scholarly journals THE ISOLATION AND PARTIAL CHARACTERIZATION OF THE PYRENOID PROTEIN OF EREMOSPHAERA VIRIDIS

1971 ◽  
Vol 51 (2) ◽  
pp. 499-513 ◽  
Author(s):  
Robert H. Holdsworth
Keyword(s):  
Specific Activity ◽  
Gradient Centrifugation ◽  
Sodium Dodecyl ◽  
High Specific Activity ◽  
Enzymatic Properties ◽  
Ultraviolet Absorption Spectrum ◽  
Fraction I Protein ◽  
Molecular Weights ◽  
Eremosphaera Viridis ◽  
Fraction I

The pyrenoids of Eremosphaera viridis, a green alga, were isolated by density gradient centrifugation and their physical and enzymatic properties were studied. The ultraviolet absorption spectrum of sodium dodecyl sulfate (SDS) extracts of pyrenoids showed a single peak at a wavelength of 277 nm, indicating the presence of protein and the probable absence of nucleic acid. Upon electrophoresis on polyacrylamide gels containing SDS, 16 bands were resolved of which two, together, accounted for 90% of the total protein on the gels. The molecular weights of these two proteins were estimated to be 59,000 and 12,300 and the ratio by weight of the larger to the smaller protein was found to be 2:1. The physical and enzymatic properties of these two proteins were found to closely resemble the properties reported in the literature for the subunits of fraction I protein. Both pyrenoids and fraction I protein are localized in the chloroplast, and both have two principal protein components. The molecular weights and relative ratio of the two pyrenoid components are very similar to those of the two components of fraction I protein. The pyrenoid was found to contain a high specific activity of ribulose-1,5-diphosphate carboxylase which is the same enzymatic activity exhibited by fraction I protein. The presence of ribose-5-phosphate isomerase and ribulose-5-phosphate kinase activities was also noted in pyrenoid preparations. It is suggested that the pyrenoid contains fraction I protein and possibly other enzymes of the Calvin-Bassham carbon dioxide fixing pathway.

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.

Low Molecular Weight Trypsin-Plasmin Inhibitors Isolated from Papain Treated Urinary Trypsin Inhibitor

1982 ◽  
Vol 47 (01) ◽  
pp. 014-018 ◽  
Author(s):  
H Sumi ◽  
N Toki ◽  
S Takasugi ◽  
S Maehara ◽  
M Maruyama ◽  
...  
Keyword(s):  
Trypsin Inhibitor ◽  
Gel Electrophoresis ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Chromatography ◽  
Urinary Trypsin Inhibitor ◽  
Plasmin Activity ◽  
Molecular Weights ◽  
Plasmin Inhibitors

SummaryPapain treatment of human urinary trypsin inhibitor (UTI67; mol. wt. 43,000 by SDS-polyacrylamide gel electrophoresis, specific activity 1,897 U/mg protein) produced four new protease inhibitors, which were highly purified by gel chromatography on Sephadex G-100 and isoelectric focusing. The purified inhibitors (UTI26, UTI9-I, UTI9-II, and UTI9-III) were shown to be homogeneous by polyacrylamide disc gel electrophoresis, and had apparent molecular weights of 26,000, 9,000, 9,000, and 9,800, respectively, by sodium dodecyl sulfate gel electrophoresis. During enzymatic degradation of UTI67, the amino acid compositions changed to more basic, and the isoelectric point increased from pH 2.0 (UTI67) to pHs 4.4, 5.2, 6.6, and 8.3 (UTI26, UTI9-I, UTI9-II, and UTI9-III), respectively. Both the parent and degraded inhibitors had anti-plasmin activity as well as antitrypsin and anti-chymotrypsin activities. Much higher anti-plasmin/anti-trypsin and anti-plasmin/anti-chymotrypsin activities were observed in the degraded inhibitors than in the parent UTI67. They competitively inhibited human plasmin with Ki values of 1.13 X 10-7 - 2.12 X 10-6 M (H-D-Val-Leu-Lys-pNA substrate). The reactions were very fast and the active site of the inhibitors to plasmin was thought to be different from that to trypsin or chymotrypsin.

