scholarly journals The Incorporation of Leucine-C14 into Microsomal Particles and other Subcellular Components of the Pea Epicotyl

1959 ◽  
Vol 5 (1) ◽  
pp. 59-68 ◽  
Author(s):  
Paul O. P. Ts'o ◽  
Clifford S. Sato
Keyword(s):  
Specific Activity ◽  
Gradient Centrifugation ◽  
High Specific Activity ◽  
Insoluble Fraction ◽  
Supernatant Fraction ◽  
Group Method ◽  
Pea Seedlings ◽  
Pea Epicotyl ◽  
Hydrolysis Of ◽  
End Group

Incorporation of leucine-C14 into subcellular fractions of the apical section of pea seedlings has been studied as a function of the length of incubation. The specific activity of the microsomes was higher than that of the supernatant for short but not for long incubations, in agreement with observations on other systems. In this developing tissue the nuclei and especially the mitochondria appear to incorporate amino acid very rapidly. An insoluble fraction of the microsome pellet, which is presumably a liponucleoprotein complex, was found to possess, after 1 hour of incubation, a specific activity much greater than that of the purified microsomal particles or the supernatant fraction. Ninety-eight per cent of the leucine-C14 in the purified microsomal particles has been shown to possess bound amino groups, presumably in peptide linkages, by the DNP-end group method. These particles liberate but little peptide or protein of very high specific activity when they are destroyed by removal of Mg or by hydrolysis of RNA. Microsomal particles were fractionated into an RNA fraction and five protein fractions by means of density gradient centrifugation. By this method 95 per cent of the RNA can be separated from 90 per cent of the protein of the particle. Furthermore, the RNA fraction has been shown to contain very little protein of high specific activity. A particular protein fraction which contains the remaining 5 per cent of the RNA, possessed after 1 hour of incubation a specific activity 2 to 9 times higher than the protein of the other fractions.

Biosynthesis of 1,2-3H and 4-14C steroid sulfates of high specific activity

1970 ◽  
Vol 48 (1) ◽  
pp. 148-150 ◽  
Author(s):  
J. Torday ◽  
G. Hall ◽  
M. Schweitzer ◽  
C. J. P. Giroud
Keyword(s):  
Rat Liver ◽  
Specific Activity ◽  
Liver Homogenate ◽  
High Specific Activity ◽  
Supernatant Fraction ◽  
Metabolic Studies

A supernatant fraction of rat liver homogenate enriched with ATP was used for the biosynthesis of the ester sulfates of several 3H and 14C steroids of the pregn-4-ene series. The method provides a simple means to prepare steroid sulfates of high specific activity for use in either metabolic studies or as reference compounds in the quantification of such conjugates by isotope assays.

scholarly journals Preliminary X-ray diffraction analysis of a thermophilic β-1,3–1,4-glucanase fromClostridium thermocellum

2014 ◽  
Vol 70 (7) ◽  
pp. 946-948 ◽  
Author(s):  
Lilan Zhang ◽  
Puya Zhao ◽  
Chun-Chi Chen ◽  
Chun-Hsiang Huang ◽  
Tzu-Ping Ko ◽  
...  
Keyword(s):  
Catalytic Domain ◽  
Specific Activity ◽  
High Specific Activity ◽  
Diffusion Method ◽  
X Ray Diffraction ◽  
Unit Cell Parameters ◽  
X Ray ◽  
Cell Parameters ◽  
Glycosidic Bonds ◽  
Hydrolysis Of

β-1,3–1,4-Glucanases catalyze the specific hydrolysis of internal β-1,4-glycosidic bonds adjacent to the 3-O-substituted glucose residues in mixed-linked β-glucans. The thermophilic glycoside hydrolase CtGlu16A fromClostridium thermocellumexhibits superior thermal profiles, high specific activity and broad pH adaptability. Here, the catalytic domain of CtGlu16A was expressed inEscherichia coli, purified and crystallized in the trigonal space groupP3121, with unit-cell parametersa=b= 74.5,c= 182.9 Å, by the sitting-drop vapour-diffusion method and diffracted to 1.95 Å resolution. The crystal contains two protein molecules in an asymmetric unit. Further structural determination and refinement are in progress.

scholarly journals Purification of the enzyme NADPH: protochlorophyllide oxidoreductase

