bluuebunny/crossref_metadata_2025 huggingface dataset:
Proteasome and thiol involvement in quality control of glycosylphosphatidylinositol anchor addition
Authors: WILBOURN Barry , NESBETH Darren N. , WAINWRIGHT Linda J. , FIELD Mark C.
Published: August 2015

Improperly processed secretory proteins are degraded by a hydrolytic system that is associated with the endoplasmic reticulum (ER) and appears to involve re-export of lumenal proteins into the cytoplasm for ultimate degradation by the proteasome. The chimaeric protein hGHDAF28, which contains a crippled glycosylphosphatidylinositol (GPI) C-terminal signal peptide, is degraded by a pathway highly similar to that for other ER-retained proteins and is characterized by formation of disulphide-linked aggregates, failure to reach the Golgi complex and intracellular degradation with a half life of ∼ 2 h. Here we show that N-acetyl-leucinal-leucinal-norleucinal, MG-132 and lactacystin, all inhibitors of the proteasome, protect hGHDAF28; hGHDAF28 is still proteolytically cleaved in the presence of lactacystin or MG-132, by the removal of ∼ 2 kDa, but the truncated fragment is not processed further. We demonstrate that the ubiquitination system accelerates ER-degradation of hGHDAF28, but is not essential to the process. Overall, these findings indicate that GPI quality control is mediated by the cytoplasmic proteasome. We also show that the presence of a cysteine residue in the GPI signal of hGHDAF28 is required for retention and degradation, as mutation of this residue to serine results in secretion of the fusion protein, implicating thiol-mediated retention as a mechanism for quality control of some GPI signals. Removal of the cysteine also prevents inclusion of hGHDAF28 in disulphide-linked aggregates, indicating that aggregate formation is an additional retention mechanism for this class of protein. Therefore our data suggest that an unpaired terminal cysteine is the retention motif of the hGHDAF28 GPI-processing signal and that additional information may be required for efficient engagement of ER quality control systems by the majority of GPI signals which lack cysteine residues.
Molecular basis of bovine red-cell protein 4.2 polymorphism in Japanese black cattle
Authors: MATSUMOTO Mitsuhito , INABA Mutsumi , ONO Ken-ichiro
Published: August 2015

Cattle were divided into three groups according to the red cell-protein 4.2 (P4.2) phenotypes P4.276, P4.275 and P4.276/75, whose red cells contained Mr 76000 (P4.2/76), 75000 (P4.2/75) and both 76000 and 75000 isoforms respectively. To elucidate the molecular basis that underlies the diversity of P4.2, the gene structures of bovine P4.2/76 and P4.2/75 were investigated. Two P4.2 cDNA clones were isolated from bone-marrow cDNAs of the animal with the P4.276/75 phenotype. These were identical in size (2.2 kb), encoding major erythroid P4.2 with 687 amino acids, but were different in three nucleotides, resulting in changes of amino acids at the 599th, 601st and 627th residues. Analysis of genomic DNA from the three phenotypes demonstrated that these two clones were derived from gene transcripts by which P4.2/76 and/or P4.2/75 were produced. In vitrotranscription and translation of P4.2/76 and P4.2/75 cDNAs indeed generated P4.2/76 and P4.2/75 identical in size to the red-cell proteins. These findings demonstrated that polymorphism of the P4.2 gene at codons 599, 601 and 627 of P4.2 cDNA was the cause of the molecular diversity of bovine red-cell P4.2. Although distinct electrophoretic mobilities suggested a structural difference in the two isoforms, this polymorphism appeared to have little effect at least on P4.2 association with band 3, since no significant difference was observed in the amount of P4.2 relative to total membrane proteins despite the phenotype difference for P4.2.
Bile salts stimulate mucin secretion by cultured dog gallbladder epithelial cells independent of their detergent effect
Authors: KLINKSPOOR J. Henriëtte , YOSHIDA Tadashi , LEE Sum P.
Published: August 2015

1. Bile salts stimulate mucin secretion by the gallbladder epithelium. We have investigated whether this stimulatory effect is due to a detergent effect of bile salts. 2. The bile salts taurocholic acid (TC) and tauroursodeoxycholic acid (TUDC) and the detergents Triton X-100 (12.5–400 µM) and Tween-20 (0.1–3.2 mM) were applied to monolayers of cultured dog gallbladder epithelial cells. Mucin secretion was studied by measuring the secretion of [3H]N-acetyl-d-glucosamine-labelled glycoproteins. We also attempted to alter the fluidity of the apical membrane of the cells through extraction of cholesterol with β-cyclodextrin (2.5–15 mM). The effect on TUDC-induced mucin secretion was studied. Cell viability was assessed by measuring lactate dehydrogenase (LDH) leakage or 51Cr release. 3. In contrast with the bile salts, the detergents were not able to cause an increase in mucin secretion without causing concomitant cell lysis. Concentrations of detergent that increased mucin release (> 100 µM Triton X-100, > 0.8 mM Tween-20), caused increased LDH release. Incubation with β-cyclodextrin resulted in effective extraction of cholesterol without causing an increase in 51Cr release. However, no effect of the presumed altered membrane fluidity on TUDC (10 mM)-induced mucin secretion was observed. 4. The stimulatory effect of bile salts on mucin secretion by gallbladder epithelial cells is not affected by the fluidity of the apical membrane of the cells and also cannot be mimicked by other detergents. We conclude that the ability of bile salts to cause mucin secretion by the gallbladder epithelium is not determined by their detergent properties.
cDNA cloning, genomic organization and chromosomal localization of human heparan glucosaminyl N-deacetylase/N-sulphotransferase-2
Authors: HUMPHRIES Donald E. , LANCIOTTI Julia , KARLINSKY Joel B.
Published: August 2015

