bluuebunny/crossref_metadata_2025 huggingface dataset:
Studies on the carbohydrate-binding characteristics of human pulmonary surfactant-associated protein A and comparison with two other collectins: mannan-binding protein and conglutinin
Authors: Haurum J S , Thiel S , Haagsman H P , Laursen S B , Larsen B , Jensenius J C
Published: August 2015

The surfactant-associated protein A (SP-A) belongs to the collectin family, a group of C-type lectins encompassing also surfactant-associated protein D, mannan-binding protein (MBP) and conglutinin. These proteins all have carbohydrate-recognition domains joined to collagen stalks. It seems likely that SP-A, like MBP and conglutinin, may mediate anti-microbial activity through binding to carbohydrates on the microorganisms and collectin receptors on phagocytic cells. We have studied the influence of carbohydrates on the binding of SP-A, MBP and conglutinin to mannan in an enzyme-linked lectin-binding assay. All sugars were of D-configuration, except fucose of which both L- and D-configurations were tested. The order of inhibiting potency on the binding of SP-A was: N-acetylmannosamine > L-fucose, maltose > glucose > mannose. The following sugars were non-inhibitory: galactose, D-fucose, glucosamine, mannosamine, galactosamine, N-acetylglucosamine, and N-acetylgalactosamine. The best inhibitor of MBP was N-acetylglucosamine. Otherwise MBP showed a selectivity similar to that of SP-A. Conglutinin binding was inhibited by all the sugars examined except N-acetylgalactosamine. For conglutinin, as for MBP, the best inhibitor was N-acetylglucosamine. Normal human SP-A, alveolar-proteinosis SP-A purified by ion-exchange chromatography, and alveolar-proteinosis SP-A purified by n-butanol extraction showed no difference in sugar selectivity. The influence of pH and of the calcium concentration was also examined. Organic solvent-extracted SP-A from patients suffering from alveolar proteinosis and normal SP-A showed different sensitivity profiles.
An insulin receptor peptide (1135-1156) stimulates guanosine 5′-[γ-thio]triphosphate binding to the 67 kDa G-protein associated with the insulin receptor
Authors: Jo H , Radding W , Anantharamaiah G M , McDonald J M
Published: August 2015

Peptides representing two putative G-protein-binding motifs (GPBP1 and GPBP2) derived from insulin-receptor sequences were tested for their ability to stimulate guanosine 5′-[gamma-thio]-triphosphate (GTP[S]; ‘GTP gamma S’) binding to a preparation containing the 41 and 67 kDa G-proteins that are associated with the insulin receptor [Jo, Cha, Davis and McDonald (1992) Endocrinology (Baltimore) 131, 2855-2861]. GPBP2 (residues 1135-1156) specifically stimulated GTP[S] binding, whereas GPBP1 (1319-1333) did not. Substitution of Arg-1152 with Gln in GPBP2 corresponding to a mutation site in insulin-resistant patients [Cocozza, Porcellini, Riccardi, Monticelli, Condorelli, Ferrera, Pianese, Miele, Capaldo, Beguinot and Varrone (1992) Diabetes 41, 521-526] attenuated the stimulatory potency of GPBP2. Size-exclusion chromatography and studies with purified 67 kDa G-protein revealed that GPBP2 stimulated GTP[S] binding only to the 67 kDa G-protein. These studies provide evidence for a potential regulatory site for G-protein interaction with the insulin receptor in the tyrosine kinase domain.
Astrocytic glucose-6-phosphatase and the permeability of brain microsomes to glucose 6-phosphate
Authors: Forsyth R J , Bartlett K , Burchell A , Scott H M , Eyre J A
Published: August 2015

Cells from primary rat astrocyte cultures express a 36.5 kDa protein that cross-reacts with polyclonal antibodies to the catalytic subunit of rat hepatic glucose-6-phosphatase on Western blotting. Glucose-6-phosphate-hydrolysing activity of the order of 10 nmol/min per mg of total cellular protein can be demonstrated in cell homogenates. This activity shows latency, and is localized to the microsomal fraction. Kinetic analysis shows a Km of 15 mM and a Vmax. of 30 nmol/min per mg of microsomal protein in disrupted microsomes. Approx. 40% of the total phosphohydrolase activity is specific glucose-6-phosphatase, as judged by sensitivity to exposure to pH 5 at 37 degrees C. Previous reports that the brain microsomal glucose-6-phosphatase system does not distinguish glucose 6-phosphate and mannose 6-phosphate are confirmed in astrocyte microsomes. However, we demonstrate significant phosphomannose isomerase activity in brain microsomes, allowing for ready interconversion between mannose 6-phosphate and glucose 6-phosphate (Vmax. 15 nmol/min per mg of microsomal protein; apparent Km < 1 mM; pH optimum 5-6 for the two-step conversion). This finding invalidates the past inference from the failure of brain microsomes to distinguish mannose 6-phosphate and glucose 6-phosphate that the cerebral glucose-6-phosphatase system lacks a ‘glucose 6-phosphate translocase’ [Fishman and Karnovsky (1986) J. Neurochem. 46, 371-378]. Furthermore, light-scattering experiments confirm that a proportion of whole brain microsomes is readily permeable to glucose 6-phosphate.
A putative glutathione-binding site in CdZn-metallothionein identified by equilibrium binding and molecular-modelling studies
Authors: Brouwer M , Hoexum-Brouwer T , Cashon R E
Published: August 2015

