bluuebunny/crossref_metadata_2025 huggingface dataset:
Structural organization and chromosomal localization of the mouse collagenase type I gene
Authors: Schorpp M , Mattei M G , Herr I , Gack S , Schaper J , Angel P
Published: August 2015

A clone containing genomic sequences of part of the murine collagenase type 1 (MMP-1) gene was isolated. It contains exons 1-6 encoding all the domains required for collagenase function and 9 kb of 5′-flanking sequences. The gene organization and exon/intron borders are highly similar to the already described human and rabbit MMP-1 genes. However, neither the intron sequences, nor the promoter region up to position -660 exhibit significant sequence homologies with rabbit and human MMP-1, except for an AP-1-binding site and two PEA-3 consensus sequences. Binding studies in vitro revealed that the AP-1-binding site is recognized by Fos/Jun heterodimers with very high affinity. By in situ hybridization the mouse MMP-1 gene was located to the A1-A2 region of chromosome 9 in proximity to the curly whiskers (cw) locus. Based on the lack of sequence homologies of the promoter and intron regions, and since the chromosomal localization of the mouse and human MMP-1 genes may not be syntenic, these data strongly support previous suggestions that the MMP-1 genes from mouse, compared with rabbit and human, have evolved from different ancestral genes. The presence of the AP-1- and PEA-3- binding sites in all mammalian MMP-1 genes isolated so far, may, however, suggest evolutionary selection for common regulatory mechanisms of MMP-1 transcription.
Photoaffinity labelling of cyanomethaemoglobin with derivatives of tryptophan and 5-bromotryptophan
Authors: Li M , Lin Z , Johnson M E
Published: August 2015

Tryptophan and 5-bromotryptophan (5-BrTrp) are relatively potent inhibitors of sickle-haemoglobin polymerization. The binding sites of these compounds to normal and sickle haemoglobin (HBA and HBS) have been suggested, but not firmly established, through the use of spin-labelled derivatives and/or computer modeling. In the present study we approached the problem by utilizing the technique of photoaffinity labelling. The cyanomet forms of HBA and HBS were subjected to photoaffinity labelling with N alpha-(4-azidotetrafluorobenzoyl)tryptophan and N alpha-(1-ethyl-2-diazomalonyl)-5-bromotryptophan respectively. Both irradiated samples of HBA and HBS were denatured, digested with trypsin, and then separated by reversed-phase HPLC. A labelled tryptic peptide was isolated from the photolabelling of HBS with N alpha-(1-ethyl-2-diazomalonyl)-5-bromotryptophan. The peptide was identified to be Val1(alpha)-Lys7(alpha), with the label attached to Val1(alpha), by virtue of amino acid analysis and sequencing, in conjunction with fast-atom-bombardment MS. The binding mode of N alpha-(1-ethyl-2-diazomalonyl)-5-bromotryptophan is proposed and its relevance to the potency of the 5-BrTrp-based anti-sickling agents is discussed.
Detection and analysis of agonist-induced formation of the complex of the stimulatory guanine nucleotide-binding protein with adenylate cyclase in intact wild-type and β2-adrenoceptor-expressing NG108-15 cells
Authors: Kim G D , Carr I C , Milligan G
Published: August 2015

Neuroblastoma x glioma hybrid, NG108-15, cells appear to express the alpha-subunit of the guanine nucleotide-binding protein Gs in a substantial molar excess over its effector adenylate cyclase [Kim, Adie and Milligan (1994) Eur. J. Biochem. 219, 135-143]. Addition of the IP prostanoid receptor agonist iloprost to intact NG108-15 cells resulted in a dose-dependent increase in formation of the complex between Gs alpha and adenylate cyclase (GSAC) as measured by specific high-affinity binding of [3H]forskolin. NG108-15 cells transfected to express either relatively high (clone beta N22) or low (clone beta N17) levels of beta 2-adrenoceptor both showed dose-dependent increases in specific [3H]forskolin binding in response to the beta-adrenoceptor agonist isoprenaline, and maximally effective concentrations of isoprenaline resulted in the generation of similar numbers of GSAC complexes in both clones. The dose-effect curve for clone beta N22, however, was some 15-fold to the left of that for clone beta N17, which is similar to that noted for isoprenaline-mediated stimulation of adenylate cyclase activity [Adie and Milligan (1994) Biochem. J. 303, 803-808]. In contrast, dose-effect curves for iloprost stimulation of [3H]forskolin binding were not different in clones beta N22 and beta N17. Basal specific [3H]forskolin binding in the absence of agonist was significantly greater in cells of clone beta N22 than clone beta N17. This was not a reflection of higher immunological levels of adenylate cyclase, indicating that the higher basal formation of GSAC probably reflects empty-receptor activation of Gs. This higher basal specific [3H]forskolin binding was partially reversed by propranolol. The addition of the opioid peptide D-Ala-D-Leu-enkephalin to NG108-15 cells did not reduce iloprost-stimulated [3H]forskolin binding even though this peptide inhibits stimulated adenylate cyclase activity by activation of a delta opioid receptor.
Expression of human all-trans-retinoic acid receptor β and its ligand-binding domain in Escherichia coli
Authors: Berggren Söderlund M , Johannesson G , Fex G
Published: August 2015

