bluuebunny/crossref_metadata_2025 huggingface dataset:
Properties of the yeast nuclear histone deacetylase
Authors: Sanchez del Pino M M , Lopez-Rodas G , Sendra R , Tordera V
Published: August 2015

A nuclear histone deacetylase from yeast was partially purified and some of its characteristics were studied. Histone deacetylase activity was stimulated in vitro by high-mobility-group nonhistone chromatin proteins 1 and 2 and ubiquitin and inhibited by spermine and spermidine, whereas n-butyrate had no significant inhibitory effect. Like the mammalian enzyme, partially purified histone deacetylase from yeast was strongly inhibited by trichostatin A. However, in crude extract preparations the yeast enzyme was not inhibited and treatment with trichostatin in vivo did not show any effect, either on the histone acetylation level or on cell viability. At low ionic strength, the enzyme can be isolated as a complex of high molecular mass that is much less inhibited by trichostatin A than is partially purified histone deacetylase activity. Furthermore, radiolabelled oligonucleosomes were more efficiently deacetylated by the complex than by the low-molecular-mass form of the enzyme. The histone deacetylase activity was separated from a polyamine deacetylase activity and its specificity studied. Using h.p.l.c.-purified core histone species as substrate, histone deacetylase from yeast is able to deacetylate all core histones with a slight preference for H3. Our results support the idea that the yeast histone deacetylase may act as a high-molecular-mass complex in vivo.
Pteridine biosynthesis and nitric oxide synthase in Physarum polycephalum
Authors: Werner-Felmayer G , Golderer G , Werner E R , Gröbner P , Wachter H
Published: August 2015

Physarum polycephalum, an acellular slime mould, serves as a model system to study cell-cycle-dependent events since nuclear division is naturally synchronous. This organism was shown to release isoxanthopterin which is structurally related to tetrahydrobiopterin, a cofactor of aromatic amino acid hydroxylases and of nitric oxide synthases (NOSs) (EC 1.14.13.39). Here, we studied Physarum pteridine biosynthesis in more detail and found that high amounts of tetrahydrobiopterin are produced and NOS activity is expressed. Physarum pteridine biosynthesis is peculiar in as much as 7,8-dihydroneopterin aldolase (EC 4.1.2.25), an enzyme of folic acid biosynthesis usually not found in organisms producing tetrahydrobiopterin, is detected in parallel. NOS purified from Physarum depends on NADPH, tetrahydrobiopterin and flavins. Enzyme activity is independent of exogenous Ca2+ and is inhibited by arginine analogues. The purified enzyme (with a molecular mass of 130 kDa) contains tightly bound tetrahydrobiopterin and flavins. During the synchronous cell cycle of Physarum, pteridine biosynthesis increases during S-phase whereas NOS activity peaks during mitosis, drops at telophase and peaks again during early S-phase. Our results characterize Physarum pteridine biosynthesis and NOS and suggest a possible link between NOS activity and mitosis.
A possible role for glyceraldehyde transport in the stimulation of HIT-T15 insulinoma cells
Authors: Davies J , Tomlinson S , Elliott A C , Best L
Published: August 2015

D-Glyceraldehyde was transported into HIT-T15 cells at a linear rate for approx. 2 min and appeared to be unsaturable up to a concentration of 50 mM. Evidence was obtained for an electrogenic component of uptake of the triose. The rate of D-glyceraldehyde transport was also reduced in the absence of Na+, suggesting that a component of uptake was Na(+)-linked. Transport of D-glyceraldehyde could be prevented by N-ethylmaleimide but not significantly by p-chloromercuribenzenesulphonic acid, L-glyceraldehyde, nor by a number of inhibitors of known transport systems. However, D-glyceraldehyde transport was inhibited by alpha-cyano-4-hydroxycinnamate, an inhibitor of some anion transport systems. D-Glyceraldehyde caused a marked depolarization of HIT-T15 cells accompanied by a rise in cytosolic [Ca2+] and [Na+] and a gradual intracellular acidification. The glyceraldehyde-induced rise in cytosolic [Na+] and intracellular acidification, but not the depolarization or rise in cytosolic [Ca2+], were reduced by dithiothreitol and 5-aminoguanidine, compounds which form chemical adducts with alpha-ketoaldehydes. Incubation of HIT cells with either D- or L-glyceraldehyde resulted in the formation of large amounts of D-lactate, the end product of methylglyoxal metabolism via the glyoxalase pathway. It is suggested that the depolarizing action of glyceraldehyde is the result, at least in part, of its electrogenic transport, probably via Na(+)-coupled entry into HIT cells involving an unidentified transport system. The intracellular acidification and a component of the increase in cytosolic [Na+] may be largely due to the presence of one or more dicarbonyl contaminants in the glyceraldehyde preparation.
Calpastatin in erythrocytes of young and old individuals
Authors: Schwarz-Benmeir N , Glaser T , Barnoy S , Kosower N S
Published: August 2015