Glutathione (GSH) has been found to form a complex with both vertebrate and invertebrate copper-metallothionein (CuMT) [Freedman, Ciriolo and Peisach (1989) J. Biol. Chem. 264, 5598-5605; Brouwer and Brouwer-Hoexum (1991) Arch. Biochem. Biophys. 290, 207-213]. In this paper we report on the interaction of GSH with CdZnMT-I and CdZnMT-II from rabbit liver and with CdMT-I from Blue crab hepatopancreas. Ultrafiltration experiments showed that all three MTs combined with GSH. The measured binding data for the three MTs could be described by a single binding isotherm. The GSH/MT stoichiometry was 1.4 +/- 0.3 and Kdiss. = 14 +/- 6 microM. Partially Zn-depleted MT does not significantly bind GSH, indicating that the GSH-binding site is located on MT's Zn-containing N-terminal domain. The putative GSH-binding site on rabbit liver MT was investigated using molecular-graphics analysis. A cleft on the MT's N-terminal domain, which has the labile Zn-2 at its base, could easily accommodate GSH. Cysteine-ligand exchange between the terminal (non-bridging) Cys-26, bound to Zn-2, and the cysteine in GSH is stereochemically possible. Based on these considerations a model of MT-GSH was built in which GSH's cysteine replaces Cys-26 as a terminal Zn-2 ligand. This complex was energy-minimized by molecular-mechanics calculations, taking into account computed partial electrostatic charges on all atoms, including Cd and Zn. These calculations showed that the MT-GSH complex was thermodynamically more stable than MT, due to favourable non-bonded, electrostatic and van der Waals interactions. Six hydrogen bonds can form between GSH and MT. The average pairwise root-mean-square deviations (RMSD) of the metals in energy-minimized MT and MT-GSH, compared with the metals in the crystal structure, were 0.0087 +/- 0.0028 nm (0.087 +/- 0.028 A) and 0.0168 +/- 0.0087 nm (0.168 +/- 0.087 A) respectively. The RMSD values for the polypeptide-backbone alpha carbons were 0.0136 +/- 0.0060 nm (0.136 +/- 0.060 A) and 0.0491 +/- 0.0380 nm (0.491 +/- 0.380 A) respectively. No other docking sites for GSH were found. The energy-minimized structure of an MT-2-mercaptoethanol complex was somewhat less stable than the native MT domain, attesting to the specificity of the MT-GSH interaction. The possible physiological significance of the MT-GSH interaction is discussed.
Nuclear factor binding to an AP-1 site is associated with the activation of pro-α 1(I)-collagen gene in dedifferentiating chondrocytes
Authors: Määttä A , Glumoff V , Paakkonen P , Liska D , Penttinen R P K , Elima K
Published: August 2015

Isolated chondrocytes grown on plastic gradually lose their differentiated phenotype upon subculturing. This dedifferentiation is manifested by an altered production of extracellular-matrix molecules (ECM): e.g., the cartilage specific type II collagen is replaced by types I and III. We have studied the regulation of ECM gene expression in dedifferentiating human and murine fetal chondrocytes. Nuclear extracts from dedifferentiated cells, human fetal fibroblasts and 3T3 cells contained a protein that bound in an electrophoretic mobility shift assay to an AP-1 site in the first intron of the human alpha 1(I) collagen gene. This binding activity was not present in freshly isolated human or murine chondrocytes, which produced type II, but not type I, collagen mRNA in culture. Thus the binding activity was induced simultaneously with alpha 1(I)-collagen-gene expression during dedifferentiation. The specific interaction was sensitive to dephosphorylation of the nuclear extract and to chemical modification of reduced cysteine residues. The AP-1 site we studied had previously been shown to be a positive transcriptional contributor in the first intron to the expression of the alpha 1(I) collagen gene. In transient transfections into dedifferentiating chondrocytes, an alpha 1(I) collagen expression plasmid carrying a mutated AP-1 site in the first intron resulted in three-times-lower reporter gene RNA levels than a plasmid carrying the respective functional AP-1 site. These data suggest that the AP-1 sequence and its respective trans-acting factors may play a role in the transcriptional regulation of the alpha 1(I) collagen gene during dedifferentiation of chondrocytes.
Suggestive evidence for two different mucin genes in rat intestine
Authors: Khatri I A , Forstner G G , Forstner J F
Published: August 2015

In the present report we describe the isolation and sequence of a partial cDNA (M2-798) for a rat intestinal mucin designated M2. A rat intestinal lambda ZAP II cDNA library was screened using a polyclonal antiserum which was prepared against deglycosylated high-molecular-mass glycopeptides of the purified mucin. Mucin cDNA clones were found to contain tandem repeats of 18 nt which encoded a threonine- and proline-rich peptide having a consensus sequence of TTTPDV. This is the same sequence reported recently by Gum, Hicks, Lagace, Byrd, Toribara, Siddiki, Fearney, Lamport and Kim [(1991) J. Biol. Chem. 266, 22733-22738] for a rat intestinal cDNA called RMUC 176. A novel feature present in the cDNA M2-798 is a 246 nt unique region at the 3′ end which encodes a hydrophobic sequence of 82 amino acids. RNA blots probed with M2-798 cDNA produced a single hybridization band between 7.5 and 9.0 kb in rat small intestine and colon. An identical hybridization pattern was obtained with a PCR-generated cDNA probe corresponding solely to the unique hydrophobic region of M2-798, demonstrating that this region is encoded by the authentic M2 mRNA. Our data suggest that the unique region of M2 has the potential to be either a transmembrane region, or a domain which mediates hydrophobic interactions of the mucin with other molecules. Since we have previously reported another rat intestinal cDNA which encodes the C-terminus of a mucin-like peptide (MLP) [Xu, Wang, Huan, Cutz, Forstner and Forstner (1992) Biochem. J. 286, 335-338], we wished to discover whether M2 was encoded by the same gene. RNA blotting experiments with probes specific for M2 and MLP showed different mRNAs for each. The message for M2 (7.5-8.5 kb) was smaller than that for MLP (> 9.5 kb) and, unlike MLP, gave no signal in human colonic LS174T cells. The results of DNA blots probed with M2-798 and an MLP-probe suggest that M2 and MLP are likely to be single-copy genes. It would appear therefore that normal rat intestine, like human intestine, may express two different mucin genes.
Purification and separation of holo- and apo-forms of Saccharopolyspora erythraea acyl-carrier protein released from recombinant Escherichia coli by freezing and thawing
Authors: Morris S A , Revill W P , Staunton J , Leadlay P F
Published: August 2015

