Separation of Adenosine Triphosphatase of HK and LK Sheep Red Cell Membranes by Density Gradient Centrifugation

Membrane fragments from high potassium (HK) and low potassium (LK) sheep red cells were separated by density gradient centrifugation. Three preparations were studied: (1) HK membranes sonicated for 20 minutes, (2) HK membranes sonicated for 3 minutes, and (3) LK membranes sonicated for 3 minutes. The adenosine triphosphatase (ATPase) activity in the maximally disrupted preparation (1) was not sensitive to Na + K and was recovered in relatively small but heavy (specific gravity 1.19) fragments which made up no more than 8 per cent of the total membrane. Both Na + K-sensitive (S) and Na + K-insensitive (I) ATPase activity were found in the more gently broken up preparations (2) and (3) but the ratio of S- to I-ATPase was much greater in HK than in LK membrane fragments. S-ATPase activity in preparation (2) was about 50 per cent that observed in HK membranes prior to sonication. S-ATPase activity was recovered from the density gradient in relatively large but light (specific gravity 1.10) fragments. As was the case with the maximally disrupted preparation (1), I-ATPase activity in both preparations (2) and (3) was recovered in small but heavy (specific gravity > 1.20) fragments. The possibility that sensitivity of sheep red cell membrane ATPase to Na + K depends on the association between units containing the enzyme(s) and large, light, phospholipid-containing components is discussed.     

Calcium Uptake and Associated Adenosine Triphosphatase Activity of Isolated Platelet Membranes

A platelet subcellular fraction, sedimenting between 14,000 and 40,000 g and consisting primarily of membrane vesicles, accumulates up to 200–400 nmoles calcium/mg protein in the presence of ATP and oxalate. Steady-state levels of calcium accumulation are attained in 40–60 min. Calcium uptake requires adenosine triphosphate (ATP), is enhanced by oxalate, and is accompanied by the release of inorganic phosphate. Calcium accumulation and phosphate release require magnesium and are inhibited by Salyrgan (10 µM) and adenosine diphosphate (ADP) (1 mM), but not by ouabain (0.1 mM). The ATPase activity is stimulated by low concentrations of calcium (5–10 µM) and is inhibited by 2 mM EGTA. Electron microscopic histochemistry using lead nitrate to precipitate released phosphate results in lead precipitates localized primarily at the inner surface of membrane vesicles. These results provide evidence for a membrane ATPase that is stimulated by low concentrations of calcium and may be involved in the transport of calcium across the membrane. It is postulated that the observed calcium uptake activity is an in vitro manifestation of a calcium extrusion pump in the intact platelet.     

Inhibition Kinetics of Sheep Brain Glutathione Reductase by Cadmium Ion

Glutathione reductase participates in preventing lipid peroxidation by oxygen radicals which results in cellular damage. The brain is among the organs most susceptible to cadmium-induced lipid peroxidation. The mechanism of free radical generation by Cd²⁺ is not well understood, but it is known that Cd²⁺ is an inhibitor of glutathione reductase. In this study, inhibition kinetics of the brain glutathione reductase by Cd²⁺ was investigated. Sheep brain enzyme (11,000-fold purified) was used for this purpose. The data were analyzed by a nonlinear curve fitting program. It was found that the inhibition was competitive with respect to oxidized glutathione and uncompetitive with respect to NADPH. Inhibition constants were found as 12.3 and 9.4 μM, respectively. These findings might contribute to the understanding of the mechanism of lipid peroxidation by Cd²⁺ in brain.     

Membrane Adenosine Triphosphatase of Escherichia coli: Activation by Calcium Ion and Inhibition by Monovalent Cations

Membrane ghost preparations of Escherichia coli K-12 obtained by osmotic lysis of lysozyme-induced spheroplasts were found to possess both Mg(++)- and Ca(++)-activated adenosine 5'-triphosphatase (ATPase, EC 3.6.1.3) activities. Maximal activities of 1.0 to 1.5 mumoles of orthophosphate released per min per mg of protein were obtained at pH 9.0 with a molar Mg(++) to adenosine 5'triphosphate (ATP) ratio of 2:5 and at pH 9.9 with a molar Ca(++) to ATP ratio of 1:5. These ATPase activities were not altered by ouabain, fluoride, N-ethylmaleimide, 2,4-dinitrophenol, cyanide, or dithionite, but were inhibited by low concentrations of azide, p-chloromercuribenzoate, and pentachlorophenol. Mg(++) ATPase was more susceptible to inhibition by azide than was Ca(++) ATPase. Fifty per cent inactivation of both activities was observed when membrane ghost preparations were preincubated at 66 C for 10 min. The Mg(++) and Ca(++) ATPase activities of these preparations were not additive, but did respond independently to inhibition by monovalent cations. Ca(++) ATPase was found to be very sensitive to inhibition by K(+), Na(+), Li(+), Rb(+), and Cs(+); Mg(++) ATPase was relatively insensitive to these ions. One possible interpretation of the results presented in this paper is that the membrane of E. coli possesses an ATPase which is activated by either Mg(++) or Ca(++) and that activation by Ca(++) increases the susceptibility of this enzyme to inhibition by monovalent cations. Increased susceptibility of E. coli membrane ATPase to inhibition by monovalent cations such as Na(+) and K(+) as a consequence of Ca(++) activation could represent a regulatory mechanism.     

