bluuebunny/crossref_metadata_2025 huggingface dataset:
Molecular cloning of Δ9 fatty acid desaturase from the protozoan Tetrahymena thermophila and its mRNA expression during thermal membrane adaptation
Authors: NAKASHIMA Shigeru , ZHAO Yutong , NOZAWA Yoshinori
Published: August 2015

In response to a decrease in its growth temperature, the protozoan Tetrahymena is known to increase the level of unsaturated fatty acids in its membrane phospholipids so as to maintain the correct physical state (fluidity) of the membranes. In this organism, synthesis of unsaturated fatty acids is initiated by Δ9 acyl-CoA desaturase. Our previous studies have shown that, during cold adaptation, the activity of microsomal palmitoyl- and stearoyl-CoA desaturase increases, reaching a maximal level at 2 h after a temperature down-shift to 15 °C. Two hypotheses have been proposed to explain this increase in desaturase activity: (1) self-regulation via a direct effect of reduced membrane fluidity, and (2) induction of desaturase mRNA. However, the precise mechanism is not clearly understood. In order to obtain further insight into the mechanism of regulation of the desaturase, we have isolated a gene that encodes Δ9 fatty acid desaturase from T. thermophila and examined its expression during cold adaptation. The nucleotide sequence indicates that the 1.4 kbp gene encodes a polypeptide of 292 amino acid residues which shows marked sequence similarity to Δ9 acyl-CoA desaturases from other sources, e.g. rat, mouse, Amblyomma americanum and Saccharomyces cerevisiae. This protein has three histidine-cluster motifs (one HXXXXH and two HXXHH), and two hydrophobic regions which are conserved among Δ9 acyl-CoA desaturases. The level of desaturase mRNA was sensitive to decreasing the temperature of the culture media, and was close to maximal immediately after the temperature was shifted down from 35 °C to 15 °C (0.8 °C/min). Thereafter, the amount of mRNA gradually decreased with time, but remained above the control level for at least 5 h. Furthermore, during the course of the cooling process to 15 °C, the increased expression of desaturase mRNA became evident at 27 °C. Nuclear run-on analysis and actinomycin D chase experiments revealed that the elevation of the mRNA level was due to increases in both transcription and mRNA stability. These results suggest that the enhanced desaturase activity is controlled, at least in part, at the transcriptional level.
Nucleation and inhibition of hydroxyapatite formation by mineralized tissue proteins
Authors: HUNTER Graeme K. , HAUSCHKA Peter V. , POOLE Robin A. , ROSENBERG Lawrence C. , GOLDBERG Harvey A.
Published: August 2015

Many proteins found in mineralized tissues have been proposed to function as regulators of the mineralization process, either as nucleators or inhibitors of hydroxyapatite (HA) formation. We have studied the HA-nucleating and HA-inhibiting properties of proteins from bone [osteocalcin (OC), osteopontin (OPN), osteonectin (ON) and bone sialoprotein (BSP)], dentine [phosphophoryn (DPP)] and calcified cartilage [chondrocalcin (CC)] over a wide range of concentrations. Nucleation of HA was studied with a steady-state agarose gel system at sub-threshold [Ca]×[PO4] product. BSP and DPP exhibited nucleation activity at minimum concentrations of 0.3 μg/ml (9 nM) and 10 μg/ml (67 nM) respectively. OC, OPN, ON and CC all lacked nucleation activity at concentrations up to 100 μg/ml. Inhibition of HA formation de novo was studied with calcium phosphate solutions buffered by autotitration. OPN was found to be a potent inhibitor of HA formation [IC50 = 0.32 μg/ml (0.01 μM)] whereas OC was of lower potency [IC50 = 6.1 μg/ml (1.1 μM)]; BSP, ON and CC all lacked inhibitory activity at concentrations up to 10 μg/ml. The effect of OPN on HA formation de novo is mainly to inhibit crystal growth, whereas OC delays nucleation. These findings are consistent with the view that BSP and DPP may play roles in the initiation of mineralization in bone and dentine respectively. OPN seems to be the mineralized tissue protein most likely to function in the inhibition of HA formation, possibly by preventing phase separation in tissue fluids of high supersaturation.
Flavodoxin 1 of Azotobacter vinelandii: characterization and role in electron donation to purified assimilatory nitrate reductase
Authors: GANGESWARAN Rathi , EADY Robert R.
Published: August 2015

Flavodoxins synthesized by Azotobacter vinelandii strain UW 136 during growth on nitrate as nitrogen source were separated by FPLC on a Mono Q column into two species, flavodoxin 1 (AvFld 1) and flavodoxin 2 (AvFld 2). Both proteins migrated as single bands on SDS/PAGE. AvFld 1 was approx. 5-fold more abundant than AvFld 2 in the unresolved flavodoxin mixture. N-terminal amino acid analysis showed the sequence of AvFld 2 to correspond to the nif F gene product, an electron donor to nitrogenase. The sequences also show that these species corresponded to the flavodoxins Fld A and Fld B isolated from N2-grown cultures of the closely related organism Azotobacter chroococcum [Bagby, Barker, Hill, Eady and Thorneley (1991) Biochem. J. 277, 313–319]. Electrospray mass spectrometry gave Mr values for the polypeptides of 19430±3 and 19533±5 respectively. 31P-NMR measurements showed that in addition to the phosphate associated with the FMN (Δ = -136.3 p.p.m. and -135.48 p.p.m.), AvFld 1 had a signal at Δ = -142.1 p.p.m. and AvFld 2 at Δ = -138.59 p.p.m. present in substoichiometric amounts with FMN. These appeared to arise from unstable species since they were readily lost on further manipulation of the proteins. The mid-point potentials of the semiquinone–hydroquinone redox couples were -330 mV and -493 mV for AvFld 1 and AvFld 2 respectively, but only AvFld 1 was competent in donating electrons to the purified assimilatory nitrate reductase of A. vinelandii to catalyse the reduction of nitrate to nitrite. Flavodoxin isolated from NH4+-grown cells (Fld 3) also functioned as electron donor at half the rate of AvFld 1, but ferredoxin 1 from A. chroococcum did not.
Panagrellus redivivus ornithine decarboxylase: structure of the gene, expression in Escherichia coli and characterization of the recombinant protein
Authors: NIEMANN Gabriele , BESSER Hans von , WALTER Rolf D.
Published: August 2015