Saccharopolyspora erythraea acyl-carrier protein, highly expressed from a T7-based expression plasmid in Escherichia coli, can be selectively released from the cells in near-quantitative yield by a single cycle of freezing and thawing in a neutral buffer. Electrospray mass spectrometry was used to confirm that the recombinant S. erythraea acyl-carrier protein over-expressed in E. coli is present predominantly as the holo-form, with variable amounts of apo-acyl-carrier protein, holo-acyl-carrier protein dimer and holo-acyl-carrier protein glutathione adduct. The holo- and apo-acyl-carrier proteins are both readily purified on a large scale from the freeze-thaw extracts and can be separated from one another by octyl-Sepharose chromatography. The holo-acyl-carrier protein obtained in this way was fully active in supporting the synthesis of acyl-acyl-carrier protein by extracts of S. erythraea.
cDNA cloning and characterization of the protein encoded by RD, a gene located in the class III region of the human major histocompatibility complex
Authors: Cheng J , Macon K J , Volanakis J E
Published: August 2015

The RD gene, initially defined in the mouse, has been mapped between the Bf and C4A genes in the human major histocompatibility complex class III region. Using the mouse cDNA as a probe, we isolated and sequenced human RD cDNA clones. The composite nucleotide sequence consisted of 1301 nucleotides, excluding a poly(A) tail at the 3′ end. It contained a single open reading frame encoding a polypeptide of 380 amino acid residues with a calculated molecular mass of 42274 Da. The most striking structural feature of the deduced amino acid sequence is a region consisting entirely of 24 tandem repeats of an Arg-Asp (or Glu) dipeptide. The human RD cDNA was expressed in Escherichia coli as a fusion protein with glutathione S-transferase and used to produce antisera in rabbits. Western blot analysis and immunoprecipitation of lysates of biosynthetically labelled HeLa cells indicated that RD is a 44 kDa nuclear protein.
High-level expression and in vitro mutagenesis of a fibrillogenic 109-amino-acid C-terminal fragment of Alzheimer's-disease amyloid precursor protein
Authors: Gardella J E , Gorgone G A , Candela L , Ghiso J , Castaño E M , Frangione B , Gorevic P D
Published: August 2015

We amplified DNA encoding the 3′ 109 codons of Alzheimer's-disease amyloid precursor protein (APP) inclusive of the beta protein (A beta) and cytoplasmic domains from cDNA using oligonucleotide primers designed to facilitate cloning into the T7 expression vector pT7Ad23K13. We also modified this construct to generate recombinant molecules incorporating two recently described APP mutants by site-directed mutagenesis. Both native C109 (deletion construct inclusive of the C-terminal 109 residues of APP) and constructs with a single mutation at codon 642 (T-->G, resulting in a substitution of glycine for valine) or a double mutation at codons 595 (G-->T, substituting asparagine for lysine) and 596 (A-->C, substituting leucine for methionine) were expressed in Escherichia coli to levels of 5-20% of total bacterial protein after induction. The major constituent of expressed C109 protein had an apparent molecular mass of 16-18 kDa by SDS/PAGE and appeared to be the full-length construct by size and N-terminal microsequencing. Also present was a 4-5 kDa species that co-purified with C109, constituting only approximately 1% of expressed protein, which was revealed by Western-blot analysis with antibodies specific for A beta epitopes and after biotinylation of purified recombinant C109. This fragment shared N-terminal sequence with, and appeared to arise by proteolysis of, full-length C109 in biosynthetic labelling experiments. C109 spontaneously precipitated after dialysis against NaCl or water, and with prolonged (> 20 weeks) standing was found by electron microscopy to contain a minor (< 5%) fibrillar component that was reactive with antibodies to a C-terminal epitope of APP. Recombinant C109 appears to duplicate some of the biochemical and physicochemical properties of C-terminal A beta-inclusive fragments of APP that have been found in transfected cells, brain cortex and cerebral microvessels.
Regulation of sphingosine-activated protein kinases: selectivity of activation by sphingoid bases and inhibition by non-esterified fatty acids
Authors: MYu Pushkareva , Bielawska A , Menaldiv D , Liotta D , Hannun Y A
Published: August 2015

Sphingosine has been shown to activate protein kinases in Jurkat T cell cytosol [Pushkareva, Khan, Alessenko, Sahyoun and Hannun (1992) J. Biol. Chem. 267, 15246-15251]. In this study, two sphingosine-activated protein kinases were distinguished by their substrate specificity, their dose-response to sphingosine and the specificity of their activation by sphingosine and dihydrosphingosine stereoisomers. A p32-sphingosine-activated protein kinase responded to low concentrations of D-erythrosphingosine with an initial activation observed at 2.5 microM and a peak activity at 10-20 microM. This kinase showed a modest specificity for D-erythro-sphingosine over other sphingosine stereoisomers, and a preference for sphingosines over dihydrosphingosines. Phosphorylation of a p18 substrate required higher concentrations of sphingosine (20-100 microM) and showed a significant preference for the erythro isomers of sphingosine and dihydrosphingosine over the threo isomers. The ability of other lipids to modulate sphingosine activation of these kinases was also examined. Oleic acid, but not oleic alcohol or the methyl ester, induced the phosphorylation of a distinct set of substrates (probably through the activation of protein kinase C), and inhibited sphingosine-induced phosphorylation with an IC50 of approximately 20 microM. Oleic anhydride failed to induce changes in basal protein phosphorylation but inhibited sphingosine-activated protein kinases, thus distinguishing the effects of fatty acids on protein kinase C from the inhibition of sphingosine-induced phosphorylation. These studies define two distinct sphingosine-activated protein kinases and reveal an important interaction between two classes of putative lipid second messengers.
Regulation of annexin I-dependent aggregation of phospholipid vesicles by protein kinase C
Authors: Johnstone S A , Hubaishy I , Waisman D M
Published: August 2015

