Histone 4 Phosphotransferase Activities in Proliferating Lymphocytes PARTIAL PURIFICATION AND CHARACTERIZATION OF AN ENZYME SPECIFIC FOR SER-47*

Two histone H4 phosphotransferase activities are observed in DE52 chromatography or sucrose gradient ultra- centrifugation of murine lymphosarcoma cell extracts. One of these enzymes (H4-Kinase-(I)) has been purified several thousandfold by ion exchange and Sephadex G-200 chromatography. H4 phosphorylated an equimolar Hl, H2A, H2B, H3, and H4 incubated H4-Kinase-(I) and The phosphorylation rates of H2A, H2B, Hl, or H3 was ~20% of that observed with H4. The apparent K,,, values for ATP and H4 were 69 and 16 PM, respectively. The molecular weight of H4-Kinase-(I), estimated by gel filtration, is 102,000, consistent with an s~~,~ of 5.2 obtained from sucrose gradient centrifugation. H4-Kinase-(I) the formation of 1.02 mol of phosphoserinelmol of H4. the two amino acid serine H4

Two histone H4 phosphotransferase activities are observed in DE52 chromatography or sucrose gradient ultracentrifugation of murine lymphosarcoma cell extracts. One of these enzymes (H4-Kinase-(I)) has been purified several thousandfold by ion exchange and Sephadex G-200 chromatography. H4 was the only phosphorylated product detected when an equimolar mixture of histones Hl, H2A, H2B, H3, and H4 was incubated with H4-Kinase- (I) and [+*PlATP. The phosphorylation rates of H2A, H2B, Hl, or H3 was ~20% of that observed with H4. The apparent K,,, values for ATP and H4 were 69 and 16 PM, respectively.
The molecular weight of H4-Kinase-(I), as estimated by gel filtration, is 102,000, consistent with an s~~,~ of 5.2 obtained from sucrose gradient centrifugation. H4-Kinase-(I) catalyzed the formation of 1.02 mol of phosphoserinelmol of H4. Peptides corresponding to the two amino acid sequences containing serine in H4 have been synthesized.
The peptide Ac-Ser-Gly-Arg-Gly-Lys-Gly-Gly (residues 1 to 7) was not a substrate for H4-Kinase- (I) when tested at concentration of up to 1 mu. In contrast, the second peptide containing residues 43 to 49 with an added COOH-terminal glycine (i.e. Val-Lys-Arg-lle-Ser-Gly-Leu-Gly) was phosphorylated with an apparent K, of 35 pm. It is concluded that H4-Kinase-(1) specifically phosphorylates Ser-47 in Histone H4. The second H4-phosphotransferase (H4-Kinase-(II)) was partially purified by ion exchange chromatography and ammonium sulfate precipitation.
H4-Kinase-(II) phosphorylated the H4 peptide containing Ser-1 (residues 1 to 7) but not the H4 peptide containing Ser-47 (residues 43 to 49). these proteins with such modifications potentially associated with DNA replication, gene expression, the orderly assembly of the v body, and/or the separation of the genome at cell division.

