bluuebunny/crossref_metadata_2025 huggingface dataset:
Pancreatic acinar-cell desensitization alters InsP3 production and Ca2+ mobilization under conditions where InsP3 receptor remains intact
Authors: Servant M , Guillemette G , Morisset J
Published: August 2015

Desensitization of rat pancreatic acinar cells with 0.1 mM carbamoylcholine (Cch) or 0.5 nM caerulein (CAE), a cholecystokinin (CCK) agonist, altered the subsequent secretory responses to these two agonists. Changes in receptor affinities, shifts in receptor populations, receptor internalization and phosphorylation are the major modifications affecting the muscarinic and CCK receptors in response to desensitization. In this study, post-receptor alterations were examined in order to explain the altered enzyme secretion. Cch or CAE desensitization resulted in decreased Ca2+ release in response to CAE and Cch respectively. Under desensitizing conditions, the biochemical and pharmacological properties of the InsP3 receptor were not affected. Control and desensitized acini had similar Bmax. and KD values. The Ca(2+)-channel property of the InsP3 receptor was not affected, either, since Ca2+ release in response to increasing concentrations of InsP3 remained comparable in both groups of saponin-permeabilized acini. Finally, the quantities of InsP3 formed in response to Cch and CAE, measured by InsP3 radioreceptor assay, were significantly decreased in the Cch- and CAE-desensitized groups, and these decreases were not due to increased InsP3 turnover. These new data indicate that desensitization of acinar cells with Cch and CAE causes post-receptor modifications resulting in decreased InsP3 formation and decreased intracellular Ca2+ mobilization. It is suggested that the attenuated Ca2+ response is related to a decreased formation of InsP3 from PtdInsP2 hydrolysis and that phospholipase C could be the immediate target of this regulation.
Partial sequence of the purified protein confirms the identity of cDNA coding for human lysosomal α-mannosidase B
Authors: Emiliani C , Martino S , Stirling J L , Maras B , Orlacchio A
Published: August 2015

Human lysosomal alpha-mannosidase has been purified by a simple and rapid method in sufficient quantities for the analysis of its subunit composition and partial protein sequencing. Analysis of the N-terminal residues of the 30 kDa polypeptide has enabled us to confirm the identity of the recently cloned cDNA that was tentatively identified as that of lysosomal alpha-mannosidase [Nebes and Schmidt (1994) Biochem. Biophys. Res. Commun. 200, 239-245] and to locate the position of this polypeptide within the total deduced amino acid sequence. This finding will therefore provide a firm foundation for the characterization of alpha-mannosidosis mutations.
Caldesmon mRNA splicing and isoform expression in mammalian smooth-muscle and non-muscle tissues
Authors: Payne A M , Yue P , Pritchard K , Marston S B
Published: August 2015

The recent determination of the genomic sequence of human caldesmon indicates that eight caldesmon mRNA species could be generated by selection of exon 1 or 1′, exon 3a or 3ab and/or exon 4. We used reverse transcriptase PCR to determine which transcripts were produced in human, rabbit and sheep artery, vein, lung, intestine, kidney and liver. In all tissues the same three transcripts were present: exons 1′-2-3a-5-6...13, exons 1′-2-3a3b-5-6-...13 and exons 1′-2-3a3b-4-5-6...13. Exon 1 was not present and exon 4 was only present when exon 3b was also present. Three protein isoforms of caldesmon can be distinguished by electrophoresis on high-porosity 6% polyacrylamide gel: 130 kDa, 120 kDa and 70 kDa. The 70 kDa isoform lacks the sequence encoded by exon 3b. We investigated whether the two high-molecular-mass isoforms correspond to the presence and absence of exon 4 using an antiserum specific to the sequence encoded by exon 4. Western-blotting and immunoprecipitation experiments showed that both the 130 kDa and the 120 kDa isoforms were expressed with and without the exon 4 sequence. We therefore propose that the molecular-mass heterogeneity arises from additional first exons, possibly with separate promoter regions, which have not yet been characterized in the genomic sequence.
Different molecular forms of rat kidney gp330, the dominant autoantigen of active Heymann nephritis
Authors: Jokhadze G G , Oleinikov A V , Kanalas J J , Makker S P
Published: August 2015

The primary structure, consisting of 1650 amino acid residues, of the C-terminal end of the dominant autoantigen of active Heymann Nephritis, gp330, from rat kidney was obtained by cloning and sequencing of cDNA clones. Comparison of this sequence with the previously published sequences of fragments of the C-terminal end of gp330 [Raychowdhury, Niles, McCluskey and Smith (1989) Science 244, 1163-1165] revealed certain differences in their primary structures. These differences included several single amino acid substitutions, replacement of a stretch of 15 amino acid residues by a different stretch of six amino acid residues, and different lengths of cytoplasmic domain (188 versus 213 amino acid residues). These findings of two different primary structures of gp330 provide direct evidence for the existence of two molecular forms of gp330.
Respiratory-deficient human fibroblasts exhibiting defective mitochondrial DNA replication
Authors: Bodnar A G , Cooper J M , Leonard J V , Schapira A H V
Published: August 2015

We have characterized cultured skin fibroblasts from two siblings affected with a fatal mitochondrial disease caused by a nuclear genetic defect. Mitochondrial respiratory-chain function was severely decreased in these cells. Southern-blot analysis showed that the fibroblasts had reduced levels of mitochondrial DNA (mtDNA). The mtDNA was unstable and was eliminated from the cultured cells over many generations, generating the rho0 genotype. As the mtDNA level decreased, the cells became more dependent upon pyruvate and uridine for growth. Nuclear-encoded subunits of respiratory-chain complexes were synthesized and imported into the mitochondria of the mtDNA-depleted cells, albeit at reduced levels compared with the controls. Mitochondrial protein synthesis directed by the residual mtDNA indicated that the mtDNA was expressed and that the defect specifically involves the replication or maintenance of mtDNA. This is a unique example of a respiratory-deficient human cell line exhibiting defective mtDNA replication.
Human eosinophil major basic protein, a mediator of allergic inflammation, is expressed by alternative splicing from two promoters
Authors: Li M S , Sun L , Satoh T , Fisher L M , Spry C J F
Published: August 2015

