bluuebunny/crossref_metadata_2025 huggingface dataset:
Analysis of the structural features of the C-terminus of GLUT1 that are required for transport catalytic activity
Authors: Muraoka A , Hashiramoto M , Clark A E , Edwards L C , Sakura H , Kadowaki T , Holman G D , Kasuga M
Published: August 2015

C-terminally truncated and mutated forms of GLUT1 have been constructed to determine the minimum structure at the C-terminus required for glucose transport activity and ligand binding at the outer and inner binding sites. Four truncated mutants have been constructed (CTD24 to CTD27) in which 24 to 27 amino acids are deleted. In addition, point substitutions of R468-->L, F467-->L and G466-->E have been produced. Chinese hamster ovary clones which were transfected with these mutant GLUT1s were shown, by Western blotting and cell-surface carbohydrate labelling, to have expression levels which were comparable with the wild-type clone. Wild-type levels of 2-deoxy-D-glucose transport activity were retained only in the clone transfected with the construct in which 24 amino acids were deleted (CTD24). The CTD25, CTD26 and CTD27 clones showed markedly reduced transport activity. From a kinetic comparison of the CTD24 and CTD26 clones it was found that the reduced transport was mainly associated with a reduced Vmax. value for 2-deoxy-D-glucose uptake but with a slight lowering of the Km. These data establish that the 24 amino acids at the C-terminus of GLUT1 are not required for the transport catalysis. However, the point mutations of F467L and G466E (26 and 27 residues from the C-terminus) did not significantly perturb the kinetics of 2-deoxy-D-glucose transport. The substitution of R468L produced a slight, but significant, lowering of the Km. The ability of the truncated GLUt1s to bind the exofacial ligand, 2-N-4-(1-zai-2,2,2-trifluoroethyl)benzoyl-1,3-bis-(D-mannos- 4-yl-oxy) -2-propylamine (ATB-BMPA), and the endofacial ligand, cytochalasin B, were assessed by photolabelling procedures. The ability to bind ATB-BMPA was retained only in the CTD24 truncated mutant and was reduced to levels comparable with those of the non-transfected clone in the other mutant clones. Cytochalasin B labelling was unimpaired in all four mutated GLUT1s. These data establish that a minimum structure at the C-terminus of GLUT1, which is required for the conformational change to expose the exofacial site, includes amino acids at positions Phe-467 and Arg-468; however, these amino acids are not individually essential.
Human diadenosine 5′,5′′′-P1,P4-tetraphosphate pyrophosphohydrolase is a member of the MutT family of nucleotide pyrophosphatases
Authors: Thorne N M H , Hankin S , Wilkinson M C , Nuñez C , Barraclough R , McLennan A G
Published: August 2015

The cDNA and derived amino acid sequence of human diadenosine 5′,5‴-P1,P4-tetraphosphate pyrophosphohydrolase have been determined with the aid of the GenBank Expressed Sequence Tag database. This enzyme possesses a modification of the MutT sequence motif found in certain nucleotide pyrophosphatases. It is unrelated to the enzymes of diadenosine tetraphosphate catabolism found in prokaryotes and fungi.
Factor IX Zutphen: a Cys18→Arg mutation results in formation of a heterodimer with α1-microglobulin and the inability to form a calcium-induced conformation
Authors: Wojcik E G C , van den Berg M , van der Linden I K , Poort S R , Cupers R , Bertina R M
Published: August 2015

Factor IX Zutphen is a variant factor IX molecule isolated from the blood of a patient with severe haemophilia B. The molecular defect in factor IX Zutphen is a Cys18-->Arg mutation as a result of a T-->C transition at residue 6427 of the factor IX gene of the patient. The mutation disrupts the disulphide bond in the Gla-domain between Cys18 and Cys23. The remaining free cysteine residue results in the formation of a 95 kDa complex with alpha 1-microglobulin through an intermolecular disulphide bond. The same complex circulates at high levels in plasma of carriers of the mutation. The variant molecule has a calcium-binding defect, which is shown not to be caused by incomplete gamma-carboxylation. Factor IX Zutphen can not bind to phospholipids and can not be activated by factor XIa or by factor VIIa-tissue factor complex. Two sequential metal ion-dependent conformational transitions (factor IX-->factor IX′-->factor IX*) have been proposed for human factor IX [Liebman (1987) J. Biol. Chem. 262, 7605-7612], based upon the metal ion requirements for binding to anti-factor IX:Mg(II) antibodies, which are specific for the factor IX′ conformation, and anti-factor IX:Ca(II) antibodies, which are specific for the factor IX* conformation. We used these conformation-specific antibodies, and antibodies raised against a synthetic peptide corresponding to residues 35-50 of human factor IX [anti-factor IX(35-50)] to study the metal ion-induced conformation of factor IX Zutphen. The disruption of the disulphide bond in the Gla-domain, maybe in combination with the complex with alpha 1-microglobulin, destabilized the factor IX′ conformation. The formation of the factor IX* conformation was prevented independent of the presence of alpha 1-microglobulin. The disulphide bond in the Gla-domain is therefore essential for the calcium-dependent conformation and function of factor IX.
Sequence and expression of the mRNA encoding HSP22, the mitochondrial small heat-shock protein in pea leaves
Authors: Lenne C , Block M A , Garin J , Douce R
Published: August 2015