The cDNA and gene encoding human heparan glucosaminyl N-deacetylase/N-sulphotransferase-2 have been cloned. The cDNA encoded a protein of 883 amino acids that was 94% similar to heparan N-sulphotransferase-2 from mouse mast cells. Comparison of the deduced amino acid sequences of human heparan N-sulphotransferase-1 and -2 showed that the enzymes were 70% similar; greater than 90% of the amino acids between residues 418 and 543 were identical. The least conserved amino acids were found in the N-terminus/putative transmembrane regions of the two enzymes. The human heparan N-sulphotransferase-2 gene was localized to chromosome arm 10q (band 10q22) by in situ fluorescent hybridization. The gene contains 13 exons spanning 6.5 kb, ranging in size from 88 bp (exon 2) to > 1 kb (exon 1), and 12 introns, which were found to occur at similar sites within the coding sequence of the human heparan N-sulphotransferase-1 gene. The structure of the two genes differed in that the heparan N-sulphotransferase-1 gene contained one additional intron. The similarity of the heparan N-sulphotransferase-1 and -2 proteins and their similar exon-intron organization suggest that they derive from a common ancestral gene.
Mapping the epitope of a functional P-selectin monoclonal antibody (LYP20) to a short complement-like repeat (SCR 4) domain: use of human–mouse chimaera and homologue-replacement mutagenesis
Authors: RUCHAUD-SPARAGANO Marie-Hélène , MALAUD Eric , GAYET Odile , CHIGNIER Elza , BUCKLAND Robin , McGREGOR John L.
Published: August 2015

P-selectin (CD62P), an adhesion molecule localized in platelet α-granules and endothelial cell Weibel–Palade bodies, is rapidly expressed on the surface of activated cells. This adhesion molecule, a member of the selectin family, mediates leucocyte interactions with activated platelets or endothelial cells. The aim of this study was to identify and characterize the epitope of a functional blocking P-selectin monoclonal antibody (mAb), LYP20. LYP20 recognizes human or rat, but not mouse, P-selectin. Human/mouse chimaeras and wild-type constructs, modified by homologue replacement mutagenesis, were expressed in COS cells. Blocking anti-(P-selectin) mAbs (G1, G3 or CLB-thromb/6) were observed, by flow cytometry, to bind to the lectin-like domain. In contrast, LYP20 was found to bind to one of the P-selectin short complement-like repeats (SCR domain 4). Homologue replacement mutagenesis of SCR domain 4 (region delineated by amino acid residues 359–457) identified three amino acids (Cys412 → Ser, Cys416 → Ser or Arg415 → Lys) as being implicated in the LYP20 epitope. Deleting the region bearing the LYP20 epitope, from a wild-type CD62P construct, showed a decrease in polymorphonuclear leucocyte (PMNL) binding to transfected COS cells. In addition, mutation of one of the three amino acids, implicated in the LYP20 epitope, markedly affected PMNL binding to transfected COS cells but did not affect the binding of mAbs G1 and CLB-thromb/6. These results are the first to indicate (1) that a functional blocking anti-P-selectin mAb binds to SCR 4, a site other than the lectin-like/epidermal growth factor-like domain, and (2) that SCR domain 4 has a functional role in P-selectin–leucocyte interactions.
Salicortin: a repeat-attack new-mechanism-based Agrobacterium faecalis β-glucosidase inhibitor
Authors: ZHU Jianjun , WITHERS Stephen G. , REICHARDT Paul B. , TREADWELL Edward , CLAUSEN Thomas P.
Published: August 2015

Salicortin, a natural product abundant in most members of the Salicaceae family, is a mechanism-based inactivator of Agrobacterium faecalis β-glucosidase. Inactivation is delayed in the presence of competitive inhibitors, thereby demonstrating the requirement for an enzyme-bound salicortin before inactivation. Product studies suggest that inactivation proceeds via a quinone methide intermediate formed by the fragmentation of the aglycone of salicortin while it is bound to the enzyme. Tryptic digest and HPLC/MS studies confirm the role of quinone methide attack and also show that the enzyme undergoes multiple modifications. In addition, when the inactivation was run in the presence of a mutant inactive form of the enzyme, HPLC/MS analyses clearly showed no modification of the mutant enzyme, demonstrating that the quinone methide does not exist in free solution and suggesting that inactivation is active-site directed.
Activation of mitogen-activated protein kinases (p38-MAPKs, SAPKs/JNKs and ERKs) by the G-protein-coupled receptor agonist phenylephrine in the perfused rat heart
Authors: LAZOU Antigone , SUGDEN Peter H. , CLERK Angela
Published: August 2015

We investigated the ability of phenylephrine (PE), an α-adrenergic agonist and promoter of hypertrophic growth in the ventricular myocyte, to activate the three best-characterized mitogen-activated protein kinase (MAPK) subfamilies, namely p38-MAPKs, SAPKs/JNKs (i.e. stress-activated protein kinases/c-Jun N-terminal kinases) and ERKs (extracellularly responsive kinases), in perfused contracting rat hearts. Perfusion of hearts with 100 µM PE caused a rapid (maximal at 10 min) 12-fold activation of two p38-MAPK isoforms, as measured by subsequent phosphorylation of a p38-MAPK substrate, recombinant MAPK-activated protein kinase 2 (MAPKAPK2). This activation coincided with phosphorylation of p38-MAPK. Endogenous MAPKAPK2 was activated 4–5-fold in these perfusions and this was inhibited completely by the p38-MAPK inhibitor, SB203580 (10 µM). Activation of p38-MAPK and MAPKAPK2 was also detected in non-contracting hearts perfused with PE, indicating that the effects were not dependent on the positive inotropic/chronotropic properties of the agonist. Although SAPKs/JNKs were also rapidly activated, the activation (2–3-fold) was less than that of p38-MAPK. The ERKs were activated by perfusion with PE and the activation was at least 50% of that seen with 1 µM PMA, the most powerful activator of the ERKs yet identified in cardiac myocytes. These results indicate that, in addition to the ERKs, two MAPK subfamilies, whose activation is more usually associated with cellular stresses, are activated by the Gq/11-protein-coupled receptor (Gq/11PCR) agonist, PE, in whole hearts. These data indicate that Gq/11PCR agonists activate multiple MAPK signalling pathways in the heart, all of which may contribute to the overall response (e.g. the development of the hypertrophic phenotype).
Growth hormone- and testosterone-dependent regulation of glutathione transferase subunit A5 in rat liver
Authors: STAFFAS Louise , ELLIS Elizabeth M. , HAYES John D. , LUNDGREN Bo , DePIERRE Joseph W. , MANKOWITZ Louise
Published: August 2015

