bluuebunny/crossref_metadata_2025 huggingface dataset:
Regulation of urea and citrulline synthesis under physiological conditions
Authors: Beliveau Carey G , Cheung C W , Cohen N S , Brusilow S , Raijman L
Published: August 2015

Information on the regulation of urea synthesis in vivo was obtained by examining the relationship between ureagenesis in vivo, citrulline synthesis in vitro, and two factors currently hypothesized to exert short-term regulation of this pathway: the liver mitochondrial content of N-acetylglutamate (NAG) and substrate availability. Rats meal-fed for 4 h every day (4-20 schedule) or for 8 h every other day (8-40 schedule) were used. (1) The citrulline-synthesizing capacity of mitochondria from livers of rats on the 8-40 schedule exceeded the corresponding velocity of urea synthesis in vivo at all time points studied. (2) Mitochondrial NAG in these livers increased from 127 +/- 32 pmol/mg of protein at 0 h to 486 +/- 205 pmol/mg at 3 h after the start of a meal, and decreased thereafter, but the correlation between NAG content and the velocity of citrulline synthesis was not simple, suggesting that NAG is not the only determinant of the state of activation of carbamoyl phosphate synthase I. (3) In rats on the 4-20 schedule killed 1 h after the start of the meal, the liver content of ornithine, citrulline, arginine, glutamate, alanine and urea increased 2.1-12-fold with respect to the values at 0 h; glutamine decreased by 39%. (4) The combined findings indicate that in vivo, moment-to-moment control of the velocity of urea synthesis is exerted by substrate availability. (5) Digestion limits the supply of substrate to the liver, and prevents its ureagenic capacity from being overwhelmed following a protein-containing meal.
Indication of possible post-translational formation of disulphide bonds in the β-sheet domain of human lysozyme
Authors: Kanaya E , Ishihara K , Tsunasawa S , Nokihara K , Kikuchi M
Published: August 2015

Lysozyme has two distinct folding domains, and in most molecules the alpha-helical domain folds more quickly than the beta-sheet domain in vitro [Radford, Dobson and Evans (1992) Nature (London) 358, 302-307]. In order to investigate the relationship between the formation of disulphide bonds and protein folding in vivo, we carried out cysteine scanning mutagenesis to shift positions of the disulphide bonds in both the alpha-helical and beta-sheet domains of human lysozyme. Of the constructed mutants (nine in the beta-sheet domain and 13 in the alpha-helical domain), the mutant L79CC81A, in which Leu-79 and Cys-81 in the beta-sheet domain were replaced by Cys and Ala respectively, was secreted by yeast. The rest of the mutants were retained in the insoluble fraction of the cell, probably because of a failure of folding. The distance between the two alpha-carbons at positions 79 and 95 in the wild-type protein is too far to form a disulphide bond, but analysis of the primary structure revealed that the major part of L79CC81A was secreted with a non-native disulphide bond Cys79-Cys95 and two free cysteine residues at positions 65 and 77 in the beta-sheet domain. These results suggest that the beta-sheet domain of human lysozyme can tolerate the shift of locations of disulphide bonds, and the non-native folding of mutated polypeptide chains in in vivo folding. The free residues Cys-65 and Cys-77 formed a disulphide bond in vitro by air oxidation, yielding two isomers. On the basis of our previous results and present study it is suggested that the formation of Cys6-Cys128 is the first step of the in vivo correct folding of human lysozyme, and disulphide bonds in the beta-sheet domain are post-translationally formed in vivo.
Intracellular degradation and reduced cell-surface expression of sucrase-isomaltase in heat-shocked Caco-2 cells
Authors: Quaroni A , Paul E C A , Nichols B L
Published: August 2015

To investigate the role of post-translational events in intestinal cell differentiation we have studied the effects of heat shock on processing and cell surface delivery of sucrase-isomaltase (SI), dipeptidylpeptidase IV (DPPIV) and aminopeptidase N (APN) in Caco-2 cells. In cells cultured at 42.5 degrees C there was a rapid decline in sucrase activity, while DPPIV and APN were unaffected over a 3-day period. Immunofluorescence staining confirmed the selective disappearance of SI from the surface membrane after only 1 day of culture at 42.5 degrees C. Cell-surface biotinylation of cells metabolically labelled with [35S]methionine 4 h after a switch from 37 degrees C to 42.5 degrees C demonstrated that newly synthesized APN and DPPIV were associated with the surface membrane, while SI was almost completely retained intracellularly. Pulse-chase experiments confirmed that, in these cells, DPPIV and APN were normally processed and vectorially delivered to the cell surface; in contrast, conversion between the two conformationally distinct high-mannose precursor forms of SI (hmP1 and hmP2) was markedly inhibited, a significant fraction of newly synthesized enzyme was degraded, probably in the ER, and an immature form of complex-glycosylated SI precursor (cP) was produced and mostly retained intracellularly. Double labelling of Caco-2 cells for SI and cathepsin D excluded an accumulation of SI in the lysosomes, suggesting that this organelle was not involved in the degradation of SI. These results indicate that the ER may play an important role in intestinal cell differentiation by regulating the conformational maturation, degradation and eventual cellular localization of some digestive enzymes.
Protein kinase C activity is not involved in N-formylmethionyl-leucyl-phenylalanine-induced phospholipase D activation in human neutrophils, but is essential for concomitant NADPH oxidase activation: studies with a staurosporine analogue with improved selectivity for protein kinase C
Authors: Kessels G C , Krause K H , Verhoeven A J
Published: August 2015