A 3 h treatment at 40 degrees C of pea (Pisum sativum var. Douce Provence) plants induces production and accumulation of a small heat-shock protein of 22 kDa apparent molecular mass, designated HSP22, in the matrix compartment of mitochondria [Lenne and Douce (1994) Plant Physiol. 105, 1255-1261]. We show here that the HSP22 precursor (i.e. the mature protein plus the transit peptide) has an apparent molecular mass of 26 kDa after in vitro translation of mRNA extracted from heat-stressed pea plants and immunodetection. We have isolated, cloned and sequenced the full-length cDNA encoding the precursor of the mitochondrial HSP22. An analysis of the amino acid sequence of the mitochondrial HSP22 reveals that this protein is a representative member of the low-molecular-mass heat shock protein (HSP) superfamily, exhibiting the specific consensus regions that are typical of the small HSPs. Most importantly, comparison of the mitochondrial HSP22 sequence with that of chloroplast small HSPs indicates that HSP22 does not contain the typical chloroplast consensus region III. We have also analysed the kinetics of HSP22 induction, and report results on the temporal expression of HSP22 at the transcriptional level. HSP22 mRNA was detected as soon as 10 min after the temperature was raised to a high temperature of 40 degrees C. Then the amount of HSP22 mRNA declined considerably even though pea plants were still submitted to the heat treatment. These results are discussed in light of the translation data previously published [Lenne and Douce (1994) Plant Physiol. 105, 1255-1261], particularly concerning the physiological behaviour of mitochondria when plants are heat-stressed. Furthermore, we have studied the dependence of HSP22 accumulation with temperature and demonstrate that the pea mitochondrial heat-shock response is only developed under extreme environmental growth conditions.
Permissive effect of ceramide on growth factor-induced cell proliferation
Authors: Sasaki T , Hazeki K , Hazeki O , Ui M , Katada T
Published: August 2015

Addition of bacterial sphingomyelinase to quiescent Swiss 3T3 cells effectively potentiated the platelet-derived growth factor (PDGF)-stimulated cell proliferation, though the enzyme by itself had little effect on the cell proliferation. Such potentiation of the cell growth could also be observed by the addition of ceramide, a product of the sphingomyelinase-catalysed reaction. In contrast, phosphocholine, another product of the enzyme reaction, had no synergistic effect on the action of PDGF. Treatment of the cells with sphingomyelinase or ceramide increased the cellular activity of mitogen-activated protein kinases (MAP kinases), which have been implicated in the regulation of cell proliferation. However, the synergistic effect of sphingomyelinase on the PDGF-induced cell growth could still be observed even when the cellular MAP kinase activity was fully activated by the growth factor alone. These results indicate that a ceramide-mediated cellular event(s) other than the MAP kinase activation is potentially involved in the regulation of cell growth.
Flexibility of zonation of fatty acid oxidation in rat liver
Authors: Guzmán M , Bijleveld C , Geelen M J H
Published: August 2015

Periportal and perivenous hepatocytes were isolated from rats subjected to different treatments that induce (starvation, cold exposure) or depress (refeeding after starvation) hepatic fatty acid oxidation. These experiments were designed to determine factors that may be involved in creating and maintaining the asymmetrical distribution of this metabolic pathway in the acinus of the liver. The uneven distribution of mitochondrial [14C]-palmitate oxidation within the acinus (i) was very flexible and changed markedly with the physiological status of the animal (periportal/perivenous ratio: 1.5, 2.0, 1.0 and 0.4 for fed, starved, refed and cold-exposed animals respectively), (ii) coincided with a similar zonation of carnitine palmitoyltransferase I activity in fed as well as in cold-exposed animals, (iii) was paralleled by a comparable zonation of mitochondrial 3-hydroxy-3-methyl-glutaryl-CoA synthase activity in starved animals, and (iv) was not determined by zonal differences in any of the following parameters: sensitivity of carnitine palmitoyltransferase I to malonyl-CoA, intracellular concentration of malonyl-CoA, fatty acid synthesizing capacity, acetyl-CoA carboxylase activity, fatty acid synthase activity or relative content of the two hepatic acetyl-CoA carboxylase isoforms. Unlike mitochondrial oxidation, peroxisomal [14C]palmitate oxidation was always zonated towards the perivenous zone of the liver irrespective of the physiological status of the animal. The data presented show that changes in the acinar distribution of mitochondrial long-chain fatty acid oxidation involve specific long-term mechanisms under different physiological conditions.
Combination plots as graphical tools in the study of enzyme inhibition
Authors: Chan W W C
Published: August 2015

Although statistical regression has become the method of choice in the analysis of enzyme kinetics, graphical methods continue to be useful on account of their illustrative capabilities. It is pointed out in this paper that enzyme inhibition data may be presented more efficiently as a single linear plot than the traditional way as a family of lines. This approach has been taken previously by Hunter and Downs [Hunter and Downs (1945) J. Biol. Chem. 157, 427-446] but has remained neglected. A new version of this type of plot (combination plot) has been devised which is linear for competitive, non-competitive, uncompetitive and linear mixed inhibition and has a characteristic appearance for each type of inhibition behaviour. The slopes and intercepts not only indicate directly the dissociation constant but also provide quantitative criteria for the nature of inhibition. This plot should serve as a useful graphical tool in enzyme research as well as in biochemical education.
The Gly-54→Asp allelic form of human mannose-binding protein (MBP) fails to bind MBP-associated serine protease
Authors: Matsushita M , Ezekowitz R A B , Fujita T
Published: August 2015