Annexin I is a member of the annexin family of Ca(2+)- and phospholipid-binding proteins. The ability of this protein to aggregate and to mediate the fusion of various types of vesicles has supported the hypothesis that this protein might be involved in intracellular membrane fusion processes such as exocytosis. Although annexin I has been described as a major in vitro substrate of both protein kinase C and the epidermal-growth-factor-receptor protein tyrosine kinase, the functional consequences of these phosphorylation events have not been investigated. In this paper we examine the effect of the phosphorylation of annexin I by protein kinase C on the phospholipid aggregation activity of the protein. The stoichiometry of phosphorylation of the protein was affected by the method of preparation of the phospholipid. Under optimal assay conditions protein kinase C catalysed the incorporation of 2.83 +/- 0.23 mol of phosphate/mol of annexin I (mean +/- S.E.M., n = 21). Studies with the Ca(2+)- and phospholipid-independent form of protein kinase C suggested that the phosphorylation of annexin I was stimulated by phospholipid in the absence of Ca2+, although maximal phosphorylation was achieved in the presence of both phospholipid and Ca2+. Phosphorylation of annexin I resulted in a dramatic decrease in the rate and extent of phospholipid vesicle aggregation, without significantly disrupting the binding of the protein to the phospholipid vesicles. The phosphorylation of annexin I increased the EC50 (Ca2+) of phospholipid vesicle aggregation from 19 +/- 10 microM (mean +/- S.D., n = 7) for the native protein to 290 +/- 95 microM (mean +/- S.D., n = 5) for the phosphorylated protein. These results suggest that protein kinase C may act to inhibit the phospholipid vesicle aggregation activity of annexin I.
Branched-chain-amino-acid biosynthesis in plants: molecular cloning and characterization of the gene encoding acetohydroxy acid isomeroreductase (ketol-acid reductoisomerase) from Arabidopsis thaliana (thale cress)
Authors: Dumas R , Curien G , DeRose R T , Douce R
Published: August 2015

Towards the goal of gaining a better understanding of the molecular mechanisms controlling branched-chain-amino-acid biosynthesis in plants, we have isolated, sequenced and characterized a gene encoding acetohydroxy acid isomero-reductase (ketol-acid reductoisomerase) from Arabidopsis thaliana (thale cress). Comparison between the acetohydroxy acid isomeroreductase cDNA and the genomic sequence has allowed us to determine the exon structure of the coding region. The isolated acetohydroxy acid isomeroreductase gene is distributed over approx. 4.5 kbp and contains nine introns (79-347 bp). The transcriptional start site was found to be 52 bp upstream of the translational initiation site. Southern-blot analysis of A. thaliana genomic DNA shows that the acetohydroxy acid isomeroreductase is encoded by a single-copy gene.
Decrease in cerebral inositols in rats and humans
Authors: Moats R A , Lien Y H H , Filippi D , Ross B D
Published: August 2015

Rats with portacaval shunts and humans with hepatic encephalopathy show severe myo-inositol depletion in the brain. The portacaval-shunted rat may therefore be a useful model for the investigation of neurochemical pathways containing myoinositol, which are modulated not only in hepatic encephalopathy but also in diabetes mellitus and Alzheimer's disease.
A novel mono-branched lipid phosphate acts as a substrate for dolichyl phosphate mannose synthetase
Authors: Wilson I B H , Taylor J P , Webberley M C , Turner N J , Flitsch S L
Published: August 2015

Dolichyl phosphate mannose synthetase (GDP-mannose: dolichyl-phosphate O-beta-D-mannosyltransferase; EC 2.4.1.83) is an enzyme that is involved in glycoconjugate biosynthesis and possesses a putatively conserved dolichol binding site. In order to probe the interaction between the enzyme and the dolichol chain, lipid phosphates varying in length and extent of branching have been tested as substrates in crude microsomal preparations from Saccharomyces cerevisiae. It was found that phytanyl (3,7,11,15-tetramethylhexadecanyl) phosphate was utilized at 60-70% of the efficiency of the natural dolichyl lipid in transfer of [3,4,-3H]mannose from GDP-Man to organic soluble material, whereas addition of S-3-methyloctadecanyl phosphate, which is of similar length to the phytanyl analogue but with only one branch, resulted in approximately 25% of the incorporation of the natural substrate. Incubations with the unbranched tetradecanyl phosphate and with the short, doubly branched R- and S-dihydrocitronellyl (3,7-dimethyloctanyl) phosphates exhibited levels of activity similar to incubations with no exogenous acceptor. These results were qualitatively confirmed with experiments on Escherichia coli harbouring the S. cerevisiae DPM1 gene. The [3H]mannosylated lipid-linked material from microsomal incubations was purified by anion-exchange chromatography. The major saccharide component recovered after hydrolysis was determined to be mannose, but a mannose-containing disaccharide was also present. It is concluded that branching of lipid phosphates is essential for substrates of dolichyl phosphate mannose synthetase and that significant transfer of mannose occurs even if only branching at C-3 is present.
Involvement of the proteasome and antizyme in ornithine decarboxylase degradation by a reticulocyte lysate
Authors: Murakami Y , Matsufuji S , Tanaka K , Ichihara A , Hayashi S
Published: August 2015