To gain knowledge about the behaviour of calpastatin (the specific inhibitor of the Ca(2+)-dependent thiol protease calpain) in the intact cell, we analysed the inhibitor by specific antibodies and determined its activity in erythrocytes from individuals 20-34 years old (young) and 70-93 years old (old). Differences between old and young in the behaviour of erythrocyte calpastatin were observed. Erythrocytes of old individuals had lower amounts of calpastatin and less calpastatin activity than those of young ones. A difference between old and young was also found in the molecular-mass distribution of calpastatin subunits. Increasing the erythrocyte Ca2+ induced changes in calpastatin in young individuals, rendering it similar to calpastatin in cells of old individuals. When calpastatin (isolated from erythrocytes of a young individual) was added to erythrocyte membranes, the initial binding and subsequent association of calpastatin with the membrane were lower in old than in young individuals. We had previously found that calpain binding and activation were enhanced in erythrocyte membranes from old individuals, along with enhanced degradation of band 3 (a major erythrocyte transmembrane anion-transport protein). The overall results indicate an interaction of calpain with calpastatin in the intact cell. Enhanced activation of erythrocyte calpain and degradation of calpastatin occur under conditions of increased cellular Ca2+ and in cells of the aged.
Purification of NADPH-dependent dehydroascorbate reductase from rat liver and its identification with 3α-hydroxysteroid dehydrogenase
Authors: Del Bello B , Maellaro E , Sugherini L , Santucci A , Comporti M , Casini A F
Published: August 2015

Rat liver cytosol has been found to reduce dehydroascorbic acid (DHAA) to ascorbic acid in the presence of NADPH. The enzyme responsible for such activity has been purified by ammonium sulphate fractionation, DEAE-Sepharose, Sephadex G-100 SF and Reactive Red column chromatography, with an overall recovery of 27%. SDS/PAGE of the purified enzyme showed one single protein band with an M(r) of 37,500. A similar value (36,800) was found by gel filtration on a Sephadex G-100 SF column. The results indicate that the enzyme is a homogeneous monomer. The Km for DHAA was 4.6 mM and the Vmax. was 1.55 units/mg of protein; for NADPH Km and Vmax. were 4.3 microM and 1.10 units/mg of protein respectively. The optimum pH was around 6.2. Several typical substrates and inhibitors of the aldo-keto reductase superfamily have been tested. The strong inhibition of DHAA reductase effected by steroidal and non-steroidal anti-inflammatory drugs, together with the ability to reduce 5 alpha-androstane-3,17-dione strongly, suggest the possibility that DHAA reductase corresponds to 3 alpha-hydroxysteroid dehydrogenase. Microsequence analysis performed on the electro-transferred enzyme band shows that the N-terminus is blocked. Internal primary structure data were obtained from CNBr-derived fragments and definitely proved the identity of NADPH-dependent DHAA reductase with 3 alpha-hydroxysteroid dehydrogenase.
Anti-diabetic biguanides inhibit hormone-induced intracellular Ca2+ concentration oscillations in rat hepatocytes
Authors: Ubl J J , Chen S , Stucki J W
Published: August 2015

Rat hepatocytes respond to glycogenolytic stimuli acting via phosphoinositide breakdown (e.g. alpha 1-adrenergic agonists, vasopressin) by oscillations of the free intracellular Ca2+ concentration ([Ca2+]i). We have investigated the action of metformin and phenformin, two anti-diabetic drugs of the biguanide type, on phenylephrine-induced [Ca2+]i oscillations. Metformin and phenformin lowered the frequency of the [Ca2+]i oscillations in a concentration-dependent manner with an IC50 of 0.1 mM and 1 microM, respectively. Simultaneous addition of the biguanides and insulin resulted in a further reduction of the frequency. By contrast, agents which increase the cellular cyclic AMP (cAMP) concentration (glucagon, forskolin, N,2′-O-dibutyryl-cAMP) reversed this inhibition. Furthermore, we investigated whether biguanides influenced the agonist-induced Ca2+ influx across the plasma membrane. When hepatocytes were loaded with the acetoxymethyl ester of fura-2 (fura-2/AM), addition of Mn2+ led to a quench of cellular fura-2, measured at the isosbestic excitation wavelength of 360 nm, until a new steady state was reached. Surprisingly, however, this addition of Mn2+ caused a marked increase of the fluorescence ratio simultaneously measured at 340 and 380 nm during the approach of the 360 nm signal to a new steady state. This observation can be understood on the basis of a compartmentalization of fura-2/AM into intracellular stores sensing the [Ca2+] therein. Subsequent application of phenylephrine resulted in a further decline of the fura-2 signal at 360 nm and a concomitant decrease of the fluorescence ratio. This second phase of the Mn2+ quench and the decrease of the fluorescence ratio could be diminished by addition of either 3 mM metformin or 30 microM phenformin. By contrast, when hepatocytes were loaded with fura-2/pentapotassium salt via a patch pipette, only the initial Mn(2+)-induced quench, measured at 360 nm, but no change of the fluorescence ratio, could be observed. The subsequent addition of phenylephrine and biguanides during the on-going quench caused no further changes, except for a fading oscillatory response. After loading hepatocytes with fluo-3 acetoxymethyl ester, the cells were permeabilized with 5 microM digitonin. Addition of inositol-1,4,5-trisphosphate (IP3) caused a rapid decrease of the remaining cellular fluorescence which could be effectively inhibited by 20 micrograms/ml heparin, indicating a release of Ca2+ from intracellular compartments mediated by IP3. This IP3-induced release of Ca2+ from intracellular stores could be diminished by prior addition of metformin and phenformin.(ABSTRACT TRUNCATED AT 400 WORDS)
Ca2+-mobilizing hormones induce sequentially ordered Ca2+ signals in multicellular systems of rat hepatocytes
Authors: Combettes L , Tran D , Tordjmann T , Laurent M , Berthon B , Claret M
Published: August 2015

