Optimal Conditions for Measurement of Na+, K+-ATPase Activity of Human Leucocytes

1985 ◽  
Vol 68 (2) ◽  
pp. 143-149 ◽  
Author(s):  
D. N. Baron ◽  
F. A. Khan
Keyword(s):  
Enzyme Activity ◽  
Atpase Activity ◽  
Coefficient Of Variation ◽  
Healthy Subjects ◽  
Inorganic Phosphate ◽  
Inhibitory Effect ◽  
Cell Disruption ◽  
Optimal Conditions ◽  
Maximal Inhibition ◽  
Hypotonic Lysis

1. The Na+, K+-ATPase activity of human leucocytes was assayed by measuring the release of inorganic phosphate (Pi) from ATP. 2. The maximum enzyme activity was achieved under the following conditions: concentration (mmol/l), Tris/HCl 50, Na 100, K 15, ATP 5, Mg 7, EDTA 1; pH 7.2 and temperature 37°C, were optimal. Ouabain showed maximal inhibition at a concentration of 10-100 μmol/l. Ethanol, the solvent for ouabain, had a dose-related inhibitory effect. 3. Heparin or citrate used as an anticoagulant gave similar results. Leucocyte samples could be stored at −20°C for up to 6 days without loss of activity. Hypotonic lysis had advantages over sonication as the technique for cell disruption. 4. The leucocyte Na+, K+-ATPase enzyme activity in healthy subjects was 186 μmol of Pi h−1 g−1 of protein (median) with a range 136-243 μmol of Pi h−1 g−1 of protein. The within-batch coefficient of variation was 6.4% and the between-batch precision was 9.6%.

scholarly journals The erythrocyte calcium pump is inhibited by non-enzymic glycation: studies in situ and with the purified enzyme

1993 ◽  
Vol 293 (2) ◽  
pp. 369-375 ◽  
Author(s):  
F L González Flecha ◽  
P R Castello ◽  
A J Caride ◽  
J J Gagliardino ◽  
J P Rossi
Keyword(s):  
Enzyme Activity ◽  
Atpase Activity ◽  
Inorganic Phosphate ◽  
Deleterious Effect ◽  
Kinetic Properties ◽  
Control Activity ◽  
End Products ◽  
Effect Of Glucose ◽  
Inside Out

In a previous paper we demonstrated that incubation of either intact erythrocytes or erythrocytes membranes with glucose decreases the activity of the membrane Ca(2+)-ATPase [González Flecha, Bermúdez, Cédola, Gagliardino and Rossi (1990) Diabetes 39, 707-711]. The aim of the present work was to obtain information about the mechanism of this inhibition. For this purpose, experiments were carried out with purified Ca(2+)-ATPase, inside-out vesicles and membranes from human erythrocytes. Incubation of the purified Ca(2+)-ATPase with glucose led to a decay in the enzyme activity of up to 50% of the control activity under the conditions used. The decrease in ATPase activity was concomitant with labelling by [6-3H]glucose of the purified Ca2+ pump; the kinetic properties of both processes were almost identical, suggesting that inhibition is a consequence of the incorporation of glucose into the Ca(2+)-ATPase molecule. In inside-out vesicles, glucose also promoted inhibition of Ca(2+)-ATPase activity as well as of active Ca2+ transport. Arabinose, xylose, mannose, ribose, fructose and glucose 6-phosphate (but not mannitol) were also able to inactive the ATPase. The activation energy for both the decrease in ATPase activity by glucose and the labelling of the pump with [6-3H]glucose was about 65 kJ/mol. Furthermore, inorganic phosphate enhanced the inactivation of the Ca(2+)-ATPase by glucose. This evidence strongly suggests that inhibition is a non-enzymically catalysed process. Inactivation of the Ca(2+)-ATPase by glucose was enhanced by reductive alkylation with sodium borohydride. Aminoguanidine, an inhibitor of the formation of the advanced end products of glycosylation, did not prevent the deleterious effect of glucose on the enzyme activity. Therefore it is concluded that inactivation of the Ca2+ pump is a consequence of the glycation of this protein.