all-trans-Retinoic acid, one of the hormonally active derivatives of vitamin A, occurs physiologically in plasma at a concentration below 10 nmol/l. The methods currently used for its quantification are based on HPLC, need about 1 ml of serum, are relatively laborious and thus not well suited for mass analysis. The affinity and specificity of retinoic acid receptors for all-trans-retinoic acid encouraged us to express both the entire human retinoic acid receptor beta (RAR-beta) and two versions of its retinoic acid-binding domain in Escherichia coli in the hope that these recombinant proteins might be used as binders in a ligand-binding assay for all-trans-retinoic acid. The recombinant receptors, the whole receptor [RAR-beta-(V7-Q448)], corresponding to domains A-F, and the ligand-binding domain [RAR-beta-(E149-Q448)], corresponding to domains D-F, were expressed in the vector pET 3d/BL21 (DE3) as inclusion bodies, solubilized with guanidinium chloride, renatured and purified by ion-exchange chromatography. RAR-beta-(P193-Q448), corresponding to domains E-F, was expressed in the vector pET 3d/BL21(DE3)pLysS, and purified by reversed-phase chromatography. Under non-denaturing conditions, the expressed whole receptor [RAR-beta-(V7-Q448)] and the D-F construct (RAR-beta-(E149-Q448)] behaved chromatographically as monomeric proteins whereas the E-F construct [RAR-beta-(P193-Q448)] had a strong tendency to aggregate. RAR-beta-(V7-Q448) and RAR-beta-(E149-Q448) had similar Kd values for all-trans-retinoic acid (1.4 and 0.6 nmol/l respectively) whereas RAR-beta-(P193-Q448) bound all-trans-retinoic acid less avidly (Kd 9.6 nmol/l). 9-cis-Retinoic acid bound to RAR-beta-(E149-Q448) and RAR-beta-(V7-Q448) as avidly as all-trans-retinoic acid. Competition experiments showed weak or no binding of 4-oxo-all-trans-retinoic acid, 4-oxo-13-cis-retinoic acid, 13-cis-retinoic acid, acitretin and retinol by RAR-beta-(E149-Q448).
Mouse aggrecan, a large cartilage proteoglycan: protein sequence, gene structure and promoter sequence
Authors: Watanabe H , Gao L , Sugiyama S , Doege K , Kimata K , Yamada Y
Published: August 2015

Seven genomic clones for mouse aggrecan core protein have been isolated including 3 kb of 5′- and 7 kb of 3′-flanking sequences. All exon sequences and their intron boundary sequences in these clones were identified and mapped by DNA sequencing. The gene spans at least 61 kb and contains 18 exons. Exon 1 encodes 5′-untranslated sequence and exon 2 contains a translation start codon, methionine. The coding sequence is 6545 bp for a 2132-amino-acid protein with calculated M(r) = 259,131 including an 18-amino-acid signal peptide. There is a strong correlation between structural domains and exons. Notably, the chondroitin sulphate domain consisting of 1161 amino acids is encoded by a single exon of 3.6 kb. Although link protein has similar structural domains and subdomains, the sequence identity and the organization of exons encoding the subdomains B and B′ of G1 and G2 domains revealed a strong similarity of mouse aggrecan to both human versican and rat neurocan. Primer extension analysis identified four transcription start sites which are close together. The promoter sequence showed high G/C content (65%) and contained several consensus binding motifs for transcription factors including Sp-1 and the glucocorticoid receptor. There are stretches of sequences similar to the promoter region of both the type-II collagen and link protein genes. These sequences may be important for cartilage gene expression.
DNase I hypersensitivity sites and nuclear protein binding on the fatty acid synthase gene: identification of an element with properties similar to known glucose-responsive elements
Authors: Foufelle F , Lepetit N , Bosc D , Delzenne N , Morin J , Raymondjean M , Ferré P
Published: August 2015

We have shown previously that fatty acid synthase (FAS) gene expression is positively regulated by glucose in rat adipose tissue and liver. In the present study, we have identified in the first intron of the gene a sequence closely related to known glucose-responsive elements such as in the L-pyruvate kinase and S14 genes, including a putative upstream stimulatory factor/major late transcription factor (USF/MLTF) binding site (E-box) (+ 292 nt to + 297 nt). Location of this sequence corresponds to a site of hypersensitivity to DNase I which is present in the liver but not in the spleen. Moreover, using this information from a preliminary report of the present work, others have shown that a + 283 nt to + 303 nt sequence of the FAS gene can confer glucose responsiveness to a heterologous promoter. The protein binding to this region has been investigated in vitro by a combination of DNase I footprinting and gel-retardation experiments with synthetic oligonucleotides and known nuclear proteins. DNase I footprinting experiments using a + 161 nt to + 405 nt fragment of the FAS gene demonstrate that a region from + 290 nt to + 316 nt is protected by nuclear extracts from liver and spleen. This region binds two ubiquitous nuclear factors, USF/MLTF and the CAAT-binding transcription factor/nuclear factor 1 (CTF/NF1). Binding of these factors is similar in nuclear extracts from liver which does or does not express the FAS gene as observed for glucose-responsive elements in the L-pyruvate kinase and S14 genes. This suggests a posttranslational modification of a factor of the complex after glucose stimulation.
Endotoxin-induced inflammation does not cause hepatic zinc accumulation in mice lacking metallothionein gene expression
Authors: Philcox J C , Coyle P , Michalska A , Choo K H A , Rofe A M
Published: August 2015

The action of endotoxin lipopolysaccharide (LPS) on hepatic Zn uptake was examined in mice lacking expression of metallothionein (MT)-1 and MT-II genes. Hepatic Zn concentrations, which in normal control mice increased by a mean 29% (MT elevated 20-fold) 16 h post-LPS exposure, did not increase in MT-null mice. Plasma Zn fell by 68% in controls and 32% in MT-null mice. The time course of LPS action in normal mice was characterized by a rapid reduction (-74% at 4 h, -81% at 8 h) and partial recovery (-39% at 24 h) in plasma Zn, with a progressive increase over 24 h in hepatic concentrations of MT (by 36-fold) and Zn (by 40%). In contrast, the MT-null mice had a linear decrease in plasma Zn (-15% at 8 h, -41% at 24 h) and early loss of Zn from the liver. The Zn changes seen in MT-null mice were largely attributable to LPS-associated anorexia. Food deprivation (20 h) alone caused respective 14% and 30% decreases in hepatic and plasma Zn concentrations and a 27% reduction in total liver Zn reserves, whereas fasted normal mice conserved Zn with a 4-fold increase in hepatic MT. This study confirms that MT synthesis is essential for endotoxin-induced liver Zn accumulation.
Study of the rat liver S-adenosylmethionine synthetase active site with 8-azido ATP
Authors: Deigner H P , Mato J M , Pajares M A
Published: August 2015

