of the appropriate resonances, were Phe2-Pro3 (35:65), Pro3-Pro4 (40:60), Val5-Pro6 (70:30), and Gly7-Pro8 (30:70). Temperature studies (25–70 °C) were used to semi-quantitatively estimate the rates of isomerization for the different prolines. In water, Pro8 undergoes rapid isomerization; Pro3 isomerizes at an intermediate rate; while Pro4 and Pro6 both appear to isomerize very slowly since no coalescence of amide resonances was observed. In DMSO, only Pro4 displayed slow isomerization. Slow kinetics combined with a similar 60:40 ratio of conformers determined by RP-HPLC and NMR suggests that isomerization of the Pro3-Pro4 bond generates the two RP-HPLC peaks. Both proximal and distal proline isomerization effects were observed in NMR experiments. All of the 16 theoretical (24 = 16) proline configurations appear to exist in equilibrium in water. The predominant (19%) conformation, trans3-trans4-cis6-trans8, may reflect the configuration of the PRM prolines in the native PRL-R. Isomerization of Pro6 from cis to trans generates an interaction between the peptide N-and C-termini, suggesting an overall pseudo-cyclic conformation. This all-trans proline configuration may play an important biochemical role in the function of cytokine/haematopoietin receptors. A model is proposed which suggests that isomerization of the PRM by an immunophilin such as the FK506-binding protein (FKBP) serves as an on–off switch for cytokine receptor activation.
Functional expression of rat GLUT 1 glucose transporter in Dictyostelium discoideum
Authors: COHEN Naomi R. , KNECHT David A. , LODISH Harvey F.
Published: August 2015

To facilitate expression of the rat GLUT 1 glucose transporter cDNA in Dictyostelium discoideum, we mutated the 5´ end of the coding sequence such that the codons for the first ten amino acids conformed to preferred Dictyostelium codon usage. As determined by Western-blot analysis, a population of Dictyostelium transformed with the mutated cDNA expressed non-glycosylated GLUT 1 protein. Cell lines expressing GLUT 1 transport radiolabelled 2-deoxy-D-glucose at a rate 6–10 times that of cell lines transformed with vector alone. The initial rate of inward transport of 2-deoxy-D-glucose was stimulated severalfold by the presence of unlabelled glucose in the Dictyostelium cytoplasm, exemplifying the trans-activation of GLUT 1 transport characteristic of GLUT 1 present in erythrocyte membranes. The Km and Ki values for 2-deoxy-D-glucose, D-glucose, D-mannose and D-galactose were 3.7 mM, 2.6 mM, 11 mM and 30 mM respectively, similar to the values for GLUT 1 expressed in mammalian cells. L-Glucose and L-galactose, which are not transported by GLUT 1, do not inhibit uptake of 2-deoxy-D-glucose in Dictyostelium expressing GLUT 1. Thus, even though GLUT 1 expressed in Dictyostelium is not N-glycosylated, it transports hexoses normally; this is the first example of functional expression of a mammalian transport protein in this lower eukaryote.
Binding and internalization of extracellular type-I phospholipase A2 in uterine stromal cells
Authors: Paolo ROSSINI Gian , FAYARD Jean-Michel , TESSIER Christian , LAUGIER Christian
Published: August 2015

The cellular uptake of extracellular type-I phospholipase A2 (PLA2) was investigated in rat uterine stromal cells (UIII) in culture, which were found to express the high-affinity binding site for mammalian type-I PLA2, with a measured KD of 6.4 nM, a Bmax of 0.1–1 pmol/mg of DNA at 4 °C, and a molecular mass of about 200 kDa. When UIII cells were treated with type-I PLA2 at 37 °C, the ligand specifically associated with the cells increased, reaching a plateau after 90 min of incubation, whose level was about 5-fold higher than that measured if cells were maintained at 4 °C. We could determine that the PLA2 was bound to plasma membrane receptors which were responsible for internalization of the ligand, and that the binding sites were still suitable for binding at the level of plasma membrane during UIII cell incubation at 37 °C. Proteolysis of internalized PLA2 could be clearly detected only after 90 min of UIII cell incubation with the ligand at 37 °C, and most of the intracellular PLA2 consisted of the apparently intact 14 kDa enzyme. By cross-linking studies, we found that most of the internalized PLA2 was not associated with the receptor, supporting the conclusion that in our experimental system a single pool of membrane receptors for mammalian type-I PLA2 undergoes cycles of ligand binding, intracellular transfer and release of PLA2, followed by restoration of binding sites on the plasma membrane. We calculated that the rate of internalization of the ligand by one receptor molecule in UIII cells at 37 °C is about three molecules of type-I PLA2 per h.
Localization of a putative second membrane association site in penicillin-binding protein 1B of Escherichia coli
Authors: WANG Chia-Chang , SCHULTZ Derk E. , NICHOLAS Robert A.
Published: August 2015