The class Alpha glutathione S-transferase (GST) subunit A5 is expressed in the livers of young male and female rats. After sexual maturation, this protein is no longer detectable in the livers of male rats, but is still expressed in female rats. We have previously demonstrated that the sexually dimorphic secretion of growth hormone regulates the levels of certain class Mu GSTs in rat liver, and this study was designed to investigate the hormonal regulation of GSTA5. Control and hypophysectomized rats of both sexes were used to study the role of growth hormone in the regulation of hepatic GSTA5; and the influence of testosterone on the expression of this same subunit was investigated in intact females and castrated males. Liver cytosols were subjected to SDS/PAGE and immunoblotting using antibodies directed towards rat (r)GSTA5, and to affinity purification on glutathione–Sepharose followed by reverse-phase HPLC in order to quantify the relative levels of rGSTA1, A2, A3, A4, M1 and M2 subunits. These analyses revealed that the expression of rGSTA5 is, indeed, regulated by both growth hormone and testosterone.
Differential activation of stress-activated protein kinase kinases SKK4/MKK7 and SKK1/MKK4 by the mixed-lineage kinase-2 and mitogen-activated protein kinase kinase (MKK) kinase-1
Authors: CUENDA Ana , DOROW Donna S.
Published: August 2015

Overexpression of the protein kinases mixed-lineage kinase-2 (MLK2) or mitogen-activated protein kinase (MAPK) kinase kinase-1 (MEKK1) is known to trigger the activation of stress-activated protein kinase (SAPK1)/c-Jun N-terminal kinase (JNK). Here we demonstrate that MLK2 activates SAPK kinase-1 (SKK1)/MAPK kinase 4 (MKK4) and SKK4/MKK7, the two known direct activators of SAPK1/JNK (both in transfection studies and in vitro). In contrast, MEKK1 activates SKK1/MKK4 more efficiently than MLK2, but barely activates SKK4/MKK7. Since SKK4/MKK7 (but not SKK1/MKK4) is activated by interleukin-1 and tumour necrosis factor in several cells and tissues, we suggest that MEKK1 does not mediate the activation of SKK4/MKK7 and SAPK1/JNK induced by these pro-inflammatory cytokines. MLK2 and MEKK1 also activated SKK2/MKK3 and SKK3/MKK6, the direct upstream activators of SAPK2a/p38.
Alteration of the isoform composition of plasma-membrane-associated rat sperm α-l-fucosidase during late epididymal maturation: comparative characterization of the acidic and neutral isoforms
Authors: ABASCAL Irene , SKALABAN Sheri R. , GRIMM Karen M. , AVILÉS Manuel , MARTÍNEZ-MENÁRGUEZ José Angel , CASTELLS Maria Teresa , BALLESTA José , ALHADEFF Jack A.
Published: August 2015

In a previous study, evidence was provided for the presence of a novel plasma-membrane-associated neutral-pH-optimum α-l-fucosidase in rat sperm. In the present study, rat sperm α-l-fucosidase was characterized during epididymal maturation. The pH 7 activity optimum of α-l-fucosidase and its subunit composition (one or two closely spaced immunoreactive protein bands of about 53±2 kDa) did not appear to change during transit through the epididymis. Isoelectric focusing of α-l-fucosidase indicated the presence of a major isoform (B) with a pI near 7 in sperm from testis, caput, corpus and the proximal half of the cauda. α-l-Fucosidase from sperm from the distal half of the cauda, which contained a significant enrichment of sperm and α-l-fucosidase activity, contained isoform B and an additional minor isoform (A) with a pI near 5.2. Isoform B and small amounts of isoform A were present in sperm from the vas deferens. The two fucosidase isoforms present in sperm from the distal cauda were separated by isoelectric focusing and comparatively characterized. They had similar pH–activity curves (with optima near pH 7) and comparable apparent KM values (0.4±0.04 mM) for 4-methylumbelliferyl α-l-fucopyranoside. Preincubation of the isoforms at different temperatures indicated that isoform A is considerably more thermostable than isoform B. Immunoprecipitation studies using polyclonal antibodies against human liver α-l-fucosidase indicated that approx. 90% of the enzymic activity for both isoforms was immunoprecipitable under conditions that immunoprecipitated essentially all the human liver enzyme. Neuraminidase treatment of sperm α-l-fucosidase from distal cauda (when compared with the appropriate heat-treated control) led to disappearance of isoform A and a concomitant increase in isoform B. The overall results suggest that isoform A is derived by sialylation of isoform B near the end of epididymal maturation.
Prenylation of Rab8 GTPase by type I and type II geranylgeranyl transferases
Authors: WILSON Amy L. , ERDMAN Robert A. , CASTELLANO Flavia , MALTESE William A.
Published: August 2015

