Anion-stimulated ATPase in locust rectal epithelium

1988 ◽  
Vol 66 (2) ◽  
pp. 431-438 ◽  
Author(s):  
R. A. Lechleitner ◽  
J. E. Phillips
Keyword(s):  
Alkaline Phosphatase ◽  
Plasma Membrane ◽  
Atpase Activity ◽  
Microsomal Fraction ◽  
Leucine Aminopeptidase ◽  
Mitochondrial Fraction ◽  
Mitochondrial Enzymes ◽  
Differential Centrifugation ◽  
Succinate Cytochrome C Reductase ◽  
Nacl Cotransport

The rectum, the main reabsorptive site in the locust excretory system, actively transports Cl−. This Cl− absorption is electrogenie, not dependent on Na+or [Formula: see text] and insensitive to inhibitors of NaCl cotransport or [Formula: see text] exchange. To determine if active Cl− transport across rectal epithelia might be due to an anion-stimulated ATPase, a microsomal fraction was obtained by differential centrifugation. Microsomal ATPase activity was stimulated in the following sequence: sulphite > bicarbonate > chloride. Maximal ATPase activity was obtained at 25 mM [Formula: see text] or 25 mM Cl−. Thiocyanate (10 mM) inhibited 90% of the anion-stimulated ATPase activity. The microsomal fraction was enriched in the plasma membrane markers, leucine aminopeptidase, alkaline phosphatase, 5′-nucleotidase, and γ-glutamyItranspeptidase, and had little contamination of the mitochondrial enzymes, succinate cytochrome c reductase and cytochrome oxidase. Na,K-ATPase was enriched in the mitochondrial fraction. Microscopic examination confirmed that basolateral membranes were associated with mitochondria following differential centrifugation, while the microsomal fraction contained little mitochondrial contamination. These results indicate the presence of an anion-stimulated ATPase activity that could be responsible for active Cl− transport across locust recta.

Anion-stimulated ATPase activity of brush border from rat small intestine.

1979 ◽  
Vol 236 (1) ◽  
pp. E70 ◽  
Author(s):  
M H Humphreys ◽  
L Y Chou
Keyword(s):  
Alkaline Phosphatase ◽  
Atpase Activity ◽  
Brush Border ◽  
Atp Hydrolysis ◽  
Intestinal Transport ◽  
Mitochondrial Enzymes ◽  
Intestinal Alkaline Phosphatase ◽  
Ph Optimum ◽  
Low Concentrations ◽  
Small Intestinal

Differential centrifugation of rat small intestinal homogenates produced a crude brush border (BB) fraction that was enriched 15-fold for the marker enzymes, alkaline phosphatase and sucrase; contamination with mitochondrial enzymes, monoamine oxidase and succinate dehydrogenase, was minimal. ATP hydrolysis by this BB fraction was stimulated by addition of several anions to the incubation medium: HCO3 and Cl were equally effective in this regard, with NO3, NO2, SO4, and acetate being less stimulatory. SCN and CNO inhibited ATPase activity, whereas the divalent anion SO3 was stimulatory at low concentrations (less than 25 mM) but inhibitory at 100 mM. Maximum anion stimulation was observed at a Mg concentration of 0.5 mM, and pH optimum was 8.5. Kinetic analysis showed that HCO3 increased the Vmax without altering the Km for ATP; the Ka for this effect of HCO3 was 35 mM. This enzyme activity was completely inhibited by 20 mM L-phenylalanine, 10 mM L-cysteine, and 3 mM EDTA, compounds that also inhibited intestinal alkaline phosphatase. These results demonstrate the presence of anion-stimulated ATPase activity in rat small intestinal brush border and suggest that this activity may be related to intestinal alkaline phosphatase. The role of this enzyme in intestinal transport is not known, but could relate to the regulation of intestinal absorption and secretion.

scholarly journals 3β-Hydroxy steroid dehydrogenase activity in the mitochondria of rat adrenal homogenates