Ornithine decarboxylase (ODC) degradation in a freshly prepared reticulocyte lysate was examined. Immunodepletion of proteasomes from the reticulocyte lysate resulted in almost complete loss of ODC degradation. In contrast with the previously reported degradation in extracts of hepatoma tissue-culture (HTC) and Chinese-hamster ovary (CHO) cells or that by the purified 26 S proteasome, efficient degradation of ODC was observed in the lysate without exogenous antizyme, an ODC protein inhibitor induced by polyamines, owing to the presence of a significant amount of antizyme in the lysate. The degradation of ODC in the lysate was strongly suppressed on inactivation of antizyme in the lysate with antizyme inhibitor, a protein which binds to the antizyme and releases ODC from the ODC-antizyme complex. Thus the main pathway for ODC degradation in a reticulocyte lysate was essentially the same as that characterized previously in extracts of HTC and CHO cells, namely an ATP- and antizyme-dependent 26 S proteasome-catalysed pathway that is presumed to be responsible for ODC degradation in whole cells.
Cytoplasmic pH regulates ATP-induced Ca2+-dependent K+-current oscillation in rat megakaryocytes
Authors: Uneyama C , Uneyama H , Takahashi M , Akaike N
Published: August 2015

The effect of cytoplasmic pH (pH(i)) on ATP-induced Ca2+ oscillation of rat megakaryocytes was investigated by the whole-cell patch-clamp technique. Megakaryocytes responded to extracellular ATP and showed the periodic activation of K+ channels, which reflects the oscillation in intracellular free Ca2+ concentration. Intracellular alkalinization by 20 mM NH4Cl resulted in inhibition of the oscillatory response, and intracellular acidification by 20 mM sodium acetate enhanced the response. NH4Cl also inhibited the Ins(1,4,5)P3-induced oscillation. Sodium acetate had no effect on the InsP3-induced oscillation, but enhanced the guanosine 5′-[gamma-thio]triphosphate-induced response. These results indicate that pH(i) modulates the ATP-induced cellular response at least at two points.
Characterization of a chelator-resistant proteinase from Thermus strain Rt4A2
Authors: Freeman S A , Peek K , Prescott M , Daniel R
Published: August 2015

The Thermus isolate Rt4A2 was found to produce an extracellular chelator-resistant proteinase. The proteinase was purified to homogeneity by (NH4)2SO4 precipitation, cation-exchange chromatography, gel-filtration chromatography, and weak anion-exchange chromatography. The Rt4A2 proteinase was found to have properties typical of an alkaline serine proteinase. It had a pH optimum of 9.0 and was specifically inhibited by phenylmethanesulphonyl fluoride. Its isoelectric point was greater than 10.25. Its molecular-mass was 31.6 kDa as determined by SDS/PAGE. N-terminal sequencing has shown it to have high sequence similarity with other serine proteinases from Thermus species. The proteinase hydrolysed a number of substrates including fibrin, casein, haemoglobin, collagen, albumin and the synthetic chromogenic peptide substrate Suc-Ala-Ala-Pro-Phe-NH-Np. The specific activity of the purified proteinase using azocasein as substrate was 313 units/mg. Substrate inhibition was observed above an azocasein concentration of 0.05% (w/v). Esterase activity was directed mainly towards those substrates containing the aliphatic or aromatic residues of alanine, glycine, tryptophan, tyrosine and phenylalanine. Thermostability half-lives of greater than 7 days at 70 degrees C, 43 h at 80 degrees C and 90 min at 90 degrees C were found in the presence of 5 mM CaCl2. At 90 degrees C increasing the CaCl2 concentration 100-fold (0.5 mM to 50 mM) caused a 4.3-fold increase in the half-life of the enzyme from 30 to 130 min. Half-lives of 19.4 min at 100 degrees C and 4.4 min at 105 degrees C were found in the presence of 50 mM CaCl2. The metal chelators EGTA and EDTA reduced the stability at higher temperatures but had no effect on the activity of the proteinase. Activity was not stimulated by common metal activators such as Ca2+, Mg2+ and Zn2+.
Dissociation, unfolding and refolding trials of pig kidney 3,4-dihydroxyphenylalanine (dopa) decarboxylase
Authors: Dominici P , Moore P S , Borri Voltattorni C
Published: August 2015

The effect of guanidinium chloride (GuCl) on enzyme activity, hydrodynamic volume, circular dichroism, and fluorescence of 3,4-dihydroxyphenylalanine (Dopa) decarboxylase from pig kidney (pkDDC) was studied under equilibrium conditions. Unfolding proceeds in at least three stages. The first transition, occurring between 0 and 1 M GuCl, gives rise to a dimeric inactive species which has lost pyridoxal 5′-phosphate (PLP), and has a high tendency to aggregate, but retains almost all of the native spectroscopic characteristics. The second equilibrium transition, between 1 and 2.2 M GuCl, involves dimer dissociation, with some loss of tertiary and secondary structure. Additionally, gross conformational changes at or near the PLP microenvironment were detected by fluorescence of NaBH4-reduced enzyme. The third step, presumably representing complete unfolding of pkDDC, appears to be complete at 4.5 M GuCl, as indicated by the lack of further substantial changes in any of the signals being studied. Attempts at refolding resulted in the findings that: (1) partial reactivation is observed only starting from enzyme denatured at concentrations below 1.5 M GuCl, and (2) starting from completely denatured protein, the refolding process is apparently reversible down to concentrations of approx. 2 M GuCl. Taken together, this would seem to indicate that the monomer-dimer transition is impaired under the experimental conditions tested. A plausible model is presented for the unfolding/refolding of pkDDC.
Matrix metalloproteinases cleave at two distinct sites on human cartilage link protein
Authors: Nguyen Q , Murphy G , Hughes C E , Mort J S , Roughley P J
Published: August 2015