The development of hormone-mediated Ca2+ signals was analysed in polarized doublets, triplets and quadruplets of rat hepatocytes by video imaging of fura2 fluorescence. These multicellular models showed dilated bile canaliculi, and gap junctions were observed by using an anti-connexin-32 antibody. They also showed highly organized Ca2+ signals in response to vasopressin or noradrenaline. Surprisingly, the primary rises in intracellular Ca2+ concentration ([Ca2+]i) did not start randomly from any cell of the multiplet. It originated invariably in the same hepatocyte (first-responding cell), and then was propagated in a sequential manner to the nearest connected cells (cell 2, then 3, in triplets; cell 2, 3, then 4 in quadruplets). The sequential activation of the cells appeared to be an intrinsic property of multiplets of rat hepatocytes. (1) In the continued presence of hormones, the same sequential order was observed up to six times, i.e. at each train of oscillations occurring between the cells. (2) The order of [Ca2+]i responses was modified neither by the repeated addition of hormones nor by the hormonal dose. (3) The mechanical disruption of an intermediate cell slowed down the speed of the propagation, suggesting a role of gap junctions in the rapidity of the sequential activation of cells. (4) The same multiplet could have a different first-responding cell for vasopressin or noradrenaline, suggesting a role of the hormonal receptors in the sequentiality of cell responses. It is postulated that a functional heterogeneity of hormonal receptors, and the presence of functional gap junctions, are involved in the existence of sequentially ordered hormone-mediated [Ca2+]i rises in the multiplets of rat hepatocytes.
Selective elongation of the oligosaccharide attached to the second potential glycosylation site of yeast exoglucanase: effects on the activity and properties of the enzyme
Authors: Basco R D , Hernández L M , Muñox M D , Olivero I , Andaluz E , Del Rey F , Larriba G
Published: August 2015

Three exoglucanases (Exgs), ExgIa, ExgIb and Exg325, are secreted by Saccharomyces cerevisiae cells. They share a common protein portion with two potential glycosylation sites (sequons) but differ in the amount of N-linked carbohydrate [Basco, R.D., Muñoz, M.D., Hernández, L.M., Váquez de Aldana, C. and Larriba, G. (1993) Yeast 9, 221-234]. ExgIb contains two short oligosaccharides attached to asparagines (Asn) 165 and 325 of the primary translation product [Hernández, L.M., Olivero, I., Alvarado, E. and Larriba, G. (1992) Biochemistry 31, 9823-9831]. Exg325 carries a single, short oligosaccharide bound to Asn325 whereas ExgIa has at least one large oligosaccharide, since it has not been produced by mutant mnn9. To address the question of the origin of ExgIa, both sequons were individually mutated by substituting Gln for Asn. An ExgIa-like isoenzyme was still secreted by mutant Exg165 but not by mutant Exg325. Additional studies on sequential deglycosylation of ExgIa with endo-beta-N-acetylglucosaminidase H (endo H), the susceptibility of both oligosaccharides to the endoglycosidase, and analysis of the presence of GlcNAc at both asparagine residues after total deglycosylation with endo H, indicated that ExgIa contained two oligosaccharides, a short one bound to Asn165 and a large one bound to Asn325, and, accordingly, originated from ExgIb. The elongation of the second oligosaccharide did not result in a higher stability towards thermal inactivation or unfolding, or in an increased resistance to proteases as compared with ExgIb; however, the affinity of the enzyme towards laminarin decreased by 50%. This site-specific elongation occurred in the oligosaccharide that was less susceptible to endo H, indicating that these properties are determined by different conformational constraints.
Cloning and functional expression of a Na+-dependent phosphate co-transporter from human kidney: cDNA cloning and functional expression
Authors: Miyamoto K , Tatsumi S , Sonoda T , Yamamoto H , Minami H , Taketani Y , Takeda E
Published: August 2015

A cDNA clone encoding a protein 69% identical in amino acid sequence with that of the Na/P(i) co-transporter NaP(i)-1 was isolated from a human kidney cDNA library. The DNA sequence was identical with that of NPT-1 cDNA published by Chong, Kristjansson, Zoghbi and Hughe (1993) (Genomics, 18, 355-359). In the present study, we have characterized the function of the encoded protein and the tissue distribution of its mRNA. Injection of RNA transcribed from NPT-1 into Xenopus oocytes resulted in expression of Na/P(i) co-transport activity showing a high affinity for P(i) transport (Km 0.29 mM). Kinetic characterization ([P(i)], [Na+]) demonstrated that the expressed transport activity has properties similar to those displayed by oocytes injected with human kidney poly(A)+ RNA. Northern blotting demonstrated that NPT-1 mRNA is expressed in renal cortex, liver and brain but not in other tissues. Hybrid depletion with antisense oligonucleotides to NaP(i)-3 and NPT-1 completely inhibited poly(A)+ RNA-induced Na(+)-dependent P(i) uptake in oocytes. These findings indicate that two high-affinity Na/P(i) cotransporters (NaP(i)-3 and NPT-1) are present in human kidney cortex.
Adrenocorticotrophic-hormone-dependent regulation of a μ-class glutathione transferase in mouse adrenocortical cells
Authors: Mankowitz L , Staffas L , Bakke M , Lund J
Published: August 2015