The active site of rat liver S-adenosylmethionine synthetase was studied using 8-azido ATP, a photolabile analogue of ATP. Both forms of the enzyme, tetramer and dimer, could be labelled by using concentrations of the analogue similar to the KmATP values for each form, 350 microM and 1 mM respectively. Labelling of both S-adenosylmethionine synthetase forms with 8-azido [alpha-32P]ATP, followed by tryptic digestion and purification by HPLC, afforded one specifically labelled peptide in each case. Identification of the labelled peptide by amino acid analysis and peptide sequencing, and comparison with the enzyme sequence, indicated that the same peptide (267-286) was modified in both enzyme forms. The results are discussed on the basis of the high degree of similarity that this peptide shows in all the known S-adenosylmethionine synthetase sequences.
Functional differences between dimeric and octameric mitochondrial creatine kinase
Authors: Kaldis P , Wallimann T
Published: August 2015

Mitochondrial creatine kinase (Mi-CK) consists of octameric and dimeric molecules that are interconvertible. In the present study, the kinetic properties of purified chicken heart Mi-CK (Mib-CK) dimers and octamers were investigated separately under highly controlled conditions. Gel-permeation chromatography was performed before and after kinetic measurements in order to clearly define the proportions of octamers and dimers. ‘Dimeric’ Mi-CK solutions consisted of > or = 90% dimers throughout the experiment whereas ‘octameric’ Mi-CK solutions consisted in the beginning of 90% octamers, but upon measuring with the highest concentrations of creatine (Cr) and ATP approximately one-third of the octamers dissociated into dimers. These proper controls enabled us to pinpoint the observed kinetic differences between dimers and octamers solely to the oligomeric state of Mib-CK. Both dimeric and octameric Mi-CK displayed synergism in substrate binding (Kd values are higher than Km values), meaning that binding of the first substrate facilities subsequent binding of the second substrate. Most interestingly, Km(Cr) and Kd(Cr) values are both 2-3 times higher for octameric than for dimeric Mi-CK. Thus, at low Cr concentrations, the dimer is kinetically favoured for the forward direction of the reaction (phosphorylcreatine synthesis) compared with the octamer. The possible physiological significance of the lower Kd(Cr) value of dimeric versus octameric Mib-CK, as well as the apparent negative cooperativity of ATP binding at higher [Cr], are discussed within the context of a possible functional role for dimeric Mib-CK in vivo.
Control analysis of transit time for free and enzyme-bound metabolites: physiological and evolutionary significance of metabolic response times
Authors: Cascante M , Meléndez-Hevia E , Kholodenko B , Sicilia J , Kacser H
Published: August 2015

Control analysis of transit time, defined as tau = delta/J, has previously been considered with the constraint of low enzyme concentrations compared with free pools of metabolites [Meléndez-Hevia, Torres, Sicilia and Kacser (1990) Biochem. J. 265, 195-202]. One of the conclusions was that the sum of the control coefficients of the transition time with respect to enzyme concentration was -1. Here we demonstrate that, if the enzyme-bound pools are taken into consideration (which would be important at high enzyme concentrations and high affinities), the sum lies between 0 and -1. The transition time between two steady states, which are frequent physiological events, is mainly governed by time constants involved in changing the enzyme concentrations. Some physiological and evolutionary aspects are discussed.
Purification and properties of d-myo-inositol 1,4,5-trisphosphate 3-kinase from bovine iris sphincter smooth muscle: effects of protein phosphorylation in vitro and in intact muscle
Authors: Wang X L , Akhtar R A , Abdel-Latif A A
Published: August 2015

Stimulation of bovine iris sphincter muscle with carbachol (10 microM) increased accumulation of Ins(1,4,5)P3 (InsP3) and Ins(1,3,4,5)P4 (InsP4) by 86 and 32% respectively. Addition of isoproterenol (5 microM) to muscle pretreated with carbachol reduced the 3H-radioactivity in InsP3 by 30% and increased that of InsP4 by 41%. InsP3 3-kinase was predominantly localized in the soluble fraction (110,000 g supernatant) of the iris sphincter. The enzyme was purified from this fraction by sequential chromatography on DEAE-cellulose, calmodulin (CAM)-agarose affinity, and Mono-Q anion-exchange columns. The specific activity of the purified enzyme was 1.94 mumol/min per mg protein with a purification of 114-fold, compared with the cytosolic fraction of the muscle. SDS/PAGE showed the enzyme to be associated with a protein band corresponding to 50 kDa. In the presence of 10 microM Ca2+, CaM dose-dependently stimulated the enzyme. InsP3 3-kinase specifically phosphorylated InsP3 with an apparent K(m) of 0.56 microM and a Vmax. of 2.5 mumol/min per mg protein. The stimulatory effect of CaM was due to a change in Vmax. and not in its K(m). The enzyme was maximally active at pH 7.0-7.5. Phosphorylation of the purified InsP3 3-kinase with protein kinase A increased its activity; in contrast, phosphorylation with protein kinase C inhibited the enzyme activity. Treatment of the intact iris sphincter with isoproterenol or phorbol 12,13-dibutyrate resulted in stimulation of InsP3 3-kinase activity in the soluble fraction and this activation was preserved on SDS/PAGE and renaturation. These results indicate that the bovine iris sphincter contains a Ca-CaM-dependent InsP3 3-kinase which can be differentially regulated, both in vitro and in intact muscle, by protein kinases A and C.
Cofactor role for 10-formyldihydrofolic acid
Authors: Baggott J E , Johanning G L , Branham K E , Prince C W , Morgan S L , Eto I , Vaughn W H
Published: August 2015