Synthesis -The peptides
Ac-Ser-Gly-Arg-Gly-Lys-Gly, corresponding to amino acids 1 to 6 in H4, and Val-Lys-Arg-Ile-Ser-Gly-Leu-Gly, corresponding to amino acids 43 to 49 of H4 plus an added COOH-terminal glycine, were synthesized by the solid state synthesis method of Gutte and Merrifield (42) as previously described (361. In this text these peptides are designated the Ser-1 peptide and the Ser-47 peptide, respectively. The peptides were purified by chromatography on Sephadex G-25 and sulfopropyl (SP)-Sephadex.
The products from the Merrifield synthesis were applied to a column (2.5 x 80 cm) of Sephadex G-25 equilibrated with 30% acetic acid. The eluted fractions containing the peptide were pooled and lyophilized, and the residue was dissolved in 2 ml of 0.2 M pyridine/acetic acid (pH 2.8). The sample was applied to a column (1.9 x 80 cm) of SP-Sephadex equilibrated with the same buffer at 50". The peptide was eluted with a concave gradient of 0.2 M pyridineiacetic acid, pH 2.8, to 2 M pyridinelacetic acid, pH 5.1, in a total volume of 500 ml. Fractions containing the peptide were pooled and lyophilized.
The peptide Leu-Arg-Arg-Ala-Ser-Leu-Gly, which correpsonds to the amino acid sequence of pyruvate kinase that is phosphorylated by the CAMP-dependent protein kinase, was synthesized as described previously (36,42). This peptide, designated PK-1, has been shown to contain the primary structure specificity determinants for the CAMP-dependent protein kinase (43, 44). was added, and the digestion was continued for 1 h at 40". The proteolytic digestion was terminated by addition of acetic acid to a final concentration of 30%. The products were applied to a column (2.5 x 80 cm) of Sephadex G-25 equilibrated with 30% acetic acid. One major $*P-peptide, comprising >80% of the total radioactivity, was eluted as a symmetrical peak between 102 and 113 ml. The fractions were pooled, concentrated by lyophilization, and rechromatographed on Sephadex G-25 under identical conditions. The peptide fraction obtained was radiochemically pure on the basis of the symmetrical elution profile from Sephadex G-25 and by paper electrophoresis at pH 1.9 but contained several nonradioactive peptides.
Other Methods -Electrophoresis of phosphorylated histone products was performed according to the procedure of Panyim and Chalkley (38) in pre-electrophoresed 13% polyacrylamide gels (8 cm) containing 2.5 M urea, 0.9 M acetic acid, pH 3.2, using a methylene green tracking dye. All gels were stained with Coomassie blue in 45% methanol, 7.5% acetic acid, 47.5% water and destained overnight in the same buffer.
Gels containing phosphorylated products were sliced horizontally into 2-mm sections. Each section was placed on a piece (1 x 1.5 cm) of Whatman No. 31 ET chromatography paper, dried for 1 h at 115", and counted in 5 ml of toluene-based scintillation fluid containing 4 g/liter of Omnifluor (New England Nuclear).
Paper electrophoresis of 32P-labeled peptides was performed at pH 1.9 on strips (57 cm) of Whatman No. 3MM chromatography paper at 3000 V for 60 min at 25" using a solvent of 1.4 M e-.:tic acid, 0.6 M formic acid, pH 1.9.
Samples (10 to 300 nmol) for amino acid analysis were hydrolyzed in 2 ml of triple-distilled 5.7 N HCl in uacuo for 30 h at 105". Amino acid analysis was performed on a Durrum D-500 amino acid analyzer as previously described (36). Protein concentrations were determined by biuret or fluorescamine assays (45). Peptide concentrations were determined by quantitative amino acid analysis. During purification of the peptide concentrations were determined by fluorescamine assays (45).
In the absence of CAMP a low level of H2B phosphorylation, probably due to spurious dissociation of the CAMP-dependent holoenzyme, is detected in the 6.8 S region of the gradient (Fig.  1, A and B), but the predominant CAMP-independent H2B phosphotransferase activity sediments at 5.8 S. A major histone phosphotransferase, sedimenting in the same region of the gradient, is also detected with H2A as substrate ( Fig. 10 (Fig. 6C). This value exceeds the Mg*+ concentration required to form the ATP*Mg2+ complex, the established substrate for other kinase-catalyzed reactions, and suggests that Mg*+ also activates the reaction by interaction with either the protein substrate or the enzyme. Under the described assay conditions, maximum activity was observed in the presence of 10 mM MgZ+; Mg2+ in excess of this concentration was inhibitory (Table II). Mn*+ can substitute for Mg2+ in the metal ion. ATP complex and is equally effective as an apparent activator of H4-Kinase-(I) ( Table II). Mn*+, as well as KCl, K2HP01, and CaCl,, was inhibitory at high concentrations (Table II)   u Measured in the presence of 10 rn~ MgCl,.