Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P00790 (PGA)
 2,475 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The hydrolysis kinetics of native and denatured haemoglobin, using pepsin immobilized on aluminium oxide, was studied in order to produce hydrolysates containing bioactive peptides. Pepsin was immobilized on acidic alumina and on 2-ethanolamine- O -phosphate (2-EAOP)-modified acidic alumina. Surface charge of the two supports was determined as a function of pH. Kinetic studies were performed at 23 degrees C in 0.1 M acetate buffer, pH 4.5. At this pH, the surface charge of the two supports was almost the same. The coating of alumina by 2-EAOP only introduced a two carbon spacer between alumina surface and the reaction medium. Adsorption on the two supports of haemoglobin, haem and peptides produced in the course of hydrolyses were compared. Fixation of 2-EAOP on a pepsin-alumina complex gave hydrolysis kinetics of urea-denatured haemoglobin close to that obtained with the same amount of pepsin in solution, but with comparatively less adsorption of peptides and complete adsorption of haem. Heterogeneous hydrolyses of haemoglobin with pepsin, immobilized on functionalized alumina, resulted in the presence of VV-haemorphin-4, VV-haemorphin-7 and neokyotorphin in the supernatants without haem, the presence of which makes further purification difficult.
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PMID:A kinetic study of bovine haemoglobin hydrolysis by pepsin immobilized on a functionalized alumina to prepare hydrolysates containing bioactive peptides. 1503 40

The allosteric enzyme ADP-Glc pyrophosphorylase (AGPase) catalyzes the synthesis of ADP-Glc, a rate-limiting step in starch synthesis. Plant AGPases are heterotetramers, most of which are activated by 3-phosphoglyceric acid (3-PGA) and inhibited by phosphate. The objectives of these studies were to test a hypothesis concerning the relative roles of the two subunits and to identify regions in the subunits important in allosteric regulation. We exploited an Escherichia coli expression system and mosaic AGPases composed of potato (Solanum tuberosum) tuber and maize (Zea mays) endosperm subunit fragments to pursue this objective. Whereas potato and maize subunits have long been separated by speciation and evolution, they are sufficiently similar to form active mosaic enzymes. Potato tuber and maize endosperm AGPases exhibit radically different allosteric properties. Hence, comparing the kinetic properties of the mosaics to those of the maize endosperm and potato tuber AGPases has enabled us to identify regions important in regulation. The data herein conclusively show that both subunits are involved in the allosteric regulation of AGPase. Alterations in the small subunit condition drastically different allosteric properties. In addition, extent of 3-PGA activation and extent of 3-PGA affinity were found to be separate entities, mapping to different regions in both subunits.
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PMID:Both subunits of ADP-glucose pyrophosphorylase are regulatory. 1512 37

The purpose of this study was to qualitatively and quantitatively determine potential cellulose acetate butyrate (CAB) extractables in a way to meaningfully predict the in vivo exposure resulting from clinical administration. Extractions of CAB-381-20 were performed in several solvent systems, consistently resulting in the detection of three extractables. The extractables have been identified as acetic acid, butyric acid, and E-2-ethyl-2-hexenoic acid (E-EHA) by LC/UV, LC/MS and NMR. Extraction studies of CAB powders in acetonitrile/phosphate buffer demonstrated quantitative extraction in 1 h for acetic acid (approximately 150 microg/g), butyric acid (approximately 200 microg/g), and EHA (approximately 20 microg/g). Subsequently, extraction studies for CAB powders and coated tablets in USP simulated gastric and intestinal fluids were performed to evaluate potential in vivo exposure. Similarly, acetic and butyric acids were quantitatively extracted from CAB-381-20 powder after 24 h exposure in both USP simulated fluids. The amounts of EHA extracted from CAB powder after 24 h were determined to be 2 and 16 microg/g in USP simulated gastric and intestinal fluids, respectively. After 24 h exposure in USP simulated fluids, the maximum amount of EHA extracted corresponds to < 0.3 microg of EHA per tablet. Pepsin and pancreatin in USP simulated fluids had no effect on EHA extraction and quantitation.
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PMID:Identification and quantitation of extractables from cellulose acetate butyrate (CAB) and estimation of their in vivo exposure levels. 1519 22