A Southern blot analysis of the Panagrellus redivivus ornithine decarboxylase (ODC) gene suggests that it is a single-copy gene that resides on a genomic 3.2 kb EcoRI fragment. Phage clones possessing ODC gene sequences were isolated from a genomic EMBL-4 library and purified. The phage DNA inserts were analysed and a 3.2 kb EcoRI fragment containing the entire ODC gene was isolated. The nucleotide sequence analysis of this fragment reveals that the gene is interrupted by two introns of 47 and 49 bp. In the 5´ non-translated region of the gene, putative AP1, VPE2 and c-Myc binding sites were identified. The ODC cDNA was expressed in a bacterial system as a His-fusion protein and the enzyme was purified by Ni2+-chelating affinity chromatography. The subunit molecular mass, as deduced from the cDNA and shown by SDS/PAGE, is 47.1 kDa. On the basis of gel filtration analyses it is shown that the active enzyme is a dimer. The specific enzyme activity was determined to be 4.2 μmol CO2/min/mg protein. The enzyme is dependent on pyridoxal 5-phosphate as a cofactor, and the presence of dithioerythritol or other thiol-reducing agents is essential for maximal activity. The Km value for l-ornithine was determined as 44 μM. The Ki values for putrescine, α-difluoromethylornithine, α-hydrazino-ornithine and α-methylornithine were calculated as 51, 34, 0.34 and 42 μM respectively.
Type-III procollagen assembly in semi-intact cells: chain association, nucleation and triple-helix folding do not require formation of inter-chain disulphide bonds but triple-helix nucleation does require hydroxylation
Authors: BULLEID Neil J. , WILSON Richard , LEES Janice F.
Published: August 2015

Procollagen assembly is initiated within the endoplasmic reticulum by three α-chains associating via their C-propeptides (C-terminal propeptides). To study the requirements for the association of procollagen monomers at synthesis we have reconstituted the initial stages in the folding, assembly and modification of procollagen using semi-permeabilized cells. By translating a type-III procollagen ‘mini-gene’ which lacks part of the triple-helical domain, we demonstrate that these cells efficiently carry out the assembly of hydroxylated, triple-helical, procollagen trimers and allow the identification of specific disulphide-bonded intermediates in the folding pathway. Mutant chains, which lack the ability to form inter-chain disulphide bonds within the C-propeptide, were still able to assemble within this system. Furthermore, characterization of the trimeric molecules formed suggested that inter-chain disulphide bonds had formed within the C-telopeptide (C-terminal telopeptide). However, when hydroxylation of prolyl and lysyl residues was inhibited no inter-chain disulphide bonds were formed in the C-telopeptide, indicating that hydroxylation is required for the initial nucleation of the triple-helical domain. Mutant chains which lacked the ability to form inter-chain disulphide bonds within the C-propeptide or the C-telopeptide could still assemble to form trimeric triple-helical molecules linked by inter-chain disulphide bonds within the N-propeptide (N-terminal propeptide). These results indicate that inter-chain disulphide bond formation within the C-propeptide or the C-telopeptide is not required for chain association and triple-helix formation.
Flavinylation in wild-type trimethylamine dehydrogenase and differentially charged mutant enzymes: a study of the protein environment around the N1 of the flavin isoalloxazine
Authors: MEWIES Martin , PACKMAN Leonard C. , MATHEWS F. Scott , SCRUTTON Nigel S.
Published: August 2015

In wild-type trimethylamine dehydrogenase, residue Arg-222 is positioned close to the isoalloxazine N1/C2 positions of the 6S-cysteinyl FMN. The positively charged guanidino group of Arg-222 is thought to stabilize negative charge as it develops at the N1 position of the flavin during flavinylation of the enzyme. Three mutant trimethylamine dehydrogenases were constructed to alter the nature of the charge at residue 222. The amount of active flavinylated enzyme produced in Escherichia coli is reduced when Arg-222 is replaced by lysine (mutant R222K). Removal or reversal of the charge at residue 222 (mutants R222V and R222E, respectively) leads to the production of inactive enzymes that are totally devoid of flavin. A comparison of the CD spectra for the wild-type and mutant enzymes revealed no major structural change following mutagenesis. Like the wild-type protein, each mutant enzyme contained stoichiometric amounts of the 4Fe-4S cluster and ADP. Electrospray MS also indicated that the native and recombinant wild-type enzymes were isolated as a mixture of deflavo and holo enzyme, but that each of the mutant enzymes have masses expected for deflavo trimethylamine dehydrogenase. The MS data indicate that the lack of assembly of the mutant proteins with FMN is not due to detectable levels of post-translational modification of significant mass. The experiments reported here indicate that simple mutagenic changes in the FMN-binding site can reduce the proportion of flavinylated enzyme isolated from Escherichia coli and that positive charge is required at residue 222 if flavinylation is to proceed.
Involvement of the N-terminal part of cyclophilin B in the interaction with specific Jurkat T-cell binding sites
Authors: MARILLER Christophe , HAENDLER Bernard , ALLAIN Fabrice , DENYS Agnès , SPIK Geneviève
Published: August 2015