The soluble adenosine triphosphatase of Thiobacillus ferrooxidans

1975 ◽  
Vol 21 (1) ◽  
pp. 1-7 ◽  
Author(s):  
Catherine Adapoe ◽  
Marvin Silver
Keyword(s):  
Enzyme Activity ◽  
Atpase Activity ◽  
Gel Electrophoresis ◽  
Inorganic Phosphate ◽  
Adenosine Triphosphatase ◽  
Thiobacillus Ferrooxidans ◽  
Polyacrylamide Gel Electrophoresis ◽  
Histochemical Analysis ◽  
Ph Optimum ◽  
Major Band

Adenosine triphosphatase (ATPase) from Thiobacilhis ferrooxidans was purified 55-fold. Polyacrylamide gel electrophoresis of the most purified fraction showed only one major band; histochemical analysis showed that the ATPase activity was associated with this band. The pH optimum is 9–10. The enzyme hydrolyzed ATP stoichiometrically to ADP and inorganic phosphate, the Km for this substrate being 7.75 × 10−3 M. GTP and ITP are alternate substrates, the Km values for these being 6.71 × 10−3 M and 3.12 × 10−3 M, respectively. ADP is slightly hydrolyzed. Magnesium, manganese, and calcium can serve as cofactors; Km values for these are 2.0 × 10−3 M, 9.4 × 10−4 M, and 8.0 × 10−4 M, respectively. The enzyme activity was not activated by either sodium or potassium, but a combination of the two ions were inhibitory. Azide and p-hydroxymercuribenzoate strongly inhibited the enzyme activity, whereas cyanide, dinitrophenol, and N, N′-dicyclohexylcarbodiimide (DCCD) were without effect. The enzyme was cold labile at 0 °C, but was more stable at 18–24 °C.

Determination of Na-K-ATPase activity in single segments of the mammalian nephron

1979 ◽  
Vol 237 (2) ◽  
pp. F105-F113 ◽  
Author(s):  
A. Doucet ◽  
A. I. Katz ◽  
F. Morel
Keyword(s):  
Enzyme Activity ◽  
Atpase Activity ◽  
Inorganic Phosphate ◽  
Rapid Freezing ◽  
Phosphate Release ◽  
Large Numbers ◽  
Hydrolysis Of ◽  
Single Tubules

A micromethod for the determination of Na-K-ATPase in discrete segments of nephrons from rabbit, rat, and mouse kidneys is described. To facilitate tubule microdissection, the kidneys were perfused with collagenase after it had been verified that collagenase had no effect on ATPase activity. Individual tubule segments were dissected under stereomicroscopic observation, exposed to a hypotonic environment followed by rapid freezing, and incubated in 1 microliter assay medium. Enzyme activity was determined by direct measurement of labeled inorganic phosphate release by the hydrolysis of [gamma-32P]ATP and was expressed as a function of tubule length. This method is technically simple enough to permit simultaneous measurement of the enzyme in large numbers of tubules and sufficiently sensitive to determine its activity in each region of the nephron. Correlation of Na-K-ATPase activity in single tubules with functional measurements obtained in the corresponding segment of the nephron with the perfused tubule or micropuncture techniques should help define the role of this enzyme in tubular ion transport.

Locally formed dopamine inhibits Na+-K+-ATPase activity in rat renal cortical tubule cells

1988 ◽  
Vol 255 (4) ◽  
pp. F666-F673 ◽  
Author(s):  
I. Seri ◽  
B. C. Kone ◽  
S. R. Gullans ◽  
A. Aperia ◽  
B. M. Brenner ◽  
...  
Keyword(s):  
Atpase Activity ◽  
Active Agent ◽  
Sodium Concentration ◽  
Inhibitory Effect ◽  
Dependent Manner ◽  
Maximal Inhibition ◽  
Concentration Dependent Manner ◽  
Order Of Magnitude ◽  
Tubule Cells ◽  
Dopa Concentration