The actions of human recombinant stromelysins-1 and -2, collagenase, gelatinases A and B and matrilysin on neonatal human proteoglycan aggregates were examined. With the exception of gelatinase B, aggrecan was degraded extensively by most metalloproteinases studied, whereas link protein showed only limited proteolysis. Sequencing studies of modified link protein components revealed that stromelysins-1 and -2, gelatinases A and B and collagenase cleaved specifically between His16 and Ile17, and matrilysin, stromelysin-2 and gelatinase A cleaved between Leu25 and Leu26. Cleavage at the former bond generated a link protein component with the same N-terminus as that isolated from newborn human cartilage. Based on previously determined in situ cleavage sites it is evident that matrix metalloproteinases are not solely responsible for the accumulation of link protein degradation products in adult human cartilage, indicating that additional proteolytic agents are involved in the normal catabolism of human cartilage matrix.
The substrate specificity of brain microsomal phospholipase D
Authors: Horwitz J , Davis L L
Published: August 2015

Neurotransmitters activate a phospholipase D that is though to specifically hydrolyse phosphatidylcholine. This enzyme has a unique property known as transphosphatidylation: in the presence of an appropriate nucleophilic receptor such as an alcohol, phospholipase D will catalyse the production of phosphatidyl-alcohol. We have studied phospholipase D using an in vitro assay that uses [3H]butanol of high specific radioactivity (15 Ci/mmol) as an acceptor. In the presence of [3H]butanol and phosphatidylcholine, a microsomal membrane fraction from rat brain catalysed the production of phosphatidyl[3H]butanol. Phospholipase D activity was dependent upon the presence of a detergent; the optimal sodium oleate concentration was between 4 and 6 mM. The RF of the phosphatidyl[3H]butanol on t.l.c. was identical to the RF of the phosphatidylbutanol formed when [3H]phosphatidylcholine was incubated with 100 mM butanol. These data confirm the identity of phosphatidyl[3H]butanol. One important advantage of this assay is that the substrate does not need to be labelled. We have used this advantage to examine the substrate specificity of phospholipase D. Microsomal phospholipase D appears to hydrolyse phosphatidylcholine most efficiently. There is a relatively small but significant activity against phosphatidylethanolamine and phosphatidylserine, and there is no significant activity against phosphatidylinositol. As the head-group becomes more like choline, the phospholipid becomes a better substrate for phospholipase D. The addition of one methyl group leads to a large increase in activity. Fatty acid composition does not play a role in determining the substrate specificity. This assay should be useful in furthering our understanding of this important enzyme.
F1F0-ATP synthase from bovine heart mitochondria: development of the purification of a monodisperse oligomycin-sensitive ATPase
Authors: Lutter R , Saraste M , van Walraven H S , Runswick M J , Finel M , Deatherage J F , Walker J E
Published: August 2015

A new procedure for the isolation of ATP synthase from bovine mitochondria has been developed, with the primary objective of producing enzyme suitable for crystallization trials. Proteins were extracted from mitochondrial membranes with dodecyl-beta-D-maltoside, and the ATP synthase was purified from the extract in the presence of the same detergent by a combination of ion-exchange and gel-filtration chromatography and ammonium sulphate precipitation. This simple and rapid procedure yields 20-30 mg of highly pure and monodisperse enzyme, evidently consisting of 14 different subunits, amongst them, in apparently stoichiometric amounts with the established subunits, subunit e, a recently discovered subunit of unknown function. The enzyme preparation has an oligomycin-sensitive ATP hydrolysis activity, and so the F1 domain is functionally associated with the membrane domain, F0. In contrast with the N-termini of some of the subunits of bovine mitochondrial F1-ATPase, those of the F1F0-ATP synthase are not degraded by proteolysis during the isolation procedure. This preparation therefore satisfies prerequisites for crystallization trials.
myo-inositol transport and metabolism in fetal-bovine aortic endothelial cells
Authors: Berry G T , Johanson R A , Prantner J E , States B , Yandrasitz J R
Published: August 2015

The myo-inositol transport system in confluent fetal-bovine aortic endothelial cells was characterized after 7-10 days in subculture, at which time the myo-inositol levels and rates of myo-[2-3H]-inositol uptake and incorporation into phospholipid had reached steady state. Kinetic analysis indicated that the uptake occurred by both a high-affinity transport system with an apparent Kt of 31 microM and Vmax. of 45 pmol/min per mg of protein, and a non-saturable low-affinity system. Uptake was competitively inhibited by phlorhizin, with a Ki of 50 microM; phloretin was a non-competitive inhibitor, with half-maximal inhibition between 0.2 and 0.5 mM. Glucose was a weak competitive inhibitor, with a Ki of 37 mM; galactose failed to inhibit uptake. A weak dependence on Na+ for the initial rate of uptake was observed at 11 microM myo-inositol. When fetal-bovine-serum (FBS)-supplemented medium, which contained 225 microM myo-inositol, was used, the cells contained about 200 nmol of myo-inositol/mg of DNA. With adult-bovine-serum (ABS)-supplemented medium, which contained 13 microM myo-inositol, the cells contained about 110 nmol/mg of DNA. Transport of 11 microM myo-[2-3]inositol was 18 and 125 pmol/min per mg of DNA for cells grown in FBS and ABS respectively. Kinetic analysis showed that for the cells grown in FBS the Vmax. of the high-affinity system was decreased by 64%, whereas the Kt remained essentially unchanged. Increased cell myo-inositol levels were not associated with an increased rate of phosphatidylinositol synthesis. After prolonged exposure of fetal endothelial cells to a myo-inositol concentration which approximated to a high fetal as opposed to a low adult blood level, cell myo-inositol levels doubled and high-affinity transport underwent down-regulation.
Induction of germ-cell alkaline phosphatase by butyrate and cyclic AMP in BeWo choriocarcinoma cells
Authors: Telfer J F , Green C D
Published: August 2015