Cyclophilin B (CyPB) is secreted in biological fluids such as blood or milk and binds to a specific receptor present on the human lymphoblastic cell line Jurkat and on human peripheral blood lymphocytes. This study was intended to specify the areas of CyPB that are involved in the interaction with the receptor. A synthetic peptide corresponding to the first 24 N-terminal amino acid residues of CyPB was shown to specifically recognize the receptor. Moreover, modification of Arg18 of CyPB by p-hydroxyphenylglyoxal led to a dramatic loss of affinity for the receptor. However, when this residue was replaced by an alanine residue using site-directed mutagenesis, no modification of the binding properties was found, suggesting that Arg18 is not directly involved but is sufficiently close to the interaction site to interfere with the binding when modified. Competitive binding experiments using a chimaeric protein made up of the 24 N-terminal amino acid residues of CyPB fused to the cyclophilin A core sequence confirmed the involvement of this region of CyPB in receptor binding.
Uncoupling effect of polyunsaturated fatty acid deficiency in isolated rat hepatocytes:effect on glycerol metabolism
Authors: PIQUET Marie-Astrid , FONTAINE Eric , SIBILLE Brigitte , FILIPPI Céline , KERIEL Christiane , LEVERVE Xavier M.
Published: August 2015

The effects of a 4-week deficiency in polyunsaturated fatty acids (PUFA) in isolated rat hepatocytes have been investigated for oxidative phosphorylation and fatty acid, dihydroxyacetone (DHA) or glycerol metabolism. Oxygen uptake was significantly increased (by 20%) with or without fatty acid addition (octanoate or oleate) in the PUFA-deficient group compared with controls. The effect persisted after oligomycin addition but not after that of potassium cyanide, leading to the conclusion that, in these intact cells, the mitochondria were uncoupled. The PUFA-deficient group exhibited a significant decrease in the cytosolic ATP/ADP ratio, whereas the mitochondrial ratio was not affected. PUFA deficiency led to a 16% decrease in DHA metabolism owing to a 34% decrease in glycerol kinase activity; the significant decrease in the ATP/ADP ratio was accompanied by an increase in the fractional glycolytic flux. In contrast, glycerol metabolism was significantly enhanced in the PUFA-deficient group. The role of the glycerol 3-phosphate dehydrogenase step in this stimulation was evidenced in hepatocytes perifused with glycerol and octanoate in the presence of increased concentrations of 2,4-dinitrophenol (Dnp): uncoupling with Dnp led to an enhancement of glycerol metabolism, as found in PUFA deficiency, although it was more pronounced than in controls. The matrix/cytosol gradients for redox potential and ATP/ADP ratio were lower in cells from PUFA-deficient rats, suggesting a decreased mitochondrial membrane potential in accordance with the uncoupling effect. Moreover, a doubling of the mitochondrial glycerol 3-phosphate dehydrogenase activity in the PUFA-deficient group compared with controls led us to conclude that the activation of glycerol metabolism is the consequence of two mitochondrial effects: uncoupling and an increase in glycerol 3-phosphate dehydrogenase activity.
Modulation of intrahepatic cholesterol trafficking: evidence by in vivo antisense treatment for the involvement of sterol carrier protein-2 in newly synthesized cholesterol transport into rat bile
Authors: PUGLIELLI Luigi , RIGOTTI Attilio , AMIGO Ludwig , NUÑEZ Liliana , GRECO Aldo V. , SANTOS Manuel J. , NERVI Flavio
Published: August 2015

Biliary cholesterol represents one of the two major excretory pathways for sterol elimination from the body and plays a central role in cholesterol gallstone formation. Biliary cholesterol originates from a precursor pool of preformed and newly synthesized free cholesterol. Although it has been suggested that newly synthesized and preformed biliary cholesterol are secreted by independent pathways, the specific cellular and molecular mechanisms are unknown. We used male Wistar rats to study the time-course of the appearance of newly synthesized cholesterol, phosphatidylcholine and protein into bile. The specific role of sterol carrier protein-2 (SCP-2) in the transport of newly synthesized biliary cholesterol was evaluated by an in vivo antisense oligonucleotide approach. In contrast to [14C]phosphatidylcholine and [35S]proteins, the time-course of [14C]cholesterol appearance into bile was rapid, and microtubule- and Golgi-independent. In vivo SCP-2 antisense treatment reduced and delayed the appearance of biliary [14C]cholesterol. Furthermore, hepatic SCP-2 expression increased more than 3-fold over control values in rats that had been treated with diosgenin to increase biliary secretion of newly synthesized cholesterol. These results suggest that SCP-2 is necessary for the rapid transport of newly synthesized cholesterol into bile and that hepatocytes can induce SCP-2 expression according to the rate of biliary secretion of newly synthesized cholesterol.
Immunological characterization of eristostatin and echistatin binding sites on αIIb β3 and αVβ3 integrins
Authors: MARCINKIEWICZ Cezary , ROSENTHAL Louis A. , MOSSER David M. , KUNICKI Thomas J. , NIEWIAROWSKI Stefan
Published: August 2015