Three different forms of glutathione transferase (GST) have been resolved in the two mouse adrenal tumour cell lines Y1 and Kin 8. Two of these belong to the mu and pi classes respectively. The third form is so far unidentified. In the Y1 cells, the levels of the mu form (mGTmu1) and the unidentified form, are both down-regulated in the presence of adrenocorticotrophic hormone (ACTH) while the pi form is unaffected. The Kin 8 cell line is derived from Y1 cells and harbours a defect in the cyclic AMP (cAMP)-dependent protein kinase, making it refractory to cAMP-dependent regulation of several enzymes. The GST levels in this cell line were unaffected by ACTH. Also, the steady-state levels of mGTmu1 mRNA were much lower in Y1 cells treated with forskolin (which activates adenylate cyclase) compared with control cells, but there was no difference in mGTmu1 mRNA levels between control and forskolin-treated Kin 8 cells. This indicates that the ACTH-dependent regulation of the mu class GST is pre-translational and that a functional cAMP-dependent protein kinase is required for the regulation. We have further shown that the difference in mRNA steady-state levels between control and forskolin-treated Y1 cells is abolished when transcription is inhibited by actinomycin D. In light of the stability of mGTmu1 mRNA, it would appear most likely that actinomycin D inhibits the transcription of short-lived factors which regulate the turn-over of mGTmu1 transcripts in response to changes in intracellular cAMP levels.
Identification of an immunodominant functional domain on human CD36 antigen using human-mouse chimaeric proteins and homologue-replacement mutagenesis
Authors: Daviet L , Buckland R , Puente Navazo M D , McGregor J L
Published: August 2015

The human CD36 antigen is a multifunctional membrane glycoprotein that acts as a receptor for thrombospondin, malaria-infected erythrocytes and oxidized low-density lipoprotein, as well as being implicated in the recognition of apoptotic neutrophils by macrophages. OKM5 and other anti-CD36 monoclonal antibodies have been shown to inhibit these CD36 adhesive functions, suggesting that the monoclonal-antibody epitopes and the domains that mediate these events are closely related. Analysis of a series of chimaeric exchanges between human and mouse CD36 shows that six anti-CD36 monoclonal antibodies (OKM5, FA6-152, L103, 5F1, SM phi and 10/5) recognize epitopes within the domain comprising amino acids 155-183. A seventh monoclonal antibody (13/10) binds to another domain that spans amino acids 30-76. Homologue-replacement mutagenesis performed within the human 155-183 immunodominant sequence identifies key residues for the binding of three functional monoclonal antibodies (OKM5, FA6-152 and L103). The fact that antibodies directed against the 155-183 domain can inhibit adhesion suggests that this domain is directly involved in CD36-ligand binding.
Regulation of the efflux of putrescine and cadaverine from rapidly growing cultured RAW 264 cells by extracellular putrescine
Authors: Tjandrawinata R R , Byus C V
Published: August 2015

Cultures of the macrophage-like RAW 264 cells were adapted to divide normally in a synthetic serum-supplemented culture medium lacking any polyamines and diamine oxidase activity. These rapidly dividing cells actively effluxed large amounts of putrescine and cadaverine, compared with the intracellular levels, into the culture medium. The efflux of putrescine was stimulated by the amino acid ornithine, whereas efflux of cadaverine was inhibited. Relatively low levels of spermidine and N1-acetyl-spermidine, compared with those of exported putrescine, were observed to accumulate in the culture medium. A careful analysis of the changes in the intracellular concentration of putrescine relative to the steady-state net rate of putrescine export, as the doubling time of the cultures increased from 16 h to 22 h, indicated that an inverse relationship existed between these two parameters. As the intracellular putrescine concentrations increased, the net rate of putrescine export decreased markedly. Determination of the rate of putrescine uptake indicated that putrescine uptake also decreased significantly as the cultures neared confluency, and at no time during the growth of the culture did the rate of putrescine uptake approximate to the high rate of putrescine efflux. The decrease in the putrescine export rate seen as the cells grew toward confluency was determined to be primarily due to the inhibitory effect of the effluxed putrescine in the medium (Ki = 2 microM), and not to contact inhibition. The data suggested that the efflux of putrescine and cadaverine is not mediated to a significant degree by a process involving simple diffusion.
Expression and DNA binding of the human 52 kDa Ro/SSA autoantigen
Authors: Frank M B , McCubbin V R , Heldermon C
Published: August 2015

The 52 kDa Ro/SSA protein is an intracellular autoantigen that is frequently recognized by antibodies in sera of patients with systemic lupus erythematosus or Sjogren's syndrome. While the function of this molecule is not known, zinc finger and leucine zipper motifs have been identified in its predicted amino acid sequence which suggest that it may interact with nucleic acids. To test this hypothesis, the human gene which encodes this protein was cloned in a baculovirus and expressed in Spodoptera frugipoda cells. Extracts from these infected insect cells were used as a source of protein for this study. The protein is similar in size and antigenicity to that expressed in human cells. This protein binds to DNA at physiological temperature and is eluted with high concentrations of sodium chloride. Striking similarities were found between the sequence in, and adjacent to, the nucleic acid-binding motifs of 52 kDa Ro/SSA and a growing family of zinc finger proteins which have been shown to bind to DNA or regulate gene expression. The findings presented here place this protein structurally and functionally in this family and demonstrate a biochemical assay which can be used to study its function.
Ultraviolet radiation stimulates a biphasic pattern of 1,2-diacylglycerol formation in cultured human melanocytes and keratinocytes by activation of phospholipases C and D
Authors: Carsberg C J , Ohanian J , Friedmann P S
Published: August 2015