Human eosinophil major basic protein (MBP) is one of the principal mediators of injury to parasites and tissues in allergic inflammation. MBP is stored in eosinophil crystalloid granules and released with other granule constituents during eosinophil action. Previous studies have identified an MBP gene promoter that generates a 1.0 kb mRNA transcript encoding MBP preproprotein which undergoes processing to the mature storage form. To investigate how the MBP gene is regulated, we have examined the identity and levels of the MBP transcripts both in precursor cells and in blood eosinophils. It was found that the gene was expressed from two upstream promoters, a distal promoter P1 in addition to the previously described promoter P2. Evidence for the second promoter was initially provided by isolation from a human HL-60 leukaemic cell cDNA library of a novel 1.6 kb MBP cDNA that was distinct from the known 1.0 kb cDNA. The complete nucleotide sequence of the 1.6 kb cDNA was determined, and showed that the two cDNAs had identical coding and 3′ untranslated regions but differed in their 5′ sequences. By isolating and sequencing MBP genomic clones from an arrayed chromosome 11 library, it was demonstrated that the MBP gene is composed of nine upstream exons and five coding exons. The 1.6 and 1.0 kb cDNAs arise by differential splicing of alternate MBP transcripts from promoters P1 and P2 respectively, located 32 kb apart in the genomic DNA. Primer extension analysis identified two transcription start sites at P1, neither associated with a typical TATA box motif. Northern blotting and reverse-transcription PCR analysis showed that the 1.0 kb mRNA was present at higher levels than the 1.6 kb species in immature cells including HL-60 and bone-marrow cells. By contrast, low levels of 1.6 kb mRNA transcripts predominated in differentiated blood eosinophils. The results are compatible with differential use of P1 and P2 promoters as a mechanism for regulation of MBP expression during eosinophil maturation.
Insulin-independent and extremely rapid switch in the partitioning of hepatic fatty acids from oxidation to esterification in starved-refed diabetic rats. Possible roles for changes in cell pH and volume
Authors: Moir A M B , Zammit V A
Published: August 2015

The requirement for a normal insulin response in mediating the starved-to-refed transition, with respect to the partitioning of hepatic fatty acids between beta-oxidation and esterification to glycerol, was studied. Diabetic rats were starved for 24 h and refed ad libitum for various periods of time. There was no increase in plasma insulin in response to the meal. However, the fatty acid oxidation:esterification ratio was very rapidly decreased from the starved to the fed value, most of the transition being achieved within the first hour of refeeding. There was a 2 h lag in the response of hepatic malonyl-CoA concentration, such that this rapid switch from oxidation to esterification could not be explained on the basis of changes in the absolute concentration of this inhibitor of carnitine palmitoyltransferase I (CPT I). Hepatic pyruvate and lactate concentrations both increased by several-fold upon refeeding and peaked after 1 h and 3 h, respectively. The hepatic lactate:pyruvate ratio increased 3.2-fold during the first 3 h of refeeding, suggesting that the cytosolic NAD(+)-NADH couple became much more highly reduced during the lag-period between the onset of inhibition of flux of fatty acids towards oxidation and the rise in malonyl-CoA concentration. This may be indicative of a lowering of intracellular pH, which would amplify greatly the sensitivity of CPT I to the inhibitor. In view of the very rapid and high food intake by these diabetic rats, the possibility is also considered that portal concentrations of amino acids and other metabolites could give rise to an increase in liver cell-volume that would inhibit CPT I acutely by an as yet unknown mechanism [M. Guzman, G. Velasco, J. Castro and V. A. Zammit (1994) FEBS Lett. 344, 239-241].
Conformational changes and in vitro core-formation modifications induced by site-directed mutagenesis of the specific N-terminus of pea seed ferritin
Authors: van Wuytswinkel O , Briat J F
Published: August 2015

Plant ferritin has a three-dimensional structure predicted to be very similar to that of animal ferritin. It has, however, an additional specific sequence of 24 amino acids at its N-terminus named extension peptide (EP). In order to determine precisely the interactions between EP and other domains of the pea seed ferritin subunit, three point mutations were performed. The mutated residues were chosen by three-dimensional computer modelling of the pea seed ferritin subunit structure [Lobréaux, Yewdall, Briat and Harrison (1992) Biochem. J. 228, 931-939]. The mutant recombinant proteins were expressed in Escherichia coli and purified to homogeneity; all the mutants were found to be assembled as 24-mers. When Ala-13 was replaced by His, as in mammalian ferritins, ferroxidase activity was significantly reduced. Moreover, in vitro iron-core formation in Pro-X-->Ala, Lys-R-->Glu and Ala-13-->His mutants was increased after denaturation by urea followed by renaturation; this was also observed with the EP deletion mutant (r delta TP/EP). The recombinant ferritins were also analysed using tryptophan fluorescence spectra. The r delta TP/EP, Pro-X-->Ala and Lys-R-->Glu mutants were found to be more susceptible to denaturation by urea than the native r delta TP pea seed ferritin.
The structural origins of the unusual specificities observed in the isolation of chymopapain M and actinidin by covalent chromatography and the lack of inhibition of chymopapain M by cystatin
Authors: Thomas M P , Verma C , Boyd S M , Brocklehurst K
Published: August 2015