Two disintegrins with a high degree of amino acid sequence similarity, echistatin and eristostatin, showed a low level of interaction with Chinese hamster ovary (CHO) cells, but they bound to CHO cells transfected with αIIbβ3 genes (A5 cells) and to CHO cells transfected with αvβ3 genes (VNRC3 cells) in a reversible and saturable manner. Scatchard analysis revealed that eristostatin bound to 816000 sites per A5 cell (Kd 28 nM) and to 200000 sites (Kd 14 nM) per VNRC3 cell respectively. However, VNRC3 cells did not bind to immobilized eristostatin. Echistatin bound to 495000 sites (Kd 53 nM) per A5 cell and to 443000 sites (Kd 20 nM) per VNRC3 cell. As determined by flow cytometry, radiobinding assay and adhesion studies, binding of both disintegrins to A5 cells and resting platelets and binding of echistatin to VNRC3 cells resulted in the expression of ligand-induced binding sites (LIBS) on the β3 subunit. Eristostatin inhibited, more strongly than echistatin, the binding of three monoclonal antibodies: OPG2 (RGD motif dependent), A2A9 (αIIbβ3 complex dependent) and 7E3 (αIIbβ3 and αvβ3 complex dependent) to A5 cells, to resting and to activated platelets and to purified αIIbβ3. Experiments in which echistatin and eristostatin were used alone or in combination to inhibit the binding of 7E3 and OPG2 antibodies to resting platelets suggested that these two disintegrins bind to different but overlapping sites on αIIbβ3 integrin. Monoclonal antibody LM 609 and echistatin seemed to bind to different sites on αvβ3 integrin. However, echistatin inhibited binding of 7E3 antibody to VNRC3 cells and to purified αvβ3, suggesting that αvβ3 and αIIbβ3 might share the same epitope to which both echistatin and 7E3 bind. Eristostatin had no effect in these systems, providing further evidence that it binds to a different epitope on αvβ3.
Characterization of pig liver glutathione S-transferases using HPLC-electrospray-ionization mass spectrometry
Authors: ROUIMI Patrick , ANGLADE Patricia , DEBRAUWER Laurent , TULLIEZ Jacques
Published: August 2015

We have characterized 11 porcine liver cytosolic glutathione S-transferase (GST) subunits from their precise molecular mass, immunoreactivity and partial amino acid sequence. Four Alpha-, six Mu- and one unexpected Pi-class GST subunits were found with average molecular masses of 24.984–25.228 kDa, 25.039–25.657 kDa and 23.510 kDa respectively. Molecular masses were established using electrospray-ionization mass spectrometry, with a precision of ±3–4 mass units. Glutathione (GSH) and S-hexylglutathione (ShGSH) were tested as affinity ligands in the purification procedure. The binding selectivity of GSH was better than that of ShGSH, although non-GST proteins were retained on both matrices. As already described in other studies, a number of non-GST proteins bound to the affinity resins. Two of them were tentatively identified as mevalonate kinase and carbonyl reductase. The characterization of pig liver cytosolic GST subunits pattern achieved in this work should constitute a useful tool for rapid evaluation of these enzymes' expression in modulation studies.
Role of an aprotinin-sensitive protease in the activation of Ca2+-ATPase by superoxide radical (O2-.) in microsomes of pulmonary vascular smooth muscle
Authors: CHAKRABORTI Tapati , GHOSH Salil K. , MICHAEL John R. , CHAKRABORTI Sajal
Published: August 2015

We have investigated the role of an aprotinin-sensitive protease in regulating Ca2+-ATPase activity and Ca2+ uptake (ATP-dependent and Na+-dependent) in microsomes of bovine pulmonary vascular smooth muscle during treatment with the O2-•-generating system hypoxanthine plus xanthine oxidase. Treatment of the smooth muscle microsomes with the O2-•-generating system produced a protease in a gelatin-containing zymogram with an apparent molecular mass of 16 kDa. This 16 kDa proteolytic protein was found to be inhibited by superoxide dismutase (SOD) and aprotinin but not by PMSF. Using polyclonal antiserum to aprotinin, we found that it is an ambient antiprotease of the smooth muscle microsomes. Treatment of the microsomes with the O2-•-generating system stimulated protease activity tested with a synthetic substrate N-benzoyl-dl-arginine p-nitroanilide and also enhanced Ca2+-ATPase activity. It also stimulated ATP-dependent Ca2+ uptake. In contrast, Na+-dependent Ca2+ uptake was found to be inhibited by the O2-•-generating system. Pretreatment of the microsomes with SOD and aprotinin preserved the increase in protease activity, Ca2+-ATPase activity and ATP-dependent Ca2+ uptake. In addition, O2-•-caused inhibition of the Na+-dependent Ca2+ uptake which was reversed by SOD and aprotinin. Pretreatment with PMSF did not cause any discernible alteration in the protease activity, Ca2+-ATPase activity, ATP-dependent Ca2+ uptake and Na+-dependent Ca2+ uptake in the microsomes caused by the O2-•-generating system. These results suggest that an aprotinin-sensitive protease plays a pivotal role in regulating Ca2+-ATPase and Ca2+-uptake activities in microsomes of pulmonary vascular smooth muscle under oxidant O2-•-triggered conditions.
Measurement of oxidized and methylated DNA bases by HPLC with electrochemical detection
Authors: KAUR Harparkash , HALLIWELL Barry
Published: August 2015

Oxidative DNA damage is thought to be an important contributor to cancer development and to be affected by dietary constituents, so its accurate measurement is important. DNA methylation is recognized as an important mechanism affecting gene expression. In the present paper we describe an HPLC-with-electrochemical-detection procedure that allows rapid and sensitive measurement of four oxidized (2,6-diamino-4-hydroxy-5-formamidopyrimidine, 5-hydroxyuracil, 8-hydroxyguanine, 8-hydroxyadenine) and three methylated (7-methylguanine, 1-methylguanine, O6-methylguanine) bases in acid hydrolysates of DNA. Guanine was also detected, but was clearly separated from the other bases.
In vivo and in vitro folding of a recombinant metalloenzyme, phosphomannose isomerase
Authors: PROUDFOOT Amanda E. I. , GOFFIN Laurence , PAYTON Mark A , WELLS Timothy N. C. , BERNARD Alain R
Published: August 2015

