scholarly journals Spectrophotometric method for the determination of renal ouabain-sensitive H+,K+-ATPase activity.

2002 ◽  
Vol 49 (2) ◽  
pp. 515-527 ◽  
Author(s):  
Jerzy Bełtowski ◽  
Grazyna Wójcicka
Keyword(s):  
Atpase Activity ◽  
Spectrophotometric Method ◽  
Inorganic Phosphate ◽  
Microsomal Fraction ◽  
Renal Cortex ◽  
Renal Medulla ◽  
Good Reproducibility ◽  
Atpase Assay ◽  
Low Activity

The aim of this work was to develop a method for renal H+,K+-ATPase measurement based on the previously used Na+,K+-ATPase assay (Beltowski et al.: J Physiol Pharmacol.; 1998, 49: 625-37). ATPase activity was assessed by measuring the amount of inorganic phosphate liberated from ATP by isolated microsomal fraction. Both ouabain-sensitive and ouabain-resistant K+-stimulated and Na+-independent ATPase activity was detected in the renal cortex and medulla. These activities were blocked by 0.2 mM imidazolpyridine derivative, Sch 28080. The method for ouabain-sensitive H+,K+-ATPase assay is characterized by good reproducibility, linearity and recovery. In contrast, the assay for ouabain-resistant H+,K+-ATPase was unsatisfactory, probably due to low activity of this enzyme. Ouabain-sensitive H+,K+-ATPase was stimulated by K+ with Km of 0.26 +/- 0.04 mM and 0.69 +/- 0.11 mM in cortex and medulla, respectively, and was inhibited by ouabain (Ki of 2.9 +/- 0.3 microM in the renal cortex and 1.9 +/- 0.4 microM in the renal medulla) and by Sch 28080 (Ki of 1.8 +/- 0.5 microM and 2.5 +/- 0.9 microM in cortex and medulla, respectively). We found that ouabain-sensitive H+,K+-ATPase accounted for about 12% of total ouabain-sensitive activity in the Na+,K+-ATPase assay. Therefore, we suggest to use Sch 28080 during Na+,K+-ATPase measurement to block H+,K+-ATPase and improve the assay specificity. Leptin administered intraperitoneally (1 mg/kg) decreased renal medullary Na+,K+-ATPase activity by 32.1% at 1 h after injection but had no effect on H+,K+-ATPase activity suggesting that the two renal ouabain-sensitive ATPases are separately regulated.

A Quick Assay for Na+-K+-ATPase Specific Activity

2002 ◽  
Vol 57 (5-6) ◽  
pp. 562-564 ◽  
Author(s):  
Pradip K. Sarkar
Keyword(s):  
Atpase Activity ◽  
Protein Content ◽  
Simultaneous Determination ◽  
Lipid Content ◽  
Present Method ◽  
Inorganic Phosphate ◽  
Specific Activity ◽  
Adult Rat ◽  
High Lipid

The method describes a simultaneous determination of inorganic phosphate (Pi) and protein content from a reaction mixture used for assay of adult rat cerebrocortical synaptosomal membrane Na+-K+-ATPase specific activity. The present method is more convenient, accurate and quicker compared to the existing methods for the determination of Na+-K+-ATPase activity. It also eliminates the possible errors in protein estimation by other classical methods in brain, which have a high lipid content.

Renal cortical Na+-K+-ATPase activity and abundance is decreased in normal pregnant rats

1998 ◽  
Vol 275 (5) ◽  
pp. F812-F817 ◽  
Author(s):  
J. Mahaney ◽  
C. Felton ◽  
D. Taylor ◽  
W. Fleming ◽  
J. Q. Kong ◽  
...  
Keyword(s):  
Atpase Activity ◽  
Brain Stem ◽  
Renal Cortex ◽  
Renal Medulla ◽  
Late Pregnancy ◽  
Sodium Retention ◽  
Plasma Volume Expansion ◽  
Pregnant Rats ◽  
Small Rise ◽  
Α1 Subunit