The human mannose-binding protein (MBP) is a pattern recognition molecule that appears to play a role in initial host defence. MBP activates the complement cascade and it may act as an opsonin both in the absence and in the presence of complement. A number of distinct MBP allelic forms exist in different population groups. An allele that occurs in 5-7% of Caucasians was identified by an inability to activate the complement system. A homozygous mutation at base pair 230 of the MBP gene results in a Gly-to-Asp substitution at the fifth collagen repeat. It appears that the resultant protein, MBPD, is able to form high-order multimers that bind bacteria but do not support complement activation. Recently a novel serine protease, the MBP-associated serine protease (MASP), has been described. MBP-MASP complexes circulate in serum and result in the direct activation of a novel complement pathway (lectin pathway) in the absence of the first complement components. In this study we demonstrate that MASP and its proenzyme proMASP are unable to bind to recombinant (r)MBPD. This lack of a MASP-rMBPD association corresponds to a failure of the Gly-54-->Asp form of MBP to activate complement. Our results provide a biochemical basis for the functional deficit in the Gly-54-->Asp allelic form of MBP and suggest that the proMASP/MASP binding site maps to the fifth collagen repeat of MBP.
Biochemical and pharmacological studies with KT7692 and LY294002 on the role of phosphatidylinositol 3-kinase in FcεRI-mediated signal transduction
Authors: Yano H , Agatsuma T , Nakanishi S , Saitoh Y , Fukui Y , Nonomura Y , Matsuda Y
Published: August 2015

Wortmannin inhibited phosphatidylinositol 3-kinase (P13-kinase) and Fc epsilon RI-mediated histamine secretion in RBL-2H3 cells to a similar degree, with IC50 values of 3 and 2 nM, respectively. Although P13-kinase is an acknowledged regulator of intracellular trafficking and secretion, wortmannin has proved to be a difficult drug to use in assessing the role of P13-kinase because it inhibits another important enzyme, myosin light-chain kinase (MLCK; IC50 = 200 nM). In the present study we synthesized a unique derivative of wortmannin, O-acetyl-delta 16-wortmannin-17-ol (KT7692), that has an inhibitory potency against PI3-kinase one-hundredth that of wortmannin, but retains a similar potency to wortmannin against MLCK. Histamine secretion was influenced 100-fold more by wortmannin than by KT7692.2-(4-Morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002), a structurally different PI3-kinase inhibitor from wortmannin, inhibited PI3-kinase with an IC50 of 2 microM but had little effect on MLCK activity in this concentration range. LY294002 also inhibited histamine secretion in RBL-2H3 cells with an IC50 of 5 microM. These results provide further evidence that PI3-kinase is involved in the signal transduction pathway responsible for histamine secretion after stimulation of Fc epsilon RI. Furthermore KT7692 in combination with wortmannin and LY294002 would be a powerful tool for clarifying the involvement of PI3-kinase as distinct from that of MLCK in signal transduction systems of various cellular responses.
The effects of the site-directed removal of N-glycosylation sites from β-1,4-N-acetylgalactosaminyltransferase on its function
Authors: Haraguchi M , Yamashiro S , Furukawa K , Takamiya K , Shiku H , Furukawa K
Published: August 2015

The amino acid sequence deduced from the cloned human cDNA of beta-1,4-N-acetylgalactosaminyltransferase (GalNAc-T; EC 2.4.1.92) gene predicted three potential sites for N-linked glycosylation. Although many glycosyltransferases isolated contain from 2 to 6 N-glycosylation sites, their significance has not been adequately demonstrated. To clarify the roles of N-glycosylation in GalNAc-T function, we generated a series of mutant cDNAs, in which some or all of the glycosylation recognition sites were eliminated by polymerase chain reaction (PCR)-mediated site-directed mutagenesis. Using transcription/translation in vitro, we confirmed that all potential N-glycosylation sites could be used. Although cell lines transfected with mutant cDNAs showed equivalent levels of GalNAc beta 1-->4(NeuAc alpha 2-->3)Gal beta 1-->4Glc-Cer (GM2) to that of the wild-type, the extracts from mutant cDNA transfectants demonstrated lower enzyme activity than in the wild-type. The decrease in enzyme activity was more evident as the number of deglycosylated sites increased, with about 90% decrease in a totally deglycosylated mutant. The enzyme kinetics analysis revealed no significant change of Km among wild-type and mutant cDNA products. The intracellular localization of GalNAc-T expressed in transfectants with wild-type or mutant cDNAs also showed a similar perinuclear pattern (Golgi pattern). These results suggest that N-linked carbohydrates on GalNAc-T are required for regulating the stability of the enzyme structure.
Activation of mitogen-activated protein kinase by heat shock treatment in Drosophila
Authors: Chen F , Torres M , Duncan R F
Published: August 2015

Heat shock treatment of Drosophila melanogaster tissue culture cells causes increased tyrosine phosphorylation of several 44 kDa proteins, which are identified as Drosophila mitogen-activated protein (MAP) kinases. Tyrosine phosphorylation occurs within 5 min, and is maintained at high levels during heat shock. It decreases to basal levels during recovery, concurrent with the repression of heat shock transcription and heat-shock-protein synthesis. The increased MAP kinase tyrosine phosphorylation is parallelled by increased MAP kinase activity. At least two MAP kinases, DmERK-A and DmERK-B, are identified whose tyrosine phosphorylation increases during heat shock. Thus MAP kinase activation is an immediate early response to heat shock, and its increased activity is maintained throughout heat shock treatment. Protracted MAP kinase activation may contribute to heat shock transcription factor phosphorylation and the numerous metabolic alterations that constitute the heat-shock response.
Intracellular reactive oxygen species as apparent modulators of heat-shock protein 27 (hsp27) structural organization and phosphorylation in basal and tumour necrosis factor α-treated T47D human carcinoma cells
Authors: Mehlen P , Kretz-Remy C , Briolay J , Fostan P , Mirault M E , Arrigo A P
Published: August 2015