The changing morphology of quenched polyglycolide (PGA) is investigated during hydrolytic degradation in phosphate buffered saline at pH 7.4. Analysis techniques include small and wide-angle X-ray scattering (SAXS and WAXS), mass measurements, DSC, pH measurement and UV-spectrophotometry. It is postulated that the degradation process can be separated into four distinct stages. In stage I, water diffuses quickly into the sample. During stage II, the polymer crystallizes by insertion crystallization, whilst the molecular weight gradually falls. This stage is characterized by a dramatic fall in the long period together with an increase in the crystallinity, minimal mass loss and minimal water uptake. At the onset of stage III, at around 10 days, a critical molecular weight is reached. Degradation products are now small enough to diffuse from the surface of the sample which begins to swell, water diffuses into the space created, and the crystals are freed from constraint. A co-operation between degradation products diffusing out of the sample and the water diffusing in causes "reaction-erosion" fronts to develop inside the sample. Ahead of these fronts, the trapped acidic degradation products remain to catalyze the hydrolysis. Stage III is characterized by swelling and an increase in the long period, together with mass loss and further water uptake. It is postulated that these reaction-erosion fronts move through the sample and meet in the centre at the beginning of stage IV, at which point the degradation again becomes homogeneous throughout the sample.
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PMID:Polyglycolide: degradation and drug release. Part I: changes in morphology during degradation. 1534 29

Considering Alcaligenes faecalis pencillin G acylase(AfPGA), which possesses the attractive characteristics for beta-lactam antibiotics conversions, the gene of PGA was cloned into an expressing vector pKKFPGA. The recombinant plasmid contained multicopy replicon(COLE 1), trc promoter, AfPGA gene, rrnB transcript terminator and ampicillin marker transformed Escherichia coli DH5alpha. As both the recombinant plasmid and the host DH5alpha had no laclq gene, the trc promoter was always active and the AfPGA could be constitutively expressed without IPTG induction in the host DH5alpha. In the shaking flask, the recombinant cell was inoculated into the fermentation medium (tryptone 10g/L, yeast extract 5g/L, MgSO4 x 7 H2O 1g, KH2 PO4 2g/L, K2HPO4 x 3H2O 5g/L, Na2HPO4 x 12H2O 7g/L, (NH4)2SO4 1.2g/L, NH4Cl 0.2 g/L, NaCl 0.1g/L, dextrin 30g/L) and cultured at 28 degrees C for 20h. The production of AfPGA reached 2,590u/L(NIPAB method), with a cell-density-specific activity of more than 300(u/L)/A600, this yield increased 432 fold higher than the native expression of Alcaligenes faecalis . Without ammonium sulphate fractionation and dialysis, the supernatant of crude extract was directly loaded on DEAE-Sepharose CL 6B column equilibrated by phosphate buffer (50mmol/L, pH7.8), and the enzyme fraction was not absorbed on the column but impurities were absorbed. Subsequently the effluent was added ammonium sulphate to 1mol/L and loaded on Butyl-Sepharose CL 4B column equilibrated by 50mmol/L phosphate buffer pH7.8-1mol/L ammonium sulphate. The enzyme was eluted as concentration of ammonium sulphate in phosphate buffer decreased to 0, PGA was eluted. After these two column chromatography, the enzyme was enriched 20 times with a 91% activity recovery. The purified enzyme had a specific activity of 68.6u/mg protein. However, the overproduction of PGA was often limited by translocation and/or periplasmic processing steps, subsequently resulted in intracellular accumulation of various types of PGA precursors and then formed inclusion bodies in the cytoplasm and/or periplasm. In this study, 5% PGA precursors formed as inclusion bodies in the cytoplasm while no inclusion bodies formed in the periplasm. It suggested most PGA precursors were transported to the periplasm and matured to active PGA and also explained why PGA gene was highly expressed in the host DH5alpha. On the other hand, inclusion bodies in the cytoplasm indicated that the maturation of PGA in the host DHSalpha was limited by the translocation step.
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PMID:[Constitutive expression and purification of Alcaligenes faecalis penicillin G acylase in Escherichia coli]. 1597

