Cellulose Phosphate Research Articles

Protein productivity of cell-free translation was improved by removing phosphatase from wheat germ extract with immunoprecipitation

The biochemical energy charge, ([ATP] + [ADP]/2)/([ATP] + [ADP] + [AMP]), is necessary to be kept at a high level close to 1.0 for protein synthesis in a cell-free translation system. Aiming at improvement of protein productivity of wheat germ extract (WGE) cell-free translation system, the authors developed a method of maintaining the energy charge at the high level by removing acid phosphatase activity of hydrolyzing ATP from the system. About 3 unit/ml activity of acid phosphatase (ACP) was detected in WGE. The detected activity of ACP accounted for 90% of ATP consumption in WGE cell-free translation system. Immunoprecipitation of ACP with ACP-specific antiserum reduced ACP activity in WGE by 60%. As the result, the energy charge in the translation system was kept above 0.97 for 150 min when the antiserum-treated WGE was used, while it was kept so for only 40 min when the untreated WGE was used. The protein productivity of the translation system increased 5-fold by the immunoprecipitation of ACP. Affinity separation of ACP with cellulose phosphate, or inhibition of ACP activity with inhibitors such as molybdate and vanadate reduced ACP activity in the translation system. However, these non-biological methods resulted in decrease rather than increase in protein productivity.

Purification of peroxidase isoenzymes from turnip roots

Simple reproducible procedures for purification of the main soluble (S) and ionically bound (IB) cationic peroxidase isoenzymes from turnip roots were established. The procedures included ammonium sulfate precipitation of the isoenzymes, chromatographic separation of the main isoenzymes using cellulose phosphate columns and purification to homogeneity by hydrophobic interaction chromatography on phenyl Sepharose columns. The specific activity of the phenyl Sepharose purified S and IB isoenzymes were 2760 and 896 units/mg protein with 140 and 4.8 fold increase over the crude extract and 38 and 13% recovery. The pH maxima and K m for phenol and H 2O 2 of purified S and IB were determined.

Thermal degradation of cellulose and cellulose esters

Cellulose, cellulose diacetate (CDA), cellulose triacetate (CTA), cellulose nitrate (CN), and cellulose phosphate (CP) were subjected to dynamic thermogravimetry in nitrogen and air. The thermostability of the cellulose and its esters was estimated, taking into account the values of initial thermal degradation temperature Td, the temperature at the maximum degradation rate Tdm, and char yield at 400°C. The results show that these polymers may be arranged in the following order of increasing thermostability: CN < CP < regenerated cellulose < filter cotton < CDA < CTA. The activation energy (E), order (n), and frequency factor (Z) of their degradation reactions were obtained following the Friedman, Chang, Coats–Redfern, Freeman–Carroll, and Kissinger methods. The dependence of Td, Tdm, E, n, Ln Z, and char yield at 400°C on molecular weight and test atmosphere is also discussed. © 1998 John Wiley & Sons, Inc. J Appl Polym Sci 68:293–304, 1998

Prevotella intermedia が産生する酸性ホスファターゼに関する研究

Prevotella intermedia (P. intermedia), an anaerobic gram-negative bacterium, has been implicated as a pathogen in adult periodontitis, in acute necrotizing ulcerative gingivitis and in pregnancy gingivitis. P. intermedia has been known to have a high acid phosphatase (ACP) activity, one of the potential virulence factors in periodontopathogenic bacteria. However, little is known about the structure and function of ACPs from oral bacteria. In this study, in order to examine the potential role of ACP, the ACP from P. intermedia ATCC 25611 was purified to homogeneity and characterized. The ACP from P. intermedia was solubilized by extraction with 1% Triton X-114, and the crude extract was purified by ion-exchange chromatography on DEAE Bio-Gel, CM Bio-Gel and cellulose phosphate, followed by hydrophobic chromatography on Phenyl-Sepharose. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the purified enzyme revealed a single protein band with a molecular weight of Mr &ap;30 kDa. The molecular weight of the native enzyme from P. intermedia was estimated to be &ap;30 kDa using a gel filtration column with TSK gel G3000SW in the HPLC system. These findings suggest that the purified ACP is a monomer. The optimum pH for the enzyme was about 4.9 and the optimum temperature was 46℃. The enzyme activity was inhibited by sodium orthovanadate, sodium molybdate, sodium fluoride and Zn^<2+>, which are known as the inhibitors of protein tyrosine phosphatase (PTP) activity. On the other hand, it was not inhibited by okadaic acid, which is known to be a specific inhibitor of protein serine/threonine phosphatase (PP) activity. The enzyme had high activities against o-phospho-L-tyrosine and phosphotyrosine-containing peptides. The purified ACP was found to dephosphorylate the tyrosine-phosphorylated protein of a molecular weight &ap;170 kDa in an A431 cell lysate. The N-terminal amino acid sequence was determined for the purified ACP, and significant homology (64.3% identical amino acid residues) was found using the Swiss Prot Protein database between the purified ACP and the Pho C, the acid phosphatase of Morganella morganii. Thus, these findings suggest the possibility that the ACP from P. intermedia constitutes a novel class of ACPs, which has a PTP-like activity.