The small stress protein heat-shock protein 27 (hsp27) is an oligomeric phosphoprotein, constitutively expressed in most human cells, which enhances cellular resistance to tumour necrosis factor alpha (TNF alpha). This phenomenon correlates with dramatic changes in hsp27 cellular location, structural organization and phosphorylation. To gain a better understanding of the molecular mechanisms regulating these properties of hsp27, we investigated whether they were a consequence of the intracellular production of reactive oxygen species (ROS) generated by TNF alpha. Here, we report that, in T47D carcinoma cell lines, the rapid burst of intracellular ROS production and changes in hsp27 locale, structural organization and phosphoisoform composition induced by TNF alpha were abolished by the overexpression of the antioxidant enzyme seleno-glutathione peroxidase (GSHPx). These effects were greatly diminished when GSHPx-expressing cells were grown in the absence of selenium, a cofactor that is essential for seleno-GSHPx activity, indicating that they are directly linked to the increased GSHPx activity. Moreover, in growing T47D cells, GSHPx expression induced intracellular redistribution of hsp27 and decreased the phosphorylation of this protein without altering its pattern of oligomerization. In contrast, the heat-mediated phosphorylation of hsp27 was not altered by decreased intracellular ROS levels. Hence, in growing and TNF-treated cells, several hsp27 properties appear to be modulated by fluctuations in intracellular ROS levels.
The lux autoinducer-receptor interaction in Vibrio harveyi: binding parameters and structural requirements for the autoinducer
Authors: Cao J G , Wei Z Y , Meighen E A
Published: August 2015

To assess the binding parameters and the structure-function relationship of the Vibrio harveyi lux autoinducer, N-(D-3-hydroxybutanoyl)homoserine lactone (D-HBHL), to light emission, a series of acylhomoserine lactone analogues were synthesized and their effects on the stimulation of luminescence of an autoinducer-deficient mutant of V. harveyi, D1, examined. Of the analogues with 3-hydroxyacyl chains, only N-(3-hydroxyvaleryl)homoserine lactone (HVHL) could act as an inducer, with about 85% of the potency of D-HBHL in stimulation of luminescence; the apparent Kd of the putative receptor for HVHL was 3.8 microM, close to that for the natural autoinducer (1.4 microM). Analogues with longer 3-hydroxyacyl chains, N-(3-hydroxyhexanoyl)homoserine lactone and N-(3-hydroxyheptanoyl)homoserine lactone, acted as competitive inhibitors of HBHL with apparent KI values of 77 and 53 microM respectively. Replacement of the 3-hydroxybutanoyl moiety with a 3-methylbutanoyl or 3-methoxybutanoyl group created weak competitive inhibitors, N-(isovaleryl)- and N-(3-methoxybutanoyl)- homoserine lactones, with apparent KI values of 150 and 360 microM respectively. Two other analogues, N-(2-hydroxybutanoyl)- and N-(4-hydroxybutanoyl)-homoserine lactone, could neither stimulate nor inhibit luminescence. The approach used in these studies to demonstrate binding of autoinducer analogues at the same site, as well as measurement of the relative dissociation constant, may be of value in analysing analogues activating or inhibiting luminescence and other processes that are under control of acylhomoserine lactone autoregulators.
Glutathionylspermidine metabolism in Escherichia coli
Authors: Smith K , Borges A , Ariyanayagam M R , Fairlamb A H
Published: August 2015

Intracellular levels of glutathione and glutathionylspermidine conjugates have been measured throughout the growth phases of Escherichia coli. Glutathionylspermidine was present in mid-log-phase cells, and under stationary and anaerobic growth conditions accounted for 80% of the total glutathione content. N1,N8-bis(glutathionyl)spermidine (trypanothione) was undetectable under all growth conditions. The catalytic constant kcat/Km of recombinant E. coli glutathione reductase for glutathionylspermidine disulphide was approx. 11,000-fold lower than that for glutathione disulphide. The much higher catalytic constant for the mixed disulphide of glutathione and glutathionylspermidine (11% that of GSSG), suggests a possible explanation for the low turnover of trypanothione disulphide by E. coli glutathione reductase, given the apparent lack of a specific glutathionylspermidine disulphide reductase in E. coli.
Protease-susceptible sites and properties of fragments of aortic smooth-muscle myosin
Authors: King L , Jiang M J , Huang T S , Sheu G C
Published: August 2015

We have examined the protease susceptibility of aortic myosin, the thermal unfolding profiles of myosin rod and light meromyosin (LMM) and the solubility properties of the LMM fragments. Two major protease-susceptible sites were found, located at the head-rod junction and the heavy meromyosin (HMM)-LMM junction. Both tryptic and chymotryptic digestion of aortic myosin rod produced the LMM (80-85 kDa) and short subfragment 2 (S-2) (40-45 kDa) segments, which were similar to those of gizzard myosin rod and differed from the short LMM (70 kDa) and long S-2 (58 kDa) segments produced from skeletal-muscle rod. The thermal unfolding profile of aortic myosin rods exhibited three helix-unfolding transitions, at 47.5, 51 and 54 degrees C, similar to those of gizzard rods yet different from those of skeletal-muscle rods. There was a dramatic difference in the solubility of aortic LMM fragments of various molecular mass, as for gizzard smooth-muscle LMM and rabbit skeletal-muscle LMM. LMM fragments of molecular mass 77 kDa or more were completely insoluble in low-ionic-strength buffer, whereas LMM fragments of molecular mass 73 kDa or less were completely soluble in low-ionic-strength buffer. Proteolytic digestion patterns of LMM showed two additional protease-susceptible sites located 13 and 30 kDa from the ends of the LMM molecule. This suggests the existence of flexible regions within the LMM molecule, which may be responsible for the folded form of aortic myosin.
Purification, characterization and specificity of chondroitin lyases and glycuronidase from Flavobacterium heparinum
Authors: Gu K , Linhardt R J , Laliberté M , Gu K , Zimmermann J
Published: August 2015