Electrospinning of poly(glycolic acid) (PGA)/chitin blend solutions in 1,1,1,3,3,3-hexafluoro-2-propanol was investigated to fabricate biodegradable and biomimetic nanostructured scaffolds for tissue engineering. The morphology of the electrospun PGA/chitin blend nanofibers was investigated with a field emission scanning electron microscope. The PGA/chitin blend fibers have average diameters of around 140 nm, and their diameters have a distribution in the range 50-350 nm. The miscibility of PGA/chitin blend fibers was examined by differential scanning calorimetry. The PGA and chitin were immiscible in the as-spun nanofibrous structure. An in vitro degradation study of PGA/chitin blend nanofibers was conducted in phosphate-buffered saline, pH 7.2. It was found that the hydrolytic cleavage of PGA in the blend nanofibers was accelerated by the coexistence of hydrophilic chitin. To assay the cytocompatability and cell behavior on the PGA/chitin blend nanofibrous scaffolds, cell attachment and spreading of normal human epidermal fibroblasts seeded on the scaffolds were studied. Our results indicate that the PGA/chitin blend nanofibrous matrix, particularly the one that contained 25% PGA and 75% chitin with bovine serum albumin coating, could be a good candidate for tissue engineering scaffolds, because it has an excellent cell attachment and spreading for normal human fibroblasts.
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PMID:Biomimetic nanofibrous scaffolds: preparation and characterization of PGA/chitin blend nanofibers. 1647 41

The development of chemical reactions in nanospaces is of paramount importance for the development of active nanodevices, particularly in nanofluidics. It has been shown in a previous paper that phospholipid vesicles can be incorporated without spontaneous bilayer rupture into poly-L-glutamic acid/poly(allylamine) (PGA/PAH) multilayered polyelectrolyte films. The aim of the present study was to use such a system as an "embedded submicronic reactor" able to trigger precipitation of calcium phosphates within closed spaces through an enzymatic reaction, the enzyme also being encapsulated in the vesicle interior. To this aim, large unilamellar vesicles (LUVs) were produced containing calcium ions as active ions in the mineralization process, spermine as an activator of crystal growth, and alkaline phosphatase as a catalyst to convert phosphate esters into phosphates. After stabilization by adding a layer of poly-(D-lysine), these vesicles were embedded in a (PGA-PAH)n film. A paranitrophenyl phosphate containing solution was then put in contact with this film. It is shown by means of infrared spectroscopy in the attenuated total reflection mode that, consecutively to this contact, calcium phosphates are growing inside the embedded vesicles. By using scanning near-field fluorescence microscopy, it is demonstrated that the alkaline phosphatase enzymes are most probably located inside the vesicles after their embedding. In addition, atomic force microscopy was used to show, after chemical removal of the organic top layer of the film, that the inorganic platelets produced after the precipitation reaction are localized in volumes of similar size and shape as that of the vesicles into which the phosphate ester hydrolysis and subsequent precipitation reaction did occur.
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PMID:Layer-by-layer self-assembled polyelectrolyte multilayers with embedded liposomes: immobilized submicronic reactors for mineralization. 1648 29

An assay was developed for simultaneous kinetic analysis of the activities of the bifunctional plant enzyme ribulose 1,5-bisphosphate carboxylase/oxygenase [EC 4.1.1.39]. [1-(14)C,5-(3)H]Ribulose 1,5-bisphosphate (RuBP) was used as the labeled substrate. Tritium enrichment of the doubly labeled 3-phosphoglycerate (3-PGA) product, common to both enzyme activities, may be used to calculate V(c)/V(o) ratios from the expression A/(B-A) where A and B represent the (3)H/(14)C isotope ratios of doubly labeled RuBP and 3-PGA, and V(c) and V(o) represent the activities of carboxylase and oxygenase, respectively. Doubly labeled substrate was synthesized from [2-(14)C]glucose and [6-(3)H]glucose using the enzymes of the pentose phosphate pathway coupled with phosphoribulokinase.The kinetic properties of a commercial preparation of fully activated spinach carboxylase were studied under approximated physiological conditions of 20% O(2) (252 micromolar), 295 mul/l CO(2) (10 micromolar), 25 C, and pH 8.19. The V(c)/V(o) ratio was, within experimental error, constant at 30 seconds and 1 minute. This double label assay method may be used to calculate V(c)/V(o) ratios for the Laing-Ogren-Hageman equation, V(c)/V(o) = (V(c)K(o)/V(o)K(c)) ([CO(2)]/[O(2)]) where V(c) and V(o) represent V(max), and K(c) and K(o) represent Michaelis constants for the carboxylase and oxygenase activities, respectively.
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PMID:Simultaneous kinetic analysis of ribulose 1,5-bisphosphate carboxylase/oxygenase activities. 1666 Dec 14

