scholarly journals A DEMONSTRATION OF SEVERAL DEOXYRIBONUCLEASE ACTIVITIES IN MAMMALIAN CELL MITOCHONDRIA

1974 ◽  
Vol 62 (3) ◽  
pp. 695-706 ◽  
Author(s):  
Michael Durphy ◽  
Paul N. Manley ◽  
Errol C. Friedberg
Keyword(s):  
Ionic Strength ◽  
Adult Rabbit ◽  
Mitochondrial Enzyme ◽  
Alkaline Ph ◽  
Sh Group ◽  
Liver And Kidney ◽  
Substrate Variation ◽  
Enzyme Markers ◽  
Triton X ◽  
Deoxyribonuclease Activity

Extracts of purified mitochondria from adult rabbit liver and kidney have been prepared by lysis with Triton X-100. Such extracts contain deoxyribonuclease activity demonstrable at alkaline pH. Studies utilizing the effects of substrate variation, differing ionic strength, nucleoside di- and triphosphates, and SH-group inhibitors reveal the existence of at least five distinguishable deoxyribonuclease activities in these extracts. Assay of lysosomal and mitochondrial enzyme markers indicates no significant lysosomal contamination of the mitochondrial extracts. Further studies also suggest that the alkaline deoxyribonuclease activity is specifically located in or in association with mitochondria.

Optimum pH and ionic strength for the assay of cytochrome c oxidase from pea cotyledon mitochondria

1977 ◽  
Vol 55 (10) ◽  
pp. 1114-1117 ◽  
Author(s):  
Gerrit H. Bomhoff ◽  
Mary Spencer
Keyword(s):  
Ionic Strength ◽  
Cytochrome C ◽  
Cytochrome C Oxidase ◽  
Ferrocytochrome C ◽  
Optimum Ph ◽  
Triton X ◽  
Assay Conditions ◽  
Nonionic Detergents

Cytochrome c oxidase (EC 1.9.3.1) has been solubilized by use of the nonionic detergents Triton X-114 and Triton X-100, from pea cotyledon mitochondria. Optimum assay conditions were determined for the oxidation of ferrocytochrome c in air. The results indicate that the plant cytochrome c oxidase resembles mammalian preparations in its sensitivity towards ionic strength and pH of the assay buffer.

Purification par séparation de phase et caractérisation du plasmalemme de l'épicotyle de pois

1986 ◽  
Vol 64 (5) ◽  
pp. 448-455 ◽  
Author(s):  
Jacques Rembur ◽  
Pierre Landré ◽  
Arlette Nougarède
Keyword(s):  
Atpase Activity ◽  
Plasma Membranes ◽  
Microsomal Fraction ◽  
Alkaline Ph ◽  
Phase Partition ◽  
Pea Epicotyl ◽  
Glucan Synthetase ◽  
Triton X ◽  
Two Sides ◽  
Slight Contamination

The validity of phase partition to obtain a substantial proportion of vesicles of plasmalemma origin from the microsomal fraction of pea epicotyl has been demonstrated. In the fractions enriched with plasma membranes, N-naphthyl phtalamic acid binding and β-glucan synthetase II activity, showed a yield of about 60% and an enrichment of 2.3 and 2.2, respectively, in comparison with the microsomal fraction. When such plasmalemmic vesicles are permabilized by Triton X-100, an intense Mg2+-ATPase activity is obtained in presence of K+ at acid as well as alkaline pH. Inhibition of Mg2+-ATPase by vanadate in presence of K+ and its variations in relation to pH were shown. Dicyclohexylcarbodiimide and diethylstilbestrol inhibit 40–55% of this enzymatic activity, both at acid and neutral pH. The data show a slight contamination of the plasmalemmic fraction by endomembranes and suggest an asymmetry of the two sides of the plasmalemma.

Membrane-binding properties of filensin, a cytoskeletal protein of the lens fiber cells

1992 ◽  
Vol 103 (3) ◽  
pp. 709-718 ◽  
Author(s):  
M. Brunkener ◽  
S.D. Georgatos
Keyword(s):  
Binding Capacity ◽  
Lens Fiber ◽  
Alkaline Ph ◽  
Trypsin Treatment ◽  
Binding Properties ◽  
Fiber Cells ◽  
Lens Membranes ◽  
Acylated Proteins ◽  
Triton X