10-Formyl-7,8-dihydrofolic acid (10-HCO-H2folate) was prepared by controlled air oxidation of 10-formyl-5,6,7,8-tetrahydrofolic acid (10-HCO-H4folate). The UV spectra of the 10-HCO-H2folate preparation has lambda max. 234, 333 nm and lambda min. 301 nm at pH 7.4, and lambda max. 257, 328 nm and lambda min. 229, 307 nm at pH 1. 1H-NMR spectroscopy of 10-HCO-H2folate (in 2H2O; 300 MHz) suggested a pure compound and gave resonances for one formyl group proton, two protons on C-7 and C-9, and no evidence for a C-6 proton, which is consistent with the structure proposed. The spectral properties indicated that the 10-HCO-H2folate preparation is not appreciably contaminated with 10-HCO-H4folate, 5,10-methenyltetrahydrofolic acid (5,10-CH = H4folate) or 10-formylfolic acid (10-HCO-folate). The above data establish that the 10-HCO-H2folate prepared here is authentic. In contrast, a folate with a UV spectrum having lambda max. 272 nm and lambda min. 256 nm at pH 7, which was prepared by 2,6-dichloro-indophenol oxidation of 10-HCO-H4folate and reported to be 97% pure [Baram, Chabner, Drake, Fitzhugh, Sholar and Allegra (1988) J. Biol. Chem. 263, 7105-7111], is apparently not 10-HCO-H2folate. 10-HCO-H2folate is utilized by Jurkat-cell (human T-cell leukaemia) and chicken liver aminoimidazolecarboxamide ribonucleotide transformylase (AICAR T'ase; EC 2.1.2.3) in the presence of excess 5-amino-imidazole-4-carboxamide ribotide (AICAR) resulting in the appearance of approximately 1 mol of H2folate product for each mol of AICAR formylated. The present 10-HCO-H2folate preparation had a kinetic advantage over 10-HCO-H4folate resulting from a difference of approx. 5-fold in K(m) values when both folates were used as cofactors for Jurkat-cell and rat bone marrow AICAR T'ase. No substantial kinetic advantage was observed using chicken liver AICAR T'ase. 10-HCO-H2folate had little or no activity with Jurkat-cell or chicken liver glycinamide ribonucleotide transformylase (GAR T'ase, EC 2.1.2.2). The existence in vivo of 10-HCO-H2folate is suggested in mammals by several reports of detectable amounts of radiolabelled 10-HCO-folate in bile and urine after administration of radiolabelled folic acid.
Regulation of the induction of ornithine decarboxylase in keratinocytes by retinoids
Authors: Zheng Z S , Xue G Z , Prystowsky J H
Published: August 2015

Treatment of SV40-transformed keratinocytes (Z114) with epidermal growth factor (EGF) resulted in an increase in ornithine decarboxylase (ODC) activity and a dose-dependent increase in ODC mRNA levels. Pretreatment of keratinocytes with all-trans-retinoic retinoic acid inhibited the EGF induction of ODC activity. In both quiescent and EGF-stimulated cells, all-trans-retinoic acid inhibited ODC gene transcription and lowered ODC mRNA levels, whereas glyceraldehyde phosphate dehydrogenase expression remained unaffected. Treatment with all-trans-retinoic acid for 24 h resulted in a dose- and time-dependent decrease of up to 52% in EGF binding to EGF receptors and a 30-75% decrease in EGF-receptor quantity. In addition, when cells were treated with both UV radiation and all-trans-retinoic acid, their effects were additive in causing a decrease in EGF binding. Blocking of EGF receptors with a neutralizing antibody for EGF receptors inhibited the induction of ODC activity by EGF. The effects of several other retinoids, including Ro15-0778, etretinate, Ro13-7410, etarotene, Ro40-8757, 13-cis-retinoic acid and acitretin, were also studied to determine their effects on EGF binding and ODC activity. Two of these other retinoids, 13-cis-retinoic acid and Ro13-7410, inhibited EGF binding the most (35-46%, P < 0.001); several others (etarotene, Ro40-8757 and etretinate) were less effective (7-16%), but significantly decreased EGF binding (P < 0.05), and two retinoids (Ro15-0778 and acitretin) showed no significant effect on EGF binding. In contrast, all of the retinoids tested inhibited the induction of ODC activity by EGF, although etretinate and Ro15-0778 were less effective. EGF signal transduction is important in ODC gene regulation, and retinoids are significant modulators of this pathway.
Non-muscle myosin II heavy chain has a cryptic cell-adhesion domain
Authors: Grinnell F , Ho C H
Published: August 2015

We have discovered a cryptic cell-adhesion domain in non-muscle myosin II heavy chain. A 205 kDa cell-adhesion-promoting polypeptide (p205) was extracted from BHK cells by Nonidet P-40 or Dounce homogenization. Adhesion to p205 was specifically inhibited by the peptide Gly-Arg-Gly-Asp-Ser-Pro, indicating a role for the Arg-Gly-Asp cell-adhesion motif. Purified p205 was identified as non-muscle myosin II heavy chain, based on sequence analysis and on the cross-reactivity of p205 with anti-(bovine trachea myosin) antibodies. Further experiments showed that the heavy chain of purified myosin II has cell-adhesion-promoting activity in a cell-blotting assay, and cross-reacted with anti-p205 antibodies. Finally, the adhesion domain was located in the tail portion of myosin II heavy chain, where an Arg-Gly-Asp-containing sequence can be found.
Differences between the catalytic properties of recombinant human PC2 and endogenous rat PC2
Authors: Bailyes E M , Shennan K I J , Usac E F , Arden S D , Guest P C , Docherty K , Hutton J C
Published: August 2015