Preconcentration of heavy metals ions from aqueous solutions by means of cellulose phosphate: an application in water analysis

Cellulose phosphate (CELLPHOS) was studied as a collector for analytical preconcentration of traces of Cd(II), Cr(III), Cu(II) and Ni(II) from aqueous sample solution. It has been proved that using chromatographic columns packed with CELLPHOS for preconcentration and 1.0 mol 1 −1 HCl for elution the adsorbed analytes are quantitatively enriched. An enrichment factor of 20 (100 ml sample, 5 ml concentrate) was achieved by this separation procedure, which was applied to a series of water analyses (river, sea, bog water).

Compression force/time-profiles of microcrystalline cellulose, dicalcium phosphate dihydrate and their binary mixtures—a critical consideration of experimental parameters

Compression force/time-profiles of microcrystalline cellulose and dicalcium phosphate dihydrate and mixtures thereof were compared on a modern rotary press by three different methods of compression: compression to a constant tablet weight, compression to a constant tablet height and compression from a constant filling depth. Compression was carried out at three different compression force levels. The differences obtained by analysing the area under the curve in the compression, dwell and decompression phase could be explained by differences of the in-die working density of the powders and their respective compression behaviour. From data of the compression phase as well as from the area-quotient-index obtained from the dwell time it was possible to estimate the percolation threshold of dicalcium phosphate dihydrate in a mixture with microcrystalline cellulose to ~ 50% (w/w) corresponding to 30% (v/v).

Substrate specificities of 3-oxoacyl-CoA thiolase A and sterol carrier protein 2/3-oxoacyl-CoA thiolase purified from normal rat liver peroxisomes. Sterol carrier protein 2/3-oxoacyl-CoA thiolase is involved in the metabolism of 2-methyl-branched fatty acids and bile acid intermediates.

The two main thiolase activities present in isolated peroxisomes from normal rat liver were purified to near homogeneity. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the first enzyme preparation displayed a single band of 41 kDa that was identified as 3-oxoacyl-CoA thiolase A (thiolase A) by N-terminal amino acid sequencing. The second enzyme preparation consisted of a 58- and a 46-kDa band. The 58-kDa polypeptide reacted with antibodies raised against either sterol carrier protein 2 or the thiolase domain of sterol carrier protein 2/3-oxoacyl-CoA thiolase (SCP-2/thiolase), formerly also called sterol carrier protein X, whereas the 46-kDa polypeptide reacted only with the antibodies raised against the thiolase domain. Internal peptide sequencing confirmed that the 58-kDa polypeptide is SCP-2/thiolase and that the 46-kDa polypeptide is the thiolase domain of SCP-2/thiolase. Thiolase A catalyzed the cleavage of short, medium, and long straight chain 3-oxoacyl-CoAs, medium chain 3-oxoacyl-CoAs being the best substrates. The enzyme was inactive with the 2-methyl-branched 3-oxo-2-methylpalmitoyl-CoA and with the bile acid intermediate 24-oxo-trihydroxycoprostanoyl-CoA. SCP-2/thiolase was active with medium and long straight chain 3-oxoacyl-CoAs but also with the 2-methyl-branched 3-oxoacyl-CoA and the bile acid intermediate. In peroxisomal extracts, more than 90% of the thiolase activity toward straight chain 3-oxoacyl-CoAs was associated with thiolase A. Kinetic parameters (Km and Vmax) were determined for each enzyme with the different substrates. Our results indicate the following: 1) the two (main) thiolases present in peroxisomes from normal rat liver are thiolase A and SCP-2/thiolase; 2) thiolase A is responsible for the thiolytic cleavage of straight chain 3-oxoacyl-CoAs; and 3) SCP-2/thiolase is responsible for the thiolytic cleavage of the 3-oxoacyl-CoA derivatives of 2-methyl-branched fatty acids and the side chain of cholesterol.

