Characterization studies on the membrane-bound adenosine triphosphatase (ATPase) of Azotobacter vinelandii

1975 ◽  
Vol 21 (11) ◽  
pp. 1807-1814 ◽  
Author(s):  
Peter Jurtshuk ◽  
John E. McEntire
Keyword(s):  
Electron Transport ◽  
Atpase Activity ◽  
Azotobacter Vinelandii ◽  
Specific Activity ◽  
Maximal Activity ◽  
Molar Ratio ◽  
Supernatant Fraction ◽  
Time Interval ◽  
Time Intervals ◽  
Membrane Bound

The adenosinetriphosphatase (ATPase) (EC 3.6.1.3) activity in Azotobacter vinelandii concentrates in the membranous R3 fraction that is directly associated with Azotobacter electron transport function. Sonically disrupted Azotobacter cells were examined for distribution of ATPase activity and the highest specific activity (and activity units) was consistently found in the particulate R3 membranous fraction which sediments on ultracentrifugation at 144 000 × g for 2 h. When the sonication time interval was increased, the membrane-bound ATPase activity could neither be solubilized nor released into the supernatant fraction. Optimal ATPase activity occurred at pH 8.0; Mg2+ ion when added to the assay was stimulatory. Maximal activity always occurred when the Mg2+:ATP stoichiometry was 1:1 on a molar ratio at the 5 mM concentration level. Sodium and potassium ions had no stimulatory effect. The reaction kinetics were linear for the time intervals studied (0–60 min). The membrane-bound ATPase in the R3 fraction was stimulated 12-fold by treatment with trypsin, and fractionation studies showed that trypsin treatment did not solubilize ATPase activity off the membranous R3 electron transport fraction. The ATPase was not cold labile and the temperature during the preparation of the R3 fraction had no effect on activity; overnight refrigeration at 4 °C, however, resulted in a 25% loss of activity as compared with a 14% loss when the R3 fraction was stored overnight at 25 °C. A marked inactivation (although variable, usually about 60%) did occur by overnight freezing (−20 °C), and subsequent sonication failed to restore ATPase activity. This indicates that membrane reaggregation (by freezing) was not responsible for ATPase inactivation. The addition of azide, ouabain, 2,4-dinitrophenol, or oligomycin to the assay system resulted in neither inhibition nor stimulation of the ATPase activity. The property of trypsin activation and that ATPase activity is highest in the R3 electron transport fraction suggests that its probable functional role is in coupling of electron transport to oxidative phosphorylation.

Oxidation–reduction titration of cytochrome components in the electron transport chain of Azotobacter vinelandii

1981 ◽  
Vol 59 (2) ◽  
pp. 137-144 ◽  
Author(s):  
Tsanyen Yang
Keyword(s):  
Electron Transport ◽  
Cytochrome B ◽  
Absorption Maximum ◽  
Azotobacter Vinelandii ◽  
Reactive Component ◽  
Midpoint Potential ◽  
Oxidation Reduction ◽  
Membrane Bound ◽  
The Individual ◽  
Cytochrome D

The multiple cytochrome components in the electron transport particle of Azotobacter vinelandii were resolved and their oxidation–reduction midpoint potentials were determined by a simultaneous potentiometric and absorption measurements under anaerobic condition with or without CO. The midpoints of the individual cytochrome component corresponding to the membrane-bound types were also determined in the solubilized fractions prepared by a differential detergent solubilization of the membrane particles of A. vinelandii. Two cytochromes of b type, one with an absorption maximum measured at 559 nm and another at 561 nm in the membrane particle, were resolved and their Em, 7.4 values determined to be −30 mV and +122 mV, respectively. Cytochrome b559 reacted with CO readily in both membrane-bound and solubilized forms, however, cytochrome b561 was inert to CO treatment. Only one cytochrome of c type (c4) measured at 575–551 nm was resolved, its midpoint potential at pH 7.4 was +322 mV in the membrane-bound form and +278 mV in the solubilized form. This c-type cytochrome had no CO reactivity. Cytochrome d, a CO-reactive component, had a midpoint of +270 mV in the membrane fraction. The midpoint of cytochrome a1 in its membrane-bound form could not be measured accurately because of its low concentration. However, in the solubilized preparations, cytochrome a1 apparently had a red shift with an absorption maximum at 613 nm, with an estimated Em, 7.4 of −45 mV, while cytochrome d was no longer detected, possibly because of denaturation.

