Abstract
We report the purification from bovine brain and describe some of the properties of a 19-kDa protein, p19, which we have previously shown to undergo hormone-dependent, cAMP-mediated phosphorylation in several peptide hormone-producing tumor cells. The procedure for purifying p19 to apparent homogeneity utilized ammonium sulfate fractionation, sequential chromatography on DEAE-cellulose and phenyl-Sepharose, followed by fast protein liquid chromatography using a Mono Q and, finally, a C8 reverse-phase column. The yield was 0.3-0.5 mg of p19/kg of brain. The molecular weight (Mr = 19,000) and frictional ratio (f/f0 = 1.87) of p19, which were derived from its Stokes radius (33 A) and sedimentation constant (s20,w = 1.4), suggest that the native form of p19 is an asymmetrically shaped monomer. We provide evidence to suggest that p19 is isolated as a mixture of molecular forms consisting of an unphosphorylated form and of three phosphoforms indicative of multisite phosphorylation. These forms cosedimented on sucrose density gradients and coeluted on gel filtration, hydrophobic chromatography, and reverse-phase fast protein liquid chromatography. They were resolved from each other by anion-exchange chromatography. The unphosphorylated form (pI 6.2) was phosphorylated by catalytic subunit of cAMP-dependent protein kinase to a stoichiometry of 0.5 mol of P/mol of p19, thereby giving rise to the three phosphoforms (pI 5.8, pI 5.6, and pI 5.2, respectively). We conclude that p19 is a novel cAMP-dependent protein kinase substrate protein that is present in brain and in peptide hormone-producing tumor cells. Its function remains to be identified.
Highlights
The procedure for purifying p19 to apparenhtomo- ized [2, 3], most of the proteins that have been shown to geneity utilized ammonium sulfate fractionation, se- undergo secretagogue-stimulated phosphorylation have not pbtpqmfirhhruooaaeaenmtinsonnaeygtl.iilcrtT-raasoSalhcplteSuheihpomtryhomo(nakmufor.e/oslTfsaeisotnhcero=uga,egaldfya1roiMriaul.el8pwsool7dhw(ne)3yoiwego3dQfhaAbst.aopy)0nn1(DaM f.d93anE,-srd0Atwf=.pis5Ehner1i-aodcc9lmtihleem,ygl0ilw,neau0onel0fCotrla)pissetaqerlinudo9edinev/dkreficgravroscineeonchddf---ro-yffocoufeocnDtuiucnrubstssireeeuoidennlnoiog.nfncttahthhhneaeedrsparecoaosttslteheutredoiszrfeieevppdsere,orpwattelwiidiytenheepahhrrhaeosovs,rspemwpehoodconrtekrteteysoiclnatt(eht4tide-oh7inira,ssi1tlnsar1eutb,thco1etoru5afstr)eoe.1crIa9rynen-tkdthihDoaensa stant, suggest that the native formof p19 phosphoproteins (p19) in hamster insulinoma cells, migrating is an asymmetricallyshaped monomer
Without the addition of exogenous protein kinase, p19 did Our interest in p19 stems from the original observation not incorporate 32P(see autoradiogram, Fig. 6C)
The inclu- that this group of slightly acidic 19-kDa cytosolic proteins sion of catalytic subunit of CAMP-dependent protein kinase undergoes phosphorylation in hamster insulinoma cells when in the reaction mixture resulted in the appearance of two they are stimulated by glucagonto release insulin [11].Submajor and one minor 32P-labeledspots
Summary
As determined by SDS-PAGE and by two-dimensional electrophoresis, the last protein peak eluting at the first step (peak I) contained, in addition to numerous other proteins, p19, which consisted predominantly of the least acidic form. Step 4: FPLC Using a Mono Q Column-After each chromatography run using phenyl Sepharose, the fractions containing p19 were pooled and precipitatedby the addition of ammonium sulfate (see above), redissolved in H20, and dialyzed against 1000 volumes of buffer Afor2h at room temperature.
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