Affinity purification of newly phosphorylated protein molecules. Thiophosphorylation and recovery of histones H1, H2B, and H3 and the high mobility group protein HMG-1 using adenosine 5'-O-(3-thiotriphosphate) and cyclic AMP-dependent protein kinase.

I.Y Sun,E.M Johnson,V.G Allfrey

doi:10.1016/s0021-9258(19)86243-9

Abstract

Affinity purification of newly phosphorylated protein molecules. Thiophosphorylation and recovery of histones H1, H2B, and H3 and the high mobility group protein HMG-1 using adenosine 5'-O-(3-thiotriphosphate) and cyclic AMP-dependent protein kinase.

Highlights

MATERIALS AND METHODSPreparation of Protein Substrates-Calf thymus histones were prepared from chromatin which had been washed with 0.14 M NaCI, 0.5 mM phenylmethylsulfonyl fluoride
THIOPHOSPHORYLATION AND RECOVERY OF HISTONES H1, H2B, AND H3 AND THE HIGH MOBILITY GROUP PROTEIN HMG-1 USING ADENOSINE 5’-0-(3-THIOTRIPHOSPHATAEN)D CYCLIC AMPDEPENDENT PROTEIN KINASE*
The present study extends the application of ATP-y-S to protein phosphorylationc, ombiningthe kinase-mediated thiophosphorylation with procedures for the recovery of the thiophosphorylated molecules by mercury affinity chromatography

Summary

MATERIALS AND METHODS

Preparation of Protein Substrates-Calf thymus histones were prepared from chromatin which had been washed with 0.14 M NaCI, 0.5 mM phenylmethylsulfonyl fluoride. The standard reaction mixture for thiophosphorylation contained 5 pgof CAMP-dependent protein kinase and 250pgof histone (or other substrate) in 0.25 ml of 10 m~ Tris-HC1 (pH 7.0), 6 mM MgC12,3 mM dithiothreitol, 0.02 mM cyclic AMP, and 0.3 mM [y-%]ATP. The incorporation of [:1"S]thiophosphate and [32P]phosphate into proteins was determined by precipitation of 50-p1 aliquots of the reaction mixture with 5%trichloroacetic acid containing 0.25%sodium tungstate and 0.06 N H2SO4 [15]. Organomercurial-Sepharose Chromatography-Radioactive proteins were applied to a column (1 X 30 cm) of Affigel 501 (Bio-Rad, Inc.) which had been equilibrated with 10 mM Tris-HC1 (pH 7.9), 0.1 M NaC1,lO mM EDTA, 0.1% NaDodS04(Buffer A). Fractions containing radioactive proteins were pooled, dialyzed against 2 mM 2mercaptoethanol, concentrated by lyophilization,and used for further analyses. DodS04 [16].The DsS-labeledH1 bands were detected by fluorography of the gels, while 32P-labeledH1 bands were detected autoradiographically

RESULTS

O 12 14 16

DISCUSSION