Properties of a bicarbonate-stimulated ATPase from rat uterus

Crude subcellular fractions from rat uterus contain a HCO₃⁻-stimulated Mg²⁺-ATPase with properties analogous to those previously reported for the enzyme in gastric mucosa, pancreas, salivary gland and liver lysosome. Estradiol-17β treatment of ovariectomized rats resulted in an increase in uterine mitochondrial (HCO₃⁻ + Mg²⁺)-ATPase and Mg²⁺-ATPase activity. In an early response (105 min) to estradiol-17β treatment of ovariectomized rats, the lysosomal enzyme, β-N-acetylglucosaminidase increased in the nuclear and mitochondrial fractions and decreased in the microsomal and supernatant fractions.     

Properties of a bicarbonate-stimulated ATPase from rat uterus.

Crude subcellular fractions from rat uterus contain a HCO-3 -stimulated Mg2+ -ATPase with properties analogous to those previously reported for the enzyme in gastric mucosa, pancreas, salivary gland and liver lyosome. Estradiol-17 beta treatment of ovariectomized rats resulted in an increase in uterine mitochondrial (HCO-3 +Mg2+)-ATPase and Mg2+ -ATPase activity. In an early response (105 min) to estradiol-17 beta treatment of ovariectomized rats, the lysosomal enzyme, beta-N-acetylglucosaminidase increased in the nuclear and mitochondrial fractions and decreased in the microsomal and supernatant fractions.     

Distribution of bicarbonate-stimulated ATPase in rat intestinal epithelium

This study reports on the distribution of bicarbonate-stimulated ATPase in rat intestinal epithelial cells. Brush-border membranes and basolateral membranes were separated from each other and from mitochondrial and other intracellular membranes by differential and density gradient centrifugation. Bicarbonate-sensitive ATPase activity followed the mitochondrial marker succinic dehydrogenase closely throughout all the centrifugation steps. The low HCO3--ATPase activity in purified brush-border and basolateral plasma membranes could be accounted for quantitatively by the small mitochondrial contamination. Consequently, there are no grounds for postulating that this enzyme has a direct role in H+ or HCO3- transport across the rat small intestine.     

The absence of bicarbonate-stimulated ATPase activity in the plasma membranes of the bicarbonate secreting ox corneal endothelial cells

Membranes of ox corneal endothelial cells were studied for their surface area by electron microscopical stereology and then separated from homogenates on self forming Percoll gradients. Enzymic analysis of the plasma membrane-enriched fraction failed to demonstrate an ATPase activity which was stimulated by the presence of bicarbonate ions. It is proposed that the well-established bicarbonate secretion of these cells may be coupled to the plasma membrane (Na+ + K+)-ATPase activity by the stoichiometry of Na+/HCO3- close to 1:1.     

Properties of ATPase activity associated with peroxisomes of rat and bovine liver.

1. Peroxisomes were isolated from bovine and rat liver by use of differential and density gradient centrifugations. 2. In the final density gradient (Nycodenz) a distinct peak of ATPase activity codistributed with the peroxisome marker catalase and was well separated from the bulk of the ATPase activity and from markers for other subcellular organelles. 3. The peroxisome-associated ATPase had a pH optimum of 7.5 and was inhibited by N-ethylmaleimide, by N,N'-dicyclohexylcarbodiimide and by 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole, but was unaffected by up to 30 microM n-tributyltin chloride. 4. Prolonged incubation with oligomycin at high concentrations indicated that 50% of peroxisomal ATPase was resistant to this inhibitor. The oligomycin-sensitive ATPase activity required at least a four-fold higher ratio of inhibitor to protein for inhibition than mitochondrial ATPase did. It was concluded that oligomycin-sensitive and oligomycin-resistant ATPase may be associated with liver peroxisomes.     

Electron-cytochemical study of the localization of ATPase activity in the brain tissue of white rats]

A localization of ATPase activity in a rat's brain was studied by the lead method at the presence of Mg2+. The highest activity was noticed in the capillar basal layer, in nucleoli and nuclear chromatin. Less intensive sedimentation of precipitate was observed on the external nuclear membrane, granular cytoplasmic reticulum, ribosomes and in lipofuscin granules. Besides, the reaction product was observed on the cytomembrane of neurons, in synaptic slits and on the vesicles of some terminals. No ATPase activity was seen in mitochondria in the experimental conditions. Equal distribution of reaction product was seen in the cortex and hypothalamus.     

Membranous localization and properties of ATPase of rat liver lysosomes

Summary Lysosomes isolated from rat liver were found to have ATPase activity (EC No. 3.6.1.3). Subfractionation of the lysosomes revealed a membranous localization of ATPase activity. The enzyme has half maximal activity at 0.2mm ATP and is inhibited by high concentrations of ATP. The apparentK _m for divalent metal is 0.2mm, and either ca²⁺ or Mg²⁺ give maximal activity.The ATPase activity has latency when lysosomes are isolated from rats treated with Triton WR-1339. This latency may be due to the presence of internalized sucrose because the activity ofL fraction lysosomes is much less latent and Triton WR-1339 itself is not inhibitory. The latency of glucosamindase, a marker enzyme for lysosomes, contrasts with the low latency of the ATPase and points to an ATPase with an exposed active site in intact lysosomes.     