1981 ◽  
Vol 195 (1) ◽  
pp. 83-92 ◽  
Author(s):  
N S Beer ◽  
W T Griffiths
Keyword(s):  
Specific Activity ◽  
Gradient Centrifugation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
High Specific Activity ◽  
Sucrose Density Gradient ◽  
Sucrose Density Gradient Centrifugation ◽  
Triton X ◽  
Etioplast Membranes

A procedure for the purification of the enzyme NADPH:protochlorophyllide oxidoreductase is described. This involves fractionation of sonicated oat etioplast membranes by discontinuous-sucrose-density-gradient centrifugation, which gives membranes in which the enzyme is present at a high specific activity. The enzyme is solubilized from the membranes with Triton X-100, followed by gel filtration of the extract; enzyme activity is eluted in fractions corresponding to a mol.wt of approx. 35000. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of the enzyme-containing fractions from gel filtration shows two peptides, of mol.wts. approx. 35000 and 37000.

Métabolisme intracellulaire et extracellulaire de l'acide 3-indolylacétique dans les cultures aseptiques ou non aseptiques de pointes de racine du Pisum sativum

1968 ◽  
Vol 46 (8) ◽  
pp. 969-978 ◽  
Author(s):  
G. F. Collet
Keyword(s):  
Absorption Spectrum ◽  
Pisum Sativum ◽  
Indoleacetic Acid ◽  
Specific Activity ◽  
High Specific Activity ◽  
Ultraviolet Absorption Spectrum ◽  
Root Tips ◽  
Pea Seedlings ◽  
Oxidase Inhibition ◽  
Layer Chromatography

Studies on indoleacetic acid (IAA) metabolism with root tips of 3-day-old pea seedlings were carried out by thin-layer chromatography and 14C-labelled auxin of high specific activity at 10−6 M. After 16-h incubation, five metabolites of IAA could be recognized in the tissues and two in solution; one metabolite was identified as o-formamidoacetophenone from its chromatographic Rf and ultraviolet absorption spectrum. Under sterile conditions the rate of breakdown of IAA is higher in the medium than under nonsterile conditions (even after removal of the tissues). We noted, too, a decrease of the action of IAA on the growth. The decreased activity of solution under nonsterile conditions is believed to be due to the production of oxidase inhibition by microorganisms.

Purification and Properties of a Neutral Protease from Soil Bacterium WM 122

1977 ◽  
Vol 37 (03) ◽  
pp. 556-565 ◽  
Author(s):  
S. E Papaioannou ◽  
W. J Marsheck
Keyword(s):  
Methyl Ester ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
High Specific Activity ◽  
Soil Bacterium ◽  
Ester Hydrochloride ◽  
Apparent Homogeneity ◽  
Purification And Properties ◽  
Hydrolysis Of

SummaryAn extracellular protease SN 687, secreted by the soil bacterium isolate WM 122, has been purified by means of gel filtration, ammonium sulfate precipitation, DEAE-Sephadex and hydroxylapatite chromatography. Apparent homogeneity was ascertained by Polyacrylamide gel electrophoresis. The protease was inactivated by ethylenediamine tetracetic acid (EDTA) but not by diisopropylfluorophosphate (DFP), and it was partially inhibited by serum inhibitors. SN 687 was shown to be of high specific activity against casein and fibrin, but it did not hydrolyze L- lysine -methyl ester dihydrochloride (LME), p-tosyl-L-arginine-methyl ester hydrochloride (TAME) and N-benzoyl-L-tyrosine-ethyl ester hydrochloride (BTEE) synthetic substrates. The optimum pH for hydrolysis of casein was 7.5 and the molecular weight, as determined by gel filtration, was 31,000.

scholarly journals Nonidet P-40 extraction of lymphocyte plasma membrane. Characterization of the insoluble residue

1984 ◽  
Vol 219 (1) ◽  
pp. 301-308 ◽  
Author(s):  
A A Davies ◽  
N M Wigglesworth ◽  
D Allan ◽  
R J Owens ◽  
M J Crumpton
Keyword(s):  
Plasma Membrane ◽  
Plasma Membranes ◽  
Specific Activity ◽  
Gradient Centrifugation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Fold Increase ◽  
Insoluble Fraction ◽  
Insoluble Residue ◽  
Protein Ratio

