Rat liver cytosol phosphoprotein: Purification and enzymatic phosphorylation and dephosphorylation

Characterization of cytosol phosphopeptides

“…The purification procedure described in the present paper provided the demonstration that the two 32p-labelled peaks obtained by Sephadex G-200 gel filtration of rat liver cytosol phosphorylated crude fraction [ 1 ] contain the same or very similar phosphorylated components, which, according to their very low molecular weight, must be regarded as large phosphopeptides rather than phosphoproteins. Such a fraction is characterized by high phosphate content -over 4% -the highest ever reported for any phosphoprotein but phosvitin.…”

“…1). As previously reported [1], gel filtration resolves the labelled fraction into two radioactive peaks with sharply different specific radioactivities (expressed as cpm/mg protein). Both peaks however, once submitted to gel electrophoresis, give rise to a single major radioactive band exhibiting the same specific radioactivity independently of the G-200 peak from which it is derived.…”

“…Moreover both contain conspicuous amounts of protein-bound iron. On the contrary the cytosol phosphopeptides differ from histones and protamines, which also must be regarded as phosphorylated protein [ 10,12], for their acidic nature, high phosphate content and enzyme specificity [ 1 ].…”

“…Thirty to 40 mg of a crude preparation of cytosol phosphoprotein obtained by DEAE-cellulose column chromatography as previously described [1] were. incubated in 12 ml of a medium containing: 0.1 M Tris-HC1 buffer pH 7.5; 6 mM ATP containing 0.4 mCi as [3,-32P] ATP; 0.5 ml of cytosol phosvitin kinase purified as previously described [2].…”

“…In a previous paper the isolation from rat liver cytosol of a phosphoprote[n fraction has been described [1 ]. Such a phosphoprotein fraction, containing both phosphorylserine and phosphorylthreonine, was found to have a phosphate content approaching 1%.…”