The chondroitin lyases from Flavobacterium heparinum (Cytophaga heparinia) have been widely used in depolymerization of glycosaminoglycan and proteoglycan chondroitin sulphates. Oligosaccharide products derived from chondroitin sulphate can be further degraded by glycuronidases and sulphatases obtained from the same organism. There has been no reported purification of these enzymes to homogeneity nor is there any information on their physical and kinetic characteristics. The absence of pure enzymes has resulted in a lack of understanding of the optimal conditions for their catalytic activity and their substrate specificity. This has limited the use of these enzymes as reagents for preparation of oligosaccharides for structure and activity studies. Reproducible schemes to purify a chondroitin AC lyase, a glycuronidase and chondroitin B lyase from Flavobacterium heparinum to apparent homogeneity are described. Chondroitin AC lyase (chondroitinase AC, EC 4.2.2.5), glycuronidase [chondro-(1-->3)-glycuronidase, no EC number] and chondroitin B lyase (chondroitinase B, no EC number) have M(r) values (assessed by SDS/PAGE) of 74,000, 41,800 and 55,200 respectively, and isoelectric points (determined by isoelectric focusing) of 8.85, 9.28 and 9.05 respectively. Chondroitin lyase AC and B contain pyroglutamic acid at their N-termini precluding their analysis by Edman degradation. Deblocking with pyroglutamate aminopeptidase facilitated the determination of their N-terminal sequences. The kinetic properties of these enzymes have been determined as well as the optimum conditions for their catalytic activity. The specificity of the glycouronidase, determined using 17 different disaccharide substrates, shows that it only acts on unsulphated or 6-O-sulphated 1-->3 linkages. The chondroitin lyases are both endolytic enzymes, and oligosaccharide mapping shows their expected specificity towards the chondroitin and dermatan sulphate polymers.
Location of smooth-muscle myosin and tropomyosin binding sites in the C-terminal 288 residues of human caldesmon
Authors: Huber P A J , Fraser I D C , Marston S B
Published: August 2015

We have produced nine recombinant fragments, H1 to H9, from a human cDNA that codes for the C-terminal 288 residues of caldesmon. The fragment H1, encompassing the 288 residues, is equivalent to domains 3 and 4 of caldesmon (amino acids 506-793 in human, 476-737 in the chicken gizzard sequence). It has been shown [Huber, Redwood, Avent, Tanner and Marston (1993) J. Muscle Res. Cell Motil. 14, 385-391] to bind to actin, Ca(2+)-calmodulin, tropomyosin and myosin. The fragments, H2 to H9, differ in length between 60 and 176 residues and cover the whole of domains 3 and 4 with many of the fragments overlapping. We have characterized the myosin and tropomyosin binding of these fragments. The binding of both tropomyosin and myosin is highly dependent on salt concentration, indicating the ionic nature of these interactions. The location of the myosin binding is an extended region encompassing the junction of domains 3/4 and domain 4a (residues 622-714, human; 566-657, chicken gizzard). Tropomyosin binds in a smaller region within domain 4a of caldesmon (residues 663-714, human; 606-657 chicken gizzard). We confirmed predictions based on sequence similarities of a tropomyosin binding site in domain 3 of caldesmon; however, this site bound to skeletal-muscle tropomyosin and had little affinity for the smooth-muscle tropomyosin isoform. None of the protein fragments H2-H9 retained the affinity of the parent fragment H1 for either myosin or tropomyosin. This indicates the need for several interaction sites scattered over an extended region to attain higher affinity. The regions interacting with caldesmon in both tropomyosin and myosin are coiled-coil structures. This is probably the reason for their shared interaction sites on caldesmon and their similar natures of binding.
Identification of polymorphism at the glutathione S-transferase, GSTM3 locus: evidence for linkage with GSTM1*A
Authors: Inskip A , Elexperu-Camiruaga J , Buxton N , Dias P S , MacIntosh J , Campbell D , Jones P W , Yengi L , Talbot J A , Strange R C , Fryer A A
Published: August 2015

Allelism in the glutathione S-transferase, GSTM3 gene has been identified using PCR with specific primers to exon 6/exon 7. Sequencing showed the mutant GSTM3*B allele to have a three-base deletion in intron 6 with a frequency of 0.158. The mutation generates a recognition sequence, 5′-AAGATA-3′, for the negative transcription factor YY1. GSTM3*B was significantly associated with GSTM1*A.
Analysis of the mitochondrial ATP synthase β-subunit gene in Drosophilidae: structure, transcriptional regulatory features and developmental pattern of expression in Drosophila melanogaster
Authors: Peña P , Ugalde C , Calleja M , Garesse R
Published: August 2015

We have cloned and determined the structure of the gene encoding the H(+)-ATP synthase beta subunit in two distantly related Drosophila species, D. melanogaster and D. virilis. The gene contains three exons that are extremely well conserved at the amino acid level, not only in the region encoding the mature protein but also in that encoding the leader peptide. Primer extension analysis indicates that the 5′ untranslated region is extremely short, and reveals the presence of multiple initiation sites of transcription in both Drosophila species. The promoters of D. melanogaster and D. virilis H(+)-ATP synthase beta-subunit genes contain a conserved region surrounding the initiation transcription sites. Nucleotide sequence analysis has revealed the absence of canonical TATA and CCAAT boxes and the presence of several putative regulatory elements in both promoter regions, including GAGA, GATA and Ets binding sites. We have analysed the pattern of gene expression during D. melanogaster development. The mRNA is stored in oocytes, and activation of transcription takes place after 10 h of development. The expression of the nuclear-encoded H(+)-ATP synthase beta subunit is strictly coordinated with the expression of subunits 6 and 8 of the same complex that are encoded in the mitochondrial genome.
Direct activation and anti-repression functions of GAL4-VP16 use distinct molecular mechanisms
Authors: Lyons J G , Chambon P
Published: August 2015