We have shown previously that the periplasmic domain of penicillin-binding protein 1B (PBP 1Bper; residues 90–844) from Escherichia coli is insoluble in the absence of detergents, and can be reconstituted into liposomes [Nicholas, Lamson and Schultz (1993) J. Biol. Chem. 268, 5632–5641]. These data suggested that native PBP 1B contains a membrane association site in addition to its N-terminal transmembrane anchor. We have studied the membrane topology of PBP 1B in greater detail by assessing detergent binding and solubility in the absence of detergents for PBP 1Bper and a set of proteolytic fragments of PBP 1B. PBP 1Bper was shown by three independent methods to bind to detergent micelles, which strongly suggests that the periplasmic domain interacts with the hydrophobic milieu of membrane bilayers. Digestion with high weight ratios of thrombin of purified PBP 1B containing an engineered thrombin cleavage site on the periplasmic side of the transmembrane anchor generated four fragments in addition to PBP 1Bper that varied in size from 71 to 48 kDa. In contrast to PBP 1Bper, all fragments of 67 kDa and smaller were eluted from a gel-filtration column in the absence of detergents and did not bind to detergent micelles. The N-terminal sequences of the four fragments were determined, allowing the cleavage sites to be located in the primary sequence of PBP 1B. These data localize the membrane association site of PBP 1B to a region comprising the first 163 amino acids of the periplasmic domain, which falls within the putative transglycosylase domain. Lipid modification does not appear to be the mechanism by which PBP 1Bper associates with membranes.
Transient activation of mitochondrial translation regulates the expression of the mitochondrial genome during mammalian mitochondrial differentiation
Authors: OSTRONOFF Luciana K. , IZQUIERDO José M. , ENRÍQUEZ José A. , MONTOYA Julio , CUEZVA José M.
Published: August 2015

Regulation of the expression of the nuclear-encoded β-subunit of H+-ATP synthase (β-F1-ATPase) gene of oxidative phosphorylation during differentiation of liver mitochondria is mainly exerted at two post-transcriptional levels affecting both the half-life [Izquierdo, Ricart, Ostronoff, Egea and Cuezva (1995) J. Biol. Chem. 270, 10342–10350] and translational efficiency [Luis, Izquierdo, Ostronoff, Salinas, Santarén and Cuezva (1993) J. Biol. Chem. 268, 1868–1875] of the transcript. Herein, we have studied the expression of the mitochondrial (mt) genome during differentiation of rat liver mitochondria in an effort to elucidate the mechanisms of nucleo-mitochondrial cross-talk during biogenesis of the organelle. Estimation of the relative cellular representation of mt-DNA in liver reveals a negligible increase in mt-DNA copy number during organelle differentiation. Concurrently, the lack of changes in transcription rates of the mt-DNA ‘in organello’, as well as in steady-state levels of the mt-transcripts, suggests that organelle differentiation is not controlled by an increase in transcription of the mt-genome. However, translation rates in isolated mitochondria revealed a transient 2-fold increase immediately after birth. Interestingly, the transient activation of mitochondrial translation at this stage of liver development is dependent on the synthesis of proteins in cytoplasmic polyribosomes. These findings support the hypothesis that the expression of nuclear and mitochondrial genes during biogenesis of mammalian mitochondria is developmentally regulated by a post-transcriptional mechanism that involves concerted translational control of both genomes.
Regulation of the mouse inducible-type nitric oxide synthase gene promoter by interferon-γ, bacterial lipopolysaccharide and NG-monomethyl-l-arginine
Authors: WEISZ Alessandro , CICATIELLO Luigi , ESUMI Hiroyasu
Published: August 2015

Cytokines and bacterial lipopolysaccharides (LPSs) stimulate nitric oxide production in macrophages by inducing transcription of the gene coding for the inducible isoform of nitric oxide synthase (iNOS). We have cloned the mouse iNOS gene promoter and analysed its structural features and its response to interferon-γ (IFN-γ) and Escherichia coli LPS in RAW 264.7 mouse macrophage-like cells. Transcription of a recombinant reporter gene including the promoter and 4 kb of its 5′-flanking DNA, linked to the bacterial chloramphenicol acetyltransferase (CAT) reporter gene, is stimulated by IFN-γ and, more efficiently, by LPS upon transient transfection in RAW 264.7 cells. Two upstream DNA regions are required for maximal promoter activation by LPS: the first maps between positions -1541 and -775 and the other between -420 and -47, with respect to the major transcriptional start site of the iNOS gene. The upstream-most region also mediates promoter trans-activation by IFN-γ. As reported earlier for transcription of the endogenous iNOS gene, combined stimulation of RAW 264.7 cells with IFN-γ and LPS results in lower activation of the transfected promoter, when compared with LPS alone. NG-Monomethyl-L-arginine, a competitive inhibitor of nitric oxide synthase activity, enhances iNOS gene mRNA induction and promoter activation by IFN-γ and LPS, indicating that nitric oxide can influence negatively the responsiveness of this gene to inducers. These results suggest the possibility of a negative regulatory feedback exerted by iNOS on the transcriptional activation of its own gene.
Inhibition of mammalian nitric oxide synthases by agmatine, an endogenous polyamine formed by decarboxylation of arginine
Authors: GALEA Elena , REGUNATHAN S. , ELIOPOULOS Vassily , FEINSTEIN Douglas L. , REIS Donald J.
Published: August 2015

