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Ca2+-dependent protein phosphorylation in brush border membranes of rat kidney proximal tubules

  • Excitable Tissues and Central Nervous Physiology
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Abstract

Ca2+-dependent protein phosphorylation was studied in isolated brush border membrane vesicles from rat kidney cortex. Phosphorylation of about 20 target proteins with γ-32P-ATP was observed after opening the vesicles transiently by detergent treatment or by hypotonic shock indicating that phosphorylation takes place from the cytoplasmic side. Five of these polypeptides (Mr 12, 19, 29, 97 and 138 kD) showed increased phosphorylation in the presence of micromolar calcium.

Addition of exogenous calmodulin did not enhance the calcium induced phosphorylation significantly, nor did trifluoperazine depress it, suggesting that Ca2+-calmodulin-dependent protein kinase is not involved in the studied Ca2+-induced phosphorylation.

12-O-tetradecanoylphorbol-13-acetate (TPA) minimized the Ca2+ dependency for the 12 and 97 kD polypeptides. Cytotoxin I inhibited the incorporation of phosphate into the 12 and 97 kD polypeptides in a dose-dependent manner. Excess phosphatidylserine could reverse this inhibition but stimulated also the phosphorylation of the 19 kD polypeptide. These findings suggest that at least the 12 and the 97 kD polypeptides are substrates for an endogenous protein kinase C.

When studied under conditions where Ca2+ increases membrane phosphorylation, no effects could be observed on the kinetic parameters (K m andV max) of the sodium-dependent phosphate uptake.

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Abbreviations

BBMV:

Brush border membrane vesicle

EDTA:

ethylenediamine-tetraacetic acid

EGTA:

ethyleneglycol (β-aminoethylether)-N,N′tetra-acetic acid

HEDTA:

N-hydroxyethylene-diamine triacetic acid

HEPES:

N-2-hxdroxyethylpiperazine-N′-2-ethanesulfonic acid

Pi :

inorganic phosphate

PTH:

parathyroid hormone

PS:

phosphatidylserine

TFP:

trifluoperazine

Tris:

Tris-(hydroxymethyl)aminomethane

TPA:

12-O-tetradecanoylphorbol-13-acetate

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  1. Institute of Physiology, University of Zurich-Irchel, Winterthurer Strasse 190, CH-8057, Zurich, Switzerland

    Kerstin Malmström & Heini Murer

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  1. Kerstin Malmström
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  2. Heini Murer
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Malmström, K., Murer, H. Ca2+-dependent protein phosphorylation in brush border membranes of rat kidney proximal tubules. Pflugers Arch. 404, 358–364 (1985). https://doi.org/10.1007/BF00585349

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  • DOI: https://doi.org/10.1007/BF00585349

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