Stimulation of human neutrophils by the receptor agonist N-formylmethionyl-leucyl-phenylalanine (fMLP) results in a respiratory burst, catalysed by an NADPH oxidase. Concomitantly, phospholipase D (PLD) is activated. To investigate the role of protein kinase C (PKC) in these neutrophil responses, we have compared the effects of staurosporine and a structural analogue of staurosporine (cgp41251), that reflects a higher selectivity towards PKC [Meyer, Regenass, Fabbro, Alteri, Rösel, Müller, Caravatti and Matter (1989) Int. J. Cancer 43, 851-856]. Both staurosporine and cgp41251 dose-dependently inhibited the production of superoxide induced by phorbol 12-myristate 13-acetate (PMA). Both compounds also caused inhibition of the fMLP-induced respiratory burst, but with a lower efficacy during the initiation phase of this response. This latter observation cannot be taken as evidence against PKC involvement in the activation of the respiratory burst, because pretreatment of neutrophils with ionomycin before PMA stimulation also results in a lower efficacy of inhibition. Activation of PLD by fMLP was enhanced in the presence of staurosporine, but not in the presence of cgp41251. Enhancement of PLD activation was also observed in the presence of H-89, an inhibitor of cyclic-AMP-dependent protein kinase (PKA). Both staurosporine and H-89 reversed the dibutyryl-cyclic-AMP-induced inhibition of PLD activation, whereas cgp41251 was without effect. These results indicate that the potentiating effect of staurosporine on PLD activation induced by fMLP does not reflect a feedback inhibition by PKC activation, but instead a feedback inhibition by PKC activation. Taken together, our results indicate that in human neutrophils: (i) PKC activity is not essential for fMLP-induced activation of PLD; (ii) PKC activity does play an essential role in the activation of the respiratory burst by fMLP, other than mediating or modulating PLD activation; (iii) there exists a negative-feedback mechanism on fMLP-induced PLD activation by concomitant activation of PKA.
Nitric oxide-generating system as an autocrine mechanism in human polymorphonuclear leucocytes
Authors: Riesco A , Caramelo C , Blum G , Montón M , Gallego M J , Casado S , López Farré A
Published: August 2015

Recent data [Lopéz-Farré, Riesco, Moliz, Egido, Casado, Hernando and Caramelo (1991) Biochem. Biophys. Res. Commun. 178, 884-891] revealed that endothelin 1 (ET-1) increases intracellular free [Ca2+] in polymorphonuclear leucocytes (PMN) by a mechanism that can be inhibited by L-arginine. The aim of the present study was to clarify the mechanisms of the interaction between the effects of ET-1 and L-arginine in human PMN. The experimental findings showed that in human PMN: (a) ET-1 and the chemoattractant peptide N-formylmethionyl- leucyl-phenylalanine (fMLP) induce both the metabolism of L-arginine to L-citrulline and cyclic GMP (cGMP) formation; (b) the ET-1-induced cGMP production is inhibitable by the L-arginine antagonist NG-monomethyl-L-arginine, therefore suggesting the involvement of NO; (c) the ET-1- or fMLP-induced NO/cGMP stimulation is critically dependent on the availability of L-arginine; (d) human PMN possess a L-arginine transport system with both Na(+)-dependent and -independent components; (e) the L-arginine transport system in PMN appears to be feedback-regulated by NO/cGMP in ET-1-stimulated conditions, but not under baseline conditions; (f) the L-arginine transport system in PMN is independent of the gamma-glutamyl cycle and is not modified by either ET-1 or fMLP. The L-arginine/NO/cGMP-dependent mechanisms characterized in the present study may be relevant in the regulation of PMN activation in pathophysiological conditions in vivo.
Enzymic characterization of murine and human prohormone convertase-1 (mPC1 and hPC1) expressed in mammalian GH4C1 cells
Authors: Jean F , Basak A , Rondeau N , Benjannet S , Hendy G N , Seidah N G , Chrétien M , Lazure C
Published: August 2015

Prohormone convertase-1 (PC1), an endopeptidase that is structurally related to the yeast subtilisin-like Kex2 gene product, has been proposed to be involved in mammalian tissue-specific prohormone processing at pairs of basic residues. To better study this enzyme, a rat somatomammotroph cell line, GH4C1, was infected with vaccinia virus recombinants of murine PC1 (mPC1) and human PC1 (hPC1). An enzymically active form of each protein was secreted into the cell medium and partially purified by anion-exchange chromatography. The 80-85 kDa enzyme was shown to be Ca(2+)-dependent and exhibited a pH optimum of 6.0 when assayed against a synthetic fluorogenic substrate, acetyl-Arg-Ser-Lys-Arg-4-methylcoumaryl-1-amide. mPC1 and hPC1 displayed identical cleavage selectivity towards a number of fluorogenic substrates, and those incorporating an Arg at the P4 site were most favoured. Synthetic peptides, encompassing the junction between the putative pro-region and the active enzyme, and between the pro-region and the biologically active parathyroid hormone, were shown to be recognized and cleaved specifically at the pair of basic residues by both enzymes. Group-specific proteinase inhibitors such as metal ion chelators and p-hydroxymercuribenzoate, but not phenylmethanesulphonyl fluoride and pepstatin, strongly inhibit the PC1-associated activity. In addition, it is shown that an enzyme activity displaying identical properties is present in the cell medium of uninfected corticotroph AtT-20 cells and that its level is increased following stimulation of secretion by the secretagogue 8-bromo cyclic AMP.
Smooth-muscle caldesmon phosphatase is SMP-I, a type 2A protein phosphatase
Authors: Pato M D , Sutherland C , Winder S J , Walsh M P
Published: August 2015