1. The selectivity observed when the potentially general technique for the isolation of fully active forms of cysteine proteinases, covalent chromatography by thiol-disulphide interchange, is applied to chymopapain M and to actinidin was investigated by a combination of experimentation and computer modelling. Neither of these enzymes is able to react with the original Sepharose-GSH-2-dipyridyl disulphide gel, but fully active forms of both enzymes are obtained by using Sepharose-2-hydroxypropyl-2′-dipyridyl disulphide gel, which is both electrically neutral and sterically less demanding than the GSH gel. Electrostatic potential calculations, minimization and molecular-dynamics simulations provide explanations for the unusual, but different, specificities exhibited by actinidin and chymopapain M in the interactions of their active centres with ligands. 2. The unique behaviour of chymopapain M in exerting an almost absolute specificity for substrates with glycine at the P1 position and in resisting inhibition by cystatin was examined by the computer-modelling techniques. A new, modelled, structure of the complete chicken egg-white cystatin molecule based on the crystal structure of a short form of cystatin was deduced as a necessary prerequisite. The results suggest that electrostatic repulsion prevents reaction of actinidin with the GSH gel, whereas a steric ‘cap’ resulting from a unique arginine-65-glutamic acid-23 interaction in chymopapain M prevents reaction of the gel with this enzyme and accounts for the lack of its inhibition by cystatin and its specificity in catalysis. 3. Use of chymopapain M as a structural variant of papain demonstrates the validity of the predictions of Lowe and Yuthavong [Biochem. J. (1971) 124, 107-115] relating to the structural requirements and binding characteristics of the S1 subsite of papain.
Sheep pancreatic microsomes as an alternative to the dog source for studying protein translocation
Authors: Kaderbhai M A , Harding V J , Karim A , Austen B M , Kaderbhai N N
Published: August 2015

A procedure is described for the preparation of rough membrane vesicles of endoplasmic-reticular origin from the pancreas of sheep. These isolated membranes translocate, process and glycosylate in vitro-translated heterologous proteins in a manner comparable with that exhibited by dog pancreatic microsomes.
Interaction of non-conjugated olefinic substrate analogues with dopamine β-monooxygenase: catalysis and mechanism-based inhibition
Authors: Sirimanne S R , May S W
Published: August 2015

The reaction of dopamine beta-monooxygenase (DBM; EC 1.14.17.1) with the prototypical non-conjugated olefinic substrate, 2-(1-cyclohexenyl)ethylamine (CyHEA) [see Sirimanne and May (1988) J. Am. Chem. Soc. 110, 7560-7561], was characterized. CyHEA undergoes facile DBM-catalysed allylic hydroxylation to form (R)-2-amino-1-(1-cyclohexenyl)ethanol (CyHEA-OH) without detectable epoxidation or allylic hydroxylation to form (R)-2-amino-1-(1-cyclohexenyl)ethanol (CyHEA-OH) without detectable epoxidation or allylic rearrangement, and with stereochemistry consistent with that of DBM-catalysed benzylic hydroxylation and sulphoxidation. The kcat. of 90 s-1 for CyHEA oxygenation is about 75% of the kcat. for tyramine, the substrate commonly used in assays of DBM activity. DBM-catalysed oxygenation of CyHEA also results in mechanism-based inactivation of DBM, with the inactivation reaction yielding kinact. = 0.3 min-1 at pH 5.0 and 37 degrees C, and a partition ratio of 16,000. Although both CyHEA turnover and inactivation exhibit normal kinetics, CyHEA processing also results in gradual depletion of copper from DBM; however, mechanism-based irreversible DBM inactivation occurs independent of this copper depletion when sufficient copper is present in the assay solution. A likely mechanism for turnover-dependent DBM inactivation by CyHEA involves initial abstraction of an allylic hydrogen to form a resonance-stabilized allylic radical, which can then either partition to product or undergo attack by an active-site residue. Acyclic, non-conjugated olefinic analogues exhibit diminished substrate activity toward DBM. Thus, kcat. for oxygenation of cis-2-hexenylamine, which also produces only allylic alcohol product, is only 14% of that for CyHEA. Similarly, kinact./KI for turnover-dependent inactivation by the acyclic olefin 2-aminomethyl-1-pentene is more than an order of magnitude smaller than that for benzylic olefins. Our results establish that DBM catalyses allylic oxygenation of a number of non-conjugated olefinic substrate analogues with neither epoxidation nor allylic rearrangement occurring. The absence of epoxide products from non-conjugated olefinic substrates implies an inability of the activated copper-oxygen species of DBM to effect radical cation formation from a non-conjugated olefinic moiety. The striking contrast between DBM and cytochrome P-450, which carries out both epoxidation and allylic oxidation with non-conjugated olefinic substrates, is probably a reflection of the differences in redox potential of the activated oxygen species operative for these two enzymes.
A novel exocytoplasmic endonuclease from Streptomyces antibioticus
Authors: Cal S , Aparicio J F , de los Reyes-Gavilan C G , Nicieza R G , Sanchez J
Published: August 2015

A new exocytoplasmic, nutritionally controlled endodeoxyribonuclease (EC 3.1.21.-) was purified to homogeneity from Streptomyces antibioticus. The enzyme showed an apparent molecular mass of 29 kDa (being active in the monomeric form) and a pI of approximately 7.8. The nuclease hydrolysed endonucleolytically double-stranded circular and linear DNA. The enzyme makes nicks in one strand of the DNA in G-rich regions, leaving either 5′ or 3′ short, single-stranded overhangs with 3′-hydroxy and 5′-phosphate termini. Breaks in the DNA occur when two nicks in opposite strands are close together. The enzyme had an optimum pH of 7.5 and an absolute requirement for bivalent cations and > or = 100 mM NaCl in the reaction buffer. Activity was greatly diminished in the presence of phosphate, Hg2+ or iodoacetate and was stimulated by dimethyl sulphoxide. Single-stranded DNA was a much poorer substrate than double-stranded DNA. The nuclease hydrolyses sequences of three or preferably more (dG).(dC) tracts in the DNA. The initial specificity shifts to other sequences (including sequences shorter than those initially hydrolysed) during the course of the reaction, giving the changing pattern of bands observed in agarose gels. 5-Methylcytosine-hemimethylated DNA is not hydrolysed by the nuclease. The properties of this novel enzyme suggest a relationship with class II restriction endonucleases and also with some eukaryotic nucleases.
The roles of multiple pathways in regulating bombesin-stimulated phospholipase D activity in Swiss 3T3 fibroblasts
Authors: Briscoe C P , Martin A , Cross M , Wakelam M J O
Published: August 2015