BeWo choriocarcinoma cells synthesize two alkaline phosphatase isoenzymes: germ-cell alkaline phosphatase and tissue-unspecific alkaline phosphatase. We have made use of the differential heat-stabilities of these two isoenzymes to study the induction of germ-cell alkaline phosphatase by sodium butyrate and cyclic AMP (cAMP). Sodium butyrate causes a large induction of germ-cell alkaline phosphatase activity (approx. 35-fold after 96 h) after an initial lag period of 12-24 h. We showed that butyrate increases germ-cell alkaline phosphatase mRNA. Dibutyryl cAMP also induces germ cell alkaline phosphatase (approx. 2.5-fold after 96 h). When optimal concentrations of butyrate and dibutyryl cAMP were added simultaneously to cells, they caused a synergistic induction of activity. This suggested that these compounds use separate mechanisms to induce germ-cell alkaline phosphatase activity and that it is the cAMP moiety of dibutyryl cAMP that induces enzyme activity. This was confirmed by the use of two additional cAMP analogues, 8-(4-chlorophenylthio) cAMP and 8-bromo cAMP, and of two compounds, 3-methyl-1-isobutylxanthine and cholera toxin, which raise the endogenous concentration of cAMP. All four compounds caused a 2-fold increase in enzyme activity. Treatment of cells with 8-(4-chlorophenylthio) cAMP, 8-bromo cAMP and cholera toxin increased germ-cell alkaline phosphatase mRNA between 2- and 7-fold. These data suggest that this alkaline phosphatase isoenzyme is regulated at the level of its mRNA by cAMP, in a manner distinct from that of butyrate.
β
3-adrenergic receptors are responsible for the adrenergic inhibition of insulin-stimulated glucose transport in rat adipocytes
Authors: Carpéné C , Chalaux E , Lizarbe M , Estrada A , Mora C , Palacin M , Zorzano A , Lafontan M , Testar X
Published: August 2015

The inhibition of insulin-stimulated glucose transport by isoprenaline, a mixed beta-adrenergic-receptor (AR) agonist, is well documented in rat adipocytes. Since it has been described that rat adipocytes possess not only beta 1- and beta 2- but also beta 3-ARs, the influence of various subtype-selective beta-AR agonists and antagonists on 2-deoxyglucose (2-DG) transport was assessed in order to characterize the beta-AR subtype involved in the adrenergic counter-regulation of the insulin effect. The stimulation of 2-DG transport by insulin was counteracted, in a dose-dependent manner, by all the beta-AR agonists tested, and the magnitude of the inhibition followed the rank order: BRL 37344 > isoprenaline = noradrenaline >> dobutamine = procaterol. The same rank order of potency was obtained for lipolysis activation. This is not in accordance with the pharmacological definition of a beta 1- or a beta 2-adrenergic effect, but agrees with the pharmacological pattern of a beta 3-adrenergic effect. The inhibitory effect of the beta 3-agonist BRL 37344 on insulin-stimulated 2-DG transport was not reversed by either the selective beta 1-antagonist ICI 89406 or the beta 2-antagonist ICI 118551. In addition, neither of these beta-antagonists was able to block the isoprenaline and noradrenaline effects, supporting major beta 3-adrenoceptor-subtype involvement in the adrenergic inhibition of insulin-stimulated 2-DG transport. Like isoprenaline, BRL 37344 inhibited (60% inhibition) insulin-stimulated glucose transport only when adenosine deaminase was present in the assay. Furthermore, the maximal inhibitory effects of isoprenaline and BRL 37344 were not additive, and were both dependent on albumin concentration in the incubation medium: they increased when the albumin concentration decreased in the medium from 3.5 to 1%. To conclude, the similarities between isoprenaline and BRL 37344 action on insulin-stimulated 2-DG transport, the poor efficacy of the beta 1-/beta 2-agonists and the lack of effect of selective beta 1- and beta 2-antagonists are compelling arguments to support the important role of beta 3-adrenoceptors in the adrenergic inhibition of glucose transport in rat adipocytes.
Stimulation of secretion in permeabilized PC12 cells by adenosine 5′-[γ-thio]triphosphate: possible involvement of nucleoside diphosphate kinase
Authors: Vu N D , Wagner P D
Published: August 2015

The addition of Ca2+, adenosine 5′-[gamma-thio]triphosphate (ATP[S]) or guanosine 5′-[gamma-thio]triphosphate (GTP[S]) to digitonin-permeabilized PC12 cells stimulates noradrenaline secretion. Both ATP[S] and GTP[S] stimulate release in the absence of Ca2+. Whereas ADP and adenosine 5′-[beta gamma-imido]triphosphate inhibited ATP[S]-stimulated release, they did not inhibit Ca(2+)-stimulated release even in the absence of added ATP. This suggests that the kinase which uses ATP[S] to induce secretion may not play an essential role in Ca(2+)-stimulated release. As GTP[S]-stimulated and ATP[S]-stimulated secretions were not additive, it seemed possible that stimulation by ATP[S] might in part result from the thiophosphorylation of GDP by nucleoside-diphosphate (NDP) kinase to form GTP[S]. The following results are consistent with this possibility. (1) A low concentration of GDP increased ATP[S]-stimulated secretion, but not GTP[S]-stimulated or Ca(2+)-stimulated secretion. (2) A variety of ribo- and deoxyribo-nucleoside di- and tri-phosphates inhibited ATP[S]-stimulated secretion, but not GTP[S]-stimulated or Ca(2+)-stimulated secretion. Thus, like NDP kinase, the kinase which uses ATP[S] to cause noradrenaline release appears to have a very low specificity for ATP. (3) Incubation of permeabilized cells in a sucrose-containing buffer resulted in the preferential loss of ATP[S]-stimulated secretion and a decrease in the level of NDP kinase. The addition of rat liver NDP kinase to those depleted cells partially restored ATP[S]-stimulated secretion.
The regulation of hepatic carbon flux by pyruvate dehydrogenase and pyruvate dehydrogenase kinase during long-term food restriction
Authors: Sugden M C , Grimshaw R M , Holness M J
Published: August 2015