Phosphomannose isomerase (PMI) catalyses the interconversion of mannose 6-phosphate and fructose 6-phosphate in prokaryotic and eukaryotic cells. The enzyme is a metalloenzyme which contains 1 mol of zinc per mol of enzyme. Heterologous expression of the cDNA coding for the Candida albicans enzyme in the prokaryotic host Escherichia coli results in an expression level of up to 30% of total E. coli protein. Ten percent of recombinant PMI is expressed in the soluble fraction and 90% in inclusion bodies. Inclusion of a high level of zinc in the fermentation medium resulted in a fourfold increase in soluble protein. Co-expression of the bacterial chaperones, GroES and GroEL, resulted in a proportional twofold increase in soluble PMI while causing an overall decrease in the PMI expression level. Folding denatured PMI in vitro required reductant and zinc ions. The yield of renatured protein was increased by folding in the presence of GroEL and DnaK in an ATP-independent manner. The refolding yield of denatured soluble enzyme from a guanidine solution was threefold higher than that of folding monomerized inclusion body protein solubilized in guanidine hydrochloride. This suggests that a proportion of recombinant protein expressed in E. coli inclusion bodies may be irreversibly denatured.
Binding of ATP and ATP analogues to the uncoating ATPase Hsc70 (70 kDa heat-shock cognate protein)
Authors: BUXBAUM Engelbert , WOODMAN Philip G
Published: August 2015

Nucleotide binding to the 70 kDa heat-shock cognate protein (Hsc70) from mung bean seeds and pig brain was investigated, as well as the clathrin uncoating activity of Hsc70 in the presence of these nucleotides. The two enzymes were found to behave identically. ATP bound to two different forms of Hsc70, with dissociation constants of 1.1±0.1 µM and 1.4±0.7 mM respectively at 25 °C. This corresponds to ΔG0´ = -34 and -16 kJ/mol respectively. From the temperature-dependence of the dissociation constant of the high-affinity site, ΔH0´ was calculated to -36±2 kJ/mol. This gives ΔS0´ = 6.7 J/mol per K. Adenosine 5´-[γ-thio]triphosphate, ADP, adenosine 5´-[β,γ-imino]triphosphate and adenosine 5´-[β,γ-methylene]triphosphate showed dissociation constants of 2.3, 11, 31 and 284 µM respectively. The order of affinities corresponded to the order of effectiveness in uncoating of pig brain coated vesicles. The implications of these findings for the mechanism of Hsc70 action are discussed.
Intracellular retention of a factor VIII protein with an Arg2307>Gln mutation as a cause of haemophilia A
Authors: VOORBERG Jan , de LAAF Rozalia T. M. , KOSTER Pauline M , MOURIK Jan A. van
Published: August 2015

Substitution of Arg2307 by Gln in factor VIII has been found to be associated with mild to moderate haemophilia A [Gitschier, Wood, Shuman and Lawn (1986) Science 232, 1415–1416]. We have introduced this particular point mutation into a B-domain-deleted factor VIII cDNA and expressed the modified cDNA in C127 cells. Cells expressing the resulting protein, termed des-(868–1562)-factor VIII-R2307Q, were compared with those expressing the previously characterized des-(868–1562)-factor VIII. No immunoreactive material could be detected in the conditioned medium of cells transfected with des-(868–1562)-factor VIII-R2307Q cDNA using assays specific for the factor VIII light chain and the factor VIII heavy chain. Analysis of metabolically labelled cells transfected with des-(868–1562)-factor VIII-R2307Q cDNA revealed that this mutant protein is synthesized at a level similar to des-(868–1562)-factor VIII. In contrast to des-(868–1562)-factor VIII, metabolically labelled des-(868–1562)-factor VIII-R2307Q was not encountered in the conditioned medium of the transfected cells, indicating that the mutant protein is not secreted from the cell. Inspection of the intracellular localization of the two proteins in the cell employing morphological analysis, endoglycosidase H and experiments with inhibitors of glucosidases I and II was consistent with localization of des-(868–1562)-factor VIII and des-(868–1562)-factor VIII-R2307Q in the endoplasmic reticulum. Taken together, our data indicate that the Arg2307 → Gln mutation results in aberrant intracellular trafficking of factor VIII, which may explain the low levels of factor VIII antigen in the plasma of haemophilia A patients that carry this particular point mutation.
Prenylation of a Rab1B mutant with altered GTPase activity is impaired in cell-free systems but not in intact mammalian cells
Authors: WILSON Amy L , SHERIDAN Kathleen M , ERDMAN Robert A , MALTESE William A
Published: August 2015

Previous studies have reached differing conclusions as to whether or not guanine-nucleotide-dependent conformational changes affect the ability of Rab proteins to undergo post-translational modification by Rab:geranylgeranyltransferase (Rab-GGTase). We now show that the ability of a Rab1B mutant [Q67L (Gln-67 → Leu)] with reduced intrinsic GTPase activity to undergo geranylgeranylation in cell-free assays depends on the guanine nucleotide composition of the system. When GTP is the predominant nucleotide in the assay, Rab1BQ67L is a poor substrate. However, when GDP is present and GTP is omitted, prenylation of the Q67L mutant is comparable with that of the wild-type (WT) protein. These studies, coupled with the poor prenylation of Rab1BWT in the presence of the non-hydrolysable GTP analogue guanosine 5´-[γ-thio]triphosphate, support the notion that Rab-GGTase prefers substrates in the GDP conformation. When the abilities of Rab1BQ67L and Rab1BWT to undergo prenylation were compared by metabolic labelling of transiently expressed proteins in cultured human 293 cells, we did not observe a decline in prenylation of the mutant protein as predicted on the basis of the cell-free assays. Moreover, the Q67L mutant was comparable with the wild-type Rab1B in its ability to associate with co-expressed Rab GDP dissociation inhibitors in 293 cells. These findings raise the possibility that unidentified proteins present in intact cells may compensate for the reduced intrinsic GTPase activity of the Q67L mutant, allowing a significant proportion of the nascent Rab1BQ67L to assume a GDP conformation. The differential prenylation of Rab1BQ67L in cell-free systems versus intact cells underscores the importance of evaluating the posttranslational modification of specific Rab mutants in vivo, where poorly characterized regulatory proteins may have a significant effect on GTPase activity or nucleotide exchange rates.
Cation-π bonding and amino-aromatic interactions in the biomolecular recognition of substituted ammonium ligands
Authors: SCRUTTON Nigel S. , RAINE Andrew R.C.
Published: August 2015