Agmatine, decarboxylated arginine, is a metabolic product of mammalian cells. Considering the close structural similarity between L-arginine and agmatine, we investigated the interaction of agmatine and nitric oxide synthases (NOSs), which use L-arginine to generate nitric oxide (NO) and citrulline. Brain, macrophages and endothelial cells were respectively used as sources for NOS isoforms I, II and III. Enzyme activity was measured by the production of nitrites or L-citrulline. Agmatine was a competitive NOS inhibitor but not an NO precursor. Ki values were approx. 660 μM (NOS I), 220 μM (NOS II) and 7.5 mM (NOS III). Structurally related polyamines did not inhibit NOS activity. Agmatine, therefore, may be an endogenous regulator of NO production in mammals.
Evidence that biliverdin-IXβ reductase and flavin reductase are identical
Authors: SHALLOE Fiona , ELLIOTT Gordon , ENNIS Orla , MANTLE Timothy J.
Published: August 2015

A search of the database shows that human biliverdin-IXβ reductase and flavin reductase are identical. We have isolated flavin reductase from bovine erythrocytes and show that the activity co-elutes with biliverdin-IXβ reductase. Preparations of the enzyme that are electrophoretically homogeneous exhibit both flavin reductase and biliverdin-IXβ reductase activities; however, they are not capable of catalysing the reduction of biliverdin-IXα. Although there is little obvious sequence identity between biliverdin-IXα reductase (BVR-A) and biliverdin-IXβ reductase (BVR-B), they do show weak immunological cross-reactivity. Both enzymes bind to 2´,5´-ADP–Sepharose.
Rapid burst kinetics in the hydrolysis of 4-nitrophenyl acetate by penicillin G acylase from Kluyvera citrophila. Effects of mutation F360V on rate constants for acylation and de-acylation
Authors: Roa A , Goble M L , García J L , Acebal C , Virden R
Published: August 2015

The kinetics of release of 4-nitrophenol were followed by stopped-flow spectrophotometry with two 4-nitrophenyl ester substrates of penicillin G acylase from Kluyvera citrophila. With the ester of acetic acid, but not of propionic acid, there was a pre-steady-state exponential phase, the kinetics of which were inhibited by phenylacetic acid (a product of hydrolysis of specific substrates) to the extent predicted from Ki values. This was interpreted as deriving from rapid formation (73 mM-1·s-1) and slow hydrolysis (0.76 s-1) of an acetyl derivative of the side chain of the catalytic-centre residue Ser-290. With the mutant F360V, which differs from the wild-type enzyme in its ability to hydrolyse adipyl-L-leucine and has a kcat for 4-nitrophenyl acetate one-twentieth that of the wild-type enzyme, the corresponding values for the rates of formation and hydrolysis of the acetyl-enzyme were 11.1 mM-1·s-1 and 0.051 s-1 respectively. The ratio of these rate constants was three times that for the wild-type enzyme, suggesting that the mutant is less impaired in the rate of formation of an acetyl-enzyme than in its subsequent hydrolysis.
Trout red blood cell arrestin (TRCarr), a novel member of the arrestin family: cloning, immunoprecipitation and expression of recombinant TRCarr
Authors: JAHNS Roland , BORGESE Franck , LINDENTHAL Sabine , STRAUB Annette , MOTAIS René , FIÉVET Bruno
Published: August 2015

Arrestins are cytosolic proteins involved in the desensitization of G-protein-coupled receptors. We report the cloning of trout red blood cell arrestin which shows 76, 82 and 52% identity with bovine β-arrestin1, β-arrestin2 and retinal arrestin respectively. Antibodies were generated against the C-terminus of trout red blood cell arrestin. These antibodies detected arrestin in erythrocyte cytosol and were able to precipitate the native protein. The Na+/H+ antiporter of trout red blood cell is activated by β-adrenergic stimulation and is then desensitized whereas the transmembrane signalling pathway is not. To investigate the subcellular distribution of arrestin on β-adrenergic activation and desensitization of the antiporter, precipitation experiments were carried out on trout erythrocytes. A desensitization-dependent shift in cytosolic arrestin to the membranes could not be detected using the immunoprecipitation technique but we cannot exclude the possibility that a small number of cytosolic arrestins might be involved in the regulation of membrane proteins in trout erythrocyte. Recombinant trout arrestin was produced in a protease-deficient Escherichia coli strain and its functionality was tested in a reconstituted rhodopsin assay. The recombinant protein provides a suitable tool for investigating the target for arrestin in trout red blood cell, which still remains to be identified.
Biogenesis of thermogenic mitochondria in brown adipose tissue of Djungarian hamsters during cold adaptation
Authors: KLINGENSPOR Martin , IVEMEYER Marc , WIESINGER Herbert , HAAS Kirsten , HELDMAIER Gerhard , WIESNER Rudolf J.
Published: August 2015