The regulation of bombesin-stimulated phospholipase D (PLD) activity in Swiss 3T3 fibroblasts was examined. Increasing protein-tyrosine phosphorylation by using pervanadate to inhibit tyrosine phosphatases was found to stimulate protein kinase C (PKC)-independent [3H]phosphatidylbutanol ([3H]PtdBut) accumulation within 5 min, which continued to increase up to 30 min. The stimulation of PLD activity in response to submaximal [bombesin] could be decreased by approx. 50% by the tyrosine kinase inhibitor genistein, whereas pretreatment with genistein and the PKC inhibitor Ro-31-8220 completely abolished the generation of [3H]PtdBut in response to a maximal concentration of bombesin. The addition of guanosine 5′-[gamma-thio]triphosphate (GTP[S]) into permeabilized cells resulted in an increase in [3H]PtdBut, which was abolished by depletion of cellular ATP. The additional presence of 30 microM GTP[S] did not increase the stimulation of PLD activity by any [bombesin] tested, whereas it was synergistic with that stimulated in response to phorbol 12-myristate 13-acetate. These findings suggest that bombesin-stimulated PLD activity is indirectly regulated by G-proteins, possibly through a kinase intermediate. Furthermore, activation of protein tyrosine kinases is proposed to account for the PKC-independent arm of bombesin-stimulated PLD activity. No evidence was obtained for a form of PLD directly regulated by tyrosine phosphorylation.
Kinetic study in vitro of Escherichia coli promoter closure during transcription initiation
Authors: Schmitt B , Reiss C
Published: August 2015

The rate of closure of two Escherichia coli promoters borne by plasmid pBR322, following transcription initiation from the open complex, was probed in vitro by the protection of unpaired thymines in the open complex against oxidation by KMnO4. Run-off transcription kinetics were also studied under identical conditions. Closure of the open promoter appears to be by far the rate-limiting step of transcription initiation and elongation for the linearized beta-lactamase gene, and is strongly dependent on template topology for the RNAI gene. It is suggested that the corresponding signals are deposited 30 bases at least downstream of transcription initiation and that promoter closure, and its clearance by elongating RNA polymerase, may occur almost simultaneously.
Cause of spectral variation in the luminescence of semisynthetic aequorins
Authors: Shimomura O
Published: August 2015

Aequorin emits light in the presence of Ca2+, decomposing into apoaequorin, coelenteramide and CO2. Semisynthetic aequorins, produced by replacing the coelenterazine moiety in aequorin with analogues of coelenterazine, showed widely different sensitivities to Ca2+ as well as certain spectral variations. A group of semisynthetic aequorins, e-type aequorins, showed bimodal luminescence, with peaks at 400-405 nm and 440-475 nm in various intensity ratios, whereas all other aequorins luminesced with only one peak, in the range 440-475 nm. The cause of the spectral variation was studied by various experiments including: (1) comparison with the fluorescence of the spent solution and the luminescence of the spent solution produced by added coelenterazine; (2) luminescence in 2H2O; (3) the rate of conformational change of apoaequorin; (4) the rates of regeneration in the presence and absence of O2. The results suggested that the spectrum of Ca(2+)-triggered luminescence is strongly affected by the ionic charge on the amide N atom of the coelenteramide that is bound to apoaequorin. When the amide N atom is negatively charged, light is emitted with a 440-475 nm peak. In the case of e-type aequorins, the negative charge on the amide N atom is less because of the structure of e-coelenterazine involved, resulting in the emission of a 400-405 nm peak from the uncharged form of coelenteramide; the intensity ratio of 400-405 nm peak to 440-475 nm peak is determined by the amount of negative charge resting on the amide N atom of e-coelenteramide at the time of light emission. Most of the spectral variations in luminescence and fluorescence can be explained on the basis of ionic and hydrophobic interaction between a coelenteramide and apoaequorin.
Inositol 1,2,3-trisphosphate and inositol 1,2- and/or 2,3-bisphosphate are normal constituents of mammalian cells
Authors: Barker C J , French P J , Moore A J , Nilsson T , Berggren P O , Bunce C M , Kirk C J , Michell R H
Published: August 2015