During late pregnancy, the rat undergoes massive plasma volume expansion due to cumulative renal sodium retention. In the present study, conducted in virgin, mid- ( days 11–13), and late-pregnant ( days 18–20) rats, we measured both Na+-K+-ATPase activity (by coupled enzyme assay) and abundance of the α-subunits of the Na+-K+-ATPase (by Western and slot blot analyses) in renal cortex, medulla, and brain stem. Unexpectedly, Na+-K+-ATPase in renal cortex, in both stages of pregnancy, is reduced versus the virgin, consistent with our finding of a reduced quantity of the α1-subunit. In renal medulla, there is a small rise in activity at midterm, but there is no difference in either activity or abundance of the α1-subunit in late pregnancy, when renal Na retention is maximal. In brain stem, where only α2- and α3-subunits are evident, pregnancy has no impact on enzyme activity or abundance of either isoform. In conclusion, the outcome of these experiments was unexpected in that we did not observe increased renal Na+-K+-ATPase activity in late pregnancy in the rat. In fact, in renal cortex, Na+-K+-ATPase activity and abundance are reduced. Whatever promotes net sodium retention in pregnancy must be capable of overwhelming this and several other strong natriuretic signals.

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.

Evaluation of a New System for the Kinetic Measurement of Serum Glutamic Oxalacetic Transaminase

1969 ◽  
Vol 15 (6) ◽  
pp. 496-504 ◽  
Author(s):  
Seymour Winsten ◽  
J Henry Wilkinson ◽  
Joseph H Boutwell
Keyword(s):  
Spectrophotometric Method ◽  
New Method ◽  
Activity Levels ◽  
Good Reproducibility ◽  
Kinetic Measurement ◽  
Glutamic Oxalacetic Transaminase ◽  
Serum Glutamic Oxalacetic Transaminase ◽  
New System

Abstract A new spectrophotometric method for the determination of serum glutamic oxalacetic transaminase§ (SGOT) is described which uses standardized technics and prepackaged reagents. A study was made of this new method in three laboratories and was compared with the routine methods, using identical serum specimens. The new procedure gave good reproducibility and precision. In all methods studied there was evidence of failure of linearity of response at the two highest activity levels examined.

scholarly journals Some properties of the Ca2+-stimulated ATPase of a rat liver microsomal fraction

1983 ◽  
Vol 210 (2) ◽  
pp. 405-410 ◽  
Author(s):  
A P Dawson ◽  
D V Fulton
Keyword(s):  
Atpase Activity ◽  
Rat Liver ◽  
Microsomal Fraction ◽  
Ionophore A23187 ◽  
Calmodulin Antagonist ◽  
Atpase Assay ◽  
Accumulation System ◽  
System 2 ◽  
Vanadate Inhibition ◽  
Assay Time

1. The heavy microsomal fraction from rat liver apparently has very little Ca2+-stimulated ATPase activity, although it has an active, ATP-driven Ca2+ accumulation system. 2. The addition of ionophore A23187 to the ATPase assay, to allow continuous Ca2+ recycling during the assay time, reveals the presence of a substantial Ca2+-stimulated ATPase with Vmax. 160 nmol of Pi/10 min per mg of protein and Km for Ca2+ 0.19 microM. 3. The Ca2+-stimulated ATPase, but not the basal Mg2+-stimulated ATPase, is potently inhibited by orthovanadate. Both the Ca2+-stimulated ATPase and the vanadate inhibition are enhanced by the presence of Mg2+. 4. Ca2+-stimulated ATPase activity is not responsive to calmodulin or the calmodulin antagonist trifluoperazine.