Human prohormone convertase PC2 was expressed in Xenopus oocytes and its properties were compared with those of the Type-2 endopeptidase of rat insulin secretory granules, previously identified as PC2 [Bennett, Bailyes, Nielson, Guest, Rutherford, Arden and Hutton (1992) J. Biol. Chem. 267, 15229-15236]. Recombinant PC2 had the same substrate specificity as the Type-2 endopeptidase, cleaving at the CA-junction (Lys64, Arg65) of human des-31,32-proinsulin to generate insulin; little activity was found toward human des-64,65-proinsulin or proinsulin itself. Recombinant PC2 was maximally active in 5-7 mM Ca2+ (K0.5 = 1.6 mM) whereas the Type-2 endopeptidase was maximally active in 0.5-1 mM Ca2+ (K0.5 = 40 microM). Both enzymes had a pH optimum of 5.0-5.5 but the Type-2 endopeptidase was active over a wider pH range. Two molecular forms of recombinant PC2 (71 kDa and 68 kDa) were found, both had an intact C-terminus but differed by the presence of the propeptide. The endogenous PC2 comprised several overlapping forms (size range 64-68 kDa), approximately two-thirds of which lacked C-terminal immunoreactivity. Part of the size difference between recombinant and endogenous PC2 was attributable to differences in N-glycosylation. The different post-translational proteolytic modifications of recombinant and endogenous PC2 did not account for the different pH and Ca2+ sensitivities shown by the enzymes. A modulating effect of carbohydrate on enzyme activity could not be excluded.
Two thermostable type II restriction endonucleases from Icelandic strains of the genus Thermus: Tsp4C I (ACN/GT), a novel type II restriction endonuclease, and Tsp8E I, an isoschizomer of the mesophilic enzyme Bgl I (GCCNNNN/NGGC)
Authors: Welch S G , Williams R A D
Published: August 2015

Sixteen isolates of thermophilic bacteria from the genus Thermus, isolated from neutral and alkaline hot water springs in the southwest region of Iceland, were tested for the presence of restriction endonucleases. Extracts from five of the isolates showed evidence of the presence of restriction endonuclease activity by producing discrete nucleotide fragments when incubated at 65 degrees C with lambda phage DNA. Two of the isolates (Tsp4C and Tsp8E) were found to have particularly high levels of restriction endonuclease activity, and the respective enzymes from these two Thermus isolates were partially purified and characterized and their recognition and cleavage sites were determined. Enzyme Tsp4C I is a novel Type II restriction endonuclease recognizing the interrupted palindromic tetranucleotide sequence ACNGT, where N can be any one of the four bases in DNA. Tsp4C I, which retains full enzyme activity when incubated for 10 min at temperatures up to 76 degrees C, hydrolyses the phosphodiester bond in both strands of a double-stranded DNA substrate between the third and fourth bases of the recognition sequence (ACN/GT), generating fragments with a single base 3′-OH overhang. Enzyme Tsp8E I is a thermostable isoschizomer of the mesophilic Type II restriction endonuclease Bgl I (GCCNNNN/NGGC) [Lee, Clanton and Chirikjiam (1979) Fed. Proc. 28, 294], generating fragments with a three base 3′-OH overhang. However, unlike Bgl I, Tsp8E I exhibits considerable thermal stability, retaining full enzyme activity when incubated for 10 min at temperatures up to 78 degrees C. Both Tsp4C I and Tsp8E I represent significant additions to the small but expanding list of the extremely thermostable restriction endonucleases.
A comparison of the secondary structure of human brain mitochondrial and cytosolic ‘malic’ enzyme investigated by Fourier-transform infrared spectroscopy
Authors: Kochan Z , Karbowska J , Bukato G , Zydowo M M , Bertoli E , Tanfani F , Swierczyński J
Published: August 2015

The secondary structure of human brain cytosolic and mitochondrial ‘malic’ enzymes purified to homogeneity has been investigated by Fourier-transform IR spectroscopy. The absorbance IR spectra of these two isoenzymes were slightly different, but calculated secondary-structure compositions were essentially similar (38% alpha-helix, 38-39% beta-sheet, 14% beta-turn and 9-10% random structure). These proportions were not affected by succinate, a positive effector of mitochondrial ‘malic’ enzyme activity. IR spectra indicate that the tertiary structures of human brain cytosolic and mitochondrial ‘malic’ enzymes are slightly different, and addition of succinate does not cause conformational changes to the tertiary structure of the mitochondrial enzyme. Thermal-denaturation patterns of the cytosolic and mitochondrial enzymes, obtained from spectra recorded at different temperatures in the absence or presence of Mg2+, suggest that the tertiary structure of both isoenzymes is stabilized by bivalent cations and that the cytosolic enzyme possesses a more compact tertiary structure.
Mutation in aspartic acid residues modifies catalytic and haemolytic activities of Bacillus cereus sphingomyelinase
Authors: Tamura H , Tameishi K , Yamada A , Tomita M , Matsuo Y , Nishikawa K , Ikezawa H
Published: August 2015