Dopamine, generated locally from L-dopa, inhibits Na+-K+-ATPase in permeabilized rat proximal tubules under maximum transport rate conditions for sodium. To determine whether locally formed dopamine inhibits Na+-K+-ATPase activity in intact cortical tubule cells we studied the effect of L-dopa on ouabain-sensitive oxygen consumption rate (QO2) and 86Rb uptake in renal cortical tubule cell suspensions. L-Dopa (10(-4) M) did not affect ouabain-insensitive QO2 or mitochondrial respiration. However, L-dopa inhibited ouabain-sensitive QO2 in a concentration-dependent manner, with half-maximal inhibition (K0.5) of 5 x 10(-7) M and a maximal inhibition of 14.1 +/- 1.5% at 10(-4) M (P less than 0.05). L-Dopa also blunted the nystatin-stimulated QO2 in a concentration-dependent manner, with a K0.5 of 5 x 10(-8) M and a maximal inhibition of 21.8 +/- 1.2% at 10(-5) M (P less than 0.05), indicating that L-dopa directly inhibits Na+-K+-ATPase activity and not sodium entry. Ouabain-sensitive 86Rb uptake was also inhibited by L-dopa (16.0 +/- 2.4%, P less than 0.05). Carbidopa (10(-4) M), an inhibitor of the conversion of L-dopa to dopamine, eliminated the effect of L-dopa on ouabain-sensitive QO2 and 86Rb uptake, indicating that dopamine rather than L-dopa was the active agent. The finding that the L-dopa concentration-response curve was shifted to the left by one order of magnitude in the presence of nystatin suggests that the inhibitory effect is enhanced when the intracellular sodium concentration is increased.(ABSTRACT TRUNCATED AT 250 WORDS)

Enzymic measurement of urinary pyrophosphate with a centrifugal analyzer.

1982 ◽  
Vol 28 (1) ◽  
pp. 134-137 ◽  
Author(s):  
J B Roullet ◽  
B Lacour ◽  
A Ulmann ◽  
M Bailly
Keyword(s):  
Coefficient Of Variation ◽  
Healthy Subjects ◽  
High Sensitivity ◽  
Inhibitory Effect ◽  
Mean Value ◽  
Good Reproducibility ◽  
Combined System ◽  
Phosphate Ions ◽  
The Mean ◽  
Automated Technique

Abstract We describe a simple, rapid, and fully automated technique for measuring urinary pyrophosphates with a centrifugal analyzer (the ENI GEMSAEC). This technique depends on the enzymic magnesium-dependent reaction with UDPG pyrophosphorylase (UTP: alpha-D-glucose-1-phosphate uridylyl transferase, EC 2.7.7.9) and spectrophotometry of the NADPH formed in a combined system of phosphorylation and reduction. Many samples of urine can be analyzed quickly without pretreatment, with high sensitivity (1.3 mA/mumol of substrate) and good reproducibility. The mean within-run coefficient of variation for a 50 mumol/L pyrophosphate solution was 1.4%. We determined the optimum enzyme and magnesium concentrations necessary for use in a 4-min reaction. Because there is no inhibitory effect of chloride and phosphate ions, pyrophosphate can be measured directly in urine, without prior extraction. With this technique, the mean value (and SD) for urinary pyrophosphate excretion by 30 healthy subjects was 39.3 (SD 17.2) mumol/24 h.

Regulation of (Ca2+-Mg2+)-ATPase activity by calcitonin binding to rat liver plasma membranes

1985 ◽  
Vol 110 (1) ◽  
pp. 124-129 ◽  
Author(s):  
Masayoshi Yamaguchi ◽  
Michiyo Ito
Keyword(s):  
Plasma Membrane ◽  
Atpase Activity ◽  
Rat Liver ◽  
Plasma Membranes ◽  
Specific Binding ◽  
Inhibitory Effect ◽  
Maximal Inhibition ◽  
Low Concentration ◽  
High Affinity ◽  
Rat Liver Plasma Membranes

Abstract. The plasma membranes isolated from rat liver bound 125I-labelled ([125I]) synthetic [Asu1,7]eel calcitonin (CT), with increasing concentrations of [125I]CT. This specific binding was completely saturated at a concentration of 0.5 nm CT. A high affinity Ca2+ -stimulated, Mg2+-dependent ATPase [(Ca2+-Mg2+)-ATPase] activity in the plasma membranes was significantly decreased by the presence of a very low concentration of CT (7.4 pm), although the hormone did not affect the activity of the plasma membrane 5′-nucleotidase. The concentration of CT needed for maximal inhibition of (Ca2+-Mg2+)-ATPase in the plasma membranes was less than 0.74 nm. The plasma membranes washed with 10−3% digitonin did not show an inhibitory effect of CT on (Ca2+-Mg2+)-ATPase activity, while the reagent did not have a significant effect on the enzyme. These results suggest that the inhibition of (Ca2+-Mg2+)-ATPase activity maybe part of the mechanism by which CT elevates cytosolic Ca2+ in liver cells.