Caldesmon phosphatase was identified in chicken gizzard smooth muscle by using as substrates caldesmon phosphorylated at different sites by protein kinase C, Ca2+/calmodulin-dependent protein kinase II and cdc2 kinase. Most (approximately 90%) of the phosphatase activity was recovered in the cytosolic fraction. Gel filtration after (NH4)2SO4 fractionation of the cytosolic fraction revealed a single major peak of phosphatase activity which coeluted with calponin phosphatase [Winder, Pato and Walsh (1992) Biochem. J. 286, 197-203] and myosin LC20 phosphatase. Further purification of caldesmon phosphatase was achieved by sequential chromatography on columns of DEAE-Sephacel, omega-amino-octyl-agarose, aminopropyl-agarose and thiophosphorylated myosin LC20-Sepharose. A single peak of caldesmon phosphatase activity was detected at each step of the purification. The purified phosphatase was identified as SMP-I [Pato and Adelstein (1980) J. Biol. Chem. 255, 6535-6538] by subunit composition (three subunits, of 60, 55 and 38 kDa) and Western blotting using antibodies against the holoenzyme which recognize all three subunits and antibodies specific for the 38 kDa catalytic subunit. SMP-I is a type 2A protein phosphatase [Pato, Adelstein, Crouch, Safer, Ingebritsen and Cohen (1983) Eur. J. Biochem. 132, 283-287; Winder et al. (1992), cited above]. Consistent with the conclusion that SMP-I is the major caldesmon phosphatase of smooth muscle, purified SMP-I from turkey gizzard dephosphorylated all three phosphorylated forms of caldesmon, whereas SMP-II, -III and -IV were relatively ineffective. Kinetic analysis of dephosphorylation by chicken gizzard SMP-I of the three phosphorylated caldesmon species and calponin phosphorylated by protein kinase C indicates that calponin is a significantly better substrate of SMP-I than are any of the three phosphorylated forms of caldesmon. We therefore suggest that caldesmon phosphorylation in vivo can be maintained after kinase inactivation due to slow dephosphorylation by SMP-I, whereas calponin and myosin are rapidly dephosphorylated by SMP-I and SMP-III/SMP-IV respectively. This may have important functional consequences in terms of the contractile properties of smooth muscle.
Sequences of members of the human gene family for the c subunit of mitochondrial ATP synthase
Authors: Dyer M R , Walker J E
Published: August 2015

Subunit c is an intrinsic membrane component of ATP synthase, and in mammals it is encoded by two expressed nuclear genes, P1 and P2. Both genes encode the same mature c subunit, but the mitochondrial import pre-sequences in the precursors of subunit c are different. The DNA sequences of the human P1 and P2 genes are described. They occupy about 3.0 and 10.9 kb respectively of the human genome, and both genes are split into five exons. The human genome also contains about 14 related spliced pseudogenes, and the sequence of one such pseudogene related to P2 is described. Sequences flanking the 5′ ends of the human P1 and P2 coding sequences each contain a CpG-rich island. Potential promoter elements (TATA and CCAAT boxes) are present in the 5′ sequences of the P1 gene, but not that of P2, although there is no direct experimental evidence to show the involvement of these sequences in transcription of the genes.
Characterization of the expressed genes for subunit c of mitochondrial ATP synthase in sheep with ceroid lipofuscinosis
Authors: Medd S M , Walker J E , Jolly R D
Published: August 2015

The human and bovine genomes each contain two expressed nuclear genes, called P1 and P2, for subunit c, a hydrophobic subunit of the membrane sector, Fo, of mitochondrial ATP synthase. Both P1 and P2 encode the same mature protein, but the associated mitochondrial import sequences are different. In sheep with the neurodegenerative disease ceroid lipofuscinosis, and also in humans with Batten's disease, unmodified subunit c accumulates in lysosome-derived organelles in a variety of tissues. However, the sequences of cDNAs for P1 and P2 from sheep with ceroid lipofuscinosis were identical to those in healthy control animals. Therefore, since there was no mutation in either of the mitochondrial import sequences of subunit c in the diseased animals, ceroid lipofuscinosis does not arise from changes in an import sequence causing mis-targeting of the c subunit to lysosomes. The levels of expression of P1 and P2 genes were approximately the same in diseased and healthy animals, and so the protein is unlikely to accumulate because of excessive transcription of either gene. Transcription of a spliced pseudogene related to P2 was detected in both a control animal and a sheep with ceroid lipofuscinosis. The transcripts encode amino acids 1-31 of the P2 mitochondrial targeting sequence. In the diseased animal, an arginine replaced a glutamine in the control sequence. However, restriction fragment analysis of genomic DNA from a further 12 sheep established that the sequence differences were not linked to ceroid lipofuscinosis.
Rat liver mitochondrial phospholipase A2 is an endotoxin-stimulated membrane-associated enzyme of Kupffer cells which is released during liver perfusion
Authors: Hatch G M , Vance D E , Wilton D C
Published: August 2015

A novel fluorescence assay for phospholipase A2 [Wilton (1990) Biochem. J. 266, 435-439] has been used to study the Group-II rat liver mitochondrial enzyme, and a number of novel properties of this enzyme were identified. (1) The enzyme activity was located in the liver macrophages (Kupffer cells) while negligible activity was associated with hepatocytes. (2) Although subcellular fractionation of whole liver confirmed the predominantly mitochondrial location of this enzyme activity, the analysis of the hepatocyte-free Kupffer-cell-enriched fraction revealed a different enzyme distribution, with the majority of activity being associated with the microsomal membrane fraction. (3) Bacterial endotoxin has been previously shown to be scavenged by Kupffer cells in rats. Treatment of rats with bacterial lipopolysaccharide (endotoxin) resulted in a dramatic time- and dose-dependent increase in liver phospholipase A2 activity. (4) It is known that injection of endotoxin into rodents results in elevated serum phospholipase A2 activity, while a similar phenomenon is seen in the condition of septic shock in man. The source of this serum enzyme was unknown. In this study perfusion of livers from rats pretreated with lipopolysaccharide with physiological saline demonstrated a 6-fold increase in phospholipase A2 activity in the perfusate compared with sham-treated controls, with only minor release of hepatic lipase. (5) Western-blot analysis confirmed an increased release of this Group-II phospholipase A2 into the perfusate of lipopolysaccharide-treated rats compared with sham-treated controls. These results suggest that liver Kupffer cells are a major source of the endotoxin-induced serum Group-II phospholipase A2 activity associated with bacterial infection and trauma.
Dystrophin is phosphorylated by endogenous protein kinases
Authors: Luise M , Presotto C , Senter L , Betto R , Ceoldo S , Furlan S , Salvatori S , Sabbadini R A , Salviati G
Published: August 2015