Lability of heavy metal species in aquatic humic substances characterized by ion exchange with cellulose phosphate

Labile metal species in aquatic humic substances (HSs) were characterized by ion exchange on cellulose phosphate (CellPhos) by applying an optimized batch procedure. The HSs investigated were pre-extracted from humic-rich waters by ultrafiltration and a resin XAD 8 procedure. The HS—metal species studied were formed by complexation with Cd(II), Ni(II), Cu(II), Mn(II) and Pb(II) as a function of time and the ratio ions to HSs. The kinetics and reaction order of this exchange process were studied. At the beginning (<3 min), the labile metal fractions are separated relatively quickly. After 3 min, the separation of the metal ions proceeds with uniform half-lives of about 12–14 min, revealing rather slow first-order kinetics. The metal exchange between HSs and CellPhos exhibited the following order of metal lability with the studied HSs: Cu > Pb > Mn > Ni > Cd. The required metal determinations were carried out by atomic absorption spectrometry.

Partial Replacement of Egg White Proteins with Whey Proteins in Angel Food Cakes

ABSTRACTThis study was conducted to investigate the effects of partial replacement of egg white proteins (EWP) with whey protein isolate (WPI) on the appearance, structure, texture, and sensory properties of angel food cakes baked in conventional and microwave/conventional ovens. Factors studied were: 1) replacement of 25% or 50% of EWP with WPI; 2) added xanthan gum (XG), methyl cellulose (MC), cupric sulfate (Cu+2) or sodium phosphate (PHOS); and 3) conventional vs microwave/conventional oven baking. EWP replacement cakes without additives were generally inferior to 100% EWP control cakes, whereas EWP replacement cakes with added XG were most similar to 100% EWP control cakes with respect to appearance, texture, and sensory properties and those with added MC exhibited air cell size distributions that most closely resembled control cakes. The other additives and microwave/ conventional vs conventional baking had minor effects on the quality of EWP replacement cakes.

Purification of Mammalian Ribonuclease Using Immobilized Human Ribonuclease Inhibitor

Affinity chromatography on immobilized ribonuclease (RNase) inhibitor was developed for purification of mammalian RNase. Human placental RNase inhibitor was conjugated to CNBr-activated Sepharose in the presence of dithiothreitol. About 80% of the immobilized RNase inhibitor was capable of binding bovine pancreatic RNase A. The bound RNase A was eluted with 3MNaCl at pH 5.0. Two 25-kDa and 18-kDa RNases, which were obtained from human liver using a cellulose phosphate column, were bound to the immobilized RNase inhibitor and recovered in a pure and active form after treatment of the resin withp-hydroxymercuribenzoate. These enzymes were considered to be nonsecretory-type RNases with different sugar contents.

Spray-Dried Rice Starch: Comparative Evaluation of Direct Compression Fillers

AbstractSpray-dried rice starch (SDRS), microcrystalline cellulose (MCC), lactose (L), pregelatinized starch (PS), and dibasic calcium phosphate (DCP) were studied for their flow behaviors and tableting properties. Both flow rate and percent compressibility values indicated that SDRS exhibited excellent flowability. The increase in magnesium stearate content reduced the hardness of MCC and SDRS tablets; however, general tablet properties were still acceptable while the PS tablets were unsatisfactory at high lubricant concentrations. The hardness of L or DCP tablets was not affected by the lubricant. The disintegration of L tablets was prolonged with the increased lubricant concentration while that of PS tablets seemed to be decreased due to softened tablets. The disintegration times of MCC and SDRS tablets seemed to be independent of the lubricant added. With respect to the dissolution, SDRS-based tablets offered fast and complete release of the drug regardless of its solubility. SDRS, L, and DCP exhibite...