Four aspartic acid residues (Asp126, Asp156, Asp233 and Asp295) of Bacillus cereus sphingomyelinase (SMase) in the conservative regions were changed to glycine by in vitro mutagenesis, and the mutant SMases [D126G (Asp126-->Gly etc.), D156G, D233G and D295G] were produced in Bacillus brevis 47, a protein-producing strain. The sphingomyelin (SM)-hydrolysing activity of D295G was completely abolished and those of D126G and D156G were reduced by more than 80%, whereas that of D233G was not so profoundly affected. Two mutant enzymes (D126G and D156G) were purified and characterized further. The hydrolytic activities of D126G and D156G toward four phosphocholine-containing substrates with different hydrophobicities, SM, 2-hexadecanoylamino-4-nitrophenylphosphocholine(HNP), lysophosphatidylcholine (lysoPC) and p-nitro-phenylphosphocholine (p-NPPC), were compared with those of the wild-type. The activity of D126G toward water-soluble p-NPPC was comparable with that of the wild-type. On the other hand, D156G catalysed the hydrolysis of hydrophilic substrates such as HNP and p-NPPC more efficiently (> 4-fold) than the wild-type. These results suggested that Asp126 and Asp156, located in the highly conserved region, may well be involved in a substrate recognition process rather than catalytic action. Haemolytic activities of the mutant enzymes were found to be parallel with their SM-hydrolysing activities. Two regions, including the C-terminal region containing Asp295, were found to show considerable sequence identity with the corresponding regions of bovine pancreatic DNase I. Structural predictions indicated structural similarity between SMase and DNase I. An evolutionary relationship based on the catalytic function was suggested between the structures of these two phosphodiesterases.
Molecular cloning of an orphan G-protein-coupled receptor that constitutively activates adenylate cyclase
Authors: Eggerickx D , Denef J F , Labbe O , Hayashi Y , Refetoff S , Vassart G , Parmentier M , Libert F
Published: August 2015

A human gene encoding an orphan G-protein-coupled receptor named ACCA (adenylate cyclase constitutive activator) was isolated from a genomic library using as a probe a DNA fragment obtained by low-stringency PCR. Human ACCA (hACCA) is a protein of 330 amino acids that exhibits all the structural hallmarks of the main family of G-protein-coupled receptors. Expression of hACCA resulted in a dramatic stimulation of adenylate cyclase, similar in amplitude to that obtained with other Gs-coupled receptors fully activated by their respective ligands. This stimulation was obtained in a large variety of stable cell lines derived from various organs, and originating from different mammalian species. hACCA was found to be the human homologue of a recently reported mouse orphan receptor (GPCR21). The mouse ACCA (mACCA) was therefore recloned by PCR, and expression of mACCA in Cos-7 cells demonstrated that the mouse receptor behaved similarly as a constitutive activator of adenylate cyclase. It is not known presently whether the stimulation of adenylate cyclase is the result of a true constitutive activity of the receptor or, alternatively, is the consequence of a permanent stimulation by a ubiquitous ligand. The tissue distribution of mACCA was determined by RNase protection assay. Abundant transcripts were found in the brain, whereas lower amounts were detected in testis, ovary and eye. Various hypotheses concerning the constitutive activity of ACCA and their potential biological significance are discussed.
Alignment of caldesmon on the actin-tropomyosin filaments
Authors: Tsuruda T S , Watson M H , Foster D B , Lin J J J C , Mak A S
Published: August 2015

We have reported previously that each smooth-muscle caldesmon binds predominantly to a region within residues 142-227 of tropomyosin, but a weaker binding site also exists at the N-terminal region of tropomyosin [Watson, Kuhn, Novy, Lin and Mak (1990) J. Biol. Chem. 265, 18860-18866]. In view of recent evidence for the presence of tropomyosin-binding sites at both the N- and C-terminal domains of caldesmon, we have studied the binding of the N- and C-terminal fragments of human fibroblast caldesmon expressed in Escherichia coli to tropomyosin and its CNBr fragments. The N-terminal fragment, CaD40 (residues 1-152), binds tropomyosin, but the interaction is mostly abolished in the presence of actin. CaD40 binds strongly to Cn1B(142-281) of tropomyosin, but weakly to Cn1A(11-127). The C-terminal fragment, CaD39, which corresponds to residues 443-736 of gizzard caldesmon, binds tropomyosin, and the interaction is enhanced by actin. CaD39 binds to both Cn1A(11-127) and Cn1B(142-281) of tropomyosin. Our results suggest that the N-terminal domain of caldesmon interacts with the C-terminal half of one tropomyosin molecule, whereas the C-terminal domain binds to both N- and C-terminal regions of the adjacent tropomyosin molecule along the actin filament. In addition, the binding of the N-terminal domain of caldesmon to the actin-tropomyosin filament is weak, which may allow this domain to project off the thin filament to interact with myosin.
The chloride effect in the human embryonic haemoglobins
Authors: Hofmann O , Carrucan G , Robson N , Brittain T
Published: August 2015

The interactions of the three human embryonic haemoglobins with chloride ions have been investigated. Each of the three embryonic haemoglobins exhibits a unique pattern of oxygen-affinity-dependence on chloride ion concentration. Human embryonic haemoglobin Portland (zeta 2 gamma 2) is found to be completely insensitive to chloride ion concentration. Haemoglobin Gower I (zeta 2 gamma 2) shows a small concentration dependence, whilst haemoglobin Gower II (alpha 2 epsilon 2) exhibits a dependence approaching that of the adult protein. The degree of co-operativity for each protein is essentially chloride concentration independent. The chloride-dependent and -independent components of the alkaline Bohr effects have been measured for each of the embryonic haemoglobins and compared with that of the adult protein. Both the chloride-binding data and the Bohr effect have been analysed in terms of the recently developed allosteric model proposed by Perutz [Perutz, Fermi, Poyart, Pagnier and Kister (1993) J. Mol. Biol. 233, 536-545].
An inverted TATA box directs downstream transcription of the bone sialoprotein gene
Authors: Li J J , Kim R H , Sodek J
Published: August 2015