cAMP increases synthesis of surfactant-associated protein A by perfused rat lung

1991 ◽  
Vol 260 (4) ◽  
pp. L226-L233 ◽  
Author(s):  
A. B. Fisher ◽  
I. Arad ◽  
C. Dodia ◽  
A. Chander ◽  
S. I. Feinstein
Keyword(s):  
Protein Fraction ◽  
Specific Activity ◽  
Gradient Centrifugation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Synthetic Medium ◽  
Protein A ◽  
Sodium Dodecyl ◽  
Lamellar Body ◽  
Surfactant Protein ◽  
Lag Phase

Synthesis and secretion of surfactant-associated protein were studied in isolated rat lungs perfused with [3H]phenylalanine or [35S]methionine in synthetic medium. Surfactant was isolated by lung lavage and density-gradient centrifugation followed by dialysis to remove unincorporated amino acid and extraction with ethanol-ether to yield a delipidated protein fraction. Incorporation of [3H]phenylalanine into the delipidated surfactant protein fraction showed a lag phase of approximately 3 h followed by progressive increase over the next 3 h at a rate of 1.6 nmol.mg protein-1.h-1. With 8-bromoadenosine 3',5'-cyclic monophosphate (8-BrcAMP, 0.1 mM) added to the perfusate, the incorporation rate between 3 and 6 h was increased by 75%. 3H specific activity in a delipidated lamellar body-rich fraction isolated from lung homogenates was unchanged by 8-BrcAMP at 3 h but was increased by 45% at 6 h. The major peak of radioactivity on sodium dodecyl sulfate-polyacrylamide gel electrophoresis of surfactant and lamellar bodies corresponded to proteins of 27–36 kDa that were identified as surfactant protein A (SP-A) by immunoblot. In the presence of 8-BrcAMP during 6 h of perfusion, specific activity of 35S-labeled SP-A in immunoprecipitated protein was increased by 93% and the SP-A mRNA content of lung was increased 145%. These results show that isolated perfused lungs synthesize and secrete surfactant-associated proteins and that the presence of a permeable cAMP analogue in the lung perfusate leads to increased secretion followed by induction of synthesis for SP-A.

Partial characterization of a novel oestrogen-induced protein in the rat adenohypophysis

1993 ◽  
Vol 10 (3) ◽  
pp. 345-357
Author(s):  
X Casabiell ◽  
J L Zugaza ◽  
C M Pombo ◽  
L Bokser ◽  
N Mulet ◽  
...  
Keyword(s):  
Incubation Medium ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Limited Proteolysis ◽  
Electrophoretic Pattern ◽  
High Specific Activity ◽  
Tumour Cell Line ◽  
Pituitary Cells ◽  
Tissue Extracts

ABSTRACT In order to detect putative markers of prolactin-secreting pituitary tumours, adult rats were subjected to long-term oestrogenization with oestradiol benzoate (OE2) on a monthly basis. At 6 months, anterior pituitaries were dissected and incubated either as tissue fragments or as dispersed cells with a S]methionine mix for labelling. Proteins released into the incubation medium and from tissue extracts were further analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and fluorography. Oestrogen induced the appearance in the incubation medium of a protein (OE2 band) with an Mr of 38 000 under reducing conditions, and high specific activity. Surprisingly, such a protein was not detected in tissue extracts. The OE2 band was detectable by 7 days after the first dose of oestrogen, and remained throughout 1 year of treatment. The tumour cell line GH3 showed a similar OE2 band which was further enhanced by oestrogens. The protein was observed similarly in both female and male pituitary donors, either intact or gonadectomized, and also in rats of different strains, suggesting that its appearance was independent of the strain of rat and gonadal status. Furthermore, the OE2 band was specific for pituitary cells and not produced by other oestrogenized tissues. No alteration in the rate of generation or the electrophoretic pattern of the OE2 band was observed when pituitary cells from oestrogenized rats were metabolically labelled while being incubated with tunicamycin. Furthermore, a system for glycan detection, adsorption to Concanavalin A or incubation with endoglycosidase F also failed to show a clear amount of glycosylation of the oestrogen-induced protein. Both immunoprecipitation experiments and time-limited proteolysis with V8 protease ruled out the possibility that the OE2 band could be structurally related to either GH or prolactin. In conclusion, oestrogens induce the generation of a new monocatenary protein with an apparent Mr of 38 000, which has at least one intramolecular disulphide loop and is not glycosylated. The OE2 band was detected only in incubation medium and never in tissue extracts.

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.