Rab GTPases are post-translationally modified by addition of geranylgeranyl moieties to carboxyl-terminal cysteine residues. For Rab proteins ending with xxCC xCxC and CCxxmotifs this modification is catalysed by geranylgeranyltransferase type II (GGTaseII), and is entirely dependent on the Rab substrate being bound to Rab escort protein (REP). Several Rab proteins contain carboxyl-terminal CaaL prenylation motifs typical of members of the Rho family, which are modified in a REP-independent manner by geranylgeranyltransferase type I (GGTaseI). The present studies show that one such Rab protein (Rab8), which ends with a CVLL motif, is uniquely able to serve as a substrate for either REP/GGTaseII or GGTaseI in cell-free assays. The modification of Rab8 by GGTaseI did not require REP, indicating that a REP-induced conformational change is not essential for exposure of the Rab carboxyl-terminal cysteine prenylation site. To determine whether one enzyme plays a predominant role in Rab8 prenylation in vivo, the incorporation of [3H]mevalonate into Rab8 was measured in human embryonal kidney 293 cells under conditions where the activity of GGTaseI, but not GGTaseII, was blocked by the peptidomimetic inhibitor GGTI-298. The GGTaseI inhibitor did not prevent prenylation of either overexpressed Myc-tagged Rab8 or endogenous Rab8, whereas prenylation of a known GGTaseI substrate with the same carboxyl-terminal motif, Cdc42Hs, was completely blocked. To rule out the possibility that the apparent prenylation of Rab8 by GGTaseII occurs only when GGTaseI activity is eliminated, metabolic labelling studies were carried out in the absence of the GGTaseI inhibitor, using a REP-binding-deficient Rab8 construct (Y78D) that cannot serve as a substrate for GGTaseII, but is indistinguishable from wild-type Rab8 as a substrate for GGTaseI. Prenylation of the Y78D mutant was reduced by 60–70% in intact cells, consistent with the conclusion that the majority of Rab8 is prenylated by the REP/GGTaseII system in vivo.
Native rat kidney mineralocorticoid receptor is a phosphoprotein whose transformation to a DNA-binding form is induced by phosphatases
Authors: GALIGNIANA Mario D.
Published: August 2015

Addition of alkaline phosphatase to rat kidney cytosol diminishes the ability of the mineralocorticoid receptor (MR) to bind aldosterone in a time-, temperature- and concentration-dependent form. A variety of phosphatase inhibitors, including levamisole, are effective in preventing this inactivation. On the other hand, when the steroid–receptor complex is incubated in the presence of alkaline phosphatase, an increment in the rate of receptor transformation is evidenced by a change in the sedimentation coefficient from 8.8 S to 5.1 S, as well as increased DNA-binding capacity. The effects of alkaline phosphatase on activation and transformation can also be observed when the MR is incubated at 20 °C in the cytosolic medium, indicating that the catalytic action of an endogenous phosphatase may be involved in the transformation process. The ability of phosphatase inhibitors such as levamisole for suppressing both alkaline phosphatase- and endogenous phosphatase-directed transformation does not correspond well between them. Evidence is presented to affirm that the endogenous phosphatase activity is not due to an alkaline phosphatase-type, but it may be due to a protein serine/threonine phosphatase, as evidenced by the inhibitory effects of okadaic acid. The experimental results also show direct evidence that the MR undergoes phosphorylation in a physiological milieu.
Stimulation of multiple mitogen-activated protein kinase sub-families by oxidative stress and phosphorylation of the small heat shock protein, HSP25/27, in neonatal ventricular myocytes
Authors: CLERK Angela , MICHAEL Ashour , SUGDEN Peter H.
Published: August 2015

We investigated the activation of three subfamilies of mitogen-activated protein kinases (MAPKs), namely the stress-activated protein kinases/c-Jun N-terminal kinases (SAPKs/JNKs), the extracellularly responsive kinases (ERKs) and p38-MAPK, by oxidative stress as exemplified by H2O2 in primary cultures of neonatal rat ventricular myocytes. The 46 and 54 kDa species of SAPKs/JNKs were activated 5- and 10-fold, respectively, by 0.1 mM H2O2 (the maximally effective concentration). Maximal activation occurred at 15–30 min, but was still detectable after 2 h. Both ERK1 and ERK2 were activated 16-fold by 0.1 mM H2O2 with a similar time course to the SAPKs/JNKs, and this was comparable with their activation by 1 µM PMA, the most powerful activator of ERKs that we have so far identified in these cells. The activation of ERKs by H2O2 was inhibited by PD98059, which inhibits the activation of MAPK (or ERK) kinases, and by the protein kinase C (PKC) inhibitor, GF109203X. ERK activation was also inhibited by down-regulation of PMA-sensitive PKC isoforms. p38-MAPK was activated by 0.1 mM H2O2 as shown by an increase in its phosphorylation. However, maximal phosphorylation (activation) was more rapid (< 5 min) than for the SAPKs/JNKs or the ERKs. We studied the downstream consequences of p38-MAPK activation by examining activation of MAPK-activated protein kinase 2 (MAPKAPK2) and phosphorylation of the MAPKAPK2 substrate, the small heat shock protein HSP25/27. As with p38-MAPK, MAPKAPK2 was rapidly activated (maximal within 5 min) by 0.1 mM H2O2. This activation was abolished by 10 µM SB203580, a selective inhibitor of certain p38-MAPK isoforms. The phosphorylation of HSP25/27 rapidly followed activation of MAPKAPK2 and was also inhibited by SB203580. Phosphorylation of HSP25/27 was associated with a decrease in its aggregation state. These data indicate that oxidative stress is a powerful activator of all three MAPK subfamilies in neonatal rat ventricular myocytes. Activation of all three MAPKs has been associated with the development of the hypertrophic phenotype. However, stimulation of p38-MAPK and the consequent phosphorylation of HSP25/27 may also be important in cardioprotection.
The broad specificity of dominant inhibitory protein kinase C mutants infers a common step in phosphorylation
Authors: GARCIA-PARAMIO Pilar , CABRERIZO Yolanda , BORNANCIN Frederic , PARKER Peter J.
Published: August 2015

