Extraction and characterization of the enzymes of fructan biosynthesis in timothy (Phleum pratense)

1986 ◽  
Vol 64 (9) ◽  
pp. 1884-1887 ◽  
Author(s):  
Michio Suzuki ◽  
Christopher J. Pollock
Keyword(s):  
Molecular Weight ◽  
Higher Plants ◽  
Molecular Size ◽  
Phleum Pratense ◽  
Ph Optimum ◽  
Diethyl Dithiocarbamate ◽  
Phleum Pratense L ◽  
Sodium Diethyl Dithiocarbamate

A preparation of phlein sucrase from seedling shoots of timothy (Phleum pratense L.) is described which catalyzed the synthesis of fructan with a mean molecular size of 34 000 using sucrose as the substrate. Activity was fully sedimentable at 25 000 × g, had a pH optimum of 7.0, and a Km for sucrose of 0.15 M. Activity was inhibited by β-mercaptoethanol and sodium diethyl dithiocarbamate. Raffinose and stachyose, but not members of the kestose series of oligofructans, could act as fructosyl donors in addition to sucrose. Formation of oligosaccharides during high molecular weight fructan synthesis was minimal, with synthesis occurring by a mechanism apparently analogous to bacterial levansucrase. These observations are discussed in relation to the in vivo patterns of fructan biosynthesis observed in different species of higher plants.

Partial Characterization of a Histone Acetyltransferase from Trout Testis

1975 ◽  
Vol 53 (7) ◽  
pp. 796-803 ◽  
Author(s):  
E. P. M. Candido
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Histone Acetyltransferase ◽  
Ph Optimum ◽  
Acetyltransferase Activity ◽  
Nuclear Extracts ◽  
Specific Activities ◽  
Histone Acetyltransferase Activity

Histone acetyltransferase activity of trout testis was studied both in intact nuclei, and in high salt nuclear extracts, With intact nuclei, the distribution of incorporated [14C]acetate in the various histones was similar to that observed in vivo; the arginine-rich histones H3 and H4 showed the highest specific activities, and lower amounts of label were detected in histones H2a and H2b. Histone H1 incorporated little or no label. Acetyltransferase activity could be detected in purified, sheared chromatin after the addition of MgCl2 or KCl, suggesting that the enzyme is bound to chromatin.Treatment of nuclei with 0,4 M NaCl caused the dissociation of acetyltransferase activity. Most of this solubilized activity failed to bind to DEAE Sephadex and behaved as a high molecular weight heterogeneous complex on Sephadex G-100, suggesting that the enzyme is present as an aggregate with other proteins in the extract. The pH optimum of this preparation was approximately 8.5, and the enzyme showed a preference for histones H3 and H4 as substrates.

Isolation and Characterization of Plasminogen Activator from Pig Leucocytes

1974 ◽  
Vol 31 (01) ◽  
pp. 072-085 ◽  
Author(s):  
M Kopitar ◽  
M Stegnar ◽  
B Accetto ◽  
D Lebez
Keyword(s):  
Molecular Weight ◽  
Plasminogen Activator ◽  
Gel Electrophoresis ◽  
Gel Filtration ◽  
Ph Optimum ◽  
Isolation And Characterization ◽  
Ph Range ◽  
Inhibition Studies ◽  
Final Purification

SummaryPlasminogen activator was isolated from disrupted pig leucocytes by the aid of DEAE chromatography, gel filtration on Sephadex G-100 and final purification on CM cellulose, or by preparative gel electrophoresis.Isolated plasminogen activator corresponds No. 3 band of the starting sample of leucocyte cells (that is composed from 10 gel electrophoretic bands).pH optimum was found to be in pH range 8.0–8.5 and the highest pH stability is between pH range 5.0–8.0.Inhibition studies of isolated plasminogen activator were performed with EACA, AMCHA, PAMBA and Trasylol, using Anson and Astrup method. By Astrup method 100% inhibition was found with EACA and Trasylol and 30% with AMCHA. PAMBA gave 60% inhibition already at concentration 10–3 M/ml. Molecular weight of plasminogen activator was determined by gel filtration on Sephadex G-100. The value obtained from 4 different samples was found to be 28000–30500.

scholarly journals In Vitro Import of a Nuclearly Encoded tRNA into Mitochondria of Solanum tuberosum