Cation-π bonds and amino-aromatic interactions are known to be important contributors to protein architecture and stability, and their role in ligand-protein interactions has also been reported. Many biologically active amines contain substituted ammonium moieties, and cation-π bonding and amino-aromatic interactions often enable these molecules to associate with proteins. The role of organic cation-π bonding and amino-aromatic interactions in the recognition of small-molecule amines and peptides by proteins is an important topic for those involved in structure-based drug design, and although the number of structures determined for proteins displaying these interactions is small, general features are beginning to emerge. This review explores the role of cation-π bonding and amino-aromatic interactions in the biological molecular recognition of amine ligands. Perspectives on the design of ammonium-ligand-binding sites are also discussed.
Predicted α-helix/β-sheet secondary structures for the zinc-binding motifs of human papillomavirus E7 and E6 proteins by consensus prediction averaging and spectroscopic studies of E7
Authors: ULLMAN Christopher G , HARIS Parvez I , GALLOWAY Denise A. , EMERY Vincent C. , PERKINS Stephen J.
Published: August 2015

The E7 and E6 proteins are the main oncoproteins of human papillomavirus types 16 and 18 (HPV-16 and HPV-18), and possess unknown protein structures. E7 interacts with the cellular tumour-suppressor protein pRB and contains a zinc-binding site with two Cys-Xaa2-Cys motifs spaced 29 or 30 residues apart. E6 interacts with another cellular tumour-suppressor protein p53 and contains two zinc-binding sites, each with two Cys-Xaa2-Cys motifs at a similar spacing of 29 or 30 residues. By using the GOR I/III, Chou-Fasman, SAPIENS and PHD methods, the effectiveness of consensus secondary structure predictions on zinc-finger proteins was first tested with sequences for 160 transcription factors and 72 nuclear hormone receptors. These contain Cys2His2 and Cys2Cys2 zinc-binding regions respectively, and possess known atomic structures. Despite the zinc- and DNA-binding properties of these protein folds, the major α-helix structures in both zinc-binding regions were correctly identified. Thus validated, the use of these prediction methods with 47 E7 sequences indicated four well-defined α-helix (α) and β-sheet (β) secondary structure elements in the order ββαβ in the zinc-binding region of E7 at its C-terminus. The prediction was tested by Fourier transform infrared spectroscopy of recombinant HPV-16 E7 in H2O and 2H2O buffers. Quantitative integration showed that E7 contained similar amounts of α-helix and β-sheet structures, in good agreement with the averaged prediction of α-helix and β-sheet structures in E7 and also with previous circular dichroism studies. Protein fold recognition analyses predicted that the structure of the zinc-binding region in E7 was similar to a ββαβ motif found in the structure of Protein G. This is consistent with the E7 structure predictions, despite the low sequence similarities with E7. This predicted motif is able to position four Cys residues in proximity to a zinc atom. A model for the zinc-binding motif of E7 was constructed by combining the Protein G coordinates with those for the zinc-binding site in transcription factor TFIIS. Similar analyses for the two zinc-binding motifs in E6 showed that they have different α/β secondary structures from that in E7. When compared with 12 other zinc-binding proteins, these results show that E7 and E6 are predicted to possess novel types of zinc-binding structure.
Conformational analysis of retinoids and restriction of their dynamics by retinoid-binding proteins
Authors: AALTEN Daan M. F. van , GROOT Bert L. de , BERENDSEN Herman J. C. , FINDLAY John B. C.
Published: August 2015

An exhaustive sampling of the configurational space of all-trans retinol using a 0.1 µs molecular-dynamics simulation is presented. The essential dynamics technique is used to describe the conformational changes in retinol using only three degrees of freedom. The different conformational states of retinol are analysed, and differences in free energy are calculated. The essential dynamics description allows a detailed comparison of free retinol and retinoids bound to retinoid-binding proteins and opens new possibilities in the small-molecule docking field. The dynamics of retinoids when complexed with their binding proteins are restricted, and they are forced into strained conformations. A ‘spring’ model for retinoid binding is proposed. This model is extended to a hypothesis for retinoid binding to visual pigments and bacteriorhodopsin.
Type VIII collagen is a product of vascular smooth-muscle cells in development and disease
Authors: MacBEATH Jonathan R. E. , KIELTY Cay M , SHUTTLEWORTH C. Adrian
Published: August 2015