Ultraviolet radiation (UVR) induces melanin synthesis by human epidermal melanocytes, and phospholipid-derived 1,2-diacylglycerols (DAGs) have been implicated in mediating this response. In previous experiments, addition of the synthetic DAG 1-oleoyl-2-acetylglycerol to cultured pigment cells stimulated melanogenesis. The purpose of the present study was to analyse the effects of UVR on the endogenous generation of DAGs. It was found that in a number of cultured cell types, including human melanocytes and B16 mouse melanoma cells, but also human keratinocytes and Swiss 3T3 fibroblasts, exposure to a single dose of UVR stimulated a biphasic increase in endogenous DAG formation. An early transient rise, over seconds, was followed by a more sustained delayed rise over minutes. The early rise in DAG levels was accompanied by a transient rise in inositol trisphosphate formation, indicating activation of phosphatidylinositol-specific phospholipase C. The delayed rise was accompanied by activation of phospholipase D. This endogenous DAG formation by pigment cells is further evidence for the involvement of DAGs in UVR-induced epidermal melanin synthesis. Since DAG formation is also seen in other cells types, it is possible that DAGs may be involved in an array of UVR-induced responses.
A novel receptor-type protein tyrosine phosphatase with a single catalytic domain is specifically expressed in mouse brain
Authors: Hendriks W , Schepens J , Brugman C , Zeeuwen P , Wieringa B
Published: August 2015

Protein tyrosine phosphatases (PTPases) are important regulatory proteins that, together with protein tyrosine kinases, determine the phosphotyrosine levels in cell signalling proteins. By PCR amplification of mouse brain cDNA fragments encoding the catalytic domains of these enzymes, we identified three novel members of the PTPase gene family. Northern-blot analysis showed that two of these novel clones represent brain-specific PTPases, whereas the third originates from a large-sized mRNA that is more ubiquitously expressed. A full-length cDNA encoding one of the brain-specific PTPases, PTP-SL, was isolated. Sequence analysis revealed a transmembrane PTPase containing a single catalytic phosphatase domain that has 45% homology to a rat cytoplasmic brain-specific PTPase named STEP. This suggests a role for PTP-SL in cell-cell signalling processes in the brain.
μ-opioids activate phospholipase C in SH-SY5Y human neuroblastoma cells via calcium-channel opening
Authors: Smart D , Smith G , Lambert D G
Published: August 2015

We have recently reported that, in SH-SY5Y cells, mu-opioid receptor occupancy activates phospholipase C via a pertussis toxin-sensitive G-protein. In the present study we have further characterized the mechanisms involved in this process. Fentanyl (0.1 microM) caused a monophasic increase in inositol 1,4,5-trisphosphate mass formation, with a peak (20.5 +/- 3.6 pmol/mg of protein) at 15 s. Incubation in Ca(2+)-free buffer abolished this response, while Ca2+ replacement 1 min later restored the stimulation of inositol 1,4,5-trisphosphate formation (20.1 +/- 0.6 pmol/mg of protein). In addition, nifedipine (1 nM-0.1 mM), an L-type Ca(2+)-channel antagonist, caused a dose-dependent inhibition of inositol 1,4,5-trisphosphate formation, with an IC50 of 60.3 +/- 1.1 nM. Elevation of endogenous beta/gamma subunits by selective activation of delta-opioid and alpha 2 adrenoceptors failed to stimulate phospholipase C. Fentanyl also caused a dose-dependent (EC50 of 16.2 +/- 1.0 nM), additive enhancement of carbachol-induced inositol 1,4,5-trisphosphate formation. In summary, we have demonstrated that in SH-SY5Y cells activation of the mu-opioid receptor allows Ca2+ influx to activate phospholipase C. However, the possible role of this mechanism in the process of analgesia remains to be elucidated.
Role of phosphodiesterase isoenzymes in regulating intracellular cyclic AMP in adenosine-stimulated smooth muscle cells
Authors: Xiong Y , Westhead E W , Slakey L L
Published: August 2015

Three phosphodiesterase (PDE) isoenzymes were separated by Mono Q h.p.l.c. column chromatography from the soluble fraction of a homogenate of pig aortic smooth muscle cells. The first peak of PDE activity was stimulated by calmodulin in the presence of calcium. The second broad peak contained at least two activities, which were sensitive to inhibition by CI-930 or rolipram respectively. The distribution of total cellular enzyme activity in different subcellular fractions was also determined. The majority (78%) of the total activity was present in the cytosolic fraction, 18% of activity was in a membrane-bound form and 4% of activity was associated with the cytoskeleton. Rolipram-sensitive PDE was present predominantly in the cytosolic fraction, whereas cyclic GMP-inhibited, CI-930-sensitive PDE was evenly distributed between the cytosolic and particulate fractions. All of the calmodulin-dependent PDE activity was found in the soluble fraction. CI-930 and rolipram enhanced, by 2-fold and 3-4-fold respectively, the adenosine-stimulated rise in cellular cyclic AMP level. The increase in cyclic AMP levels due to CI-930 or rolipram was dose-dependent. Removal of adenosine once cyclic AMP had risen resulted in a rapid fall in cyclic AMP levels even in the presence of rolipram and CI-930. M&B 22,948, the calmodulin-dependent PDE inhibitor, caused less than a 25% increase of the adenosine-stimulated cyclic AMP levels by itself, but it contributed substantially to controlling the cyclic AMP levels after the removal of adenosine when used together with CI-930 and rolipram. These phenomena suggested that all three PDE isoenzymes participated in modulating cellular cyclic AMP levels after adenosine stimulation, and that differential importance of the individual isoenzymes depends on cellular cyclic AMP levels.
src-homology 2 (SH2) domain ligation as an allosteric regulator: modulation of phosphoinositide-specific phospholipase Cγ1 structure and activity
Authors: Koblan K S , Schaber M D , Edwards G , Gibbs J B , Pompliano D L
Published: August 2015

