An ATP-inhibited soluble 5'-nucleotidase of rat kidney

Hir, Michel Le; Dubach, U. C.

doi:10.1152/ajprenal.1988.254.2.f191

Cited by 8 publications

(4 citation statements)

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“…The apparent Km for 5'-AMP obtained by least-square analysis of initial velocity-substrate concentration data was 0.32 mM. It is in the same range than that measured with 5'-nucleotidase of some other cells [ 1.24], but higher than that reported for the enzyme of brush border membranes [3] or for the soluble purified enzyme [4] from rat renal cortex. The activity with /j-nitrophenylphosphate.…”

Section: Discussionsupporting

confidence: 51%

“…ADP was a more powerful inhibitor than ATP. Concentrations of ATP in the kidney in situ approximate 5 mM [4], During renal ischemia, the fall in ATP concentration would be expected to stimulate 5'-nucleotidase activity. However, a fairly active ectoATPase which splits ATP to ADP and inor ganic phosphate is present on mesangial cells (unpublished results).…”

Section: Discussionmentioning

confidence: 99%

“…5'-Nucleotidase (5'-ribonucleotide phosphohydrolase, EC3.1.3.5) has been character ized as an ectoenzyme, the catalytic site of which is located at the external surface of the plasma membrane, in a variety of normal and transformed cells [1,2], and particularly in the kidney [3,4], It converts its substrate, 5'-AMP. a metabolite resulting from the cata bolism of ATP.…”

Section: Introductionmentioning

confidence: 99%

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Ecto-5’-Nucleotidase of Cultured Rat Mesangial Cells

Stefanović

Savić²,

Vlahović³

et al. 1988

Kidney Blood Press Res

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Because adenosine plays a role in the regulation of glomerular filtration rate and of the release of renin, we examined the possibility of a local source for this mediator. We found that rat cultured glomerular mesangial cells converted 5’-AMP into adenosine. The properties of the enzyme involved in the reaction were those of an ecto-5’nucleotidase: (1) the products of the reaction were generated in the extracellular fluid although no 5’-nucleotidase was released by the cells into the medium; (2) identical activities were found for cultured cells in situ and sonicated cells; (3) the diazonium salt of sulfanilic acid which is a nonpenetrating reagent inhibited up to 75% of the enzyme activity. Ecto-5’-nucleotidase activity of intact cells obeyed Michaelis-Menten kinetics. Apparent K_m for 5’-AMP was 0.32 mM. 5’-UMP was a strictly competitive inhibitor. ADP exerted a very powerful inhibitory effect and behaved also as a competitive inhibitor. ATP was inhibitory both by increasing K_m and by decreasing V_max. Ecto-5’-nucleotidase was active in the absence of divalent cations. However, Mg²⁺, Ca²⁺, Co²⁺ and Mn²⁺ were stimulatory. Zn²⁺ and Cu²⁺ suppressed the activity. Concanavalin A, a plant lectin, was markedly inhibitory, suggesting that a glycoprotein moiety was necessary to express enzyme activity. Ecto-5’-nucleotidase activity was not modified during phagocytosis of serum-treated zymosan by mesangial cells. Rat cultured glomerular epithelial cells exhibited a 5’-nucleotidase activity which was 4 times lower than that of the mesangial cells in primary culture. Activity was also present in freshly isolated glomeruli and total cortex, but at lower concentrations than in mesangial cells. These findings suggest that ecto-5’-nucleotidase of mesangial cells may be the main source of adenosine within the glomeruli. Therefore, activity of this enzyme should be essential in the regulation of the glomerular microcirculation.

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Section: Discussionsupporting

confidence: 51%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Ecto-5’-Nucleotidase of Cultured Rat Mesangial Cells

Stefanović

Savić²,

Vlahović³

et al. 1988

Kidney Blood Press Res

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“…Adenosine was eluted with 10 ml of the same buffer. One millilitre of the eluate was mixed with 4 ml scintillation liquid (Ultima Gold Packard, Downers Grove, IL) and radioactivity was counted (Le Hir & Dubach, 1988). Results were corrected for the amount of radioactivity present in samples that were not incubated with cells.…”

Section: J-nucleotidase Measurementmentioning

confidence: 99%

Autoradiography-based cytochemical detection of ecto-ATPase, ecto-ADPase, 5?-nucleotidase, and extracellular adenosine production, employing 141Ce3+ as a capturing agent

et al. 1995

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A method for the visualization of the ecto-nucleotidase enzyme activities present on the cell surface, employing 141Ce3+ as a capturing and labelling agent, is described. Phosphate ions precipitated at the cell surface can be detected by coating the cells with an autoradiographic emulsion, followed by light microscopical inspection of the formed silver grains. The activities of ecto-ATPase, ecto-ADPase and 5'-nucleotidase were detected by this approach in four different cell lines. Parallel biochemical measurements of the activities of the corresponding enzymes were carried out in order to validate, evaluate, and optimize the cytochemical detection. The finding that Ce3+ ions are inhibitory to ecto-ATPase provided evidence for the necessity of carefully establishing appropriate reaction conditions for the cytochemical determination of ecto-nucleotidases. The application of this method to the indirect detection of extracellular adenosine production from substrates like ATP has also been documented. It allows a cytochemical determination of adenosine formed through cascade nucleotide dephosphorylation. This newly described method is of high sensitivity and potentially of value for a variety of applications, including not only cytochemistry but also cell biology, and molecular biology studies.

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Adenosine production and energy metabolism in ischaemic and metabolically stimulated rat heart

Headrick¹

1989

Journal of Molecular and Cellular Cardiology

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An ATP-inhibited soluble 5'-nucleotidase of rat kidney

Cited by 8 publications

References 0 publications

Ecto-5’-Nucleotidase of Cultured Rat Mesangial Cells

Ecto-5’-Nucleotidase of Cultured Rat Mesangial Cells

Autoradiography-based cytochemical detection of ecto-ATPase, ecto-ADPase, 5?-nucleotidase, and extracellular adenosine production, employing 141Ce3+ as a capturing agent

Adenosine production and energy metabolism in ischaemic and metabolically stimulated rat heart

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