After cold exposure, cytochrome c oxidase (COX) activity increased about 2.5-fold within 2 weeks in the brown adipose tissue (BAT) of Djungarian hamsters. The mRNAs for COX subunits I and III and the 12 S rRNA, encoded on mitochondrial DNA (mtDNA), as well as mRNAs for COX subunits IV, Va and mitochondrial transcription factor A, encoded in the nucleus, were unchanged when expressed per unit of total tissue RNA. However, since total tissue RNA doubled per BAT depot, while total DNA remained unchanged, the actual levels of these transcripts were increased within BAT cells. In contrast, the abundance of mRNA for uncoupling protein was increased 10-fold, indicating specific activation of this gene. In addition, the maximal rate of protein synthesis analysed in a faithful in organello system was increased 2.5-fold in mitochondria isolated from BAT after 7 days of cold exposure. We conclude from these data that the biogenesis of thermogenic mitochondria in BAT following cold adaptation is achieved by increasing the overall capacity for synthesis of mitochondrial proteins in both compartments, by increasing their mRNAs as well as the ribosomes needed for their translation. In addition, the translational rate for COX subunits as well as all other proteins encoded on mtDNA is increased. Thus the pool of subunits encoded on mtDNA required for assembly of respiratory chain complexes is provided. By comparison with other models of increased mitochondrial biogenesis, we propose that thyroid hormone (generated within BAT cells by 5´-deiodinase, and induced upon sympathetic stimulation), which is a well known regulator of the biogenesis of mitochondria in many tissues, is also the major effector of these adaptive changes in BAT.
Structural environment of an essential cysteine residue of xylanase from Chainia sp. (NCL 82.5.1)
Authors: RAO Mala , KHADILKAR Suvarna , BANDIVADEKAR Kavita R. , DESHPANDE Vasanti
Published: August 2015

N-(2,4-Dinitroanilino)maleimide (DAM) reacts covalently with the thiol group of the xylanase from Chainia leading to complete inactivation in a manner similar to N-ethylmaleimide, but provides a reporter group at the active site of the enzyme. Increasing amounts of xylan offered enhanced protection against inactivation of the xylanase by DAM. Xylan (5 mg) showed complete protection, providing evidence for the presence of cysteine at the substrate-binding site of the enzyme. Kinetics of chemical modification of the xylanase by DAM indicated the involvement of l mol of cysteine residue per mol of enzyme, as reported earlier [Deshpande, Hinge and Rao (1990) Biochim. Biophys. Acta 1041, 172–177]. The second-order rate constant for the reaction of DAM with the enzyme was 3.61×103 M-1·min-1. The purified xylanase was alkylated with DAM and digested with pepsin. The peptides were separated by gel filtration. The specific modified cysteinyl peptide was further purified by reverse-phase HPLC. The active-site peptide was located visually by its predominant yellow colour and characterized by a higher A340 to A210 ratio. The modified active-site peptide has the sequence: Glu-Thr-Phe-Xaa-Asp. The sequence of the peptide was distinctly different from that of cysteinyl peptide derived from a xylanase from a thermotolerant Streptomyces species, but showed the presence of a conserved aspartic acid residue consistent with the catalytic regions of other glucanases.
The genes YNI1 and YNR1, encoding nitrite reductase and nitrate reductase respectively in the yeast Hansenula polymorpha, are clustered and co-ordinately regulated
Authors: BRITO Nélida , AVILA Julio , PEREZ M. Dolores , GONZALEZ Celedonio , SIVERIO José M.
Published: August 2015

The nitrite reductase-encoding gene (YNI1) from the yeast Hansenula polymorpha was isolated from a lambda EMBL3 H. polymorpha genomic DNA library, using as a probe a 481 bp DNA fragment from the gene of Aspergillus nidulans encoding nitrite reductase (niiA). An open reading frame of 3132 bp, encoding a putative protein of 1044 amino acids with high similarity with nitrite reductases from fungi, was located by DNA sequencing in the phages λNB5 and λJA13. Genes YNI1 and YNR1 (encoding nitrate reductase) are clustered, separated by 1700 bp. Northern blot analysis showed that expression of YNI1 and YNR1 is co-ordinately regulated; induced by nitrate and nitrite and repressed by sources of reduced nitrogen, even in the presence of nitrate. A mutant lacking nitrite reductase activity was obtained by deletion of the chromosomal copy of YNI1. The mutant does not grow in nitrate or in nitrite; it exhibits a similar level of transcription of YNR1 to the wild type, but the nitrate reductase enzymic activity is only about 50% of the wild type. In the presence of nitrate the Δyni1::URA3 mutant extrudes approx. 24 nmol of nitrite/h per mg of yeast (wet weight), about five times more than the wild type.
Differential regulation of neuronal nicotinic acetylcholine receptor subunit gene promoters by Brn-3 POU family transcription factors
Authors: MILTON Nathaniel G. N. , BESSIS Alain , CHANGEUX Jean-Pierre , LATCHMAN David S.
Published: August 2015

The regulatory region of the neuronal nicotinic acetylcholine (nACh) receptor α2 subunit gene is activated by the Brn-3b POU family transcription factor but not by the closely related factors Brn-3a and Brn-3c. This pattern of regulation has not previously been observed for other neuronally expressed genes, several of which, such as those encoding α-internexin or SNAP-25, are activated by Brn-3a and Brn-3c but repressed by Brn-3b. The α3 nACh receptor subunit gene is also shown to be activated by Brn-3a but is repressed by Brn-3b and Brn-3c. In contrast, the Brn-3 POU family transcription factors have no effects on either the α7 or β4 nACh receptor subunit genes. The actions of Brn-3b on the α2 subunit are thus in contrast to the inhibitory actions of Brn-3b on several promoters that are activated by Brn-3a. The different actions of the Brn-3 POU factors on the range of nACh receptor genes tested suggests that the novel stimulation of the α2 subunit by Brn-3b is specific to this subunit and not a general feature of nACh receptor genes.
The mechanism of inhibition of the Ca2+-ATPase of skeletal-muscle sarcoplasmic reticulum by the cross-linker o-phthalaldehyde
Authors: KHAN Yamin M. , STARLING Anthony P. , EAST J. Malcolm , LEE Anthony G.
Published: August 2015