A particulate preparation (MgP) capable of photosynthetic CO(2) assimilation without the addition of stromal protein was obtained by rupturing whole spinach (Spinacia oleracea var. America) chloroplasts in 15 millimolar MgCl(2) buffered with Tricine at pH 8.5. This CO(2) assimilation was dependent upon light, inorganic phosphate, ferredoxin, ADP, NAD or NADP, and primer. Excepting glycolate, the products of CO(2) fixation by MgP were similar to those found with whole chloroplasts.Glycerate-3-phosphate (PGA), fructose-1, 6-bisphosphate (FBP), and ribose-5-phosphate (R5P) but not fructose-6-P (F6P) functioned as primers. Concentrations of PGA and FBP but not of R5P higher than 2 millimolar were inhibitory to CO(2) fixation. A lag of CO(2) fixation was observed with PGA and FBP but not with R5P. This lag as well as inhibition by NADP, ADP, and ATP in the FBP-primed preparation was eliminated by an equimolar mixture of FBP plus F6P indicating FBPase as the sensitive site. NADP, ADP, and ATP also blocked CO(2) fixation by the PGA-fortified preparation but inhibition was even more sensitive than that observed when FBP was added. Inhibition by AMP in the PGA and FBP-primed preparations was not affected by the addition of F6P. When R5P was the starting primer, inhibition of CO(2) fixation was relatively insensitive to the adenylates and NADP. In contrast to the parent whole chloroplast, CO(2) fixation by MgP was insensitive to high (5 millimolar) inorganic phosphate. Depending upon the ferredoxin concentration, NAD was as effective as NADP in supporting CO(2) fixation.
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PMID:Characterization of a Photosynthesizing Reconstituted Spinach Chloroplast Preparation : REGULATION BY PRIMER, ADENYLATES, FERREDOXIN, AND PYRIDINE NUCLEOTIDES. 1666 54

The conversion of fructose-1,6-bisphosphate to glycerate-3-phosphate (PGA) was studied in a reconstituted spinach (Spinacia oleracea L.) chloroplast preparation to determine whether a chloroplast-localized NAB(P)H-oxidizing system (Kow, Smyth, Gibbs 1982 Plant Physiol 69: 72-76 with substrates of ascorbate, NAD(P)H, and H(2)O(2) could serve as a coupling enzyme in the recycling of NAD(P)H. The rate of PGA formation was monitored as an indicator of NAD(P) generation. With NAD as a cofactor, ascorbate enhanced PGA formation, and an additional increase resulted upon addition of glucose-glucose oxidase, a H(2)O(2)-generating enzyme. This increase in PGA formation due to H(2)O(2) was eliminated by the addition of catalase. With NADP and ferredoxin as cofactors, the recycling of NADP apparently was catalyzed both by ferredoxin-NADP reductase coupled to O(2) and by the NAD(P)H-oxidizing system.It was concluded that the oxidation of NAD(P)H by a system using ascorbate and H(2)O(2) can serve as a means of recycling NAD(P)H but that another reaction involving ascorbate and NAD(P)H may also function in the spinach chloroplast.
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PMID:Oxidation of NAD(P)H in a Reconstituted Spinach Chloroplast Preparation Using Ascorbate and Hydrogen Peroxide. 1666 86


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