Type VIII collagen is a short-chain collagen with considerable similarity to type X collagen. We have generated chain-specific antibodies to the α1 and α2 chains of type VIII collagen, and used them as probes to examine the synthesis of type VIII collagen by vascular smooth muscle cells (VSMC). In addition, chain-specific oligonucleotides have been used in reverse transcriptase-PCR (RT-PCR) reactions with RNA extracted from cultured smooth muscle cells in culture and from freshly isolated vascular tissues. Radiolabelling of VSMC in culture and immunoprecipitation with chain-specific antibodies showed that both chains were expressed. Lower levels of type VIII collagen were found in adult VSMC than in neonatal VSMC. RT-PCR showed that both chains were expressed in tissues as well as cells in culture. The results indicate that type VIII collagen is a product of VSMC of normal adult vessels and is expressed at high levels by VSMC in vascular lesions.
Evidence for two types of phosphate translocators in sweet-pepper (Capsicum annum L.) fruit chromoplasts
Authors: QUICK Paul W. , NEUHAUS H. Ekkehard
Published: August 2015

We have investigated whether there is evidence for the presence of different types of phosphate translocators in envelopes purified from pepper-fruit chromoplasts. A method was developed that allowed the purification of envelope membranes from isolated pepper-fruit chromoplasts. Proteoliposomes containing envelope-membrane proteins are able to import inorganic phosphate (Pi) or glucose 6-phosphate (Glc6P). In both cases, the rate of import is strongly dependent upon preloading of proteoliposomes with either Pi, dihydroxyacetone phosphate (DHAP) or Glc6P. This demonstrates the presence of a phosphate translocator activity catalysing a counter exchange of phosphorylated intermediates. Interestingly, a high external concentration of Glc6P does not strongly inhibit Pi uptake into proteoliposomes preloaded with DHAP, whereas external Glc6P strongly inhibits Pi uptake into proteoliposomes preloaded with Glc6P. This observation strongly indicates that two types of phosphate translocator are present in chromoplast envelopes from red-pepper fruits. These data are discussed with respect to the possible physiological function of two types of phosphate translocator in one type of plastid.
Changes in the nuclear matrix of chicken erythrocytes that accompany maturation
Authors: CHEN Hou Y , SUN Jian-Min , HENDZEL Michael J , RATTNER Jerome B. , DAVIE James R.
Published: August 2015

The protein composition and structure of nuclear matrices isolated from adult chicken immature and mature erythrocytes were analysed. Visualization of nuclear matrices by electron microscopy showed that immature-erythrocyte nuclear matrices had internal structures, while most mature-erythrocyte nuclear matrices did not. Both mature- and immature-erythrocyte nuclear matrices were surrounded by a fibrous network of intermediate filaments. Two-dimensional gel electrophoretic analysis of proteins obtained from fractionated nuclear matrices led to the assignment of the proteins as components of the nuclear pore-lamina, internal matrix, or cytoskeleton. Common and different proteins belonging to one of the three groups were identified in nuclear matrices of immature and mature erythrocytes. Investigation of the partitioning of histone deacetylase activity, an enzyme associated with the internal matrix, among the erythroid nuclear matrix fractions provided evidence that mature- and immature-erythrocyte nuclear matrices have internal structures. However, the activity of histone deacetylase and level of internal matrix proteins from mature-erythrocyte nuclear matrices were less than those from immature-erythrocyte matrices. The low levels of nuclear RNA and internal matrix proteins may account for lack of visual evidence for an internal matrix in mature erythrocytes.
Subcellular distribution of glycosylphosphatidylinositol-specific phospholipase D in rat liver
Authors: HARI Thomas , KUNZE Hans , BOHN Ernst , BRODBECK Urs , BÜTIKOFER Peter
Published: August 2015

Glycosylphosphatidylinositol (GPI)-hydrolysing enzymes have been described in many mammalian tissues and body fluids; however, their site(s) of action and in vivo functions have remained unclear. In order to identify a possible intracellular site of GPI hydrolysis, we studied the subcellular distribution of GPI-hydrolysing activity in rat liver. We found that purified fractions from rat liver hydrolysed the GPI moieties of two GPI-anchored proteins with the specificity of a phospholipase D. This GPI-specific phospholipase D (GPI-PLD) activity was found to be highly enriched in a lysosomal fraction and showed a similar intracellular distribution to that of typical lysosomal enzymes. Our results indicate that lysosomes may represent a possible intracellular site of GPI-PLD action.
Characterization of heterotrimeric G-proteins in adult Acanthocheilonema viteae
Authors: Karen R. GRANT , HARNETT Margaret M. , MILLIGAN Graeme , HARNETT William
Published: August 2015

Heterotrimeric G-proteins have been found in eukaryotic cells, from yeast to humans, but have received little attention, to date, with respect to parasitic organisms. We now present the first report of the characterization of heterotrimeric G-proteins expressed in a filarial nematode, Acanthocheilonema viteae. Using a combination of (i) affinity labelling with [α-32P]GTP; (ii) ADP-ribosylation with cholera toxin and pertussis toxin; (iii) Western blotting with a panel of anti-G-protein antibodies; and (iv) reverse transcriptase-PCR with degenerate G-protein oligonucleotide primers followed by hybridization analysis using oligonucleotides specific for individual G-protein subunits, we demonstrate that adult A. viteae expresses homologues of the β1-and/or β2-like subunits and α-subunits of the Gs, Gi, Gq and G12 subfamilies found in mammals. The role which these G-proteins may play in the biology of the organism is discussed.
Partial characterization of the C-terminal non-collagenous domain (NC1) of collagen type X
Authors: BARBER Ruth E. , KWAN Alvin P. L.
Published: August 2015