An Adenosine Triphosphatase in Agrobacterium tumefaciens

Cell-free extracts from the phytopathogen Agrobacterium tumefaciens contained an enzyme(s) capable of cleaving nucleoside triphosphates, showing highest specific activity toward adenosine triphosphate. Differences are indicated between the adenosine triphosphate phospbohydrolase described hero and other microbial adenosine triphosphatases previously reported. The adenosine trit)hosphatasc activity was stimulated by Mg²⁺ and Mn²⁺ and inhibited by other divalent cations. Enzymatic activity was inhibited 100% by mercuric chloride, 38% by p-chloromercuribenzoate, 22% by potassium fluoride, 12% by sodium azide, and 10% by iodoacetic acid, all at a final concentration of 10^?2 M. The enzyme (s) had an optimal PH range of 7.8 to 8.0, and an optimal temperature for hydrolysis at 40deg;C.     

Energy-Transducing Adenosine Triphosphatase from Escherichia coli: Purification, Properties, and Inhibition by Antibody

The membrane adenosine triphosphatase (E.C. 3.6.1.3) from Escherichia coli has been solubilized with Triton X-100 and purified to near homogeneity. The purified enzyme has a sedimentation coefficient of 12.9S in a sucrose gradient, corresponding to a molecular weight of about 360,000. On electrophoresis in gels containing sodium dodecyl sulfate, it dissociates into subunits with apparent molecular weights of 60,000, 56,000, 35,000, and 13,000. The purified enzyme loses activity and breaks down into subunits when stored in the cold. Guanosine 5'-triphosphate and inosine 5'-triphosphate are alternative substrates. Ca(2+) and, to a small extent, Co(2+) or Ni(2+) will substitute for Mg(2+) in the reaction. The K(m) for Mg-adenosine triphosphate of the membrane-bound enzyme is 0.23 mM, and for the pure enzyme it is 0.29 mM. Azide is a noncompetitive inhibitor of both the membrane-bound enzyme and the pure enzyme. P(i) is a noncompetitive inhibitor of the solubilized enzyme. An antibody to the purified enzyme was obtained from rabbits. The antibody inhibits the solubilized enzyme and virtually all of the adenosine triphosphate hydrolysis by membranes from cells grown aerobically or anaerobically. The antibody also inhibits the adenosine triphosphate-stimulated pyridine nucleotide transhydrogenase (E.C. 1.6.1.1) of the E. coli membrane.     

Inhibition of Adenosine Triphosphatase Activity from a Plasma Membrane Fraction of Acer pseudoplatanus Cells by Carbanilate Derivatives

Thirty-one carbanilate derivatives were assayed for their capabilities to inhibit the ATPase activity of a plasma membrane fraction from Acer pseudoplatanus cells. At a concentration of 100 micromolar, nine compounds strongly inhibited the ATPase activity, with I₅₀ ranging from 14.5 micromolar to 35 micromolar. These molecules were also inhibitory to plasma membrane ATPases of other origins: plant (maize shoot), yeast (Schizosaccharomyces pombe), and animal (dog kidney). The most efficient molecule appeared to be 2,2,2-trichloroethyl 3,4-dichlorocarbanilate.     

Membrane-bound Adenosine Triphosphatase Activities of Oat Roots

Homogenates of oat (Avena sativa cv. Goodfield) roots contained at least five membrane-associated adenosine triphosphatase (ATPase) activities. The membrane-bound ATPases were separated on sucrose gradients and distinguished by membrane density, pH optima, sensitivity to monovalent salts, and substrate specificity.     