Microcharacterization of a new injectable polymer/ceramic composite as bone substitute in spine surgery

The results of microscopic and spectroscopic analysis of a mixture of biphasic calcium phosphate (BCP) powder and cellulose ether aqueous sol are reported in this study. This new composite is injectable and is aimed to be used as bone substitute for spine surgery. The influence of the polymer on the physico-chemical properties of BCP was investigated by complementary techniques such as scanning electron microscopy (SEM), high-resolution transmission electron microscopy (HR-TEM), X-ray diffraction (XRD), infrared and Raman spectroscopies and X-ray photoelectron spectroscopy (XPS). When brought into contact with the polymer solution, the ceramic is modified in its granulometry, crystallinity, and surface chemistry. A dissolution/precipitation reaction occurs, leading to a phosphorus-rich and amorphous outer layer of the ceramic surface. Moreover, the bonding state of the composite involves new features that could be assigned to calcium complexes (calcium carbonate or calcium hydroxide).

Determining the site and nature of DNA mutations with the cloned MutY mismatch repair enzyme.

The Escherichia coli MutY gene was cloned into a modified pET-11 plasmid which was then transfected into an E.coli HMS174 host for overproduction of the MutY mismatch repair (MR) enzyme. Approximately 30-50% of the total cellular protein in the transformed HMS174 cells was isopropyl-beta-D-thiogalactoside-induced MutY protein, as estimated from the staining intensity on an SDS-PAGE gel following electrophoresis. The MutY protein was purified to near homogeneity by cellulose phosphate ion-exchange chromatography followed by gel filtration chromatography. The purified MutY protein had enzyme activities which cleaved the A of a G/A mismatch at the 3' end of the first phosphodiester bond and then the 5' end of the second phosphodiester bond of the A. It also cut the A of a C/A mismatch, but to a much lesser extent, and the activity was DNA sequence-dependent. The reliability of the assay in determining the site and nature of a DNA mutation was examined in human tumor DNA samples with known or unknown p53 mutations. In the assay, polymerase chain reaction-amplified DNA fragments from normal and mutated p53 genes were mixed, denatured and annealed to generate mismatches of G/A or C/A for cleavage by the MutY MR enzyme. The assay results revealed the site and nature of known G:C<-->T:A mutations. In addition, a previously unknown G:C to T:A mutation, which was misread in the sequencing analysis of a tumor DNA preparation, was identified by this assay.

Human mast cell tryptase isoforms: separation and examination of substrate-specificity differences.

Tryptases are trypsin-like enzymes found in mast cell granules that appear to exist as tetramers. These enzymes are not controlled by blood plasma proteinase inhibitors and only cleave a few physiological substrates in vitro, including high-molecular-mass kininogen (HMMK) and vasoactive intestinal peptide (VIP). Purified human lung mast cell tryptase (HLT) contained two bands of approx. molecular mass 29 and 33 kDa on SDS/PAGE. These two forms of HLT have been separated by chromatography on a cellulose phosphate column, with the high-molecular-mass form (high-HLT) being eluted with 10 microM heparin and the low-molecular-mass form (low-HLT) subsequently eluted with 1 M NaCl. Removal of asparagine-linked carbohydrate caused both isoforms to run as single sharp bands on SDS/PAGE, differing slightly in molecular mass. Separation of these two isoforms of tryptase shows that tetramers consist of four homologous subunits rather than mixtures of the two isoforms. Using HMMK and VIP as substrates, these two forms of HLT were found to differ with regard to specificity and rate of cleavage. High-HLT initially cleaved HMMK at Arg-431 within the C-terminal anionic binding region of the molecule, whereas low-HLT cleaved HMMK simultaneously at multiple sites within the C-terminal portion of the molecule. On the basis of HPLC peptide mapping, each isoform also cleaved VIP at different sites. Comparison of cleavage rates based on the active-site concentrations of titrated isoforms showed that low-HLT cleaved HMMK more rapidly than did high-HLT. These two isoforms may represent different gene products or they may result from post-translational modification.