Dystrophin, the protein coded by the gene missing in Duchenne muscular dystrophy, is assumed to be a component of the membrane cytoskeleton of skeletal muscle. Like other cytoskeletal proteins in different cell types, dystrophin bound to sarcolemma membranes was found to be phosphorylated by endogenous protein kinases. The phosphorylation of dystrophin was activated by cyclic AMP, cyclic GMP, calcium and calmodulin, and was inhibited by cyclic AMP-dependent protein kinase peptide inhibitor, mastoparan and heparin. These results suggest that membrane-bound dystrophin is a substrate of endogenous cyclic AMP- and cyclic GMP-dependent protein kinases, calcium/calmodulin-dependent kinase and casein kinase II. The possibility that dystrophin could be phosphorylated by protein kinase C is suggested by the inhibition of phosphorylation by staurosporin. On the other hand dystrophin seems not to be a substrate for protein tyrosine kinases, as shown by the lack of reaction of phosphorylated dystrophin with a monoclonal antiphosphotyrosine antibody. Sequence analysis indicates that dystrophin contains seven potential phosphorylation sites for cyclic AMP- and cyclic GMP-dependent protein kinases (all localized in the central rod domain of the molecule) as well as several sites for protein kinase C and casein kinase II. Interestingly, potential sites of phosphorylation by protein kinase C and casein kinase II are located in the proximity of the actin-binding site. These results suggest, by analogy with what has been demonstrated in the case of other cytoskeletal proteins, that the phosphorylation of dystrophin by endogenous protein kinases may modulate both self assembly and interaction of dystrophin with other cytoskeletal proteins in vivo.
Cell-cycle-dependent phosphorylation and activity of Chinese-hamster ovary topoisomerase II
Authors: Burden D A , Goldsmith L J , Sullivan D M
Published: August 2015

Cell-cycle-dependent protein levels and phosphorylation of DNA topoisomerase II in relation to its catalytic and cleavage activities were studied in Chinese-hamster ovary cells. Immunoreactive topoisomerase II protein levels were maximal in G2-phase cells, intermediate in S- and M-phase cells, and minimal in a predominantly G1-phase population. When the phosphorylation of topoisomerase II in vivo was corrected for differences in specific radioactivity of intracellular ATP, the apparent phosphorylation of S- and M-phase topoisomerase II was altered significantly. Relative phosphorylation in vivo was found to be greatest in M-phase cells and decreased in the other populations in the order: S > G2 > asynchronous. Phosphoserine was detected in every phase of the cell cycle, with a minor contribution of phosphothreonine demonstrated in M-phase cells. Topoisomerase II activity measured in vivo as 9-(4,6-O-ethylidene-beta-D-glucopyranosyl)-4′-demethylepipodophylloto xin (VP-16)-induced DNA double-strand breaks (determined by neutral filter elution) increased in the order: asynchronous < S < G2 < M. Topoisomerase II cleavage activity, assayed in vitro as the formation of covalent enzyme-DNA complexes, was lowest in S phase, intermediate in asynchronous and G2-phase cells, and maximal in M phase. Topoisomerase II decatenation activity was 1.6-1.8-fold greater in S-, G2- and M-phase populations relative to asynchronous cells. Therefore DNA topoisomerase II activity measured both in vivo and in vitro is maximal in M phase, that phase of the cell cycle with an intermediate level of immunoreactive topoisomerase II but the highest level of enzyme phosphorylation. The discordance between immunoreactive topoisomerase II protein levels, adjusted relative phosphorylation, catalytic activity, cleavage activity and amino acid residue(s) modified, suggests that the site of phosphorylation may be cell-cycle-dependent and critical in determining catalytic and cleavage activity.
Band 3 HT, a human red-cell variant associated with acanthocytosis and increased anion transport, carries the mutation Pro-868→Leu in the membrane domain of band 3
Authors: Bruce L J , Kay M M , Lawrence C , Tanner M J
Published: August 2015

1. We have studied band 3 HT, a human red-cell band 3 variant with increased M(r), which is associated with abnormal red-cell shape (acanthocytosis) and increased anion-transport activity. 2. We have shown that the increased M(r) does not result from the presence of the band 3 Memphis mutation, and that the variant band 3 is covalently labelled by 4,4′-di-isothiocyanato-1,2-diphenylethane-2,2′-disulphonic acid (H2DIDS) less readily than normal. 3. cDNA cloning studies show that band 3 HT results from the mutation Pro-868-->Leu, and the possible significance of the mutation in the altered anion-transport activity and cytoskeleton binding properties of band 3 HT is discussed.
Predominant expression of type-VI adenylate cyclase in C6-2B rat glioma cells may account for inhibition of cyclic AMP accumulation by calcium
Authors: Debernardi M A , Munshi R , Yoshimura M , Cooper D M , Brooker G
Published: August 2015

