Adenosine 3':5'-cyclic phosphate-dependent and -independent protein kinases of renal brush border membranes. Solubilization, separation, and characterization of multiple forms

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

Archives of Biochemistry and Biophysics 184(2): 391-399

1977


ISSN/ISBN: 0003-9861
PMID: 202197
DOI: 10.1016/0003-9861(77)90366-6
Accession: 068523875

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Abstract
Multiple protein kinase activities were found in the luminal segment of the . cAMP binding activity eluted with histone kinase activity. Rechromatography of IIa + b on diethylaminoethyl cellulose containing 1 .mu.M cAMP resulted in passage through the column of most of the histone kinase activity (IIa) prior the the salt gradient, but retention of kinase IIb, which again eluted in its original position. Characterization of the separated enzymes revealed that kinase I was highly specific for protamine and totally insensitive to cAMP and a specific protein inhibitor of cAMP-dependent kinases. Kinase IIa was relatively specific for histones and was completely inhibited by the protein inhibitor. Kinase IIb was nonspecific, catalyzing phosphorylation of protamine, casein, histones and phosvitin in decreasing order of activity, and was insensitive to cAMP and the protein inhibitor. Exposure of intact brush border membranes to elevated temperatures revealed that phosphorylation of intrinsic membrane proteins and protamine was thermolabile, whereas cAMP-dependent histone kinase activity was relatively thermostable. These findings implicate cAMP-independent protamine kinases in the cAMP-independent autophosphorylation of the brush border membrane.