The 40 kDa 63Ni 2+-binding protein (pNiXc) on Western blots of Xenopus laevis oocytes and embryos is the monomer of fructose-1,6-bisphosphate aldolase A

A Ni 2+-binding protein (pNiXc, 40 kDa), present in Xenopus laevis oocytes and embryos, was isolated from mature oocytes by chromatography on DEAE-cellulose and cellulose phosphate, followed by FPLC on Ni-iminodiacetate-Agarose, or reverse-phase HPLC on a C-4 column. Size-exclusion HPLC showed that intact pNiXc is ≈ 155 kDa, consistent with tetrametric structure, After cleavage with Lys-C proteinase or cyanogen bromide, six peptides were separated by HPLC and sequenced by Edman degradation, providing sequence data for 83 residues. Data-base search showed similarity of pNiXc to eukaryotic aldolases, with 96% identity to human aldolase A. pNiXc demonstrated aldolase activity with fructose 1,6-bisphosphate as substrate ( K m, 30 μM V max 26 μmol min −1mg −1); the aldolase activity was inhibited non-competitively by Cu 2+, Cd 2+, Co 2+, or Ni 2+. Equilibrium dialysis showed high affinity binding ( K D, 7μM) of 1 mole of Ni per mole of 40 kDa subunit. Based on metal-blot competition assays, the abilities of metals to compete with 63Ni 2+ for binding to pNiXc were ranked: Cu 2+ ⪢ Zn 2+ > Cd 2+ > Co 2+. This study identifies pNiXc as the monomer of fructose-1,6-bisphosphate aldolase A, and raises the possibility that aldolase A is a target enzyme for metal toxicity.

Thermophysical properties of some pharmaceutical excipients compressed in tablets.

Thermophysical properties of three tableting excipients; microcrystalline cellulose, lactose and dicalcium phosphate dihydrate were observed to evaluate their ability to resist temperature induced changes in tablet form. Two thermophysical parameters, thermal diffusivity and specific heat, were measured by a pulse heating method. The materials were also evaluated by differential scanning calorimetry (DSC). Microcrystalline cellulose in tablet form was found to be rather insensitive to heating and cooling treatments, even though the tablets seemed to remain in a stressed state four weeks after tableting. This stress, indicated by low temperature anomalies, was observed by the pulse method, but not by DSC. When magnesium stearate was incorporated as a lubricant within the microcrystalline cellulose powder, the thermophysical parameters indicated that the internal structure of the tablets changed with heating and cooling. Magnesium stearate eliminated the low temperature anomalies as well. The heat treatment changed the thermophysical properties of tablets made of the crystalline excipients lactose and dicalcium phosphate dihydrate, permanently causing irreversible structural changes. The melting of the lubricant together with enhanced stress relaxation in the structure of microcrystalline cellulose most probably caused the improved thermal diffusivity. The observed thermophysical changes with the crystalline excipients were due to changes in tablet's structure and material. The combination of methods used was found to be an accurate and reliable way to obtain useful information on the structural changes and material relaxations of intact tablets during temperature treatment and age-related changes in material properties.

Evolution of carbon structure in chemically activated wood

13C NMR and FTIR analyses have been employed to follow the evolution of chemical structure in relation to porosity development, as a function of heat treatment temperature (HTT), for activated carbons produced from white oak by phosphoric acid activation. The chemical changes effected by acid treatment at low HTT are: by 50 °C there is significant alteration of the lignin structure; by 100 °C a significant portion of the cellulose has reacted, with the formation of ketones and esters; the formation of phosphate esters becomes apparent around 150 °C; crosslinking reactions are initiated below 150 °C, consistent with the higher carbon yield obtained in chemical activation; and generally there is an increase in aromaticity and loss of aliphatic, carboxyl, and carbonyl groups. The low temperature phenomena precede, and relate to, the development of porosity and structural dilation that commences around 250 °C, and attains a maximum between 350 and 450 °C. Up to 450 °C, pore volume is found to correlate with crosslink density. Above 450 °C, there is a dimensional contraction and a reduction in porosity. Among the accompanying phenomena are: the elimination of cellulose phosphates and oxygen functionalities; and a dramatic increase in the estimated aromatic cluster size. The latter would require a reduction in crosslink density to facilitate cluster growth, and the resulting structural rearrangement and increased alignment of clusters would produce a more densely packed structure with reduced porosity.

5-enol-Pyruvyl-Shikimate-3-Phosphate Synthase from Zea mays Cultured Cells (Purification and Properties).