scholarly journals Factors influencing the activity of succinate dehydrogenase in membrane preparations from Micrococcus lysodeikticus

1970 ◽  
Vol 120 (2) ◽  
pp. 237-243 ◽  
Author(s):  
Peter Owen ◽  
John H. Freer
Keyword(s):  
Succinate Dehydrogenase ◽  
Specific Activity ◽  
Sodium Deoxycholate ◽  
Maximal Activity ◽  
Spectrophotometric Assay ◽  
Enzyme Activation ◽  
Succinate Dehydrogenase Activity ◽  
Micrococcus Lysodeikticus ◽  
Membrane Bound ◽  
Phenazine Methosulphate

1. Some properties of succinate dehydrogenase [succinate–(acceptor) oxidoreductase, EC 1.3.99.1] in membrane preparations from Micrococcus lysodeikticus (N.C.T.C. 2665) were investigated. 2. In the spectrophotometric assay system adopted the reaction velocity was shown to be proportional to the amount of membrane added. Dichlorophenol-indophenol, reduced photochemically in the presence of phenazine methosulphate, or enzymically by the membrane-bound enzyme, was shown to undergo reoxidation in the dark. 3. The membrane-bound enzyme was found to be inactivated at temperatures above 10°C. 4. The specific activity of membrane-bound succinate dehydrogenase was found to increase between two- and three-fold in diluted membrane preparations equilibrated at 0°C for 6h. Membranes treated with sodium deoxycholate showed no enzyme activation on dilution but displayed maximal activity, all activity being sedimentable at 103000g. The increase in specific activity observed on dilution could be partially inhibited by fixation with glutaraldehyde, or by the presence of bovine serum albumin. 5. The addition of Mg2+ or Ca2+ ions to membrane suspensions caused an overall depression of enzyme activity. 6. The results suggest the presence of an `inhibitor' that affects the expression of membrane bound succinate dehydrogenase activity.

scholarly journals The presence and activity in normal and regenerating rat liver postmicrosomal supernatant fraction of an enzyme with properties similar to those of membrane-bound 5′-nucleotidase

1986 ◽  
Vol 239 (1) ◽  
pp. 185-190 ◽  
Author(s):  
P Fritzson ◽  
T B Haugen ◽  
H Tjernshaugen
Keyword(s):  
Alkaline Phosphatase ◽  
Rat Liver ◽  
Specific Activity ◽  
Ph Dependence ◽  
Gel Permeation Chromatography ◽  
Total Activity ◽  
Soluble Form ◽  
Supernatant Fraction ◽  
Distinct Fraction ◽  
Membrane Bound

An alkaline 5′-nucleotidase with properties similar to those of membrane-bound 5′-nucleotidase was recovered in soluble form in the postmicrosomal supernatant fraction (cytosol) of rat liver. The enzyme seems to constitute a quantitatively distinct fraction, since the activity in postmicrosomal supernatants was increased by a further 10% by additional homogenization of livers. Lysosomal acid phosphatase activity increased similarly, whereas other membrane-bound marker enzymes alkaline phosphatase, phosphodiesterase I and glucose-6-phosphatase showed no increase when homogenization of liver tissue was continued. Gel-permeation chromatography and pH-dependence studies indicated that enzyme activity in the supernatant fraction with 0.3 mM-UMP or -AMP as substrate at pH 8.1 was about 85 or 100% specific respectively. In regenerating liver the enzyme recovered in soluble form showed decreased specific activity, in contrast with alkaline phosphatase measured for comparison. The nucleotidase activity per mg of cytosolic protein was 2.1 nmol/min with AMP as substrate. The total activity measured in the postmicrosomal supernatant was 1.5% of the homogenate activity measured in the presence of detergent.

scholarly journals Ca2+ uptake by corpus-luteum plasma membranes. Evidence for the presence of both a Ca2+-pumping ATPase and a Ca2+-dependent nucleoside triphosphatase