Dominant negative properties are conferred on protein kinase (PK) Cα by mutation of the phosphorylation site in the activation loop of the kinase domain. To address the universality and/or specificity of such mutations, analogous alterations were introduced in other members of the PKC family and tested for their effects on the function of co-transfected activated PKC. For all three subclasses of the PKC family, mutations of the predicted activation loop phosphorylation sites resulted in dominant negative properties. These properties were not restricted to the cognate PKC isotypes, but were effective across the different subclasses. For example, two PKCζ mutants (atypical isotype) inhibited both PKCα (classical isotype) and PKCε (novel isotype). For all these mutants, inhibition correlated with an ability to prevent the accumulation of phosphorylated PKCα, consistent with the expected mode of action. In the case of the PKCα mutant, it was shown that inhibition required the full-length mutant protein. The results provide evidence for the involvement of a common step in the phosphorylation of all PKC isotypes.
Tyrosinase kinetics: failure of the auto-activation mechanism of monohydric phenol oxidation by rapid formation of a quinomethane intermediate
Authors: COOKSEY Christopher J. , GARRATT Peter J. , LAND Edward J. , RAMSDEN Christopher A. , RILEY Patrick A.
Published: August 2015

When 3,4-dihydroxybenzylcyanide (DBC) is oxidized by mushroom tyrosinase, the first visible product, identified as the corresponding quinomethane, exhibits an absorption maximum at 480 nm. Pulse-radiolysis experiments, in which the o-quinone is formed by disproportionation of semiquinone radicals generated by single-electron oxidation of DBC, showed that the quinomethane (A480 6440 M-1·cm-1) is formed through the intermediacy of the o-quinone with a rate constant at neutral pH of 7.5 s-1. The oxygen stoichiometry of the formation of the quinomethane by tyrosinase-catalysed oxidation of DBC was 0.5:1. On the basis of oxygen utilization rates the calculated Vmax was 4900 nmol·min-1 and the apparent Km was 374 µM. The corresponding monohydric phenol, 4-hydroxybenzylcyanide (HBC), was not oxidized by tyrosinase unless the enzyme was pre-exposed to DBC, the maximum acceleration of HBC oxidation being obtained by approximately equimolar addition of DBC. These results are consistent with tyrosinase auto-activation on the basis of the indirect formation of the dihydric phenol-activating cofactor. The rapid conversion of the o-quinone to the quinomethane prevents the formation of the catechol by reduction of the o-quinone product of monohydric phenol oxidation from occurring in the case of the compounds studied. In the absence of auto-activation, the kinetic parameters for HBC oxidation by tyrosinase were estimated as Vmax 70 nmol·min-1 and Km 309 µM. The quinomethane was found to decay with a rate constant of 2k 38 M-1·s-1, as determined both by pulse-radiolysis and tyrosinase experiments. The second-order kinetics indicate that a dimer is formed. In the presence of tyrosinase, but not in the pulse-radiolysis experiments, the quinomethane decay was accompanied by a steady-state oxygen uptake concurrently with the generation of a melanoid product measured by its A650, which is ascribed to the formation of an oligomer incorporating the oxidized dimer.
Trichomonas vaginalis thymidine kinase: purification, characterization and search for inhibitors
Authors: STROSSELLI Silvia , SPADARI Silvio , WALKER Richard T. , BASNAK Ivan , FOCHER Federico
Published: August 2015

We report that a thymidine kinase (TK) activity is present in Trichomonas vaginalis and can be separated from the deoxyribonucleoside phosphotransferase. T. vaginalis TK, purified 11200-fold to apparent homogeneity, has a molecular mass of 31500 Da. It phosphorylates not only thymidine (Km 0.18 µM) but also deoxycytidine (Km 0.88 µM) and deoxyuridine (Km 0.14 µM). In contrast with T. vaginalis deoxyribonucleoside phosphotransferase, the TK activity is strongly inhibited by novel deoxyuridine analogues such as 5-methyl-4´-thio-2´-deoxyuridine (MTdU) (Ki 20 nM) and 5-iodo-4´-thio-2´-deoxyuridine (ITdU) (Ki 24 nM). MTdU and ITdU are phosphorylated by T. vaginalis TK in vitro. In vivo they inhibit [3H]thymidine incorporation in T. vaginaliscultured cells and T. vaginalis growth (IC50 7.5 and 24 µM respectively; minimal lethal dose 100 µM). Thus the TK inhibitors described here demonstrate the key role of T. vaginalis TK for protozoal growth and viability and indicate TK as a new target for the design of antitrichomonal drugs.
Haem propionates control oxidative and reductive activities of horseradish peroxidase by maintaining the correct orientation of the haem
Authors: ADAK Subrata , BANERJEE Ranajit K.
Published: August 2015

The role of haem propionates in oxidative and reductive reactions catalysed by horseradish peroxidase (HRP) was studied after successful reconstitution of ferric protoporphyrin IX dimethyl ester (PPDME) into the apoperoxidase. The reconstituted enzyme oxidizes neither guaiacol (aromatic electron donor) nor iodide or thiocyanate (inorganic donor). Although the reconstituted enzyme binds guaiacol with a similar Kd (13 mM) to that of the native enzyme (10 mM), the Kd for SCN- binding (5 mM) is decreased 20-fold compared with that of the native enzyme (100 mM). This indicates that haem propionates hinder the entry or binding of inorganic anion to the active site of the native HRP. However, the reconstituted enzyme is catalytically inactive as it does not form spectroscopically detectable compound II with H2O2. CD measurements indicate a significant loss of haem CD spectrum of the reconstituted enzyme at 409 nm, suggesting a loss of asymmetry of the haem–protein interaction. Thus the inability of the reconstituted enzyme to form catalytic intermediates results from the change in orientation of the haem due to loss of interactions via the haem propionates. HRP also catalyses reductive reactions such as reduction of iodine (I+) in the presence of EDTA and H2O2. The reconstituted enzyme cannot catalyse I+ reduction because of the loss of I+ binding to the haem propionate. Since I+ reduction requires formation of the catalytically active enzyme–I+–EDTA ternary complex, the loss of reductive activity is primarily due to the loss of active enzyme formation. Haem propionates thus play a vital role in the oxidative and reductive reactions of HRP by favouring the formation of catalytic intermediates with H2O2 by maintaining the correct orientation of the haem with respect to the surrounding residues.
Thermal inactivation and chaperonin-mediated renaturation of mitochondrial aspartate aminotransferase
Authors: LAWTON James M. , DOONAN Shawn
Published: August 2015