1971 ◽  
Vol 125 (4) ◽  
pp. 983-989 ◽  
Author(s):  
R S Basch ◽  
M J Finegold
Keyword(s):  
Electron Microscopy ◽  
Microsomal Fraction ◽  
Total Activity ◽  
Mitochondrial Fraction ◽  
Differential Centrifugation ◽  
Radioisotopic Method ◽  
Cell Fractions ◽  
Kinetics Of ◽  
Hydroxy Steroid ◽  
Steroid Dehydrogenase

The activity of 3β-hydroxy steroid dehydrogenase (EC 1.1.1.51) in the mitochondrial fraction of rat adrenal homogenates was approx. 31% of the total activity recovered after differential centrifugation and washing of the particulate fractions. Some 45% of the total activity was found in the microsomal fraction. The activity was assayed by a radioisotopic method devised in this laboratory for the purpose of studying small quantities of tissue and cell fractions. Satisfactory separation of the two fractions was demonstrated by electron microscopy of the pellets and by comparative recoveries of RNA, steroid 21-hydroxylase and cytochrome c oxidase in the various compartments. Analyses of the kinetics of the enzyme activity in the two fractions revealed no significant differences in apparent Km for pregnenolone, dehydroepiandrosterone or NAD+, but demonstrated a distinct difference in the Km for NADP+. pH optima and susceptibility to cyanoketone inhibition were similar in both fractions.

Synthetic [Asu1,7] eel calcitonin increases phosphorus content in the hepatic mitochondria of rats

1983 ◽  
Vol 104 (3) ◽  
pp. 352-356 ◽  
Author(s):  
Masayoshi Yamaguchi
Keyword(s):  
Body Weight ◽  
Plasma Membrane ◽  
Oxidative Phosphorylation ◽  
Atpase Activity ◽  
Phosphorus Content ◽  
Mitochondrial Fraction ◽  
Liver Cells ◽  
Significant Alteration ◽  
Intracellular Phosphorus

Abstract. The change of subcellular phosphorus content in the liver was investigated after a single sc administration of synthetic [Asu1,7] eel calcitonin (CT) to fed rats. Administration of CT (80 MRC mU/100 g body weight) produced a significant increase in phosphorus content in the mitochondrial fraction, while this increase was not observed in fractions containing plasma membrane, nuclei, microsomes and cytosol. A significant increase in the mitochondrial phosphorus content was observed even at the lowest dose of CT (40 MRC mU/100 g body weight). A single ip administration of 2,4-dinitrophenol (0.1 mg/100 g body weight), an inhibitor of oxidative phosphorylation, did not prevent significant increases in phosphorus contents of the homogenate and the mitochondria caused by administration of CT (80 MRC mU/100 g body weight), although the drug markedly inhibited ATPase activity in the mitochondria. Administration of CT did not produce a significant alteration in the mictochondrial ATPase activity. These results suggest that phosphorus taken up by the liver cells after CT administration is largely located in the mitochondria, and that this increase is not related to oxidative phosphorylation. Presumably the hepatic mitochondria play a role in the storage of intracellular phosphorus increased by CT.

Cl- -HCO3- -stimulated ATPase in intestinal mucosa of Aplysia

1985 ◽  
Vol 248 (2) ◽  
pp. R241-R248 ◽  
Author(s):  
G. A. Gerencser ◽  
S. H. Lee
Keyword(s):  
Plasma Membrane ◽  
Atpase Activity ◽  
Intestinal Mucosa ◽  
Membrane Fraction ◽  
Transport Mechanism ◽  
Plasma Membranes ◽  
Sucrose Density Gradient ◽  
Disulfonic Acid ◽  
Differential Centrifugation ◽  
Optimum Ph

