Renal brush border membrane phosphorylation: influence of pH, cAMP and ATP concentrations, parathyroid hormone status, and dietary phosphate.
It is known that parathyroidectomy, administration of parathyroid hormone (PTH), and dietary phosphate depletion or excess result in variations in phosphaturia and in phosphate transport through brush border membrane vesicles isolated from the kidneys of various animals. Parathyroid hormone has been shown to ultimately phosphorylate some brush border membrane proteins and it has been postulated that the resulting phosphaturia is related to this phosphorylation. However, it is not known whether the regulation of phosphate transport by the diet is affected through similar pathways. Our experiments were designed to study the phosphorylation of brush border membrane with [gamma-32P]ATP using the intrinsic protein kinase of the membranes. Five groups of rats were used: normal, phosphate loaded, phosphate depleted, and thyroparathyroidectomized and acutely loaded with parathyroid hormone. In each series of animals, the proteins whose phosphorylation was cAMP dependent were detected by sodium dodecyl sulfate polyacrylamide gel electrophoresis, and their phosphorylation with various concentrations of ATP, in the presence or absence of cAMP in the incubation medium, was quantified. In the normal rat, 17 proteins were phosphorylated, the phosphorylation of two of them (Mr, 71 000 and 84 000) being cAMP dependent. Maximal response to cAMP for these two proteins was obtained with 10 microM cAMP. The peaks of phosphorylation were observed at pH 7 for protein 71 000 and pH 10 for protein 84 000. When brush border membranes from normal rats were incubated with 10-100 microM ATP, cAMP-dependent phosphorylation increased to reach a maximal phosphorylation of 4.44 +/- 0.90 pmol/mg protein for protein 71 000 and 1.32 +/- 0.15 pmol/mg protein for protein 84 000.(ABSTRACT TRUNCATED AT 250 WORDS)
D000255:Adenosine Triphosphate / Q000494:pharmacology*; D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D000242:Cyclic AMP / Q000494:pharmacology*; D004032:Diet; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D007668:Kidney / Q000378:metabolism* / Q000648:ultrastructure; D007700:Kinetics; D008297:Male; D008871:Microvilli / Q000378:metabolism; D010280:Parathyroid Glands / Q000502:physiology; D010281:Parathyroid Hormone / Q000494:pharmacology*; D010710:Phosphates / Q000494:pharmacology*; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism; D051381:Rats; D011919:Rats, Inbred Strains; D013965:Thyroidectomy
D010281:Parathyroid Hormone; D010710:Phosphates; D000255:Adenosine Triphosphate; D000242:Cyclic AMP; D011494:Protein Kinases