Phosphoinositide-specific phospholipase C gamma 1 (PI-PLC gamma 1) catalyses the hydrolysis of PtdIns(4,5)P2 to generate the second messengers diacylglycerol and Ins(1,4,5)P3. PI-PLC gamma 1, an src-homology 2/3 (SH2/SH3)-domain-containing enzyme, is activated in response to growth-factor-induced tyrosine phosphorylation, and, in vivo, is translocated from the cytosol to the particulate cell fraction. Here we report the bacterial expression of rat brain PI-PLC gamma 1 under the control of the T7 promoter. Production of the active enzyme in amounts suitable for structure-function analysis depended on coupling the translation of PLC gamma 1 to the expression of the phage-phi 10 coat protein. Purification of the enzyme was facilitated by the presence of a three-amino-acid C-terminal antibody epitope tag (Glu-Glu-Phe) engineered into the cloned PLC gamma 1. Examination of the specific activity, pH-rate profile, [Ca2+]-dependence and substrate specificity of bacterially expressed PLC gamma indicated that it had kinetic properties similar to those of PLC gamma isolated from bovine brain. The substrate specificity was dependent on [Ca2+]: at low [Ca2+] (1-10 microM) PtdIns(4,5)P2 was a better substrate than PtdIns. Addition of phosphotyrosine-containing peptides (12-mers) with the cognate sequence of the high-affinity binding site for PLC gamma 1 on the activated epidermal-growth-factor (EGF) receptor (Tyr-992) increased enzyme activity (up to 85%) in vitro. Cognate non-phosphorylated peptides had no effect on activity. When c.d. spectroscopy was used to monitor the effect of added phosphotyrosine-containing peptide on the structure of recombinant PLC gamma 1, significant spectral shifts, indicative of a conformational change, were observed upon complexation with the EGF-receptor phosphotyrosine-containing 12-residue peptide (Tyr*-992). How SH2 domains from PLC gamma 1 can mediate structural rearrangements and modulate enzymic activity on their ligation by growth-factor receptors is discussed.
Topological organization of the Rieske iron-sulphur protein and subunit IV in the cytochrome bc1 complex of Rhodobacter sphaeroides
Authors: Wu J , Niederman R A
Published: August 2015

The ubiquinol-cytochrome c2 oxidoreductases (cytochrome bc1 complex) of Rhodobacter sphaeroides contains highly conserved cytochrome b, cytochrome c1 and Rieske FeS subunits, as well as a unique 14 kDa polypeptide, designated as subunit IV, thought to function as a ubiquinol-binding protein [Yu and Yu (1991) Biochemistry 30, 4934-4939]. As the topology of subunit IV is unknown and that of the FeS subunit remains a matter of debate, both the inner (cytoplasmic) and outer (periplasmic) surfaces of the intracytoplasmic membrane (ICM) were digested with proteinase K, and cleavage products were identified by immunoblotting. In uniformly oriented chromatophore vesicles (inner ICM surface exposed), fragments of approx. 4 and 1 kDa were removed from subunit IV and the FeS protein respectively. Neither subunit IV nor the FeS protein was cleaved from the outer ICM surface as exposed in osmotically protected spheroplasts or as presented to proteinase K after microencapsulation of the protease in unilamellar liposomes and fusion of these structures to chromatophore vesicles. Studies with the isolated bc1 complex, however, suggested that the C-terminal domain of the Rieske FeS, thought to reside on the periplasmic side of the ICM, was resistant to proteinase K. Overall, these results suggest a single N-terminal transmembrane helix for the FeS protein, with exposure of the N-terminus to the cytoplasm and an orientation in which a major, N-terminal portion of subunit IV is located in the cytoplasm with the predicted C-terminal transmembrane domain anchoring this polypeptide to the membrane.
Activation of hepatic glutaminase by spermine
Authors: Kovacevic Z , Day S H , Collett V , Brosnan J T , Brosnan M E
Published: August 2015

Glutaminase activity in intact mitochondria from rat liver is activated by spermine, as indicated both by increased glutamate production from glutamine and by increased respiration with glutamine as sole substrate. Glutaminase activity assayed in membranes from frozen-thawed mitochondria, is activated by spermine about 6-fold at physiological concentrations of its other effectors (NH4+ at 0.7 mM, Pi 5 mM) and at pH 7.4. Spermine decreased the apparent Km for glutamine from 38 to 15 mM at 5 mM Pi, and increased the sensitivity of the enzyme for phosphate activation so that the concentration required for 50% stimulation decreased from 15 to 4 mM. Half-maximal spermine effects occurred at 0.15 mM, which is in the physiological range. Spermine was effective in the presence of physiological concentrations of Mg2+. We suggest that spermine may be a physiological activator of hepatic glutaminase.
Comparative study of the structural and functional properties of a bovine plasma C-type lectin, collectin-43, with other collectins
Authors: Holmskov U , Laursen S B , Malhotra R , Wiedemann H , Timpl R , Stuart G R , Tornøe I , Madsen P S , Reid K B M , Jensenius J C
Published: August 2015