The localization of the anion-sensitive ATPase activity in corneal endothelium

The localization of the anion-sensitive ATPase (EC 3.6.1.3) of bovine corneal endothelium has been investigated. Homogenates were fractionated by differential and density gradient centrifugation, into fractions enriched in plasma membranes and mitochondria. (Na+ + K+)-ATPase (EC 3.6.1.3) and cytochrome oxidase (EC 1.9.3.1) were used as marker enzymes for these two cell components, and glucose-6-phosphatase (EC 3.1.3.5) was used to identify endoplasmic reticulum. 5'-Nucleotidase (EC 3.1.3.5) was also measured but was found not to be exclusively associated with any one cell component. The activity of the anion-sensitive ATPase (HCO3--ATPase) was measured in suspensions that were frozen and thawed before assay in order to expose latent enzyme activity. The fraction containing the greatest amount of (Na+ + K+)-ATPase (35%) contained only 6% of the cytochrome oxidase and HCO3--ATPase. Conversely, the mitochondrial fraction, containing 40% of the cytochrome oxidase, contained about 40% of the HCO3--ATPase, but only 7% of the (Na+ + K+)-ATPase. The recoveries and relative degree of purification of the cytochrome oxidase and HCO3--ATPase were also nearly identical in the other fractions examined. It was concluded that the anion-sensitive ATPase activity of the corneal endothelium is located solely in the mitochondria and not in the plasma membrane. Consequently, any role that the enzymes may have in the transport of bicarbonate across this tissue, which had been suggested in earlier studies, must be an indirect one.     

Cytochemical localization of plasma membrane ATPase activity in plant cells

Summary The cytochemical localization of ATPase activity has been investigated in maize root cells using both lead and cerium-based capture methods. With both methods, staining at the plasma membrane was observed in all cells of the root, although the precipitate obtained with cerium was more uniform and granular than that with lead. Controls using no substrate or no magnesium, -glycerophosphate to replace ATP, vanadate or boiled tissue generally showed little or no staining. However, biochemical studies on purified plasma membrane fractions showed that ATPase activity was markedly inhibited by fixation, particularly by glutaraldehyde, and also by lead and cerium ions. Non-enzymic hydrolysis of ATP by cerium was greater than that by lead. The value and limitations of these procedures for the localization of plasma membrane H⁺-ATPase activity are summarized in relation to previous criticisms of these methods.Abbreviations DTT dithiothreitol - EDTA ethylene diaminetetraacetic acid - GP B-glycerophosphate - PCMBS p-chloromercuribenzene sulphonic acid - PMSF phenylmethylsulphonyl fluoride     

Biochemical and cytochemical comparison of intestinal bicarbonate-stimulated Mg2+ dependent ATPase and alkaline phosphatase activities in rat, rabbit and guinea pig

Summary Bicarbonate-stimulated Mg²⁺ dependent ATPase activity was demonstrated both biochemically and cytochemically, in brush border membranes from rat, rabbit and guinea pig duodenum. There was no correlation between enzyme activity and basal HCO ₃ ^- secretion rates in the different species. The concentration of HCO ₃ ^- necessary for optimal stimulation of ATPase activity, degree of stimulation and total activity was higher in the rat than in other species. Activity was higher in rat duodenum than in the ileum. This is consistent with the proposed electrogenic HCO ₃ ^- secretion in the duodenum. Distribution of activities of alkaline phosphatase and HCO ₃ ^- -stimulated Mg²⁺-ATPase along the duodenal villus showed significant differences, suggesting that the two activities reflect, at least in part, distinct enzymes.     

Cytochemical localization of ATPase activity in phloem tissues ofRicinus communis

Summary Standard lead precipitation procedures have been used to examine the localization of ATPase activity in phloem tissues ofRicinus communis. Reaction product was localized on the plasma membrane of the companion cells associated with sieve elements and of parenchyma cells in phloem tissues from the leaf, petiole, stem and root. ATPase activity was also present on the plasma membrane and dispersed P-protein of sieve elements in petiole, stem and root tissue, but was absent from the plasma membrane of these cells in the leaf minor veins. Substitution of-glycerophosphate for ATP produced no change in the localization of reaction product in leaf tissue. These findings are discussed in relation to current theories on the mechanism of sugar transport and phloem loading.     