Labelling the Ca2+-ATPase of skeletal-muscle sarcoplasmic reticulum with o-phthalaldehyde (OPA) results in loss of ATPase activity at a 1:1 molar ratio of label to ATPase. The affinity of the ATPase for Ca2+ is unaffected, as is the E1/E2 equilibrium constant. The rate of dissociation of Ca2+ from the Ca2+-bound ATPase is also unaffected and Mg2+ increases the rate of dissociation, as for the unlabelled ATPase. Effects of Mg2+ on the fluorescence intensity of the ATPase labelled with 4-(bromomethyl)-6,7-dimethoxycoumarin are also unaffected by labelling with OPA, consistent with the fluorescence change reporting on Mg2+ binding at the gating site on the ATPase. The affinity of the ATPase for ATP is reduced by labelling, as is the rate of phosphorylation. The rate of phosphorylation is independent of the concentration of ATP above 25 μM ATP, so that the slow step is the first-order rate constant for phosphorylation by bound ATP. The rate of the back reaction between phosphorylated ATPase and ADP is little affected, suggesting that the slow step in phosphorylation could be the slow conformation step before phosphoryl transfer. The rate of dephosphorylation of the phosphorylated ATPase is also decreased, suggesting that a similar conformation change could be involved in the dephosphorylation step. The rate of the Ca2+ transport step appears to be unaffected by labelling. The net result of these changes is that the labelled ATPase is present predominantly in a Ca2+-free, phosphorylated form at steady state in the presence of ATP.
Genetic analysis of the phenobarbital regulation of the cytochrome P-450 2b-9 and aldehyde dehydrogenase type 2 mRNAs in mouse liver
Authors: DAMON Marie , FAUTREL Alain , GUILLOUZO André , CORCOS Laurent
Published: August 2015

The aim of this study was to investigate the effect of the genetic background on the phenobarbital inducibility of cytochrome P-450 2b-9, cytochrome P-450 2b-10 and aldehyde dehydrogenase type 2 mRNAs in mice. We analysed the basal expression and the phenobarbital inducibility of both cytochrome P-450 mRNAs by semi-quantitative specific reverse transcription-PCR analyses in five inbred mouse strains (A/J, BALB/cByJ, C57BL/6J, DBA/2J and SWR/J). Male mice constitutively expressed cytochrome P-450 2b-9 and cytochrome P-450 2b-10 mRNAs, but a number of differences in their response to phenobarbital were observed. In all these mouse strains, phenobarbital induced cytochrome P-450 2b-10 mRNA whereas it could have either a positive or a negative effect on cytochrome P-450 2b-9 expression, depending on the strain and the sex of the mice. Specifically, phenobarbital increased cytochrome P-450 2b-9 expression in C57BL/6J males while it decreased it in DBA/2J mice. Interestingly, dexamethasone was able to mimic the phenobarbital effect on both cytochromes P-450 in these two strains. Aldehyde dehydrogenase type 2 mRNA was always induced by phenobarbital, except in the C57BL/6J strain. Genetic analysis revealed that the phenobarbital-inducible phenotype was either a semi-dominant or a recessive trait in F1 animals from a C57BL/6J×DBA/2J cross for the cytochrome P-450 2b-9 and the aldehyde dehydrogenase type 2 genes, respectively. This study suggests that the genetic basis for phenobarbital induction in mice depends on the target gene, and that more than one regulatory step would be involved in this response pathway.
Characterization of the stilbenedisulphonate binding site on band 3 Memphis variant II (Pro-854→Leu)
Authors: SALHANY James M. , SLOAN Renee L. , SCHOPFER Lawrence M.
Published: August 2015

Band 3 Memphis variant II is a mutant anion-exchange protein associated with the Diego a+ blood group antigen. There are two mutations in this transporter: Lys-56 → Glu within the cytoplasmic domain, and Pro-854 → Leu within the membrane-bound domain. The Pro-854 mutation, which is thought to give rise to the antigenicity, is located within the C-terminal subdomain of the membrane-bound domain. Yet, there is an apparent enhancement in the rate of covalent binding of H2DIDS (4,4´-di-isothiocyanatodihydro-2,2´-stilbenedisulphonate) to ‘lysine A’ (Lys-539) in the N-terminal subdomain, suggesting widespread conformational changes. In this report, we have used various kinetic assays which differentiate between conformational changes in the two subdomains, to characterize the stilbenedisulphonate site on band 3 Memphis variant II. We have found a significantly higher H2DIDS (a C-terminal-sensitive inhibitor) affinity for band 3 Memphis variant II, due to a lower H2DIDS ‘off’ rate constant, but no difference was found between mutant and control when DBDS (4,4´-dibenzamido-2,2´-stilbenedisulphonate) (a C-terminal-insensitive inhibitor) ‘off’ rates were measured. Furthermore, there were no differences in the rates of covalent binding to lysine A, for either DIDS (4,4´-di-isothiocyanato-2,2´-stilbenedisulphonate) or H2DIDS. However, the rate of covalent intrasubunit cross-linking of Lys-539 and Lys-851 by H2DIDS was abnormally low for band 3 Memphis variant II. These results suggest that the Pro-854 → Leu mutation causes a localized conformational change in the C-terminal subdomain of band 3.
Effects of steroids and verapamil on P-glycoprotein ATPase activity: progesterone, desoxycorticosterone, corticosterone and verapamil are mutually non-exclusive modulators
Authors: ORLOWSKI Stéphane , MIR Lluis M. , BELEHRADEK Jean , GARRIGOS Manuel
Published: August 2015