Collectin-43 (CL-43) is a recently described bovine plasma protein containing both collagenous regions and C-type-lectin domains [Holmskov, Teisner, Willis, Reid and Jensenius (1993) J. Biol. Chem. 268, 10120-10125; Lim, Willis, Reid, Lu, Laursen, Jensenius and Holmskov (1994) J. Biol. Chem. 269, 11820-11824]. CL-43 was purified by affinity chromatography on mannan-Sepharose. On SDS/PAGE under reducing conditions the purified lectin showed a double band at about 43 kDa, with the upper band representing the intact molecule and the lower band a truncated form that lacked the N-terminal nine amino acid residues. Under non-reducing conditions, only one band was seen at 120 kDa. Analytical gel chromatography and sucrose-density-gradient centrifugation of the purified molecule, showed a Stokes radius of 9.1 +/- 0.3 nm (91 +/- 3 A) and a sedimentation coefficient (s20,w) of 3.6 +/- 0.1 S. These values correspond to a molecular mass of 119-138 kDa under non-denaturing condition in solution. The frictional coefficient (f/f0) was 2.7, indicating extreme elongation due to the collagenous segment. Only monomer subunits, with 37.4 +/- 1.7-nm-long rods, were seen by electron microscopy. These findings indicate that CL-43, in contrast with the other circulating collectins, is found only as a single subunit composed of three polypeptide chains. Two-dimensional gel electrophoresis showed that CL-43 has two isoforms, with pI values of 4.9 and 5.3, corresponding to the native form and the truncated form of the molecule respectively. CL-43, like conglutinin, lung surfactant protein A and mannan-binding protein (MBP), was shown to bind to the collectin receptor. Bovine MBP caused the activation of the complement system as revealed by the deposition of complement component C4 upon incubation of diluted serum in wells containing MBP bound to solid-phase mannan. CL-43, lung surfactant protein D (SP-D) and conglutinin showed no complement-activating properties under the same conditions. Conglutinin binds fluid- and solid-phase iC3b, while CL-43 and MBP do not show such reactivity.
Increased response to cholesterol feeding in apolipoprotein C1-deficient mice
Authors: van Ree J H , Hofker M H , van den Broek W J A A , van Deursen J M A , van der Boom H , Frants R R , Wieringa B , Havekes L M
Published: August 2015

The function of apolipoprotein (apo) C1 in vivo is not well understood. From in vitro studies it has been reported that an excess of apoC1 relative to apoE inhibits receptor-mediated uptake of remnant lipoproteins [Sehayek and Eisenberg (1991) J. Biol. Chem. 266, 22453-22459]. In order to gain a better understanding of the role of apoC1 in lipoprotein metabolism in vivo, we have generated apoC1-deficient mice by gene targeting in embryonic stem cells. Homozygous mutant mice are viable and do not show overt abnormalities. Serum triacylglycerol levels are increased by 60% on both a standard mouse diet and a mild hypercholesterolaemic diet compared with controls. Total serum cholesterol levels are similar to controls on the two diets. However, the level of high-density lipoprotein cholesterol in the apoC1-deficient mice fed on the mild hypercholesterolaemic diet is slightly decreased, which is accompanied by a 3-fold increase in very-low-density plus low-density lipoprotein (VLDL+LDL) cholesterol. On a severe atherogenic diet, the homozygous apoC1-deficient mice become hypercholesterolaemic, with a serum cholesterol level of 10.7 +/- 3.3 mM compared with 6.7 +/- 1.8 mM and 5.1 +/- 1.6 mM in heterozygous and control mice respectively. The increase in cholesterol is mainly confined to the VLDL+LDL-sized fractions. Binding experiments revealed that lipoproteins lacking apoC1 with d < 1.006 g/ml are poor competitors for 125I-labelled LDL binding to the LDL receptor on HepG2 cells. This suggests that total apoC1 deficiency leads to impaired receptor-mediated clearance of remnant lipoproteins rather than enhanced uptake, as was expected from data reported in the literature.
Dissociation of the AT-specific bifunctional intercalator [N-MeCys3,N-MeCys7]TANDEM from TpA sites in DNA
Authors: Fletcher M C , Olsen R K , Fox K R
Published: August 2015

We have examined the dissociation of [N-MeCys3,N-MeCys7]TANDEM, an AT-selective bifunctional intercalator, from TpA sites in mixed-sequence DNAs by a modification of the footprinting technique. Dissociation of complexes between the ligand and radiolabelled DNA fragments was initiated by adding a vast excess of unlabelled calf thymus DNA. Portions of this mixture were subjected to DNAse I footprinting at various times after adding the competitor DNA. Dissociation of the ligand from each site was seen by the time-dependent disappearance of the footprinting pattern. Within a natural DNA fragment (tyrT) the ligand dissociates from TTAT faster than from ATAT. We found that the stability of complexes with isolated TpA steps decreases in the order ATAT > TTAA > TATA. Dissociation from each of these sites is much faster than from longer regions of (AT)n. These results confirm the requirement for A and T base-pairs surrounding the TpA step and suggest that the interaction is strongest with regions of alternating AT, possibly as a result of its unusual structure. The ligand dissociates more slowly from the centre of (AT)n tracts than from the edges, suggesting that variations in dissociation rate arise from sequence-dependent variations in local DNA structure.
Identification of a novel form of the α3 integrin subunit: covalent association with transferrin receptor
Authors: Coppolino M , Migliorini M , Argraves W S , Dedhar S
Published: August 2015

The alpha 3 beta 1 integrin is a cell-surface receptor for laminin, entactin, collagen, fibronectin and epiligrin. On some prostatic-carcinoma cell lines that express the alpha 3 beta 1 heterodimer we have identified a novel form of the alpha 3 subunit. Whereas the prototypic alpha 3 subunit has a molecular mass of approximately 155 kDa, we have isolated a approximately 225 kDa protein (p225) which is recognized by monoclonal antibodies to the alpha 3 subunit. Protein sequence analysis revealed that p225 consists of two polypeptides, namely integrin alpha 3 heavy chain (approximately 130 kDa) disulphide-bonded to a monomer of the transferrin receptor (approximately 95 kDa) instead of the typical alpha 3 light chain (approximately 25 kDa). The p225 seems to be directly associated with beta 1 subunit, since it was immunoprecipitable with anti-(beta 1 subunit) antibodies. The association of transferrin receptor and integrin alpha 3 was apparently not the result of spurious disulphide-bond formation occurring during the protein purification, as iodoacetamide and GSH did not block the formation of the complex. The transferrin receptor is normally a homodimer that is involved in the internalization of iron-bound transferrin into cells and can be expressed at relatively high levels in the cell lines which we have studied. The p225 is not found on all cell types examined to date and therefore it may represent a unique complex between the integrin alpha 3 subunit and the transferrin receptor, a covalent association which may play a role in the adherence and/or proliferation of some types of tumour cells.
Effect of inhibitor time-dependency on selectivity towards cyclooxygenase isoforms
Authors: Ouellet M , Percival M D
Published: August 2015