1. An inositol trisphosphate (InsP3) distinct from Ins(1,4,5)P3 and Ins(1,3,4)P3, which we previously observed in myeloid and lymphoid cells [French, Bunce, Stephens, Lord, McConnell, Brown, Creba and Michell (1991) Proc R. Soc. London B 245, 193-201; Bunce, French, Allen, Mountford, Moore, Greaves, Michell and Brown (1993) Biochem. J. 289, 667-673], is present in WRK1 rat mammary tumour cells and pancreatic endocrine beta-cells. 2. It has been identified as Ins(1,2,3)P3 by a combination of oxidation to ribitol, a structurally diagnostic polyol, and ammoniacal hydrolysis to identified inositol monophosphates. 3. Ins(1,2,3)P3 concentration in HL60 cells changed little during stimulation by ATP or fMetLeuPhe or during neutrophilic or monocytic differentiation, and Ins(1,2,3)P3 was unresponsive to vasopressin in WRK1 cells. 4. Ins(1,2,3)P3 was usually more abundant than Ins(1,4,5)P3, often being present at concentrations between approximately 1 microM and approximately 10 microM. 5. HL60, WRK-1 and lymphoid cells also contain Ins(1,2)P2 or Ins(2,3)P2, or a mixture of these two enantiomers, as a major InsP2 species. 6. Ins(1,2,3)P3 and Ins(1,2)P2/Ins(2,3)P2 are readily detected in cells labelled for long periods, but not in acutely labelled cells. This behaviour resembles that of InsP6, the most abundant cellular inositol polyphosphate that includes the 1,2,3-trisphosphate motif, which also achieves isotopic equilibrium with inositol only slowly. 7. Ins(1,2,3)P3 is the major InsP3 that accumulates during metabolism of InsP6 by WRK-1 cell homogenates. 8. Possible metabolic relationships between Ins(1,2,3)P3, Ins(1,2)P2/Ins(2,3)P2 and other inositol polyphosphates in cells, and a possible role for Ins(1,2,3)P3 in cellular iron handling, are considered.
Solubilization, molecular forms, purification and substrate specificity of two acetylcholinesterases in the medicinal leech (Hirudo medicinalis)
Authors: Talesa V , Grauso M , Giovannini E , Rosi G , Toutant J P
Published: August 2015

Two acetylcholinesterases (AChE) differing in substrate and inhibitor specificities have been characterized in the medical leech (Hirudo medicinalis). A ‘spontaneously-soluble’ portion of AChE activity (SS-AChE) was recovered from haemolymph and from tissues dilacerated in low-salt buffer. A second portion of AChE activity was obtained after extraction of tissues in low-salt buffer alone or containing 1% Triton X-100 [detergent-soluble (DS-) AChE). Both enzymes were purified to homogeneity by affinity chromatography on edrophonium- and concanavalin A-Sepharose columns. Denaturing SDS/PAGE under reducing conditions gave one band at 30 kDa for purified SS-AChE and 66 kDa for DS-AChE. Sephadex G-200 chromatography indicated a molecular mass of 66 kDa for native SS-AChE and of 130 kDa for DS-AChE. SS-AChE showed a single peak sedimenting at 5.0 S in sucrose gradients with or without Triton X-100, suggesting that it was a hydrophylic monomer (G1). DS-AChE sedimented as a single 6.1-6.5 S peak in the presence of Triton X-100 and aggregated in the absence of detergent. A treatment with phosphatidylinositol-specific phospholipase C suppressed aggregation and gave a 7 S peak. DS-AChE was thus an amphiphilic glycolipid-anchored dimer. Substrate specificities were studied using p-nitrophenyl esters (acetate, propionate and butyrate) and corresponding thiocholine esters as substrates. SS-AChE displayed only limited variations in Km values with charged and uncharged substrates, suggesting a reduced influence of electrostatic interactions in the enzyme substrate affinity. By contrast, DS-AChE displayed higher Km values with uncharged than with charged substrates. SS-AChE was more sensitive to eserine and di-isopropyl fluorophosphate (IC50 5 x 10(-8) and 10(-8) M respectively) than DS-AChE (5 x 10(-7) and 5 x 10(-5) M.
Nitrone spin traps and a nitroxide antioxidant inhibit a common pathway of thymocyte apoptosis
Authors: Slater A F G , Nobel C S I , Maellaro E , Bustamante J , Kimland M , Orrenius S
Published: August 2015

Oxidative stress has recently been suggested to be a mediator of apoptotic cell death [Buttke and Sandstrom (1994) Immunology Today 15, 7-10], although evidence that this phenomenon is a widespread component of apoptosis is lacking. When rat thymocytes were exposed to the glucocorticoid methylprednisolone (MPS), a progressive increase in intracellular peroxides and a decrease in glutathione (GSH) were observed to accompany the onset of apoptosis. Using Percoll density gradients to isolate subpopulations of thymocytes at different stages of apoptosis, the increase in peroxide content was found to be restricted to apoptotic cells, while a significant depletion of GSH and reduced protein thiol was detected in both pre-apoptotic and fully apoptotic cells. To investigate the biological significance of these redox changes, the free radical spin traps 5,5-dimethyl-1-pyrroline-1-oxide (DMPO) and 3,3,5,5-tetramethyl-1-pyrroline-1-oxide (TMPO), and the related nitroxide-radical antioxidant 2,2,6,6-tetramethyl-1-piperidinyl-1-oxyl (TEMPO) were tested as inhibitors of thymocyte apoptosis. The cell shrinkage and DNA fragmentation induced by four different initiators of apoptosis were reduced by each compound. TEMPO inhibition of both etoposide- and MPS-induced thymocyte DNA fragmentation was also found to correlate with an increase in intracellular GSH, providing support for the proposal that its antioxidant properties were responsible for the observed protective activity. We conclude that some form of intracellular oxidation (here measured indirectly by changes in intracellular GSH and peroxide levels) is required during thymocyte apoptosis even when this process is initiated by an agent that does not exert a direct oxidant action.
Importance of the glutamate residue of KDEL in increasing the cytotoxicity of Pseudomonas exotoxin derivatives and for increased binding to the KDEL receptor
Authors: Kreitman R J , Pastan I
Published: August 2015