In C6-2B cells, agonist-stimulated cyclic AMP accumulation is inhibited when the cytosolic Ca2+ concentration is increased. We now demonstrate that in C6-2B cells: (i) the early kinetics of the cyclic AMP inhibition by substance K (t1/2 = 35 s) and thapsigargin (t1/2 = 1.6 min) closely mimic the kinetics of the cytosolic Ca2+ increase evoked by either agent (t1/2 = 25 s and 1.5 min respectively); (ii) the Ca2+ rise and cyclic AMP inhibition by substance K or thapsigargin are similarly affected in EGTA-containing medium; (iii) PCR detects type-III and type-VI adenylate cyclase cDNAs, and RNAase protection assays show that the mRNA for type-VI adenylate cyclase, an isoform inhibitable by submicromolar Ca2+ concentrations, is the predominant species, strongly suggesting that type-VI adenylate cyclase is probably the target molecule for Ca(2+)-mediated inhibition of cyclic AMP accumulation.
Turnover of inositol pentakisphosphates, inositol hexakisphosphate and diphosphoinositol polyphosphates in primary cultured hepatocytes
Authors: Glennon M C , Shears S B
Published: August 2015

We have used a non-transformed cell model, the primary cultured hepatocyte, to explore the turnover of inositol hexakisphosphate, multiple isomers of inositol pentakisphosphate and two novel diphosphoinositol polyphosphates. All of these compounds gradually accumulated radioactivity throughout a 70 h period of labelling with [3H]inositol. However, a rapid metabolic rate was revealed upon inhibition of diphosphoinositol polyphosphate biphosphatase(s) with 1 mM fluoride for 40 min: this treatment elevated levels of [3H]diphosphoinositol polyphosphates up to 10-fold, indicating that their cellular pools were normally turning over at least 10 times every 40 min. This was accompanied by a turnover of about 10% of the pool of inositol hexakisphosphate. Control experiments established that 200 nM vasopressin brought about a typical activation of phospholipase C in hepatocytes after 62 h of primary culture. This agonist treatment did not affect steady-state levels of [3H]inositol pentakisphosphates, [3H]inositol hexakisphosphate or [3H]diphosphoinositol polyphosphates. However, prolonged treatment of hepatocytes with 2 microM thapsigargin reduced steady-state levels of [3H]diphosphoinositol polyphosphates by 50-70%. This effect of thapsigargin was also observed in the presence of fluoride, indicating that thapsigargin inhibited the rate of synthesis of diphosphoinositol polyphosphates.
Structure of the CAMPATH-1 antigen, a glycosylphosphatidylinositol-anchored glycoprotein which is an exceptionally good target for complement lysis
Authors: Xia M Q , Hale G , Lifely M R , Ferguson M A J , Campbell D , Packman L , Waldmann H
Published: August 2015

CAMPATH-1 antibodies recognize a unique molecule on human lymphocytes and are unusually efficient at causing cell lysis with homologous complement. They have been successfully used for lymphocyte depletion in vivo in a variety of diseases. We find that the antigen is a very small glycosylphosphatidylinositol (GPI)-anchored glycoprotein with a mature peptide comprising only 12 amino acids. It can be separated into two distinct antigenic fractions which differ in their susceptibility to phosphatidylinositol-specific phospholipase C. There is one N-linked glycosylation site, but no evidence for O-glycosylation despite the presence of several serine and threonine residues. The antibodies were found to bind, albeit with a generally reduced affinity, to a proteolytic fragment containing the C-terminal tripeptide and the GPI anchor. We postulate that one of the reasons why the CAMPATH-1 antibodies are so good for cell lysis is because they bind to an epitope which is likely to be very close to the lipid bilayer.
Adenosine 5′-[α β-methylene]triphosphate potentiates the oscillatory cytosolic Ca2+ responses of hepatocytes to ATP, but not to ADP
Authors: Dixon C J , Cobbold P H , Green A K
Published: August 2015

Single rat hepatocytes microinjected with aequorin generate oscillations in cytosolic free Ca2+ concentration ([Ca2+]i) when stimulated with agonists acting through the phosphoinositide signalling pathway. The duration of these transients has been shown to be characteristic of the stimulating agonist, so that transients of very different duration can be induced in the same individual hepatocyte by different agonists. In a previous study we have shown that ADP and ATP, which are believed to act through a single P2y-purinoceptor species, elicit very different [Ca2+]i responses in most of the hepatocytes. We have interpreted this as evidence for two Ca(2+)-mobilizing purinoceptors. The methylated derivative of ATP, adenosine 5′-[alpha beta-methylene]-triphosphate (pp[CH2]pA), is only a weak P2y-purinoceptor agonist. When 100 microM pp[CH2]pA was supplied to aequorin-injected hepatocytes, there was no effect on [Ca2+]i. However, 25 microM pp[CH2]pA co-supplied with ATP causes a potentiation of the [Ca2+]i response in most of the hepatocytes. The effect was specific for ATP-induced transients; [Ca2+]i transients induced by other agonists, and importantly by ADP, were not affected by addition of pp[CH2]pA. This further illustrates differences in the actions of ADP and ATP, strengthening the argument for separate receptors for these nucleotides.
New families in the classification of glycosyl hydrolases based on amino acid sequence similarities
Authors: Henrissat B , Bairoch A
Published: August 2015

301 glycosyl hydrolases and related enzymes corresponding to 39 EC entries of the I.U.B. classification system have been classified into 35 families on the basis of amino-acid-sequence similarities [Henrissat (1991) Biochem. J. 280, 309-316]. Approximately half of the families were found to be monospecific (containing only one EC number), whereas the other half were found to be polyspecific (containing at least two EC numbers). A > 60% increase in sequence data for glycosyl hydrolases (181 additional enzymes or enzyme domains sequences have since become available) allowed us to update the classification not only by the addition of more members to already identified families, but also by the finding of ten new families. On the basis of a comparison of 482 sequences corresponding to 52 EC entries, 45 families, out of which 22 are polyspecific, can now be defined. This classification has been implemented in the SWISS-PROT protein sequence data bank.
N.m.r. and molecular-modelling studies of the solution conformation of heparin
Authors: Mulloy B , Forster M J , Jones C , Davies D B
Published: August 2015