Filensin is a 100/110 kDa membrane-associated protein found in lens fiber cells. Previous studies have shown that this protein polymerizes in vitro and binds strongly to vimentin and to another 47 kDa lens membrane protein. Using cosedimentation assays, flotation assays and immunoelectron microscopy, we have examined the properties of purified filensin and measured its binding to lens membranes. Filensin behaves as a ureaextractable, hydrophilic protein which does not partition with Triton X-114 and is not affected by 1 M hydroxylamine at alkaline pH, an agent known to release fatty-acylated proteins from the membrane. Immunoblotting of urea-extracted lens membranes with two different affinity-purified antibodies reveals that, unlike intact filensin, a COOH-terminal filensin degradation product (51 kDa) remains tightly associated with the membranes. Purified filensin binds directly to urea-stripped lens membranes, but not to protein-free vesicles reconstituted from total lens lipids. The binding of filensin is not significantly influenced by the purified 47 kDa protein. Interestingly, the filensin-binding capacity of urea-extracted membranes is increased at least two-fold after trypsin treatment, which removes entirely the 51 kDa peptide from the membranes and presumably unmasks additional filensin-acceptor sites. Consistent with this, filensin binds to trypsinized and non-trypsinized membranes with similar affinities (2 × 10(−7) and 4 × 10(−7) M, respectively). Treatment of the membranes with thrombin, which also eliminates the 51 kDa peptide, does not increase their binding capacity, apparently because filensin-acceptor sites are also destroyed during proteolysis.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals The mechanism of assembly of Acanthamoeba myosin-II minifilaments: minifilaments assemble by three successive dimerization steps.

1989 ◽  
Vol 109 (4) ◽  
pp. 1537-1547 ◽  
Author(s):  
J H Sinard ◽  
W F Stafford ◽  
T D Pollard
Keyword(s):  
Electron Microscopy ◽  
Light Scattering ◽  
Ionic Strength ◽  
Analytical Ultracentrifugation ◽  
Myosin Ii ◽  
Alkaline Ph ◽  
Predominant Species ◽  
Assembly Mechanism ◽  
Rapid Equilibrium ◽  
Low Ionic Strength

We used 90 degrees light scattering, analytical ultracentrifugation, and electron microscopy to deduce that Acanthamoeba myosin-II minifilaments, composed of eight molecules each, assemble by a novel mechanism consisting of three successive dimerization steps rather than by the addition of monomers or parallel dimers to a nucleus. Above 200 mM KCl, Acanthamoeba myosin-II is monomeric. At low ionic strength (less than 100 mM KCl), myosin-II polymerizes into bipolar minifilaments. Between 100 and 200 mM KCl, plots of light scattering vs. myosin concentration all extrapolate to the origin but have slopes which decrease with increasing KCl. This indicates that structures intermediate in size between monomers and full length minifilaments are formed, and that the critical concentrations for assembly of these structures is very low. Analytical ultracentrifugation has confirmed that intermediate structures exist at these salt concentrations, and that they are in rapid equilibrium with each other. We believe these structures represent assembly intermediates and have used equilibrium analytical ultracentrifugation and electron microscopy to identify them. Polymerization begins with the formation of antiparallel dimers, with the two tails overlapping by approximately 15 nm. Two antiparallel dimers then associated with a 15-nm stagger to form an antiparallel tetramer. Finally, two tetramers associate with a 30-nm stagger to form the completed minifilament. At very low ionic strengths, the last step in the assembly mechanism is largely reversed and antiparallel tetramers are the predominant species. Alkaline pH, which can also induce minifilament disassembly, produces the same assembly intermediates as are found for salt induced disassembly.

scholarly journals Deoxyribonuclease from Salmon Testes

1962 ◽  
Vol 45 (4) ◽  
pp. 77-92 ◽  
Author(s):  
Margaret R. McDonald
Keyword(s):  
Ionic Strength ◽  
Maximal Activity ◽  
Optimum Concentration ◽  
Heat Denaturation ◽  
Acid Extraction ◽  
Fractional Precipitation ◽  
Temperature Optimum ◽  
Monovalent Ions ◽  
Ph Range ◽  
Deoxyribonuclease Activity

A procedure is described for the purification of salmon testis deoxyribonuclease II by means of acid extraction, fractional precipitation with ammonium sulfate, heat denaturation of extraneous proteins, and ethanol fractionation. This process separates the deoxyribonuclease activity from that of ribonuclease, phosphatase, phosphodiesterase, and protease. Over 50 per cent of the activity is retained with an over-all enrichment of 20,000-fold. The enzyme degrades both native and heat-denatured DNA, but the rate of degradation of the latter is only one-tenth that of the former. It does not hydrolyze apurinic acid. The enzyme is most stable in the pH range 4 to 5. Electrolytes are essential for the expression of its activity: monovalent ions satisfy the requirement, but divalent ones are much more effective. Above a certain optimum concentration, each electrolyte is inhibitory. The pH of maximal activity, under conditions of optimal ionic strength, is 4.8; the temperature optimum is near to 55°C.

scholarly journals A polypeptide of 59 kDa is associated with bundles of cytoplasmic filaments in Neurospora crassa

1990 ◽  
Vol 268 (3) ◽  
pp. 649-655 ◽  
Author(s):  
A L Rosa ◽  
M E Alvarez ◽  
D Lawson ◽  
H J F Maccioni
Keyword(s):  
Monoclonal Antibody ◽  
Ionic Strength ◽  
Neurospora Crassa ◽  
High Ionic Strength ◽  
Differential Centrifugation ◽  
Main Constituent ◽  
Total N ◽  
Cytoplasmic Filaments ◽  
Triton X ◽  
Isolation Buffer