2003 ◽  
Vol 23 (11) ◽  
pp. 4000-4012 ◽  
Author(s):  
Ludovic Delage ◽  
André Dietrich ◽  
Anne Cosset ◽  
Laurence Maréchal-Drouard
Keyword(s):  
Solanum Tuberosum ◽  
Higher Plants ◽  
Plant Mitochondria ◽  
Protein Translation ◽  
Trna Genes ◽  
Vitro Assay ◽  
Trna Import

ABSTRACT Some of the mitochondrial tRNAs of higher plants are nuclearly encoded and imported into mitochondria. The import of tRNAs encoded in the nucleus has been shown to be essential for proper protein translation within mitochondria of a variety of organisms. Here, we report the development of an in vitro assay for import of nuclearly encoded tRNAs into plant mitochondria. This in vitro system utilizes isolated mitochondria from Solanum tuberosum and synthetic tRNAs transcribed from cloned nuclear tRNA genes. Although incubation of radioactively labeled in vitro-transcribed tRNAAla, tRNAPhe, and tRNAMet-e with isolated potato mitochondria resulted in importation, as measured by nuclease protection, the amount of tRNA transcripts protected at saturation was at least five times higher for tRNAAla than for the two other tRNAs. This difference in in vitro saturation levels of import is consistent with the in vivo localization of these tRNAs, since cytosolic tRNAAla is naturally imported into potato mitochondria whereas tRNAPhe and tRNAMet-e are not. Characterization of in vitro tRNA import requirements indicates that mitochondrial tRNA import proceeds in the absence of any added cytosolic protein fraction, involves at least one protein component on the surface of mitochondria, and requires ATP-dependent step(s) and a membrane potential.

scholarly journals Characterization of proteins from the cytoskeleton of Giardia lamblia

1983 ◽  
Vol 59 (1) ◽  
pp. 81-103 ◽  
Author(s):  
R. Crossley ◽  
D.V. Holberton
Keyword(s):  
Molecular Weight ◽  
Sodium Dodecyl Sulphate ◽  
Giardia Lamblia ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Molecular Size ◽  
Molecular Weights

Proteins from the axonemes and disc cytoskeleton of Giardia lamblia have been examined by sodium dodecyl sulphate/polyacrylamide gel electrophoresis. In addition to tubulin and the 30 X 10(3) molecular weight disc protein, at least 18 minor components copurify with the two major proteins in Triton-insoluble structures. The most prominent minor bands have the apparent molecular weights of 110 X 10(3), 95 X 10(3) and 81 X 10(3). Protein of 30 X 10(3) molecular weight accounts for about 20% of organelle protein on gels. In continuous 25 mM-Tris-glycine buffer it migrates mostly as a close-spaced doublet of polypeptides, which are here given the name giardins. Giardia tubulin and giardin have been purified by gel filtration chromatography in the presence of sodium dodecyl sulphate. Well-separated fractions were obtained that could be further characterized. Both proteins are heterogeneous when examined by isoelectric focusing. Five tubulin chains were detected by PAGE Blue 83 dye-binding after focusing in a broad-range ampholyte gel. Giardin is slightly less acidic than tubulin. On gels it splits into four major and four minor chains with isoelectric points in the pI range from 5.8 to 6.2. The amino acid composition of the giardin fraction has been determined, and compared to Giardia tubulin and a rat brain tubulin standard. Giardins are rich in helix-forming residues, particularly leucine. They have a low content of proline and glycine; therefore they may have extensive alpha-helical regions and be rod-shaped. As integral proteins of disc microribbons, giardins in vivo associate closely with tubulin. The properties of giardins indicate that in a number of respects - molecular size, charge, stoichiometry - their structural interaction with tubulin assemblies will be different from other tubulin-accessory protein copolymers studied in vitro.

Isolation and Characterization of Human Platelet β-Thromboglobulin

1975 ◽  
Author(s):  
D. S. Pepper ◽  
S. Moore ◽  
J. D. Cash
Keyword(s):  
Molecular Weight ◽  
Plasma Proteins ◽  
Human Platelet ◽  
Weight Fraction ◽  
Human Platelets ◽  
Low Molecular Weight ◽  
Purification Procedure ◽  
Isolation And Characterization