In order to determine whether the molecular mechanisms used for direct activation by GAL4-VP16 are the same as those used for anti-repression, we have employed monoclonal antibodies specific for the VP16 activation domain. In the absence of added repressors, GAL4-VP16 was able to stimulate transcription from a template containing GAL4-binding sites, and the antibodies raised against the VP16 activation domain failed to inhibit this direct activation. GAL4-VP16 also was able to prevent histone H1-mediated repression by a mechanism that was strongly dependent on the presence of specific GAL4-binding elements in the promoter. However, in contrast to the assays conducted in the absence of repressors, the antibodies were strong inhibitors of GAL4-VP16-activated transcription in the presence of histone H1. Thus the binding of the antibodies distinguished between the direct activation and anti-repression functions of GAL4-VP16, indicating that these functions operate through distinct molecular mechanisms. The anti-repression-specific mechanism that is inhibitable by the antibodies acted at an early stage of preinitiation complex formation. Deletions of individual subdomains of the VP16 activation domain demonstrated that there was not a discrete subdomain responsible for the anti-repression function of GAL4-VP16. Thus, the inhibitory effect of the antibodies appeared to be due to the location of the epitope within the activator protein rather than to some inherent biochemical property of that region of the protein that is required specifically for anti-repression. The inhibitory effect of the antibodies also ruled out the possibility that steric exclusion of repressor proteins from the promoter was the sole means of anti-repression by the transcriptional activator.
Investigation of the active site of oligosaccharyltransferase from pig liver using synthetic tripeptides as tools
Authors: Bause E , Breuer W , Peters S
Published: August 2015

Oligosaccharyltransferase (OST), an integral component of the endoplasmic-reticulum membrane, catalyses the transfer of dolichyl diphosphate-linked oligosaccharides to specific asparagine residues forming part of the Asn-Xaa-Thr/Ser sequence. We have studied the binding and catalytic properties of the enzyme from pig liver using peptide analogues derived from the acceptor peptide N-benzoyl-Asn-Gly-Thr-NHCH3 by replacing either asparagine or threonine with amino acids differing in size, stereochemistry, polarity and ionic properties. Acceptor studies showed that analogues of asparagine and threonine with bulkier side chains impaired recognition by OST. Reduction of the beta-amide carbonyl group of asparagine yielded a derivative that, although not glycosylated, was strongly inhibitory (50% inhibition at approximately 140 microM). This inhibition may be due to ion-pair formation involving the NH3+ group and a negatively charged base at the active site. Hydroxylation of asparagine at the beta-C position increased Km and decreased Vmax, indicating an effect on both binding and catalysis. The threo configuration at the beta-C atom of the hydroxyamino acid was essential for substrate binding. A peptide derivative obtained by replacement of the threonine beta-hydroxy group with an NH2 group was found to display acceptor activity. This shows that the primary amine is able to mimic the hydroxy group during transglycosylation. The pH optimum with this derivative is shifted by approximately 1 pH unit towards the basic region, indicating that the neutral NH2 group is the reactive species. The various data are discussed in terms of the catalytic mechanism of OST, particular emphasis being placed on the role of threonine/serine in increasing the nucleophilicity of the beta-amide of asparagine through hydrogen-binding.
Transcriptional activation of the chicken lysozyme gene by NF-κ Bp65 (RelA) and c-Rel, but not by NF-κ Bp50
Authors: VAN PHI Loc
Published: August 2015

The lysozyme gene is expressed at a low level in myeloblasts and is progressively activated to constitutively high expression in mature macrophages. The binding activity of the newly defined NF-ĸB/Rel family of transcription factors increases during the terminal differentiation of macrophages. In this study, I show that NF-ĸB/Rel-like proteins bind to the nuclear factor kappa B (ĸB)-like sequence of the lysozyme promoter. These binding activities were induced by treatment of HD11 cells with lipopolysaccharide. Immunomobility shift assays show that c-Rel is possibly a factor in the complexes that bind to the ĸB-like sequence lysĸB. Binding activity to one of the protein complexes seems to be regulated by phosphorylation. In fact, overexpression of p65 and c-Rel stimulates expression of the chloramphenicol acetyltransferase gene controlled by the lysozyme promoter. Furthermore, co-transfection experiments reveal that the ĸB-like sequence within the lysozyme promoter mediates the transactivation by p65 and c-Rel. These results indicate that the p65 and c-Rel could be components of the protein complexes that bind to the ĸB-like sequence and this binding could contribute to the progressively activated expression of the lysozyme gene during the terminal differentiation of macrophages.
The low-density-lipoprotein receptor-related protein (LRP) is processed by furin in vivo and in vitro
Authors: WILLNOW Thomas E. , MOEHRING Joan M. , INOCENCIO Noel M. , MOEHRING Thomas J. , HERZ Joachim
Published: August 2015