Inhibition of Membrane-Bound Adenosine Triphosphatase Activity from Nicotiana tabacum L. Leaves with Alkyltrimethylammonium Bromide

Cetyltrimethylammonium bromide (CTAB), a cationic detergent,more effectively inhibited the activity of membrane-bound epidermaladenosine triphosphatase (ATPase) of tobacco (Nicotiana tabacumL. cv. Samsun) leaves than anionic or non-ionic detergents.The inhibition of ATPase activity was highly dependent on thelength of the alkyl chain of alkyltrimethylammonium: CTAB >dodecyltrimethylammonium bromide > n-octyltrimethylammoniumbromide trimethylammonium chloride cetyl bromide, comparedat 10^–4 M. The last three derivatives hardly inhibitedthe activity. CTAB inhibition was equivalent to that due toother cationic detergents, cetylpyridinium bromide and cetylamine, but less than that by gramicidin S and tyrocidine andstronger than that by N,N'-dicyclohexylcarbodiimide and vanadate. These results show that a certain length of the alkyl chain(C_n>12) and the combination of both hydrophobic and chargedgroups of a detergent moiety are indispensable for inhibitingthe membrane-bound epidermal ATPase activity. (Received January 26, 1982; Accepted April 10, 1982)     

Activation of the Adenosine Triphosphatase of Limulus polyphemus Actomyosin by Tropomyosin

Purified actin does not stimulate the adenosine triphosphatase (ATPase) activity of Limulus myosin greatly. The ATPase activity of such reconstituted preparations is only about one-fourth the ATPase of myofibrils or of natural actomyosin. Actin preparations containing tropomyosin, however, activate Limulus myosin fully. Both the tropomyosin and the actin preparations appear to be pure when tested by different techniques. Tropomyosin combines with actin but not with myosin and full activation is reached at a tropomyosin-to-actin ratio likely to be present in muscle. Tropomyosin and actin of several different animals stimulate the ATPase of Limulus myosin. Tropomyosin, however, is not required for the ATPases of scallop and rabbit myosin which are fully activated by pure actin alone. Evidence is presented that Limulus myosin, in the presence of ATP at low ionic strength, has a higher affinity for actin modified by tropomyosin than for pure actin.     

Inhibition of Adenosine Triphosphatase Activity from a Plasma Membrane Fraction of Acer pseudoplatanus Cells by 2,2,2-Trichloroethyl 3,4-Dichlorocarbanilate

2,2,2-Trichloroethyl 3,4-dichlorocarbanilate (SW26) is toxic for Acer pseudoplatanus cell cultures. It inhibited the cellular proton extrusion and depolarized the plasmalemma. In vitro, it inhibited the plasma membrane ATPase. SW 26 was also inhibitory to membrane ATPases of other origins—plant (maize shoot), fungus (Schizosaccharomyces pombe), and animal (dog kidney)—with about the same efficiency (7.5 micromolar < I₅₀ < 22 micromolar). It did not inhibit the oligomycin-sensitive ATPase from purified plant mitochondria, nor molybdate-sensitive soluble phosphatases. SW26 was more specific for plasma membrane ATPases than diethylstilbestrol or vanadate. A Lineweaver-Burk plot analysis showed that inhibition kinetics were purely noncompetitive (K_i = 14.7 micromolar) below 20 micromolar. Above this concentration, the inhibition pattern was not consistent with Michaelis-Menten kinetics, and a Hill plot representation revealed a positive cooperativity.     

Flexibility of an Active Center in Sodium-Plus-Potassium Adenosine Triphosphatase

In plasma membranes of intact cells an enzymatic pump actively transports sodium ions inward and potassium ions outward. In preparations of broken membranes it appears as an adenosine triphosphatase dependent on magnesium, sodium, and potassium ions together. In this adenosine triphosphatase a phosphorylated intermediate is formed from adenosine triphosphate in the presence of sodium ions and is hydrolyzed with the addition of potassium ions. The normal intermediate was not split by adenosine diphosphate. However, selective poisoning by N-ethylmaleimide or partial inhibition by a low magnesium ion concentration yielded an intermediate split by adenosine diphosphate and insensitive to potassium ions. Pulse experiments on the native enzyme supported further a hypothesis of a sequence of phosphorylated forms, the first being made reversibly from adenosine triphosphate in the presence of sodium ion and the second being made irreversiblyfrom the first and hydrolyzed in the presence of potassium ion. The cardioactive steriod inhibitor, ouabain, appeared to combine preferentially with the second form. Phosphorylation was at the same active site according to electrophoretic patterns of proteolytic phosphorylated fragments of both reactive forms. It is concluded that there is a conformational change in the active center for phosphorylation during the normal reaction sequence. This change may be linked to one required theoretically for active translocation of ions across the cell membrane.     

Adaptation of the Blood Antioxidant Defence Mechanisms of Sheep with a Genetic Lesion Resulting in Low Red Cell Glutathione Concentrations

Finnish Landrace sheep with a genetic lesion which results in restricted cysteine transport across the red cell membrane have total glutathione concentrations in their red blood cells that are approximately 40% of those in normal sheep of the same breed. However, dimethyldisulphide-challenged red blood cells from both phenotypes produce an ESR-spin adduct at similar rates. The resistance of the low glutathione phenotype red cells to oxidant challenge is reflected by increases in the activities of antioxidant enzymes. Sheep with a genotypic disorder in cysteine transport may be a suitable model for studying the genetic expression of antioxidant enzymes in response to oxidant loads.     