The thrombin released products from washed human platelets were separated by filtration on 4% agarose in 0.15 M NaCl. The high molecular weight PF4 complex was dissociated and re-chromatographed in 0.75 M NaCl. The low molecular weight fraction, including β thromboglobulin and a low MW anti-heparin was freed of plasminogen anti-activator by dissociation and chromatography in pH 3.5 pyridine acetic acid. The anti-activator was irreversibly denatured and albumin was removed in the void volume of the column. A more suitable purification procedure for recovery of all activities was affinity chromatography on heparin-agarose. The anti-activator was excluded and could be obtained free of plasma proteins by Sephadex G-200 chromatography. The βTG eluted at 0.3 M NaCl and the low MW anti-heparin at 1.5 M NaCl. The pure βTG (MW 36,000) was injected into rabbits and the resulting antiserum used to produce a radioimmunoassay for the release reaction in vivo.

scholarly journals Characterization of the complete chloroplast genome of Phleum pratense L. cv. Minshan

2019 ◽  
Vol 4 (2) ◽  
pp. 4180-4181
Author(s):  
Guangxin Cui ◽  
Yuan Lu ◽  
Xiaoxing Wei ◽  
Xiaoli Wang ◽  
Chunmei Wang ◽  
...  
Keyword(s):  
Chloroplast Genome ◽  
Phleum Pratense ◽  
Complete Chloroplast Genome ◽  
Phleum Pratense L

Biliary obstruction dissipates bioelectric sinusoidal-canalicular barrier without altering taurocholate uptake

1989 ◽  
Vol 256 (2) ◽  
pp. G312-G318
Author(s):  
J. Cotting ◽  
T. Zysset ◽  
J. Reichen
Keyword(s):  
Molecular Weight ◽  
Biliary Obstruction ◽  
Molecular Size ◽  
Base Line ◽  
Hepatic Extraction ◽  
Short Term ◽  
Extrahepatic Cholestasis ◽  
Large Molecular Weight ◽  
Unidirectional Transfer

To study immediate events during extrahepatic cholestasis, we investigated the effect of short-term biliary obstruction on the bioelectrical sinusoidal-canalicular barrier in the rat using molecular weight-matched uncharged and negatively charged inert solute pairs. The bioelectrical barrier averaged -22 +/- 5 and -18 +/- 4 mV (NS) using the pair carboxy-/methoxyinulin and ferrocyanide/sucrose, respectively. After a 20-min biliary obstruction both decreased by 61 and 11%, respectively, but only the large molecular weight pair (the inulins) returned to base line after release of the obstruction. Inert solute clearances were increased after short biliary obstruction depending on molecular size and negative charge (ferrocyanide greater than sucrose greater than carboxyinulin greater than inulin), suggesting that both permeability and bioelectrical barriers were affected by obstruction. The hepatic extraction in vivo of a passively transported drug not excreted into bile (D-propranolol) was not affected by obstruction, whereas that of an actively transported drug (glycocholate) decreased from 66 +/- 8 to 41 +/- 20% during biliary obstruction (P less than 0.01). Unidirectional transfer of glycocholate was not affected by short-term biliary obstruction in the situ perfused rat liver; however, 2 min after [14C]glycocholate administration, increased return was observed in hepatic venous effluent in obstructed animals. Our findings demonstrate a loss of the bioelectrical barrier immediately after short-term biliary obstruction. Decreased hepatic extraction in the view of unaltered sinusoidal uptake demonstrates regurgitation of bile into blood during short-term biliary obstruction.

scholarly journals Characterization of the Two Neurospora crassa Cellobiose Dehydrogenases and Their Connection to Oxidative Cellulose Degradation

2012 ◽  
Vol 78 (17) ◽  
pp. 6161-6171 ◽  
Author(s):  
Christoph Sygmund ◽  
Daniel Kracher ◽  
Stefan Scheiblbrandner ◽  
Kawah Zahma ◽  
Alfons K. G. Felice ◽  
...  
Keyword(s):  
Neurospora Crassa ◽  
Enzyme System ◽  
Cellulose Degradation ◽  
Cellulose Hydrolysis ◽  
Carbohydrate Binding ◽  
Ph Optimum ◽  
Content Type ◽  
Polysaccharide Monooxygenase