The orientation of the TATA box is thought to direct downstream transcription of eukaryotic genes by RNA polymerase II. However, the putative TATA box in the promoter of the bone sialoprotein (BSP) gene, which codes for a tissue-specific and developmentally regulated bone matrix protein, is inverted (5′-TTTATA-3′) relative to the consensus TATA box sequence (5′-TATAAA-3′) and is overlapped by a vitamin D3-response element. Here we show that the inverted TATA sequence in the rat BSP gene binds to recombinant TATA-box-binding protein (TBP) with an affinity similar to that observed with the consensus TATA box, and site-directed point mutations in the inverted TATA sequence (mutating TTTATA into TCTCTA) abrogate both TBP binding and BSP promoter activity. However, when the inverted TATA sequence is changed to a canonical TATAAA, the TBP- and vitamin D3 receptor-binding properties together with the BSP promoter activity are retained. In addition, we found that the TBP is required to reconstitute in vitro transcription driven by the BSP promoter. These studies, which have revealed a naturally occurring inverted TATA box that can bind TBP and direct downstream transcription, demonstrate that the orientation of the TATA box does not determine the direction of transcription in higher eukaryotic genes. Consequently, the inverted TATA box that is conserved in the human, rat and mouse BSP gene promoters will provide an excellent in vivo model to investigate the polarity of the transcription factor IID-DNA complex and its relation to downstream transcription.
The mRNAs for cyclin A, c-myc and ribosomal proteins L4 and S6 are associated with cytoskeletal-bound polysomes in HepG2 cells
Authors: Hovland R , Campbell G , Pryme I , Hesketh J
Published: August 2015

Cytosolic, cytoskeleton and membrane fractions were extracted from HepG2 cells by a sequential detergent/salt extraction procedure. The cytosolic fraction contained 93% of the lactic dehydrogenase activity while the cytoskeleton fraction was enriched in actin and vimentin. The distribution of mRNAs for c-myc, glucose transporter 1, ribosomal proteins L4 and S6 and cyclin A were investigated by Northern hybridization of total RNA extracted from polysomes isolated from cytosolic, cytoskeleton and membrane fractions. The membrane-bound polysomes were enriched in the glucose transporter 1 mRNA and the cytoskeleton-bound polysomes were enriched in the mRNAs for the two ribosomal proteins, c-myc and cyclin A. The results suggest that the mRNAs for nuclear proteins are one class of mRNAs which are translated on polysomes associated with the cytoskeleton; this may be related to the requirement to transport the newly synthesized protein to the nucleus.
Agonist-induced internalization and recycling of the glucagon-like peptide-1 receptor in transfected fibroblasts and in insulinomas
Authors: Widmann C , Dolci W , Thorens B
Published: August 2015

Glucagon-like peptide-1 (GLP-1) is the most potent stimulator of glucose-induced insulin secretion and its pancreatic beta-cell receptor is a member of a new subfamily of G-protein-coupled receptors which includes the receptors for vasoactive intestinal polypeptide, secretin and glucagon. Here we studied agonist-induced GLP-1 receptor internalization in receptor-transfected Chinese hamster lung fibroblasts using three different approaches. First, iodinated GLP-1 bound at 4 degrees C to transfected cells was internalized with a t 1/2 of 2-3 min following warming up of the cells to 37 degrees C. Secondly, exposure to GLP-1 induced a shift in the distribution of the receptors from plasma membrane-enriched to endosomes-enriched membrane fractions, as assessed by Western blot detection of the receptors using specific antibodies. Thirdly, continuous exposure of GLP-1 receptor-expressing cells to iodinated GLP-1 led to a linear accumulation of peptide degradation products in the medium following a lag time of 20-30 min, indicating a continuous cycling of the receptor between the plasma membrane and endosomal compartments. Potassium depletion and hypertonicity inhibited transferrin endocytosis, a process known to occur via coated pit formation, as well as GLP-1 receptor endocytosis. In contrast to GLP-1, the antagonist exendin-(9-39) did not lead to receptor endocytosis. Surface re-expression following one round of GLP-1 receptor endocytosis occurred with a half-time of about 15 min. The difference in internalization and surface re-expression rates led to a progressive redistribution of the receptor in intracellular compartments upon continuous exposure to GLP-1. Finally, endogenous GLP-1 receptors expressed by insulinoma cells were also found to be internalized upon agonist binding. Together our data demonstrate that the GLP-1 receptor is internalized upon agonist binding by a route similar to that taken by single transmembrane segment receptors. The characterization of the pathway and kinetics of GLP-1-induced receptor endocytosis will be helpful towards understanding the role of internalization and recycling in the control of signal transduction by this receptor.
Identification of periplasmic nitrate reductase Mo(V) EPR signals in intact cells of Paracoccus denitrificans
Authors: Sears H J , Bennett B , Spiro S , Thomson A J , Richardson D J
Published: August 2015

EPR spectroscopy has been successfully used to detect signals due to molybdenum (V) and ferric iron in intact cells of aerobically grown Paracoccus denitrificans. The signals are ascribed to the catalytic molybdenum centre and to the haem iron of the periplasmic nitrate reductase. These signals are absent from a mutant strain deficient in this enzyme. The Mo(V) signal is due to the High-g Split species which has been well characterized in the purified enzyme. This confirms that the High-g Split is the physiologically relevant signal of a number observed in the previous work on the purified enzyme.
Early Ca2+ signalling events in neutrophils detected by rapid confocal laser scanning
Authors: Pettit E J , Hallett M B
Published: August 2015