The solution conformations of heparin and de-N-sulphated, re-N-acetylated heparin have been determined by a combination of n.m.r. spectroscopic and molecular-modelling techniques. The 1H- and 13C-n.m.r. spectra of these polysaccharides have been assigned. Observed 1H-1H nuclear Overhauser enhancements (n.O.e.s) have been simulated using the program NOEMOL [Forster, Jones and Mulloy (1989) J. Mol. Graph. 7, 196-201] for molecular models derived from conformational-energy calculations; correlation times for the simulations were chosen to fit experimentally determined 13C spin-lattice relaxation times. In order to achieve good agreement between calculated and observed 1H-1H n.O.e.s it was necessary to assume that the reorientational motion of the polysaccharide molecules was not isotropic, but was that of a symmetric top. The resulting model of heparin in solution is similar to that determined in the fibrous state by X-ray-diffraction techniques [Nieduszynski, Gardner and Atkins (1977) Am. Chem. Soc. Symp. Ser. 48, 73-80].
Control analysis applied to single enzymes: can an isolated enzyme have a unique rate-limiting step?
Authors: Brown G C , Cooper C E
Published: August 2015

Control analysis is used to analyse and quantify the concept of a rate-limiting step within an enzyme. The extent to which each rate constant within the enzyme limits the steady-state rate of the enzyme and the levels of enzyme intermediate species are quantified as flux and concentration control coefficients. These coefficients are additive and obey summation theorems. The control coefficients of triose phosphate isomerase, carbamate kinase and lactate dehydrogenase are calculated from literature values of the rate constants. It is shown that, contrary to previous assumption, these enzymes do not have a unique rate-limiting step, but rather flux control is shared by several rate constants and varies with substrate, product and effector concentrations, and with the direction of the reaction. Thus the general assumption that an enzyme will have a unique rate-limiting step is unjustified.
The kinetics of slow-binding and slow, tight-binding inhibition: the effects of substrate depletion
Authors: Waley S G
Published: August 2015

Inhibitors with dissociation constants in the micromolar to nanomolar range are important, but hard to characterize kinetically, especially when the substrate concentration in the assay is less than Km. When inhibition increases during the course of the assay (slow-binding inhibition) the concentration of substrate may decrease appreciably. Methods that take substrate depletion into account are described for analysing experiments in which the initial substrate concentration is below Km. Fitting progress curves gives the rate constants for the second (slow) step in a two-step mechanism. An approximate value for the overall dissociation constant may be determined from measurements of rates when the reaction is treated as a first-order process. When the concentrations of inhibitor and enzyme are comparable numerical methods are required. Procedures, suitable for implementation on a microcomputer, for the solution of the differential equations and the fitting of progress curves are described.
Photoaffinity labelling of cyclic GMP-binding proteins in human platelets
Authors: Tang K M , Sherwood J L , Haslam R J
Published: August 2015

The photoaffinity labelling of platelet cyclic GMP (cGMP)-binding proteins by [32P]cGMP was studied; at least five labelled proteins (110, 80, 55, 49 and 38 kDa) were detected in platelet cytosol and four (80, 65, 49 and 38 kDa) in platelet membranes. The 110 kDa species was identified as cGMP-inhibited cyclic AMP (cAMP) phosphodiesterase (PDE III) by immunoprecipitation and by the inhibition of photolabelling by specific inhibitors of this enzyme. Similarly, the 80 kDa species was identified as cGMP-dependent protein kinase by immunoprecipitation and by the effects of cGMP analogues on photolabelling. Addition of cAMP greatly enhanced the labelling of this 80 kDa protein, implying the existence of a potentially important interaction between the effects of cGMP and cAMP. The 65 kDa photolabelled protein appears to be a novel platelet cyclic-nucleotide-binding protein. In contrast, the 49 and 55 kDa photolabelled species are probably the RI and RII regulatory subunits of cAMP-dependent protein kinase, and the 38 kDa protein(s) may be proteolytic fragment(s) of RI and/or RII.
Interleukin-1 activates transcription factor NFκ B in glial cells
Authors: Moynagh P N , Williams D C , O'Neill L A J
Published: August 2015

Recombinant human interleukin-1 (IL-1) alpha and beta were found to activate a latent cytosolic form of the transcription factor NF kappa B in rat C6 glioma. IL-1 beta was 10 times more potent than IL-1 alpha for this activity and both were inhibited by the IL-1 receptor antagonist. The activation was detectable from 20 min and remained sustained for up to 24 h. The electrophoretic mobility of the activated complex was shown to be different from that of the corresponding complexes in another IL-1-responsive cell line, the murine thymoma line EL4.NOB-1. C6 cells, when transiently transfected with five NF kappa B consensus sequence repeats linked to the reporter gene chloramphenicol acetyltransferase (CAT), demonstrated increased CAT activity in response to IL-1 beta treatment. The activation of NF kappa B in glial cells may thus represent an early step in IL-1 signalling in brain and is likely to have consequences for IL-1-induced gene expression in these cells.
Molecular cloning and sequence analysis of the cDNA for ancrod, a thrombin-like enzyme from the venom of Calloselasma rhodostoma
Authors: Au L C , Lin S B , Chou J S , Teh G W , Chang K J , Shih C M
Published: August 2015