Bicarbonate-activated ATPase activity in renal cortex of chronically acidotic rats

1979 ◽  
Vol 57 (3) ◽  
pp. 271-276 ◽  
Author(s):  
Craig H. Cole
Keyword(s):  
Atpase Activity ◽  
Active Transport ◽  
Wistar Rats ◽  
Microsomal Fraction ◽  
Renal Cortex ◽  
Subcellular Fractionation ◽  
Renal Tissue ◽  
Control Group ◽  
Cortical Tissue ◽  
Tissue Homogenates

The effect of chronic NH4Cl-induced acidosis on the activity of a bicarbonate-activated component of ATPase was studied in homogenates of renal tissue from Wistar rats. This particular component of ATPase, which is maximally stimulated by 50 mM bicarbonate, and is insensitive to the action of ouabain, has been implicated in the active transport of bicarbonate in various tissues. The activity of this enzyme in cortical homogenates from an acidotic group of animals was 4.3 ± 0.4 μmol Pi/mg protein per hour compared with 5.8 ± 0.3 μmol Pi/mg protein per hour in a control group (p < 0.02). No significant change in bicarbonate ATPase activity was observed in medullary homogenates, and NaK-ATPase activity remained the same in cortex and medulla of both groups. Subcellular fractionation of the cortical tissue homogenates revealed that bicarbonate ATPase activity in a microsomal fraction from acidotic animals was 6.5 ± 1.1 μmol Pi/mg protein per hour compared with 9.4 ± 1.2 μmol Pi/mg protein per hour in control animals (p < 0.02). Bicarbonate ATPase activity in other subcellular fractions was not different in the two groups of animals. These findings are compatible with the hypothesis that a certain percentage of bicarbonate reabsorption in the nephron is mediated by a bicarbonate-activated component of ATPase.

scholarly journals Time-dependent effect of leptin on renal Na+,K+-ATPase activity.

2005 ◽  
Vol 52 (4) ◽  
pp. 803-810 ◽  
Author(s):  
Andrzej Marciniak ◽  
Anna Jamroz-Wiśniewska ◽  
Ewelina Borkowska ◽  
Jerzy Bełtowski
Keyword(s):  
Atpase Activity ◽  
Renal Cortex ◽  
Specific Inhibitor ◽  
Renal Medulla ◽  
Oxidase Inhibitor ◽  
Janus Kinases ◽  
Extracellular Signal ◽  
Time Dependent Effect ◽  
Time Periods ◽  
Diabetes And Obesity

Leptin, secreted by adipose tissue, is involved in the pathogenesis of arterial hypertension, however, the mechanisms through which leptin increases blood pressure are incompletely elucidated. We investigated the effect of leptin, administered for different time periods, on renal Na(+),K(+)-ATPase activity in the rat. Leptin was infused under anesthesia into the abdominal aorta proximally to the renal arteries for 0.5-3 h. Leptin administered at doses of 1 and 10 microg/min per kg for 30 min decreased the Na(+),K(+)-ATPase activity in the renal medulla. This effect disappeared when the hormone was infused for > or =1 h. Leptin infused for 3 h increased the Na(+),K(+)-ATPase activity in the renal cortex and medulla. The stimulatory effect was abolished by a specific inhibitor of Janus kinases (JAKs), tyrphostin AG490, as well as by an NAD(P)H oxidase inhibitor, apocynin. Leptin increased urinary excretion of hydrogen peroxide (H(2)O(2)) between 2 and 3 h of infusion. The effect of leptin on renal Na(+),K(+)-ATPase and urinary H(2)O(2) was augmented by a superoxide dismutase mimetic, tempol, and was abolished by catalase. In addition, infusion of H(2)O(2) for 30 min increased the Na(+),K(+)-ATPase activity. Inhibitors of extracellular signal regulated kinases (ERKs), PD98059 or U0126, prevented Na(+),K(+)-ATPase stimulation by leptin and H(2)O(2). These data indicate that leptin, by acting directly within the kidney, has a delayed stimulatory effect on Na(+),K(+)-ATPase, mediated by JAKs, H(2)O(2) and ERKs. This mechanism may contribute to the abnormal renal Na(+) handling in diseases associated with chronic hyperleptinemia such as diabetes and obesity.

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