Ultrastructural localization of ATPase activity in cotton fiber during elongation

Summary The ultrastructural distribution of potassium chloride stimulated adenosine triphosphatase activity was investigated in the outer integument of a linted cultivar of cotton and a lintless (naked seed) mutant from one day preanthesis to eight days postanthesis by using a heavy metal simultaneous capture reaction technique. No enzyme activity other than in mitochondria was observed in the lintless mutant. In the linted cultivar no ATP-specific enzyme activity was seen in non-elongating epidermal cells, subepidermal cells of the outer integuments or any controls. As fiber initials started elongating, enzyme activity gradually appeared on the tonoplasts of enlarging vacuoles. Heavier lead phosphate deposits were observed on the membrane of small vacuoles compared to the tonoplast. This activity continued at least to eight days postanthesis. The enzyme inhibitor, N,N-dicyclohexylcarbodiimide inhibited, while KCl stimulated, tonoplast ATPase activity. The gradual increase of ATPase activity on the tonoplast of expanding fibers, but not on the tonoplasts of non-fiber cells, suggests the active transport of osmotically active compounds, presumably potassium and malate, into the vacuoles of expanding fibers. Fusion of smaller vacuoles with the large central vacuole indicates that these structures contribute additional membrane components along with their enzyme activity to the tonoplast of expanding fibers. The occurrence of ATPase activity, of ER-derived vesicular structures, and the organized pattern of deposition of these structures on the tonoplast indicate ER-originated ATPase activity. This study supports the theory of osmoregulation in cotton fiber where ATPase provides the energy for active accumulation of osmotically active compounds, (K⁺, malate) into the vacuoles, thereby generating and maintaining the turgor pressure required for fiber expansion.Abbreviations ATPase Adenosine triphosphatase - DCCD N,N-Di-cyclohexylcarbodiimide - EM Electron microscope - ER Endoplasmic reticulum - GP -Glycerophosphate - LC Lead citrate - PEP-Case Phosphoenolpyruvate carboxylase - UA Uranyl acetate     

Inhibition of calcium ATPase by phencyclidine in rat brain

Phencyclidine (PCP) is a potent psychotomimetic drug of abuse and has profound effect on the functioning of the central nervous system (CNS). Many of the CNS functions are known to be mediated by calcium (Ca2+). In the present study we have investigated the effects of PCP on Ca2+ ATPase activity in rat brain both in vitro and in vivo. For in vitro studies, synaptic membrane fractions prepared from normal rat brain were incubated with PCP at different concentrations (25-100 M) before the addition of substrate. For n vivo studies, rats were treated with a single moderate dose of PCP (10 mg/kg, IP) and animals were sacrificed at 1,2, 6 and 12 h after treatment. Ca2+ ATPase activity in synaptic membrane fractions was assayed by estimation of inorganic phosphate. PCP inhibited the Ca2+ ATPase in vitro in a concentration dependent manner with significant effect at 50 and 100 M. A significant time-dependent reduction of the Ca2+ ATPase activity was evident in vivo. As early as 2 h after the treatment of rats with PCP the ATPase activity was significantly reduced. The reduction of Ca2+ ATPase observed even at 12 h after treatment suggesting a prolonged presence of the drug in the brain tissue. Further, kinetic studies in vitro indicated PCP to be a competitive inhibitor of Ca2+ ATPase with respect to the substrate, ATP. The present findings indicate that PCP inhibits synaptic membrane Ca2+ ATPase thus altering cellular Ca2+ homeostasis in CNS which may partially explain the pharmacological effects of the drug and/or its neurotoxicity.     

Uncompetitive stimulation of rat brain Na-K ATPase activity by rapid eye movement sleep deprivation

Rapid eye movement sleep deprivation is associated with an increase in Na-K ATPase activity. In order to understand the possible biochemical mechanism of this increase, the kinetics of Na-K ATPase was studied. Although the enzyme activity increased after the deprivation, the catalytic efficiency of the enzyme remained unaltered. The rapid eye movement sleep deprivation increased both the Vmax and the Km suggesting an uncompetitive stimulation of the enzyme. While increase in norepinephrine resulted in an increased Vmax, that of calcium increased the Km. Since an increase in norepinephrine has been suggested after deprivation, the increased Vmax is attributed to increased norepinephrine level following deprivation. However, since rapid eye movement sleep deprivation is reported to be associated with a decrease in calcium levels, the increase in Km following deprivation may be attributed to changes in factor(s) other than calcium.     

Membranous localization and properties of ATPase of rat liver lysosomes.

Lysosomes isolated from rat liver were found to have ATPase activity (EC No 3.6.1.3). Subfractionation of the lysosomes revealed a membranous localization of ATPase activity. The enzyme has half maximal activity at 0.2 mM ATP and is inhibited by high concentrations of ATP. The apparent Km for divalent metal is 0.2 mM, and either Ca2+ or Mg2+ give maximal activity. The ATPase activity has latency when lysosomes are isolated from rats treated with Triton WR-1339. This latency may be due to the presence of internalized sucrose because the activity of L fraction lysosomes is much less latent and Triton WR-1339 itself is not inhibitory. The latency of glucosaminidase, a marker enzyme for lysosomes, contrasts with the low latency of the ATPase and points to an ATPase with an exposed active site in intact lysosomes.