Collagen type X is composed of three identical α1(X) chains of 59 kDa, each containing a triple-helical region of 45 kDa flanked by a short N-terminal sequence and a larger non-collagenous C-terminal (NC1) domain of approx. 15 kDa. Collagen type X molecules can associate via their C-termini to form a regular hexagonal lattice in vitro, which in vivo may provide a modified extracellular matrix for the events of endochondral ossification. The NC1 domain of chick collagen type X was isolated and purified from a highly purified bacterial collagenase digest of hypertrophic chondrocyte medium proteins. The structure and aggregation properties of the NC1 domain of collagen X were investigated, independently of the triple helix. A trimer, a dimer and a monomer of the individual α-chain NC1 polypeptides were identified from a bacterial collagenase digest of cartilage collagens using [14C]tyrosine labelling, N-chlorosuccinimide peptide mapping and N-terminal sequencing. The trimer (50 kDa) remained intact in Laemmli sample buffer unless boiled, upon which it dissociated into the dimer (38 kDa) and the monomer (20 kDa). The dimer persisted even after prolonged periods of heating or reduction with β-mercaptoethanol, and in preparations obtained from chondrocyte cultures treated with β-aminoproprionitrile, indicating the presence of non-reducible, non-lysine-derived, covalent cross-links. Hexamers of the individual C-termini were observed in rotary-shadowed preparations of purified NC1 domain, reflecting the ability of collagen type X to self-assemble via its C-termini under appropriate conditions.
Ca2+ influx does more than provide releasable Ca2+ to maintain repetitive spiking in human umbilical vein endothelial cells
Authors: J. MORGAN Anthony , JACOB Ron
Published: August 2015

We investigated why oscillations of intracellular Ca2+ concentrations ([Ca2+]i) in endothelial cells challenged by submaximal histamine run down in Ca2+-free medium despite stores retaining most of their Ca2+. One explanation is that only a small subpopulation of the Ca2+ stores oscillate and are completely emptied of Ca2+. To investigate if influx refills an empty store subpopulation, we differentiated between cations entering the cell and those released from internal stores by using extracellular Sr2+ as a Ca2+ surrogate; we distinguished between [Sr2+]i and [Ca2+]i by using the larger effect of Sr2+ on fura 2 fluorescence at 360 nm (F360). Ca2+ was still available for release when oscillations had run down since oscillations promptly reappeared on addition of Sr2+o and these were predominantly of Ca2+ (indicated by F360 changes). Also, totally depleting Ca2+ stores inhibited Sr2+-induced oscillations, suggesting that Sr2+ entry leads to Ca2+ release. In contrast, Ba2+o was unable to stimulate oscillations. Finally, oscillations generated by photolytic release of inositol trisphosphate (IP3) analogues were similarly sensitive to extracellular Ca2+ and Sr2+. We conclude that stores (or a subpopulation) are not completely depleted of Ca2+ when oscillations run down in Ca2+-free medium. Bivalent cation entry therefore maintains sensitivity to IP3, possibly by maintaining luminal bivalent cation levels.
Identification of phosphorylation sites in keratinocyte transglutaminase
Authors: RICE Robert H. , MEHRPOUYAN Majid , QIN Qin , PHILLIPS Marjorie A. , LEE Young M.
Published: August 2015

Phosphorylation of keratinocyte transglutaminase occurs in its N-terminal extension and is stimulated several-fold by the protein kinase C agonist phorbol myristate acetate. In the present work, this stimulation was prevented by simultaneous treatment of the cells with the protein kinase C-selective inhibitor GF109203X. In contrast, phosphorylation occurring in the absence of phorbol ester was essentially unaffected by GF109203X, although it was decreased dramatically by the non-selective kinase inhibitor staurosporine. Four serine residues that are subject to phosphorylation have been identified by sequencing of radioactive tryptic peptides. Serines at positions 24 and 92, each containing 2–8% of the total radioactivity with or without phorbol ester stimulation, were minor sites of phosphorylation. Serine-82 was by far the dominant site of phosphorylation in the absence of phorbol ester treatment, and was also the major site in its presence. Serine-85 was phosphorylated to a high degree in the presence but not in the absence of phorbol ester stimulation. Thus the data indicate the influence of at least two different kinase activities in transglutaminase phosphorylation.
Lactase phlorhizin hydrolase turnover in vivo in water-fed and colostrum-fed newborn pigs
Authors: DUDLEY Mary A. , BURRIN Douglas G. , QUARONI Andrea , ROSENBERGER Judy , COOK Gary , NICHOLS Buford L. , REEDS Peter J.
Published: August 2015

We have estimated the synthesis rates in vivo of precursor and brush-border (BB) polypeptides of lactase phlorhizin hydrolase (LPH) in newborn pigs fed with water or colostrum for 24 h post partum. At the end of the feeding period, piglets were anaesthetized and infused intravenously for 3 h with l-[4-3H]-phenylalanine. Blood and jejunal samples were collected at timed intervals. The precursor and BB forms of LPH were isolated from jejunal mucosa by immunoprecipitation followed by SDS/PAGE, and their specific radioactivity in Phe determined. The kinetics of precursor and BB LPH labelling were analysed by using a linear compartmental model. Immunoisolated LPH protein consisted of five polypeptides [high-mannose LPH precursor (proLPHh), complex glycosylated LPH precursor (proLPHc), intermediate complex glycosylated LPH precursor (proLPHi) and two forms of BB LPH]. The fractional synthesis rate (Ks) of proLPHh and proLPHc (approx. 5%/min) were the same in the two groups but the absolute synthesis rate (in arbitrary units, min-1) of proLPHh in the colostrum-fed animals was twice that of the water-fed animals. The Ks values of proLPHi polypeptides were significantly different (water-fed, 3.89%/min; colostrum-fed, 1.6%/min), but the absolute synthesis rates did not differ. The Ks of BB LPH was not different between experimental treatment groups (on average 0.037%/min). However, the proportion of newly synthesized proLPHh processed to BB LPH was 48% lower in colostrum-fed than in water-fed animals. We conclude that in neonatal pigs, the ingestion of colostrum stimulates the synthesis of proLPHh but, at least temporarily, disrupts the processing of proLPH polypeptides to the BB enzyme.