It was previously shown that amino acids 609-613 (REDLK) at the C-terminus of Pseudomonas exotoxin (PE) are necessary for cytotoxicity, presumably by directing the toxin to the endoplasmic reticulum (ER) [Chaudhary, Jinno, FitzGerald and Pastan (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 308-312]. Using the anti-[interleukin 2 receptor (IL2R)] immunotoxin anti-Tac(Fv)-PE38 (AT-PE38REDLK), it was found that removing the terminal lysine did not alter the activity, but replacing REDL with KDEL, the most common ER retention sequence, increased activity. To determine which amino acid in KDEL was responsible for the increase in activity, we tested eight C-terminal mutants of AT-PE38REDLK. Using IL2R-bearing MT-1 cells, we found that the glutamate residue of KDEL was required for high activity, as the cytotoxicity of AT-PE38 ending in KDEL, RDEL, KEEL or REEL was much greater than that of AT-PE38 ending in REDL, KEDL, RDDL or KDDL. Using freshly isolated lymphocytic leukaemia cells, AT-PE38 ending in KDEL, REEL or RDEL was more than 100-fold more cytotoxic than AT-PE38 ending in KEDL, REDL, RDDL or the native sequence REDLK. The RDEL sequence also improved the cytotoxic activity of an interleukin 4-PE38 toxin fusion protein. Improved cytotoxic activity correlated with improved binding of the C-termini to the KDEL receptor on rat Golgi membranes. These data indicate that the glutamate residue of KDEL improves the cytotoxicity of PE by increasing binding to a sorting receptor which transports the toxin from the transreticular Golgi apparatus to the ER, where it is translocated to the cytosol and inhibits protein synthesis.
Anti-(human immunodeficiency virus) activity of polyoxotungstates and their inhibition of human immunodeficiency virus reverse transcriptase
Authors: Moore P S , Jones C J , Mahmood N , Evans I G , Goff M , Cooper R , Hay A J
Published: August 2015

Heteropolyoxotungstates of the Keggin class containing different heteroatoms were tested for inhibition of two strains of human immunodeficiency virus 1 (HIV-1); they exhibited varying antiviral activity. Compounds containing boron were inactive, only one of those containing phosphorus showed selective anti-viral activity, whereas all silicon-containing compounds exhibited significant anti-viral activity in C8166 cells infected with the IIIB strain. Their effectiveness was some 10-fold higher in JM cells with selectivity indices of about 2000. The silicotungstates were effective inhibitors of HIV reverse transcriptase, showing greater inhibition with RNA/DNA template primers than with DNA/DNA template.primer. Kinetic analysis demonstrated that they inhibit the enzyme by different mechanisms, as, of the four compounds examined, two competed with template.primer and two competed with deoxynucleoside triphosphate. Inhibition of DNA polymerase activity by these compounds was compared using polymerases from different sources, including human; although not necessarily most specific for HIV-1 reverse transcriptase, they did not inhibit all DNA polymerases to a similar degree.
The binding of natural and fluorescent lysophospholipids to wild-type and mutant rat liver fatty acid-binding protein and albumin
Authors: Thumser A E A , Wilton D C
Published: August 2015

Rat liver fatty acid-binding protein (FABP) is able to bind a wide range of non-polar anionic ligands, including lysophospholipids. In order to understand the nature of lysophospholipid interactions with liver FABP, the binding of natural lysophospholipids and two fluorescent analogues, N-(5-dimethylaminonaphthalenesulphonyl)-1-palmitoyl-sn-glycero-3- phosphoethanolamine (dansyl lysoPE) and 1-(O-[11-(5-dimethylaminonaphthalene-sulphonyl)amino]undecyl)-sn-glycero -3- phosphocholine (dansyl-C11-lysoPAF), has been investigated. The results confirmed the ability of liver FABP to bind lysophospholipids with KD values in the range of 1-2 microM, and a 1:1 binding stoichiometry was indicated. Binding of fluorescent lysophospholipids was enhanced with the FABP mutant, R122Q, possibly due to increased flexibility of the binding cavity as a result of reduced hydrogen-bonding constraints. The fluorescent lysophospholipids also bound to albumin, with KD values in the range 0.1-1.0 microM, and could be displaced by oleic acid. The fluorescence characteristics of the dansyl lysophospholipid analogue dansyl-C11-lyso-PAF suggested that this probe binds to the same site(s) on albumin as the fluorescent fatty acid probe 11-(5-dimethylaminonaphthalene-sulphonylamino)-undecanoic acid (DAUDA).
Fatty acyl-CoA esters inhibit glucose-6-phosphatase in rat liver microsomes
Authors: Fulceri R , Gamberucci A , Scott H M , Giunti R , Burchell A , Benedetti A
Published: August 2015

In native rat liver microsomes glucose 6-phosphatase activity is dependent not only on the activity of the glucose-6-phosphatase enzyme (which is lumenal) but also on the transport of glucose-6-phosphate, phosphate and glucose through the respective translocases T1, T2 and T3. By using enzymic assay techniques, palmitoyl-CoA or CoA was found to inhibit glucose-6-phosphatase activity in intact microsomes. The effect of CoA required ATP and fatty acids to form fatty acyl esters. Increasing concentrations (2-50 microM) of CoA (plus ATP and 20 microM added palmitic acid) or of palmitoyl-CoA progressively decreased glucose-6-phosphatase activity to 50% of the control value. The inhibition lowered the Vmax without significantly changing the Km. A non-hydrolysable analogue of palmitoyl-CoA also inhibited, demonstrating that binding of palmitoyl-CoA rather than hydrolysis produces the inhibition. Light-scattering measurements of osmotically induced changes in the size of rat liver microsomal vesicles pre-equilibrated in a low-osmolality buffer demonstrated that palmitoyl-CoA alone or CoA plus ATP and palmitic acid altered the microsomal permeability to glucose 6-phosphate, but not to glucose or phosphate, indicating that T1 is the site of palmitoyl-CoA binding and inhibition of glucose-6-phosphatase activity in native microsomes. The type of inhibition found suggests that liver microsomes may comprise vesicles heterogeneous with respect to glucose-6-phosphate translocase(s), i.e. sensitive or insensitive to fatty acid ester inhibition.
Mutation-deletion analysis of a Ca2+-dependent phospholipid binding (CaLB) domain within p120 GAP, a GTPase-activating protein for p21 ras
Authors: Gawler D J , Zhang L J W , Moran M F
Published: August 2015