The 1.54 kb cDNA for ancrod, a thrombin-like enzyme, was cloned from a lambda ZAP cDNA library derived from the venom glands of Calloselasma (Agkistrodon) rhodostoma. The cDNA sequence reveals that ancrod is synthesized as a pre-zymogen of 258 amino acids, including a putative secretory peptide of 18 amino acids and a proposed zymogen peptide of 6 amino-acid residues. The amino-acid sequence of the predicted active form of the enzyme exhibits a high degree of sequence similarity to those of mammalian serine proteases (trypsin and pancreatic kallikrein) and other thrombin-like enzymes (batroxobin and flavoxobin). Key amino-acid residues (His43, Asp88, Ser182 and Asp176) that are thought to be involved in the substrate cleavage and in the substrate-binding reaction are conserved. Ancrod contains 13 cysteine residues. Based on alignment with the amino-acid sequences of trypsin and batroxobin, six disulphide bridges can be predicted to be present in the ancrod protein. The existence of a free cysteine, which changes the common sequence surrounding the Ser182 active site from Gly-Asp-Ser-Gly-Gly-Pro to Cys-Asp-Ser-Gly-Gly-Pro, is unusual for a serine protease.
Hydralazine induces Z-DNA conformation in a polynucleotide and elicits anti(Z-DNA) antibodies in treated patients
Authors: Thomas T J , Seibold J R , Adams L E , Hess E V
Published: August 2015

We studied the effect of hydralazine, an antihypertensive drug with lupus-inducing side effects, on the conformation of poly(dG-m5dC).poly(dG-m5dC) and a plasmid with a 23 bp insert of (dG-dC)n.(dG-dC)n sequences. Using an e.l.i.s.a. with a monoclonal anti-(Z-DNA) antibody Z22, we found that hydralazine provoked the Z-DNA conformation in poly(dG-m5dC).poly(dG-m5dC) at 250-500 microM concentration. The supercoiled form of hydralazine-treated plasmid bound to Z22 in a gel-retardation assay. To examine further whether Z-DNA could act as an inciting agent in anti-nuclear antibody production in patients, we analysed 65 sera from 25 hypertensive patients taking hydralazine and found anti-(Z-DNA) antibodies in 82% of these sera. Sera from age-matched normal controls showed no binding to Z-DNA. Data on sera drawn sequentially from four hypertensive patients showed that antibodies were present after the drug treatment. These data demonstrate the presence of a high incidence of anti-(Z-DNA) antibodies in patients treated with hydralazine and suggest that a possible mechanism for the production of autoantibodies in drug-related lupus might involve the induction and stabilization of Z-DNA by drugs.
Human thimet oligopeptidase
Authors: Dando P M , Brown M A , Barrett A J
Published: August 2015

We have purified human thimet oligopeptidase to homogeneity from erythrocytes, and compared it with the enzyme from rat testis and chicken liver. An antiserum raised against rat thimet oligopeptidase also recognized the human and chicken enzymes, suggesting that the structure of the enzyme has been strongly conserved in evolution. Consistent with this, the properties of the human enzyme were very similar to those for the other species. Thus human thimet oligopeptidase also is a thiol-dependent metallo-oligopeptidase with M(r) about 75,000. Specificity for cleavage of a number of peptides was indistinguishable from that of the rat enzyme, but Ki values for the four potent reversible inhibitors tested were lower. In discussing the results, we consider the determinants of the complex substrate specificity of thimet oligopeptidase. We question whether substrates containing more than 17 amino acid residues are cleaved, as has been suggested. We also point out that the favourable location of a proline residue and a free C-terminus in the substrate may be as important as the hydrophobic residues in the P2, P1 and P3′ positions that have been emphasized in the past.
Preparation and characterization of basolateral membrane vesicles from pig and human colonocytes: the mechanism of glucose transport
Authors: Pinches S A , Gribble S M , Beechey R B , Ellis A , Shaw J M , Shirazi-Beechey S P
Published: August 2015

Membrane vesicles were isolated from the basolateral domains of pig and normal human colonocytes. The activity of the ouabain-sensitive K(+)-activated phosphatase, the basolateral membrane marker, was enriched 13-fold in these membrane vesicles over the original homogenate. The membranes displayed cross-reactions with antibodies to the (Na+/K+)ATPase and the RLA class I major histocompatibility antigen, both known indicators of the basolateral membrane. There was negligible contamination by other organelles and the luminal membrane, as revealed by marker-enzyme analysis and Western blotting, using an antibody to villin. The vesicles transported D-glucose in a cytochalasin B-inhibitable Na(+)-independent manner, with a Km of 28.1 +/- 0.8 mM and Vmax. of 3.1 +/- 0.4 nmol/s per mg of protein. The transport was inhibited by 2-deoxy-D-glucose and 3-O-methyl-D-glucose, but not by L-glucose or methyl-alpha-D-glucose. Probing the colonocyte basolateral membranes with an antibody against the C-terminus of the human liver GLUT 2 produced a cross-reaction at 52 kDa. These properties indicate the presence of a GLUT 2 isoform on the basolateral membranes of human and pig colonocytes.
Co-expression of collagens II and XI and alternative splicing of exon 2 of collagen II in several developing human tissues
Authors: Sandberg M M , Hirvonen H E , Elima K J M , Vuorio E I
Published: August 2015