P-glycoprotein (P-gp) is a membranous ATPase responsible for the multidrug resistance (MDR) phenotype. Using membrane vesicles prepared from the highly resistant cell line DC-3F/ADX we studied the influence on P-gp ATPase activity of four progesterone derivatives which specifically bind to P-gp and reverse MDR. Progesterone and desoxycorticosterone stimulate P-gp ATPase activity with, respectively, apparent concentrations giving half-maximal activation of 20–25 μM and 40–50 μM, and activation factors of 2.3 (at 100 μM progesterone) and 1.8 (at 170 μM desoxycorticosterone). Hydrocortisone above 100 μM stimulates P-gp ATPase activity while corticosterone has no apparent stimulating effect. Our data are consistent with the location of the binding sites for the progesterone derivatives on the P-gp membranous domain. The effects of these steroids on verapamil-stimulated P-gp ATPase activity support a non-competitive mechanism, i.e. the binding sites for verapamil and steroids are mutually non-exclusive for P-gp ATPase modulation. A similar non-competitive inhibition of progesterone-stimulated P-gp ATPase activity by desoxycorticosterone or by corticosterone leads to the conclusion that these steroids, although sharing related structures, have distinct modulating sites on P-gp. As expected from their mutually non-exclusive interactions on P-gp, progesterone and verapamil when mixed induce a synergistic modulation of P-gp ATPase activity. Since drug transport by P-gp is believed to be coupled to its ATPase activity, a corresponding synergistic effect of these two modulators for the inhibition of P-gp-mediated drug resistance can be expected.
Activation of protein kinase C by lysophosphatidic acid: dependence on composition of phospholipid vesicles
Authors: SANDO Julianne J. , CHERTIHIN Olga I.
Published: August 2015

Lysophosphatidic acid (LPA) has attracted recent attention as a major serum-derived regulator implicated in responses to vascular injury and inflammation, in tumour invasiveness and in neuronal signalling and remodelling. Although the possibility of a specific G-protein-coupled LPA receptor protein has been suggested, characterization of such a receptor is lacking. Since LPA can activate protein kinase C (PKC) pathways in many cells and PKC activators mimic many LPA effects, the possibility of more direct LPA effects on PKC was investigated. Phosphatidylcholine (PC)/phosphatidylserine (PS)/diacylglycerol (DAG) lipid vesicles of defined acyl chain composition were used to activate the enzyme. At total concentrations of saturated PC/PS+DAG vesicles (2–3 mM) that provided maximal PKC activation, 1–10 mol% [18:1]-LPA led to a further approx. 2-fold activation of PKCα. At lower lipid concentrations, a greater increase was observed with LPA concentrations up to 16–20 mol%. Higher concentrations of LPA were inhibitory. The LPA activation of PKC was dependent on the presence of DAG, PS and Ca2+. [18:1]-Lysophosphatidylcholine produced similar PKC activation in PC/PS/DAG vesicles. [14:0]-LPA was less effective, and longer-chain saturated lysolipids were ineffective. In unsaturated PC/PS vesicles, very little to no effect of LPA was discernable. These results suggest that physiologically or pathologically relevant concentrations of LPA can contribute to PKC activation depending on the composition of the lipid membrane. We hypothesize that LPA may affect the formation of lipid domains that are recognized by the enzyme.
Intracellular dissociation and reassembly of prolyl 4-hydroxylase:the α-subunits associated with the immunoglobulin-heavy-chain binding protein (BiP) allowing reassembly with the β-subunit
Authors: JOHN David C. A. , BULLEID Neil J.
Published: August 2015

Prolyl 4-hydroxylase (P4-H) consists of two distinct polypeptides; the catalytically more important α-subunit and the β-subunit, which is identical to the multifunctional enzyme protein disulphide isomerase. The enzyme appears to be assembled in vivo into an α2β2 tetramer from newly synthesized α-subunits associating with an endogenous pool of β-subunits. Using a cell-free system, we have shown previously that enzyme assembly is redox-dependent and that assembled α-subunits are intramolecularly disulphide-bonded [John and Bulleid (1994) Biochemistry 33, 14018–14025]. Here we have studied this assembly process within intact cells by expressing both subunits in COS-1 cells. Newly synthesized α-subunits were shown to assemble with the β-subunit, to form insoluble aggregates, or to remain soluble but not associate with the β-subunit. Treatment of cells with dithiothreitol (DTT) led to dissociation of P4-H into subunits and on removal of DTT the enzyme reassembled. This reassembly was ATP-dependent, suggesting an interaction with an ATP-dependent chaperone. This was confirmed when immunoglobulin-heavy-chain binding protein (BiP) and α-subunits were co-immunoprecipitated with antibodies against the α-subunit and BiP, respectively. These results indicate that unassembled α-subunits are maintained in an assembly-competent form by interacting with the molecular chaperone BiP.
Reversal of the temperature-shift-induced growth restriction of a temperature-sensitive simian virus 40 T-antigen-transformed human fibroblast cell line by treatment with retinoic acid
Authors: TSAO Yeou-Ping , LI Shu-Fang , KUO Shu-Wen , LIU Jiang-Chuan , CHEN Show-Li
Published: August 2015