The serosa negative transepithelial potential difference across Aplysia intestine is generated by a Na+-independent, active electrogenic Cl- absorptive mechanism. In an attempt to clarify the Cl- absorptive mechanism an anion-stimulated ATPase was prepared from plasma membranes from Aplysia enterocytes utilizing differential centrifugation and sucrose density gradient techniques. ATPase activity, which could be activated by either Cl- or HCO3-, was found in the plasma membrane fraction. Maximal anion-ATPase activity was achieved with either 25 mM Cl- or 25 mM HCO3-. The apparent Km for Cl- activation of the ATPase was 10.3 mM, whereas apparent Km for HCO3- was 9.7 mM. ATP was the most effective nucleotide substrate for both HCO3- and Cl- -ATPase activities, whereas optimum pH for both activities was 7.8. These enzyme activities were inhibited more than 30% by thioacyanate (10 mM). Acetazolamide and vanadate were also found to strongly inhibit both Cl- and HCO3- -ATPase activities, whereas 10 microM 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid, 1 mM furosemide, or 1 mM ouabain had little or no effect. These results are consistent with the hypothesis that the active Cl- transport mechanism in Aplysia intestine could be a Cl- -HCO3- -stimulated ATPase found in the enterocyte plasma membrane.

A Cyanobacterial ATPase Distinct from the Coupling Factor of Photophosphorylation

1982 ◽  
Vol 37 (7-8) ◽  
pp. 658-664 ◽  
Author(s):  
Wolfgang Lockau ◽  
Susanne Pfeffer
Keyword(s):  
Alkaline Phosphatase ◽  
Plasma Membrane ◽  
Atpase Activity ◽  
Cytochrome Oxidase ◽  
Oxidase Activity ◽  
Maximal Activity ◽  
Coupling Factor ◽  
Anabaena Variabilis ◽  
Cytochrome Oxidase Activity ◽  
Low Concentrations

Abstract A particle-bound, Mg2+ -dependent ATPase activity is investigated in cell-free extracts of the tyanobacterium Anabaena variabilis. The enzyme can be clearly distinguished from the cyanobacterial coupling factor of photophosphorylation and from the alkaline phosphatase. It requires low concentrations of Ca2+ for maximal activity and is inhibited by ortho-vanadate, indicating that the enzyme may form a phosphorylated intermediate in its catalytic cycle. The distribution of the ATPase in sucrose density gradients does not follow that of thylakoids. It is concluded from these characteristics that the enzyme is bound to the plasma membrane. The cytochrome oxidase activity of the extracts appears to be restricted to the thylakoids.

Localization of Adenosine Triphosphatase Activity in the Phleom of Nicotiana Tabacum

1972 ◽  
Vol 30 ◽  
pp. 230-231
Author(s):  
James Cronshaw ◽  
Jamison E. Gilder
Keyword(s):  
Plasma Membrane ◽  
Atpase Activity ◽  
Adenosine Triphosphatase ◽  
Higher Plants ◽  
High Surface ◽  
Root Tip ◽  
Animal Cells ◽  
Physiological Processes ◽  
Tip Cells ◽  
Atpase Localization

Adenosine triphosphatase (ATPase) activity has been shown to be associated with numerous physiological processes in both plants and animal cells. Biochemical studies have shown that in higher plants ATPase activity is high in cell wall preparations and is associated with the plasma membrane, nuclei, mitochondria, chloroplasts and lysosomes. However, there have been only a few ATPase localization studies of higher plants at the electron microscope level. Poux (1967) demonstrated ATPase activity associated with most cellular organelles in the protoderm cells of Cucumis roots. Hall (1971) has demonstrated ATPase activity in root tip cells of Zea mays. There was high surface activity largely associated with the plasma membrane and plasmodesmata. ATPase activity was also demonstrated in mitochondria, dictyosomes, endoplasmic reticulum and plastids.