Mitochondrial aspartate aminotransferase is inactivated irreversibly on heating. The inactivated protein aggregates, but aggregation is prevented by the presence of the chaperonin 60 from Escherichia coli (GroEL). The chaperonin increases the rate of thermal inactivation in the temperature range 55–65 °C but not at lower temperatures. It has previously been shown [Twomey and Doonan (1997) Biochim. Biophys. Acta 1342, 37–44] that the enzyme switches to a modified, but catalytically active, conformation at approx. 55–60 °C and the present results show that this conformation is recognized by and binds to GroEL. The thermally inactivated protein can be released from GroEL in an active form by the addition of chaperonin 10 from E. coli (GroES)/ATP, showing that inactivation is not the result of irreversible chemical changes. These results suggest that the irreversibility of thermal inactivation is due to the formation of an altered conformation with a high kinetic barrier to refolding rather than to any covalent changes. In the absence of chaperonin the unfolded molecules aggregate but this is a consequence, rather than the cause, of irreversible inactivation.
Selective loss of substrate recognition induced by the tumour-associated D294G point mutation in protein kinase Cα
Authors: PRÉVOSTEL Corinne , ALVARO Véronique , VALLENTIN Alice , MARTIN Annick , JAKEN Susan , JOUBERT Dominique
Published: August 2015

The tumour-associated D294G mutant of protein kinase Cα (PKCα) was recently shown not to be translocated to the plasma membrane on stimulation with PMA, in contrast with the wild-type enzyme. Using recombinant wild-type and mutant PKCα, we establish here that, although the PKCα intrinsic lipid-dependent catalytic activity remains unaltered by the D294G mutation, the mutant enzyme exhibits a selective loss of substrate recognition. Indeed, whereas the mutant enzyme is still able to phosphorylate histone IIIS with comparable efficiency to that of the wild-type enzyme, it exhibits a lack of kinase activity towards the previously cloned 35F and 35H substrates for PKC. Overlay experiments demonstrate that this selective loss of kinase activity is correlated with a decrease in binding of D294G PKCα to the 35F and 35H proteins compared with that of the wild-type enzyme. Because the 35H and 35F proteins are predicted to be PKCα-anchoring proteins, these findings suggest a selective loss of PKCα–protein interactions that might fail to stabilize the location of the PKCα mutant at the plasma membrane.
A glutamate residue contributes to the exopeptidase specificity in aminopeptidase A
Authors: VAZEUX Gilles , ITURRIOZ Xavier , CORVOL Pierre , LLORENS-CORTES Catherine
Published: August 2015

Aminopeptidase A (EC 3.4.11.7, APA) is a 130 kDa membrane-bound aminopeptidase that contains the consensus sequence HEXXH (385–389) found in the zinc metalloprotease family, the zincins. Sequence alignment of the mouse APA with other monozinc-aminopeptidases indicates the presence of a highly conserved glutamate residue (Glu352 in APA) found in the conserved motif GAMEN (349–353). In monozinc-aminopeptidases, the negative charge of the glutamate side-chain carboxylate may constitute the anionic binding site involved in the recognition of the free amino group of substrates or inhibitors. The functional role of Glu352 in APA was investigated by substituting this residue with an aspartate (Asp352), a glycine (Gly352), a glutamine (Gln352) or an arginine (Arg352) residue by site-directed mutagenesis. Kinetic studies showed that the Km values of the mutant enzymes were unaffected, whereas kcat values were decreased 10–250-fold, resulting in a 10-, 30- 260- and 400-fold reduction in cleavage efficiencies for the mutants Asp352, Gly352, Gln352 and Arg352 respectively. The inhibitory potency of two different classes of inhibitors, a thiol and a phosphonate compound, was significantly (P< 0.05) decreased by 10- and 4-fold respectively in the mutated enzymes. Moreover, the inhibitory potency of angiotensin I, used as a competitor of the synthetic substrate α-l-glutamyl β-naphthylamide, displayed a 4-fold reduction (P< 0.01) in the mutated enzymes, whereas the Ki values of its N-acetyl derivative were unchanged. These data strongly suggest that Glu352 is involved in the catalytic process of APA and contributes to the exopeptidase activity of this enzyme through interaction with the N-terminal part of substrates or inhibitors.
Restricting mobility of Gsα relative to the β2-adrenoceptor enhances adenylate cyclase activity by reducing Gsα GTPase activity
Authors: WENZEL-SEIFERT Katharina , LEE Tae Weon , SEIFERT Roland , KOBILKA Brian K.
Published: August 2015