1987 ◽  
Vol 242 (3) ◽  
pp. 889-894 ◽  
Author(s):  
J Minami ◽  
J T Penniston
Keyword(s):  
Atpase Activity ◽  
Corpus Luteum ◽  
Decomposition Rate ◽  
Plasma Membranes ◽  
Specific Activity ◽  
Membrane Vesicles ◽  
Maximal Activity ◽  
Ionophore A23187 ◽  
Plasma Membrane Vesicles ◽  
Bivalent Cation

Plasma-membrane vesicles from rat corpus luteum showed an ATP-dependent uptake of Ca2+. Ca2+ was accumulated with a K1/2 (concn. giving half-maximal activity) of 0.2 microM and was released by the bivalent-cation ionophore A23187. A Ca2+-dependent phosphorylated intermediate (Mr 100,000) was detected which showed a low decomposition rate, consistent with it being the phosphorylated intermediate of the transport ATPase responsible for Ca2+ uptake. The Ca2+ uptake and the phosphorylated intermediate (E approximately P) displayed several properties that were different from those of the high-affinity Ca2+-ATPase previously observed in these membranes. Both Ca2+ uptake and E approximately P discriminated against ribonucleoside triphosphates other than ATP, whereas the ATPase split all the ribonucleoside triphosphates equally. Both Ca2+ uptake and E approximately P were sensitive to three different Hg-containing inhibitors, whereas the ATPase was inhibited much less. Ca2+ uptake required added Mg2+ (Km = 2.2 mM), whereas the ATPase required no added Mg2+. The maximum rate of Ca2+ uptake was about 400-fold less than that of ATP splitting; under different conditions, the decomposition rate of E approximately P was 1,000 times too slow to account for the ATPase activity observed. All of these features suggested that Ca2+ uptake was due to an enzyme of low activity, whose ATPase activity was not detected in the presence of the higher-specific-activity Ca2+-dependent ATPase.

Biochemical Basis of Heart Function. X. Reduction in the Na+–K+-Stimulated ATPase Activity in Failing Rat Heart Due to Hypoxia

1973 ◽  
Vol 51 (7) ◽  
pp. 504-510 ◽  
Author(s):  
V. Balasubramanian ◽  
D. B. McNamara ◽  
J. N. Singh ◽  
N. S. Dhalla
Keyword(s):  
Atpase Activity ◽  
Specific Activity ◽  
Heart Function ◽  
Perfusion Medium ◽  
Biochemical Basis ◽  
Exogenous Glucose ◽  
Hypoxic Heart ◽  
Rat Hearts ◽  
Membrane Bound ◽  
Assay Conditions

The activities of the membrane bound enzyme Na+–K+-stimulated ATPase were studied in control and hypoxic isolated rat hearts. The control hearts were perfused with medium gassed with 95% O2 and 5% CO2, whereas hypoxia was induced by perfusing the hearts with medium gassed with 95% N2 and 5% CO2. The specific activity of the Na+–K+ ATPase was lower in the hypoxic hearts than in the control preparations. The enzyme activity of the hypoxic hearts was also lower when determined under different assay conditions obtained by changing the concentration of Na+, K+, H+, and MgATP in the incubation medium. The ouabain-induced inhibition of Na+–K+ ATPase activity in the hypoxic hearts was similar to that in the control. The depression in Na+–K+ ATPase activity due to hypoxia was reversible on perfusing the hypoxic heart with aerobic medium. The decreased Na+–K+ ATPase activity in the hypoxic heart preparations supports the concept of membrane abnormality associated with heart failure due to lack of oxygen. Since the depression in the activity of Na+–K+ ATPase in the hypoxic heart was markedly pronounced on omitting the substrate from the perfusion medium, it is likely that exogenous glucose plays an important role in maintaining the integrity of hypoxic heart membranes.