Secretagogue-induced changes in subcellular Ca2+ distribution in isolated pancreatic acini

1981 ◽  
Vol 240 (2) ◽  
pp. G130-G140
Author(s):  
R. L. Dormer ◽  
J. A. Williams
Keyword(s):  
Atomic Absorption Spectrometry ◽  
High Speed ◽  
Microsomal Fraction ◽  
Absorption Spectrometry ◽  
Subcellular Fractionation ◽  
Differential Centrifugation ◽  
Zymogen Granules ◽  
Pancreatic Acini ◽  
Induced Changes ◽  
Stimulation Of

In a prior study, we demonstrated that pancreatic secretagogues increased both the uptake into and washout of 45Ca2+ from isolated mouse pancreatic acini. The net result of these processes was an initial fall in total acinar cell Ca2+ content. In the present study, we have employed subcellular fractionation of acini under conditions that minimized posthomogenization redistribution of Ca2+ in order to localize those organelles involved in intracellular Ca2+ fluxes. Homogenization and differential centrifugation of acini, preloaded with 45Ca2+ and subjected to a period of washout, showed that carbachol induced an increased loss of 45Ca2+ from all fractions isolated. The high-speed microsomal fraction lost 45Ca2+ to a greater extent than did whole acini; measurement of total Ca2+ by atomic absorption spectrometry showed a net loss of Ca2+ from this fraction. Purification of the lower-speed fractions indicated that carbachol increased 45Ca2+ exchange with both zymogen granules and mitochondria, but net Ca2+ levels in these organelles were unchanged. It was concluded that stimulation of pancreatic acini by carbachol results in the release of calcium from a microsomal compartment leading to a rise in cytoplasmic Ca2+, increased exchange with granule and mitochondrial Ca2+, and increased efflux of Ca2+ from the cell.

scholarly journals Potassium depletion increases proton pump (H+-ATPase) activity in intercalated cells of cortical collecting duct

2000 ◽  
Vol 279 (1) ◽  
pp. F195-F202 ◽  
Author(s):  
Randi B. Silver ◽  
Sylvie Breton ◽  
Dennis Brown
Keyword(s):  
Plasma Membrane ◽  
Atpase Activity ◽  
Collecting Duct ◽  
Proton Pumps ◽  
Intercalated Cells ◽  
Cortical Collecting Duct ◽  
Collecting Ducts ◽  
Acid Load ◽  
Collecting Tubules ◽  
Atpase Staining

Intercalated cells (ICs) from kidney collecting ducts contain proton-transporting ATPases (H+-ATPases) whose plasma membrane expression is regulated under a variety of conditions. It has been shown that net proton secretion occurs in the distal nephron from chronically K+-depleted rats and that upregulation of tubular H+- ATPase is involved in this process. However, regulation of this protein at the level of individual cells has not so far been examined. In the present study, H+-ATPase activity was determined in individually identified ICs from control and chronically K+-depleted rats (9–14 days on a low-K+ diet) by monitoring K+- and Na+-independent H+ extrusion rates after an acute acid load. Split-open rat cortical collecting tubules were loaded with the intracellular pH (pHi) indicator 2′,7′-bis(2-carboxyethyl)-5(6)-carboxyfluorescein, and pHiwas determined by using ratiometric fluorescence imaging. The rate of pHi recovery in ICs in response to an acute acid load, a measure of plasma membrane H+-ATPase activity, was increased after K+ depletion to almost three times that of controls. Furthermore, the lag time before the start of pHirecovery after the cells were maximally acidified fell from 93.5 ± 13.7 s in controls to 24.5 ± 2.1 s in K+-depleted rats. In all ICs tested, Na+- and K+-independent pHi recovery was abolished in the presence of bafilomycin (100 nM), an inhibitor of the H+-ATPase. Analysis of the cell-to-cell variability in the rate of pHi recovery reveals a change in the distribution of membrane-bound proton pumps in the IC population of cortical collecting duct from K+-depleted rats. Immunocytochemical analysis of collecting ducts from control and K+-depleted rats showed that K+-depletion increased the number of ICs with tight apical H+ATPase staining and decreased the number of cells with diffuse or basolateral H+-ATPase staining. Taken together, these data indicate that chronic K+ depletion induces a marked increase in plasma membrane H+ATPase activity in individual ICs.

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