Purified preparations of lymphocyte plasma membrane were extracted exhaustively with Nonidet P-40 in Dulbecco's phosphate-buffered saline medium. The insoluble fraction, as defined by sedimentation at 10(6) g-min, contained about 10% of the membrane protein as well as cholesterol and phospholipid. The lipid/protein ratio, cholesterol/phospholipid ratio and sphingomyelin content were increased in the residue. Density-gradient centrifugation suggested that the lipid and protein form a common entity. As judged by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, the Nonidet P-40-insoluble fractions of the plasma membranes of human B lymphoblastoid cells and pig mesenteric lymph-node lymphocytes possessed similar qualitative polypeptide compositions but differed quantitatively. Both residues comprised major polypeptides of Mr 28 000, 33 000, 45 000 and 68 000, together with a prominent band of Mr 120 000 in the human and of Mr 200 000 in the pig. The polypeptides of Mr 28 000, 33 000, 68 000 and 120 000 were probably located exclusively in the Nonidet P-40-insoluble residue, which also possessed a 4-fold increase in 5′-nucleotidase specific activity. The results indicate that a reproducible fraction of lymphocyte plasma membrane is insoluble in non-ionic detergents and that this fraction possesses a unique polypeptide composition. By analogy with similar studies with erythrocyte ghosts, it appears likely that the polypeptides are located on the plasma membrane's cytoplasmic face.

scholarly journals Identification of an Invertase With High Specific Activity for Raffinose Hydrolysis and Its Application in Soymilk Treatment

2021 ◽  
Vol 12 ◽  
Author(s):  
Juanjuan Liu ◽  
Jing Cheng ◽  
Min Huang ◽  
Chen Shen ◽  
Ke Xu ◽  
...  
Keyword(s):  
Nutritional Value ◽  
Specific Activity ◽  
High Specific Activity ◽  
Maximum Activity ◽  
Beneficial Bacteria ◽  
Human Gut ◽  
Mild Conditions ◽  
Alternative Approach ◽  
Hydrolysis Of

The hydrolyzation of raffinose into melibiose by using invertases under mild conditions improves the nutritional value of soybean products. However, this strategy has received little attention because a suitable invertase remains lacking. In this study, a novel invertase named InvDz13 was screened and purified from Microbacterium trichothecenolyticum and characterized. InvDz13 was one of the invertases with the highest specific activity toward raffinose. Specifically, it had a specific activity of 229 U/mg toward raffinose at pH 6.5 and 35°C. InvDz13 retained more than 80% of its maximum activity at pH 5.5–7.5 and 25–40°C and was resistant to or stimulated by most cations that presented in soymilk. In soymilk treated with InvDz13 under mild conditions, melibiose concentration increased from 3.1 ± 0.2 to 6.1 ± 0.1 mM due to raffinose hydrolyzation by InvDz13. Furthermore, the prebiotic property of InvDz13-treated soymilk was investigated via in vitro fermentation by human gut microbiota. Results showed that InvDz13 treatment increased the proportion of the beneficial bacteria Bifidobacterium and Lactobacillus by 1.6- and 3.7-fold, respectively. By contrast, the populations of Escherichia and Collinsella decreased by 1.8- and 11.7-fold, respectively. Thus, our results proved that the enzymatic hydrolysis of raffinose in soymilk with InvDz13 was practicable and might be an alternative approach to improving the nutritional value of soymilk.

scholarly journals Characterization of a Novel Zinc-Containing, Lysine-Specific Aminopeptidase from the Hyperthermophilic Archaeon Pyrococcus furiosus

2005 ◽  
Vol 187 (6) ◽  
pp. 2077-2083 ◽  
Author(s):  
Sherry V. Story ◽  
Claudia Shah ◽  
Francis E. Jenney ◽  
Michael W. W. Adams
Keyword(s):  
Metal Ion ◽  
Specific Activity ◽  
Pyrococcus Furiosus ◽  
High Specific Activity ◽  
Native Form ◽  
Genome Database ◽  
Hyperthermophilic Archaeon ◽  
Cell Extracts ◽  
Hydrolysis Of

