Spectrophotometric method for the determination of renal ouabain-sensitive Hplus, Kplus-ATPase activity

• Autorzy: Beltowski J. , Wojcicka G.
• Języki publikacji: EN

Abstrakty

EN

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 (Bełtowski 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 uM in the renal cortex and 1.9 ± 0.4 uM in the renal medulla) and by Sch 28080 (Ki of 1.8 ± 0.5 uM and 2.5 ± 0.9 uM 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.

Słowa kluczowe

EN

determination; homeostasis; potassium balance; spectrophotometry; kidney; ATPase activity; sodium homeostatis; urinary excretion

• Wydawca: -
• Czasopismo: Acta Biochimica Polonica
• Rocznik: 2002
• Tom: 49
• Numer: 2
• Opis fizyczny: p.515-527,fig.

Twórcy

autor

Beltowski J.

• Medical University, K.Jaczewskiego 8, 20-090 Lublin, Poland

autor

Wojcicka G.

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