We previously established a human fibroblast cell line, HFL 6-2, that contains a temperature sensitive simian virus 40 (SV40) T antigen, permitting cell growth at 35 °C but restricting growth at 39 °C. p21 (Waf1/Cip1) was significantly induced by temperature shifts in HFL 6-2 cells. Here we show that all-trans-retinoic acid (RA) treatment prevented the growth restriction of HFL 6-2 cells at 39 °C. In the presence of RA, HFL 6-2 cells proliferated into sizeable colonies even at 39 °C. [3H]Thymidine incorporation and flow cytometry analysis revealed that cells exposed to RA maintained DNA synthesis at 39 °C. Prevention of growth restriction by RA was correlated with a lack of induction of p21 at the transcription level. These observations suggest that RA may prevent the senescence process by repressing p21 gene expression, and perturb the growth regulation of somatic cells.
Identification of the cysteine residues implicated in the formation of α2 and α/β dimers of rat meprin
Authors: CHEVALLIER Sylvie , AHN Jinhi , BOILEAU Guy , CRINE Philippe
Published: August 2015

Meprin (endopeptidase-24.18; EC 3.4.24.18) is a multisubunit zinc-metallopeptidase found in the brush-border membranes of rodent kidney and human intestine. The α and β subunits of meprin are disulphide-linked to form either soluble α2 homodimers or membrane-associated α/β heterodimers. The aim of the present study was to identify the cysteine residue(s) implicated in the formation of α2 and α/β dimers and to investigate the effects of dimerization on intracellular transport and processing of the α subunit. Three cysteine residue candidates for the formation of disulphide bonds in the α subunit were selected by hydrophobic cluster analysis. These residues, located at positions 309, 560 and 562, were mutated to serine residues. When the resulting α subunit mutants were expressed alone in COS-1 cells, the αC560S and αC562S mutants were found to be secreted as α2 homodimers whereas the αC309S mutant was found as monomers in the culture medium. In double-transfection experiments with the wild-type β subunit, the αC560S and αC562S mutants behaved exactly as the wild-type α subunit and formed membrane-bound α/β heterodimers. In contrast, the αC309S mutant was not retained at the cell surface but rather secreted as monomers in the culture medium, as was found in the simple transfection experiment. These results show that, despite the normal expression level and folding of the protein in a transport-competent form, the αC309S mutant is unable to form α2 homodimers or α/β heterodimers. This suggests that Cys309 is the unique residue of the α subunit implicated in the α2 and α/β dimerizations. Hydrophobic cluster analysis of the α and β subunit sequences predicts that Cys309 is similar to Cys306 of the β subunit. We mutated the latter residue to a serine and expressed the βC306S mutant and the wild-type α subunit in the same COS-1 cells. No β2 or α/β dimers were observed on immunoblotting, showing that Cys306 of the β subunit is required for the formation of intermolecular disulphide bonds both in β2 homodimers and in α/β heterodimers. Taken together, these results suggest that the α/β heterodimeric form of meprin is held together by a single disulphide bond linking Cys309 in the α subunit to Cys306 in the β subunit.
The properties of a subtype of the inositol 1,4,5-trisphosphate receptor resulting from alternative splicing of the mRNA in the ligand-binding domain
Authors: LIÈVREMONT Jean-Philippe , LANCIEN Haud , HILLY Mauricette , MAUGER Jean-Pierre
Published: August 2015

Subtypes of the type-1 inositol 1,4,5-trisphosphate (InsP3) receptor differ at the mRNA level in two small variably spliced segments. One segment (SI) encodes for a sequence within the InsP3-binding domain, thus its presence or absence could affect the functions of the receptor. We have used anti-peptide antibodies to confirm the existence of different subtypes of the InsP3 receptor (InsP3R) protein. The antibody against residues 322–332, within the SI region, recognized a 260 kDa polypeptide in membranes prepared from rat cerebellum or cerebral cortex. The cerebellum contained a few percent of the InsP3R protein having the SI region, whereas the cerebral cortex contained a high proportion of receptors with the SI region. These two tissues were representative of both isoforms, SI- or SI+, and displayed the same [3H]InsP3-binding characteristics. Thus, the SI region was not involved in the basic properties of the receptor. Deletion of the peptide 316–352 containing the SI segment greatly reduced InsP3 binding [Miyawaki, Furuichi, Ryou, Yoshikawa, Nakagawa, Saitoh and Mikoshiba (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 4911–4915]. The antibodies against the SI region or against residues 337–349 did not modify the binding of [3H]InsP3 in the cortical membranes rich in the SI+ isoform or in cerebellar membranes. These results suggested that the SI region was not part of the binding site. The subcellular distribution of these two isoforms was then investigated in rat liver. The two isoforms were identified in different membrane fractions and they followed the same subcellular distribution. We suggest that the domain with the SI region may be involved in a function other than InsP3-induced Ca2+ release.
Enzyme-to-enzyme channelling of Krebs cycle metabolic intermediates in Caco-2 cells exposed to [2-13c]propionate
Authors: MALAISSE Willy J. , ZHANG Tie-Mei , VERBRUGGEN Ingrid , WILLEM Rudolph
Published: August 2015