Northern analyses, RNAase protection assays and in situ hybridizations were used to study the expression of the mRNA for the alpha 2 chain of collagen XI and the two different mRNAs generated from the collagen II gene through alternative splicing of exon 2 in several different tissues of 15-19-week-old fetuses. The highest expression levels of procollagen alpha 2(XI) and alpha 1(II) mRNAs were detected in cartilage, but, using long exposure times, Northern hybridization revealed the presence of the approximately 5.3 kb procollagen alpha 1(II) mRNA in most tissues analysed: calvarial and diaphyseal bone, striated and cardiac muscle, skin, brain, lung, kidney, liver, small intestine and colon. Both alternatively spliced forms of the mRNA were present in these tissues. In cartilage, the short form of the procollagen alpha 1(II) mRNA (without exon 2 sequences) was clearly more abundant, whereas in most of the non-cartilaginous tissues the long form was the predominant one. Low levels of procollagen alpha 2(XI) mRNA were also seen in non-cartilaginous tissues: calvarial and diaphyseal bone, kidney, skin, muscle, intestine, liver, brain, and lung. In all the other positive tissues except brain cortex, both collagen II and XI transcripts were observed. The localization of collagen II and XI signals was identical in cartilage, kidney and skin. However, in cartilage the signal with collagen II probe was much higher than that with the collagen alpha 2(XI) probe. In epidermis the situation was reversed. Our results show considerable co-expression and co-localization of procollagen alpha 1(II) and alpha 2(XI) mRNAs in many tissues of developing human fetuses. Since the collagen alpha 1(II) gene also codes for the alpha 3(XI) chain of collagen XI we propose that some, but not all, of the expression of the collagen II gene in non-cartilaginous tissues relates to collagen XI production.
Acyloin production from aldehydes in the perfused rat heart: the potential role of pyruvate dehydrogenase
Authors: Montgomery J A , Jetté M , Huot S , Des Rosiers C
Published: August 2015

Aldehydes represent an important class of cytotoxic products derived from free radical-induced lipid peroxidation which may contribute to reperfusion injury following myocardial infarct. Metabolism of aldehydes in the heart has not been well characterized aside from conjugation of unsaturated aldehydes with glutathione. However, aliphatic aldehydes like hexanal do not form stable glutathione conjugates. We have recently demonstrated in vitro that pig heart pyruvate dehydrogenase catalyses a reaction between pyruvate and saturated aldehydes to produce acyloins (3-hydroxyalkan-2-ones). In the present study, rat hearts were perfused with various aldehydes and pyruvate. Acyloins were generated from saturated aldehydes (butanal, hexanal or nonanal), but not from 2-hexanal (an unsaturated aldehyde) or malondialdehyde. Hearts perfused with 2 mM pyruvate and 10-100 microM hexanal rapidly took up hexanal in a dose-related manner (140-850 nmol/min), and released 3-hydroxyoctan-2-one (0.7-30 nmol/min), 2,3-octanediol (0-12 nmol/min) and hexanol (10-200 nmol/min). Small quantities of hexanoic acid (about 10 nmol/min) were also released. The rate of release of acyloin metabolites rose with increased concentration of hexanal, whereas hexanol release attained a plateau when hexanal infusion concentrations rose above 50 microM. Up to 50% of hexanal uptake could be accounted for by metabolite release. Less than 0.5% of hexanal uptake was found to be bound to acid-precipitable macromolecules. When hearts perfused with 50 microM hexanal and 2 mM pyruvate were subjected to a 15 min ischaemic period, the rates of release of 2,3-octanediol, 3-hydroxyoctan-2-one, hexanol and hexanoate during the reperfusion period were not significantly different from those in the pre-ischaemic period. Our results indicate that saturated aldehydes can be metabolically converted by the heart into stable diffusible compounds.
The role of high-density lipoprotein and lipid-soluble antioxidant vitamins in inhibiting low-density lipoprotein oxidation
Authors: Mackness M I , Abbott C , Arrol S , Durrington P N
Published: August 2015

1. The oxidation of low-density lipoprotein (LDL) is believed to play a central role in atherogenesis. We have compared the effect of antioxidant vitamins and high-density lipoprotein (HDL) on the Cu(2+)-catalysed oxidation of LDL. 2. Antioxidant vitamin supplementation significantly reduced conjugated diene formation but did not affect the formation of lipid peroxides. 3. Conversely, HDL did not affect conjugated diene formation but inhibited the formation of lipid peroxides by up to 90%. 4. The inhibition by HDL of lipid peroxide formation in oxidized LDL was dependent on the concentration of HDL and was not due to HDL chelating Cu2+. 5. Large interindividual variations in the inhibition of lipid peroxide formation by autologous HDL were evident, which were related to the rate of lipid peroxide generation in the LDL. 6. We conclude that HDL is a powerful antioxidant or more probably inhibitor of LDL oxidation in vitro and may play an important role in vivo in preventing atherosclerosis by inhibiting LDL oxidation in the artery wall.
Purification and properties of the enzyme arylamine N-acetyltransferase from the housefly Musca domestica
Authors: Whitaker D P , Goosey M W
Published: August 2015

The enzyme arylamine N-acetyltransferase (ANAT) from the housefly (Musca domestica) has been purified. The M(r) of the purified enzyme was 27,600 +/- 1700 as estimated by gel filtration. SDS/PAGE yielded a value of 26,000 +/- 300, clearly indicating a monomeric structure. The purified enzyme had apparent Km values for acetyl-CoA and tyramine of 8.4 microM and 8.8 microM respectively, a pH optimum of 7.2 in 10 mM potassium phosphate buffer and an apparent pI of 5.8. ANAT activity showed a strong dependency on the presence of 2-mercaptoethanol during the purification stages. The enzyme could be completely inactivated by treatment with p-chloromercuribenzoate although the enzyme activity was protected by preincubation with acetyl-CoA. One or more cysteine residues are clearly required for catalytic activity, as demonstrated for the mammalian enzyme. In contrast, partial sequencing of the enzyme has yielded a number of peptide sequences, including the N-terminal sequence, which show no similarity with those reported for the mammalian and avian enzymes.