Rat intestinal maltase–glucoamylase. Purification of the detergent-solubilized enzyme in the presence of protease inhibitors: properties and identification of a protease-sensitive subunit

1984 ◽  
Vol 62 (1) ◽  
pp. 36-43 ◽  
Author(s):  
Lilian Lee ◽  
Gordon Forstner
Keyword(s):  
Protease Inhibitors ◽  
Gradient Centrifugation ◽  
Sodium Dodecyl ◽  
Prolonged Storage ◽  
Affinity Column ◽  
Molecular Weights ◽  
Clear Cut ◽  
Maltase Activity ◽  
Continuous Presence ◽  
Monomeric Proteins

Failure to develop clear-cut, distinguishing characteristics for hydrophobic and hydrophilic forms of maltase–glucoamylase led us to attempt the purification of the detergent-extracted enzyme in the continuous presence of protease inhibitors (phenylmethylsulfonyl fluoride and N-ethylmaleimide). The enzyme was purified by molecular exclusion, anion-exchange, and affinity column chromatography to a final specific maltase activity of 80 U/mg protein, comparable to previously solubilized enzymes. Both detergent (d-maltase) and proteolytically (p-maltase) solubilized enzymes had identical Km's for maltose and similar glycogenase activity. d-Maltase was clearly amphipathic. Whereas 95% of p-maltase was eluted with aqueous buffer from an octyl-Sepharose CL-4B column, the elution of d-maltase required solutions containing Triton X-100 and ethylene glycol. On density gradient centrifugation and sodium dodecyl sulfate (SDS) – polyacrylamide gels, p-maltase migrated as one high molecular weight species of 500 000. In contrast d-maltase migrated heterogeneously and the smallest maltase-active forms delineated by these two techniques, as well as by high pressure liquid chromatography, had molecular weights which ranged from 120 000 to 150 000. Both p- and d-maltase were dissociated by heat in SDS, forming five prominent species as we have previously described. In contrast to p-maltase, in which the smallest species, band 1, equalled 36.7% of the total mass, band 1 of d-maltase accounted for 66.5%. Band 1 was separable when smaller amounts of enzyme were applied to slab gels and stained with silver, into two proteins of 130 000 and 145 000 daltons. The 145 000 dalton protein was absent in p-maltase and was replaced by a faint band of 140 000 daltons. The 140 000 dalton band, plus a new N-terminal glycine, were also generated from d-maltase during prolonged storage at −20 °C. These data suggest that rat intestinal maltase–glucoamylase contains two monomeric proteins. The largest monomer contains an additional peptide segment at the N-terminus, which is removed by proteolysis and presumably anchors the enzyme to the microvillus membrane. After removal from the membrane, the two monomers of the d-enzyme are apparently partially dissociated to account for maltase activity within the 120 000 to 150 000 dalton range. Conversely, removal of the anchor segment favours a polymeric structure.

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 Partial Purification and Characterization of Proteases in Tobacco Leaf and Callus

1984 ◽  
Vol 12 (4) ◽  
pp. 219-226
Author(s):  
D. Liu ◽  
S. J. Sheen
Keyword(s):  
Cation Exchange ◽  
Specific Activity ◽  
Partial Purification ◽  
Fraction I Protein ◽  
Ph Optimum ◽  
Tobacco Leaf ◽  
Exchange Column ◽  
Gel Permeation ◽  
Cation Exchange Column ◽  
Fraction I

AbstractAmmonium sulfate fra.ctionation, gel permeation and cation-exchange column chromatography were employed for panial purification of proteases from leaf laminae and callus tissues of Samsun NN tobacco (Nicotiana tabacum L). The predominant proteases in the leaf and callus are acidic sulfhydryl proteases which are activated by 2-mercaptoethanol and ethylenediamine tetraacetic acid, completely inhibited- by iodoacetic acid, and partially inhibited by phenylmethane sulfonyl fluoride and pepstatin A. With hemoglobin and tobaccl:l Fraction I protein as substrates, leaf and callus proteases showed a pH optimum of 5. However, specific activity was significandy higher in the callus than in the leaf. Tobacco proteases digested hemoglobin more effectively than Fraction I protein and showed the least activity with casein. Gel permeation resolved three -protease variants in leaf extracts but only two in callus samples. Rechromatography of the large molecular weight fraction in a cation-exchange column produced three and two variants for leaf and callus, respectively. The present results suggest that there are at least five variants of sulfhydryl protease in tobacco leaf and three in callus tissue and that tobacco Fraction I protein can be metabolized by both leaf and callus proteases.