The low-density-lipoprotein receptor-related protein (LRP) is a multifunctional receptor involved in the clearance of a large number of diverse ligands, including proteases, protease-inhibitor complexes and lipoproteins. The mature receptor is composed of a 515 kDa and a 85 kDa subunit generated by proteolytic cleavage from a 600 kDa precursor polypeptide in a trans-Golgi compartment. Proteolytic processing occurs C-terminal to the tetrabasic amino acid sequence RHRR, a consensus recognition site for precursor processing endoproteases or convertases. In this study we have identified furin, a subtilisin-type protease, to be necessary for efficient processing of LRP in cells. Furin-deficient RPE.40 cells exhibited an impaired processing of endogenous LRP and of a recombinant soluble form of the receptor containing the processing site. The processing defect in RPE.40 cells could be complemented by expression of furin from a transfected cDNA in cultured cells and by purified furin in vitro. The impaired maturation of LRP in RPE.40 cells did not affect its intracellular transport, and correlated with a slight but consistent reduction in the endocytosis of LRP-specific ligands. These data suggest that proteolytic processing of LRP by furin is not necessary for intracellular trafficking but might be required for normal receptor activity.
A single gene encodes two different transcripts for the ADP-glucose pyrophosphorylase small subunit from barley (Hordeum vulgare)
Authors: THORBJØRNSEN Tine , VILLAND Per , KLECZKOWSKI Leszek A. , OLSEN Odd-Arne
Published: August 2015

ADP-glucose pyrophosphorylase (AGPase), a heterotetrameric enzyme composed of two small and two large subunits, catalyses the first committed step of starch synthesis in plant tissues. In an attempt to learn more about the organization and expression of the small-subunit gene of AGPase, we have studied the small-subunit transcripts as well as the structure of the gene encoding these transcripts in barley (Hordeum vulgare L. cv. Bomi). Two different transcripts (bepsF1 and blps14) were identified: bepsF1 was abundantly expressed in the starchy endosperm but not in leaves, whereas blps14 was isolated from leaves but was also found to be present at a moderate level in the starchy endosperm. The sequences for the two transcripts are identical over approx. 90% of the length, with differences being confined solely to their 5ʹ ends. In blps14, the unique 5ʹ end is 259 nt long and encodes a putative plastid transit peptide sequence. For the 178-nt 5ʹ end of bepsF1, on the other hand, no transit peptide sequence could be recognized. A lambda clone that hybridized to the AGPase transcripts was isolated from a barley genomic library and characterized. The restriction map has suggested a complex organization of the gene, with alternative exons encoding the different 5ʹ ends of the two transcripts followed by nine exons coding for the common part of the transcripts. The sequence of a portion of the genomic clone, covering the alternative 5ʹ-end exons as well as upstream regions, has verified that both transcripts are encoded by the gene. The results suggest that the small-subunit gene of barley AGPase transcribes two different mRNAs by a mechanism classified as alternative splicing.
Glycine decarboxylase and pyruvate dehydrogenase complexes share the same dihydrolipoamide dehydrogenase in pea leaf mitochondria: evidence from mass spectrometry and primary-structure analysis
Authors: BOURGUIGNON Jacques , MERAND Véronique , RAWSTHORNE Stephen , FOREST Eric , DOUCE Roland
Published: August 2015

In order to compare the dihydrolipoamide dehydrogenase associated with the pyruvate dehydrogenase complex (E3) with that associated with the glycine decarboxylase complex (L-protein), we report for the first time the purification and characterization of the E3 component from pea leaf mitochondria. The first 30 amino acids of the N-terminal sequence of the mature E3 protein are identical with those of the mature L-protein of the glycine decarboxyase complex. Electrospray ionization-mass spectrometric analysis of E3 and the L-protein gave exactly the same molecular mass of 49753±5 Da. We have also confirmed the primary structure of the L-protein, in particular the C-terminal sequence, deduced from the cDNA published by Bourguignon, Macherel, Neuburger and Douce [(1992) Eur. J. Biochem. 204, 865-873]. Western-blot analysis shows that specific polyclonal antibodies raised against the L-protein recognize specifically both E3 and L-protein but not the porcine dihydrolipoamide dehydrogenase. We conclude that, in pea leaf mitochondria, the pyruvate dehydrogenase and glycine decarboxylase complexes share the same dihydrolipoamide dehydrogenase. We have also confirmed by MS analysis that the FAD is not covalently bound to the enzyme.
PEST motifs are not required for rapid calpain-mediated proteolysis of c-fos protein
Authors: CARILLO Serge , PARIAT Magali , STEFF Ann-Muriel , JARIEL-ENCONTRE Isabelle , POULAT Francis , BERTA Philippe , PIECHACZYK Marc
Published: August 2015

Cytoplasmic degradation of c-fos protein is extremely rapid. Under certain conditions, it is a multi-step process initiated by calcium-dependent and ATP-independent proteases called calpains. PEST motifs are peptide regions rich in proline, glutamic acid/aspartic acid and serine/threonine residues, commonly assumed to constitute built-in signals for rapid recognition by intracellular proteases and particularly by calpains. Using a cell-free degradation assay and site-directed mutagenesis, we report here that the three PEST motifs of c-fos are not required for rapid cleavage by calpains. Testing the susceptibility of PEST motif-bearing and non-bearing transcription factors including GATA1, GATA3, Myo D, c-erbA, Tal-1 and Sry, demonstrates that PEST sequences are neither necessary nor sufficient for specifying degradation of other proteins by calpains. This conclusion is strengthened by the observation that certain proteins, reportedly known to be cleavable by calpains, are devoid of PEST motifs
The effect of intracellular pH on long-chain fatty acid uptake in 3T3-L1 adipocytes: evidence that uptake involves the passive diffusion of protonated long-chain fatty acids across the plasma membrane
Authors: TRIGATTI Bernardo L. , GERBER Gerhard E.
Published: August 2015