Complex arrangements of filamentous structures have been isolated from vegetative cells of the fungus Neurospora crassa. They were enriched by differential centrifugation and purified by permeation chromatography. The filamentous structures are made up of units of 8-10 nm diameter and were isolated in bundles of up to six to nine units. The main constituent of these structures is a polypeptide with an apparent molecular mass of 59 kDa (P59Nc), which represents 4-5% of the total N. crassa proteins. The filamentous structures are cold-stable and are not affected by high-ionic-strength solutions or by the presence of 10 mM-EDTA or 1% (w/v) Triton X-100; they were disassembled by raising the pH of the solution or by using Tris-based buffers. The disassembled form assembled into structures sedimentable at 105,000 g after dialysis against the isolation buffer. The sedimentable structures were organized in the form of regular aggregates of 42-45 nm polypeptides and reacted weakly with anti-IFA, a monoclonal antibody which recognizes an epitope common to many of the higher-eukaryote intermediate-filament polypeptides. Immunofluorescence examination of wall-digested hyphae of N. crassa using affinity-purified antibodies prepared against P59Nc showed immunostaining of abundant filamentous and dot-shaped structures distributed in the cytoplasm.

Intracellular poly(3-hydroxybutyrate) depolymerase inAlcaligenes eutrophus

1995 ◽  
Vol 41 (13) ◽  
pp. 187-191 ◽  
Author(s):  
Terumi Saito ◽  
Kazuaki Takizawa ◽  
Haruhisa Saegusa
Keyword(s):  
Alcaligenes Eutrophus ◽  
Cell Extract ◽  
Supernatant Fraction ◽  
Alkaline Ph ◽  
Ph Optimum ◽  
Ph Profile ◽  
Phb Depolymerase ◽  
Diisopropyl Fluorophosphate ◽  
Triton X ◽  
Two Peaks

The intracellular depolymerization of poly(3-hydroxybutyrate) (PHB) in Alcaligenes eutrophus was investigated. PHB granules of A. eutrophus released D-(−)-3-hydroxybutyrate when incubated at 37 °C. The pH profile of autodigestion of the PHB granules revealed two peaks of activity, one at pH 6–7 and the other at a more alkaline pH. Autodigestion of native PHB granules was inhibited in the presence of Triton X-100. The PHB depolymerase activity was detected in the supernatant from centrifugation at 100 000 × g of the cell extract by using the protease-treated native PHB granules as substrate that lost most of their autodigestive activity. The soluble PHB depolymerase activity increased by about 20-fold or more during PHB synthesis compared with that of PHB-deficient cells, and there was a stringent correlation between PHB content and PHB depolymerase activity in A. eutrophus cells. A soluble PHB depolymerase was partially purified and was inhibited strongly in the presence of diisopropyl fluorophosphate. The pH optimum of the PHB depolymerase in the supernatant fraction was about 8–9. These results indicate that two types of PHB depolymerase may be associated with the native granules, and there was an enzyme with an alkaline pH optimum in the supernatant of the cell extract that seemed to be coregulated with PHB synthesis in the cells.Key words: Alcaligenes eutrophus, poly(3-hydroxybutyrate) depolymerase, poly(3-hydroxybutyrate) granules, biodegradation.

scholarly journals The activity of phosphoenolpyruvate carboxykinase in rat tissues. Assay techniques and effects of dietary and hormonal changes

1975 ◽  
Vol 152 (2) ◽  
pp. 401-408 ◽  
Author(s):  
Christopher I. Pogson ◽  
Stephen A. Smith
Keyword(s):  
Adipose Tissue ◽  
Phosphoenolpyruvate Carboxykinase ◽  
Alloxan Diabetes ◽  
Forward Direction ◽  
Epididymal Adipose Tissue ◽  
Rat Tissues ◽  
Hormonal Changes ◽  
Mitochondrial Enzyme ◽  
Forward Reaction ◽  
Liver And Kidney

1. Phosphoenolpyruvate carboxykinase was assayed by three methods: (i) incorporation of H14CO3- into oxaloacetate: (ii) conversion of oxaloacetate into phosphoenolpyruvate, subsequently assayed enzymically; and (iii) transfer of 32P from [γ-32P]GTP to oxaloacetate. 2. Enzyme activity is increased in liver and epididymal adipose tissue in alloxan-diabetes and starvation, and in kidney in starved, acidotic and steroid-treated animals. 3. The ratios of the ‘back’ to the ‘forward’ reactions in liver, kidney and epididymal adipose tissue are different and characteristic of each tissue; they differ markedly from values reported for the purified mitochondrial enzyme. 4. The ratio of the ‘back’ to ‘forward’ reaction in any one tissue is constant in adrenalectomized, diabetic, acidotic and steroid-treated animals. 5. In starved animals, the ratio is increased in liver and kidney, but decreased in epididymal adipose tissue. 6. Administration of l-tryptophan results in an acute (1h) increase in activity measured in the ‘forward’ direction alone in liver and epididymal adipose tissue, but not in kidney.

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