The present study investigated the effects of chronic food restriction (achieved by limiting access to food to 2 h daily for up to 8 weeks) on the activity of the active form of pyruvate dehydrogenase (PDHa) in liver. Accelerated and exaggerated activation of hepatic PDH in response to a meal, previously demonstrated to occur within 10 days of food restriction, was demonstrated to persist for 4 and 8 weeks of food restriction, despite a food intake of only 50-60% of controls. Activation of hepatic PDH during feeding in rats subjected to food restriction for 4 weeks was dependent on continued food intake. As a consequence, hepatic PDHa activities in food-restricted rats were suppressed relative to controls for 19 h of the 24 h daily cycle. Curve-fitting by second-order polynomial regression analysis demonstrated a significant positive correlation between hepatic PDHa activity and lipogenic rate over the range of PDHa activities observed during the 2 h feeding period. Increased lipogenesis during feeding in food-restricted rats was not at the expense of hepatic glycogen synthesis or deposition; measurement of concurrent rates of glycogenesis and lipogenesis revealed simultaneous flux through both pathways, but specific activation of lipogenesis. The accelerated re-activation of hepatic PDH observed within 1 h of feeding in rats subjected to 4 weeks of food restriction was facilitated by a failure of the 22 h interprandial fasting period to induce a stable increase in hepatic PDH kinase activity. The present study indicates differential regulation of hepatic PDH kinase activity during periods of food withdrawal between food-restricted rats and starved/re-fed control rats. Such regulation occupies a critical role in determining the rate of activation of hepatic PDH during feeding. In turn, increased activity of hepatic PDHa during feeding in food-restricted rats bears a close positive relationship with hepatic lipogenic rate.
Lipopeptides activate Gi-proteins in dibutyryl cyclic AMP-differentiated HL-60 cells
Authors: Klinker J F , Höer A , Schwaner I , Offermanns S , Wenzel-Seifert K , Seifert R
Published: August 2015

Synthetic lipopeptides activate superoxide-anion (O2-) formation in human neutrophils in a pertussis-toxin (PTX)-sensitive manner, suggesting the involvement of G-proteins of the Gi family in the signal-transduction pathway. We compared G-protein activation by lipopeptides and the chemotactic peptide N-formylmethionyl-leucyl-phenylalanine (fMLP) in dibutyryl-cyclic-AMP-differentiated HL-60 cells. The lipopeptide (2S)-2-palmitoylamino-6-palmitoyloxymethyl-7-palmitoyloxy heptanoyl-SK4 (Pam3AhhSK4) and fMLP activated high-affinity GTPase, i.e. the enzymic activity of G-protein alpha-subunits, in HL-60 membranes in a time- and protein-dependent manner, but they had no effect on Mg(2+)-ATPase and Na+/K(+)-ATPase. Pam3AhhSK4 and fMLP increased Vmax. of GTP hydrolysis. Pam3AhhSK4 activated GTP hydrolysis with half-maximal and maximal effects at about 2 microM and 10 microM respectively. Other lipopeptides activated GTP hydrolysis as well. Lipopeptides were less effective than fMLP to activate GTPase. In membranes from PTX-treated cells, the stimulatory effects of lipopeptides and fMLP on GTPase were abolished. In N-ethylmaleimide-treated membranes, the relative stimulatory effect of Pam3AhhSK4 on GTP hydrolysis was enhanced, whereas that of fMLP was diminished. fMLP and Pam3AhhSK4 activated GTPase in an over-additive manner in N-ethylmaleimide-treated membranes. Unlike fMLP, Pam3AhhSK4 did not enhance incorporation of GTP azidoanilide into, and cholera-toxin-catalysed ADP-ribosylation of Gi-protein alpha-subunits in, HL-60 membranes and did not induce rises in cytosolic Ca2+ concentration. Pam3AhhSK4 and fMLP stimulated phosphatidic acid formation in a PTX-sensitive manner. Pam3AhhSK4 itself did not activate O2- formation, but potentiated the stimulatory effects of fMLP. Our data suggest that (i) lipopeptides activate the GTPase of Gi-proteins, (ii) lipopeptides and fMLP activate Gi-proteins differently, (iii) lipopeptides stimulate phospholipase D via Gi-proteins, and (iv) phosphatidic acid formation is not sufficient for activation of O2- formation.
Purification of highly intact plastids from various heterotrophic plant tissues: analysis of enzymic equipment and precursor dependency for starch biosynthesis
Authors: Neuhaus H E , Batz O , Thom E , Scheibe R
Published: August 2015

Starting with a protocol originally developed for the purification of intact plastids from cauliflower buds [Journet and Douce (1985) Plant Physiol. 79, 458-467] we have modified this method to obtain intact heterotrophic plastids from etiolated barley leaves (Hordeum vulgare) and pea (Pisum sativum) and maize (Zea mays) endosperm. Two subsequent centrifugation steps on Percoll gradients were performed, the first as an isopycnic, the second as zonal, centrifugation step in a swing-out rotor. Percoll density and centrifugation time were adjusted for the various tissues. The obtained plastid preparations are characterized by a low degree of contamination with other cellular components and an intactness of at least 90%. In isolated maize endosperm amyloplasts, starch synthesis is driven by exogenously applied hexose phosphates (glucose 6-phosphate and glucose 1-phosphate) rather than by dihydroxyacetone phosphate. The hexose-phosphate-dependent starch synthesis is strictly dependent upon the intactness of the plastids and is increased up to 9-fold when ATP and 3-phosphoglyceric acid are added to the incubation medium. The occurrence of fructose-1,6-bisphosphatase and malate dehydrogenases in some plastid types is discussed in relation to their possible role in starch synthesis.