Light-dependent Reduction of Oxidized Glutathione by Ruptured Chloroplasts

Crude extracts of pea shoots (Pisum sativum) catalyzed oxidized glutathione (GSSG)-dependent oxidation of NADPH which was attributed to NADPH-specific glutathione reductase. The pH optimum was 8 and the K_m values for GSSG and NADPH were 23 μm and 4.9 μm, respectively. Reduced glutathione (GSH) inhibited the reaction. Crude extracts also catalyzed NADPH-dependent reduction of GSSG; the ratio of the rate of NADPH oxidized to GSH formed was 0.49. NADH and various substituted mono- and disulfides would not substitute for NADPH and GSSG respectively. Per mg of chlorophyll, enzyme activity of isolated chloroplasts was 69% of the activity of crude extracts.     

Strain Energy Function of Red Blood Cell Membranes

The several widely different values of the elastic modulus of the human red blood cell membrane which have been reported in the literature are incorporated into a single strain energy function consisting of two terms. One term gives the small stresses and low elastic modulus which is observed when the red cell membrane is deformed at constant area. The second term contributes a large isotropic stress dependent on the change of area. The strain energy function is applied to the process of sphering of red blood cells in a hypotonic solution. It is shown that a nearly perfect sphere can result even though the red blood cell membrane is homogeneous in all areas of the cell. Results pertinent to sieving and micropipette experiments are also explored.     

Salt-stimulated Adenosine Triphosphatase from Smooth Microsomes of Turnip

The turnip (Brassica rapa L.) microsome fraction contains both a Mg²⁺-inhibited acid phosphatase and a salt-stimulated Mg²⁺-activated ATPase. However, as the pH optimum of the ATPase was 8.0 to 8.5, the acid phosphatase activity could be eliminated by assaying at or above pH 7.8. The ATPase was concentrated in a fraction equivalent to the smooth microsomal membranes and was not due to fragments of mitochondria. The salt-stimulated activity showed specificity for anions rather than cations. The activity was further stimulated by carbonyl cyanide m-chloro-phenylhydrazone (CCCP), 2,4-dinitrophenol, valinomycin, nigericin, and NH₄Cl. There was a synergistic effect between CCCP and valinomycin. Activity was insensitive to oligomycin phlorizin, ouabain, and atractylate. Based on similarity to the chloroplast ATPase, it was proposed that this ATPase was situated on the outside of the vesicle.     

Characterization of the H Translocating Adenosine Triphosphatase and Pyrophosphatase of Vacuolar Membranes Isolated by Means of a Perfusion Technique from Chara corallina

Sealed tonoplast vesicles were isolated from single cells of Chara corallina with the aid of an intracellular perfusion technique in combination with a 3/10% Percoll two step gradient centrifugation. The isolated tonoplast fraction was free from plasmalemma and chloroplasts, and showed no activities of cytochrome c oxidase, and latent IDPase, but had about 10% of the NADH-cytochrome c reductase activity. The vesicles had both ATPase and PPase activities, which could be stimulated in the presence of 10 micromolar gramicidin by 170 and 130%, respectively, demonstrating the existence of sealed vesicles. Furthermore, ATP- and PPi-dependent H⁺ pumping through the membrane into the vesicles was shown. Both ATPase and PPase had pH optima around pH 8.5. At the physiological pH, 7.3, they still had more than 80% of their maximal activities. Ammonium molybdate, azide, and vanadate had no or little effect on the activities of both enzymes or their associated H⁺ pumping activities. N,N′-dicyclohexylcarbodiimide inhibited the ATPase strongly (I₅₀ = 20 micromolar) but the PPase only weakly. The ATPase was also more sensitive to N-ethylmaleimide than the PPase. 4,4′-Stilbenedisulfonic acid affected both enzyme activities and their associated H⁺ pumping activities. This is in contrast to the H⁺-PPase of higher plants which is 4,4′-stilbenedisulfonic acid insensitive.     

Dependence of Delayed Luminescence upon Adenosine Triphosphatase Activity in Chlorella

Delayed luminescence and fluorescence yield after illumination by a short flash were measured in Chlorella pyrenoidosa in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea. Addition of tri-N-butyl-tin (TNBT), a specific inhibitor of ATPase, drastically increases the life-time of the reduced photosystem II primary acceptor Q⁻ and decreases the intensity of delayed luminescence. This indicates a slowing of the charge recombination between the oxidized donor and reduced acceptor of photosystem II centers. No inhibition is observed in isolated chloroplasts when the membrane is permeable to ions, i.e. in the presence of Gramicidin D and KCl.