Evidence for the presence of a glycosphingolipid-transfer protein in rat brain cytosol

1987 ◽  
Vol 65 (5) ◽  
pp. 493-500 ◽  
Author(s):  
Sankar N. Sanyal
Keyword(s):  
Rat Brain ◽  
Lipid Fraction ◽  
Active Material ◽  
Supernatant Fraction ◽  
Appreciable Effect ◽  
Divalent Metal Ions ◽  
Time Intervals ◽  
Ph Optimum ◽  
Red Cell Membrane ◽  
Membrane Bound

Proteins in the postmicrosomal supernatant fraction of rat brain catalyzed the transfer of bovine brain galactocerebroside, sulfatide, and ganglioside GM1 from unilamellar liposomes to the rat erythrocytes or ghosts. The vesicles were made with egg yolk lecithin, cholesterol, 3H-labelled glycolipid, and a trace of [14C]triolein as a nonexchangeable marker. The routine assay of the glycosphingolipid transfer consisted of incubation of the donor liposomes with erythrocytes in the presence or absence of supernatant protein in physiological buffer at 37 °C for various time intervals. After the incubation, the erythrocytes were separated from the vesicles by centrifugation and the extent of protein-catalyzed transfer of labelled glycolipid in the membrane-bound total lipid fraction was determined by scintillation spectrometry. The fraction of [3H]glycosphingolipid transferred is represented by a change in the 3H/14C ratios at initial and subsequent time intervals. The glycosphingolipid transfer catalyzed by the supernatant protein was found to be logarithmic, whereas the protein-independent transfer was linear over a period of 3–4 h. The rate constant (K) and half time (t1/2) of the protein-catalyzed transfer reaction of cerebrosides and sulfatides were almost the same, while the transfer of ganglioside GM1 occurred at a slightly faster rate, probably owing to the greater aqueous solubility of this lipid. The transfer activity was also increased in a manner dependent on the amount of supernatant protein added up to 10 mg. The catalytic activity of the protein was lost when heated at 70 °C for 5 min. The pH optimum of the activity was around 7.4. Divalent metal ions Ca2+, Mg2+, and Mn2+ at a concentration of 0.1–2.0 mM had no appreciable effect on the transfer of cerebroside. However, Ca2+ at the concentration tested notably inhibited sulfatide transfer. Approximately, 3–5 pmol of the glycosphingolipids was transferred from the vesicles to the erythrocytes per milligram of supernatant protein per hour. The transferred radioactivity can be exclusively recovered in the red cell membrane bound glycolipid fraction, as analyzed by high performance liquid chromatography. The active material was partially purified (over 30-fold) by ammonium sulfate precipitation and gel permeation chromatography on Sephadex G-75, with an indicated molecular weight of about 21 000.

A High Resolution Cytochemical Study of Nuclear ATPase in Human Spermatozoa

1975 ◽  
Vol 33 ◽  
pp. 594-595
Author(s):  
A. Sosa ◽  
L. Calzada
Keyword(s):  
High Resolution ◽  
Atpase Activity ◽  
Nuclear Membrane ◽  
Human Sperm ◽  
Sperm Nucleus ◽  
Human Spermatozoa ◽  
Cytochemical Study ◽  
Membrane Bound ◽  
Sperm Nuclei ◽  
Interchromatin Space

The dependence of nuclear metabolism on the function of the nuclear membrane is not well understood. Whether or not the function of the nuclear membrane is partial or totally responsible of the repressed template activity of human sperm nucleus has not at present been elucidated. One of the membrane-bound enzymatic activities which is concerned with the mechanisms whereby substances are thought to cross cell membranes is adenosintriphosphatase (ATPase). This prompted its characterization and distribution by high resolution photogrammetry on isolated human sperm nuclei. Isolated human spermatozoa nuclei were obtained as previously described. ATPase activity was demonstrated by the method of Wachstein and Meisel modified by Marchesi and Palade. ATPase activity was identified as dense and irregularly distributed granules confined to the internal leaflet of the nuclear membrane. Within the nucleus the appearance of the reaction product occurs as homogenous and dense precipitates in the interchromatin space.

