Cyclic adenosine monophosphate-dependent and -independent protein kinase activity of renal brush border membranes

George, E.R.; Balakir, R.A.; Filburn, C.R.; Sacktor, B.

Archives of Biochemistry and Biophysics 180(2): 429-443

1977


ISSN/ISBN: 0003-9861
PMID: 195523
DOI: 10.1016/0003-9861(77)90057-1
Accession: 068523610

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Abstract
Kinase(s) in brush border membranes, isolated from rabbit renal proximal tubules, phosphorylated proteins intrinsic to the membrane and exogenous proteins. cAMP stimulated phosphorylation of histone; phosphorylation of protamine was cAMP independent. cAMP-dependent increased in phosphorylation of endogenous membrane protein were small, but highly reproducible. Most of the 32P incorporated into membranes represented phosphorylation of serine residues, with phosphorylthreonine comprising a minor component. cAMP did not alter the electrophoretic pattern of 32P-labeled membrane polypeptides. The small cAMP-dependent phosphorylation of brush border membrane proteins was not due to membrane phosphodiesterase or adenylte cyclase activities. Considerable cAMP was found endogenously bound to the membranes as prepared. This did not result in preactivation of the kinase since activity was not inhibited by a heat-stable protein inhibitor of cAMP-dependent protein kinases. With intrinsic membrane protein as phosphate acceptor, the relationship between rate of phosphorylation and ATP concentration appeared to follow Michaelis-Menton kinetics. With histone the relationship was complex. cAMP did not affect the apparent Km for histone. One-half maximal stimulation of the rate of histone phosphorylation was obtained with 7 .times. 10-8 M cAMP. the Ka values for dibutyryl cAMP, cIMP, and cGMP were 1-2 orders of magnitude greater. Treatment of brush border membranes with detergent greatly increased the dependency of histone phosphorylation on cAMP. Phosphorylations of intrinsic membrane protein and histone were nonlinear with time, due in part to the lability of the protein kinase, the hydrolysis of ATP and minimally to the presence of phosphoprotein phosphatase in the border membrane. The membrane phosphoprotein phosphatase was unaffected by cyclic nucleotides. Protein kinase activity was also found in cystolic and crude particulate fractions of the renal cortex. Activity was enriched in the brush border membrane relative to that in the crude membrane preparation. The kinase activities in the different loci were distinct both in relative activities toward different substrates and in responsiveness to cAMP.