The β2-adrenoceptor (β2AR) activates the G-protein Gsα to stimulate adenylate cyclase (AC). Fusion of the β2AR C-terminus to the N-terminus of Gsα (producing β2ARGsα) markedly increases the efficiency of receptor/G-protein coupling compared with the non-fused state. This increase in coupling efficiency can be attributed to the physical proximity of receptor and G-protein. To determine the optimal length for the tether between receptor and G-protein we constructed fusion proteins from which 26 [β2AR(Δ26)Gsα] or 70 [β2AR(Δ70)Gsα] residues of the β2AR C-terminus had been deleted and compared the properties of these fusion proteins with the previously described β2ARGsα. Compared with β2ARGsα, basal and agonist-stimulated GTP hydrolysis was markedly decreased in β2AR(Δ70)Gsα, whereas the effect of the deletion on binding of guanosine 5´-[γ-thio]triphosphate (GTP[S]) was relatively small. Surprisingly, deletions did not alter the efficiency of coupling of the β2AR to Gsα as assessed by GTP[S]-sensitive high-affinity agonist binding. Moreover, basal and ligand-regulated AC activities in membranes expressing β2AR(Δ70)Gsα and β2AR(Δ26)Gsα were higher than in membranes expressing β2ARGsα. These findings suggest that restricting the mobility of Gsα relative to the β2AR results in a decrease in G-protein inactivation by GTP hydrolysis and thereby enhanced activation of AC.
Increased choline transport in erythrocytes from mice infected with the malaria parasite Plasmodium vinckei vinckei
Authors: STAINES Henry M. , KIRK Kiaran
Published: August 2015

Parasitized erythrocytes from mice infected with the murine malaria parasite Plasmodium vinckei vinckei showed a marked increase in the rate of influx of choline compared with erythrocytes from uninfected mice. In contrast, uninfected erythrocytes from P. vinckei-infected animals transported choline at the same rate as those from uninfected mice. The increased influx of choline into parasitized cells was via two discrete routes. One was a saturable pathway with a Km similar to that of the choline carrier of normal erythrocytes but a Vmax approx. 20-fold higher than that observed in uninfected cells. The other was a non-saturable pathway inhibited by furosemide. At choline concentrations within the normal physiological plasma concentration range, the former pathway contributed approx. two-thirds and the latter approx. one-third of the influx of choline into parasitized cells. The characteristics of the furosemide-sensitive pathway were similar to those of a broad-specificity pathway that is induced in human erythrocytes infected in vitro with Plasmodium falciparum. The results of this study rule out the possibility that the induced transport pathway of P. falciparum-infected erythrocytes is an artifact arising in vitro from the long-term culture of parasitized cells and provide evidence that this pathway makes a significant contribution to the uptake of choline into the parasitized cells of malaria-infected animals.
Structure and organization of the human Theta-class glutathione S-transferase and d-dopachrome tautomerase gene complex
Authors: COGGAN Marjorie , WHITBREAD Lel , WHITTINGTON Angela , BOARD Philip
Published: August 2015

The structure and organization of the human Theta-class glutathione S-transferase (GST) genes have been determined. GSTT1 and GSTT2 are separated by approx. 50 kb. They have a similar structure, being composed of five exons with identical exon/intron boundaries. GSTT1 is 8.1 kb in length, while GSTT2 is only 3.7 kb. The GSTT2 gene lies head-to-head with a gene encoding d-dopachrome tautomerase (DDCT), which extends over 8.5 kb and contains four exons. The sequence between GSTT2 and DDCT may contain a bidirectional promoter. The GSTT2 and DDCT genes have been duplicated in an inverted repeat. Sequence analysis of the duplicated GSTT2 gene has identified an exon 2/intron 2 splice site abnormality and a premature translation stop signal at codon 196. These changes suggest that the duplicate gene is a pseudogene, and it has been named GSTT2P.
Mechanism for basal expression of rat mitochondrial branched-chain-2-oxo-acid dehydrogenase
Authors: HUANG Yi-Shuian , CHUANG David T.
Published: August 2015

The rat branched-chain-2-oxo-acid dehydrogenase (BCOD) kinase mRNA is transcribed from a TATA-less promoter that has GC-rich sequences and two putative Sp1 binding sites near the transcription start site. We demonstrated previously that the 5´ region of the kinase gene, base pairs -128 to +264, contained promoter activity when assayed using luciferase as a reporter (Huang and Chuang (1996) Biochem. J. 313, 603–609). To define DNA elements required for efficient expression of the kinase gene, nested deletion constructs of the above promoter region fused with a luciferase reporter gene were transfected into cultured H4IIE (hepatoma) and NRK-52E (kidney) cells. The results showed that the region between nucleotides -58 and +21 was indispensable for the kinase basal promoter activity. Methylation-interference and mutagenesis-promoter assays identified nucleotides -50 to -40 (ACAACTCCCA) as cis-acting DNA sequences that are required for nuclear protein binding and efficient promoter activity. Gel-supershift analysis with anti-Sp1 antibody suggested that the nuclear protein capable of binding to the -58 oligonucleotide (bp -58 to -34) was immunologically related to the Sp1 protein. The -58 oligonucleotide formed a DNA–protein complex with recombinant Sp1 protein with an affinity approximately ten-fold lower than that of the consensus Sp1 oligonucleotide. Co-transfection of the Sp1 expression plasmid and the -58 promoter construct into Drosophila Schneider cells revealed that Sp1 contributed to the kinase basal promoter activity by binding to the non-consensus site in the -58 region. Deletion of two consensus Sp1 binding sites (bases -150 to -140 and bases +29 to +38) in the kinase gene did not affect the basal promoter activity. Therefore binding of Sp1 or Sp1-like proteins to the above single non-consensus Sp1 sequence in the -58 region plays a major role of transactivating basal expression of the BCOD kinase.
Evidence that mammalian phosphatidylinositol transfer protein regulates phosphatidylcholine metabolism
Authors: MONACO Marie E. , ALEXANDER Richard J. , SNOEK Gerry T. , MOLDOVER Nancy H. , WIRTZ Karel W. A. , WALDEN Paul D.
Published: August 2015