Stability of antioxidant vitamins in whole human blood during overnight storage at 4°C and frozen storage up to 6 months

2018 ◽  
Vol 88 (3-4) ◽  
pp. 151-157 ◽  
Author(s):  
Scott W. Leonard ◽  
Gerd Bobe ◽  
Maret G. Traber
Keyword(s):  
Ascorbic Acid ◽  
Whole Blood ◽  
Healthy Subjects ◽  
Frozen Storage ◽  
Blood Collection ◽  
Plasma Samples ◽  
Optimal Conditions ◽  
Plasma Ascorbic Acid ◽  
Blood Samples ◽  
Sample Handling

Abstract. To determine optimal conditions for blood collection during clinical trials, where sample handling logistics might preclude prompt separation of erythrocytes from plasma, healthy subjects (n=8, 6 M/2F) were recruited and non-fasting blood samples were collected into tubes containing different anticoagulants (ethylenediaminetetra-acetic acid (EDTA), Li-heparin or Na-heparin). We hypothesized that heparin, but not EDTA, would effectively protect plasma tocopherols, ascorbic acid, and vitamin E catabolites (α- and γ-CEHC) from oxidative damage. To test this hypothesis, one set of tubes was processed immediately and plasma samples were stored at −80°C, while the other set was stored at 4°C and processed the following morning (~30 hours) and analyzed, or the samples were analyzed after 6 months of storage. Plasma ascorbic acid, as measured using HPLC with electrochemical detection (LC-ECD) decreased by 75% with overnight storage using EDTA as an anticoagulant, but was unchanged when heparin was used. Neither time prior to processing, nor anticoagulant, had any significant effects upon plasma α- or γ-tocopherols or α- or γ-CEHC concentrations. α- and γ-tocopherol concentrations remained unchanged after 6 months of storage at −80°C, when measured using either LC-ECD or LC/mass spectrometry. Thus, refrigeration of whole blood at 4°C overnight does not change plasma α- or γ-tocopherol concentrations or their catabolites. Ascorbic acid is unstable in whole blood when EDTA is used as an anticoagulant, but when whole blood is collected with heparin, it can be stored overnight and subsequently processed.

Synchronized Inhibition of the Phospholipid Mediated Autoactivation of Factor XII in Plasma by β2-glycoprotein I and Anti-β2-glycoprotein I

1995 ◽  
Vol 73 (05) ◽  
pp. 798-804 ◽  
Author(s):  
Inger Schousboe ◽  
Margit Søe Rasmussen
Keyword(s):  
Lupus Erythematosus ◽  
Lupus Anticoagulant ◽  
Response Curve ◽  
Inhibitory Effect ◽  
Factor Xii ◽  
High Antibody ◽  
Contact Activation ◽  
Maximal Inhibition ◽  
Lupus Anticoagulants ◽  
Glycoprotein I

SummaryLupus anticoagulants are a group of antibodies commonly found in patients with autoimmune diseases such as systemic lupus erythematosus. Lupus anticoagulants inhibit phospholipid dependent coagulation and may bind to negatively charged phospholipids. Recent studies have suggested an association between anti-β2-glycoprotein I and a lupus anticoagulant, whose activity is frequently dependent on the presence of β2-glycoprotein I. Based on these observations, the effect of anti-β2-glycoprotein I on the autoactivation of factor XII in plasma was investigated. Autoactivation initiated by the presence of negatively charged phospholipids, but not by sulfatide, was strongly inhibited by immunoaffinity purified anti-β2-glycoprotein I. The dose-response curve of anti-β2-gly coprotein I was identical with that of a precipitating antibody, showing no inhibition at low and high antibody dilutions and maximal inhibition at an intermediate dilution. At high antibody concentrations, an increased rate of factor Xlla activation was observed. This increase was of the same magnitude as the decreased rate observed in plasma supplemented with the same amount of β2-glycoprotein I as in the plasma itself. This confirms the inhibitory effect of β2-GP-I on the contact activation and shows that inhibition is effective on the autoactivation of factor XII in plasma. The inhibitory action of β2-glycoprotein I was independent of the inhibition caused by the anti- β2-glycoprotein I/β2-glycoprotein I complex suggesting a synchronized inhibition of factor XII autoactivation by β2-glycoprotein I and anti-β2-gly coprotein I. The inhibition caused by the antibody is suggested to be caused by a reduced availability of negatively charged phospholipids due to the binding of the anti-β2- GP-I/β2-GP-I complex. This complex may be a lupus anticoagulant.