The generation of 13C-labelled lactate by colon carcinoma cells of the Caco-2 line incubated for 120 min in the presence of [2-13C]propionate (10 mM) was assessed by 13C NMR. About 10% of the total amount of 13C-labelled lactate was recovered in the cell pellet and displayed a [2-13C]lactate/[3-13C]lactate isotopomer ratio of 1.18±0.01. An even higher isotopomer ratio of 1.53±0.14 was observed in the case of 13C-labelled lactate released by the cells into the incubation medium. These findings indicate that, in the Caco-2 cells, metabolic intermediates of the Krebs cycle undergo enzyme-to-enzyme channelling in the sequence of reactions catalysed by succinyl-CoA synthetase, succinate dehydrogenase and fumarase.
Human surfactant protein A with two distinct oligomeric structures which exhibit different capacities to interact with alveolar type II cells
Authors: HATTORI Akiko , KUROKI Yoshio , SOHMA Hitoshi , OGASAWARA Yoshinori , AKINO Toyoaki
Published: August 2015

The lung lavage fluids from patients with pulmonary alveolar proteinosis have been generally used as a source for human surfactant protein A (SP-A). We have recently found that a multimerized form of SP-A oligomer (alveolar proteinosis protein-I, APP-I) exists besides the normal-sized octadecamer (APP-II) in SP-As isolated from the patients. When analysed by Bio-Gel A15m column chromatography in 5 mM Tris buffer (pH 7.4), the apparent molecular masses of APP-I and APP-II were 1.65 MDa and 0.93 MDa, respectively. Gel-filtration analysis also revealed that APP-II is clearly separated from APP-I in the presence of 2 mM Ca2+ and 150 mM NaCl. We investigated the abilities of both SP-A oligomers to regulate phospholipid secretion and to bind to alveolar type II cells. Although APP-I inhibited lipid secretion, it was clearly a less effective inhibitor than APP-II. IC50 for inhibition of lipid secretion was apparently 0.23±0.08 µg/ml (0.14±0.05 nM) and 0.055±0.019 µg/ml (0.059±0.020 nM) for APP-I and APP-II, respectively. Both proteins bound to monolayers of type II cells in a concentration-dependent manner; however, APP-I clearly had a lower affinity to bind to type II cells. The apparent dissociation contants were, Kd = 2.31±0.70 µg/ml (1.40±0.43 nM) and 0.89±0.22 µg/ml (0.95±0.24 nM) for APP-I and APP-II, respectively. Excess unlabelled rat SP-A replaced 45% of 125I-APP-I and 77% of 125I-APP-II for type II cell binding. Although 125I-APP-II competed with excess unlabelled APP-I or APP-II, 125I-APP-I failed to compete and instead its binding rather increased in the presence of unlabelled APPs. The biotinylated APP-I bound to APP-I and APP-II coated on to microtitre wells in a concentration-dependent manner, indicating that APP-I interacts with APPs. This study demonstrates that the multimerized form of human SP-A oligomer exhibits the following attributes: (1) the reduced capacity to regulate phospholipid secretion from type II cells, and (2) lower affinity to bind to type II cells, and that the integrity of a flower-bouquet-like octadecameric structure of SP-A oligomer is important for the expression of full activity of this protein, indicating the importance of the oligomeric structure of mammalian lectins with collagenous domains.
Muscle-specific overexpression of lipoprotein lipase in transgenic mice results in increased α-tocopherol levels in skeletal muscle
Authors: SATTLER Wolfgang , LEVAK-FRANK Sanja , RADNER Herbert , KOSTNER Gerhard M. , ZECHNER Rudolf
Published: August 2015

Lipoprotein lipase (LPL) has been implicated in the delivery of chylomicron-located α-tocopherol (α-TocH) to peripheral tissues. To investigate the role of LPL in the cellular uptake of α-TocH in peripheral tissue in vivo, three lines of transgenic mice [mouse creatine kinase- (MCK) L, MCK-M and MCK-H] expressing various amounts of human LPL were compared with regard to α-TocH levels in plasma, skeletal muscle, cardiac muscle, adipose tissue and brain. Depending on the copy number of the transgene, LPL activity was increased 3- to 27-fold in skeletal muscle and 1.3- to 3.7-fold in cardiac muscle. The intracellular levels of α-TocH in skeletal muscle were significantly increased in MCK-M and MCK-H animals and correlated highly with the tissue-specific LPL activity (r = 0.998). The highest levels were observed in MCK-H (21.4 nmol/g) followed by MCK-M (13.3 nmol/g) and MCK-L (8.2 nmol/g) animals when compared with control mice (7.3 nmol/g). Excellent correlation was also observed between intracellular α-TocH and non-esterified fatty acid (NEFA) levels (r = 0.998). Although LPL activities in cardiac muscle were also increased in the transgenic mouse lines, α-TocH concentrations in the heart remained unchanged. Similarly, α-TocH levels in plasma, adipose tissue and brain were unaffected by the tissue specific overexpression of LPL in muscle. The transgenic model presented in this report provides evidence that the uptake of α-TocH in muscle is directly dependent on the level of LPL expression in vivo. Increased intracellular α-TocH concentrations with increased triglyceride lipolysis and NEFA uptake might protect the myocyte from oxidative damage during increased β-oxidation.