To understand the mechanism of long-chain fatty acid permeation of the plasma membrane in mammalian cells, the effects of changes in the cytoplasmic pH on the internalization of physiologically relevant, submicromolar concentrations of uncomplexed long-chain fatty acids were investigated in 3T3-L1 adipocytes. The acidification of the cytoplasm upon NH4Cl prepulsing of intact cells was accompanied by a rapid reduction of cellular long-chain fatty acid uptake (measured as the total accumulation of [9,10-3H]oleate). This was followed by a slow recovery to normal levels of uptake as the cytoplasmic pH recovered. Conventional filtration assays do not distinguish between fatty acid movement across the plasma membrane and intracellular steps, such as binding to cytoplasmic fatty acid-binding proteins or metabolism. While the in vitro binding of a photoreactive fatty acid, 11-m-diazirinophenoxy[11-3H]undecanoate, to a cytoplasmic fatty acid-binding protein was insensitive to changes in pH from pH 7.5 to 5.5, the in vitro conversion of oleate into oleoyl-CoA by cellular acyl-CoA synthetase decreased dramatically. Therefore, the labelling of the 15 kDa cytoplasmic fatty acid-binding protein in intact cells by the photoreactive fatty acid was used as a more direct measure of the permeation of the probe across the plasma membrane. Acidification of the cytoplasm resulted in an immediate reduction in the labelling of this protein in intact adipocytes. Its photolabelling recovered, however, upon the recovery of the cytoplasmic pH to normal levels. This was due to effects of the cytoplasmic pH on the permeation of the photoreactive fatty acid across the plasma membrane rather than its binding to the 15 kDa protein or metabolism in vivo. This is the first demonstration that the movement of physiologically relevant, submicromolar concentrations of uncomplexed long-chain fatty acids across the plasma membrane of intact cells is coupled to the cytoplasmic pH and suggests that it occurs by the diffusion of the protonated long-chain fatty acid through the lipid bilayer.
Structural organization of the rat branched-chain 2-oxo-acid dehydrogenase kinase gene and partial characterization of the promoter-regulatory region
Authors: HUANG Yi-shuian , CHUANG David T.
Published: August 2015

The gene encoding the rat branched-chain 2-oxo-acid dehydrogenase kinase (EC 2.7.1.115) has been isolated and partially characterized. The entire gene, including the promoter-regulatory region, spans 6 kb and contains 11 exons. The 5ʹ-untranslated region comprising 264 bp is interrupted by intron 1 which is 581 bp in size. The complete in-frame sequence of intron 7 encodes the 49 amino acid insert previously reported to be present in the larger isoform of the rat kinase (Harris, Popov, Shimomura, Zhao, Jaskiewicz, Nanaumi and Suzuki (1992) Adv. Enzyme Regul. 32, 267-284). Sequencing of the 679 bp of the 5ʹ-flanking region showed the absence of a canonical TATA box, similar to other branched-chain 2-oxo-acid dehydrogenase-complex genes. Several candidate cis-acting elements are present. These include CAAT boxes, Sp-1-binding sites, GCN-4 sites, CCAAT enhancer binding-protein sites (C/EBP) and glucocorticoid-responsive element (GRE) sites. Also present are a pair of direct repeats of unknown function. The luciferase-reporter assay showed that promoter activity is markedly higher in normal rat kidney (NRK-52E) cells than in rat hepatoma (FTO-2B) cells, and that the 5ʹ-flanking region between bases -449 and +264 is both necessary and sufficient for basal transcription of the kinase gene.
Ability of methotrexate to inhibit translocation to the cytosol of dihydrofolate reductase fused to diphtheria toxin
Authors: KLINGENBERG Olav , OLSNES Sjur
Published: August 2015

A fusion protein consisting of dihydrofolate reductase and diphtheria toxin A-fragment was made by genetically linking cDNA for the two proteins followed by in vitro transcription and translation in a rabbit reticulocyte lysate system. The dihydrofolate reductase in the fusion protein exhibited enzyme activity and, in the presence of methotrexate which imposes a tight structure on dihydrofolate reductase, it was trypsin resistant, indicating that it was correctly folded. When reconstituted with diphtheria toxin B-fragment, it bound specifically to diphtheria toxin receptors and was translocated into cells upon exposure to low pH. Methotrexate prevented the translocation. Protein synthesis was inhibited in cells incubated with the reconstituted fusion protein, but the inhibition was reduced in the presence of methotrexate. We also made a fusion protein containing a mutated dihydrofolate reductase with much lower affinity to methotrexate. Methotrexate did not prevent translocation of this protein. The data indicate that methotrexate prevents translocation of the fusion protein containing wild-type dihydrofolate reductase by imposing a tight structure on to the enzyme.
Effects of lipids on nucleotide inhibition of wheat-germ aspartate transcarbamoylase: evidence of an additional level of control?
Authors: KHAN Ashan , CHOWDHRY Babur Z. , YON Robert J.
Published: August 2015

Wheat-germ aspartate transcarbamoylase, a monofunctional trimer, is strongly inhibited by uridine 5ʹ-monophosphate (UMP), which shows kinetic interactions with the substrate, carbamoyl phosphate, suggesting a classical allosteric mechanism of regulation. Inhibition of the purified enzyme by UMP was amplified in the presence of a variety of ionic lipids at concentrations low enough to preclude denaturation. In the absence of UMP, most of these compounds had no kinetic effect or were slightly activating. Two phospholipids did not show the effect. In a homologous series of fatty acids (C6-C16), the potentiating effect was only seen with homologues greater than C8, reaching a maximum at C12. The effect of dodecanoate (C12) on kinetic cooperativity (UMP as variable ligand) was studied. At each of several fixed concentrations of carbamoyl phosphate, dodecanoate had a pronounced effect on the half-saturating concentration of UMP, which was reduced by about half in every case, indicating substantially tighter binding of UMP. However, dodecanoate had relatively little effect on the kinetic Hill coefficient for the cooperativity of UMP. The possible metabolic significance of these effects is discussed.