Effect of human galanin on the response of circulating catecholamines to hypoglycemia in man

1995 ◽  
Vol 133 (6) ◽  
pp. 723-728 ◽  
Author(s):  
Ettore C degli Uberti ◽  
Maria R Ambrosio ◽  
Marta Bondanelli ◽  
Giorgio Transforini ◽  
Alberto Valentini ◽  
...  
Keyword(s):  
Heart Rate ◽  
Healthy Subjects ◽  
Sympathetic Nerve Activity ◽  
Heart Rate Response ◽  
Statistical Significance ◽  
Inhibitory Effect ◽  
Amino Acid Residues ◽  
Nerve Activity ◽  
Receptor Stimulation

degli Uberti EC, Ambrosio MR, Bondanelli M, Trasforini G, Valentini A, Rossi R, Margutti A, Campo M. Effect of human galanin on the response of circulating catecholamines to hypoglycemia in man. Eur J Endocrinol 1995;133:723–8. ISSN 0804–4643 Human galanin (hGAL) is a neuropeptide with 30 amino acid residues that has been found in the peripheral and central nervous system, where it often co-exists with catecholamines. In order to clarify the possible role of hGAL in the regulation of sympathoadrenomedullary function, the effect of a 60 min infusion of hGAL (80 pmol·kg−1 · min−1) on plasma epinephrine and norepinephrine responses to insulin-induced hypoglycemia in nine healthy subjects was investigated. Human GAL administration significantly reduced both the release of basal norepinephrine and the response to insulin-induced hypoglycemia, whereas it attenuated the epinephrine response by 26%, with the hGAL-induced decrease in epinephrine release failing to achieve statistical significance. Human GAL significantly increased the heart rate in resting conditions and clearly exaggerated the heart rate response to insulin-induced hypoglycemia, whereas it had no effect on the blood pressure. We conclude that GAL receptor stimulation exerts an inhibitory effect on basal and insulin-induced hypoglycemia-stimulated release of norepinephrine. These findings provide further evidence that GAL may modulate sympathetic nerve activity in man but that it does not play an important role in the regulation of adrenal medullary function. Ettore C degli Uberti, Chair of Endocrinology, University of Ferrara, Via Savonarola 9, I-44100 Ferrara, Italy

Phosphate Binding to Isolated Chloroplast Coupling Factor (CF1)

1988 ◽  
Vol 43 (3-4) ◽  
pp. 213-218 ◽  
Author(s):  
Bernhard Huchzermeyer
Keyword(s):  
Atpase Activity ◽  
Binding Site ◽  
Inorganic Phosphate ◽  
Coupling Factor ◽  
Binding Domain ◽  
Atp Binding ◽  
Phosphate Binding ◽  
Chloroplast Coupling Factor ◽  
Single Binding Site ◽  
Site Binding

A single binding site for phosphate was found on isolated chloroplast coupling factor in the absence of nucleotides. In our experiments the phosphate binding site showed a Kd of 170 μᴍ. We did not observe any differences whether the ATPase activity of CF] had been activated or not. If the enzyme was incubated with [γ-32P]ATP the amount of 32P bound per CF1 depended on the pretreatment of the enzyme: In the presence of ADP no ATP or phosphate was bound to CF,. After activation of ATPase activity one mol of ATP per mol CF, was rapidly bound and hydrolyzed while there was a slowly occurring binding of another phosphate without concomitant nucleotide binding. We conclude that there are two different types of phosphate binding observed in our experiments: 1) Inorganic phosphate can be bound by one catalytic site per mol of CF1 2) The γ-phosphate of ATP is able to bind to an ATP binding domain of the enzyme if this domain can exchange substrates with the incubation medium. This ATP binding domain appears to differ from the site binding inorganic phosphate, because at least a portion of the coupling factor contains more than one labelled phosphate during our ATPase tests.

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