p120 GAP is a GTPase activating protein for p21 ras. It is a multidomain protein which exhibits sequence similarity with other GTPase-activating proteins, src, pleckstrin and a central portion of the protein kinase C conserved region 2 domain known as CaLB (Ca(2+)-dependent phospholipid-binding). The presence of this CaLB motif has led to the speculation that p120 GAP may be a member of a family of structurally related proteins containing a Ca(2+)-dependent membrane/lipid-binding domain. Here we have studied the in vitro Ca(2+)-dependent phospholipid-binding properties of the isolated proposed CaLB sequence in human GAP and deduce that a phospholipid-binding sequence is indeed located between amino acids 606 and 648. Binding of phosphatidylserine and phosphatidylinositol, but not phosphatidylcholine, within this sequence is Ca(2+)-dependent, with an estimated EC50 for Ca2+ of approx. 1 microM. Using deletion-mutation analysis we have further defined the minimal boundaries for this in vitro phospholipid-binding activity. p120 GAP amino acids 612-643 exhibit full phospholipid-binding activity, but further deletion of either amino acids 612-617 or amino acids 633-648 significantly decreased or abolished phospholipid binding. These studies establish that amino acids 612-643 of p120 GAP indeed constitute a functional CaLB domain and thereby imply a role for Ca2+ in the regulation of p120 GAP association with cellular (membrane) phospholipids.
Expression and site-directed mutagenesis of mouse prostaglandin E2 receptor EP3 subtype in insect cells
Authors: Huang C , Tai H H
Published: August 2015

A cDNA encoding for mouse prostaglandin E2 (PGE2) receptor EP3 subtype was cloned from a mouse kidney cDNA library by PCR using terminal primers derived from the known sequence of mouse lung EP3 receptor cDNA. The cloned cDNA was confirmed by sequencing and was expressed in Trichoplusia ni (MG1) insect cells using a baculovirus expression system. A specific protein of 60 kDa was detected by immunoblot with antibodies generated against a unique decapeptide sequence present in the second extracellular loop of the EP3 receptor. Specific binding of [3H]PGE2 with a Kd of 3 nM was also found in the membrane fraction of the insect cells. Ligand binding of the receptor was further studied by site-directed mutagenesis. Arg-309 of the receptor was separately mutated to lysine, glutamate and valine. cDNAs of the wild-type and mutant EP3 receptors were respectively expressed and studied in MG1 insect cells. Binding studies indicated that both glutamate and valine mutant EP3 receptors had no binding of [3H]PGE2. On the contrary, the lysine mutant receptor exhibited an even tighter binding (Kd = 1.3 nM) than the wild-type EP3 receptor. Immunoblot studies indicated that these receptors were expressed in a comparable amount in MG1 insect cells. These results suggest that Arg-309 of EP3 receptor may be essential in ligand binding through ionic interaction.
Characterization of a cDNA clone for human NAD+-specific isocitrate dehydrogenase α-subunit and structural comparison with its isoenzymes from different species
Authors: Kim Y O , Oh I U , Park H S , Jeng J , Song B J , Huh T L
Published: August 2015

A 0.6 kb cDNA fragment encoding the human NAD(+)-specific isocitrate dehydrogenase alpha-subunit (H-IDH alpha) was amplified by PCR using oligonucleotide primers synthesized on the basis of pig tryptic peptide sequences [Huang and Colman (1990) Biochemistry 29, 8266-8273]. With the amplified cDNA as a probe, cDNA clones for IDH alpha were isolated from a human heart lambda gt11 cDNA library. The deduced protein sequence of the largest cDNA clone (2628 bp) rendered a precursor protein of 366 amino acids (39,591 Da) and a mature protein of 339 amino acids (36,640 Da). The deduced H-IDH alpha protein sequence is highly similar to the partial peptide sequences of the pig enzyme. It is 55, 43 and 44% identical with yeast NAD(+)-specific IDH2, yeast NAD(+)-specific IDH1 and monkey NAD(+)-specific IDH gamma-subunit (IDH gamma) respectively. However, it has less similarity (about 30%) to NADP(+)-specific IDH from Escherichia coli and bovine mitochondria. These results indicate that the structure of IDH alpha closely resembles that of IDH2, the catalytic subunit of the yeast enzyme. Structural analysis of the deduced H-IDH alpha protein revealed that the amino acids responsible for the binding of isocitrate, Mg2+ and NAD+ are highly conserved. It also has two conserved motifs for the binding sites of ATP and ADP, but a canonical Ca(2+)-binding motif was not recognized. Unusual penta-(ATTTA) and tri-(TAA or ATT) nucleotides which are respectively believed to interact with RNA-binding proteins and be near the endonuclease cleavage sites were frequently recognized in its 3′ untranslated region, indicating the possibility of an additional method of regulation of this enzyme. Northern-blot analysis suggests that one mRNA transcript (2.8 kb) exists in cultured HeLa cells. Genomic DNA Southern-blot analysis indicates that the IDH alpha gene is not closely related to that of the other IDH isoenzymes, and IDH alpha appears to be encoded by a single gene.
Mass spectrometric analysis of rat liver cytosolic glutathione S-transferases: modifications are limited to N-terminal processing
Authors: Yeh H I , Hsieh C H , Wang L Y , Tsai S P , Hsu H Y , Tam M F
Published: August 2015