ABSTRACT Cell extracts of the proteolytic, hyperthermophilic archaeon Pyrococcus furiosus contain high specific activity (11 U/mg) of lysine aminopeptidase (KAP), as measured by the hydrolysis of l-lysyl-p-nitroanilide (Lys-pNA). The enzyme was purified by multistep chromatography. KAP is a homotetramer (38.2 kDa per subunit) and, as purified, contains 2.0 ± 0.48 zinc atoms per subunit. Surprisingly, its activity was stimulated fourfold by the addition of Co2+ ions (0.2 mM). Optimal KAP activity with Lys-pNA as the substrate occurred at pH 8.0 and a temperature of 100°C. The enzyme had a narrow substrate specificity with di-, tri-, and tetrapeptides, and it hydrolyzed only basic N-terminal residues at high rates. Mass spectroscopy analysis of the purified enzyme was used to identify, in the P. furiosus genome database, a gene (PF1861) that encodes a product corresponding to 346 amino acids. The recombinant protein containing a polyhistidine tag at the N terminus was produced in Escherichia coli and purified using affinity chromatography. Its properties, including molecular mass, metal ion dependence, and pH and temperature optima for catalysis, were indistinguishable from those of the native form, although the thermostability of the recombinant form was dramatically lower than that of the native enzyme (half-life of approximately 6 h at 100°C). Based on its amino acid sequence, KAP is part of the M18 family of peptidases and represents the first prokaryotic member of this family. KAP is also the first lysine-specific aminopeptidase to be purified from an archaeon.

scholarly journals CHARACTERIZATION OF POSTREPLICATION REPAIR IN MUTAGEN-SENSITIVE STRAINS OF DROSOPHILA MELANOGASTER

Genetics ◽  
1976 ◽  
Vol 84 (3) ◽  
pp. 507-526
Author(s):  
J B Boyd ◽  
R B Setlow
Keyword(s):  
Drosophila Melanogaster ◽  
Uv Radiation ◽  
Specific Activity ◽  
Gradient Centrifugation ◽  
High Specific Activity ◽  
X Rays ◽  
Postreplication Repair ◽  
Strand Breaks ◽  
Single Strand Breaks ◽  
Complementation Groups

ABSTRACT Mutants of Drosophila melanogaster, with suspected repair deficiencies, were analyzed for their capacity to repair damage induced by X-rays and UV radiation. Analysis was performed on cell cultures derived from embryos of homozygous mutant stocks. Postreplication repair following UV radiation has been analyzed in mutant stocks derived from a total of ten complementation groups. Cultures were irradiated, pulse-labeled, and incubated in the dark prior to analysis by alkaline sucrose gradient centrifugation. Kinetics of the molecular weight increase in newly synthesized DNA were assayed after cells had been incubated in the presence or absence of caffeine. Two separate pathways of postreplication repair have been tentatively identified by mutants derived from four complementation groups. The proposed caffeine sensitive pathway (CAS) is defined by mutants which also disrupt meiosis. The second pathway (CIS) is caffeine insensitive and is not yet associated with meiotic functions. All mutants deficient in postreplication repair are also sensitive to nitrogen mustard. The mutants investigated display a normal capacity to repair single-strand breaks induced in DNA by X-rays, although two may possess a reduced capacity to repair damage caused by localized incorporation of high specific activity thymidine-3H. The data have been employed to construct a model for repair of UV-induced damage in Drosophila DNA. Implications of the model for DNA repair in mammals are discussed.

Patterns of development of glycosidase activities in the pea epicotyl

1970 ◽  
Vol 48 (6) ◽  
pp. 1165-1169 ◽  
Author(s):  
Anne Harmon Datko ◽  
G. A. Maclachlan
Keyword(s):  
Indoleacetic Acid ◽  
Specific Activity ◽  
Fresh Weight ◽  
Glucosidase Activity ◽  
Time Treatment ◽  
Glycosidase Activity ◽  
Specific Localization ◽  
Weight Protein ◽  
Pea Epicotyl ◽  
Hydrolysis Of

Rates of hydrolysis of α- and β-linked glucosides and galactosides were compared in reaction mixtures containing nitrophenol derivatives, maltose, or cellobiose as substrates, plus enzyme derived from various parts of the third internode of the pea epicotyl. β-Glucosidase activity (EC. 3.2.1.21) per unit fresh weight, protein, or DNA was concentrated in meristematic tissues (plumule and hook) and nearly absent from adjacent growing and maturing regions in the internode. The other glycosidase activities showed no such specific localization. β-Glucosidase was also the only one of these enzymes that increased in specific activity in the decapitated epicotyl after treatment with indoleacetic acid. The increase (about twofold in 2 days) occurred if cell division was evoked at the same time. Treatment with gibberellic acid had little effect on any glycosidase activity. It is concluded that β-glucosidase activity may have been especially useful in cells during or shortly after cytokinesis, but none of the glycosidase activities were needed specifically for cell elongation or expansion.

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