ABSTRACTThe genome ofNeurospora crassaencodes two different cellobiose dehydrogenases (CDHs) with a sequence identity of only 53%. So far, only CDH IIA, which is induced during growth on cellulose and features a C-terminal carbohydrate binding module (CBM), was detected in the secretome ofN. crassaand preliminarily characterized. CDH IIB is not significantly upregulated during growth on cellulosic material and lacks a CBM. Since CDH IIB could not be identified in the secretome, both CDHs were recombinantly produced inPichia pastoris. With the cytochrome domain-dependent one-electron acceptor cytochromec, CDH IIA has a narrower and more acidic pH optimum than CDH IIB. Interestingly, the catalytic efficiencies of both CDHs for carbohydrates are rather similar, but CDH IIA exhibits 4- to 5-times-higher apparent catalytic constants (kcatandKmvalues) than CDH IIB for most tested carbohydrates. A third major difference is the 65-mV-lower redox potential of the hemebcofactor in the cytochrome domain of CDH IIA than CDH IIB. To study the interaction with a member of the glycoside hydrolase 61 family, the copper-dependent polysaccharide monooxygenase GH61-3 (NCU02916) fromN. crassawas expressed inP. pastoris. A pH-dependent electron transfer from both CDHs via their cytochrome domains to GH61-3 was observed. The different properties of CDH IIA and CDH IIB and their effect on interactions with GH61-3 are discussed in regard to the proposedin vivofunction of the CDH/GH61 enzyme system in oxidative cellulose hydrolysis.

Isolation and Partial Characterization of Equine Antithrombin III.

1979 ◽  
Author(s):  
D.W. Estry ◽  
T.G. Bell ◽  
G.H. Tishkoff ◽  
J.C. Mattson ◽  
S.C. Estry
Keyword(s):  
Molecular Weight ◽  
Antithrombin Iii ◽  
Stable Complex ◽  
Sds Page ◽  
At Iii ◽  
Human Thrombin ◽  
Horse Plasma ◽  
Stoichiometric Complex

A protein analogous to human antithrombin III was isolated from fresh horse plasma. The procedure for purification was a modification of Thaler and Schmer’s two-step isolation procedure. The horse protein was homogeneous on 7.5% SDS-PAGE gels and had a molecular weight of 62,000 to 64,000 daltons in both reducing and non-reducing systems (human; 62,300). Rabbit anti-human antithrombin III was used to demonstrate a line of partial identity by Immunoelectrophoresis between the horse and human protein. The horse protein rapidly neutralizes human thrombin (34,000 daltons) and the reaction appears to be greatly potentiated by heparin. In order to establish the formation of 1:1 covalent stoichiometric complex between horse AT III and thrombin (IIa), time studies were run in the presence and absence of heparin. AT III (62,000) at 15 seconds, 2, 5, 10 and 60 minutes formed a stable complex with thrombin (32,000) having a molecular weight of 86,000 daltons. Additional bands developing with time are due to the autolytic capabilities of the uncomplexed IIa. The major autolytic band had a molecular weight of 70,000 daltons. Addition of heparin potentiated the interaction although it did not change the stoichio-metry of the complexes formed. The data accumulated to date demonstrates the similarities between the human and horse protein and the possibilities of using the horse as a model system for the evaluation of AT III replacement therapy in vivo.

scholarly journals MR imaging of model drug distribution in simulated vitreous

2015 ◽  
Vol 1 (1) ◽  
pp. 236-239 ◽  
Author(s):  
Sandra Stein ◽  
Christian Simroth-Loch ◽  
Sönke Langner ◽  
Stefan Hadlich ◽  
Oliver Stachs ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Ex Vivo ◽  
Drug Distribution ◽  
Injection Technique ◽  
Innovative Therapy ◽  
Age Related ◽  
The Impact

AbstractThe in vitro and in vivo characterization of intravitreal injections plays an important role in developing innovative therapy approaches. Using the established vitreous model (VM) and eye movement system (EyeMoS) the distribution of contrast agents with different molecular weight was studied in vitro. The impact of the simulated age-related vitreal liquefaction (VL) on drug distribution in VM was examined either with injection through the gel phase or through the liquid phase. For comparison the distribution was studied ex vivo in the porcine vitreous. The studies were performed in a magnetic resonance (MR) scanner. As expected, with increasing molecular weight the diffusion velocity and the visual distribution of the injected substances decreased. Similar drug distribution was observed in VM and in porcine eye. VL causes enhanced convective flow and faster distribution in VM. Confirming the importance of the injection technique in progress of VL, injection through gelatinous phase caused faster distribution into peripheral regions of the VM than following injection through liquefied phase. VM and MR scanner in combination present a new approach for the in vitro characterization of drug release and distribution of intravitreal dosage forms.

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