Phosphatidylinositol transfer proteins (PITPs) and their yeast counterpart (SEC14p) possess the ability to bind phosphatidylinositol (PtdIns) and transfer it between membranes in vitro. However, the biochemical function of these proteins in vivo is unclear. In the present study, the physiological role of PITP was investigated by determining the biochemical consequences of lowering the cellular content of this protein. WRK-1 rat mammary tumour cells were transfected with a plasmid containing a full-length rat PITPα cDNA inserted in the antisense orientation and the resultant cell clones were analysed. Three clones expressing antisense mRNA for PITPα were compared with three clones transfected with the expression vector lacking the insert. The three antisense clones had an average of 25% less PITPα protein than control clones. Two of the three antisense clones also exhibited a decreased rate of growth. All three antisense clones exhibited a significant decrease in the incorporation of labelled precursors into PtdCho during a 90-min incubation period. Under the same conditions, however, there was no change in precursor incorporation into PtdIns. Further experimentation indicated that the decrease in precursor incorporation seen in antisense clones was not due to an increased rate of turnover. When choline metabolism was analysed more extensively in one control (2-5) and one antisense (4-B) clone using equilibrium-labelling conditions (48 h of incubation), the following were observed: (1) the decrease in radioactive labelling of PtdCho seen in short-term experiments was also observed in long-term experiments, suggesting that the total amount of PtdCho was lower in antisense-transfected clones (this was confirmed by mass measurements); (2) a similar decrease was seen in cellular sphingomyelin, lysoPtdCho and glycerophosphorylcholine; (3) an average two-fold increase in cellular phosphorylcholine was observed in the antisense-transfected clone; (4) cellular choline was, on average, decreased; and (5) cellular CDPcholine was not significantly altered.
The insertion of human apolipoprotein H into phospholipid membranes: a monolayer study
Authors: WANG Shao-Xiong , CAI Guo-ping , SUI Sen-fang
Published: August 2015

Apolipoprotein H (ApoH) is a plasma glycoprotein isolated from human serum. The interactions of ApoH with lipid membrane were reported to be essential for its physiological and pathogenic roles. In this paper we studied the ability of ApoH to insert into phospholipid membranes using the monolayer approach. The results show that ApoH is surface active and can insert into the lipid monolayers. The insertion ability of ApoH is stronger when a higher content of negatively charged lipids is present in the membrane. The acidic-pH and low-ionic-strength conditions will also enhance ApoH insertion, but these factors may not have much influence on the final insertion ability of ApoH, suggesting that, in the mechanism of ApoH insertion, not only electrostatic forces, but also hydrophobic interactions, are evidently involved. Modification by heat inactivation and reduction/alkylation does not change the critical insertion pressure (πc) of ApoH, suggesting a stable domain, maybe a linear sequence motif, but not the native three-dimensional structure of ApoH, is responsible for its insertion. The extent to which insertion of ApoH into phospholipid membranes may facilitate the ‘immune cleaning ’ of plasma liposomes is discussed.
The mouse ADP-ribosylation factor-like 4 gene: two separate promoters direct specific transcription in tissues and testicular germ cell
Authors: JACOBS Stephan , SCHÜRMANN Annette , BECKER Walter , BÖCKERS Tobias M. , COPELAND Neal G. , JENKINS Nancy A. , JOOST Hans-Georg
Published: August 2015

ADP-ribosylation factor-like protein 4 (ARL4) is a Ras-related GTPase that has been cloned from the 3T3-L1 preadipocyte cell line as an adipocyte-specific cDNA [Schürmann, Breiner, Becker, Huppertz, Kainulainen, Kentrup and Joost (1994) J. Biol. Chem. 269, 15683–15688]. The Arl4 gene maps to the proximal region of mouse chromosome 12 linked to Lamb1-1, Hfhbf1 and Sos2. Compared with all other known genes of Ras-related GTPases, the genomic organization of Arl4 is unusual in that its entire coding region, the 3´ untranslated region (UTR) and most of the 5´ UTR are located on a single exon. This structure suggests that Arl4 has evolved by retroposition of an Arf (ADP-ribosylation factor) or Arf-like gene. Isolation of the 5´ UTR by rapid amplification of cDNA ends (RACE)-PCR revealed heterogeneous transcription initiation sites in alternative exons 1. Both 5´-flanking regions exhibited promoter activity when expressed in COS-7 cells, indicating that the expression of Arl4 is directed by two separate promoters. mRNA transcribed under the control of the downstream promoter was isolated by RACE-PCR from all investigated tissues. In contrast, the upstream promoter seems to drive specifically the expression of Arl4 in adult testis. Hybridization of rat testis in situ indicated that Arl4 is expressed in germ cells of puberal and adult testis, but not in prepuberal testis, suggesting that Arl4 is involved in sperm production.
Functional analysis of the T-cell-restricted protein tyrosine kinase Txk
Authors: ELLIS Jonathan H. , SUTMULLER Roger P. M. , SIMS Martin J. , COOKSLEY Susan
Published: August 2015

T lymphocytes express a range of tyrosine kinases that are involved in signalling processes driving cell activation, proliferation and differentation. Two tyrosine kinases expressed only in T cells, the Itk/Emt and Txk gene products, are members of the Tec family of kinases. The role of Tec kinases in cellular function is poorly understood, although a Tec kinase specific to B cells, Btk, is essential for B-cell development. To explore the contribution of the T-cell-specific Tec kinases to lymphocyte function, we have expressed human Txk in the baculovirus system and conducted the first characterization of its activity. We find that Txk exhibits a substrate preference in vitro quite distinct from that of the major T-cell kinases Lck and ZAP70, suggesting that Tec-family kinases might act on a distinct range of substrates. We also investigated the interactions of Txk with the cytoplasmic domains of the key signalling molecules CD3ζ, CD28 and CTLA4 and find that none of these are phosphorylated by Txk, nor are they ligands for the SH2 or SH3 domains of Txk. We conclude that it is unlikely that Txk has a role in the early signal transduction events associated with these key pathways controlling T-cell activation.