scholarly journals Properties of a Thermostable Nitrate Reductase from the Hyperthermophilic Archaeon Pyrobaculum aerophilum

2001 ◽  
Vol 183 (19) ◽  
pp. 5491-5495 ◽  
Author(s):  
Sepideh Afshar ◽  
Eric Johnson ◽  
Simon de Vries ◽  
Imke Schröder
Keyword(s):  
Nitrate Reductase ◽  
Specific Activity ◽  
Reductase Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
High Specific Activity ◽  
Competitive Inhibitor ◽  
Pyrobaculum Aerophilum ◽  
Benzyl Viologen ◽  
Hyperthermophilic Archaeon

ABSTRACT The nitrate reductase of the hyperthermophilic archaeonPyrobaculum aerophilum was purified 137-fold from the cytoplasmic membrane. Based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis, the enzyme complex consists of three subunits with apparent molecular weights of 130,000, 52,000, and 32,000. The enzyme contained molybdenum (0.8-mol/mol complex), iron (15.4-mol/mol complex) and cytochrome b (0.49-mol/mol complex) as cofactors. The P. aerophilum nitrate reductase distinguishes itself from nitrate reductases of mesophilic bacteria and archaea by its very high specific activity using reduced benzyl viologen as the electron donor (V max with nitrate, 1,162 s−1 (326 U/mg);V max with chlorate, 1,348 s−1 (378 U/mg) [assayed at 75°C]). The Km values for nitrate and chlorate were 58 and 140 μM, respectively. Azide was a competitive inhibitor and cyanide was a noncompetitive inhibitor of the nitrate reductase activity. The temperature optimum for activity was >95°C. When incubated at 100°C, the purified nitrate reductase had a half-life of 1.5 h. This study constitutes the first description of a nitrate reductase from a hyperthermophilic archaeon.

scholarly journals The structure and subunit composition of the particulate NADH-ubiquinone reductase of bovine heart mitochondria

1976 ◽  
Vol 154 (2) ◽  
pp. 295-305 ◽  
Author(s):  
C. I Ragan
Keyword(s):  
Molecular Weight ◽  
Complex I ◽  
Gradient Centrifugation ◽  
Sodium Dodecyl ◽  
Water Soluble ◽  
Heart Mitochondria ◽  
Molar Ratio ◽  
Polypeptide Composition ◽  
Molecular Weights ◽  
Bovine Heart

Preparations of NADH-ubiquinone reductase from bovine heart mitochondria (Complex I) were shown to contain at least 16 polypeptides by gel electrophoresis in the presence of sodium dodecyl sulphate. 2. High-molecular-weight soluble NADH dehydrogenase prepared from Triton X-100 extracts of submitochondrial particles [Baugh & King (1972) Biochem. Biophys. Res. Commun. 49, 1165-1173] was similar to Complex I in its polypeptide composition. 3. Solubilization of Complex I by phospholipase A treatment and subsequent sucrose-density-gradient centrifugation did not alter the polypeptide composition. 4. Lysophosphatidylcholine treatment of Complex I caused some selective solubilization of a polypeptide of mol.wt. 33000 previosuly postulated to be the transmembrane component of Complex I in the mitochondrial membrane [Ragan (1975) in Energy Transducing Membranes: Structure, Function and Reconstitution (Bennun, Bacila & Najjar, eds.), Junk, The Hague, in the press]. 5. Chaotropic resolution of Complex I caused solubilization of polypeptides of molecular weights 75000, 53000, 29000, 26000 and 15500 and traces of others in the 10000-20000-mol.wt.range. 6. The major components of the iron-protein fraction from chaotropic resolution had molecular weights of 75000, 53000 and 29000, whereas the flavoprotein contained polypeptides of molecular weights 53000 and 26000 in a 1:1 molar ratio. 7. Iodination of Complex I by lactoperoxidase indicated that the water-soluble polypeptides released by chaotropic resolution, in particular those of the flavoprotein fraction, were largely buried in the intact Complex. 8. The polypeptides of molecular weights 75000, 53000, 42000, 39000, 33000, 29000 and 26000 were present in 1:2:1:1:1:1:1 molar proportions. The two subunits of molecular weight 53000 are probably non-identical.

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