Cytosolic glutathione S-transferases (GSTs) from rat livers were purified using an S-hexylglutathione affinity column. The GST subunits were resolved by reverse-phase HPLC and their molecular masses were determined by electrospray mass spectrometry. The major hepatic GSTs detected were subunits 1, 1′, 2, 3 and 4, with molecular mass of 25,520, 25,473, 25,188, 25,782 and 25,571 Da respectively. Subunits 6, 7 and 10 are minor components, with molecular mass of 25,551, 23,308 and 25,211 Da respectively. Alternatively, the hepatic GSTs were purified using a glutathione affinity column. Subunits 1, 1′, 2, 8 and 10 were eluted from this column with GSSG, the oxidized form of glutathione. Subunit 8 has a molecular mass of 25,553 Da. The remaining proteins on the glutathione affinity column were removed with glutathione and S-hexylglutathione. Subunits 2, 3, 4 and 6 could be detected in the eluate. We could not detect any significant difference in molecular mass between GSTs isolated from male and female rat livers. Cytosolic GSTs were isolated from livers of buthionine sulphoximine-treated female rats for MS analysis. The molecular masses obtained were identical to those determined for the controls.
Identification of cytochrome P-450 1A (CYP1A) genes from two teleost fish, toadfish (Opsanus tau) and scup (Stenotomus chrysops), and phylogenetic analysis of CYP1A genes
Authors: Morrison H G , Oleksiak M F , Cornell N W , Sogin M L , Stegeman J J
Published: August 2015

Cytochrome P-450-mediated responses to environmental challenges are well known in diverse animal taxa, but the evolution of the complex gene superfamily coding for these enzymes is poorly understood. Here we report a phylogenetic analysis of the cytochrome P-450 1A (CYP1A) genes including two new sequences determined from teleost fish, toadfish (Opsanus tau) and scup (Stenotomus chrysops). Degenerate PCR primers were used to amplify a 1.2 kbp fragment from liver cDNA. The toadfish PCR product was used as a probe to identify a full-length CYP1A clone from a toadfish liver cDNA library. The entire coding region of the scup CYP1A was obtained by rapid amplification of cDNA ends (RACE) using specific primers based on the sequence of the partial PCR product. The predicted protein sequences for toadfish and scup CYP1A shared 78% and 83% amino acid identity with rainbow trout CYP1A1 respectively. Amino acid identity with mammalian CYP1A proteins ranged from 51 to 60% for 505 aligned positions. Phylogenetic analysis of four teleost fish CYP1A genes (trout, toadfish, scup and plaice) and 12 mammalian CYP1A genes suggests a monophyletic origin of the teleost genes, with the trout gene being most divergent, and indicates three distinct groupings: mammalian 1A1, mammalian 1A2, and fish 1A. This supports the idea that the gene duplication event which gave rise to CYP1A1 and CYP1A2 occurred after the divergence of the lines leading to mammals and fish. These results establish a molecular phylogeny within the CYP1A subfamily, the first such detailed phylogenetic analysis within a cytochrome P-450 family.
In Dictyostelium discoideum inositol 1,3,4,5-tetrakisphosphate is dephosphorylated by a 3-phosphatase and a 1-phosphatase
Authors: Van Dijken P , Lammers A A , Van Haastert P J M
Published: August 2015

The degradation of Ins(1,3,4,5)P4 in Dictyostelium was investigated using a mixture of [3H]Ins(1,3,4,5)P4 and [3-32P]Ins-(1,3,4,5)P4. After incubation of this mixture with a Dictyostelium homogenate the 32P/3H ratio found in the InsP3 product was reduced to 24% of the ratio in the substrate. 32P-labelled inorganic phosphate was found as well, whereas hardly any InsP2 was detected. This indicates that Ins(1,3,4,5)P4 is mainly degraded by a 3-phosphatase. The other enzyme was characterized by identification of the 32P-labelled InsP3 isomer. This isomer did not co-elute with Ins(1,3,4)P3, indicating that no 5-phosphatase was present in Dictyostelium. The 32P-labelled InsP3 could be oxidized using NaIO4. The only InsP3 isomer that has these characteristics is Ins(3,4,5)P3, indicating 1-phosphatase activity. The 1-phosphatase appeared to be dependent on MgCl2, whereas the 3-phosphatase was still active in the absence of MgCl2. An analogue of Ins(1,3,4,5)P4 with a thiophosphate substitution at the 1-position was found to be almost completely resistant to hydrolysis by the 1-phosphatase, but was degraded by the 3-phosphatase.
Identification of transient intermediates in the bisphosphatase reaction of rat liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase by 31P-NMR spectroscopy
Authors: Okar D A , Kakalis L T , Narula S S , Armitage I M , Pilkis S J
Published: August 2015

31P-NMR spectroscopy was used to identify reaction intermediates during catalytic turn-over of the fructose-2,6-bisphosphatase domain (Fru-2,6-P2ase) of the bifunctional enzyme, 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase. When fructose-2,6-bisphosphate (Fru-2,6-P2) was added to the enzyme, the 31P-NMR spectrum showed three resonances in addition to those of free substrate: the phosphohistidine (His-P) intermediate, the C-6 phosphoryl group of fructose-6-phosphate bound to the phosphoenzyme, and phosphate generated by the hydrolysis of substrate. Direct analysis of the alkali-denatured phospho-enzyme intermediate by 1H-31P heteronuclear multiple quantum-filtered coherence spectroscopy confirmed the formation of 3-N-phosphohistidine. Binding of fructose 6-phosphate to the bisphosphatase was detected by a down-field shift and broadening of the C-6 phosphoryl resonance. The down-field shift was greater in the presence of the phosphoenzyme intermediate. Inhibition of Fru-2,6-P2 hydrolysis by fructose 6-phosphate and Fru-2,6-P2 was shown to involve binding of the sugar phosphates to the phosphoenzyme. This study provides new experimental evidence in support of the reaction mechanism of Fru-2,6-P2ase and suggests that the steady-state His-P intermediate exists primarily in the E-P.fructose 6-phosphate complex. These results lay a solid foundation for the use of 31P-NMR magnetization transfer studies to provide an in-depth analysis of the bisphosphatase reaction mechanism.