The shikimate pathway enzyme 5-enol-pyruvyl-shikimate-3-phosphate (EPSP) synthase (3-phosphoshikimate-1-carboxyvinyl transferase, EC 2.5.1.19) was purified from cultured maize (Zea mays L. var Black Mexican Sweet) cells. Homogeneous enzyme preparations were obtained by a four-step procedure using ammonium sulfate fractionation, anion- and cation-exchange chromatography, and substrate elution from a cellulose phosphate column. The last step resulted in two well-separated activities of about the same molecular weight. A 2000- to 3000-fold purification, with an overall recovery of one-fourth of the initial activity, was achieved. Both EPSP synthase isoforms were characterized with respect to structural, kinetic, and biochemical properties. Only slight differences are seen in molecular mass, activation energy, and apparent affinities for the two substrates. A more pronounced difference was found between their thermal inactivation rates. Two EPSP synthase isoforms were also elucidated in crude homogenates by anion-exchange fast protein liquid chromatography. This allowed us to follow their expression during a culture growth cycle. One form was found at substantial levels throughout, whereas the other increased in exponentially growing cells and declined in late-logarithmic phase. The analysis of highly purified plastid preparations demonstrated a plastidial localization of both proteins. Possible functional roles for maize EPSP synthase isozymes, with regard to the dual-pathway hypothesis and to the recent findings on defense-related aromatic biosynthesis in higher plants, are discussed.

Determination of Molybdenum and Vanadium in Seawater by Ion-Exchange Preconcentration-Inductively Coupled Plasma Atomic Emission Spectrometry

A simple and rapid method has been developed for the determination of molybdenum and vanadium in seawater using ion-exchange preconcentration and inductively coupled plasma atomic emission spectrometry (ICP-AES). One hundred milliliters of seawater prepared as 0.05 M hydrochloric acid solution is passed through a cellulose phosphate column, and molybdenum and vanadium adsorbed on the cellulose are eluted simultaneously with dilute ammonia solution. The effluent collected is evaporated to a small volume, in which molybdenum and vanadium can be determined by ICP-AES. The overall recoveries of molybdenum and vanadium are 93.6 and 80.4%, respectively, at the level of 1 μgMo/100 ml and 0.1 μgV/100 ml. The proposed method has been successfully applied to the determination of the two elements in several seawater samples. Relative standard deviations (n = 3) are 2.0-2.9 and 0.3-4.3% for molybdenum and vanadium, respectively.

Purification and characterization of calmodulin (lysine 115) N-methyltransferase from Paramecium tetraurelia

Calmodulin (lysine 115) N-methyltransferase was purified from the cytosolic fraction of Paramecium tetraurelia by sequential dialysis, cellulose phosphate chromatography, Reactive Red 120 agarose chromatography, and calmodulin-Sepharose affinity chromatography. The enzyme was purified 6800-fold with a 15% yield. SDS-PAGE analysis of the purified enzyme invariably revealed a major protein of 37 kDa that was reproducibly obtained and minor proteins of 35 and 28 kDa that were sometimes obtained in variable yields. The enzyme formed a mixture of mono-, di-, and trimethyllysine residues at lysine 115 of calmodulin in vitro, had a K m for the methyl donor, S-adenosyl methionine (AdoMet), of about 1 μM and a pH optimum of about 7.5. The purified enzyme had an absolute requirement for the reductant DTT for activity, whereas the enzyme in crude fractions did not. The enzyme is a monomer with an estimated molecular mass of 33 kDa. Ca 2+, Mg 2+, Mn 2+, and Ni 2+ stimulated calmodulin N-methyltransferase activity but Zn 2+ did not. Calmodulin N-methyltransferase was inhibited by its reaction product S-adenosyl homocysteine (SAH), but not by sinefungin and tubercidin. The calmodulin antagonists calmidazolium and mellitin were inhibitory out W7 was not. The enzyme was not stimulated by Triton X-100 nor by NaCl. Only calmodulins with an unmethylated lysine at residue 115, including cam2 calmodulin, were substrates. Histones and calcium-binding proteins from Paramecium other than calmodulin did not act as substrates for the purified calmodulin N-methyltransferase and no other substrates in the cytosolic fraction were observed.