Cyclooxygenase (Cox) is a key enzyme in the biosynthesis of prostaglandins and, as such, is the target of non-steroidal anti-inflammatory drugs (NSAIDs). Two isoforms exist, being expressed constitutively (Cox-1), or inducibly in response to inflammatory mediators (Cox-2). Currently available NSAIDs inhibit both isoforms somewhat equipotently but selective Cox-2 inhibition may eliminate unwanted side effects. We have characterized the kinetic mechanisms of the interactions of purified recombinant human cyclooxygenase-1 and -2 (hCox-1, hCox-2) with the selective Cox-2 inhibitor N-(2-cyclohexyloxy-4-nitrophenyl)methanesulphonamide (NS-398) and some classical non-selective NSAIDs. NS-398, flurbiprofen, meclofenamic acid and indomethacin are time-dependent, irreversible inhibitors of hCox-2. The inhibition is consistent with a two-step process, involving an initial rapid equilibrium binding of enzyme and inhibitor, characterized by Ki, followed by the slow formation of a tightly bound enzyme-inhibitor complex, characterized by a first-order rate constant kon. NS-398 is a time-independent inhibitor of hCox-1, consistent with the formation of a reversible enzyme-inhibitor complex. Flurbiprofen, meclofenamic acid and indomethacin are also time-dependent inhibitors of hCox-1 and hence show little selectivity for one isoform over the other. Flufenamic acid is time independent towards both isoforms and is also non-selective. The high degree of selectivity of NS-398 towards Cox-2 results therefore from the difference in the nature of the time-dependency of inhibition of the two isoforms.
Primary structures of seven metallothioneins from rabbit tissue
Authors: Hunziker P E , Kaur P , Wan M , Känzig A
Published: August 2015

Metallothionein from tissues of rabbits exposed to cadmium chloride was separated into seven distinct isoforms by reverse-phase liquid chromatography and their complete amino acid sequences were determined. Five of the seven isometallothioneins showed structural features so far not identified in other mammalian metallothioneins. Thus, two isoproteins contain a polypeptide with a chain length of 62 rather than 61 amino acid residues. Two isoforms are characterized by an additional positive charge and one by the presence of an isopeptide bond between aspartic acid and serine in the N-terminal half of the protein. The isoproteins characterized were identified from different sources: rabbit liver and kidney and a rabbit kidney cell-line (RK-13). In all three, the structural characteristics of the individual isoforms are retained, indicating that in the different tissues the same mechanisms control the synthesis and the stability of the different cadmium-induced isoMTs.
The characterization of two diazepam binding inhibitor (DBI) transcripts in humans
Authors: Kolmer M , Rovio A , Alho H
Published: August 2015

We have investigated the expression of diazepam binding inhibitor (DBI) (also called acyl-CoA-binding protein or endozepine) transcripts in different human tissues and tissue culture cell lines by reverse-transcriptase assisted PCR and RNase protection assay. Two different DBI transcripts capable of encoding polypeptides of 86 and 104 amino acids were detected in all the human tissues and cell lines studied. The transcript coding for the 86 amino acid DBI polypeptide was found to represent the majority of the total DBI transcript pool.
Effect of magnetic field strength on the linewidth and spin-lattice relaxation time of the thiocyanate carbon of cyanylated β-lactoglobulin B: optimization of the experimental parameters for observing thiocyanate carbons in proteins
Authors: Malthouse J P G , Phelan P
Published: August 2015

The linewidths and spin-lattice relaxation times of the 13C-n.m.r. signal at 109.7 p.p.m. due to the thiocyanate carbon of intact [cyanato-13C]cyanylated-beta-lactoglobulin-B have been determined at magnetic field strengths of 1.88, 6.34 and 11.74 T as well as the spin-lattice relaxation times of its backbone alpha-carbon atoms. The linewidths were directly proportional to the square of the magnetic field strength and we conclude that, at magnetic field strengths of 6.34 T or above, more than 70% of the linewidth will be determined by chemical-shift anisotropy. We estimate that the spin-lattice relaxation time resulting from the chemical-shift anisotropy of the thiocyanate carbon is 1.52 +/- 0.1 s and we conclude that for magnetic field strengths of 6.34 T and above the observed spin-lattice relaxation time of the thiocyanate carbon will be essentially independent of magnetic field strength. Using the rigid-rotor model we obtain estimates of the rotational correlation time of [cyanato-13C]cyanylated-beta-lactoglobulin-B and of the chemical-shift anisotropy shielding tensor of its thiocyanate carbon. We have calculated the linewidths and spin-lattice relaxation times of thiocyanate carbons at magnetic field strengths of 1.88-14.1 T in proteins with M(r) values in the range 10,000-400,000. The effects of magnetic field strength on the resolution and signal-to-noise ratios of the signals due to thiocyanate carbons attached to proteins of M(r) greater than 10,000 are discussed.