A New Method for Plasminogen Standardization

1968 ◽  
Vol 20 (03/04) ◽  
pp. 548-554
Author(s):  
J Gajewski ◽  
G Markus
Keyword(s):  
Proteolytic Activity ◽  
Specific Activity ◽  
Molar Ratio ◽  
Sharp Transition ◽  
Equivalence Point ◽  
Constant Amount ◽  
Residual Level ◽  
Activity Measurements ◽  
Complete Saturation ◽  
Human Plasminogen

SummaryA method for the standardization of human plasminogen is proposed, based on the stoichiometric interaction between plasminogen and streptokinase, resulting in inhibition of proteolytic activity. Activation of a constant amount of plasminogen with increasing amounts of streptokinase yields linearly decreasing activities, as a function of streptokinase, with a sharp transition to a constant residual level. The point of transition corresponds to complete saturation of plasmin with streptokinase in a 1:1 molar ratio, and is therefore a measure of the amount of plasminogen present initially, in terms of streptokinase equivalents. The equivalence point is independent of the kind of protein substrate used, buffer, pH, length of digestion and, within limits, temperature. The method, therefore, is not subject to the variations commonly encountered in the usual determination based on specific activity measurements.

METABOLIC STUDIES WITH 131I-LABELED THYROXINE. II.

1963 ◽  
Vol 44 (3) ◽  
pp. 475-480 ◽  
Author(s):  
R. Grinberg
Keyword(s):  
Spinal Cord ◽  
Central Nervous System ◽  
Nervous System ◽  
Optic Nerve ◽  
Pituitary Gland ◽  
Time Interval ◽  
Time Intervals ◽  
Pituitary Glands ◽  
The Central Nervous System ◽  
Tentative Explanation

ABSTRACT Radiologically thyroidectomized female Swiss mice were injected intraperitoneally with 131I-labeled thyroxine (T4*), and were studied at time intervals of 30 minutes and 4, 28, 48 and 72 hours after injection, 10 mice for each time interval. The organs of the central nervous system and the pituitary glands were chromatographed, and likewise serum from the same animal. The chromatographic studies revealed a compound with the same mobility as 131I-labeled triiodothyronine in the organs of the CNS and in the pituitary gland, but this compound was not present in the serum. In most of the chromatographic studies, the peaks for I, T4 and T3 coincided with those for the standards. In several instances, however, such an exact coincidence was lacking. A tentative explanation for the presence of T3* in the pituitary gland following the injection of T4* is a deiodinating system in the pituitary gland or else the capacity of the pituitary gland to concentrate T3* formed in other organs. The presence of T3* is apparently a characteristic of most of the CNS (brain, midbrain, medulla and spinal cord); but in the case of the optic nerve, the compound is not present under the conditions of this study.

scholarly journals Hepatic endosome fractions contain an ATP-driven proton pump

1985 ◽  
Vol 225 (1) ◽  
pp. 51-58 ◽  
Author(s):  
T Saermark ◽  
N Flint ◽  
W H Evans
Keyword(s):  
Endoplasmic Reticulum ◽  
Golgi Apparatus ◽  
Atpase Activity ◽  
Proton Pump ◽  
Subcellular Distribution ◽  
Plasma Membranes ◽  
Specific Activity ◽  
Ph Gradients ◽  
Subcellular Organelle ◽  
Liver Homogenates

Endosome fractions were isolated from rat liver homogenates on the basis of the subcellular distribution of circulating ligands, e.g. 125I-asialotransferrin internalized by hepatocytes by a receptor-mediated process. The distribution of endocytosed 125I-asialotransferrin 1-2 min and 15 min after uptake by liver and a monensin-activated Mg2+-dependent ATPase activity coincided on linear gradients of sucrose and Nycodenz. The monensin-activated Mg2+-ATPase was enriched relative to the liver homogenates up to 60-fold in specific activity in the endosome fractions. Contamination of the endosome fractions by lysosomes, endoplasmic reticulum, mitochondria, plasma membranes and Golgi-apparatus components was low. By use of 9-aminoacridine, a probe for pH gradients, the endosome vesicles were shown to acidify on addition of ATP. Acidification was reversed by addition of monensin. The results indicate that endosome fractions contain an ATP-driven proton pump. The ionophore-activated Mg2+-ATPase in combination with the presence of undegraded ligands in the endosome fractions emerge as linked markers for this new subcellular organelle.

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