Characterization of early Ca2+ signalling events is crucial for understanding the mechanisms which lead to cell signalling. Rapid confocal laser scanning of Fluo3-loaded neutrophils was used to provide spatially resolved cytosolic free Ca2+ measurements from neutrophils stimulated with formyl-Met-Leu-Phe with a time resolution of 12.5 ms. Heterogeneity of the magnitude and timing of the response was observed between individual neutrophils. There was always a delay between contact with the stimulus and onset of the Ca2+ signal, with a minimum delay of 75 ms and a mean delay of 530 ms (n = 150). There was no delay in the cytosolic free Ca2+ rise induced by ionomycin. The earliest Ca2+ event detected following stimulation with formyl-Met-Leu-Phe (1 microM) was a localized rise in cytosolic free Ca2+ from a location within the cell, pointing to Ca2+ store release as the initial event for triggering whole-cell Ca2+ changes in these cells.
Ligand-dependent and -independent activation of the transcription factor γRF-1 in a cell-free system
Authors: Feghali C A , Wright T M
Published: August 2015

gamma RF-1 is a recently identified transcription factor induced by interferon-gamma (IFN-gamma) which binds to a unique palindromic enhancer, gamma RE-1, in the promoter of the mig gene. This paper describes the ligand-dependent and ligand-independent activation of gamma RF-1 in a cell-free system. gamma RF-1 activity was induced by IFN-gamma in a time-dependent manner from 5 to 60 min in lysates prepared from the human monocytic leukaemia line THP-1 and the human epidermoid carcinoma line A431. The activation of gamma RF-1 in vitro required both ATP and an inhibitor of tyrosine phosphatases (sodium orthovanadate or pervanadate). In the presence of limiting concentrations (micromolar) of ATP, activation was also dependent upon stimulation with IFN-gamma, whereas at millimolar concentrations of ATP, gamma RF-1 was activated by either sodium orthovanadate or pervanadate in the absence of ligand. Based on cell fractionation studies, both membrane and cytosol components were essential for activation of gamma RF-1 in vitro. Consistent with a role for one or more tyrosine kinases in the activation of gamma RF-1, its DNA binding activity was blocked by monoclonal anti-phosphotyrosine antibodies and by the tyrosine kinase inhibitors genistein, lavendustin A and herbimycin A. A comparison with recently described pathways of IFN-mediated transcription factor regulation indicates that the in vitro activation of gamma RF-1 is unique, requiring both membrane and cytosol fractions and inhibition of endogenous tyrosine phosphatase activity.
Relationship between Rap1 protein phosphorylation and regulation of Ca2+ transport in platelets: a new approach
Authors: Magnier C , Corvazier E , Aumont M C , Le Jemtel T H , Enouf J
Published: August 2015

Although the interrelationship between the two messengers Ca2+ and cyclic AMP in platelet function is well documented, its mechanism of action still remains to be established. We investigated here the question of the regulation of platelet Ca(2+)-ATPases by cyclic AMP through the phosphorylation of the Rap1 protein using a pathological model. We first found experimental conditions where Ca(2+)-transport by platelet membrane vesicles appeared to be dependent on the phosphorylation of the Rap1 protein. Then, we studied platelets of patients with congestive heart failure for their expression of the potential 97 kDa Ca(2+)-ATPase target of regulation through the Rap1 protein as well as the phosphorylation of the Rap1 protein using the catalytic subunit of the cyclic AMP-dependent protein kinase (C. Sub.). In the first patients studied, we found no significant modification in the expression of the 97 kDa Ca(2+)-ATPase by Western blotting using the PL/IM 430 monoclonal antibody which specifically recognized this isoform. In contrast, the Rap1 protein was differentially phosphorylated when using 15 micrograms/ml of the C. Sub. These results allowed us to use these pathological platelets to study the relationship between the expression of Rap1 protein and the regulation of Ca2+ transport by selecting a patient with severe heart failure. We could show a decrease in the expression as well as in the phosphorylation of Rap1 protein and demonstrate a lower effect of C. Sub. on Ca2+ transport. Finally, by studying a further series of patients, we could confirm that the decrease in Rap1 protein expression in heart failure, whatever its extent, was variable, and could strictly correlate the expression of Rap1 protein with the stimulatory effect of C. Sub. on Ca2+ transport. Besides the evidence for regulation of the expression of the Rap1 protein in platelets from patients with heart failure, these findings constitute a new approach in favour of the regulation of platelet Ca2+ transport through the phosphorylation of the Rap1 protein.
Isocitrate dehydrogenase from bovine heart: primary structure of subunit 3/4
Authors: Zeng Y , Weiss C , Yao T T , Huang J , Siconolfi-Baez L , Hsu P , Rushbrook J I
Published: August 2015

Bovine NAD(+)-dependent isocitrate dehydrogenase was shown previously to contain four subunits of approx. 40 kDa (subunits 1-4) possessing different peptide maps and electrophoretic properties [Rushbrook and Harvey (1978) Biochemistry 17, 5339-5346]. In this study the heterogeneity is confirmed using enzyme purified by updated methods and from single animals, ruling out allelic variability. Subunits 1 and 2 were differentiated from each other and from subunits 3 and 4 by N-terminal amino acid sequencing. Subunits 3 and 4 (subunits 3/4) were identical in sequence over 30 residues. The N-terminal residues of subunits 1 and 2 were homologous but not identical with the beta- and gamma-subunits respectively of the comparable pig heart enzyme. Subunits 3/4 were identical over 30 residues with the N-terminus of the pig heart alpha-subunit. Full-length sequence, including that for mitochondrial import, is presented for a protein with the processed N-terminus of subunits 3/4, deduced from cloned cDNA obtained utilizing the N-terminal sequence information. The derived amino acid sequence for the mature protein contains 339 amino acids and has a molecular mass of 36,685 Da. Complete identity with N-terminal and Cys-containing peptides totalling 92 residues from the alpha-subunit of the pig heart enzyme [Huang and Colman (1990) Biochemistry 29, 8266-8273] suggests that maintenance of a particular three-dimensional structure in this subunit is crucial to the function of the enzyme. An electrophoretic heterogeneity within the pig heart alpha-subunit, similar to that shown by bovine subunits 3/4, was demonstrated. One reordering of the Cys-containing peptides of the pig heart alpha-subunit is indicated. Sequence comparison with the distantly related NADP(+)-dependent enzyme from Escherichia coli, for which the three-dimensional structure is known [Stoddard, Dean and Koshland (1993) Biochemistry 32, 9310-9316] shows strong conservation of residues binding isocitrate, Mg2+ and the NAD+ moiety of NADP+, consistent with a catalytic function.